Wan-Chen Wei

Rab11 regulates E-cadherin expression and induces cell transformation in colorectal carcinoma

BackgroundIn the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation.MethodsFor tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy.ResultsIn immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and cell membrane protrusion was induced, which resulted in cell transformation and cell migration.ConclusionsThis study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment.

Caveolin-1 Dependent Endocytosis Enhances the Chemosensitivity of HER-2 Positive Breast Cancer Cells to Trastuzumab Emtansine (T-DM1)

The humanized monoclonal antibody-drug conjugate trastuzumab emtansine (T-DM1, Kadcyla) has been approved by the U.S. FDA to treat human epidermal growth factor receptor 2 (HER-2)-positive metastatic breast cancer. Despite its effectiveness in most patients, some are initially resistant or develop resistance. No biomarker of drug resistance to T-DM1 has been identified. Antibody-drug efficacy is associated with antibody internalization in the cell; therefore, cellular sensitivity of cells to the drug may be linked to cellular vesicle trafficking systems. Caveolin-1 is a 22 KD protein required for caveolae formation and endocytic membrane transport. In this study, the relationship between caveolin-1 expression and the chemosensitivity of HER-2-positive breast cancer cells to T-DM1 was investigated. Samples from 32 human breast cancer biopsy and normal tissue specimens were evaluated immunohistochemically for caveolin-1 expression. Caveolin-1 was shown to be expressed in 68% (22/32) of the breast cancer specimens. In addition, eight (72.7%, 8/11) HER-2 positive breast cancer specimens had a higher caveolin-1 expression than normal tissues. HER-2-positive BT-474 and SKBR-3 breast cancer cells that express low and moderate levels of caveolin-1, respectively, were treated with trastuzumab or its conjugate T-DM1. Cell viability and molecular localizations of caveolin-1, antibody and its conjugate were examined. Confocal microscopy showed that T-DM1 and caveolin-1 colocalized in SKBR-3 cells, which also were five times more sensitive to the conjugate in terms of cell survival than BT-474 cells, although T-DM1 also showed improved drug efficacy in BT-474 cells than trastuzumab treatment. Caveolin-1 expression in these lines was manipulated by transfection of GFP-tagged caveolin-1 or caveolin-1 siRNA. BT-474 cells overexpressing caveolin-1 were more sensitive to T-DM1 treatment than mock-transfected cells, whereas the siRNA-transfected SKBR-3 cells had decreased sensitivity to T-DM1 than mock-transfected SKBR-3 cells. The expression of caveolin-1 could mediate endocytosis and promote the internalization of T-DM1 into HER-2 positive cancer cells. Thus, caveolin-1 protein may be an effective predictor for determining the outcome of T-DM1 treatment in breast cancer patients.

Rab11 collaborates E‐cadherin to promote collective cell migration and indicates a poor prognosis in colorectal carcinoma

Collective cell migration, whereby the cell–cell contacts such as E‐cadherin are maintained during migration, has only recently emerged, and its detailed mechanisms are still unclear. In this study, the role of Rab11, which functions in recycling endosomes, and its relationship to E‐cadherin in colorectal carcinoma were identified, and the role of Rab11 in the collective cell migration of colon cancer cells was clarified.

Metformin-induced caveolin-1 expression promotes T-DM1 drug efficacy in breast cancer cells

Trastuzumab emtansine (T-DM1) is an antibody drug conjugate (ADC) that was recently approved for the treatment of HER-2-positive metastatic breast cancer. The drug sensitivity of ADCs depends mainly on the internalization efficiency of the drug. Caveolin-1 was shown to promote T-DM1 internalization and enhance drug sensitivity. Whether caveolin-1 can be overexpressed to improve T-DM1 efficacy is interesting and has the potential for clinical application. In this study, diabetes drug metformin was investigated in terms of induction of caveolin-1 expression for increased efficacy of subsequent T-DM1 application. BT-474 cells were pretreated with metformin, followed by combined therapy with metformin and T-DM1. The T-DM1 internalization and drug efficacy were determined, and the protein expressions for signal transduction were also monitored. Caveolin-1 shRNA was applied to suppress endogenous caveolin-1 expression, and the ability of metformin to promote T-DM1 efficacy was investigated. Result showed that in BT-474 cells pretreated with metformin, cellular caveolin-1 overexpression was induced, which then promoted drug efficacy by enhancing T-DM1 internalization. As cellular caveolin-1 was suppressed by shRNA, the effect of metformin-enhanced T-DM1 cytotoxicity was decreased. This study demonstrated that metformin can be applied prior to T-DM1 treatment to improve the clinical efficacy of T-DM1 by enhancing caveolin-1-mediated endocytosis.

Abstract 1716: The role of CD44v9+ colorectal cancer stem cells in the resistance to cetuximab treatment

Recent studies found that conventional therapy combined with cetuximab treatment (EGFR inhibitor) to treat metastatic colorectal cancer, had no improvement of patient survival rate in some population. One of possibilities might be cancer stem cells (CSCs) population resist to cetuximab treatment and modulate metastasis, however, the detail mechanism has not been addressed in colorectal cancer. Thus, our study is to decipher the mechanism of resistance to cetuximab in CD44v9+ specific colorectal cancer stem cells. First, to reveal clinical significance of CD44v9, the tumor samples of colorectal cancer patients were analyzed by immunohistochemistry to evaluate CD44v9 expression status in patients. To further investigate the biological mechanism, single cell analysis in CD44v9+ population of HT29 cells was performed with Fluidigm Biomark™ HD system. The results showed that 67% patients had highly expressed CD44v9 with lower survival rate. In vitro studies showed that when HT29 cells were in 5uM cetuximab treatment for 48 hours, CD44v9, Oct4, CD133, CD24, Lgr5, CSCs markers, were up-regulated in RNA level. Furthermore, CD44v9+/Oct4+ cancer cell population was increased but CD44v9+/pEGFR+ cancer cells were eliminated according to immunofluorescence results. The results of single cell analysis demonstrated that CD44v9+/Oct4+ cancer cells highly expressed proliferation markers consistently with cetuximab treatment. Trans-well migration results showed that CD44v9+/Oct4+ cancer cells migrated collectively via mediating E-cadherin dynamics. Furthermore, in clinical outcome, CD44v9 and E-cadherin co-expression were associated with recurrence and metastasis and decreased survival rate significantly. In conclusion, cetuximab failed to target CD44v9+/Oct4+ cancer cells, which characterized as cancer stem cells and might modulate collective cell migration. This finding suggested that new therapeutic strategy to against CSCs would be the potential clinical treatment in colon cancer. Citation Format: Chia-Nung Hung, Ching-Yu Chang, Jou Hsiao, Wan-Chen Wei, Wei-Ting Chao. The role of CD44v9+ colorectal cancer stem cells in the resistance to cetuximab treatment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1716.

Abstract 1544: Overexpression of Rab11 and E-cadherin promotes collective cell migration and indicates a poor prognosis in colorectal carcinoma

Collective cell migration, whereby the cell-cell contacts such as E-cadherin are maintained during migration, has recently emerged, and the detailed mechanisms of this process are still unclear. The present study identified the role of Rab11 which functions in recycling endosome and the relation to E-cadherin in colorectal carcinoma and clarified the mechanism of Rab11 in the collective cell migration of cancer cells. The relationships between the overexpression of Rab11 and E-cadherin and survival were evaluated from colorectal carcinoma patients. A total of 107 patients with surgically resected colorectal carcinoma were enrolled in immunohistochemical study. The relationships between the overexpression of Rab11 and E-cadherin and survival were evaluated. GFP tagged Rab11 or Rab11 shRNA was introduced into HT-29 colon cancer cells for overexpression or knockdown. The interaction between E-cadherin and Rab11 was determined by immunoprecipitation. Rac1 and matrix metalloproteinase (MMP) activities were evaluated as functional effectors of collective cell migration. In the results, the expression of Rab11 and E-cadherin was not correlated with the stages of cancer or lymph node metastasis. However, the overall survival was poor in the group of 60 patients with duo-positive Rab11 and E-cadherin expression compared to the group (47 patients) without duo-positive expression (p = 0.038). In the cell biology assay, Rab11 was demonstrated through its physical interaction with E-cadherin, and overexpression of Rab11 was found to promote collective cell migration through the increased distribution of E-cadherin, which enhanced cell-cell connections. In addition, Rac1 activation and MMP2 expression were up-regulated upon Rab11 expression. This study demonstrated that Rab11and E-cadherin expression are poor indicators of survival time in colorectal carcinoma, but that Rab11 overexpression may contribute to increased collective cell invasion in colorectal carcinoma. Citation Format: Yuan-Chiang Chung, Wan-Chen Wei, Chia-Nung Hung, Chih-Ping Hsu, Hsiang-Ling Chiu, King-Jen Chang, Wei-Ting Chao. Overexpression of Rab11 and E-cadherin promotes collective cell migration and indicates a poor prognosis in colorectal carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1544.

Abstract 3166: Rab11 regulates E-cadherin expression and induces cell transformation in colorectal carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In the process of epithelial mesenchymal transition EMT, the disassembly of junctional adhesion complexes such as E-cadherin is a remarkable sign during changes in cell morphology and polarity. However, E-cadherin expression is dynamic, and is regulated by the cellular endocytic system; it is also involved in cell signaling mechanisms. In this study, we investigated the role of E-cadherin in colorectal tumors and the relationship with recycling endosome protein Rab11 in colon cell transformation. For tissue screening, the expressions of E-cadherin and Rab11 in colorectal tumors were identified by immunohistochemistry in 113 patients with colorectal carcinoma. For the in vitro cell experiment, GFP-tagged Rab11 plasmid was transfected into HT29 colon cells, E-cadherin expression and cell transformation were monitored by Western blot and confocal microscopy. In immunohistochemistry, the mean score of E-cadherin in tumor and normal tissues was 1.41 ± 0.06 and 1.08 ± 0.06 (p < 0.05). The mean score of Rab11 in tumor and normal tissues was 0.51 ± 0.05 and 0.18 ± 0.02 (p < 0.05). Synchronous overexpression of E-cadherin and Rab11 was noted in 74 patients (66.5%) with colorectal carcinoma. When GFP-tagged Rab11 plasmid was overexpressed in cultured colon cell line HT-29, the E-cadherin expression was up-regulated, and a fibroblast-like cell polarity was induced, which resulted in cell transformation. This study demonstrated the importance of the overexpression of Rab11 and E-cadherin in colorectal cancer. The results indicated that Rab11 together with E-cadherin might be potential markers for colorectal cancer progression and treatment. Citation Format: Yuan-Chiang Chung, Wan-Chen Wei, Shin-Han Huang, Chi-Min Shih, Chih-Ping Hsu, King-Jen Chang, Wei-Ting Chao. Rab11 regulates E-cadherin expression and induces cell transformation in colorectal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3166. doi:10.1158/1538-7445.AM2014-3166