KRAS Activation in Gastric Adenocarcinoma Stimulates Epithelial-to-Mesenchymal Transition to Cancer Stem-Like Cells and Promotes Metastasis.
ABSTRACT: Our previous work showed that in a mouse model of gastric adenocarcinoma with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a. Tcon mice) accelerates tumorigenesis and metastasis. Here, we sought to examine KRAS activation in epithelial-to-mesenchymal transition (EMT) and generation of cancer stem-like cells (CSC). Transduction of nontransformed HFE-145 gastric epithelial cells with oncogenic KRASG12V significantly decreased expression of the epithelial marker E-cadherin, increased expression of the mesenchymal marker vimentin and the EMT transcription factor Slug, and increased migration and invasion by 15- to 17-fold. KRASG12V also increased expression of self-renewal proteins such as Sox2 and increased spheroid formation by 2.6-fold. In tumor-derived organoids from Tcon mice, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown or MEK inhibition in patient tumor-derived gastric adenocarcinoma cell lines (AGS and KATOIII). KRAS inhibition in gastric adenocarcinoma spheroid cells led to reduced AGS flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. In a tissue microarray of human gastric adenocarcinomas from 115 patients, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival. In conclusion, KRAS activation in gastric adenocarcinoma cells stimulates EMT and transition to CSCs, thus promoting metastasis. IMPLICATIONS: This study provides rationale for examining inhibitors of KRAS to block metastasis and reverse chemotherapy resistance in gastric adenocarcinoma patients.
Project description:Substantial evidence suggests that the epithelial-mesenchymal transition (EMT) phenotype is associated with the invasive characteristics of cancer stem cells (CSCs),which possess an EMT phenotype that may predominate in tumor invasion and metastasis. However, the mechanisms for the generation and regulation of these CSCs have not been clearly defined. As hypoxia and EMT-related factors may have important functions in EMT-like CSCs, the aim of this study was to investigate the effects of hypoxia on these cells. CSCs were established from the gastric cancer cell lines MGC-803 and SGC7901, and the relationship between hypoxia and EMT-like CSCs was investigated in gastric cancer. After hypoxia treatment, some gastric CSCs exhibited a marked increase in hypoxia-inducible factor-1? (HIF-1?)expression and increased migration and invasion capabilities compared with the normoxic control. These CSCs were defined by activation of the mesenchymal cell marker Vimentin and by inhibition of the epithelial cell marker E-cadherin. Our analyses also show that HIF-1? was responsible for activating EMT via increased expression of the transcription factor Snail in gastric CSCs. Moreover, inhibition of Snail by shRNA reduced HIF-1?-induced EMT in gastric CSCs. The results demonstrated that hypoxia-induced EMT-like CSCs rely on HIF-1?to activate Snail, which may result in recurrence and metastasis of gastric cancer.
Project description:The KRAS mutations have been an obstacle to identify therapeutic targets in cancer treatment. In this work, we clarified the distinct metastasis pattern of non-small-cell lung carcinoma (NSCLC) induced by KRASG12V/KRASG12D mutations and inhibited the KRASG12V mediated metastasis by Wnt inhibitor. First, we found that KRASG12V induced more aggressive phenotype in vitro and in vivo experiments. The Gene Set Enrichment Analysis (GSEA) results of H838 KRASG12V cells showed a significant negative correlation with RhoA-related signaling. Following this clue, we observed KRASG12D induced higher activation of RhoA and suppressed activation of Wnt/?-catenin in H838KRASG12D cells. The restored activation of Wnt/?-catenin in H838KRASG12D cells could be detected when expression with a dominant-negative mutant of RhoA or treatment with RhoA inhibitor. Furthermore, the Wnt inhibitor abolished the KRASG12V-induced migration. We elucidated the importance of the axis of RhoA/Wnt in regulatory NSCLC metastasis driven by KRAS mutations. Our data indicate that KRASG12V driven NSCLC metastasis is Wnt-dependent and the mechanisms of NSCLC metastasis induced by KRASG12V/KRASG12D is distinct.
Project description:OBJECTIVES:Evidence has indicated that ovarian epithelial cancer-type cells under serum-free culture conditions can form spheroid cells and exhibit characteristics expected of cancer stem-like cells (CSCs). However, the mechanism by which differentiated ovarian cancer cells acquire stem-cell properties during CSC enrichment has needed to be elucidated. Recent studies have demonstrated that induction of epithelial to mesenchymal transition (EMT) can generate CSCs and be associated with tumour aggressiveness and metastasis. MATERIALS AND METHODS:Ovarian epithelial cancer cell lines, SKOV3 and HO8920, were cultured for spheroid cells and adherent cells. CSC enrichment was investigated using MTT assay, flow cytometery and qRT-PCR and expression level of PI3K/AKT pathway components was analysed by western blotting. RESULTS:Compared to adherent cells, the spheroid cells expressed mesenchymal markers highly and exhibited significantly more motility; we also observed increases in phosphate AKT1 levels in the spheroid cells. Moreover, transfection of miR-20a or miR-200c led to corresponding reduction in endogenous PTEN protein, while AKT1 and phosphate AKT1 levels were upregulated in miRNAs-transfected cells. Finally, PI3K/AKT pathway inhibitor LY294002 reduced expressions of mesenchymal markers and stem-cell gene activity in spheroid cells, enhancing sensitivity of spheroid cells to paclitaxel treatment. CONCLUSIONS:Our findings demonstrate that EMT contributed to enrichment of ovarian CSCs in vitro, making EMT targeting in epithelial ovarian cancer a novel therapeutic option.
Project description:BACKGROUND: Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology) GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A), Rac1 (Ras-related C3 botulinum toxin substrate 1) and Cdc42 (cell division cycle 42) in cancer progression induced by each of the three oncogenes is described. METHODS: Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities. RESULTS: Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT) were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGF?-1 pathway to provide cells with additional transforming properties. CONCLUSION: This study discriminates oncogene-specific cell migration and invasion pathways mediated by Rho GTPases in colon cancer cells and reveals potential new oncogene-specific characteristics for targeted therapeutics.
Project description:Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and metastasis. It is evident from growing reports that PI3K/Akt/mTOR and Sonic Hedgehog (Shh) signaling pathways are aberrantly reactivated in pancreatic CSCs. Here, we examined the efficacy of combining NVP-LDE-225 (PI3K/mTOR inhibitor) and NVP-BEZ-235 (Smoothened inhibitor) on pancreatic CSCs characteristics, microRNA regulatory network, and tumor growth. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting pancreatic CSC's characteristics and tumor growth in mice by acting at the level of Gli. Combination of NVP-LDE-225 and NVP-BEZ-235 inhibited self-renewal capacity of CSCs by suppressing the expression of pluripotency maintaining factors Nanog, Oct-4, Sox-2 and c-Myc, and transcription of Gli. NVP-LDE-225 co-operated with NVP-BEZ-235 to inhibit Lin28/Let7a/Kras axis in pancreatic CSCs. Furthermore, a superior interaction of these drugs was observed on spheroid formation by pancreatic CSCs isolated from Pankras/p53 mice. The combination of these drugs also showed superior effects on the expression of proteins involved in cell proliferation, survival and apoptosis. In addition, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting EMT through modulation of cadherin, vimentin and transcription factors Snail, Slug and Zeb1. In conclusion, these data suggest that the combined inhibition of PI3K/Akt/mTOR and Shh pathways may be beneficial for the treatment of pancreatic cancer.
Project description:Postoperative recurrence and metastasis have crucial roles in the poor prognosis of gastric cancer patients. Previous studies have indicated that gastric cancer originates from cancer stem cells (CSCs), and some investigators have found that a particular subset of CSCs possesses higher metastatic capacity. However, the specific mechanism remains uncertain. In the present study, we aimed to explore the biological functions of the inflammatory cytokine interleukin-17 (IL-17) in gastric cancer metastasis and the distinct IL-17-induced transformation of quiescent gastric CSCs. Our results showed that invasive gastric CSCs were CD26+ and CXCR4+ and were closely associated with increased metastatic ability. The quiescent gastric CSCs, which were CD26- and CXCR4-, were exposed to appropriate concentrations of IL-17; this resulted in the decreased expression of E-cadherin and the increased expression of vimentin and N-cadherin. In addition, the upregulation of IL-17 both in vitro and in vivo resulted in a significant induction of invasion, migration and tumor formation ability in gastric CSCs compared with the control group, which was not treated with IL-17. Further experiments indicated that the activation of the downstream phosphorylated signal transducer and activator of transcription 3 (STAT3) transcription factor pathway was facilitated by IL-17. On the contrary, the downregulation of STAT3 by the specific inhibitor Stattic significantly reversed the IL-17-induced epithelial-mesenchymal transition (EMT)-associated properties of quiescent gastric CSCs. Moreover, tumorigenesis and metastasis were suppressed. Taken together, we suggest that IL-17 is positively correlated with the transformation of quiescent gastric CSCs into invasive gastric CSCs and that targeting IL-17 may emerge as a possible novel therapeutic strategy for gastric cancer.
Project description:The Wnt1 protein, a secreted ligand that activates Wnt signaling pathways, contributes to the self-renewal of cancer stem cells (CSCs) and thus may be a major determinant of tumor progression and chemoresistance. In a series of gastric cancer specimens, we found strong correlations among Wnt1 expression, CD44 expression, and the grade of gastric cancer. Stable overexpression of Wnt1 increased AGS gastric cancer cells' proliferation rate and spheroids formation, which expressed CSC surface markers Oct4 and CD44. Subcutaneous injection of nude mice with Wnt1-overexpressing AGS cells resulted in larger tumors than injection of control AGS cells. Salinomycin, an antitumor agent, significantly reduced the volume of tumor caused by Wnt1-overexpressing AGS cells in vivo. This is achieved by inhibiting the proliferation of CD44+Oct4+ CSC subpopulation, at least partly through the suppression of Wnt1 and ?-catenin expression. Taken together, activation of Wnt1 signaling accelerates the proliferation of gastric CSCs, whereas salinomycin acts to inhibit gastric tumor growth by suppressing Wnt signaling in CSCs. These results suggest that Wnt signaling might have a critical role in the self-renewal of gastric CSCs, and salinomycin targeting Wnt signaling may have important clinical applications in gastric cancer therapy.
Project description:There is an urgent need to identify targeting molecules to control invasion and metastasis in cancer patients. We first isolated cancer stem cells (CSCs) from SKOV3 ovarian cancer cells and then investigated the role of melatonin in invasiveness and migration of CSCs compared to SKOV3 cells. The proportion of CSCs in SKOV3 cells was as low as 1.28% with overexpression of both CD133 and CD44. The ability of spheroid formation along with SOX2 overexpression revealed a high self-renewal potential in isolated cells. Melatonin (3.4?mM) inhibited proliferation of CSCs by 23% which was confirmed by a marked decrease in protein expression of Ki67, as a proliferation marker. Applying luzindole, a melatonin receptor 1, 2 inhibitor, partially abolished anti-proliferative effect of melatonin. Melatonin also decreased Epithelial mesenchymal transition (EMT) related gene expressions including ZEB1, ZEB2, snail and vimentin with increase in E-cadherin as a negative EMT regulator. Incubation of CSCs with melatonin showed a marked decrease in matrix metalloproteinase 9 (MMP9) expression and activity. Melatonin also inhibited CSCs migration in a partially receptor dependent and PI3k and MAPK independent manner. Melatonin can be considered as an important adjuvant to control invasion and metastasis especially in patients with high melatonin receptor expression.
Project description:Lung cancer is the leading cause of cancer-related mortality worldwide. Recent evidence indicates that tumors contain a subpopulation of cancer stem cells (CSCs) that are responsible for tumor maintenance and spread. CSCs have recently been linked to the occurrence of epithelial-to-mesenchymal transition (EMT). Neurotrophins (NTs) are growth factors that regulate the biology of embryonic stem cells and cancer cells, but still little is known about the role NTs in the progression of lung cancer. In this work, we investigated the role of the NTs and their receptors using as a study system primary cell cultures derived from malignant pleural effusions (MPEs) of patients with adenocarcinoma of the lung. We assessed the expression of NTs and their receptors in MPE-derived adherent cultures vs. spheroids enriched in CSC markers. We observed in spheroids a selectively enhanced expression of TrkB, both at the mRNA and protein levels. Both K252a, a known inhibitor of Trk activity, and a siRNA against TrkB strongly affected spheroid morphology, induced anoikis and decreased spheroid forming efficiency. Treatment with neurotrophins reversed the inhibitory effect of K252a. Importantly, TrkB inhibition caused loss of vimentin expression as well as that of a set of transcription factors known to be linked to EMT. These ex vivo results nicely correlated with an inverse relationship between TrkB and E-cadherin expression measured by immunohistochemistry in a panel of lung adenocarcinoma samples. We conclude that TrkB is involved in full acquisition of EMT in lung cancer, and that its inhibition results in a less aggressive phenotype.