Epithelial-to-Mesenchymal Transition and Resistance to Ingenol 3-Angelate, a Novel Protein Kinase C Modulator, in Colon Cancer Cells

Abstract

Acquired resistance to protein kinase C (PKC) modulators may explain the failure of clinical trials in patients with cancer. Herein, we established a human colon cancer cell line resistant to PEP005, a drug that inhibits PKCA and activates PKCD. Colo205-R cells, selected by stepwise exposure to PEP005, were >300-fold more resistant to PEP005 than parental Colo205-S cells and were cross-resistant to phorbol 12-myristate 13-acetate, bryostatin, bistratene A, and staurosporine. No PKCA or PKCD mutation was detected in Colo205-S and Colo205-R cells. Changes in Colo205-R cells were reminiscent of the epithelial-to-mesenchymal transition (EMT) phenotype. Accordingly, Colo205-R cells were more invasive than Colo205-S in Matrigel assays and in mouse xenografts. We also found an increased mRNA expression of several EMT genes, such as those encoding for transforming growth factor-B and vimentin, along with a decreased mRNA expression of genes involved in epithelial differentiation, such as CDH1 (E-cadherin), CLDN4 (claudin 4), S100A4, and MUC1, in Colo205-R compared with Colo205-S cells in vitro and in vivo. Interestingly, high expression of ET-1 was shown in Colo205-R cells and correlated with low sensitivity to PEP005 and staurosporine in a panel of 10 human cancer cell lines. Inhibition of the ET-1 receptor ETR-A with bosentan restored the antiproliferative effects of PEP005 in Colo205-R cells and decreased the invasive properties of this cell line. Exogenous exposure to ET-1 and silencing ET-1 expression using small interfering RNA modulated cell signaling in Colo205-S and Colo205-R. In summary, acquired resistance to PEP005 was associated with expression of EMT markers and activates the ET-1/ETR-A cell signaling. [Cancer Res 2009;69(10):4260-9]

Epithelial-to-Mesenchymal Transition and Oncogenic Ras Expression in Resistance to the Protein Kinase Cβ Inhibitor Enzastaurin in Colon Cancer Cells

“…Oncogenic Ras was previously reported as a key factor activating several signaling pathways triggering EMT in human cancer cells (13,(17)(18)(19). Furthermore, we and others showed that resistance to PKC modulators was associated with expression of mesenchymal transition markers (13,20,21). Therefore, we investigated the expression of several EMT markers in cancer cells sensitive and resistant to enzastaurin.…”

“…HCT116, COLO205-S, HCC2998, HOP62, HOP92, IGROV1, and MDA-MB-435 cell lines were obtained from the National Cancer Institute collection. COLO205-R cells were developed in our laboratory (13). Cells were grown as monolayers in RPMI 1640 supplemented with 10% FCS (Invitrogen), 2 mmol/L glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified 5% CO 2 atmosphere and regularly checked for the absence of Mycoplasma.…”

“…Mechanisms by which cancer cells may become resistant to drugs that target PKC are still poorly understood and may require further investigations to help improving patient selection in clinical trials (1,12). In previous experiments, we showed that resistance to PKC inhibitors was associated with changes in cellular morphology, invasion, and gene expression that were reminiscent of a mesenchymal phenotype (13,14). In this study, we further investigated the respective roles of oncogenic K-Ras and epithelialto-mesenchymal transition (EMT) in the sensitivity and resistance of cancer cells to enzastaurin.…”

Acquisition of Chemoresistance and EMT Phenotype Is Linked with Activation of the Endothelin A Receptor Pathway in Ovarian Carcinoma Cells

“…Given the ability of ET-1 to promote EMT machinery in ovarian tumor cells and the association between chemoresistance and acquisition of EMT phenotype in different tumor cells (11,(19)(20)(21), we analyzed whether the chemoresistance in A2780 and 2008 cells was associated with molecular changes consistent with EMT and whether the ET A R pathway is involved in this process. To this end we examined the expression of E-cadherin and its transcriptional repressors, Snail, Slug, and Twist, and other mesenchymal markers, such as vimentin and N-cadherin.…”

Evaluation of glioma-associated oncogene 1 expression and its correlation with the expression of sonic hedgehog, E-cadherin and S100a4 in human hepatocellular carcinoma

“…owing to the dramatic changes, cancer cells undergoing eMT easily dissociate from epithelial tissue (or primary malignancy) and migrate to other organs. The key feature in the initiation and execution of eMT is the downregulation of epithelial markers such as e-cadherin (5), and the up-regulation of mesenchymal markers such as S100a4 (6). Studies have confirmed that EMT frequently occurs in Hcc and is involved in tumorigenesis and metastasis (7)(8)(9).…”