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EGFR ligands induce LuCSC EMT-like differentiation. (A) Boyden chamber evaluation of NCI-H1568 parent cells and subclones, as well as mixed populations of LuCSCs with progeny cells. ½ LuCSCs + ½ 1 st DF: LuCSCs and 1 st DF cells were mixed at equal proportions and then added into the Boyden chamber inserts. ½ LuCSCs + ½ 2 nd DF: LuCSCs and 2 nd DF cells were mixed at equal proportions and then added into the Boyden chamber inserts. NC, negative control. (B) Quantification of cell invasiveness shown in (A). ns, not significant. (C) 1 st and 2 nd DF progeny cells 72 hrs after transfection with siRNA-EGFR and siRNA-scramble were lysed and protein was prepared for western blot analysis of pEGFR and EGFR. Tubulin served as a loading control. Representative images show reduced cell invasiveness of 1 st DF (D) and 2 nd DF (F) cells due to EGFR knockdown compared to control cells. NC, negative control. (E and G) Quantification of cell invasiveness shown in (E) and (G). **P < 0.01. (H) Representative images of LuCSCs stimulated with EGF (5 ng/ ml) with/without 3.3 μM Afatinib pretreatment. Scale bar: 20 μm. (I) Real-time PCR analysis of EGFR and mesenchymal markers. Gene expression levels of LuCSCs were measured upon EGF stimulation at different time points as indicated. (J) Comparison of invasive activity of LuCSCs after stimulation with EGF and conditioned medium derived from 2 nd DF cells (20% as chemoattractant) for 48 hrs. *P < 0.05.(K) Quantification of invasive activity of LuCSCs upon EGF stimulation at different time points as indicated. The values were indicated in % of control 2 nd DF cells. *P < 0.05. (L) Whole-cell lysates from LuCSCs, 2 nd DF cells, EGF stimulated LuCSCs with/without Afatinib (3.3 μM) pretreatment were collected for human phospho-kinase antibody array analysis. Each membrane contains kinase specific (number indicated) and positive control (P). (M) Relative phosphorylation of spots was quantified by normalizing pixel density of the positive control to 100. Each bar is represented as mean of duplicate spots.
Source publication
The lung cancer stem cell (LuCSC) model comprises an attractive framework to explore acquired drug resistance in non-small cell lung cancer (NSCLC) treatment. Here, we used NSCLC cell line model to translate cellular heterogeneity into tractable populations to understand the origin of lung cancers and drug resistance. The epithelial LuCSCs, presuma...
Contexts in source publication
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... and metastasis are the most insidious and life-threatening aspects of cancer [26]. NCI-H1568 parent cells were initially checked to possess high capability of invasion ( Figure 4A, top left). After sub cloning we wondered how invasiveness was associated with defined clones. ...
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... sub cloning we wondered how invasiveness was associated with defined clones. Strikingly, we found that epithelial LuCSCs had no invasion capacity, whereas 2 nd DF cells exhibited a greater than 2 and 4 fold increase in invasion compared to parent cells and 1 st DF cells, respectively (Figure 4A, Figure 4B). Our data argues against the EMT nature and aggressive behavior of CSCs [27,28]. ...
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... address the potential influence of LuCSCs on their progeny invasiveness, we co-cultured LuCSCs with 1 st and 2 nd DF cells at equal proportions for 36 hrs. Surprisingly, the invasion was increased approximately 2 fold because of co-culture of either 1 st or 2 nd DF cells with LuCSCs ( Figure 4A, two right panels). ...
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... we examined whether endogenous EGFR signaling plays a role in mediating the invasive behavior of poorly differentiated adenocarcinoma progeny. To this end we utilized EGFR-siRNA and observed effective EGFR knockdown in the highly invasive 1 st and 2 nd DF cells ( Figure 4C). Notably an equal inhibition of pEGFR was seen in both cell lines ( Figure 4C). ...
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... this end we utilized EGFR-siRNA and observed effective EGFR knockdown in the highly invasive 1 st and 2 nd DF cells ( Figure 4C). Notably an equal inhibition of pEGFR was seen in both cell lines ( Figure 4C). As expected, the invasiveness of both cell lines was almost abrogated by EGFR-siRNA specific inhibition ( Figure 4D-4G). ...
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... an equal inhibition of pEGFR was seen in both cell lines ( Figure 4C). As expected, the invasiveness of both cell lines was almost abrogated by EGFR-siRNA specific inhibition ( Figure 4D-4G). Taken together, our data demonstrated EGFR signaling is critical for the invasiveness of 1 st and 2 nd DF cells and possibly for the initiation of paracrine mediated LuCSC differentiation into aggressive mesenchymal cells. ...
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... further identified EGFR ligands in the role of stimulating LuCSC differentiation. As expected, EGF overnight application induced profound LuCSC differentiation ( Figure 4H, middle); however, pretreatment of LuCSCs with the EGFR inhibitor Afatinib abrogated this stimulatory effect ( Figure 4H, right). The blocking effect by Afatinib confirmed that EGFR activation is critical for the initiation of LuCSC differentiation. ...
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... further identified EGFR ligands in the role of stimulating LuCSC differentiation. As expected, EGF overnight application induced profound LuCSC differentiation ( Figure 4H, middle); however, pretreatment of LuCSCs with the EGFR inhibitor Afatinib abrogated this stimulatory effect ( Figure 4H, right). The blocking effect by Afatinib confirmed that EGFR activation is critical for the initiation of LuCSC differentiation. ...
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... PCR was employed simultaneously to examine the expression of a selection of genes, with potential relation to mesenchymal cells. The upregulation of EGFR, snail, and slug, together with N-cadherin, went along with the stimulation time ( Figure 4I). We also observed that other EGF family ligands (Amphiregulin, TGFα, HB-EGF and Heregulin-β1) and cytokines (IL6 and Oncostatin M), as well as the serum free medium optimized for the culture of mammospheres, all triggered LuCSC differentiation towards a mesenchymal phenotype (Supplementary Figure 3A). ...
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... short time incubation of the ligands EGF, TGFα and HB-EGF with their specific antibodies completely abrogates the stimulatory effect (data not shown). EGF which induced phenotypical changes did also enhance LuCSC invasiveness, but to a significantly lesser extent than conditioned medium derived from 2 nd DF cells ( Figure 4J). Additionally, the invasive ability of LuCSCs after EGF stimulation was inversely correlated to the cultivation time ( Figure 4K). ...
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... which induced phenotypical changes did also enhance LuCSC invasiveness, but to a significantly lesser extent than conditioned medium derived from 2 nd DF cells ( Figure 4J). Additionally, the invasive ability of LuCSCs after EGF stimulation was inversely correlated to the cultivation time ( Figure 4K). ...
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... we used human phospho-kinase arrays to analyze and understand how cells recognize and respond to changes in their environment. The data revealed a range of signaling molecules that were phosphorylated in response to EGF treatment in comparison with Afatinib blockade, and significant ones were highlighted by numbers ( Figure 4L). In LuCSC-holoclones, AKT1/2/3 was weakly expressed and no activation (AKT pS473) was observed after EGF stimuli, indicating at EGFR silent and stemness states EGFR-PI3K-AKT pathway does not play a significant role for survival and metabolic homeostasis. ...
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... LuCSC-holoclones, AKT1/2/3 was weakly expressed and no activation (AKT pS473) was observed after EGF stimuli, indicating at EGFR silent and stemness states EGFR-PI3K-AKT pathway does not play a significant role for survival and metabolic homeostasis. However, the AKT pathway was strongly activated in progeny 2 nd DF cells ( Figure 4M). Upon EGF stimulation, pCREB is increased in the LuCSCs in conjunction with β-catenin, Stat3 (Supplementary Figure 3B-3D), GSK3 (a/b) and p70S6 activations. ...
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... pretreatment with Afatinib dramatically inhibited these protein activities, except CREB which was partially inhibited to basal level. Conversely, P53 was downregulated after EGF stimulation ( Figure 4M). Overall, data indicates that growth response of LuCSCs mediated by EGF stimulation partially mimics early stage of differentiation. ...
Context 15
... and metastasis are the most insidious and life-threatening aspects of cancer [26]. NCI-H1568 parent cells were initially checked to possess high capability of invasion ( Figure 4A, top left). After sub cloning we wondered how invasiveness was associated with defined clones. ...
Context 16
... sub cloning we wondered how invasiveness was associated with defined clones. Strikingly, we found that epithelial LuCSCs had no invasion capacity, whereas 2 nd DF cells exhibited a greater than 2 and 4 fold increase in invasion compared to parent cells and 1 st DF cells, respectively (Figure 4A, Figure 4B). Our data argues against the EMT nature and aggressive behavior of CSCs [27,28]. ...
Context 17
... address the potential influence of LuCSCs on their progeny invasiveness, we co-cultured LuCSCs with 1 st and 2 nd DF cells at equal proportions for 36 hrs. Surprisingly, the invasion was increased approximately 2 fold because of co-culture of either 1 st or 2 nd DF cells with LuCSCs ( Figure 4A, two right panels). ...
Context 18
... we examined whether endogenous EGFR signaling plays a role in mediating the invasive behavior of poorly differentiated adenocarcinoma progeny. To this end we utilized EGFR-siRNA and observed effective EGFR knockdown in the highly invasive 1 st and 2 nd DF cells ( Figure 4C). Notably an equal inhibition of pEGFR was seen in both cell lines ( Figure 4C). ...
Context 19
... this end we utilized EGFR-siRNA and observed effective EGFR knockdown in the highly invasive 1 st and 2 nd DF cells ( Figure 4C). Notably an equal inhibition of pEGFR was seen in both cell lines ( Figure 4C). As expected, the invasiveness of both cell lines was almost abrogated by EGFR-siRNA specific inhibition ( Figure 4D-4G). ...
Context 20
... an equal inhibition of pEGFR was seen in both cell lines ( Figure 4C). As expected, the invasiveness of both cell lines was almost abrogated by EGFR-siRNA specific inhibition ( Figure 4D-4G). Taken together, our data demonstrated EGFR signaling is critical for the invasiveness of 1 st and 2 nd DF cells and possibly for the initiation of paracrine mediated LuCSC differentiation into aggressive mesenchymal cells. ...
Context 21
... further identified EGFR ligands in the role of stimulating LuCSC differentiation. As expected, EGF overnight application induced profound LuCSC differentiation ( Figure 4H, middle); however, pretreatment of LuCSCs with the EGFR inhibitor Afatinib abrogated this stimulatory effect ( Figure 4H, right). The blocking effect by Afatinib confirmed that EGFR activation is critical for the initiation of LuCSC differentiation. ...
Context 22
... further identified EGFR ligands in the role of stimulating LuCSC differentiation. As expected, EGF overnight application induced profound LuCSC differentiation ( Figure 4H, middle); however, pretreatment of LuCSCs with the EGFR inhibitor Afatinib abrogated this stimulatory effect ( Figure 4H, right). The blocking effect by Afatinib confirmed that EGFR activation is critical for the initiation of LuCSC differentiation. ...
Context 23
... PCR was employed simultaneously to examine the expression of a selection of genes, with potential relation to mesenchymal cells. The upregulation of EGFR, snail, and slug, together with N-cadherin, went along with the stimulation time ( Figure 4I). We also observed that other EGF family ligands (Amphiregulin, TGFα, HB-EGF and Heregulin-β1) and cytokines (IL6 and Oncostatin M), as well as the serum free medium optimized for the culture of mammospheres, all triggered LuCSC differentiation towards a mesenchymal phenotype (Supplementary Figure 3A). ...
Context 24
... short time incubation of the ligands EGF, TGFα and HB-EGF with their specific antibodies completely abrogates the stimulatory effect (data not shown). EGF which induced phenotypical changes did also enhance LuCSC invasiveness, but to a significantly lesser extent than conditioned medium derived from 2 nd DF cells ( Figure 4J). Additionally, the invasive ability of LuCSCs after EGF stimulation was inversely correlated to the cultivation time ( Figure 4K). ...
Context 25
... which induced phenotypical changes did also enhance LuCSC invasiveness, but to a significantly lesser extent than conditioned medium derived from 2 nd DF cells ( Figure 4J). Additionally, the invasive ability of LuCSCs after EGF stimulation was inversely correlated to the cultivation time ( Figure 4K). ...
Context 26
... we used human phospho-kinase arrays to analyze and understand how cells recognize and respond to changes in their environment. The data revealed a range of signaling molecules that were phosphorylated in response to EGF treatment in comparison with Afatinib blockade, and significant ones were highlighted by numbers ( Figure 4L). In LuCSC-holoclones, AKT1/2/3 was weakly expressed and no activation (AKT pS473) was observed after EGF stimuli, indicating at EGFR silent and stemness states EGFR-PI3K-AKT pathway does not play a significant role for survival and metabolic homeostasis. ...
Context 27
... LuCSC-holoclones, AKT1/2/3 was weakly expressed and no activation (AKT pS473) was observed after EGF stimuli, indicating at EGFR silent and stemness states EGFR-PI3K-AKT pathway does not play a significant role for survival and metabolic homeostasis. However, the AKT pathway was strongly activated in progeny 2 nd DF cells ( Figure 4M). Upon EGF stimulation, pCREB is increased in the LuCSCs in conjunction with β-catenin, Stat3 (Supplementary Figure 3B-3D), GSK3 (a/b) and p70S6 activations. ...
Context 28
... pretreatment with Afatinib dramatically inhibited these protein activities, except CREB which was partially inhibited to basal level. Conversely, P53 was downregulated after EGF stimulation ( Figure 4M). Overall, data indicates that growth response of LuCSCs mediated by EGF stimulation partially mimics early stage of differentiation. ...
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Background:
Lung cancer stem cells (LCSCs) are endowed with high aldehyde dehydrogenase (ALDH) expression and play roles in tumor proliferation, metastasis, and drug resistance. Their elusive nature may allow them to escape the immune response by tumor-infiltrating lymphocytes (TILs), which can positively affect the outcome in non-small cell lung...
Citations
... Another modulator, the transforming growth factor-β-induced factor homeobox 2 (TGIF2) was activated through the EGFR/RAS/ERK signalling pathway and enhanced stemness that was believed to be the driver of tumour progression and metastasis [106]. The overactivation of EGFR with suppression of a tumour suppressor, mitogen-inducible gene 6 protein (MIG6) is believed to cause lung CSCs to differentiate into malignant lineage and give rise to drug resistance [107]. ...
The role of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) has been vastly studied over the last decade. This has led to the rapid development of many generations of EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, patients treated with third-generation TKIs (osimertinib, avitinib and rociletinib) targeting the EGFR T790M mutation have shown emerging resistances and relapses. Therefore, further molecular understanding of NSCLC mutations, bypass signalling, tumour microenvironment and the existence of cancer stem cells to overcome such resistances is warranted. This will pave the way for designing novel and effective chemotherapies to improve patients' overall survival. In this review, we provide an overview of the multifaceted mechanism of resistances towards EGFR-TKIs, as well as the challenges and perspectives that should be addressed in strategising chemotherapeutic treatments to overcome the ever evolving and adaptive nature of NSCLC.
... Conversely, ectopic expression of miR-200 reverses the resistance of bladder cancer cells to EGFR inhibition therapy by regulating EMT [161]. Gene 33 may also contribute to resistance of lung cancer stem cells to the EGFR TKI by suppressing their transition from self-renewal to malignant differentiation, a process dependent on EGFR signaling [162]. Gene 33 has also been shown to participate in the resistance to inhibitors of the members of the MAP kinase pathway. ...
Gene 33 (also named Mig6, RALT, and ERRFI1) is an adapter/scaffold protein with a calculated molecular weight of about 50 kD. It contains multiple domains known to mediate protein–protein interaction, suggesting that it has the potential to interact with many cellular partners and have multiple cellular functions. The research over the last two decades has confirmed that it indeed regulates multiple cell signaling pathways and is involved in many pathophysiological processes. Gene 33 has long been viewed as an exclusively cytosolic protein. However, recent evidence suggests that it also has nuclear and chromatin-associated functions. These new findings highlight a significantly broader functional spectrum of this protein. In this review, we will discuss the function and regulation of Gene 33, as well as its association with human pathophysiological conditions in light of the recent research progress on this protein.
... These results suggest that MIG6 could variably influence cellular homeostasis through growth inhibition, apoptosis, and senescence induction [38][39][40][41]. A recent study confirms that lung cancer stem cells inversely express EGFR and MIG6 genes [42]. In endometrial carcinoma, MIG6 plays a tumor-suppressor role by promoting epithelial cell apoptosis through the inhibition of ERK2 phosphorylation [33]. ...
Epidermal growth factor receptor (EGFR) signalling is a highly regulated process with a tight balance between receptor activation and inactivation in invasive breast carcinomas (IBCs) particularly in triple-negative carcinomas (TNC). Clinical trials using anti-EGFR therapies are actually performed although no activating alterations (mutations, amplifications, or rearrangements) of EGFR have been clearly recognized in order to identify new targeted modalities for IBCs. We explored mammary-derived growth inhibitor (MDGI), estrogen-induced gene-121 (EIG121), and mitogen-induced gene-6 (MIG6), three posttranslational EGFR trafficking molecules implicated in EGFR spatiotemporal regulatory pathway. We quantified MDGI, EIG121, and MIG6 at mRNA levels by using real-time quantitative RT-PCR in a series of 440 IBCs and at protein levels by using immunohistochemistry in a series of 88 IBCs. Results obtained by RT-PCR showed that in IBCs, MDGI, MIG6, and EIG121 mRNA were mainly underexpressed (25.7%, 45.0%, and 16.1%, respectively) particularly in the TNC subtype for EIG121 (60.3%). We also observed mRNA overexpression of MDGI and EIG121, respectively, in 12.7% and 22.3% of IBCs. These altered mRNA expressions were confirmed at the protein level. Some links were found between expression patterns of these three genes and several classical pathological and clinical parameters. Only EIG121 was found to have a prognostic significance (p=0.0038). Altered expression of these three major EGFR posttranslational negative regulators could create an aberrant EGFR-mediated oncogenic signalling pathway in IBCs. MDGI, MIG6, and EIG121 expression status also may be potential useful biomarkers (sensitivity or resistance) in targeted EGFR therapy.
... Lung cancer stem cells (CSCs) are a cell subset with properties of persistence or immortalization, self-renewal, and the potential to differentiate phenotypically in lung cancer or supporting tissues [4]. Lung CSCs may be involved in the occurrence and development of lung cancer and can provide novel therapeutic targets for treatment [4]. ...
... Lung cancer stem cells (CSCs) are a cell subset with properties of persistence or immortalization, self-renewal, and the potential to differentiate phenotypically in lung cancer or supporting tissues [4]. Lung CSCs may be involved in the occurrence and development of lung cancer and can provide novel therapeutic targets for treatment [4]. For example, Wu et al. recently showed that depletion of the oncogene, prostate tumor overexpressed gene 1 (PTOV1), sensitized lung cancer cells to chemotherapy by reducing lung CSC-like traits [5]. ...
Background
Cancer stem cells (CSCs) behave as their malignant counterparts, but persist after treatment, and possess properties that allow them to interact with their environment. Itraconazole, an antifungal agent, also has a role in suppressing tumor progression, but its effects in regulating tumor cell stemness remain unclear. This study aimed to evaluate the effects of itraconazole on A549 and NCI-H460 human lung cancer cell stemness in vitro.
Material/Methods
A549 and NCI-H460 human lung cancer cells and BEAS-2B normal bronchial epithelial cells were cultured with and without itraconazole. Cell viability was evaluated. The expression of stem cell markers, CD133, ATP binding cassette subfamily G member 2 (ABCG2), and aldehyde dehydrogenase 1 (ALDH1), were measured by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Sphere-forming cells were evaluated in vitro.
Results
Itraconazole reduced the expression of stemness molecules CD133, ABCG2, and ALDH1 in A549 and NCI-H460 human lung cancer cells, and the numbers of sphere-forming cells were reduced. However, itraconazole had little effect on cell viability but enhanced the chemosensitivity of A549 and NCI-H460 cells. Itraconazole inhibited Wnt signaling. Re-activation of Wnt signaling restored itraconazole-mediated inhibition on A549 and NCI-H460 cell stemness.
Conclusions
Itraconazole altered the stemness characteristics of A549 and NCI-H460 human lung cancer cells by suppressing Wnt signaling but did not affect cell viability.
The role of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) has been vastly studied over the last decade. This has led to the rapid development of many generations of EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, patients treated with third-generation TKIs (osimertinib, avitinib and rociletinib) targeting the EGFR T790M mutation have shown emerging resistances and relapses. Therefore, further molecular understanding of NSCLC mutations, bypass signalling, tumour microenvironment and the existence of cancer stem cells to overcome such resistances is warranted. This will pave the way for designing novel and effective chemotherapies to improve patients' overall survival. In this review, we provide an overview of the multifaceted mechanism of resistances towards EGFR-TKIs, as well as the challenges and perspectives that should be addressed in strategising chemotherapeutic treatments to overcome the ever evolving and adaptive nature of NSCLC.
The roles of long noncoding RNA (lncRNA) MCF2L‐AS1 have been identified in colorectal cancer, however, its roles in lung cancer progression have never been revealed. Here, we found that lncRNA MCF2L‐AS1 was highly expressed in lung cancer tissues and cells, especially in non‐small cell lung cancer (NSCLC). Functional experiments showed that MCF2L‐AS1 knockdown suppressed the cancer stem cell (CSC)‐like traits of NSCLC cells through qRT‐PCR, western blot, tumor‐sphere formation, and ALDH activity detection. Additionally, bioinformatic assay combined with RNA pull down and luciferase reporter analysis confirmed that MCF2L‐AS1 downregulated miR‐873‐5p level. Furthermore, miR‐873‐5p expression exhibited a lower level in lung cancer tissues and cells, and a negative correlation with MCF2L‐AS1 expression in lung cancer tissues. Moreover, miR‐873‐5p inhibition partially reversed the suppression of MCF2L‐AS1 on the CSC‐like traits on NSCLC cells. Thus, this work identifies a novel MCF2L‐AS1/miR‐873‐5p regulatory axis responsible for the CSC‐like traits of NSCLC cells.
Lung adenocarcinoma (LAC) is a common and aggressive form of lung cancer that is increasing in incidence among never smokers at younger age. Current treatment of patients with LAC is insufficient and there is a need for identification of effective biomarkers and development of therapy targets. These demands require also improved models for in vivo and in vitro experimentation. In this study, we describe the establishment of two LAC cell lines, termed LuCa-3 and LuCa-6. Both were derived from pleural effusion (PE) cells of LAC patients (L3 and L6) and both are readily propagated as tumor xenografts in immune-deficient mice. PE cells from the patient L6 exhibited also capacity for in vitro growth and were cultured in two forms: (i) as a suspension growing cell population, termed LuCa-6S, composed of non-clumping single cells; and (ii) as a monolayer-like culture, labeled LuCa-6A, exhibiting tight cell-to-cell and to culture surface adherence. Unique features of these two sublines and their cell clones are the capacity to convert from a non-clumping single-cell suspension into the adherent growth pattern and vice versa. Immunostaining of patients' tumor tissue xenografts and cultured subline cells displayed markers specific for the phenotype of human LAC. LuCa-6S and LuCa-6A cells did not reveal a noticeable disparity in quantitative growth characteristics. However, a number of differences were detected between these two cell populations manifested in detection or intensities of antigen expressions on cell surface (CD133, SFTPC) and in the nucleus (TTF-1) including pluripotent (OCT-4, SOX-2, NANOG) genes in cancer stem-like cells (CSCs). Dissimilarities between these two sublines were also detected in N-glycan profiles and in the sensitivity to natural killer cells. Salient features of these subline cell populations are responsiveness to selective upregulation of the pluripotent genes in subsets of CSCs via conversion of their growth patterns and/or by using culture stem media with growth factors. The described in vivo/in vitro model enables broader experimental approaches in studies of lung adenocarcinoma.
The hypoxic microenvironment can facilitate the tumor progression, and transcription factor YY1 holds promoting effects in various tumors. This work aims to investigate whether YY1 is involved in hypoxia‐induced stemness of lung cancer cells. We showed that hypoxic microenvironment induced the expression of HIF‐1α and YY1, and the stemness of lung cancer cells, which was attenuated by YY1 knockdown. Additionally, we found that YY1 regulates the hypoxia‐induced stemness in a HIF‐1α‐dependent manner, but independent on p53 expression. Further analysis revealed that YY1 physically interacted with HIF‐1α protein and stabilized HIF‐1α protein. Our work indicates a novel YY1/HIF‐1α axis regulating the stemness of lung cancer cells.
