PO-063 Triterpenoids isolated from natural product regulates TNF(tumour necrosis factor)-mediated RIP(receptor-interacting serine/threonine-protein kinase)1-dependent apoptosis

Abstract

IntroductionThe ability to resist anoikis, a form of apoptosis triggered by detachment of non-malignant epithelial cells from the extracellular matrix (ECM), is thought to be critical for breast tumour invasion and metastasis. ErbB2/Her2, an oncoprotein often overproduced by breast cancer cells, blocks anoikis of breast cancer cells via mechanisms that are not well understood. Our goal is to understand these mechanisms.Material and methodsWe studied the expression of a transcription factor Irf6, an important mediator of the mammary gland homeostasis, in non-malignant breast epithelial cells MCF-10A and non-malignant primary human mammary epithelial cells (HMEC) before and after their detachment from the ECM. We also examined Irf6 expression in detached ErbB2-overproducing breast cancer cells. We used small interfering RNAs (siRNAs) to knock down Irf6 in the non-malignant cells, infected breast tumour cells with an Irf6-encoding retrovirus to overexpress Irf6 and measured detachment-induced apoptosis of the cells before and after the indicated changes in Irf6 expression.Results and discussionsWe found that detachment of MCF-10A or HMEC cells upregulates Irf6 and that Irf6 upregulation promotes their anoikis. We established that ErbB2 downregulates Irf6 in detached breast cancer cells. We also found that an anti-ErbB2 antibody trastuzumab and a small molecule ErbB2 inhibitor lapatinib used for ErbB2-positive breast cancer treatment upregulate Irf6 in ErbB2-overproducing detached human breast cancer cells but not in the isogenic variants of these cells selected for, respectively, trastuzumab and lapatinib resistance. Moreover, we demonstrated that ectopic Irf6 causes anoikis of ErbB2-overproducing breast cancer cells and blocks their anchorage-independent growth. We found that the effect of ErbB2 on Irf6 requires the activity of a protein kinase Mek, a mediator of ErbB2 signalling. We also observed that detachment-induced Irf6 upregulation in MCF10A cells is mediated by a transcription factor deltaNp63 and that deltaNp63 expression is blocked by ErbB2 or an activated Mek mutant in detached ErbB2-overexpressing breast cancer cells.ConclusionOur data indicate that deltaNp63-dependent Irf6 upregulation causes anoikis of non-malignant breast epithelial cells, and that ErbB2/Mek-driven deltaNp63 downregulation and the resulting Irf6 loss blocks this anoikis. Thus, we have identified a novel mechanism by which ErbB2, a major oncoprotein, promotes anchorage-independent growth of breast cancer cells.