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CXCL14 is a direct target gene of HIC1 (A) Left: relative RT-qPCR analysis of 8 differentially expressed genes (CCL2, CXCL14, CX3CL1, IL1B, IL24, IL8, TGFA and VEGFA) after HIC1 deletion in MCF7 and T47D cells (n = 3). Right: relative RT-qPCR analysis of Hic1, Cxcl14 and Sirt1 mRNA levels in the mammary glands of Hic1-/-or Hic1 +/+ mice (n = 5). (B) ELISA analysis of CXCL14 levels in the CM of MCF7 sgHIC1 /MCF7 Ctrl and T47D sgHIC1 /T47D Ctrl cells. The supernatants were collected after culture of the cells for 24 h or 48 h (n = 4). (C) Schematic of the CXCL14 promoter region. The positions of selected consensus binding sites are indicated above the diagram; the lengths of the promoter constructs used in the reporter assays are shown below. (D) CXCL14 promoter activity after transfection of the full-length construct (-2000/+136) alone or together with HIC1 expression vectors. pGL3Basic, control for promoter constructs; pc3.1, control for the HIC1 expression vector. The results are expressed as the ratio of firefly luciferase to Renilla luciferase (n = 3). (E) CXCL14 promoter activity after cotransfection with 100 ng of the HIC1 expression vector and each of the promoter constructs. The-41/+136
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Breast cancer (BrCa) is the malignant tumor that most seriously threatens female health; however, the molecular mechanism underlying its progression remains unclear. Here, we found that conditional deletion of HIC1 in the mouse mammary gland might contribute to premalignant transformation in the early stage of tumor formation. Moreover, the chemoki...
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... to identify potential downstream targets of HIC1, we analyzed the published data on the genome-wide transcriptome profiles of MDA-MB-231 HIC1 /MDA-MB-231 GFP and HBL-100 shHIC1 /HBL-100 shNC cells using Agilent Whole Human Genome Microarrays (21) (Supplemental Figure 2A). Among the differentially expressed genes encoding cytokines, eight genes that may participate in the activation of mammary fibroblasts were identified ( Figure 3A and Supplemental Figure 2B). RT-qPCR was performed to assay the expression of these eight genes at the mRNA level in MCF7 sgHIC1 /MCF7 Ctrl , T47D sgHIC1 /T47D Ctrl and MDA-MB- 231 HIC1 /MDA-MB-231 GFP cells. ...
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... was performed to assay the expression of these eight genes at the mRNA level in MCF7 sgHIC1 /MCF7 Ctrl , T47D sgHIC1 /T47D Ctrl and MDA-MB- 231 HIC1 /MDA-MB-231 GFP cells. Chemokine CXCL14 was chosen as a potentially relevant cytokine based on results showing that altering HIC1 expression in these cells markedly modulated CXCL14 expression at both the mRNA and protein levels compared with the controls ( Figure 3A and 3B, and Supplemental Figure 2B). CXCL14 expression was also greatly increased in Hic1 -/-mice at the mRNA and protein levels ( Figure 3A and Supplemental Figure 2C). ...
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... CXCL14 was chosen as a potentially relevant cytokine based on results showing that altering HIC1 expression in these cells markedly modulated CXCL14 expression at both the mRNA and protein levels compared with the controls ( Figure 3A and 3B, and Supplemental Figure 2B). CXCL14 expression was also greatly increased in Hic1 -/-mice at the mRNA and protein levels ( Figure 3A and Supplemental Figure 2C). Similarly, the expression of Sirt1, a classical downstream gene of HIC1, was also observed to be enhanced in Hic1 -/-mice ( Figure 3A). ...
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... expression was also greatly increased in Hic1 -/-mice at the mRNA and protein levels ( Figure 3A and Supplemental Figure 2C). Similarly, the expression of Sirt1, a classical downstream gene of HIC1, was also observed to be enhanced in Hic1 -/-mice ( Figure 3A). These findings suggest that CXCL14 expression is modulated by HIC1. ...
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... that HIC1 functions as a repressive transcription factor and directly binds to the promoter regions of target genes (20), we inferred that CXCL14 is a potential downstream target induced by HIC1. Indeed, as shown in Figure 3C, 11 putative HIC1-binding sites (TGCC/GGCA) were identified in the CXCL14 promoter. ...
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... constructed a series of CXCL14-truncated promoter/reporter fusion plasmids containing progressive deletions of the 5' region of the gene from -2000 to +136 ( Figure 3C). These constructs were then transfected together with the pcDNA3.1-His ...
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... vector into 293T and MCF7 cells, and the promoter activities were measured using luciferase reporter assays. Figure 3D shows that when transfected into 293T and MCF7 cells, the construct containing the full-length CXCL14 promoter resulted in higher activity than the basic construct. Moreover, transient transfection of the cells with HIC1 markedly inhibited CXCL14 promoter activity ( Figure 3D) in a dose-dependent manner (Supplemental Figure 2D). ...
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... 3D shows that when transfected into 293T and MCF7 cells, the construct containing the full-length CXCL14 promoter resulted in higher activity than the basic construct. Moreover, transient transfection of the cells with HIC1 markedly inhibited CXCL14 promoter activity ( Figure 3D) in a dose-dependent manner (Supplemental Figure 2D). The inhibitory effect of HIC1 on CXCL14 promoter activity in both cell lines was maintained in all the truncated constructs, including -2000/+136, -726/+136, -276/+136, and -41/+136 ( Figure 3E). ...
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... transient transfection of the cells with HIC1 markedly inhibited CXCL14 promoter activity ( Figure 3D) in a dose-dependent manner (Supplemental Figure 2D). The inhibitory effect of HIC1 on CXCL14 promoter activity in both cell lines was maintained in all the truncated constructs, including -2000/+136, -726/+136, -276/+136, and -41/+136 ( Figure 3E). These results suggest that the regulatory region in the HIC1-mediated repression may be located within the -41 bp to +136 bp region of the CXCL14 promoter, which contains two putative HIC1 binding sites, M1 and M2 (Supplemental Figure 2E). ...
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... to further determine whether the CXCL14 promoter is indeed directly bound by HIC1, ChIP assays were performed in MCF7 and T47D cells using an antibody against HIC1. The pulled-down DNA was amplified by ordinary PCR and RT-qPCR ( Figure 3F) with primers that were designed based on the -41/+136 region of the CXCL14 promoter (Supplemental Figure 2E, underlined sequences). As shown in Figure 3F, the CXCL14 promoter was markedly enriched in the HIC1-immunoprecipitated MCF7 and T47D chromatin but absent from the chromatin that was immunoprecipitated by the control rabbit IgG. ...
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... pulled-down DNA was amplified by ordinary PCR and RT-qPCR ( Figure 3F) with primers that were designed based on the -41/+136 region of the CXCL14 promoter (Supplemental Figure 2E, underlined sequences). As shown in Figure 3F, the CXCL14 promoter was markedly enriched in the HIC1-immunoprecipitated MCF7 and T47D chromatin but absent from the chromatin that was immunoprecipitated by the control rabbit IgG. Taken together, these results demonstrate that HIC1 directly represses CXCL14 transcription. ...
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... immortalized NAF10 cells stably overexpressing CXCL14 were observed to be activated ( Figure 4A). Moreover, CXCL14 treatment had an activating effect on NAF6 cells similar to that of CXCL12, but the effect was weaker than the effect observed after TGF- treatment (Supplemental Figure 3A). Both CXCL12 and TGF- are usually considered to be activators of mammary fibroblasts. ...
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... to investigate the effect of the HIC1/CXCL14 axis on BrCa progression in vivo, three MCF7 cell lines (Ctrl-NC, sgHIC1-NC, and sgHIC1-shCXCL14) were injected bilaterally into the fourth mammary fat pads of female BALB/c nude mice (Supplemental Figure 3B). Figures 4D and 4E show that both the volumes and the weights of the resulting tumors were greatly increased in the MCF7 sgHIC1-NC group compared with the control MCF7 Ctrl-NC group. ...
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... results were further confirmed by quantitative analysis ( Figure 4F). Finally, using two Oncomine datasets, we found that the CXCL14 mRNA levels were higher in BrCa tissues (n = 93) than they were in normal breast tissues (n = 9) (Supplemental Figure 3C). Altogether, these data suggest that CXCL14 derived from HIC1-deleted BrCa cells mediates the activation of mammary fibroblasts and that this activation is responsible for BrCa progression. ...
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... to identify potential downstream targets of HIC1, we analyzed the published data on the genome-wide transcriptome profiles of MDA-MB-231 HIC1 /MDA-MB-231 GFP and HBL-100 shHIC1 /HBL-100 shNC cells using Agilent Whole Human Genome Microarrays (21) (Supplemental Figure 2A). Among the differentially expressed genes encoding cytokines, eight genes that may participate in the activation of mammary fibroblasts were identified ( Figure 3A and Supplemental Figure 2B). RT-qPCR was performed to assay the expression of these eight genes at the mRNA level in MCF7 sgHIC1 /MCF7 Ctrl , T47D sgHIC1 /T47D Ctrl and MDA-MB-231 HIC1 /MDA-MB-231 GFP cells. ...
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... was performed to assay the expression of these eight genes at the mRNA level in MCF7 sgHIC1 /MCF7 Ctrl , T47D sgHIC1 /T47D Ctrl and MDA-MB-231 HIC1 /MDA-MB-231 GFP cells. Chemokine CXCL14 was chosen as a potentially relevant cytokine based on results showing that altering HIC1 expression in these cells markedly modulated CXCL14 expression at both the mRNA and protein levels compared with the controls ( Figure 3A and 3B, and Supplemental Figure 2B). CXCL14 expression was also greatly increased in Hic1 -/-mice at the mRNA and protein levels ( Figure 3A and Supplemental Figure 2C). ...
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... CXCL14 was chosen as a potentially relevant cytokine based on results showing that altering HIC1 expression in these cells markedly modulated CXCL14 expression at both the mRNA and protein levels compared with the controls ( Figure 3A and 3B, and Supplemental Figure 2B). CXCL14 expression was also greatly increased in Hic1 -/-mice at the mRNA and protein levels ( Figure 3A and Supplemental Figure 2C). Similarly, the expression of Sirt1, a classical downstream gene of HIC1, was also observed to be enhanced in Hic1 -/-mice ( Figure 3A). ...
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... expression was also greatly increased in Hic1 -/-mice at the mRNA and protein levels ( Figure 3A and Supplemental Figure 2C). Similarly, the expression of Sirt1, a classical downstream gene of HIC1, was also observed to be enhanced in Hic1 -/-mice ( Figure 3A). These findings suggest that CXCL14 expression is modulated by HIC1. ...
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... that HIC1 functions as a repressive transcription factor and directly binds to the promoter regions of target genes (20), we inferred that CXCL14 is a potential downstream target induced by HIC1. Indeed, as shown in Figure 3C, 11 putative HIC1-binding sites (TGCC/GGCA) were identified in the CXCL14 promoter. ...
Context 20
... constructed a series of CXCL14-truncated promoter/reporter fusion plasmids containing progressive deletions of the 5' region of the gene from -2000 to +136 ( Figure 3C). These constructs were then transfected together with the pcDNA3.1-His ...
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... vector into 293T and MCF7 cells, and the promoter activities were measured using luciferase reporter assays. Figure 3D shows that when transfected into 293T and MCF7 cells, the construct containing the full-length CXCL14 promoter resulted in higher activity than the basic construct. Moreover, transient transfection of the cells with HIC1 markedly inhibited CXCL14 promoter activity ( Figure 3D) in a dose-dependent manner (Supplemental Figure 2D). ...
Context 22
... 3D shows that when transfected into 293T and MCF7 cells, the construct containing the full-length CXCL14 promoter resulted in higher activity than the basic construct. Moreover, transient transfection of the cells with HIC1 markedly inhibited CXCL14 promoter activity ( Figure 3D) in a dose-dependent manner (Supplemental Figure 2D). The inhibitory effect of HIC1 on CXCL14 promoter activity in both cell lines was maintained in all the truncated constructs, including -2000/+136, -726/+136, -276/+136, and -41/+136 ( Figure 3E). ...
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... transient transfection of the cells with HIC1 markedly inhibited CXCL14 promoter activity ( Figure 3D) in a dose-dependent manner (Supplemental Figure 2D). The inhibitory effect of HIC1 on CXCL14 promoter activity in both cell lines was maintained in all the truncated constructs, including -2000/+136, -726/+136, -276/+136, and -41/+136 ( Figure 3E). These results suggest that the regulatory region in the HIC1-mediated repression may be located within the -41 bp to +136 bp region of the CXCL14 promoter, which contains two putative HIC1 binding sites, M1 and M2 (Supplemental Figure 2E). ...
Context 24
... to further determine whether the CXCL14 promoter is indeed directly bound by HIC1, ChIP assays were performed in MCF7 and T47D cells using an antibody against HIC1. The pulled-down DNA was amplified by ordinary PCR and RT-qPCR ( Figure 3F) with primers that were designed based on the -41/+136 region of the CXCL14 promoter (Supplemental Figure 2E, underlined sequences). As shown in Figure 3F, the CXCL14 promoter was markedly enriched in the HIC1-immunoprecipitated MCF7 and T47D chromatin but absent from the chromatin that was immunoprecipitated by the control rabbit IgG. ...
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... pulled-down DNA was amplified by ordinary PCR and RT-qPCR ( Figure 3F) with primers that were designed based on the -41/+136 region of the CXCL14 promoter (Supplemental Figure 2E, underlined sequences). As shown in Figure 3F, the CXCL14 promoter was markedly enriched in the HIC1-immunoprecipitated MCF7 and T47D chromatin but absent from the chromatin that was immunoprecipitated by the control rabbit IgG. Taken together, these results demonstrate that HIC1 directly represses CXCL14 transcription. ...
Context 26
... immortalized NAF10 cells stably overexpressing CXCL14 were observed to be activated ( Figure 4A). Moreover, CXCL14 treatment had an activating effect on NAF6 cells similar to that of CXCL12, but the effect was weaker than the effect observed after TGF- treatment (Supplemental Figure 3A). Both CXCL12 and TGF- are usually considered to be activators of mammary fibroblasts. ...
Context 27
... to investigate the effect of the HIC1/CXCL14 axis on BrCa progression in vivo, three MCF7 cell lines (Ctrl-NC, sgHIC1-NC, and sgHIC1-shCXCL14) were injected bilaterally into the fourth mammary fat pads of female BALB/c nude mice (Supplemental Figure 3B). Figures 4D and 4E show that both the volumes and the weights of the resulting tumors were greatly increased in the MCF7 sgHIC1-NC group compared with the control MCF7 Ctrl-NC group. ...
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... results were further confirmed by quantitative analysis ( Figure 4F). Finally, using two Oncomine datasets, we found that the CXCL14 mRNA levels were higher in BrCa tissues (n = 93) than they were in normal breast tissues (n = 9) (Supplemental Figure 3C). Altogether, these data suggest that CXCL14 derived from HIC1-deleted BrCa cells mediates the activation of mammary fibroblasts and that this activation is responsible for BrCa progression. ...
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... Recently, there has been increased focus on chemokine ligands such as Chemokine ligand 17 (CCL17), for which the targeted drug mogamulizumab has been developed (Maeda et al. 2019a). Previous research has shown that aberrantly high expression of CCL17 in various cancers, including leukemia, lung cancer, bladder cancer, breast cancer, gastric cancer, and liver cancer (Bouchet et al. 2020;Gao et al. 2020;Higuchi et al. 2019;Lim et al. 2014;Mishalian et al. 2014;Wang et al. 2018Wang et al. , 2019Ye et al. 2022), serves as a primary driver for recruiting CCR4-expressing Tregs into the TME. This, in turn, extensively suppresses the anti-tumor immune response and promotes tumor progression (Gao et al. 2020). ...
Purpose
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Methods
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Conclusion
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... HIC1 is a sequence-specific transcriptional repressor [17]. It binds to a specific sequence named HiRE, which consists of a TGCC or GGCA core motif [18]. To the best of our knowledge, the role of renal HIC1 using in vivo models such as CKO mice remains unclear. ...
Nicotinamide adenine dinucleotide (NAD) is involved in renal physiology and is synthesized by nicotinamide mononucleotide adenylyltransferase (NMNAT). NMNAT exists as three isoforms, namely, NMNAT1, NMNAT2, and NMNAT3, encoded by Nmnat1, Nmnat2, and Nmnat3, respectively. In diabetic nephropathy (DN), NAD levels decrease, aggravating renal fibrosis. Conversely, sodium–glucose cotransporter-2 inhibitors increase NAD levels, mitigating renal fibrosis. In this regard, renal NAD synthesis has recently gained attention. However, the renal role of Nmnat in DN remains uncertain. Therefore, we investigated the role of Nmnat by establishing genetically engineered mice. Among the three isoforms, NMNAT1 levels were markedly reduced in the proximal tubules (PTs) of db/db mice. We examined the phenotypic changes in PT-specific Nmnat1 conditional knockout (CKO) mice. In CKO mice, Nmnat1 expression in PTs was downregulated when the tubules exhibited albuminuria, peritubular type IV collagen deposition, and mitochondrial ribosome (mitoribosome) excess. In CKO mice, Nmnat1 deficiency-induced mitoribosome excess hindered mitoribosomal translation of mitochondrial inner membrane-associated oxidative phosphorylation complex I (CI), CIII, CIV, and CV proteins and mitoribosomal dysfunction. Furthermore, the expression of hypermethylated in cancer 1, a transcription repressor, was downregulated in CKO mice, causing mitoribosome excess. Nmnat1 overexpression preserved mitoribosomal function, suggesting its protective role in DN.
... [43] Previously, we identified two distinct fibroblast clusters in the PCa microenvironment. [13] We have also reported that CAFs crosstalk with tumor cells to promote breast cancer [44] and PCa progression. [27] A recent study revealed the conservation of three supercluster CAF phenotypes across species and tissues of origin. ...
Prostate cancer (PCa) is an extensive heterogeneous disease with a complex cellular ecosystem in the tumor microenvironment (TME). However, the manner in which heterogeneity is shaped by tumors and stromal cells, or vice versa, remains poorly understood. In this study, single‐cell RNA sequencing, spatial transcriptomics, and bulk ATAC‐sequence are integrated from a series of patients with PCa and healthy controls. A stemness subset of club cells marked with SOX9highARlow expression is identified, which is markedly enriched after neoadjuvant androgen‐deprivation therapy (ADT). Furthermore, a subset of CD8⁺CXCR6⁺ T cells that function as effector T cells is markedly reduced in patients with malignant PCa. For spatial transcriptome analysis, machine learning and computational intelligence are comprehensively utilized to identify the cellular diversity of prostate cancer cells and cell‐cell communication in situ. Macrophage and neutrophil state transitions along the trajectory of cancer progression are also examined. Finally, the immunosuppressive microenvironment in advanced PCa is found to be associated with the infiltration of regulatory T cells (Tregs), potentially induced by an FAP⁺ fibroblast subset. In summary, the cellular heterogeneity is delineated in the stage‐specific PCa microenvironment at single‐cell resolution, uncovering their reciprocal crosstalk with disease progression, which can be helpful in promoting PCa diagnosis and therapy.
... As expected, siRNAs targeting QKI reversed the reduced level of α-SMA induced by miR-5100 inhibition in CAF1 cells (Fig. 4I, J). A recent study has identified that AKT and STAT3 pathways are indispensable for CAFs activation [21,22]. Thus, we detected the activation of AKT and STAT3 in the recipient CAF1 cells. ...
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... However, in breast cancer, melanoma, cervical cancer and colorectal cancer, expression of CXCL14 inhibits cell migration 42 . In contrast, other studies have reported that it enhances cancer cell migration and invasion in paclitaxel resistant breast cancer cells 43,44 . On the other hand, elevated CXCL6 exhibited metastatic properties in osteosarcoma cells 45 . ...
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... Tissue Microarray and Immunohistochemistry: Immunohistochemistry was performed as described previously. [52] The expression of GRP75 was analyzed in the tissue microarray samples. In brief, the tissue microarray was incubated with GRP75 antibody (1:100 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4°C followed by a corresponding biotinylated secondary antibody. ...
Tumors often overexpress glucose‐regulated proteins, and agents that interfere with the production or activity of these proteins may represent novel cancer treatments. The chlorpromazine derivative JX57 exhibits promising effects against endometrial cancer with minimal extrapyramidal side effects; however, its mechanisms of action are currently unknown. Here, glucose‐regulated protein 75 kD (GRP75) is identified as a direct target of JX57 using activity‐based protein profiling and loss‐of‐function experiments. The findings show that GRP75 is necessary for the biological activity of JX57, as JX57 exhibits moderate anticancer properties in GRP75‐deficient cancer cells, both in vitro and in vivo. High GRP75 expression is correlated with poor differentiation and poor survival in patients with endometrial cancer, whereas the knockdown of GRP75 can significantly suppress tumor growth. Mechanistically, the direct binding of JX57 to GRP75 impairs the structure of the mitochondria‐associated endoplasmic reticulum membrane and disrupts the endoplasmic reticulum–mitochondrial calcium homeostasis, resulting in a mitochondrial energy crisis and AMP‐activated protein kinase activation. Taken together, these findings highlight GRP75 as a potential prognostic biomarker and direct therapeutic target in endometrial cancer and suggest that the chlorpromazine derivative JX57 can potentially be a new therapeutic option for endometrial cancer.
... Although it did not induce β-arrestin recruitment via the atypical chemokine receptor 2 (AKCR2) 29 , CXCL14 was implicated in breast cancer progression mediated by ACKR2 28,33 . CXCL14 was also found to promote breast cancer cell progression by another orphan GPCR, GPR85 34 . ...
... Among a large pool of 238 investigated GPCRs, MRGPRX2 was the only one activated by CXCL14 (1 µM) in β-arrestin recruitment assays, indicating the chemokine's specificity for MRGPRX2, at least among the tested GPCRs (see Supplementary Data 2). Previous studies had claimed that CXCL14 may interact with GPR85, ACKR2, and CXCR4, although there have been some controversies [30][31][32][33][34] . In the present study we found that CXCL14 (1 µM) acts as an inverse agonist of CXCR4 blocking CXCL12-induced β-arrestin-2 recruitment (see Supplementary Data 2, Supplementary Fig. 2), whereas we observed no effect of CXCL14 at GPR85. ...
Patients with idiopathic pulmonary fibrosis show a strongly upregulated expression of chemokine CXCL14, whose target is still unknown. Screening of CXCL14 in a panel of human G protein-coupled receptors (GPCRs) revealed its potent and selective activation of the orphan MAS-related GPCR X2 (MRGPRX2). This receptor is expressed on mast cells and − like CXCL14 − upregulated in bronchial inflammation. CXCL14 induces robust activation of MRGPRX2 and its putative mouse ortholog MRGPRB2 in G protein-dependent and β-arrestin recruitment assays that is blocked by a selective MRGPRX2/B2 antagonist. Truncation combined with mutagenesis and computational studies identified the pharmacophoric sequence of CXCL14 and its presumed interaction with the receptor. Intriguingly, C-terminal domain sequences of CXCL14 consisting of 4 to 11 amino acids display similar or increased potency and efficacy compared to the full CXCL14 sequence (77 amino acids). These results provide a rational basis for the future development of potential idiopathic pulmonary fibrosis therapies.
... The receptor of CXCL14 remains controversial, and ACKR2, CXCR4, and GPR85 have all been proposed as potential receptors for CXCL14. [35][36][37][38] To identify the potential receptor of CXCL14 in RCC, we analyzed the level of these three receptors in the single-cell datasets. The levels of ACKR2 and GPR85 were exceedingly low in all single-cell sequencing datasets. ...
Background
Epigenetic mechanisms play vital roles in the activation, differentiation, and effector function of immune cells. The breast and kidney-expressed chemokine (CXCL14) mainly contributes to the regulation of immune cells. However, its role in shaping the tumor immune microenvironment (TIME) is yet to be elucidated in renal cell carcinoma (RCC).
Objectives
This study aimed to elucidate the role of CXCL14 in predicting the efficacy of immunotherapy in patients with RCC.
Methods
CXCL14 expression and RNA-sequencing, single-cell RNA-sequencing (scRNA-seq), and survival datasets of RCC from public databases were analyzed, and survival was compared between different CXCL14 levels. The correlation between CXCL14 and immune infiltration and human leukocyte antigen (HLA) gene expression was analyzed with TIMER2.0 and gene expression profiling interactive analysis. Institutional scRNA-seq and immunohistochemical staining analyses were used to verify the relationship between CXCL14 expression level and the efficacy of immunotherapy.
Results
CXCL14 was expressed in fibroblast and malignant cells in RCC, and higher expression was associated with better survival. Enrichment analysis revealed that CXCL14 is involved in immune activation, primarily in antigen procession, antigen presentation, and major histocompatibility complex assemble. CXCL14 expression was positively correlated with T-cell infiltration as well as HLA-related gene expression. Among the RCC cohort receiving nivolumab in Checkmate 025, the patients with CXCL14 high expression had better overall survival than those with CXCL14 low expression after immunotherapy. scRNA-seq revealed a cluster of CXCL14+ fibroblast in immunotherapy responders. Immunohistochemistry analysis verified that the patients with high CXCL14 expression had an increased proportion of high CD8 expression simultaneously. The expression level of CXCL14 was associated with CXCR4 expression in RCC.
Conclusion
CXCL14 expression is associated with immunotherapy response in RCC. It is a promising biomarker for immunotherapy response prediction and may be an effective epigenetic modulator in combination with immunotherapy approaches.
... Recent evidence suggested that CXCL14 receptor activation is a complex and cell type-dependent process (41,42). Chemokine receptors, including CXCR4 (43,44), GPR85 (45), and ACKR2 (46,47) have been implicated in CXCL14 function. CXCR4 is a member of the G protein-coupled receptor family and ERK1/2 is part of CXCR4 signal pathways (48). ...
Immune checkpoint therapy has limited efficacy for patients with bone metastatic castrate-resistant prostate cancer (bmCRPC). In this study, we revealed a novel mechanism that may account for the relative resistance of bmCRPC to immune checkpoint therapy. We found that prostate cancer (PCa)-induced bone via endothelial-to-osteoblast (EC-to-OSB) transition causes an ingress of M2-like macrophages, leading to an immunosuppressive bone tumor microenvironment (bone-TME). Analysis of a bmCRPC RNA-seq dataset revealed shorter overall survival in patients with an M2-high versus M2-low signature. Immunohistochemical (IHC) analysis showed CD206+ M2-like macrophages were enriched in bmCRPC specimens compared with primary tumors or lymph node metastasis. In osteogenic PCa xenografts, CD206+ macrophages were enriched adjacent to tumor-induced bone. FACS analysis showed an increase in CD206+ cells in osteogenic tumors compared to non-osteogenic tumors. Genetic or pharmacological inhibition of the EC-to-OSB transition reduced aberrant bone and M2-like macrophages in osteogenic tumors. RNAseq analysis of tumor-associated macrophages from osteogenic (bone-TAMs) versus non-osteogenic (ctrl-TAMs) tumors showed high expression of an M2-like gene signature, canonical and non-canonical Wnt pathways, and a decrease in an M1-like gene signature. Isolated bone-TAMs suppressed T-cell proliferation while ctrl-TAMs did not. Mechanistically, EC-OSB hybrid cells produced paracrine factors, including Wnts, CXCL14 and LOX, which induced M2 polarization and recruited M2-like TAMs to bone-TME. Our study thus links the unique EC-to-OSB transition as an upstream event that drives downstream immunosuppression in the bone-TME. These studies suggest that therapeutic strategies that inhibit PCa-induced EC-to-OSB transition may reverse immunosuppression to promote immunotherapeutic outcomes in bmCRPC.
... SREB receptors are still considered orphan GPCRs despite different studies suggesting potential endogenous and synthetic ligands [18][19][20][21][22][23][43][44][45]. The lack of validated agonists able to activate SREBs limits the identification of the signaling cascades triggered by these receptors. ...
The Super-Conserved Receptors Expressed in the Brain (SREBs) form a subfamily of orphan G protein-coupled receptors, highly conserved in evolution and characterized by a predominant expression in the brain. The signaling pathways activated by these receptors (if any) are presently unclear. Given the strong conservation of their intracellular loops, we used a BioID2 proximity-labeling assay to identify protein partners of SREBs that would interact with these conserved domains. Using streptavidin pull-down followed by mass spectrometry analysis, we identified the amino acid transporter SLC3A2, the AKAP protein LRBA, and the 4.1 protein EPB41L2 as potential interactors of these GPCRs. Using co-immunoprecipitation experiments, we confirmed the physical association of these proteins with the receptors. We then studied the functional relevance of the interaction between EPB41L2 and SREB1. Immunofluorescence microscopy revealed that SREB1 and EPB41L2 co-localize at the plasma membrane and that SREB1 is enriched in the β-catenin-positive cell membranes. siRNA knockdown experiments revealed that EPB41L2 promotes the localization of SREB1 at the plasma membrane and increases the solubilization of SREB1 when using detergents, suggesting a modification of its membrane microenvironment. Altogether, these data suggest that EPB41L2 could regulate the subcellular compartmentalization of SREBs and, as proposed for other GPCRs, could affect their stability or activation.
