Epithelial-mesenchymal Transition In Colon Cancer Cells Research Articles

Genistein induces apoptosis of colon cancer cells by reversal of epithelial-to-mesenchymal via a Notch1/NF-\u03baB/slug/E-cadherin pathway

BackgroundGenistein has been known to inhibit proliferation and induce apoptosis in several kinds of cancer cells. While knowledge of genistein in regulating epithelial mesenchymal transition (EMT) of colon cancer cells is unknown.MethodsTo investigate the effects and mechanisms of genistein on EMT of colon cancer cells, HT-29 cells were used and treated by genistein and TNF-α in this paper. EMT was determined by cell invasion assays using a transwell chamber and the expression changes of EMT-related markers were confirmed by RT–PCR, Western blotting, and immunofluorescence staining.ResultsGenistein inhibited cell migration at 200 μmol/L. Genistein reversed the EMT of colon cancer cells by upregulation of E-cadherin and downregulation of N-cadherin, accompanied by the suppression of EMT related makers, such as Snail2/slug, ZEB1, ZEB2, FOXC1, FOXC2 and TWIST1. Moreover, genistein can inhibit the expression of notch-1, p-NF-κB and NF-κB, while promote the expression of Bax/Bcl-2 and caspase-3 in HT-29 cells.ConclusionThe present study demonstrated that genistein suppressed the migration of colon cancer cells by reversal the EMT via suppressing the Notch1/NF-κB/slug/E-cadherin pathway. Genistein may be developed as a potential antimetastasis agent to colon cancer.

Insulin-like growth factor-1 activates different catalytic subunits p110 of PI3K in a cell-type-dependent manner to induce lipogenesis-dependent epithelial-mesenchymal transition through the regulation of ADAM10 and ADAM17.

The activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) is critical for the induction of epithelial-mesenchymal transition (EMT) by growth factors, including insulin-like growth factor 1 (IGF-1). The activation of intracellular lipogenesis provides proliferative and survival signals for cancer cells. In this study, we investigated the connection between lipogenesis-related EMT processes and IGF-1-mediated PI3K p110 isoform activation in primary (SW480 cells) and metastatic (SW620) colon carcinoma cells. We also examined the underlying signaling pathway that promotes fatty acid synthesis in IGF-1-activated colon cancer cells. IGF-1 stimulation upregulated the expression of lipogenic enzymes as well as the activation of Nardilysin (N-arginine dibasic convertase, NRD1) and its downstream targets, a disintegrin and metalloproteases 10 (ADAM10) and ADAM17. The upregulation of the Lyn/Syk-mediated PI3K p110δ isoform in SW480 cells and the Lyn-dependent PI3K p110α isoform in SW620 cells triggered fatty acid production and cell motility in IGF-1-activated colon cancer cells. Pharmacological inhibition with A66 (PI3K p110α specific inhibitor) and CAL-101 (PI3K p110δ specific inhibitor) efficiently inhibited EMT in colon cancer cells by blocking the NRD1/ADAM family protein signaling pathway. Gene silencing of NRD1 and ADAM family proteins attenuated the generation of intracellular fatty acid and the migratory activity of colon cancer cells. Our results suggest that the different isoforms of the PI3K p110 subunit could be therapeutic targets for primary and metastatic colon cancer and that regulation of the NRD1/ADAM signaling pathway controls lipogenesis-mediated EMT in IGF-1-stimulated colon cancer cells.

Abstract 1879: Transcriptional regulation of KAI1 by p73 in colorectal cancer metastasis

Abstract Background: p73 is a member of the p53 family transcription factors and known tumor suppressor. However, its transcriptional activity is complex owing to its multiple isoforms, and limited information is available regarding the transcriptional and functional regulation of the p73 gene in colorectal cancer. In this study, we examined the role of p73 in colorectal cancer. Methods: We established a TAp73 overexpressing cell line using the Tet-on inducible expression system and analyzed global RNA expression by RNA-sequencing (RNA-seq). We studied the effects of TAp73 expression on the migration, invasiveness, and EMT of colon cancer cells. We conducted experiments in animals with liver metastasis of colorectal cancer. To investigate the roles of p73 in colon cancer, we examined the samples from patients, who had undergone surgical resection for colorectal cancer between 2009 and 2013. mRNA expression of TAp73 and KAI1 was analyzed in 108 colorectal cancer specimen and adjacent non-cancerous tissues by real-time RT-PCR. Results: RNA-seq showed the association between TAp73 and KAI1 expression. Migration and invasiveness of colon cancer cells were reduced by TAp73 expression, but induced by KAI1 knockdown. TAp73 regulates KAI1 expression in colon cancer cell lines. TAp73-induced decrease in invasiveness and epithelial-mesenchymal transition (EMT) was abrogated by KAI1 knockdown in TAp73 overexpressing cells. Migration ability of TAp73 overexpressing cells was recovered by KAI1 knockdown. Hepatic metastases were significantly reduced in mice injected with TAp73 overexpressing cells and increased in those injected with TAp73 overexpressing cells with KAI1 knockdown. mRNA expression of TAp73 and KAI1 was higher in colorectal cancer tissues than in adjacent non-cancerous tissues. Significant correlation between TAp73 and KAI1 mRNA expression was detected in early stage primary colorectal cancer, but not in the advanced stage. Conclusions: This study suggests that p73 (TAp73) acts as a tumor suppressor in the progression of colorectal cancer and regulates the expression of KAI1, which is a key target of p73 in colorectal cancer liver metastases. Note: This abstract was not presented at the meeting. Citation Format: Woo Kyun Bae, Kyung Hyun Yoo, Keunsoo Kang, Lothar Hennighausen, Ik-Joo Chung. Transcriptional regulation of KAI1 by p73 in colorectal cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1879. doi:10.1158/1538-7445.AM2017-1879

Abstract 854: Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer

Abstract Background: Kallikrein-related peptidase 6 (KLK6) belongs to the family of human tissue kallikrein genes, majority of which are shown to be differentially expressed in cancers. Clinical studies have demonstrated that upregulation of KLK6 in primary colorectal tumors and lymph nodes correlates with serosal invasion, liver metastasis and indicative of poor prognosis in patients. It has been reported that KLK6 protein is involved in regulation of the epithelial-mesenchymal transition (EMT) program in an organ-specific context. The aim in this study was to investigate contribution of KLK6 enzyme in the EMT during neoplastic transformation in the colon. Results: We expressed enzymatically active or inactive KLK6, using pcDNA3.1(+)preproKLK6 and pcDNA3.1(+)preproKLK6 Ser197Ala mutant plasmids, in Caco-2 colon cancer cell line, which has been characterized before as a very low KLK6 expresser with an undetectable secreted KLK6. Stable isogenic clones were selected and further evaluated for their ability to migrate and invade using in vitro assays and to metastasize in vivo using SCID orthotopic mouse model. We found no effect of KLK6 enzyme activity on migration of Caco-2 cells, expressing the empty vector (Caco-2 mock), and Caco-2 cells, expressing an enzymatically active KLK6 (Caco-2 KLK6 wt) or inactive KLK6 (Caco-2 KLK6 mut). But Caco-2 KLK6 wt cells demonstrated the invasive phenotype in Matrigel invasion assays (p<0.001, compared to Caco-2 mock and Caco2 KLK6 mut cells). The Caco-2 mock and Caco-2 KLK6 mut cells, injected in SCID mice orthotopically, developed primary colon tumors but no metastatic lesions were identified. In contrast, Caco-2 KLK6 wt cells formed primary colon tumors and metastasized locally, although they failed to form the distant metastasis (lung and mesentery). Animals, growing the Caco-2 KLK6 wt tumors, displayed a significant decrease in their survival rates, compared to other groups (p=0.02). In Caco-2 KLK6 wt cells TGF-β protein expression and secretion was induced, which resulted in activation of TGF-β-SMAD2/3 signaling pathway. This phenotype was associated with the elevated expression of known regulator of the EMT, zinc-finger protein Snail. In addition, the expression of a high-mobility group AT-hook 2 (HMGA2) protein was induced in Caco-2 KLK6 wt cells. The HMGA2 expression is implicated in the EMT program, acting through the TGF-β signaling pathway and is associated with a poor survival in colorectal cancer. Conclusion. These findings demonstrate that KLK6 enzyme activity is required for colon cancer progression via induction of the EMT program. We identified the TGF-β- signaling pathway as a mechanism driving the EMT in colon cancer cells expressing KLK6 enzyme. Citation Format: Hwudaurw Chen, Earlphia Sells, Haiyan Cui, Ritu Pandey, George Pampalakis, Georgia Sotiropoulou, Thomas Doetschman, Natalia A. Ignatenko. Human tissue Kallikrein 6 enzyme activity regulates epithelial-mesenchymal transition in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 854. doi:10.1158/1538-7445.AM2017-854

Mesenchymal Stem Cells Induce Epithelial to Mesenchymal Transition in Colon Cancer Cells through Direct Cell-to-Cell Contact

We previously reported that in an orthotopic nude mouse model of human colon cancer, bone marrow–derived mesenchymal stem cells (MSCs) migrated to the tumor stroma and promoted tumor growth and metastasis. Here, we evaluated the proliferation and migration ability of cancer cells cocultured with MSCs to elucidate the mechanism of interaction between cancer cells and MSCs. Proliferation and migration of cancer cells increased following direct coculture with MSCs but not following indirect coculture. Thus, we hypothesized that direct contact between cancer cells and MSCs was important. We performed a microarray analysis of gene expression in KM12SM colon cancer cells directly cocultured with MSCs. Expression of epithelial-mesenchymal transition (EMT)–related genes such as fibronectin (FN), SPARC, and galectin 1 was increased by direct coculture with MSCs. We also confirmed the upregulation of these genes with real-time polymerase chain reaction. Gene expression was not elevated in cancer cells indirectly cocultured with MSCs. Among the EMT-related genes upregulated by direct coculture with MSCs, we examined the immune localization of FN, a well-known EMT marker. In coculture assay in chamber slides, expression of FN was seen only at the edges of cancer clusters where cancer cells directly contacted MSCs. FN expression in cancer cells increased at the tumor periphery and invasive edge in orthotopic nude mouse tumors and human colon cancer tissues. These results suggest that MSCs induce EMT in colon cancer cells via direct cell-to-cell contact and may play an important role in colon cancer metastasis.

Nesfatin-1/Nucleobindin-2 enhances cell migration, invasion, and epithelial-mesenchymal transition via LKB1/AMPK/TORC1/ZEB1 pathways in colon cancer.

Recent studies indicate that a high level of nesfatin-1/Nucleobindin-2 (NUCB-2) is associated with poor outcome and promotes cell migration in breast cancer and prostate cancer. However, the role of NUCB2 is not well known in colon cancer. In this study, NUCB-2 level in colon cancer tissue was higher than that in non-tumor tissue. Suppression of NUCB-2 in a colon cancer cell line SW620 inhibited migration and invasion. The microarray analysis showed that low expression level of transcription factor ZEB1 in NUCB-2 knockdowned SW620 cells. In addition, expression level of epithelial-mesenchymal transition (EMT)-related molecules including N-cadherin, E-cadherin, β-catenin, Slug and Twist was affected by NUCB-2 suppression and ZEB1-denepdent pathway. The signaling pathway liver kinase B1(LKB1)/AMP-dependent protein kinase (AMPK)/target of rapamycin complex (TORC) 1 was involved in regulation of NUCB-2-mediated metastasis and EMT properties. Suppression of NUCB-2 inhibited tumor nodules formation in a murine colon tumor model as well. In summary, nesfatin-1/NUCB-2 enhanced migration, invasion and EMT in colon cancer cells through LKB1/AMPK/TORC1/ZEB1 pathways in vitro and in vivo.

Eukaryotic translation initiation factor 5A2 (eIF5A2) regulates chemoresistance in colorectal cancer through epithelial mesenchymal transition.

BackgroundChemoresistance is a major obstacle to successful chemotherapy for colorectal cancer. Eukaryotic translation initiation factor 5A2 (eIF5A2), one of the two isoforms in the eIF5A family, has been reported to be a new oncogene in many types of human cancer. In the present study, we aimed to investigate whether eIF5A2 was involved in the chemoresistance to doxorubicin in colorectal cancer.MethodsCell viability was measured by CCK-8 assay with or without doxorubicin treatment. Protein expression was detected by western blot. Tumor cells were transfected with eIF5A2 siRNA or plasmid encoding eIF5A2 to down- or up regulate the expression of eIF5A2.ResultsWe found that eIF5A2-negtive colon cancer cells (HCT116 and HT29) were more sensitive to doxorubicin compare with the eIF5A2-positive cells (LOVO and SW480). Downregulation of eIF5A2 in LOVO and SW480 cells enhanced the chemosensitivity to doxorubicin. On the contrary, overexpression of eIF5A2 reduced doxorubicin sensitivity in colon cancer cells. In addition, eIF5A2 knockdown increased the protein level of E-cadherin and reduced vimentin expression in LOVO and SW480 cells. Meanwhile, upregulation of eIF5A2 potentiated epithelial mesenchymal transition (EMT) in colon cancer cells. Moreover, blockade of EMT with Twist siRNA abolished eIF5A2-regulated chemoresistance in colon cancer cells.ConclusionOur present study demonstrated that eIF5A2 promoted the chemoresistance to doxorubicin via regulation of EMT in colon cancer cells. Therefore, eIF5A2 inhibition may be a new potential strategy for the reversal of drug resistance in colorectal cancer therapy.

Abstract 1479: Modulating AKT induced epithelial mesenchymal transition (EMT) for prevention and treatment of colon cancer.

Abstract Activation of serine threonine kinase-AKT transduces signals that regulate multiple biological processes including cellular proliferation, survival, apoptosis, gene expression, angiogenesis, migration and EMT in many cancer types. Recent studies have shown oncogenic activation of AKT has emerged as a central feature of EMT, endowing the cancer cells with migratory and invasive capability. In our results over expression of AKT in HCT-116 (AKT/HCT-116) (express low levels of pAKT as compared to the other colon cancer cell lines) showed significantly higher cell proliferation and aggressive phenotype with increase in migration and invasion when compared to the vector transfected control cells (pCMV/HCT-116). Further in vivo studies revealed highly aggressive, angiogenic with large tumor volume in AKT/HCT-116 xenografts compared to the control tumors. Down-regulating constitutively active Akt (by a PI3 kinase inhibitor, such as wortmannin or LY 294002) reverses both cell survival and resistance to chemotherapeutic agents, however, these inhibitors causes severe toxicity in animal models. Hence, it is exigent to identify novel molecules, to inhibit Akt signaling, which could be developed as a new therapeutics, as well as preventive agents for colon cancer. Our small molecules screening studies revealed, Withaferin-A (WA) as a potent molecule which significantly inhibited AKT activity that resulted in inhibition of cell proliferation in AKT over expressing HCT-116 cells. At molecular level inhibition of AKT resulted in suppression of its downstream pro-survival signaling such as NFκB and BCL-2 in AKT/HCT-116 cells and pCMV/HCT-6 cells. Oral treatment of WA significantly suppressed AKT-induced tumor growth, invasion and migration in cell culture and xenograft models. These results correlated well at molecular level with significant inhibition of the important EMT markers i.e. snail, slug, β-catenin, MMP-2 and MMP-9. Immunohistochemistry of tumor tissues showed WA treatment resulted in increase in E-cadherin expression with concomitant decrease in the expression and nuclear localization of β-catenin in AKT overexpressing tumors. Decreased levels of pAKT, NFkB, Snail, Slug, vimentin expression were seen in WA treated both pCMV/HCT-116 and AKT/HCT-116 tumors. Interestingly, significant inhibition of micro-vessel formation and length of vessels were evident in WA-treated tumors which correlated with low expression of angiogenic markers like Factor VIII and RETIC. In conclusion, the present study underscores the crucial role of AKT activation in inducing cell proliferation, angiogenesis and EMT in colon cancer cells and WA could be a potent compound for the chemoprevention and/or chemotherapy of colon cancer. Citation Format: Suman Suman, Vittal Kurisetty, Trinath P. Das, Aditi Vadodkar, Ramadevi Subramani Reddy, Rajkumar Lakshmanaswamy, Chendil Damodaran. Modulating AKT induced epithelial mesenchymal transition (EMT) for prevention and treatment of colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1479. doi:10.1158/1538-7445.AM2013-1479

Abstract LB-326: Zeb1 regulates inflammation-induced invasion and self-renewal of colon cancer cells.

Abstract Up-regulation of inflammatory cytokines such as IL-1β in tumor microenvironment has been associated with progression of human colorectal cancer, although the exact molecular mechanisms are still unclear. To understand how IL-1β influences colon cancer cell behaviors, we used a human colon cancer cell line HCT-116 as well as primary human colon cancer cells derived from a patient, which more closely reflect the properties of the primary tumor than established cell lines. Here, we demonstrate that IL-1β promotes epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) development in colon cancer. IL-1β treatment resulted in loss of E-cadherin and gain of a mesenchymal phenotype in both HCT-116 cells and primary colon cancer cells. Among E-cadherin suppressors, Zeb1 was up-regulated in IL-1β-induced EMT cells. Consistent with EMT properties, IL-1β-treated HCT-116 cells exhibited higher cell migratory ability. Moreover, IL-1β induced expression of stem-cell marker Bmi-1 in both HCT-116 cells and primary colon cancer cells. IL-1β increased these cells’ self-renewal ability to form spheres in serum-free conditions and chemo-resistance; properties associated with colon CSCs. Taken together, these results indicate that IL-1β can induce EMT and promote generation of CSCs in colon cancer. More importantly, we found that ZEB1, but not other E-cadherin transcriptional repressors (such as Zeb2, Snail and Twist), was consistently up-regulated in IL-1β-induced EMT cells and sphere cells. This suggests that Zeb1 links IL-1β-induced CSC self-renewal and EMT in colon cancer cells. Moreover, knockdown of Zeb1 in HCT-116 cells using shRNAs reversed the IL-1β effects on these two processes, further indicating that Zeb1 is essential for IL-1β-induced stem cell and EMT phenotypes in colon cancer cells. Our finding indicates that IL-1β promotes colon tumor growth and invasion through activation of CSC self-renewal and EMT, while Zeb1 plays a critical role in activation of these two processes. Thus, targeting tumor microenvironment cytokines or Zeb1 might form the basis of a promising treatment for colon cancer. Citation Format: Lei Wang, Yijing Li, Amy Beckley, Lorretta Pappan, Jishu Shi. Zeb1 regulates inflammation-induced invasion and self-renewal of colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-326. doi:10.1158/1538-7445.AM2013-LB-326 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

IL-1β promotes stemness and invasiveness of colon cancer cells through Zeb1 activation

BackgroundIL-1β is a pleiotropic pro-inflammatory cytokine and its up-regulation is closely associated with various cancers including gastrointestinal tumors. However, it remains unclear how IL-1β may contribute to the initiation and development of these inflammation-associated cancers. Here we investigated the role of IL-1β in colon cancer stem cell (CSC) development.MethodsUsing self-renewal assay, soft-agar assay, invasion assay, real-time PCR analysis, immunoblot assay and shRNA knockdown, we determined the effects of IL-1β on cancer stem cell development and epithelial-mesenchymal transition (EMT) in human primary colon cancer cells and colon cancer cell line HCT-116.ResultsWe found that IL-1β can increase sphere-forming capability of colon cancer cells in serum-free medium. IL-1β-induced spheres displayed an up-regulation of stemness factor genes (Bmi1 and Nestin) and increased drug resistance, hallmarks of CSCs. Importantly, expression of EMT activator Zeb1 was increased in IL-1β-induced spheres, indicating that there might be a close association between EMT and IL-1β-induced CSC self-renewal. Indeed, IL-1β treatment led to EMT of colon cancer cells with loss of E-cadherin, up-regulation of Zeb1, and gain of the mesenchymal phenotype. Furthermore, shRNA-mediated knockdown of Zeb1 in HCT-116 cells reversed IL-1β-induced EMT and stem cell formation.ConclusionOur findings indicate that IL-1β may promote colon tumor growth and invasion through activation of CSC self-renewal and EMT, and Zeb1 plays a critical role in these two processes. Thus, IL-1β and Zeb1 might be new therapeutic targets against colon cancer stem cells.

Transcription factors c-Myc and CDX2 mediate E-selectin ligand expression in colon cancer cells undergoing EGF/bFGF-induced epithelial–mesenchymal transition

Sialyl Lewis x (sLe(x)) and sialyl Lewis a (sLe(a)) glycans are expressed on highly metastatic colon cancer cells. They promote extravasation of cancer cells and tumor angiogenesis via interacting with E-selectin on endothelial cells. Recently, epithelial-mesenchymal transition (EMT) has been noted as a critical phenotypic alteration in metastatic cancer cells. To address the association between sLe(x/a) expression and EMT, we assessed whether sLe(x/a) are highly expressed on colon cancer cells undergoing EMT. Treatment of HT29 and DLD-1 cells with EGF and/or basic FGF (bFGF) induced EMT and significantly increased sLe(x/a) expression resulting in enhanced E-selectin binding activity. The transcript levels of the glycosyltransferase genes ST3GAL1/3/4 and FUT3 were significantly elevated and that of FUT2 was significantly suppressed by the treatment. We provide evidence that ST3GAL1/3/4 and FUT3 are transcriptionally up-regulated by c-Myc with probable involvement of Ser62 phosphorylation, and that FUT2 is transcriptionally down-regulated through the attenuation of CDX2. The contribution of c-Myc and CDX2 to the sLe(x/a) induction was proved to be significant by knockdown or forced expression experiments. Interestingly, the cells undergoing EMT exhibited significantly increased VEGF secretion, which can promote tumor angiogenesis in cooperation with sLe(x/a). Finally, immunohistological study indicated high E-selectin ligand expression on cancer cells undergoing EMT in vivo, supporting their coexistence observed in vitro. These results suggest a significant link between sLe(x/a) expression and EMT in colon cancer cells and a pivotal role of c-Myc and CDX2 in regulating sLe(x/a) expression during EMT.