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BRAK/CXCL14 expression suppresses tumor growth in vivo in human oral carcinoma cells
Abstract
In order to find a suppressor(s) of tumor progression in vivo for oral carcinoma (OC), we searched for molecules down-regulated in OC cells when the cells were treated with epidermal growth factor (EGF), whose receptor is frequently over-activated in OC. The expression of BRAK, which is also known as CXC chemokine ligand14 (CXCL14), was down-regulated significantly by the treatment of OC cells with EGF as observed by cDNA microarray analysis followed by reverse-transcriptase polymerase chain reaction analysis. The EGF effect was attenuated by the co-presence of a MEK inhibitor. The rate of tumor formation in vivo of BRAK-expressing vector-transfected tumor cells in athymic nude mice was significantly lower than that of mock vector-transfected ones. In addition tumors formed in vivo by the BRAK-expressing cells were significantly smaller than those of the mock-transfected ones. These results indicate that BRAK/CXCL14 is a chemokine, having suppressive activity toward tumor progression of OC in vivo.
... Despite mentioned pro-metastatic effect of CXCL14, in some cancer types, such as tongue cancer, it has been observed that CXCL14 upregulation reduces tumor cell migration and cell detachments [60,61]. Elongation in cell attachment after CXCL14 administration is attributed to the activation of Ras-related protein 1 (Rap1) [61]. ...
... MEK/ERK is a group of tyrosine kinases that mediates EGF/EGFR signal transduction. This pathway inhibits mRNA expression of CXCL14 [33,60]. Gefitinib is a tyrosine kinase inhibitor that can interfere with EGF/EGFR signal transduction by inhibiting MEK/ERK. ...
... Moreover, it has been shown that fibroblast-derived CXCL14 mainly promotes tumor development and invasion while epithelial-derived CXCL14 mediates tumor inhibition. However, all investigations unanimously agree that CXCL14 has tumor-suppressive activity in head and neck cancers such as oral carcinoma and tongue sarcoma [60,63]. To elucidate the definitive role of CXCL14 in cancer, it is suggested that mechanistic studies should be implemented. ...
Cancer is a leading cause of death worldwide and imposes a substantial financial burden. Therefore, it is essential to develop cost-effective approaches to inhibit tumor growth and development. The imbalance of cytokines and chemokines play an important role among different mechanisms involved in cancer development. One of the strongly conserved chemokines that is constitutively expressed in skin epithelia is the chemokine CXCL14. As a member of the CXC subfamily of chemokines, CXCL14 is responsible for the infiltration of immune cells, maturation of dendritic cells, upregulation of major histocompatibility complex (MHC)-I expression, and cell mobilization. Moreover, dysregulation of CXCL14 in several cancers has been identified by several studies. Depending on the type or origin of the tumor and components of the tumor microenvironment, CXCL14 plays a conflicting role in cancer. Although fibroblast-derived CXCL14 has a tumor-supportive role, epithelial-derived CXCL14 mainly inhibits tumor progression. Hence, this review will elucidate what is known on the mechanisms of CXCL14 and its therapeutic approaches in tumor treatment. CXCL14 is a promising approach for cancer immunotherapy.
... To identify promising target molecules for applications in the treatment of head and neck squamous cell carcinoma (HNSCC), we employed tongue carcinoma-derived HSC-3 cells in culture. Epidermal growth factor (EGF) signaling is frequently overactivated in various carcinoma cells, including HNSCC cells; thus, we cultured HSC-3 cells under serum-free conditions, treated them with EGF to mimic tumor progression in vivo, and identified genes whose expression was significantly altered using DNA chip analysis followed by quantitative real-time polymerase chain reaction [7]. Expression levels of mRNAs for matrix metalloproteinase-1 (MMP-1), also known as animal collagenase, and vimentin were significantly increased. ...
... In contrast, the expression levels of the tissue inhibitor of matrix metalloproteinase 3 (TIMP3), which inhibits the activity of various MMPs, and insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), which suppresses the activities of IGF-1 and-2 by binding to these growth factors with high affinity, were significantly down-regulated. However, the down-regulation of the chemokine CXCL14 [8] was the most prominent [7]. ...
... The rate of tumor formation in vivo in CXCL14-expressing vector-transfected tumor cells (HSC-3-CXCL14) in athymic nude mice or T and B cell-deficient SCID mice was significantly lower than that of mock vector-transfected control cells, although the growth rates of the expression vector-transfected and mock vector-transfected cells were not different under in vitro culture conditions [7,9,10]. In addition, the volume of tumors formed in vivo by CXCL14-expressing cells was significantly smaller than that of mock-transfected cells [7,[9][10][11]. ...
Cancer is a leading cause of death and disease worldwide, with a tremendous financial impact. Thus, the development of cost-effective novel approaches for suppressing tumor growth and progression is essential. In an attempt to identify the mechanisms responsible for tumor suppression, we screened for molecules downregulated in a cancer progression model and found that the chemokine CXCL14, also called BRAK, was the most significantly downregulated. Increasing the production of CXCL14 protein by transfecting tumor cells with a CXCL14 expression vector and transplanting the cells into the back skin of immunodeficient mice suppressed tumor cell growth compared with that of parental tumor cells, suggesting that CXCL14 suppressed tumor growth in vivo. However, some studies have reported that over-expression of CXCL14, especially in stromal cells, stimulated the progression of tumor formation. Transgenic mice expressing 10-fold more CXCL14 protein than wild-type C57BL/6 mice showed reduced rates of chemical carcinogenesis, transplanted tumor growth, and metastasis without apparent side effects. CXCL14 also acts as an antimicrobial molecule. In this review, we highlight recent studies involving the identification and characterization of CXCL14 in cancer progression and discuss the reasons for the context-dependent effects of CXCL14 on tumor formation.
... CXCL14 is an ELR (Glu-Leu-Arg)-negative chemokine that is abundantly expressed in most normal tissues [9][10][11][12]. However, many epithelial cancer cell lines and primary carcinomas do not express CXCL14, suggesting that it may have a tumor suppressor function [10,[12][13][14]. ...
... CXCL14 is an ELR (Glu-Leu-Arg)-negative chemokine that is abundantly expressed in most normal tissues [9][10][11][12]. However, many epithelial cancer cell lines and primary carcinomas do not express CXCL14, suggesting that it may have a tumor suppressor function [10,[12][13][14]. CXCL14 expression is suppressed by epidermal growth factor (EGF) and can be restored by treatment with an EGF receptor (EGFR) tyrosine kinase inhibitor in head and neck squamous cell carcinoma (HNSCC) cells [15]. ...
... EGFR is highly expressed in several types of carcinomas including HNSCC and high expression levels of EGFR reduce recurrence-free or overall survival rates in 70% of patients and act as a strong prognostic indicator [32]. The activation of EGFR often coincides with the down-regulation of CXCL14 in HNSCC [10,15]. It has to take into account that most studies on the role of CXCL14 in malignancies have been based on results of the CXCL14 mRNA level [11,33,34]. ...
Background: Chemokines selectively attract and activate leukocytes and play roles in a variety of homeostatic and disease processes. Explore the biological properties of CXCL14 seems complicated due to unknown functional characteristics of CXCL14 in cancer.
Methods: To study the multistep process of oral cancer development, we analyzed oral samples spanning normalcy, dysplasia and cancer from multiple perspectives, revealing a cascade of progressive changes.
Results: CXCL14 protein was expressed in the cytoplasm adjacent to tumors. T classification (P<0.001), clinical stage (P=0.0013) and nodal metastasis (P=0.0035) were significantly associated with CXCL14 in relationships between CXCL14 expression levels and tumor and patient characteristics. Compared with non-tumor tissue, expression of the epidermal growth factor receptor (EGFR) gene was increased in dysplasia and was further sustained in cancer. Our data show an inverse relationship between CXCL14 and EGFR expression levels in tumor cells indicating that CXCL14 expression is beneficial for tumor suppression. To explore epigenetic regulation and the impact of CXCL14 on oral cancer, analysis of CpG islands methylation in the CXCL14 promoter region indicated that the abnormal hypermethylation of that promoter region in tumor cells and tissues is one of the mechanisms causing the reduced expression. Restoration of CXCL14 expression was induced by treatment with 5-aza-2′-deoxycytidine. Using in vivo mouse models, we demonstrate that the restoration of CXCL14 expression in irradiation-induced oral carcinoma cells induces the expression of Late Cornified Envelope (LCE) genes.
Conclusions: Our data suggest that LCE genes are a novel target of CXCL14 and are likely to have a tumor suppressor function through the modulation of CXCL14 expression. In conclusion, CXCL14 might play a pivotal role in the pathobiology of oral cancer, probably by regulating DNA methylation and leukocyte migration. The level of CXCL14 expression may be a valuable adjuvant parameter to predict the prognosis of patients with oral carcinoma and may be a potential therapeutic target.
... derived cells for xenotransplantation in mice. The rate of tumor formation in athymic nude mice or in severe combined immunodeficiency (SCID) mice following xenotransplantation was significantly lower for the CXCL14-expressing cells than for the mocktransfected cells, though no differences were observed in the growth rates of these cells under in vitro culture conditions [31,32]. These data indicate that CXCL14 expression in tumor cells functions to suppress the growth of these cells in vivo [31][32][33]. ...
... The rate of tumor formation in athymic nude mice or in severe combined immunodeficiency (SCID) mice following xenotransplantation was significantly lower for the CXCL14-expressing cells than for the mocktransfected cells, though no differences were observed in the growth rates of these cells under in vitro culture conditions [31,32]. These data indicate that CXCL14 expression in tumor cells functions to suppress the growth of these cells in vivo [31][32][33]. Next, in order to confirm whether CXCL14 would have a tumorsuppressing effect on cells of other tissue origins, we produced transgenic (Tg) mice expressing a 10-fold higher level of CXCL14 compared with the level produced by isogenic wild-type C57BL/6 (Wt) mice. Therefore, the Tg mice showed significantly less size increase in tumors formed by transplanted melanoma cells or Lewis lung carcinoma (LLC) cells compared with tumor-bearing Wt mice [24,34]. ...
... Further, the survival rates for Tg mice after tumor cell injection were significantly increased (Fig. 2i). Since the Tg mice showed no obvious abnormality, and CXCL14 reportedly suppresses the growth of transplanted tumors of different cellular origins, such as squamous cell carcinoma [32][33][34], adenocarcinoma [24], melanoma [24,34], and fibrosarcoma [35], and also suppresses the metastasis of melanoma and adenocarcinoma cells in the lungs of mice [24], we propose CXCL14 as a promising extracellular multistep tumor suppressor with clinical potential for cancer suppression/prevention [24]. ...
Background The multi-step nature of cancer that involves carcinogenesis, an increase in tumor size, and invasion and/or metastasis is well recognized. These respective steps depend on the mutation of several genes, epigenetic changes in cells, and interactions between cancer cells and other cells in the microenvironment. To identify novel intercellular tumor suppressors, we screened for genes whose expression was down-regulated in oral cancer cells. Highlight The chemokine, CXCL14, was down-regulated in oral carcinoma cells, and its up-regulation suppressed tumor cell growth in vivo, indicating that CXCL14 is a tumor growth suppressor. Moreover, to investigate whether CXCL14 suppresses tumors originating from the other cells in a paracrine or endocrine fashion, we generated CXCL14 transgenic (Tg) mice. The Tg mice exhibited a suppressed rate of carcinogenesis, decreased volume of transplanted tumors, and reduced pulmonary metastasis, as well as an increased survival rate following tumor cell injection, compared with wild-type mice. The CXCL14-expressing Tg mice showed no apparent abnormality when observed up to 2 years of age, and, interestingly, in a normal human population an individual was found to have a blood level of CXCL14 protein 10-fold higher than the average but did not present any apparent abnormalities. Conclusion These data indicate that CXCL14 expressed at high levels does not have severe side effects, and, thus, we propose CXCL14 as a promising molecular target for cancer suppression/prevention. © 2015 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
... As a unique member of chemokine family, CXCL14 is currently suggested as a chemoattractant for dendritic cells (DC), activated monocytes, neutrophils, and natural killer cells (NK) under different pathophysiological conditions (Kurth et al., 2001;Shellenberger et al., 2004;Shurin et al., 2005;Starnes et al., 2006;Salogni et al., 2009). It has been also reported that CXCL14 was a potent mediators of neoangiogenesis and tumor growth, invasion, metastasis Ozawa et al., 2006;Augsten et al., 2009), which shares many similarities with the process of trophoblast invasion during early pregnancy (Fitzgerald et al., 2008). To date, the specific receptor of CXCL14 has not yet been found. ...
... Given the abundant expression of CXCL14 on trophectoderm and trophoblast of periimplantation embryo, and several reports showing CXCL14 as a tumor migration regulator Ozawa et al., 2006;Pelicano et al., 2009), here we want to address the question as to whether CXCL14 participated in regulating trophoblast invasion during embryo implantation, a process similar to tumor invasion in many aspects. Here an in vitro culture system was utilized to assess the ability of blastocyst attachment and outgrowth in the presence of rmCXCL14 or CXCL14 antibody. ...
... To our best knowledge, most of the currently studied chemokines with a regulatory role during early pregnancy shows a promontory effect on trophoblast migration. However, our results showed that CXCL14 played an evidenced inhibitory role on trophoblast attachment and outgrowth, this unique feature of CXCL14 on trophoblast migration were also in accordance with several reports that CXCL14 have a tumor suppressive function Ozawa et al., 2006). Furthermore, our parallel study in human pregnancy also showed an inhibitory effect of CXCL14 when culturing human trophoblast in vitro (unpublished data in our lab). ...
CXCL14, a member of chemokine family, was previously known to participate in many pathophysiological events, such as leukocytes recruitment and tumor suppression. However, it remained largely unknown whether CXCL14 is a physiological player during early pregnancy. In this regard, our recent global gene microarray analysis has observed an implantation-specific expression profile of CXCL14 mRNA during early pregnancy in mice, showing its higher levels at implantation sites compared to inter-implantation sites, implicating a potential role of CXCL14 in the periimplantation events. In the present investigation, using Northern blot, in situ hybridization and immunostaining, we further demonstrated that uterine CXCL14 expression was specifically induced at embryo implantation site and expanded with subsequent decidualization process in a spatiotemporal manner. The implanting embryo also showed a highlighted expression of CXCL14 in the blastocyst trophectoderm and its derived ectoplacental cones (EPCs) during postimplantation development. In vitro functional study revealed that CXCL14 could significantly inhibit both primary and secondary trophoblast attachment and outgrowth, correlated with a stage-dependant downregulation of MMP-2 and/or MMP-9 activity. Moreover, it was found that biotinylated CXCL14 could specifically bind to trophoblast cells in vitro and in vivo, suggesting trophoblast cell, perhaps expressing the unidentified CXCL14 receptor, is a bioactive target of CXCL14. Collectively, our findings provide evidences supporting the contention that CXCL14 is an important paracrine/autocrine modulator regulating trophoblast outgrowth at the maternal–fetal interface during the process of pregnancy establishment. This study is clinically related since CXCL14 is also highly expressed in human receptive endometrium and trophoblasts.
... CXCL14, also known as BRAK, is a member of the CXC chemokine family preferentially expressed in liver, small intestine, and uterus without the necessity of inflammatory stimulation (9 -11). The expression of CXCL14 in tumors was heterogeneous, the majority of malignant tissues showing loss of CXCL14 expression (9,(12)(13)(14)(15). It was also demonstrated that CXCL14 was a potent mediators of neoangiogenesis and tumor growth, invasion, metastasis (13,(15)(16)(17)(18)(19)(20). ...
... The expression of CXCL14 in tumors was heterogeneous, the majority of malignant tissues showing loss of CXCL14 expression (9,(12)(13)(14)(15). It was also demonstrated that CXCL14 was a potent mediators of neoangiogenesis and tumor growth, invasion, metastasis (13,(15)(16)(17)(18)(19)(20). In situ hybridization analyses revealed that CXCL14 mRNA was expressed in human placenta and that its expression is regulated during gestation (21,22). ...
... Given the evident expression of CXCL14 on trophoblast cells and established function in tumor growth and metastasis (13,17), we next investigated whether CXCL14 can regulate trophoblast outgrowth of villous explants on Matrigel. Culture medium and Matrigel were supplemented with increasing doses (0 -100 ng/ml) of recombinant human CXCL14, and the outgrowth distance of villous explant trophoblast at the Matrigel surface was monitored at 24, 48, and 72 h. ...
... CXCL14, initially called BRAK for its isolation from breast and kidney cells, was first described in 1999 as a chemokine preferentially expressed in normal peripheral tissue cells (15). Reduced expression in progressing cancer suggested the involvement of CXCL14 in antitumor activity (16,17). The role of CXCL14 in tumorigenesis, however, is controversial as more recent studies pointed to its upregulation in cancer-associated fibroblasts of prostate cancer and in pancreatic cancer (18,19). ...
... Proteolytic processing with proteinase 3 generates the bactericidal peptide CXCL14 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] Because we found that different peptides derived from the N-terminal portion of CXCL14 have antimicrobial activity, we FIGURE 4. Structure and primary sequence of human CXCL14. Sequences in red of the ribbon diagram correspond to the N-terminal region of (CXCL14 1-13 ), the core structure containing three antiparallel b-strands (CXCL14 , and the C-terminal a-helix (CXCL14 55-77 ). ...
... In vitro cleavage experiments showed that proteinase 3, one of the main proteases secreted by activated neutrophils, specifically and efficiently processed CXCL14, and several proteolytic fragments were already detectable after 1 min of protease exposure (Fig. 7A). Mass-spectrometry analysis and Edman degradation of the fragments identified a mass corresponding to CXCL14 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] . Of note, the antibacterial activity of synthesized CXCL14 1-17 was highly effective and comparable with full-length CXCL14 against E. coli and P. aeruginosa but was ineffective against the Grampositive S. mitis and S. pneumoniae, which is in accordance to the restricted activity of CXCL14 1-13 and related peptides. ...
CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.
Copyright © 2015 by The American Association of Immunologists, Inc.
... In order to further investigate whether CXCL14 has a tumoursuppressing effect in vivo, we prepared and cloned CXCL14-expression vector-transfected and mock vector-transfected tongue tumourderived cells. The rate of tumour formation in athymic nude mice or in severe combined immunodeficiency (SCID) mice following xenotransplantation was significantly lower for the CXCL14-expressing cells than for the mock-transfected cells, even though no differences were observed in the growth rates of these cells under in vitro culture conditions 18,19 . These data indicate that CXCL14 expression in tumour cells functioned to suppress the growth of these cells in vivo [18][19][20] . ...
... The rate of tumour formation in athymic nude mice or in severe combined immunodeficiency (SCID) mice following xenotransplantation was significantly lower for the CXCL14-expressing cells than for the mock-transfected cells, even though no differences were observed in the growth rates of these cells under in vitro culture conditions 18,19 . These data indicate that CXCL14 expression in tumour cells functioned to suppress the growth of these cells in vivo [18][19][20] . Next, in order to confirm whether CXCL14 would have a tumor suppressing effect on cells of other tissue origins, we produced transgenic (Tg) mice expressing 10-fold higher blood CXCL14 compared with the level produced by isogenic wild-type C57BL/6 (Wt) mice and found that these Tg mice significantly suppressed increase in the size of tumours formed by transplanted B16 melanoma cells or Lewis lung carcinoma (LLC) cells compared with Wt mice 17,21 . ...
... CXCL14 is expressed ubiquitously and constitutively in epithelia throughout the body 45 , and there are apparently contradictory data in the literature regarding the relationship between CXCL14 expression and tumour formation. For example, down-regulation of CXCL14 expression has been associated with multiple adenocarcinomas, such as those of the prostate 46 and lungs 47 , as well as colon carcinomas 48 and HNSCC [17][18][19][20] . On the other hand, in some other reports heightened expression of this chemokine was observed in these same types of carcinomas and adenocarcinomas [49][50][51] . ...
Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention. S ide effects are the most serious obstacles in the case of cancer therapeutics 1–4. Thus, prevention of cancer remains the most promising strategy for reducing its incidence and associated mortality due to this disease 5,6. Tumour progression has been shown to be largely dependent on the expression of tumour-promoting and tumour-suppressing genes, with the balance being in favour of the former at each step 7. The protein products of these oncogenes and tumour suppressor genes function as regulatory intracellular signalling molecules during this process. Recently, it was revealed that the cancer microenvironment also influences carcinogenesis and cancer progression 8,9
... Promotion of trafficking of natural killer cells to the sites of inflammation [24] and macrophage infiltration into white adipose tissue in obese mice fed a high-fat diet [25], as well as inhibition of angiogenesis [26], were also reported as functions of this chemokine. We found that expression of BRAK/CXCL14 is downregulated significantly by the treatment of HNSCC cells with EGF as observed by cDNA microarray analysis (Table 2) [1] followed by reversetranscriptase polymerase chain reaction analysis. In order toFigure 1 : CXCL14/BRAK expression in HNSCC cells suppresses growth of tumor cell xenografts in athymic nude mice. ...
... Difference between mock-transfected and BRAK-expressing cells are shown. Significant difference in the sizes of tumors was observed (Cited from [1]). investigate whether CXCL14/BRAK has a tumor-suppressing effect in vivo, we prepared CXCL14/BRAK-expression vectortransfected and mock vector-transfected tongue tumor cells. ...
... investigate whether CXCL14/BRAK has a tumor-suppressing effect in vivo, we prepared CXCL14/BRAK-expression vectortransfected and mock vector-transfected tongue tumor cells. The rate of in vivo tumor formation by BRAK-expressing vector-transfected tumour cells in athymic nude mice (Figure 1) or SCID (Figure 2) mice was significantly lower than that of mock vector-transfected ones; and, in addition, the tumors formed in vivo by these BRAK-expressing cells are significantly smaller than those of the mock vectortransfected ones [1, 2]. Interestingly, the oral administration of gefitinib, an inhibitor of EGF receptor, significantly (P < 0.001) reduces tumor growth of xenografts of three HNSCC cell lines (HSC-2, HSC-3, and HSC-4) in female athymic nude mice accompanied by an increase in CXCL14/BRAK expression specifically in the tumor tissue (Figures 3(a), 3(b), 3(c), and 3(e)). ...
In order to find a suppressor(s) of tumor progression in vivo for head and neck squamous cell carcinoma (HNSCC), we searched for molecules downregulated in HNSCC cells when the cells were treated with epidermal growth factor (EGF), whose receptor is frequently overactivated in HNSCC. The expression of BRAK, which is also known as CXC chemokine ligand 14 (CXCL14), was downregulated significantly by the treatment of HNSCC cells with EGF as observed by cDNA microarray analysis followed by reverse-transcriptase polymerase chain reaction analysis and western blotting. The EGF effect on the expression of CXCL14/BRAK was attenuated by the copresence of inhibitors of the EGF receptor, MEK, and ERK. The rate of tumor formation in vivo of BRAK-expressing vector-transfected tumor cells in athymic nude mice or SCID mice was significantly lower than that of mock vector-transfected ones. In addition tumors formed in vivo by the BRAK-expressing cells were significantly smaller than those of the mock-transfected ones. These results indicate that CXCL14/BRAK is a chemokine having suppressive activity toward tumor progression of HNSCC in vivo. Our approach will be useful to find new target molecules to suppress progression of tumors of various origins in addition to HNSCC.
... [2][3][4][5][6] In head and neck squamous cell carcinoma (HNSCC), CXCL14 expression has been demonstrated to be reduced in tumor tissues relative to adjacent normal or oral dysplasia tissues. 7 CXCL14 has also previously been associated with decreased growth in HNSCC xenograft models, [8][9][10] supporting its role as a tumor suppressor in HNSCC. This decreased expression has been attributed to epigenetic silencing of CXCL14 by promoter hypermethylation in tumor tissues, 7 11 and CXCL14 expression was shown to be restored by treatment with 5-azacytidine, 7 12 a demethylating agent. ...
... Of these, CXCL14 was the most highly downregulated gene (2.9-fold change, p=10 −43 ) in lymph nodes relative to primary tumors, suggesting it may play a role in suppressing metastasis in HNSCC. Given the previously described associations of CXCL14 with reduced tumor growth in HNSCC, [8][9][10] we focused further investigations on the role of this chemokine as a tumor suppressor and, in particular, on its ability to induce the host immune response. ...
Objectives:
To explore lymphocyte infiltration as a potential mechanism behind CXCL14-mediated tumor growth suppression in oral cavity squamous cell carcinoma (OSCC).
Methods:
We analyzed single cell RNA-sequencing (scRNA-seq) data from OSCC to identify expression changes among malignant cells in lymph nodes (LN) versus primary tumors. CXCL14 expression in murine OSCC cell lines was quantified using qRT-PCR. Short hairpin RNA knockdown of CXCL14 was performed in mouse oral cavity (MOC)1 cells, and CXCL14 overexpression was performed in MOC2 cells. Cells in each condition were injected into C57BL/6 mice with and without T cell depletion, and tumor volume was measured. At 30 days, tumors were dissociated and analyzed by flow cytometry for CD45+CD3+ T cells. CXCL14 expression was correlated with gene expression signatures of tumor infiltrating lymphocytes (TIL) in scRNA-seq data, as well as TCGA tumors.
Results:
scRNA-seq revealed CXCL14 as the most significantly downregulated gene among malignant cells in LNs relative to primary tumor, supporting a role in preventing invasion and/or metastasis. In a murine immunocompetent model, CXCL14 expression was higher in indolent MOC1 cells than in more aggressive MOC2 cells. Tumor growth in vivo was significantly increased by CXCL14 knockdown in MOC1 cells relative to control, with a corresponding decrease in TIL. In MOC2 cells, tumor growth was significantly reduced by CXCL14 overexpression relative to control and TIL were increased. Both effects were lost with T cell depletion. In a human tumor scRNA-seq cohort, we found that only malignant cell CXCL14, but not non-malignant cell or fibroblast CXCL14, was associated with TIL. Bulk CXCL14 from the TCGA cohort had no association with TIL.
Conclusions:
Higher CXCL14 expression by tumor cells is associated with reduced tumor growth and increased TIL, supporting immune-mediated suppression of tumor growth in OSCC. Given that CXCL14 is downregulated in LN metastases compared with primary tumors, our data raise the possibility that CXCL14-mediated immune infiltration may discourage invasion and metastasis. In human scRNA-seq data, only malignant cell-specific CXCL14 was associated with TIL, suggesting a critical context-dependent effect of CXCL14 expression.
... These findings further support an important role for CXCL14 in the control of immune cell function in peripheral tissues during the steady-state. Although numerous reports have implicated CXCL14 in cancer, its expression is increased in some forms of cancer and decreased in others, leading to confusion on whether CXCL14 displays anti-tumour or tumour-promoting properties (Augsten et al., 2009, Frederick et al., 2000, Ozawa et al., 2006, Shurin et al., 2005, Wente et al., 2008. ...
... Previous studies into the function of CXCL14 have reported roles in diverse processes such as killing of microorganisms , Maerki et al., 2009) and tumour progression (Augsten et al., 2009, Ozawa et al., 2006, Shurin et al., 2005, Wente et al., 2008, as well as roles unrelated to immune function including regulation of body weight and glucose metabolism Tanegashima, 2012, Tanegashima et al., 2010a). Our research group has an invested interest in the immune surveillance of skin during the steady-state. ...
The human chemokine family consists of around 50 peptides that control the
migratory patterns and positioning of all leukocytes. One such member of this family
is CXCL14. Very highly expressed in many healthy tissues including skin, gut and
kidney, loss of CXCL14 expression in chronic inflammatory conditions and certain
forms of cancer has led to a proposed role for CXCL14 in immune surveillance at
these sites. The function and target cells of CXCL14 are poorly defined however,
largely because the identity of its receptor remains unknown.
Here, I have combined the evaluation of chemotactic responses toward CXCL14 with
detection of putative CXCL14 receptor(s) on the surface of cells using a synthetic,
fluorochrome-conjugated CXCL14, to definitively identify CXCL14 target cells in
human. Monocytes were identified as the major target cells in peripheral blood, 28.4
± 6.1% Monocytes migrating toward 1 µM CXCL14 in ex vivo transwell chemotaxis
assays compared to 3.01 ± 0.65% toward buffer alone (p=0.0031). Responses to
CXCL14 also identified tissue phagocytes extracted from healthy human skin,
including an apparently novel population of skin-resident CD14+ cells characterised
by lack of CD45 expression. Screening of CXCL14-responsive cells by RNA
sequencing for expression of G protein-coupled receptors revealed five major
candidates for the CXCL14 receptor, all of which are orphan receptors; GPR35,
GPR68, GPR84, GPR141 and GPR183. At present, I am in the process of testing
these candidates in functional assays. Finally, I report on a novel ability of CXCL14 to
potently synergise with other chemokines, particularly CXCL12. This ‘synergy’ with
CXCL12 likely occurs via a direct interaction between CXCL14 and the receptor for
CXCL12, CXCR4, which is broadly expressed on immune cells.
This work identifies mononuclear phagocytes in blood and tissue as the primary
targets for CXCL14, providing new and exciting insights into the role played by
CXCL14 in immune surveillance of peripheral tissues.
... Note: NA, no published data available.Upregulated in stromal cells and downregulated in epithelial cells.We and other groups have revealed tumor suppressor functions of CXCL14 involved in immune responses, cell motility, and angiogenesis. Several studies have consistently shown that restoration of CXCL14 expression in cancer cells suppresses tumor growth in mouse models of HNC, melanoma, colon, liver, and lung cancers.12,32,34,[63][64][65] CXCL14 is expressed at a high level in normal oral epithelial cells, but frequently absent in oral carcinoma cells.10,66 ...
... CXCL14 is expressed at a high level in normal oral epithelial cells, but frequently absent in oral carcinoma cells.10,66 However, human oral squamous carcinoma cells, in which CXCL14 expression is restored by transfection of a CXCL14 expression vector, showed significantly slower tumor growth in athymic nude or severe combined immunodeficiency mice compared with vectortransfected cells.63,64 Similarly, our previous study showed that HPV-positive mouse HNC cells engineered to express physiological levels of CXCL14 considerably suppressed tumor growth in immunocompetent syngeneic mice.12 ...
The chemokine CXCL14 is a highly conserved, homeostatic chemokine that is constitutively expressed in skin epithelia. Responsible for immune cell recruitment and maturation, as well as impacting epithelial cell motility, CXCL14 contributes to the establishment of immune surveillance within normal epithelial layers. Furthermore, CXCL14 is critical to upregulating major histocompatibility complex class I expression on tumor cells. Given these important roles, CXCL14 is often dysregulated in several types of carcinomas including cervical, colorectal, endometrial, and head and neck cancers. Its disruption has been shown to limit critical antitumor immune regulation and is correlated to poor patient prognosis. However, other studies have found that in certain cancers, namely pancreatic and some breast cancers, overexpression of stromal CXCL14 correlates with poor patient survival due to increased invasiveness. Contributing to the ambiguity CXCL14 plays in cancer is that the native CXCL14 receptor remains uncharacterized, although several candidate receptors have been proposed. Despite the complexity of CXCL14 functions, it remains clear that this chemokine is a key regulatory factor in cancer and represents a potential target for future cancer immunotherapies.
... The non-ELR-motif chemokine CXCL14, 27 which lacks a Glu-Leu-Arg tripeptide sequence adjacent to the CXC motif, is a homoeostatic chemokine that reportedly stimulates the chemotaxis of B cells and monocytes, 28 dendritic cells 29,30 and natural killer cells, 31,32 and also suppresses angiogenesis. 29,33 CXCL14 is known to function as a tumour suppressor in HNSCC, 34,35 breast cancer, 36 lung cancer 37 and hepatocellular carcinoma. 38 In a previous study, we demonstrated that CXCL14 expression is significantly downregulated by the activation of EGFR signalling, 34 and that the restoration of CXCL14 expression contributes to the tumour-suppressive effect of gefitinib, a selective tyrosine kinase inhibitor of EGFR. ...
... 29,33 CXCL14 is known to function as a tumour suppressor in HNSCC, 34,35 breast cancer, 36 lung cancer 37 and hepatocellular carcinoma. 38 In a previous study, we demonstrated that CXCL14 expression is significantly downregulated by the activation of EGFR signalling, 34 and that the restoration of CXCL14 expression contributes to the tumour-suppressive effect of gefitinib, a selective tyrosine kinase inhibitor of EGFR. 39 Recently, CXCL14 expression was demonstrated to be silenced by DNA hypermethylation in many malignant tumours, including lung cancer, 37 colon cancer, 40 stomach cancer 41 and acute myeloid leukaemia. ...
Cetuximab, a monoclonal antibody against the epidermal growth factor receptor (EGFR), has been successfully used to treat some patients with colorectal cancer and those with head and neck squamous cell carcinoma (HNSCC). For the effective treatment, it is essential to first identify cetuximab-responsive patients. The level of EGFR expression and/or the presence of mutations in signalling molecules downstream of the EGFR pathway have been reported to be determining factors for cetuximab responsiveness in colorectal cancer patients; however, limited data have been reported for HNSCC patients. We previously reported that the chemokine CXCL14 exhibits tumour-suppressive effects against xenografted HNSCC cells, which may be classified into two groups, CXCL14-expressing and non-expressing cells under serum-starved culture conditions. Here we employed CXCL14-expressing HSC-3 cells and CXCL14-non-expressing YCU-H891 cells as representatives of the two groups and compared their responses to cetuximab and their CXCL14 expression under various conditions. The growth of xenografted tumours initiated by HSC-3 cells, which expressed CXCL14 in vivo and in vitro, was suppressed by the injection of cetuximab into tumour-bearing mice; however, neither the expression of the chemokine nor the cetuximab-dependent suppression of xenograft tumour growth was observed for YCU-H891 cells. Both types of cells expressed EGFR and neither type harboured mutations in signalling molecules downstream of EGFR that have been reported in cetuximab-resistant colon cancer patients. The inhibition of the extracellular signal-regulated kinase (ERK) signalling increased the levels of CXCL14 messenger RNA (mRNA) in HSC-3 cells, but not in YCU-H891 cells. We also observed that the CXCL14 promoter region in YCU-H891 cells was hypermethylated, and that demethylation of the promoter by treatment with 5-aza-2'-deoxycytidine restored CXCL14 mRNA expression and in vivo cetuximab-mediated tumour growth suppression. Finally, we observed in vivo tumour growth suppression when YCU-H891 cells were engineered to express CXCL14 ectopically in the presence of doxycycline. These results indicate that CXCL14 expression may be a good predictive biomarker for cetuximab-dependent tumour suppression.
... CXCL14 has been implicated in the maintenance of tissue macrophages, cancer progression, and metabolic regulation [6]. Interestingly, CXCL14 is downregulated in malignant tumors [7,8], and it has been shown to exhibit tumor suppressor activity [9,10]. CXCL14 chemoattracts tissue macrophages [11], iDCs [12,13] and natural killer cells [14], but not T cells. ...
... Since the activity of CXCL14 is analogous to the CXCL12 dimer, CXCL14 could suppress the tumor growth via specific inhibition of the CXCL12-CXCR4 signaling. In support of this hypothesis, CXCL14 possesses the tumor suppressive activity against certain types of carcinoma cells [9,10]. ...
Activation of the CXCL12-CXCR4 pathway is crucial for the migration of hematopoietic stem cells, various immune cells, and malignant tumor cells. Here, we show that another CXC chemokine, CXCL14, specifically binds to CXCR4 with high affinity and inhibits the CXCL12-mediated chemotaxis of human leukemia-derived cell lines and CD34+ hematopoietic progenitor cells. Thus, CXCL14 functions as a natural inhibitor of CXCL12. Our observations suggest that CXCL14 represents, along with CXCR7, molecules that co-evolved with the CXCL12-CXCR4 axis to modulate important physiological processes in development, stem cell maintenance, and immune responses.
... MAS-related GPR family member X3 (MRGPRX3), a member of the mas-related/sensory neuron specific subfamily of G protein coupled receptors, is down-regulated in human airway epithelial cells exposed to smoke from electronic cigarettes 31 . In oral cancers, lung cancers, and head and neck cancers, C-X-C Motif chemokine ligand 14 (CXCL14) functions as a tumor suppressor; it also induces growth of prostate and breast cancers [32][33][34][35][36] . Protocadherin 7 (PCDH7) is involved in cell-cell recognition and adhesion 37 . ...
During 2020, understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) became a scientific priority due to the devastating effects of the COVID-19. Many researchers have studied the effect of this viral infection on lung epithelial transcriptomes and deposited data in public repositories. Comprehensive analysis of such data could pave the way for development of efficient vaccines and effective drugs. In the current study, we obtained high-throughput gene expression data associated with human lung epithelial cells infected with respiratory viruses such as SARS-CoV-2, SARS, H1N1, avian influenza, rhinovirus and Dhori, then performed comparative transcriptome analysis to identify SARS-CoV-2 exclusive genes. The analysis yielded seven SARS-CoV-2 specific genes including CSF2 [GM-CSF] (colony-stimulating factor 2) and calcium-binding proteins (such as S100A8 and S100A9), which are known to be involved in respiratory diseases. The analyses showed that genes involved in inflammation are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein–protein interaction analyses were consistent with a functional role of CSF2 and S100A9 in COVID-19 disease. In conclusion, our analysis revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.
... Notably increased PF-4 concentration in OSCC saliva was firstly described in the current research. In contrast, decreased PF-4 expression has been found in OSCC cells and its induced up-regulation resulted in suppressed activity toward oral cancer tumor progression in vivo [46,47]. ...
This study aimed to investigate the role of a panel of salivary cytokines as biomarkers for early detection oral squamous cell carcinoma (OSCC), comparing their levels among healthy individuals, patients with oral leukoplakia (OL), and malignant lesions. Cytokine profiling analysis performed in a minimally invasive sample was correlated with clinicopathological variables in our patient cohorts. Unstimulated saliva was obtained from subjects with OSCC at early (n = 33) and advanced (n = 33) disease, OL with homogeneous (n = 33) and proliferative verrucous (n = 33) clinical presentations, and healthy controls (n = 25). Salivary IL-1α, IL-6, IL-8, IP-10, MCP-1, TNF-α, HCC-1, and PF-4 levels were analyzed by a sensitive bead-based multiplex immunoassay. Mean levels of IL-6, IL-8, TNF-α, HCC-1, MCP-1, and PF-4 differed significantly between OSCC, OL, and control saliva (p < 0.05). We found notably higher IL-6 and TNF-α in advanced compared to early OSCC stages. The area under the curve (AUC) for OSCC vs. control was greater than 0.8 for IL-6, IL-8, TNF-α, and HCC-1, and greater than 0.7 for PF-4. The presence of neck metastases (NM) was associated with increased IL-6 and TNF-α levels. Our findings suggest that salivary IL-6, IL-8, TNF-α, HCC-1, and PF-4 may discriminate between OSCC, OL, and healthy controls. IL-6 and TNF-α may indicate OSCC progression, being distinctive in the presence of NM.
... MAS-related GPR family member X3 (MRGPRX3), a member of the mas-related/sensory neuron specific subfamily of G protein coupled receptors, is down-regulated in human airway epithelial cells exposed to smoke from electronic cigarettes 31 . In oral cancers, lung cancers, and head and neck cancers, C-X-C Motif chemokine ligand 14 (CXCL14) functions as a tumor suppressor; it also induces growth of prostate and breast cancers [32][33][34][35][36] . Protocadherin 7 (PCDH7) is involved in cell-cell recognition and adhesion 37 . ...
Understanding the molecular mechanism of SARS-CoV-2 infection (the cause of COVID-19) is a scientific priority for 2020. Various research groups are working toward development of vaccines and drugs, and many have published genomic and transcriptomic data related to this viral infection. The power inherent in publicly available data can be demonstrated via comparative transcriptome analyses. In the current study, we collected high-throughput gene expression data related to human lung epithelial cells infected with SARS-CoV-2 or other respiratory viruses (SARS, H1N1, rhinovirus, avian influenza, and Dhori) and compared the effect of these viruses on the human transcriptome. The analyses identified fifteen genes specifically expressed in cells transfected with SARS-CoV-2; these included CSF2 (colony-stimulating factor 2) and S100A8 and S100A9 (calcium-binding proteins), all of which are involved in lung/respiratory disorders. The analyses showed that genes involved in the Type1 interferon signaling pathway and the apoptosis process are commonly altered by infection of SARS-CoV-2 and influenza viruses. Furthermore, results of protein-protein interaction analyses were consistent with a functional role of CSF2 in COVID-19 disease. In conclusion, our analysis has revealed cellular genes associated with SARS-CoV-2 infection of the human lung epithelium; these are potential therapeutic targets.
... Many normal tissues, such as breast and kidney [12], constitutively express CXCL14 at high level, while it is reduced or absent in most cancer cells, including head-and-neck carcinoma, lung adenocarcinoma, and prostate cancer [13][14][15]. CXCL14 was very early reported to target tissue macrophages, promote white adipose tissue macrophage migration, and regulate glucose metabolism mainly in the skeletal muscle [16]. Several previous studies [17,18] have also noticed that CXCL14 was not expressed in THP-1 cells; however, the relationship between CXCL14 and atherosclerosis has been rarely investigated. ...
CXC chemokine family has been related to atherogenesis for long. However, the relationship between CXCL14 and atherogenesis is still unclear. This study preliminarily detected CXCL14 expression at foam cells in atherosclerosis specimens by immunohistochemistry. In vitro foam cells were derived from THP-1 after phorbol-12-myristate-13-acetate (PMA) and oxidized low-density lipoprotein (ox-LDL) stimulation. Immunoblotting and qPCR convinced CXCL14 expression variation during foam cell formation. We further demonstrated that ox-LDL regulated CXCL14 expression by AP-1. AP-1 could bind to CXCL14 promoter and up-regulate CXCL14 mRNA expression. Besides, CXCL14 promoted THP-1 migration, macrophage lipid phagocytosis, and smooth muscle cell migration as well as proliferation mainly via the ERK1/2 pathway. Additionally, a CXCL14 peptide-induced immune therapy showed efficacy in ApoE−/− mouse model. In conclusion, our study demonstrated that CXCL14 is highly up-regulated during foam cell formation and promotes atherogenesis in various ways. CXCL14 may be a potent therapeutic target for atherosclerosis.
... Consistently, many studies have reported that CXCL14 was highly expressed in normal tissues, especially in normal kidney tissues, but absent in the tumor cell lines and primary tumors [18,20]. CXCL14 is generally considered a tumor suppressor [21,22], but several recent studies have reported the opposite results. For example, the upregulation of CXCL14 in pancreatic cancer promotes the invasion of tumor cells [23]. ...
To identify the mechanism and functions of IRX1 in heart failure (HF) and provide evidence for new therapies. Bioinformatic analysis was performed to select target genes in HF cells compared to normal groups. Experimental rats were treated in a controllable manner to explore how IRX1 methylation accounted for this disease in vivo. Cardiac ultrasonic and morphologic examinations were conducted to test the mouse heart and evaluate the degree of cardiac impairment at in the level of organization. GSEA analysis revealed the relative enrichment of functions. Immunofluorescent assays, western blotting and qRT-PCR were used to determine the DNA methylation and expression levels. IRX1 was hypermethylated in heart failure and identified as a target gene by bioinformatic analysis. Transverse aortic constriction (TAC) induced heart failure in rats, while 5-aza-2ʹ-deoxycytidine (5-Aza) alleviated heart failure in rats according to medical cardiac indexes. Western blotting and qRT-PCR revealed that a conspicuous difference in the expression of IRX1 and CXCL14 between HF and normal cardiac cells. As a result of gene methylation, left ventricular hypertrophy and cardiac fibrosis is usually accompanied by heart failure. Moreover, is the results implied that the demethylation of IRX1 improves heart failure in vivo and in vitro. IRX1 methylation induced damaged cardiac function and even heart failure, which has important implications for HF treatment and diagnosis.
... Previous studies showed that the expression of CXCL14 is remarkably decreased in OSCC cells, and its up-regulation suppressed tumor cell growth in vivo [24,25], but the underlying mechanism still needs to be understood. To explore whether CXCL14 serves a role as a tumor suppressor in OSCC, overexpression of CXCL14 was employed to detect the proliferation of OSCC cells. ...
Oral squamous cell carcinoma (OSCC) is an aggressive malignancy which is the most common type of head and neck cancer. The study aimed to investigate the role of CXCL14 in cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) of OSCC and its potential mechanisms. In the present study, overexpression of CXCL14 was employed in TCA-8113 cells. Cell proliferation was detected using Cell Counting Kit-8 assay and flow cytometry. Scratch wound healing and Transwell assays were applied for the analysis of cell migration and invasion, respectively. Quantitative real-time polymerase chain reaction analysis was used to explore the mRNA expression of CXCL14 and PD-L1. Moreover, the protein expression levels of CXCL14, E-cadherin, N-cadherin, vimentin, PD-L1, NF-κB p65, and phospho-IκB-α (p-IκB-α) were examined by Western blot. The results revealed that the overexpression of CXCL14 was associated with suppressed proliferation of TCA-8113 cells coupled with decreasing the percentages of cells in the S phase. Moreover, the levels of cell migration and invasion were attenuated as well. Additionally, the expression level of EMT relative protein E-cad was increased notably, whereas N-cad and Vimentin were decreased. Importantly, the mRNA and protein expression of PD-L1, a crucial factor for OSCC, were lowered after overexpression of CXCL14. At the same time, the expression of NF-κB p65 and p-IκB-α was down-regulated significantly. Taken together, we speculate that CXCL14 inhibited OSCC cell proliferation, invasion, migration and EMT via suppressing PD-L1 expression and NF-κB mediated EMT, which indicated that CXCL14 may be an underlying target for OSCC treatment.
... Although a number of studies have demonstrated a tumor-suppressive role for CXCL14 [10][11][12][13], others showed a pro-tumor one [14]. In particular, in prostate and breast cancer, CXCL14 is produced by cancer associated fibroblasts (CAFs) in the tumor microenvironment and contributes to tumor growth and invasion [15,16]. ...
Glioblastoma (GBM) is a primary brain tumor whose prognosis is inevitably dismal, leading patients to death in about 15 months from diagnosis. Tumor cells in the mass of the neoplasm are in continuous exchange with cells of the stromal microenvironment, through the production of soluble molecules, among which chemokines play prominent roles. CXCL14 is a chemokine with a pro-tumor role in breast and prostate carcinoma, where it is secreted by cancer associated fibroblasts, and contributes to tumor growth and invasion. We previously observed that CXCL14 expression is higher in GBM tissues than in healthy white matter. Here, we study the effects of exogenously supplemented CXCL14 on key tumorigenic properties of human GBM cell lines. We show that CXCL14 enhances the migration ability and the proliferation of U87MG and LN229 GBM cell lines. None of these effects was affected by the use of AMD3100, an inhibitor of CXCR4 receptor, suggesting that the observed CXCL14 effects are not mediated by this receptor. We also provide evidence that CXCL14 enhances the sphere-forming ability of glioblastoma stem cells, considered the initiating cells, and is responsible for tumor onset, growth and recurrence. In support of our in vitro results, we present data from several GBM expression datasets, demonstrating that CXCL14 expression is inversely correlated with overall survival, that it is enriched at the leading edge of the tumors and in infiltrating tumor areas, and it characterizes mesenchymal and NON G-CIMP tumors, known to have a particularly bad prognosis. Overall, our results point to CXCL14 as a protumorigenic chemokine in GBM.
... CXCL14 inhibits angiogenesis and directly recruits several types of immune cells such as dendritic, natural killer (NK), and T cells [54,189,190]. We and other groups have shown antitumor activity of CXCL14 in cancers of the lung, head and neck, colon, and liver [54,[191][192][193][194][195]. Consistently, the levels of CXCL14 expression are reduced in these and other cancers [54,187,193,[195][196][197]. ...
Viruses have evolved various mechanisms to evade host immunity and ensure efficient viral replication and persistence. Several DNA tumor viruses modulate host DNA methyltransferases for epigenetic dysregulation of immune-related gene expression in host cells. The host immune responses suppressed by virus-induced aberrant DNA methylation are also frequently involved in antitumor immune responses. Here, we describe viral mechanisms and virus–host interactions by which DNA tumor viruses regulate host DNA methylation to evade antiviral immunity, which may contribute to the generation of an immunosuppressive microenvironment during cancer development. Recent trials of immunotherapies have shown promising results to treat multiple cancers; however, a significant number of non-responders necessitate identifying additional targets for cancer immunotherapies. Thus, understanding immune evasion mechanisms of cancer-causing viruses may provide great insights for reversing immune suppression to prevent and treat associated cancers.
... Interestingly, restoration of Cxcl14 expression recruits DCs into tumors in vivo and in vitro (15,16) and induces tumor necrosis (17). Importantly, Cxcl14 expression in HNC cells suppresses tumor growth from xenografts in athymic nude and SCID mice (18,19). In addition, the rates of colorectal tumor formation and metastasis were significantly lower in Cxcl14 transgenic mice than in isogenic wild-type mice (20). ...
Unlabelled:
High-risk human papillomaviruses (HPVs) are causally associated with multiple human cancers. Previous studies have shown that the HPV oncoprotein E7 induces immune suppression; however, the underlying mechanisms remain unknown. To understand the mechanisms by which HPV deregulates host immune responses in the tumor microenvironment, we analyzed gene expression changes of all known chemokines and their receptors using our global gene expression data sets from human HPV-positive and -negative head/neck cancer and cervical tissue specimens in different disease stages. We report that, while many proinflammatory chemokines increase expression throughout cancer progression, CXCL14 is dramatically downregulated in HPV-positive cancers. HPV suppression of CXCL14 is dependent on E7 and associated with DNA hypermethylation in the CXCL14 promoter. Using in vivo mouse models, we revealed that restoration of Cxcl14 expression in HPV-positive mouse oropharyngeal carcinoma cells clears tumors in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Further, Cxcl14 reexpression significantly increases natural killer (NK), CD4(+) T, and CD8(+) T cell infiltration into the tumor-draining lymph nodes in vivo In vitro transwell migration assays show that Cxcl14 reexpression induces chemotaxis of NK, CD4(+) T, and CD8(+) T cells. These results suggest that CXCL14 downregulation by HPV plays an important role in suppression of antitumor immune responses. Our findings provide a new mechanistic understanding of virus-induced immune evasion that contributes to cancer progression.
Importance:
Human papillomaviruses (HPVs) are causally associated with more than 5% of all human cancers. During decades of cancer progression, HPV persists, evading host surveillance. However, little is known about the immune evasion mechanisms driven by HPV. Here we report that the chemokine CXCL14 is significantly downregulated in HPV-positive head/neck and cervical cancers. Using patient tissue specimens and cultured keratinocytes, we found that CXCL14 downregulation is linked to CXCL14 promoter hypermethylation induced by the HPV oncoprotein E7. Restoration of Cxcl14 expression in HPV-positive cancer cells clears tumors in immunocompetent syngeneic mice, but not in immunodeficient mice. Mice with Cxcl14 reexpression show dramatically increased natural killer and T cells in the tumor-draining lymph nodes. These results suggest that epigenetic downregulation of CXCL14 by HPV plays an important role in suppressing antitumor immune responses. Our findings may offer novel insights to develop preventive and therapeutic tools for restoring antitumor immune responses in HPV-infected individuals.
... Cxcl14 is a chemokine that is constitutively expressed in normal tissues, however its receptor selectivity still remains largely unclear. Several reports indicate that it may play an anti-cancer role [53][54][55]. Additionally, it is suggested to have broad anti-microbial activity [56,57] and is down-regulated by virulent pathogens in order to create a protected ecological niche during infection [58]. ...
During Mycobacterium tuberculosis (M.tb) infection, the initial interactions between the
pathogen and the host cell determines internalization and innate immune response events.
It is established that detergents such as Tween alter the mycobacterial cell wall and solubilize
various lipids and proteins. The implication of this is significant since induced changes
on the cell wall affect macrophage uptake and the immune response to M.tb. Importantly,
during transmission between hosts, aerosolized M.tb enters the host in its native form, i.e.
in a detergent-free environment, thus in vitro and in vivo studies should mimic this as closely
as possible. To this end, we have optimized a procedure for growing and processing detergent-
free M.tb and assessed the response of murine macrophages (BMDM) infected with
multi drug-resistant M.tb (R179 Beijing 220 clinical isolate) using RNAseq. We compared
the effects of the host response to M.tb cultured under standard laboratory conditions
(Tween 80 containing medium -R179T), or in detergent-free medium (R179NT). RNAseq
comparisons reveal 2651 differentially expressed genes in BMDMs infected with R179T M.
tb vs. BMDMs infected with R179NT M.tb. A range of differentially expressed genes
involved in BMDM receptor interaction with M.tb (Mrc1, Ifngr1, Tlr9, Fpr1 and Itgax) and
pro-inflammatory cytokines/chemokines (Il6, Il1b, Tnf, Ccl5 and Cxcl14) were selected for
analysis through qPCR. BMDMs infected with R179NT stimulate a robust inflammatory
response. Interestingly, R179NT M.tb induce transcription of Fpr1, a receptor which detects
bacterial formyl peptides and initiates a myriad of immune responses. Additionally we show
that the host components Cxcl14, with an unknown role in M.tb infection, and Tlr9, an
emerging role player, are only stimulated by infection with R179NT M.tb. Taken together,
our results suggest that the host response differs significantly in response to Tween 80 cultured
M.tb and should therefore not be used in infection experiments.
... Ultraviolet irradiation or serum deprivation of oral squamous cell carcinoma cells increases CXCL14 expression by stimulating the phosphorylation of the mitogen-activated protein kinase (MAPK) p38, which reduces cell viability [28]. In this study, the suppression of ERK phosphorylation was also seen, in line with the previous report of MEK-ERK induced downregulation of CXCL14 by activation of epidermal growth factor receptor (EGFR) in oral carcinoma cells [29,30]. It is also known that the phosphorylation of p38MAPK leads to activation of AP-1 [31], which was shown to be essential for elevation of CXCL14 expression as stimulated by ROS in breast cancer cells. ...
CXCL14, a relatively novel chemokine, is a non-ELR (glutamic acid-leucine-arginine) chemokine with a broad spectrum of biological activities. CXCL14 mainly contributes to the regulation of immune cell migration, also executes antimicrobial immunity. The identity of the receptor for CXCL14 still remains obscure and therefore the intracellular signaling pathway is not entirely delineated. The present review summarizes the contribution of CXCL14 in these two aspects and discusses the biological mechanisms regulating CXCL14 expression and potential CXCL14 mediated functional implications in a variety of cells.
... Of course, there is always the potential for repression of malignant properties by chemokines, as some have noted anti-tumor effects. The chemokine CXCL14, also known as BRAK to signify the tissues (breast and kidney) from which it was originally isolated (69), was shown to be expressed in normal squamous epithelium but reduced or absent in malignant oral tissues (70), and is also repressed by EGFR signaling (71). Ectopic expression of CXCL14 completely blocked OSCC growth in vivo (72), suggesting that loss of expression may contribute significantly to the pathogenesis of oral cancer. ...
The chemotactic cytokines, or chemokines, comprise a superfamily of polypeptides with a wide range of activities that include recruitment of immune cells to sites of infection and inflammation, as well as stimulation of cell proliferation. As such, they function as antimicrobial molecules and play a central role in host defenses against pathogen challenge. However, their ability to recruit leukocytes and potentiate or prolong the inflammatory response may have profound implications for the progression of oral diseases such as chronic periodontitis, where tissue destruction may be widespread. Moreover, it is increasingly recognized that chronic inflammation is a key component of tumor progression. Interaction between cancer cells and their microenvironment is mediated in large part by secreted factors such as chemokines, and serves to enhance the malignant phenotype in oral and other cancers. In this article, we will outline the biological and biochemical mechanisms of chemokine action in host–microbiome interactions in periodontal disease and in oral cancer, and how these may overlap and contribute to pathogenesis.
... Chemokine (C-X-C motif) ligand 14 (CXCL14), also known as BRAK, is a small cytokine belonging to the CXC chemokine family (11), and the gene encoding CXCL14 is commonly recognized as a tumor-suppressor gene (12)(13)(14). However, recent studies indicated that CXCL14 might actually promote tumor progression in some types of cancer (15)(16)(17)(18). ...
Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis-free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis.
... The PPAR signaling pathway has previously been considered a useful prognostic marker and a potential therapeutic target for TSCC (39), however, there have been no reports regarding its molecular mechanism and miRNA regulation. Numerous studies have demonstrated that cytokine interaction is involved in the process of tumor growth, which is important for TSCC (40,41). The miR-34a-sirtuin 6 axis was previously found to be involved in various types of squamous cell cancer, which also demonstrates the impact of the p53 signaling pathway on TSCC (42). ...
Tongue squamous cell carcinoma (TSCC) is a rare and aggressive type of cancer, which is associated with a poor prognosis. Identification of patients at high risk of TSCC tumorigenesis may provide information for the early detection of metastases, and for potential treatment strategies. MicroRNA (miRNA; miR) and mRNA expression profiling of TSCC tissue samples and normal control tissue samples were obtained from three Gene Expression Omnibus (GEO) data series. Bioinformatics analyses, including the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes were used to identify genes and pathways specifically associated with miRNA‑associated TSCC oncology. A total of 25 miRNAs and 769 mRNAs were differentially expressed in the two groups assessed, and all the differentially expressed miRNA and mRNA target interactions were analyzed. The miRNA target genes were predominantly associated with 38 GO terms and 13 pathways. Of the genes differentially expressed between the two groups, and confirmed in another GEO series, miRNA‑494, miRNA‑96, miRNA‑183, runt‑related transcription factor 1, programmed cell death protein 4 and membrane‑associated guanylate kinase were the most significantly altered, and may be central in the regulation of TSCC. Bioinformatics may be used to analyze large quantities of data in microarrays through rigorous experimental planning, statistical analysis and the collection of complete data on TSCC. In the present study, a novel differential miRNA‑mRNA expression network was constructed, and further investigation may provide novel targets for the diagnosis of TSCC.
... When a CXCL14 expression vector was stably introduced into head-and-neck carcinoma-derived HSC-3 and lung carcinoma cell-derived H23 cell lines of human origin, the tumor-forming activities of these cells in immunodeficient mice were dramatically suppressed (43,45). The growth of mouse melanoma and lung carcinomaderived cells was also impaired in CXCL14-transgenic mice (46). ...
Abstract CXCL12 and CXCL14 are evolutionarily conserved members of the CXC-type chemokine family. CXCL12 binds specifically to the G-protein-coupled receptor CXCR4 to induce the migration of primordial germ cells, hematopoietic stem cells, and inflammation-associated immune cells. In addition, CXCL12-CXCR4 signaling is often enhanced in malignant tumor cells and facilitates increased proliferation as well as metastasis. Although macrophage migration inhibitory factor and extracellular ubiquitin interact with CXCR4 as agonistic factors, CXCL12 was believed to be the sole chemokine ligand for CXCR4. However, a very recent report revealed that CXCL14 binds to CXCR4 with high affinity and efficiently inhibits CXCL12-mediated chemotaxis of hematopoietic progenitor and leukemia-derived cells. CXCL14 does not directly cross-compete with CXCL12 for the CXCR4 binding but instead inactivates CXCR4 via receptor internalization. Because both CXCL12 and CXCL14 are expressed during embryogenesis and brain development in mice, these two chemokines could function in an interactive fashion. We propose that the CXCL14 gene has been conserved from fish to man due to its role in fine-tuning the strength of CXCL12-mediated signal transduction. In addition to its biological implications, the above finding will be important for designing anti-cancer compounds targeting the CXCL12-CXCR4 signaling axis. In fact, a stabilized dimeric peptide containing the C-terminal 51-77 amino acid residues of CXCL14 has been shown to have stronger CXCL12 antagonistic activity than full-length CXCL14.
... Also, expression of CXCL14 in oral cancer cells inhibited tumor growth [144]. Moreover, treatment of oral cancer cells with EGF has been shown to reduce CXCL14 expression and the same group also demonstrated that treatment of head and neck cancer cell lines with the EGF-inhibitor gefitinib increase the levels of CXCL14 and inhibit xenograft growth [145,146]. Furthermore, epigenetic silencing of CXCL14 is frequently found in human lung cancer samples, and restoration of CXCL14 expression reduces growth of lung cancer xenografts [147]. ...
... Immunohistochemical staining for CXCL14 suggested that CXCL14 the immunoreactivity overlapped with the location of uNK cells. CXCL14 is a chemoatractant for peripheral blood monocytes, immature dendritic cells and active peripheral blood NK cells (Kurth et al., Shellenberger et al., 2004; Ozawa et al., 2006). We therefore tested whether CXCL14 has a role in uNK cell chemotaxis. ...
The emergence of microarray technology has enabled a thorough study of the level of transcripts in the human body. A high density micoarray analysis revealed a comprehensive list of transcripts, which were significantly different between mid-proliferative and mid-secretory phase endometrium.² An EST identified from the HG_U95B chip is identical to the 3′UTR of CXCL14 or macrophage inflammatory protein 2γ (MIP 2γ). The level is 19-fold higher in the mid-secretory compared to the mid-proliferative phase of menstrual cycle. This has suggested that the transcript level of CXCL14 may be directly regulated by progesterone. Northern hybridisation and in situ hybridisation confirmed that the transcript level of CXCL14 (MIP 2γ) was high in the mid-to-late secretory endometrium and its mRNA was localised in the glandular epithelium of this tissue.¹In silico analysis has predicted six progesterone response elements (PREs) within 2040 bp upstream from the ATG site. To investigate the possible functions of these PREs, a dual luciferase assay was performed on the ishikawa cell line transfected with five deletion constructs of the gene promoter. Cells were co-transfected with progesterone receptor B (PRB) and treated with 10–6 M progesterone. Luciferase activities of these constructs have localised two fragments that were most likely to contain the active PREs, i.e. PRE1 and PRE2. An electrophoretic mobility shift assay showed that PRE oligonulcleotides within these two regions were able to bind PRB that was synthesised in vitro, although there was a stronger signal seen in the PRE2 region. A dose competition study revealed PRE1/PRB and PRE2/PRB protein binding could be competed with different concentrations of cold wild-type competitor oligonucleotides. Mutagenesis of PRE1 and PRE2 analysed by luciferase reporter assay reduced the inductive effect of progesterone treatment. This study indicates that progestegen induced transcript encoding a chemokine in the human endometrium may likely act as a chemoattractant for leucocytes during the secretory phase of the menstrual cycle.
(1) Mokhtar NM, Smith SK, Charnock-Jones DS. (2003). Characterisation of chemokine macrophage inflammatory protein 2gamma mRNA in human endometrium. 50th Society for Gynaecologic Investigation. Washington DC, USA. March 2003.
(2) Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillip SC, Smith SK. (2003). Determination of the transcript profile of human endometrium. Mol. Hum. Reprod. 9, 19–33.
... 7 Regulation of tumor formation by the chemokine has also been reported. 8,9 For the first step to investigate the mechanism of action of BRAK/CXCL14 in vivo in humans, we modified the enzyme-linked immunosorbent assay (ELISA) to determine serum levels of BRAK/CXCL14 protein and applied this modified ELISA to serum samples from healthy volunteers. ...
Background: BRAK/CXCL14 is a chemokine expressed in many normal cells and tissues. Several papers have reported tissue distribution, but no study has examined the serum protein level of this chemokine. Methods: We evaluated serum levels of BRAK/CXCL14 by enzyme-linked immunosorbent assay, after determining the conditions necessary to increase the sensitivity and reproducibility of the method Results: The average level of males was significantly higher than that of females Two percent of the volunteers showed a value 2 S.D. higher than the average value without any obvious abnormality, but no person was lower than 1 S D of the average value The serum BRAK/CXCL14 levels of individual subjects were constant Conclusion: These data indicate that the serum BRAK/CXCL14 level is highly conserved and suggest that the levels are determined by the genetic background and/or lifestyle of the individual.
... Based on the available data, the relationship between CXCL14 expression and tumorigenesis is complex, tissue-specific, and cancer type-specific. In head, neck, and cervical squamous-cell carcinomas (34,46) and lung and breast carcinoma cell lines (34,35), CXCL14 expression is down-regulated, suggesting a role in tumor inhibition. In contrast, data from AdCas of the pancreas and breast show a stimulatory role for CXCL14 in tumorigenesis (34,35). ...
CXCL14, a recently described epithelial cytokine, has multiple putative roles in inflammation and carcinogenesis. In the context that chronic obstructive pulmonary disease (COPD) and lung cancer are both smoking-related disorders associated with airway epithelial disorder and inflammation, we hypothesized that the airway epithelium responds to cigarette smoking with altered CXCL14 gene expression contributing to the disease-relevant phenotype. Using genome-wide microarrays with subsequent immunohistochemistry analysis, the data demonstrates that the expression of CXCL14 is up-regulated in the airway epithelium of healthy smokers and further increased in COPD smokers, especially within hyperplastic/metaplastic lesions, in association with multiple genes relevant to epithelial structural integrity and cancer. In vitro studies revealed that expression of CXCL14 is induced in the differentiated airway epithelium by cigarette smoke extract in an epidermal growth factor (EGF) receptor-dependent manner, and EGF mediates CXCL14 up-regulation in the differentiated airway epithelium through its effect on basal stem/progenitor cell population. Analysis of two independent lung cancer cohorts revealed dramatic up-regulation of CXCL14 expression in adenocarcinoma and squamous cell carcinoma. High expression of the COPD-associated CXCL14-correlating cluster of genes correlated in lung adenocarcinoma with poor survival. These data suggest that smoking-induced expression of CXCL14 in the airway epithelium represents a novel potential molecular link between smoking-associated airway epithelial injury, COPD and lung cancer.
... Immunohistochemical staining for CXCL14 suggested that CXCL14 the immunoreactivity overlapped with the location of uNK cells. CXCL14 is a chemoatractant for peripheral blood monocytes, immature dendritic cells and active peripheral blood NK cells (Kurth et al., 2001; Shellenberger et al., 2004; Ozawa et al., 2006). We therefore tested whether CXCL14 has a role in uNK cell chemotaxis. ...
The aim of the present study was to identify differentially expressed genes and their related biological pathways in the secretory phase endometrium from patients with recurrent miscarriage (RM) and fertile subjects. Endometrial samples from RM and fertile patients were analyzed using the Affymetrix GeneChip® ST Array. The bioinformatic analysis using the Partek Genomic Suite revealed 346 genes (175 up-regulated and 171 down-regulated) that were differentially expressed in the endometrium of RM patients compared to the fertile subjects (fold change ≥1.5, p<0.005). Validation step using quantitative real-time polymerase chain reaction (qPCR) confirmed a similar expression pattern of four exemplary genes: one up-regulated gene (fibroblast growth factor 9, FGF9) and three down-regulated genes: integrin β3 (ITGB3), colony stimulating factor 1 (CSF1) and matrix-metalloproteinases 19 (MMP19). The Gene Set Enrichment Analysis (GSEA) and the Pathway Studio software have found 101 signaling pathways (p<0.05) associated with the affected genes including the FGFR3 /signal transducer and activator of transcription (STAT) pathway and the CSF1R/STAT pathway. Cell adhesion, cell differentiation and angiogenesis were among biological processes indicated by this system. In conclusion, microarray technique is a useful tool to study gene expression in the secretory phase-endometrium of RM patients. The differences in endometrial gene expressions between healthy and RM subjects contribute to an increase in our knowledge on molecular mechanisms of RM development and may improve the outcome of pregnancies in high-risk women with RM.
... Our studies revealed that CXCL14 is a low potent chemoattractant for human blood monocytes (Kurth et al., 2001) whereas other groups provided evidence for a role in DC migration (Sleeman et al., 2000; Shellenberger et al., 2004; Shurin et al., 2005; Salogni et al., 2009). Numerous reports have implicated CXCL14 in cancer but findings disagree whether this chemokines displays anti-tumor as opposed to tumor-promoting activity (Frederick et al., 2000; Shurin et al., 2005; Ozawa et al., 2006; Wente et al., 2008; Augsten et al., 2009). A hallmark of CXCL14 is its constitutive expression at high levels in many epithelial tissues, most notably in the skin and gastrointestinal tract (Schaerli et al., 2005; Meuter and Moser, 2008; Maerki et al., 2009). ...
The large family of chemoattractant cytokines (chemokines) embraces multiple, in part unrelated functions that go well beyond chemotaxis. Undoubtedly, the control of immune cell migration (chemotaxis) is the single, unifying response mediated by all chemokines, which involves the sequential engagement of chemokine receptors on migrating target cells. However, numerous additional cellular responses are mediated by some (but not all) chemokines, including angiogenesis, tumor cell growth, T-cell co-stimulation, and control of HIV-1 infection. The recently described antimicrobial activity of several chemokines is of particular interest because antimicrobial peptides are thought to provide an essential first-line defense against invading microbes at the extremely large body surfaces of the skin, lungs, and gastrointestinal-urinary tract. Here we summarize the current knowledge about chemokines with antimicrobial activity and discuss their potential contribution to the control of bacterial infections that may take place at the earliest stage of antimicrobial immunity. In the case of homeostatic chemokines with antimicrobial function, such as CXCL14, we propose an immune surveillance function in healthy epithelial tissues characterized by low-level exposure to environmental microbes. Inflammatory chemokines, i.e., chemokines that are produced in tissue cells in response to microbial antigens (such as pathogen-associated molecular patterns) may be more important in orchestrating the cellular arm in antimicrobial immunity.
... Pioneering studies revealed that CXCL14 mRNA expression is repressed in various malignant cancer samples and several tumour cell lines (35). The in vivo tumour-forming activity of head-and-neck carcinoma HSC-3 cells was abrogated when CXCL14 was introduced, indicating that CXCL14 has a tumoursuppressing function (24). In addition, recent studies demonstrated that CXCL14 is epigenetically silenced in several lung adenocarcinoma and prostate cancer cells of human origin (25,26). ...
CXCL14 is a member of the CXC chemokine family. CXCL14 possesses chemoattractive activity for activated macrophages, immature dendritic cells and natural killer cells. CXCL14-deficient mice do not exhibit clear immune system abnormalities, suggesting that the function of CXCL14 can be compensated for by other chemokines. However, CXCL14 does appear to have unique biological roles. It suppresses the in vivo growth of lung and head-and-neck carcinoma cells, whereas the invasiveness of breast and prostate cancer cells appears to be promoted by CXCL14. Moreover, recent evidence revealed that CXCL14 participates in glucose metabolism, feeding behaviour-associated neuronal circuits, and anti-microbial defense. Based on the expression patterns of CXCL14 and CXCL12 during embryonic development and in the perinatal brain in mice, the functions of these two chemokines may be opposite or interactive. Although CXCL14 receptors have not yet been identified, the intracellular activity of CXCL14 in breast cancer cells suggests that the CXCL14 receptor(s) and signal transduction pathway(s) may be different from those of conventional CXC-type chemokines.
Aims: To evaluate BRAK and APRIL in serum samples from healthy patients and an ovarian tumor group and analyze their effective value as biomarkers. Materials & methods: BRAK and APRIL were measured in 197 serum samples including 34 healthy controls, 48 patients with benign ovarian cysts and 115 patients with ovarian cancer, and the best statistical cutoff values were calculated. Then, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value for selected cutoff points were assessed. Results: The healthy control group had statistically significant higher BRAK and lower APRIL than the ovarian tumor group. BRAK was excellent for differentiating healthy patients from patients with ovarian tumors, showing area under the receiver operating characteristic curve 0.983, 98.16% sensitivity and 100% specificity. When BRAK was combined with APRIL and CA-125, it also played a role in distinguishing benign cysts from malignancies with area under the curve 0.864, 81.74% sensitivity and 79.17% specificity. Conclusions: BRAK and APRIL are good candidates for ovarian tumor biomarkers.
Ovarian cancer is one of the common malignant tumours of female reproductive organs. Due to early diagnosis difficulties and lack of effective treatment in the late stage, ovarian cancer has the highest mortality rate in female reproductive system malignancies. Therefore, finding reliable early diagnosis indicators and new therapeutic targets for ovarian cancer is an urgent problem to be solved. Chemokine (C‐X‐C motif) ligand 14 (CXCL14) is a small cytokine belonging to the CXC chemokine family, which has been found to possess multi‐effects in tumourigenesis and development. Here, we reported that CXCL14 was preferentially expressed in ovarian cancer. By analysing the TCGA database, we found that CXCL14 was highly expressed in advanced ovarian cancer patients and correlated with poor prognosis. In addition, the abnormal high CXCL14 levels were observed in serum and ovarian tissue of ovarian cancer patients by qRT‐PCR and ELISA. In vitro and in vivo experiments both confirmed that overexpression of CXCL14 promoted the ovarian cancer cell proliferation. Moreover, transfection of CXCL14 increased the phosphorylation level of signal transducer and activator of transcription 3 (STAT3), and administration of STAT3 inhibitor III inhibited the tumour‐promoting effects of CXCL14. Therefore, our study suggests that CXCL14 could be utilised as a novel adjunct biomarker for early diagnosis of ovarian cancer and provides new targets and ideas for the treatment of advanced ovarian cancer.
Significance paragraph
CXCL14 could be utilised as a novel adjunct biomarker for early diagnosis of ovarian cancer and provides new targets and ideas for the treatment of advanced ovarian cancer.
Sutherlandia frutescens (S. frutescens) has been traditionally used as an herbal medicine to ameliorate symptoms associated with cancer, infectious diseases, as well as inflammation. The objective of this investigation was to explore the impact of S. frutescens on the expression of genes in a murine macrophage cell line (i.e., RAW 264.7). We found that treatment with an ethanolic-extract of S. frutescens (SFE) 1 h prior to the stimulation with LPS and IFNγ for 24 h significantly affected the expression of 715 genes in RAW 264.7 cells. When the post-stimulation period was shortened to 8 h, the number of genes that were significantly impacted by SFE diminished to 50. Pathway analysis revealed that inflammatory signaling pathways, such as NF-κB, MAPK, and TNF, as well as signaling pathways associated with immune-related responses, were inhibited by SFE treatment. These findings are consistent with previously reported anti-inflammatory activity of SFE and enable better understanding of the immune-modulating properties of this botanical. To our knowledge, this represents the first report on the impact of S. frutescens on global gene expression in an immune cell population.
Under natural conditions, some embryos cannot implant successfully because of the dysfunction of receptive endometrium (RE). Thus, it is imperative for us to study the molecular mechanisms involved in the formation of the RE from pre-receptive endometrium (PE). In this study, the endometrium from gestational day 5 (D5, PE) and gestational day 15 (D15, RE) dairy goats were selected to systematically analyze the transcriptome using strand-specific Ribo-Zero RNA-Seq, >120 million high-quality paired-end reads were generated and 47,616 transcripts were identified in the endometrium of dairy goats. A total of 810 mRNAs were differentially expressed genes (DEGs) between the RE and PE meeting the criteria of P-values<0.05. Bioinformatics analysis of the DEGs revealed that a number of biological processes and pathways were potentially involved in the establishment of the RE, notably energy metabolism and amino acid metabolism. Furthermore, we speculated that CXCL14, IGFBP3, and LGALS15 potentially participated in the development of endometrium. What's more, putative SNPs, InDels and AS events were identified and analyzed in the endometrium. In a word, this resulting view of the transcriptome greatly enhances the comprehensive transcript catalog and uncovers the global trends in gene expression during the formation of receptive endometrium in dairy goats.
CXCL14 is a CXC-type chemokine that exhibits chemotactic activity for immature dendritic cells, activated macrophages, and activated natural killer cells. However, its specific receptor and signaling pathway remain obscure. Recently, it was reported that CXCL14 binds to CXCR4 with high affinity and inhibits CXCL12-mediated chemotaxis. Furthermore, the CXCL14 C-terminal α-helical region is important for binding to its receptor. In this context, we chemically synthesized CXCL14 and its derivative with a one-pot method using N-sulfanylethylanilide peptide as a thioester equivalent. The synthetic CXCL14 proteins possessed inhibitory activities to CXCL12-mediated chemotaxis comparable with that of recombinant CXCL14. Moreover, we proved that chemically biotinylated CXCL14 binds to CXCR4 on cells by flow cytometry analysis.
Copyright © 2015 Elsevier Ltd. All rights reserved.
CXCL14/BRAK (BRAK) is a secreted chemokine with anti-tumor activity, and its expression is suppressed in tumor cells. We previously reported the anti-tumor activity of BRAK in cell lines of head and neck squamous cell carcinoma (HNSCC) and the suppression of BRAK secretion in these cells. BRAK secretion in fibrosarcoma cells is restored by Fasudil, which is a Rho-kinase (ROCK) inhibitor. In this study, we examined the anti-tumor effect of BRAK by evaluating its gene expression and protein secretion in HNSCC cell lines. We found that BRAK mediated the suppressive effect of Fasudil against HNSCC cells. Tumor development in female BALB/cAJclnu/nu mice was suppressed by Fasudil. Also secretion of BRAK protein by tumor cell lines in vitro was significantly stimulated by Fasudil treatment. Similarly, the production of BRAK protein was significantly increased by the addition of Fasudil to cultured tumor cells. Furthermore Fasudil significantly increased BRAK gene expression at the mRNA level in HNSCC cell line. Inhibition of the RhoA/ROCK pathway by siRNAs significantly stimulated BRAK gene expression. These results show that the tumor-suppressive effect of Fasudil was mediated by BRAK, suggesting that Fasudil may therefore be useful for the treatment of HNSCC.
Purpose: Photodynamic therapy (PDT) induced singlet oxygen (1O2) can cause rapid necrosis of solid tumors. Using a new near-infrared photomultiplier tube system, the 1O2 production was monitored in real-time for HSC-3 tongue cancer cells and in vivo tissue, and the relationship between the 1O2 production and anti-tumor effects was investigated.
Experimental Design: Using 5-aminolevulinic acid (5-ALA), 1O2 generation was induced with a variety of laser parameters (fluence and irradiation) in vivo. During this time, tumor death was evaluated. Based on the 1O2 production, the optimal irradiation conditions and the long-term outcome were investigated on a subcutaneous tongue cancer model over 90 days.
Methods and Results: The 1O2 production showed a strong correlation with the laser output in a dose-dependent manner. At high laser output, the 1O2 levels were high and caused gradual but strong anti-tumor effects, whereas at low laser outputs, the 1O2 levels were low and the anti-tumor effects were rapid but superficial. We then performed PDT in an experimental tongue cancer model at an irradiance lower than 150 mW/cm2, which was identified in the first part of the study as the optimal irradiance value based on the 1O2 production. Within the 90 days post PDT, PDT-induced hemorrhagic necrosis was observed in 8 mice out of 10 within 5 days. In the other 2 mice, no sign of necrosis was seen until 25 days after PDT. The tumor volume of the PDT group continued to decline except for 2 mice which showed some slight growth over 70 days after PDT. In the control group, tumor size increased dramatically over 70 days.
Conclusions: The present studies established 1O2 levels as an indicator for optimal irradiation during PDT, and showed a relationship between the 1O2 production and the photodynamic effects. Although 5-ALA mediated PDT showed a strong anti-tumor effect on larger subcutaneous tongue tumors, it might be more effective for small tumors of oral cavity.
ECCO15 and 34th ESMO Multidisciplinary Congress
Berlin, 20-24 September 2009
Expression of BRAK/CXCL14 is associated with antitumor efficacy of gefitinib in head and neck squamous cell carcinoma
Ryu-Ichiro Hata,1,6 Shigeyuki Ozawa,2,6 Yasumasa Kato,1,6 Shin Ito,1,6 Reika Komori,1,3 Naoto Shiiki,4 Keiichi Tsukinoki,4,6 Yojiro Maehata,5,6 Eiro Kubota2,6
Departments of 1Biochemistry and Molecular Biology, 2Oral and Maxillofacial Surgery, 3Pediatric Dentistry, 4Pathology, and 5Pharmacology; 6Oral Health Science Research Center, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan
Background: The clinical efficacy of gefitinib (ZD1839, Iressa), which is an inhibitor specific for the epidermal growth factor (EGF) receptor tyrosine kinase, has been demonstrated in non-small cell lung carcinoma patients with EGF receptor mutations, and so these mutations are a useful marker(s) to find responders to this drug. However recent studies showed that the EGF receptor gene mutation is rare in squamous cell carcinomas of the esophagus and head and neck regions. In the present study we investigated the relationship between BRAK expression and gefitinib efficacy for tumor suppression.
Material and methods: HNSCC cells were cultured in Dulbecco's Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum. Nearly confluent cells were cultured overnight in serum-free DMEM. After starvation, they were incubated with or without EGF (10 ng/ml) and/or gefitinib (1 micro M). HSC-3 cells were subcutaneously injected into athymic nude mice. Tumor cell-xenografted mice were daily administered gefitinib (50 mg/kg) orally. In some experiments, tumor cells were introduced BRAK ShRNA expressing vector to knockdown BRAK mRNA expression and established stable transformants.
Results: Gefitinib attenuated the effect of EGF, or even stimulated BRAK mRNA expression of HNSCC cells in vitro. Oral administration of gefitinib significantly (P<0.001) reduced tumor growth of xenografts in female athymic nude mice accompanied by increased in BRAK expression specifically in tumor tissue. Introduction of BRAK ShRNA vector reduced both the expression of BRAK in the cells and the antitumor efficacy of gefitinib in vivo.
Conclusions: Our results indicate that oral administration of gefitinib reduced tumor size, at least in part, through elevation of BRAK expression. Thus, the use of gefitinib for treatment of patients with HNSCC in whom there is an inducing effect of the drug on the BRAK expression in cancer cells in culture may be advantageous. Furthermore, BRAK may be a promising molecule for gene therapy of HNSCC.
This work was performed in collaboration with Drs. Takahide Taguchi, Yukari Imagawa-Ishiguro and Mamoru Tsukuda, Department of Biology and Function in the Head and Neck, Yokohama City University Graduate School of Medicine.
To detect the expression of CXCL14 in human osteosarcoma cell lines and tissues and investigate its association with the prognosis of the patients.
RT-PCR, enzyme-linked immunosorbnent assay (ELISA) and real-time PCR were used to detect the expression of CXCL14 in 4 osteosarcoma cell lines and in 40 pairs of osteosarcoma tissues and adjacent muscular tissues. CCK8 assay and colony formation assay was used to assess the effect of CXCL14 suppression mediated by two specific siRNAs on the proliferation of U2OS osteosarcoma cells. Immunohistochemistry was performed to detect the expression of CXCL14 in 58 osteosarcoma tissues, and Kaplan-Meier method and log-rank test were performed for survival analysis of the patients.
Significant up-regulation of CXCL14 expression was found in the osteosarcoma cell lines and in osteosarcoma tissues compared with the adjacent muscles (P<0.01). In U2OS cell, suppression of CXCL14 expression by siRNA significantly inhibited the cell proliferation (P<0.01) and colony formation rate (P<0.05). Kaplan-Meier survival analysis indicated that patients with high CXCL14 expression had worse prognosis than those with low CXCL14 expression (P=0.02).
CXCL14 is up-regulated in both osteosarcoma cell lines and primary osteosarcoma tissues to promote the proliferation of osteosarcoma cells. A high CXCL14 expression in osteosarcoma tissues is associated with a poor prognosis, suggesting the that CXCL14 serve as a potential therapeutic target for osteosarcoma.
We previously reported that chemokine CXCL14/BRAK (BRAK) has antitumor activity in several carcinoma cells indicating that BRAK secretion suppresses carcinoma cells. Ras-homologous small GTPase (RhoA) and Rho-associated coiled-coil-containing protein kinase (ROCK) are important regulators of secretory processes, and activation of the RhoA/ROCK signaling pathway stimulates tumor invasion and metastasis. We investigated the effects of fasudil, a specific ROCK inhibitor, on BRAK secretion and tumor progression in mesenchymal fibrosarcoma cells (MC57). We demonstrated the antitumor activity of secreted BRAK using MC57 transplantation of BRAK in overexpressed transgenic mice. Further, to eliminate the influence of change in the mRNA expression of endogenous BRAK, we produced stable MC57 cell lines expressing BRAK (MC57-BRAK) or mock vector (MC57-MOCK). Fasudil significantly increased BRAK secretion by MC57-BRAK cells in a dose-dependent manner. To determine the effect of fasudil on tumor growth, MC57-BRAK and MC57-MOCK cells were transplanted into wild-type mice. Fasudil treatment suppressed tumor growth only in mice that had received MC57-BRAK cell transplants. These results indicate that fasudil inhibits fibrosarcoma growth by stimulating BRAK secretion and suggests that fasudil therapy might have clinical efficacy.
There is a structural and a functional classification of chemokines. The former includes four groups: CXC, CC, C and CX3C chemokines. There is a redundancy and binding promiscuity between chemokine receptors and their ligands. Recently, a functional
classification distinguishing between inflammatory and homeostatic chemokines has been introduced. However, numerous effects
of these chemokines overlap. For example, numerous homeostatic chemokines, which are involved in lymphocyte recruitment and
lymphoid tissue organization, may also play a role in B cell migration underlying germinal center formation within the inflamed
synovium. Anti-chemokine and anti-chemokine receptor targeting may be therapeutically used in future biological therapy of
arthritis. In addition to the clear clinical benefit, we can learn a lot from these trials about the actions of the targeted
chemokines and their receptors. Today, most data in this field are obtained from experimental models of arthritis; however,
results of some human trials have also become available. Thus, it is possible that a number of specific chemokine and chemokine
receptor antagonists will be administered to arthritis patients in the near future. Hopefully, some of these potential treatment
modalities will be used to control inflammation, prevent joint destruction and thus will benefit our patients.
The most accurate predictor of disease recurrence in patients treated for head and neck squamous cell carcinoma is, at present, the extent of regional lymph node metastasis. Since elevated levels of epidermal growth factor receptor (EGFR) and of its ligand, transforming growth factor-alpha (TGF-alpha), have been detected in primary tumors of patients with head and neck squamous cell carcinoma, we determined whether tumor levels of these proteins were of prognostic importance.
Monoclonal antibodies specific for EGFR and TGF-alpha were used for immunohistochemical detection of each protein in tissue sections of primary tumors from 91 patients who were treated by surgical resection. Levels of immunoreactive EGFR and TGF-alpha were quantified by use of a computerized image analysis system and were normalized to appropriate standards. The logrank test and proportional hazards regression analysis were used to calculate the probability that EGFR and TGF-alpha levels were associated with disease-free survival (i.e., no recurrence of cancer) and cause-specific survival (i.e., patients do not die of their disease). All P values were two-sided.
When tumor levels of EGFR or TGF-alpha were analyzed as continuous variables, disease-free survival and cause-specific survival were reduced among patients with higher levels of EGFR (both P = .0001) or TGF-alpha (both P = .0001). In a multivariate analysis, tumor site, tumor level of EGFR, and tumor level of TGF-alpha were statistically significant predictors of disease-free survival; in a similar analysis, regional lymph node stage and tumor levels of EGFR and of TGF-alpha were significant predictors of cause-specific survival.
Quantitation of EGFR and TGF-alpha protein levels in primary head and neck squamous cell carcinomas may be useful in identifying subgroups of patients at high risk of tumor recurrence and in guiding therapy.
A novel alpha-chemokine, designated KS1, was identified from an EST database of a murine immature keratinocyte cDNA library. The EST has 94% similarity to a recently cloned human gene, BRAK, that has no demonstrated function. Northern analysis of mouse and human genes showed detectable mRNA in brain, intestine, muscle and kidney. Tumour panel blots showed that BRAK was down-regulated in cervical adenocarcinoma and uterine leiomyoma, but was up-regulated in breast invasive ductal carcinoma. KS1 bound specifically to B cells and macrophages, as well as two B cell lines, CESS and A20, and a monocyte line, THP-1. KS1 showed no binding to naive or activated T cells. In addition, KS1 stimulated the chemotaxis of CESS and THP-1 cells but not T cells. The s.c. injection of KS1 creates a mixed inflammatory response in Nude and C3H/HeJ mice. The above data indicates that KS1 and its human homologue represents a novel non-ELR alpha-chemokine that may have important roles in trafficking of B cells and monocytes. We propose the name B cell- and monocyte-activating chemokine (BMAC) for this molecule to reflect the described biological functions.
Although numerous chemokines act on monocytes, none of them is specific for these cells. Here, we show that breast and kidney-expressed chemokine (BRAK) is a highly selective monocyte chemoattractant. Migration efficacy and Bordetella pertussis toxin-sensitive Ca(2+) mobilization responses to BRAK were strongly enhanced after treatment of monocytes with the cyclic AMP-elevating agents prostaglandin E(2) and forskolin. BRAK is the first monocyte-selective chemokine, as other types of blood leukocytes or monocyte-derived dendritic cells and macrophages did not respond. Expression in normal skin keratinocytes and dermal fibroblasts as well as lamina propria cells in normal intestinal tissues suggests a homeostatic rather than an inflammatory function for this chemokine. In addition, macrophages were frequently found to colocalize with BRAK-producing fibroblasts. We propose that BRAK is involved in the generation of tissue macrophages by recruiting extravasated precursors to fibroblasts, which are known to secrete essential cytokines for macrophage development.
Recent investigations have revealed that growth factors may influence the invasive activity of tumor cells. Expression of laminin-5 gamma2 chain (LN-5 gamma2) and epidermal growth factor receptor (EGFR) in squamous cell carcinomas of the tongue in 104 patients with stage II, III, and IVA, B (excluding the cases with distant metastasis) was examined immunohistochemically to determine the correlation between the two molecules and the associations with the clinicopathological features of each tumor. LN-5 gamma2 expression was clearly demonstrated in the cytoplasm and EGFR in the cell membranes of cancer cells. A significant increase in positivity for LN-5 gamma2 was observed in tumors showing poor differentiation (p < 0.001), infiltrative growth (p < 0.001), and deep invasion (p = 0.038). In a multivariate analysis, increased positivity for LN-5 gamma2 was an independent predictor of an unfavorable outcome (p < 0.001). A significant increase in positivity for EGFR was observed in tumors showing infiltrative growth (p = 0.032) and poor prognosis (p = 0.008). The LN-5 gamma2 expression was correlated significantly with EGFR expression (p < 0.001). Patients with tumor positivity for both molecules showed the worst prognosis (p < 0.001). LN-5 gamma2 overexpression and EGFR overexpression is evident in tumors showing infiltrative growth, suggesting that EGFR may influence the invasive activity of tumor cells through overexpression of LN-5 gamma2.
A correlative study was performed to address the impact of epidermal growth factor receptor (EGFR) overexpression on survival and pattern of failure in patients with advanced head and neck squamous cell carcinomas (HNSCCs) enrolled in a Phase III trial and randomized to receive conventional radiotherapy. The study population comprised 155 of 268 (58%) randomized patients with sufficient pretreatment biopsy specimens for immunohistochemical assay. The specimens were dewaxed and incubated after standard preparation with mouse monoclonal antibodies recognizing the extracellular domain of the EGFR molecule. The catalyzed product was visualized with 3,3'-diaminobenzidine Chromogen Kit and lightly counterstained with Mayer's hematoxylin. Quantitative EGFR immunohistochemistry (IHC) was done with SAMBA 4000 Cell Image Analysis System, without knowledge of the clinical outcome, to yield mean absorbance (MOD), staining index (SI), and quick score (QS). These EGFR IHC parameters were correlated with the T stage, N stage, combined stage grouping, and recursive partitioning analysis classes. Subsequently, the EGFR parameters were correlated with the outcome end points, i.e., overall survival (OS), disease-free survival (DFS), local-regional (LR) relapse, and distant metastasis rates. We found that HNSCCs exhibited a wide variation in EGFR expression (MOD, 0.2-66.0; SI, 0.3-97.0; QS, 0.01-69.9) with a relatively strong but nonlinear correlation between MOD and SI (r = 0.79). There was no correlation between EGFR expression and T stage, N stage, stage grouping, and recursive partitioning analysis classes (r = -0.07 to 0.17). The OS and DFS rates of patients with high EGFR-expressing HNSCCs (>median MOD) were highly significantly lower (P = 0.0006 and P = 0.0016, respectively) and the LR relapse rate was highly significantly higher (P = 0.0031) compared with those of patients with low EGFR-expressing HNSCCs. However, there was no difference in the distant metastasis rate between the two groups (P = 0.96). Significant correlations, although somewhat less robust than MOD, were also observed between SI and QS and the OS, DFS, and LR relapse rates. Multivariate analysis showed that EGFR expression was an independent determinant of survival and a robust independent predictor of LR relapse. In summary, this correlative study in a large series of patients revealed that EGFR expression, which varied considerably among HNSCCs, was a strong independent prognostic indicator for OS and DFS and a robust predictor for LR relapse but not for distant metastasis. The data suggest that EGFR IHC should be considered for selecting patients for more aggressive combined therapies or enrollment into trials targeting EGFR signaling pathways.
Although several studies have found overexpression of epidermal growth factor receptor (EGFR) proteins EGFR and Her-2 in head and neck cancers, the clinical relevance of the finding varies. We examined the expression and clinical association of these molecules with oral squamous cell carcinoma in an area where betel chewing is prevalent. EGFR and Her-2 proteins were measured in 59 paired (grossly normal and cancer) tissues by an enzyme immunoassy method. The cutoff value for gene overexpression was defined as the level of mean expression in normal tissue plus two s.d. A total of 59% of the patients consumed alcohol, 90% smoked tobacco, and 90% chewed betel quid. Of the patients assayed, 34 (58%) and 24 (41%) had EGFR and Her-2 overexpression, with average 3.5- and 1.5-fold elevations. EGFR overexpression has been shown to be statistically associated with T stage, N stage, overall TMN stage, primary tumour depth, lymph node extra-capsular spread, and poor survival. Her-2 overexpression, however, did not demonstrate a similar association with clinicopathological parameters or therapeutic outcome. On multivariant analysis, EGFR overexpression (P=0.041) and N stage (P=0.024) were the only independent factors for overall survival. These results indicate that the molecular targeting therapy to EGFR may be a treatment for oral cavity cancer in the betel quid-chewing prevalent area.
Receptor tyrosine kinase genes were sequenced in non–small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations
of the epidermal growth factor receptor gene EGFR were found in 15of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor
gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung
adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors
or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.
The activity of membrane-type 1 matrix metalloproteinase (MT1-MMP) is a double-edged sword--it is crucial for both physiological processes and disease progression. MT1-MMP modifies various cellular functions and it is, sthus, regulated precisely as a proteinase and as a membrane protein. Recent studies have further revealed that the function of MT1-MMP is modified and regulated by O-glycosylation, interaction with CD44, internalization and recycling. Such multidimensional mechanisms enable MT1-MMP to be regulated spatially and temporally, and are essential for its proper functioning on the cell surface.
Head and neck squamous cell carcinomas (HNSCCs) are characterized by up-regulation of the epidermal growth factor receptor (EGFR). We previously reported that a gastrin-releasing peptide/gastrin-releasing peptide receptor (GRP/GRPR) autocrine growth pathway is activated early in HNSCC carcinogenesis. GRP can induce rapid phosphorylation of EGFR and p42/44 mitogen-activated protein kinase (MAPK) activation in part via extracellular release of transforming growth factor alpha (TGF-alpha) by matrix metalloproteinases (MMPs). It has been reported that Src family kinases are activated by G-protein-coupled receptors (GPCRs), followed by downstream EGFR and MAPK activation. To further elucidate the mechanism of activation of EGFR by GRP in HNSCC, we investigated the role of Src family kinases. Blockade of Src family kinases using an Src-specific tyrosine kinase inhibitor A-419259 decreased GRP-induced EGFR phosphorylation and MAPK activation. GRP also failed to induce MAPK activation in dominant-negative c-Src-transfected HNSCC cells. Invasion and growth assays showed that c-Src was required for GRP-induced proliferation or invasion of HNSCC cells. In addition to TGF-alpha release, GRP induced amphiregulin, but not EGF, secretion into HNSCC cell culture medium, an effect that was blocked by the MMP inhibitor marimastat. TGF-alpha and amphiregulin secretion by GRP stimulation also was inhibited by blockade of Src family kinases. These results suggest that Src family kinases contribute to GRP-mediated EGFR growth and invasion pathways by facilitating cleavage and release of TGF-alpha and amphiregulin in HNSCC.
The extracellular pH (pHe) of tumor tissues is often acidic, which can induce the expression of several proteins. We previously showed that production
of matrix metalloproteinase-9 (MMP-9) was induced by culturing cells at acidic pHe (5.4–6.5). Here we have investigated the signal transduction pathway by which acidic pHe induces MMP-9 expression. We found that acidic pHe (5.9) activated phospholipase D (PLD), and inhibition of PLD activity by 1-butanol and Myr-ARF6 suppressed the acidic pHe-induced MMP-9 expression. Exogenous PLD, but not phosphatidylinositol-specific PLC or PLA2, mimicked MMP-9 induction by acidic pHe. Western blot analysis revealed that acidic pHe increased the steady-state levels of phosphorylated extracellular signal-regulated kinases 1/2 and p38 and that the PLD inhibitors
suppressed these increases. Using 5′-deletion mutant constructs of the MMP-9 promoter, we found that the acidic pHe-responsive region was located at nucleotide –670 to –531, a region containing the NFκB binding site. A mutation into the
NFκB binding site reduced, but not completely, the acidic pHe-induced MMP-9 promoter activity, and NFκB activity was induced by acidic pHe. Pharmacological inhibitors specific for mitogen-activated protein kinase kinase 1/2 (PD098059) and p38 (SB203580) attenuated
the acidic pHe-induced NFκB activity and MMP-9 expression. These data suggest that PLD, mitogen-activated protein kinases (extracellular
signal-regulated kinases 1/2 and p38), and NFκB mediate the acidic pHe signaling to induce MMP-9 expression. A transcription factor(s) other than NFκB may also be involved in the MMP-9 expression.
Breast and kidney-expressed chemokine (BRAK) CXCL14 is a new CXC chemokine with unknown function and receptor selectivity. The majority of head and neck squamous cell carcinoma (HNSCC) and some cervical squamous cell carcinoma do not express CXCL14 mRNA, as opposed to constitutive expression by normal oral squamous epithelium. In this study, we demonstrate that the loss of CXCL14 in HNSCC cells and at HNSCC primary tumor sites was correlated with low or no attraction of dendritic cell (DC) in vitro, and decreased infiltration of HNSCC mass by DC at the tumor site in vivo. Next, we found that recombinant human CXCL14 and CXCL14-positive HNSCC cell lines induced DC attraction in vitro, whereas CXCL14-negative HNSCC cells did not chemoattract DC. Transduction of CXCL14-negative HNSCC cell lines with the human CXCL14 gene resulted in stimulation of DC attraction in vitro and increased tumor infiltration by DC in vivo in chimeric animal models. Furthermore, evaluating the biologic effect of CXCL14 on DC, we demonstrated that the addition of recombinant human CXCL14 to DC cultures resulted in up-regulation of the expression of DC maturation markers, as well as enhanced proliferation of allogeneic T cells in MLR. Activation of DC with recombinant human CXCL14 was accompanied by up-regulation of NF-kappaB activity. These data suggest that CXCL14 is a potent chemoattractant and activator of DC and might be involved in DC homing in vivo.
ERBB receptor tyrosine kinases have important roles in human cancer. In particular, the expression or activation of epidermal growth factor receptor and ERBB2 are altered in many epithelial tumours, and clinical studies indicate that they have important roles in tumour aetiology and progression. Accordingly, these receptors have been intensely studied to understand their importance in cancer biology and as therapeutic targets, and many ERBB inhibitors are now used in the clinic. We will discuss the significance of these receptors as clinical targets, in particular the molecular mechanisms underlying response.
Angiogenesis, the formation of new blood vessels, is required for many pathologic processes, including invasive tumor growth as well as physiologic organ/tissue maintenance. Angiogenesis during development and adulthood is likely regulated by a balance between endogenous proangiogenic and antiangiogenic factors. It is speculated that tumor growth requires disruption of such balance; thus, the angiogenic switch must be turned "on" for cancer progression. If the angiogenic switch needs to be turned on to facilitate the tumor growth, the question remains as to what the physiologic status of this switch is in the adult human body; is it "off," with inhibitors outweighing the stimulators, or maintained at a fine "balance," keeping the proangiogenic properties of many factors at a delicate "activity" balance with endogenous inhibitors of angiogenesis. The physiologic status of this balance is important to understand as it might determine an individual's predisposition to turn the switch on during pathologic events dependent on angiogenesis. Conceivably, if the physiologic angiogenesis balance in human population exists somewhere between off and even balance, an individual's capacity and rate to turn the switch on might reflect their normal physiologic angiogenic status. In this regard, although extensive knowledge has been gained in our understanding of endogenous growth factors that stimulate angiogenesis, the activities associated with endogenous inhibitors are poorly understood. In this review, we will present an overview of the knowledge gained in studies related to the identification and characterization of 27 different endogenous inhibitors of angiogenesis.
Nature Rev. Cancer 5, 341–354 (2005)The authors would like to correct Figure 4b of this article.
An experimental model of tumor dormancy therapy for advanced head and neck carcinoma was developed. After transplantation of KB cells into nude mice, the mice were given tiracoxib, a selective cyclooxygenase (COX)-2 inhibitor, probucol, an antioxidant, and S-1, an oral pro-drug of 5-fluorouracil (5-FU), or combinations of two of them. The combined administration of tiracoxib with probucol significantly inhibited the tumor growth. The angiogenesis in this group was markedly reduced. Tiracoxib and probucol did not affect the intratumoral concentration of 5-FU when coadministered with S-1. The combined use of tiracoxib and probucol is thus a candidate for use in maintenance therapy after the primary therapy for patients with advanced head and neck carcinoma.
A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.
The squamous mucosa of patients who develop head and neck cancer is "condemned" or predisposed to disregulated growth as reflected by the high incidence of synchronous and metachronous primary tumors. We hypothesized that transformed and nontransformed mucosa from head and neck cancer patients would produce increased levels of transforming growth factor alpha (TGF-alpha) and its cell surface receptor, the epidermal growth factor receptor (EGFR), thereby contributing to this predisposition. Using molecular biological techniques, we examined the incidence and mechanism of TGF-alpha and EGFR overproduction in tumors and histologically normal mucosa excised from patients with squamous cell carcinoma of the head and neck (SCCHN) to test this hypothetical mechanism of field cancerization. Northern blot hybridization was used to evaluate the frequency of increased TGF-alpha and EGFR mRNA production in tissue excised from 24 patients with SCCHN and 10 cell lines compared with 7 control patients without cancer or a history of alcohol and tobacco use. Southern blot hybridization was used to examine for gene amplification. In patients with SCCHN, TGF-alpha mRNA was elevated by a mean of 5-fold in 95% of histologically "normal" mucosa samples (P = 0.001) and by a mean of 5-fold in 87.5% of tumors (P = 0.0001) while EGFR mRNA was elevated by a mean of 29-fold in 91% of histologically normal mucosa specimens (P = 0.0005) and by a mean of 69-fold in 92% of tumors (P = 0.0005), compared with mRNA levels in control normal mucosa. In 10 SCCHN cell lines, TGF-alpha mRNA was increased by a mean of 16-fold and EGFR mRNA levels were increased by a mean of 77-fold. Increased production of TGF-alpha and EGFR mRNA in the histologically normal mucosa of patients at risk for a primary or secondary head and neck cancer may serve both as a marker for malignant transformation and as a target for preventive therapies.
Interleukin 8, the first chemokine to be characterized, was discovered nearly ten years ago. Today, more than 30 human chemokines are known. They are often upregulated in inflammation and act mainly on leukocytes inducing migration and release responses. The present review deals largely with the new developments of the last three years. Several structural studies have shown that most chemokines form dimers. The dimers, however, dissociate upon dilution, and the monomers constitute the biologically active form. Chemokine activities are mediated by seven-transmembrane-domain, G protein coupled receptors, five of which were discovered in the past three years. The primary receptor-binding domain of all chemokines is near the NH2 terminus, and antagonists can be obtained by truncation or substitutions in this region. Major progress has been made in the understanding of chemokine actions on T lymphocytes that respond to several CC chemokines but also to IP10 and Mig, two CXC chemokines that selectively attract T cells via a novel receptor. Effects of chemokines on angiogenesis and tumor growth have been reported, but the data are still contradictory and the mechanisms unknown. Of considerable interest is the recent discovery that some chemokines function as HIV-suppressive factors by interacting with chemokine receptors which, together with CD4, were recognized as the binding sites for HIV-1.
Chemokines are a superfamily of pro-inflammatory polypeptide cytokines that selectively attract and activate different cell types. Many patho-physiological conditions require the participation of chemokines, including inflammation, infection, tissue injury, allergy, cardiovascular diseases, as well as malignant tumors. Chemokines activate cells through their binding to shared or unique cell surface receptors which belong to the seven-transmembrane, G-protein-coupled Rhodopsin superfamily. The role of chemokines in malignant tumors is complex: while some chemokines may enhance innate or specific host immunity against tumor implantation, others may favor tumor growth and metastasis by promoting tumor cell proliferation, migration or neovascularization in tumor tissue. In this review, the authors summarize some of the recent advances in chemokine research and emphasis is made on the effect of chemokines in tumor growth and metastasis.
Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play important roles in many disease processes. Chemokines are divided into two major groups, CC or CXC, based on their sequence around the amino terminal cysteines. We report the PCR cloning of a novel human chemokine termed BRAK for its initial isolation from breast and kidney cells. This novel chemokine is distantly related to other CXC chemokines (30% identity with MIP-2alpha and beta) and shares several biological activities. BRAK is expressed ubiquitously and highly in normal tissue. However, it was expressed in only 2 of 18 cancer cell lines. BRAK is located on human chromosome 5q31.
Craniofacial malformations are among the most frequent congenital birth defects in humans; cleft palate, that is inadequate fusion of the palatal shelves, occurs with an annual incidence of 1 in 700 to 1 in 1,000 live births among individuals of European descent. The secondary palate arises as bilateral outgrowths from the maxillary processes, and its formation depends on the coordinated development of craniofacial structures including the Meckel's cartilage and the mandible. Cleft lip and palate syndromes in humans are associated with polymorphisms in the gene (TGFA) encoding transforming growth factor-alpha (TGF-alpha), an epidermal growth factor receptor (EGFR) ligand made by most epithelia. Here we have characterized craniofacial development in Egfr-deficient (Egfr-/-) mice. Newborn Egfr-/- mice have facial mediolateral defects including narrow, elongated snouts, underdeveloped lower jaw and a high incidence of cleft palate. Palatal shelf explants from Egfr-/- mice fused, but frequently had residual epithelium in the midline. In addition, morphogenesis of Meckel's cartilage was deficient in cultured mandibular processes from Egfr-/- embryos. The secretion of matrix metalloproteinases (MMPs) was diminished in Egfr-/- explants, consistent with the ability of EGF to increase MMP secretion and with the decreased MMP expression caused by inhibition of Egfr signalling in wild-type explants. Accordingly, inactivation of MMPs in wild-type explants phenocopied the defective morphology of Meckel's cartilage seen in Egfr-/- explants. Our results indicate that EGFR signalling is necessary for normal craniofacial development and that its role is mediated in part by its downstream targets, the MMPs, and may explain the genetic correlation of human cleft palate with polymorphisms in TGFA.
Using differential display, we cloned a gene with reduced expression in short-term explants of head and neck squamous cell carcinoma (HNSCC) tumors compared to cultured normal oral epithelial cells. The differentially expressed gene was identical to the recently cloned CXC chemokine BRAK, which is ubiquitously expressed in normal tissue extracts but is absent from many tumor cell lines in vitro. To define the cell populations expressing BRAK in vivo, in situ mRNA hybridization was performed on normal and cancerous tissues from six different histological sites. The predominant normal cell type constitutively expressing BRAK in vivo was squamous epithelium. Expression in tumors was heterogeneous, with the majority of HNSCCs and some cervical squamous cell carcinomas (SCCs) showing loss of BRAK mRNA. Although absent in unstimulated peripheral blood mononuclear cells, high levels of BRAK were consistently found in infiltrating inflammatory cells (with lymphocyte morphology) in nearly all cancers examined. Furthermore, BRAK expression was demonstrated in B cells and monocytes, after stimulation of peripheral blood mononuclear cells with lipopolysaccharide. This study demonstrates for the first time up-regulation of BRAK mRNA by inflammatory cells in the tumor microenvironment and lost expression from certain cancers in vivo. The data suggest that BRAK may have a role in host-tumor interactions.
An experimental model of tumor dormancy therapy for advanced head and neck carcinoma was developed. After transplantation of KB cells into nude mice, the mice were given tiracoxib, a selective cyclooxygenase (COX)-2 inhibitor, probucol, an antioxidant, and S-1, an oral pro-drug of 5-fluorouracil (5-FU), or combinations of two of them. The combined administration of tiracoxib with probucol significantly inhibited the tumor growth. The angiogenesis in this group was markedly reduced. Tiracoxib and probucol did not affect the intratumoral concentration of 5-FU when coadministered with S-1. The combined use of tiracoxib and probucol is thus a candidate for use in maintenance therapy after the primary therapy for patients with advanced head and neck carcinoma.
When epidermal growth factor and its relatives bind the ErbB family of
receptors, they trigger a rich network of signalling pathways, culminating
in responses ranging from cell division to death, motility to adhesion. The
network is often dysregulated in cancer and lends credence to the mantra that
molecular understanding yields clinical benefit: over 25,000 women with breast
cancer have now been treated with trastuzumab (Herceptin®), a recombinant
antibody designed to block the receptor ErbB2. Likewise, small-molecule enzyme
inhibitors and monoclonal antibodies to ErbB1 are in advanced phases of clinical
testing. What can this pathway teach us about translating basic science into
clinical use?
SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.
The epidermal growth factor receptor (EGFR) autocrine pathway contributes to a number of processes important to cancer development and progression, including cell proliferation, apoptosis, angiogenesis, and metastatic spread. The critical role the EGFR plays in cancer has led to an extensive search for selective inhibitors of the EGFR signaling pathway. The results of a large body of preclinical studies and the early clinical trials thus far conducted suggest that targeting the EGFR could represent a significant contribution to cancer therapy. A variety of different approaches are currently being used to target the EGFR. The most promising strategies in clinical development include monoclonal antibodies to prevent ligand binding and small molecule inhibitors of the tyrosine kinase enzymatic activity to inhibit autophosphorylation and downstream intracellular signaling. At least five blocking monoclonal antibodies have been developed against the EGFR. Among these, IMC-225 is a chimeric human-mouse monoclonal IgG1 antibody that has been the first anti-EGFR targeted therapy to enter clinical evaluation in cancer patients in Phase II and III studies, alone or in combination with conventional therapies, such as radiotherapy and chemotherapy. A number of small molecule inhibitors of the EGFR tyrosine kinase enzymatic activity is also in development. OSI-774 and ZD1839 (Iressa) are currently in Phase II and III development, respectively. ZD1839, a p.o. active, selective quinazoline derivative has demonstrated promising in vitro and in vivo antitumor activity. Preliminary results from Phase I and II trials in patients with advanced disease demonstrate that ZD1839 and OSI-774 have an acceptable tolerability profile and promising clinical efficacy in patients with a variety of tumor types. This mini-review describes the EGFR inhibitors in clinical development.
The interactions between cancer cells and their micro- and macroenvironment create a context that promotes tumour growth and protects it from immune attack. The functional association of cancer cells with their surrounding tissues forms a new 'organ' that changes as malignancy progresses. Investigation of this process might provide new insights into the mechanisms of tumorigenesis and could also lead to new therapeutic targets.
Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that is frequently expressed in malignant cancer cells and has potent invasion-promoting activity. When expressed on the cell surface, MT1-MMP degrades the extracellular matrix (ECM) barrier adjacent to the cells to maintain the migration route to traverse the tissue. But MT1-MMP is not just an enzyme that degrades ECM. MT1-MMP also introduces limited cleavage into proteins at the cell-ECM interspaces and converts their functions. The target molecules are ECM components, cell adhesion molecules, and latent forms of MMPs. Through these processing events MT1-MMP modulates the migratory and invasive behavior of the cells.
Cell-cell adhesion determines the polarity of cells and participates in the maintenance of the cell societies called tissues. Cell-cell adhesiveness is generally reduced in human cancers. Reduced intercellular adhesiveness allows cancer cells to disobey the social order, resulting in destruction of histological structure, which is the morphological hallmark of malignant tumors. Reduced intercellular adhesiveness is also indispensable for cancer invasion and metastasis. A tumor-suppressor gene product, E-cadherin, and its undercoat proteins, catenins, which connect cadherins to actin filaments, are located at lateral borders, concentrating on adherens junctions, of epithelial cells and establish firm cell-cell adhesion. The E-cadherin cell adhesion system in cancer cells is inactivated by various mechanisms that reflect the morphological and biological characteristics of the tumor. Silencing of the E-cadherin gene by DNA hypermethylation around the promoter region occurs frequently, even in precancerous conditions. In diffuse infiltrating cancers, mutations are found in the genes for E-cadherin and alpha- and beta-catenins. At the invading front of cancers, the E-cadherin cell adhesion system is inactivated by tyrosine phosphorylation of beta-catenin; an oncogene product, c-erbB-2 protein, is found to associate directly with beta-catenin. The E-cadherin cell adhesion system cross-talks with the Wingless/Wnt signaling pathway through beta-catenin, and expression of genes, which participate in cancer morphogenesis, may be regulated in conjunction with the Wingless/Wnt signaling pathway. Dysadherin, a newly identified cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes cancer metastasis. In conclusion, inactivation of the E-cadherin cell adhesion system by both genetic and epigenetic mechanisms plays a significant role during multistage human carcinogenesis.
Antisense approaches targeting the epidermal growth factor receptor (EGFR) have been demonstrated to inhibit the growth of squamous cell carcinoma of the head and neck (SCCHN). Docetaxel is an effective chemotherapeutic agent in the treatment of SCCHN. The present study was undertaken to evaluate the antitumor mechanisms of EGFR antisense (AS) oligonucleotides administered in combination with docetaxel in preclinical models of SCCHN.
SCCHN cells lines and xenografts were treated with an EGFR AS oligonucleotide targeting region 760-779 of EGFR mRNA (GenBank accession XM_004738) alone and in combination with docetaxel. Proliferation in vitro and tumor growth in vivo were examined in addition to determinations of EGFR expression and signaling pathways to evaluate antitumor mechanisms.
A combination of docetaxel with EGFR AS resulted in increased cytotoxicity compared with treatment with docetaxel plus EGFR sense oligonucleotides or docetaxel alone after 24 h. Tumor volumes were significantly reduced in the mice treated with a combination of intratumoral EGFR AS and systemic docetaxel compared with mice receiving monotherapy. The combination of docetaxel plus EGFR AS resulted in decreased expression levels of EGFR, phosphotyrosine signal transducers and activators of transcription 3, vascular endothelial growth factor, and pAKT compared with expression levels after either treatment alone.
A combination of EGFR AS and docetaxel may be effective in the treatment of SCCHN with a reduced toxicity profile compared with standard chemotherapy regimens.
Receptor tyrosine kinases are a subclass of cell-surface growth-factor receptors with an intrinsic, ligand-controlled tyrosine-kinase activity. They regulate diverse functions in normal cells and have a crucial role in oncogenesis. Twenty years ago, the first primary structure of a receptor tyrosine kinase, the epidermal growth factor receptor, was elucidated. The characterization of both the molecular architecture of receptor tyrosine kinases and the main functions of these proteins and their ligands in tumorigenesis opened the door to a new era in molecular oncology and paved the way to the development of the first target-specific cancer therapeutics.
Here we describe the comprehensive gene expression profiles of each cell type composing normal breast tissue and in situ and invasive breast carcinomas using serial analysis of gene expression. Based on these data, we determined that extensive gene expression changes occur in all cell types during cancer progression and that a significant fraction of altered genes encode secreted proteins and receptors. Despite the dramatic gene expression changes in all cell types, genetic alterations were detected only in cancer epithelial cells. The CXCL14 and CXCL12 chemokines overexpressed in tumor myoepithelial cells and myofibroblasts, respectively, bind to receptors on epithelial cells and enhance their proliferation, migration, and invasion. Thus, chemokines may play a role in breast tumorigenesis by acting as paracrine factors.
The revolution in cancer research can be summed up in a single sentence: cancer is, in essence, a genetic disease. In the last decade, many important genes responsible for the genesis of various cancers have been discovered, their mutations precisely identified, and the pathways through which they act characterized. The purposes of this review are to highlight examples of progress in these areas, indicate where knowledge is scarce and point out fertile grounds for future investigation.
This study was undertaken to determine whether low intratumoral doses of the epidermal growth factor receptor ligand-transforming growth factor alpha (TGF-alpha) fused to Pseudomonas exotoxin (TGF-alpha-PE38)-abrogated head and neck squamous cell carcinoma (HNSCC) tumor growth in vitro and in vivo.
In vitro cytotoxicity assays were carried out to determine the sensitivity of HNSCC cells to TGF-alpha-PE38. TGF-alpha-PE38-treated HNSCC cells were examined by immunoblotting for cleaved poly(ADP-ribose) polymerase to evaluate apoptosis. Nude mice bearing established HNSCC xenografts were treated with several doses of TGF-alpha-PE38 to evaluate the antitumor efficacy in vivo. Tumor sections were stained with terminal deoxynucleotidyl transferase-mediated nick end labeling for apoptosis. To determine the effect of oral administration of TGF-alpha-PE38, gavage injections of TGF-alpha-PE38 were administered, and the esophagus and surrounding soft tissue were then stained for apoptotic cells.
HNSCC cell lines examined were sensitive to low doses of TGF-alpha-PE38 (EC(50) in the range of 1.6 to 10 ng/mL). HNSCC cells treated with TGF-alpha-PE38 undergo apoptosis. Antitumor effects were observed using 0.1 and 0.03 microg of TGF-alpha-PE38 administered intratumorally. At these doses, the treatment was well tolerated. Tumors treated with the toxin had a higher number of apoptotic cells compared with the control tumors. No apoptotic cells were observed in the pharyngoesophageal tissues of the mice after gavage administration of the toxin suggesting that the toxin could be orally administered without toxicity.
These results indicate that topical or intratumoral administration of low doses of TGF-alpha-PE38 may demonstrate antitumor effects in HNSCC without associated systemic toxicity.
Head and neck squamous cell carcinoma (HNSCC) is the most common malignant neoplasm arising in the mucosa of the upper aerodigestive tract. Nearly two thirds of patients present with advanced (stage III and IV) disease. Fifty percent of HNSCC patients die of their disease, and 5% of HNSCC patients per year will develop additional second primary tumors. Currently used therapeutic modalities (surgery, radiation, and/or chemotherapy) have been associated with rather modest improvements in patient survival. The Head and Neck Cancer: Research and Therapeutic Opportunities Workshop (held in Washington, DC, May 24-26, 2004) was organized by the Division of Cancer Biology at the National Cancer Institute to identify research areas and directions that will advance understanding of HNSCC biology and accelerate clinical translation. The primary goal of the workshop was to identify the barriers that impede basic science discovery and the translation of these developments to the clinical setting. Over a 2.5-day period, experts in both HNSCC and other cancer-related fields met to identify and prioritize the key areas for future research. The overall consensus was that HNSCC is a relatively understudied malignancy and that investigations that focus on the biology of this tumor have the potential to impact significantly on the prevention and treatment of epithelial malignancies. The chief objective is to communicate these research goals to the cancer biology community and encourage more interest in HNSCC as a tumor model to test translational research hypotheses.
BRAK/CXCL14 is a CXC chemokine constitutively expressed at the mRNA level in certain normal tissues but absent from many established tumor cell lines and human cancers. Although multiple investigators cloned BRAK, little is known regarding the physiologic function of BRAK or the reason for decreased expression in cancer. To understand the possible significance associated with loss of BRAK mRNA in tumors, we examined the pattern of BRAK protein expression in normal and tumor specimens from patients with squamous cell carcinoma (SCC) of the tongue and used recombinant BRAK (rBRAK) to investigate potential biological functions. Using a peptide-specific antiserum, abundant expression of BRAK protein was found in suprabasal layers of normal tongue mucosa but consistently was absent in tongue SCC. Consistent with previous in situ mRNA studies, BRAK protein also was expressed strongly by stromal cells adjacent to tumors. In the rat corneal micropocket assay, BRAK was a potent inhibitor of in vivo angiogenesis stimulated by multiple angiogenic factors, including interleukin 8, basic fibroblast growth factor, and vascular endothelial growth factor. In vitro, rBRAK blocked endothelial cell chemotaxis at concentrations as low as 1 nmol/L, suggesting this was a major mechanism for angiogenesis inhibition. Although only low affinity receptors for BRAK could be found on endothelial cells, human immature monocyte-derived dendritic cells (iDCs) bound rBRAK with high affinity (i.e., K(d), approximately 2 nmol/L). Furthermore, rBRAK was chemotactic for iDCs at concentrations ranging from 1 to 10 nmol/L. Our findings support a hypothesis that loss of BRAK expression from tumors may facilitate neovascularization and possibly contributes to immunologic escape.
Recent studies suggest inflammatory processes may be involved in the development or progression of prostate cancer. Chemokines are a family of cytokines that can play several roles in cancer progression including angiogenesis, inflammation, cell recruitment, and migration.
Real-time quantitative RT-PCR, in situ RNA hybridization, laser capture microscopy, immunohistochemistry, and cDNA array based technologies were used to examine CXCL14 (BRAK) expression in paired normal and tumor prostate. To determine the role CXCL14 expression has on cancer progression, LAPC4 cells were engineered to overexpress mouse or human CXCL14, and xenograft studies were performed.
CXCL14 RNA expression was observed in normal and tumor prostate epithelium and focally in stromal cells adjacent to cancer. CXCL14 mRNA was significantly upregulated in localized prostate cancer and positively correlated with Gleason score. CXCL14 levels were unchanged in BPH specimens. LAPC4 cells expressing CXCL14 resulted in a 43% tumor growth inhibition (P = 0.019) in vivo compared to vector only xenografts.
CXCL14 mRNA upregulation is a common feature in prostate cancer. The finding that CXCL14 expression inhibits tumor growth suggests this gene has tumor suppressive functions.
Squamous cell carcinoma (SCC) of the tongue is sometimes associated with local recurrence and regional metastases despite adequate surgical excision. The present study sought to determine if epidermal growth factor receptor (EGFR) and Cerb-B2 expression has prognostic or predictive value in SCC of the tongue, as with other epithelial malignancies. The study sample comprised of 27 patients with carcinoma of the tongue who underwent partial glossectomy and neck dissection between 1990 and 1999. Average follow-up was 54 months. Specimens from 23 patients were analyzed for growth factor expression using monoclonal antibodies specific for EGFR and Cerb-B2. Findings were correlated with the clinical course. EGFR and Cerb-B2 were expressed in 34% and 17% of the specimens, respectively. There was a statistically significant correlation between EGFR expression and tumor differentiation, and a borderline correlation between Cerb-B2 expression and T stage. No association was found between growth factor expression and tumor depth, lymph node status, extracapsular invasion, recurrence, or survival. Overexpression of EGFR and Cerb-B2 cannot serve as a prognostic factor or predictor of survival and treatment success in SCC of the tongue.
In order to investigate the mechanisms by which 1alpha,25(OH)2 vitamin D3 (VD3) stimulates the differentiation of human osteoblasts, we cultured MG-63, which is a human osteoblastic cell line, in the presence or absence of VD3 and/or L-ascorbic acid 2-phosphate (Asc 2-P), a long-acting vitamin C derivative. The cell growth rate was decreased by the presence of VD3 in the culture medium. Type I collagen synthesis and alkaline phosphatase (ALP) activity, which are markers of early stage osteoblast differentiation, were stimulated by the presence of VD3 as well as by that of Asc 2-P. The co-presence of Asc 2-P and VD3 had a synergistic effect on the collagen synthesis and ALP activity of the cells. Inhibition of collagen synthesis by the addition of inhibitors of collagen synthesis to the medium attenuated the stimulative effect of VD3 and Asc 2-P on the ALP activity. Transfection of the cells with siRNA-expressing vectors for COL1A1 decreased the expression level of ALP mRNA in addition to that of COL1A1. On the other hand, ALP activity was significantly increased, and the growth rate was decreased, when the cells were cultured on type I collagen-coated dishes. These effects were not seen when the cells were cultured on dishes coated with heat-denatured collagen. VD3 also increased the mRNA levels for Runx2 and osterix, which are transcription factors critical for osteoblast differentiation, as well as those of differentiation markers such as bone/liver/kidney type ALP, COL1A1, (the gene for the alpha1 chain of type I collagen), and osteocalcin, in the cells. Normal human osteoblasts and human bone marrow-derived mesenchymal stem cells (hBMSC) showed quite similar responses to VD3. These results indicate that VD3-stimulated gene expression of type I collagen and that mature type I collagen produced in the presence of Asc 2-P mediates at least a part of the stimulative effects of Asc 2-P and VD3 on the differentiation of these human osteoblastic cells. Levels of mRNAs for ALP and COL1A1 were increased, but the level of Runx2 was decreased, by the expression of osterix in MG-63 cells. These results also suggest that VD3 controls the growth and differentiation of human osteoblastic cells by regulating the gene expression of osteoblast-related transcription factors as well as that of type I collagen, and that the co-presence of both signals is essential for VD3 to express full activity toward the differentiation of human osteoblasts.
Development of cDNA microarray for analysis of gene expression profiles during the differentiation process in the craniomandibular system and for molecular diagnosis of craniomandibular disorders
- K Izukuri
- S Ozawa
- Y Maehata
- S Takamizawa
- E Kubota
- S Sato
- R Hata
K. Izukuri, S. Ozawa, Y. Maehata, S. Takamizawa, E. Kubota, S.
Sato, R. Hata, Development of cDNA microarray for analysis of
gene expression profiles during the differentiation process in the
craniomandibular system and for molecular diagnosis of craniomandibular disorders, Bull. Kanagawa Dent. Coll. 30 (2002) 137-140.
Development of cDNA microarray for analysis of gene expression profiles during the differentiation process in the craniomandibular system and for molecular diagnosis of craniomandibular disorders
- Izukuri
Cancer genes and the pathways they control
- Vogelstein
A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor
- Ciardiello