αVβ3 integrin regulates heregulin (HRG)-induced cell proliferation and survival in breast cancer

Abstract

Alpha(v)beta(3) integrin-overexpression in tumor associated vasculature is a marker of poor prognosis in breast cancer. A positive correlation between alpha(v)beta(3) integrin and overexpression of Heregulin (HRG), a growth factor associated with breast cancer aggressiveness was recently demonstrated. Here, we addressed the role of alpha(v)beta(3) in the proliferation and survival of HRG-overexpressing breast cancer models. Expression of the RGD-binding integrins alpha(v)beta(3), alpha(v)beta(5) and alpha(v)beta(6) was assessed in the HRG-overexpressing breast cancer cells MDA-MB-231, Hs578T (231/WT and Hs578T/WT, respectively) and derived cells transfected with the antisense orientation of the HRG-beta2 full-length cDNA (231/ASPOOL, 231/AS31 and Hs578T/AS15). Interestingly, only alpha(v)beta(3) expression was noticeably decreased by blockade of HRG expression in the 231/ASPOOL, 231/AS31 and Hs578T/AS15 cells. Small RGD-based peptidomimetic alpha(v)beta(3) antagonists significantly decreased cell viability and anchorage-dependent cell growth of HRG-overexpressing cells, while the low-HRG-expressing 231/AS31 cells did not show a significant response. Mechanistically, functional blockade of alpha(v)beta(3) impaired HRG-promoted hyperactivation of ERK1/ERK2 MAPK without altering the activation of AKT. Flow cytometric analysis of the cell cycle demonstrated that alpha(v)beta(3) antagonists significantly decreased S- and G2/M-phase subpopulations of 231/WT and control 231/VEC cells. Comparable, this effect was linked to an increase in the levels and nuclear translocation of the CDKs inhibitor p27(Kip1). Besides downregulating alpha(v)beta(3) and its driven signaling, HRG blockade led to decreased levels of CYR61 in 231/ASPOOL and 231/AS31 cells. Considering that CYR61 is sufficient to upregulate the expression of alpha(v)beta(3), we then assessed alpha(v)beta(3) levels in MDA-MB-231 cell derivatives expressing the antisense orientation of the CYR61 cDNA (231/CYR61AS-5 and 231/CYR61AS-8). Remarkably, downregulation of CYR61 drastically decreased the levels of alpha(v)beta(3) in the 231/CYR61-5 and 231/CYR61-8 cells, providing further evidence of a key role for CYR61 in HRG-dependent alpha(v)beta(3) overexpression. Moreover, blockade of CYR61 expression impaired the HRG-induced hyperactivation of ERK1/ERK2 MAPK without altering the activation status of AKT in MDA-MB-231 cells, thus resembling the effects exerted by the downregulation of HRG expression as well as by functional blockade of alpha(v)beta(3). These results indicate that HRG is regulating alpha(v)beta(3) levels as well as alpha(v)beta(3)-triggered signaling through its downstream effector, CYR61, in highly invasive breast cancer cells. Altogether, the data presented here provide evidence of a CYR61-regulatory role on alpha(v)beta(3) integrin expression in the modulation of uncontrolled growth of HRG-overexpressing breast carcinomas. This work supports additional studies concerning the use of integrin antagonists as dual therapeutic agents in breast cancer, targeting both, endothelial and tumor cells.