Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature

Introduction

Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin.

Methods

An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed.

Results

Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines.

Conclusion

Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer.     

Schisandrin B attenuates cancer invasion and metastasis via inhibiting epithelial-mesenchymal transition

Background

Metastasis is the major cause of cancer related death and targeting the process of metastasis has been proposed as a strategy to combat cancer. Therefore, to develop candidate drugs that target the process of metastasis is very important. In the preliminary studies, we found that schisandrin B (Sch B), a naturally-occurring dibenzocyclooctadiene lignan with very low toxicity, could suppress cancer metastasis.

Methodology

BALB/c mice were inoculated subcutaneously or injected via tail vein with murine breast cancer 4T1 cells. Mice were divided into Sch B-treated and control groups. The primary tumor growth, local invasion, lung and bone metastasis, and survival time were monitored. Tumor biopsies were examined immuno- and histo-pathologically. The inhibitory activity of Sch B on TGF-β induced epithelial-mesenchymal transition (EMT) of 4T1 and primary human breast cancer cells was assayed.

Principal Findings

Sch B significantly suppressed the spontaneous lung and bone metastasis of 4T1 cells inoculated s.c. without significant effect on primary tumor growth and significantly extended the survival time of these mice. Sch B did not inhibit lung metastasis of 4T1 cells that were injected via tail vein. Delayed start of treatment with Sch B in mice with pre-existing tumors did not reduce lung metastasis. These results suggested that Sch B acted at the step of local invasion. Histopathological evidences demonstrated that the primary tumors in Sch B group were significantly less locally invasive than control tumors. In vitro assays demonstrated that Sch B could inhibit TGF-β induced EMT of 4T1 cells and of primary human breast cancer cells.

Conclusions

Sch B significantly suppresses the lung and bone metastasis of 4T1 cells via inhibiting EMT, suggesting its potential application in targeting the process of cancer metastasis.     

Metformin Inhibits the IL-6-Induced Epithelial-Mesenchymal Transition and Lung Adenocarcinoma Growth and Metastasis

Objective

Epithelial-mesenchymal transition (EMT) plays an important role in cancer tumorigenesis. However, the underlying mechanisms of EMT in lung adenocarcinoma, and how this process might be inhibited, remain to be explored. This study investigated the role of IL-6 in lung adenocarcinoma cell EMT and explored the potential effects of metformin on this process.

Methods

Invasion assay and MTT assay was performed to determine cell invasion and cell proliferation. Western blotting, immunofluorescence, real-time PCR, ELISA, and immunohistochemistry were performed to detect the expression of IL-6, E-cadherin, Vimentin, and p-STAT3.

Results

We discovered that IL-6, via STAT3 phosphorylation, could promote lung adenocarcinoma cell invasion via EMT in vitro. This was supported by the inverse correlation between E-cadherin and IL-6 expression, positive correlation between IL-6 and vimentin mRNA expression and between STAT3 phosphorylation and IL-6 expression in tumor tissues. Importantly, metformin inhibited tumor growth and distant metastases in tumor-bearing nude mice and reversed IL-6-induced EMT both in vitro and in vivo. Furthermore, we found that blockade of STAT3 phosphorylation might be the underlying mechanism of metformin inhibition of IL-6-induced EMT.

Conclusions

Collectively, our present results show that enhanced IL-6 expression, via STAT3 phosphorylation, is a mechanism of EMT in lung adenocarcinoma. We found that metformin could inhibit IL-6-induced EMT possibly by blocking STAT3 phosphorylation.     

Inhibition of Hedgehog signaling sensitizes NSCLC cells to standard therapies through modulation of EMT-regulating miRNAs

Background

Epidermal growth factor receptor- tyrosine kinase inhibitors (EGFR-TKIs) benefit Non-small cell lung cancer (NSCLC) patients, and an EGFR-TKIi erlotinib, is approved for patients with recurrent NSCLC. However, resistance to erlotinib is a major clinical problem. Earlier we have demonstrated the role of Hedgehog (Hh) signaling in Epithelial-to-Mesenchymal transition (EMT) of NSCLC cells, leading to increased proliferation and invasion. Here, we investigated the role of Hh signaling in erlotinib resistance of TGF-β1-induced NSCLC cells that are reminiscent of EMT cells.

Methods

Hh signaling was inhibited by specific siRNA and by GDC-0449, a small molecule antagonist of G protein coupled receptor smoothened in the Hh pathway. Not all NSCLC patients are likely to benefit from EGFR-TKIs and, therefore, cisplatin was used to further demonstrate a role of inhibition of Hh signaling in sensitization of resistant EMT cells. Specific pre- and anti-miRNA preparations were used to study the mechanistic involvement of miRNAs in drug resistance mechanism.

Results

siRNA-mediated inhibition as well as pharmacological inhibition of Hh signaling abrogated resistance of NSCLC cells to erlotinib and cisplatin. It also resulted in re-sensitization of TGF-β1-induced A549 (A549M) cells as well the mesenchymal phenotypic H1299 cells to erlotinib and cisplatin treatment with concomitant up-regulation of cancer stem cell (CSC) markers (Sox2, Nanog and EpCAM) and down-regulation of miR-200 and let-7 family miRNAs. Ectopic up-regulation of miRNAs, especially miR-200b and let-7c, significantly diminished the erlotinib resistance of A549M cells. Inhibition of Hh signaling by GDC-0449 in EMT cells resulted in the attenuation of CSC markers and up-regulation of miR-200b and let-7c, leading to sensitization of EMT cells to drug treatment, thus, confirming a connection between Hh signaling, miRNAs and drug resistance.

Conclusions

We demonstrate that Hh pathway, through EMT-induction, leads to reduced sensitivity to EGFR-TKIs in NSCLCs. Therefore, targeting Hh pathway may lead to the reversal of EMT phenotype and improve the therapeutic efficacy of EGFR-TKIs in NSCLC patients.

     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

Pancreatic cancer cells enhance the ability of collagen internalization during epithelial-mesenchymal transition

Background

Extracellular matrix (ECM) remodeling is predominantly mediated by fibroblasts using intracellular and extracellular pathways. Although it is well known that extracellular degradation of the ECM by proteases derived from cancer cells facilitates cellular invasion, the intracellular degradation of ECM components by cancer cells has not been clarified. The aim of this study was to characterize collagen internalization, which is the initial step of the intracellular degradation pathway in pancreatic cancer cells, in light of epithelial–mesenchymal transition (EMT).

Methodology/Principal Findings

We analyzed the function of collagen internalization in two pancreatic cancer cell lines, SUIT-2 and KP-2, and pancreatic stellate cells (PSCs) using Oregon Green 488-gelatin. PSCs had a strong ability for collagen uptake, and the pancreatic cancer cells also internalized collagen although less efficiently. The collagen internalization abilities of SUIT-2 and KP-2 cells were promoted by EMT induced by human recombinant transforming growth factor β1 (P<0.05). Expression of Endo180, a collagen uptake receptor, was high in mesenchymal pancreatic cancer cell lines, as determined by EMT marker expression (P<0.01). Quantitative RT-PCR and western blot analyses showed that Endo180 expression was also increased by EMT induction in SUIT-2 and KP-2 cells. Endo180 knockdown by RNA interference attenuated the collagen uptake (P<0.01) and invasive abilities (P<0.05) of SUIT-2 and KP-2 cells.

Conclusions/Significance

Pancreatic cancer cells are capable of collagen internalization, which is enhanced by EMT. This ECM clearance system may be a novel mechanism for cellular invasion and a potential therapeutic target in pancreatic cancer.     

Over-expression of LSD1 promotes proliferation, migration and invasion in non-small cell lung cancer

Background

Lysine specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics, but the pathological roles of its dysfunction in lung cancer remain to be elucidated. The aim of this study was to evaluate the prognostic significance of LSD1 expression in patients with non-small cell lung cancer (NSCLC) and to define its exact role in lung cancer proliferation, migration and invasion.

Methods

The protein levels of LSD1 in surgically resected samples from NSCLC patients were detected by immunohistochemistry or Western blotting. The mRNA levels of LSD1 were detected by qRT-PCR. The correlation of LSD1 expression with clinical characteristics and prognosis was determined by statistical analysis. Cell proliferation rate was assessed by MTS assay and immunofluorescence. Cell migration and invasion were detected by scratch test, matrigel assay and transwell invasion assay.

Results

LSD1 expression was higher in lung cancer tissue more than in normal lung tissue. Our results showed that over-expression of LSD1 protein were associated with shorter overall survival of NSCLC patients. LSD1 was localized mainly to the cancer cell nucleus. Interruption of LSD1 using siRNA or a chemical inhibitor, pargyline, suppressed proliferation, migration and invasion of A549, H460 and 293T cells. Meanwhile, over-expression of LSD1 enhanced cell growth. Finally, LSD1 was shown to regulate epithelial-to-mesenchymal transition in lung cancer cells.

Conclusions

Over-expression of LSD1 was associated with poor prognosis in NSCLC, and promoted tumor cell proliferation, migration and invasion. These results suggest that LSD1 is a tumor-promoting factor with promising therapeutic potential for NSCLC.     

MicroRNA-147 Induces a Mesenchymal-To-Epithelial Transition (MET) and Reverses EGFR Inhibitor Resistance

Background

The epithelial-mesenchymal transition (EMT) is a key developmental program that is often activated during cancer progression and may promote resistance to therapy. An analysis of patients (n = 71) profiled with both gene expression and a global microRNA assessment (∼415 miRs) identified miR-147 as highly anti-correlated with an EMT gene expression signature score and postulated to reverse EMT (MET).

Methods and Findings

miR-147 was transfected into colon cancer cells (HCT116, SW480) as well as lung cancer cells (A-549). The cells were assessed for morphological changes, and evaluated for effects on invasion, motility, and the expression of key EMT markers. Resistance to chemotherapy was evaluated by treating cells with gefitinib, an EGFR inhibitor. The downstream genes regulated by miR-147 were assayed using the Affymetrix GeneChip U133 Plus2.0 platform. miR-147 was identified to: 1. cause MET primarily by increasing the expression of CDH1 and decreasing that of ZEB1; 2. inhibit the invasion and motility of cells; 3. cause G1 arrest by up-regulating p27 and down-regulating cyclin D1. miR-147 also dramatically reversed the native drug resistance of the colon cancer cell line HCT116 to gefitinib. miR-147 significantly repressed Akt phosphorylation, and knockdown of Akt with siRNA induced MET. The morphologic effects of miR-147 on cells appear to be attenuated by TGF-B1, promoting a plastic and reversible transition between MET and EMT.

Conclusion

miR-147 induced cancer cells to undergo MET and induced cell cycle arrest, suggesting a potential tumor suppressor role. miR-147 strikingly increased the sensitivity to EGFR inhibitor, gefitinib in cell with native resistance. We conclude that miR-147 might have therapeutic potential given its ability to inhibit proliferation, induce MET, as well as reverse drug sensitivity.     

Genes suppressed by DNA methylation in non-small cell lung cancer reveal the epigenetics of epithelial–mesenchymal transition

Background

DNA methylation is associated with aberrant gene expression in cancer, and has been shown to correlate with therapeutic response and disease prognosis in some types of cancer. We sought to investigate the biological significance of DNA methylation in lung cancer.

Results

We integrated the gene expression profiles and data of gene promoter methylation for a large panel of non-small cell lung cancer cell lines, and identified 578 candidate genes with expression levels that were inversely correlated to the degree of DNA methylation. We found these candidate genes to be differentially methylated in normal lung tissue versus non-small cell lung cancer tumors, and segregated by histologic and tumor subtypes. We used gene set enrichment analysis of the genes ranked by the degree of correlation between gene expression and DNA methylation to identify gene sets involved in cellular migration and metastasis. Our unsupervised hierarchical clustering of the candidate genes segregated cell lines according to the epithelial-to-mesenchymal transition phenotype. Genes related to the epithelial-to-mesenchymal transition, such as AXL, ESRP1, HoxB4, and SPINT1/2, were among the nearly 20% of the candidate genes that were differentially methylated between epithelial and mesenchymal cells. Greater numbers of genes were methylated in the mesenchymal cells and their expressions were upregulated by 5-azacytidine treatment. Methylation of the candidate genes was associated with erlotinib resistance in wild-type EGFR cell lines. The expression profiles of the candidate genes were associated with 8-week disease control in patients with wild-type EGFR who had unresectable non-small cell lung cancer treated with erlotinib, but not in patients treated with sorafenib.

Conclusions

Our results demonstrate that the underlying biology of genes regulated by DNA methylation may have predictive value in lung cancer that can be exploited therapeutically.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1079) contains supplementary material, which is available to authorized users.     

Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells

Background

mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways.

Methods

Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR.

Results

IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes.

Conclusions

In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.     

Epithelial to mesenchymal transition of a primary prostate cell line with switches of cell adhesion modules but without malignant transformation

Background

Epithelial to mesenchymal transition (EMT) has been connected with cancer progression in vivo and the generation of more aggressive cancer cell lines in vitro. EMT has been induced in prostate cancer cell lines, but has previously not been shown in primary prostate cells. The role of EMT in malignant transformation has not been clarified.

Methodology/Principal Findings

In a transformation experiment when selecting for cells with loss of contact inhibition, the immortalized prostate primary epithelial cell line, EP156T, was observed to undergo EMT accompanied by loss of contact inhibition after about 12 weeks in continuous culture. The changed new cells were named EPT1. EMT of EPT1 was characterized by striking morphological changes and increased invasion and migration compared with the original EP156T cells. Gene expression profiling showed extensively decreased epithelial markers and increased mesenchymal markers in EPT1 cells, as well as pronounced switches of gene expression modules involved in cell adhesion and attachment. Transformation assays showed that EPT1 cells were sensitive to serum or growth factor withdrawal. Most importantly, EPT1 cells were not able to grow in an anchorage-independent way in soft agar, which is considered a critical feature of malignant transformation.

Conclusions/Significance

This work for the first time established an EMT model from primary prostate cells. The results show that EMT can be activated as a coordinated gene expression program in association with early steps of transformation. The model allows a clearer identification of the molecular mechanisms of EMT and its potential role in malignant transformation.     

Targeting mTOR to Overcome Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance in Non-Small Cell Lung Cancer Cells

Aims

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic clinical benefits in advanced non-small cell lung cancer (NSCLC); however, resistance remains a serious problem in clinical practice. The present study analyzed mTOR-associated signaling-pathway differences between the EGFR TKI-sensitive and -resistant NSCLC cell lines and investigated the feasibility of targeting mTOR with specific mTOR inhibitor in EGFR TKI resistant NSCLC cells.

Methods

We selected four different types of EGFR TKI-sensitive and -resistant NSCLC cells: PC9, PC9GR, H1650 and H1975 cells as models to detect mTOR-associated signaling-pathway differences by western blot and Immunoprecipitation and evaluated the antiproliferative effect and cell cycle arrest of ku-0063794 by MTT method and flow cytometry.

Results

In the present study, we observed that mTORC2-associated Akt ser473-FOXO1 signaling pathway in a basal state was highly activated in resistant cells. In vitro mTORC1 and mTORC2 kinase activities assays showed that EGFR TKI-resistant NSCLC cell lines had higher mTORC2 kinase activity, whereas sensitive cells had higher mTORC1 kinase activity in the basal state. The ATP-competitive mTOR inhibitor ku-0063794 showed dramatic antiproliferative effects and G1-cell cycle arrest in both sensitive and resistant cells. Ku-0063794 at the IC₅₀ concentration effectively inhibited both mTOR and p70S6K phosphorylation levels; the latter is an mTORC1 substrate and did not upregulate Akt ser473 phosphorylation which would be induced by rapamycin and resulted in partial inhibition of FOXO1 phosphorylation. We also observed that EGFR TKI-sensitive and -resistant clinical NSCLC tumor specimens had higher total and phosphorylated p70S6K expression levels.

Conclusion

Our results indicate mTORC2-associated signaling-pathway was hyperactivated in EGFR TKI-resistant cells and targeting mTOR with specific mTOR inhibitors is likely a good strategy for patients with EGFR mutant NSCLC who develop EGFR TKI resistance; the potential specific roles of mTORC2 in EGFR TKI-resistant NSCLC cells were still unknown and should be further investigated.