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

¹Institut National de la Sante et de la Recherche Medicale U728, RayLab, and Departments of Medical Oncology, Beaujon and Saint-Louis University Hospital (AP-HP-Paris 7 Diderot); ²Laboratory of Molecular Genetics, Beaujon University Hospital-Paris 7 Diderot, Clichy, France and ³Department of Clinical Pharmacology, René Huguenin Cancer Center, Saint-Cloud, France

^* To whom correspondence should be addressed. E-mail: eric.raymond{at}bjn.aphp.fr.

	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 PKC and activates PKC. 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 PKC or PKC 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- 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]

Key Words: Endothelin 1, ET1, ETR-A, invasion, adhesion, staurosporine, PMA, bryostatin, bistratene A, bosentan