1. ¹Department of Medicine, Breast Cancer Translational Research Laboratory, Evanston Northwestern Healthcare Research Institute, 1001 University place, Evanston, IL 60201, USA
2. ²Northwestern University Feinberg School of Medicine, Chicago, IL, USA

Correspondence: R Lupu, E-mail: [email protected]

Received 14 September 2004; Revised 08 December 2004; Accepted 08 December 2004; Published online 14 March 2005.

Abstract

_v3 integrin-overexpression in tumor associated vasculature is a marker of poor prognosis in breast cancer. A positive correlation between _v3 integrin and overexpression of Heregulin (HRG), a growth factor associated with breast cancer aggressiveness was recently demonstrated. Here, we addressed the role of _v3 in the proliferation and survival of HRG-overexpressing breast cancer models. Expression of the RGD-binding integrins _v3, _v5 and _v6 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-2 full-length cDNA (231/ASPOOL, 231/AS31 and Hs578T/AS15). Interestingly, only _v3 expression was noticeably decreased by blockade of HRG expression in the 231/ASPOOL, 231/AS31 and Hs578T/AS15 cells. Small RGD-based peptidomimetic _v3 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 _v3 impaired HRG-promoted hyperactivation of ERK1/ERK2 MAPK without altering the activation of AKT. Flow cytometric analysis of the cell cycle demonstrated that _v3 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 _v3 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 _v3, we then assessed _v3 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 _v3 in the 231/CYR61-5 and 231/CYR61-8 cells, providing further evidence of a key role for CYR61 in HRG-dependent _v3 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 _v3. These results indicate that HRG is regulating _v3 levels as well as _v3-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 _v3 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.