Squalamine blocks tumor-associated angiogenesis and growth of human breast cancer cells with or without HER-2/neu overexpression

Highlights

• Squalamine (SQ) is an aminosterol that blocks VEGF-induced angiogenesis.

• HER2-overexpressing tumor cells secrete high levels of VEGF.

• Dual therapy with SQ and Herceptin markedly stops HER2+ breast tumor growth in vivo.

• SQ alone also reduces progression of HER2-negative breast tumor cells in vivo.

Abstract

Angiogenesis is critical for breast cancer progression. Overexpression of HER-2/neu receptors occur in 25–30% of breast cancers, and treatment with trastuzumab inhibits HER-2-overexpressing tumor growth. Notably, HER-2-mediated signaling enhances vascular endothelial growth factor (VEGF) secretion to increase tumor-associated angiogenesis. Squalamine (aminosterol compound) suppresses VEGF-induced activation of kinases in vascular endothelial cells and inhibits tumor-associated angiogenesis. We assessed antitumor effects of squalamine either alone or with trastuzumab in nude mice bearing breast tumor xenografts without (MCF-7) or with HER2-overexpression (MCF-7/HER-2). Squalamine alone inhibited progression of MCF-7 tumors lacking HER2 overexpression, and squalamine combined with trastuzumab elicited marked inhibition of MCF-7/HER2 growth exceeding that of trastuzumab alone. MCF-7/HER-2 cells secrete higher levels of VEGF than MCF-7 cells, but squalamine elicited no growth inhibition of either MCF-7/HER-2 or MCF-7 cells in vitro. However, squalamine did stop growth of human umbilical vein endothelial cells (HUVECs) and reduced VEGF-induced endothelial tube-like formations in vitro. These effects correlated with blockade of focal adhesion kinase phosphorylation and stress fiber assembly in HUVECs. Thus, squalamine effectively inhibits growth of breast cancers with or without HER-2-overexpression, an effect due in part to blockade of tumor-associated angiogenesis.

Introduction

Despite an increase in early detection, improved surgical treatment, radiotherapy and drug therapy, breast cancer remains a major cause of mortality among women worldwide. The overexpression of HER-2/neu proto-oncogene, which encodes a 185 kDa transmembrane receptor tyrosine kinase with homology to epidermal growth factor receptor [1,2], has been found in 25–30% of human breast cancers and correlates with poor clinical outcome [[3], [4], [5], [6], [7], [8]]. Trastuzumab (Herceptin^®), a humanized monoclonal antibody specific for the extracellular domain of HER-2 receptor, has shown effectiveness as a single agent as well as in combination with chemotherapeutic agents [9,10]. HER-2 receptor-mediated signaling is also known to enhance secretion of vascular endothelial growth factor (VEGF), eliciting increased tumor-associated angiogenesis that is critical for tumor growth and progression [[11], [12], [13], [14], [15], [16], [17], [18], [19]]. Consequently, the use of antiangiogenic therapy such as bevacizumab (Avastin), a humanized monoclonal antibody that inhibits VEGF, alone and in combination with HER2-targeted therapies has been investigated [[11], [12], [13],[15], [16], [17],19]. To date, randomized clinical trials of dual therapy with bevacizumab and trastuzumab have not demonstrated an additional overall survival benefit of adding bevacizumab to trastuzumab and/or docetaxel chemotherapy despite some improvement in progression-free survival [11,12,17,19,20]. However, alternative antiangiogenic agents that have a different mechanism of action have shown significant antitumor activity in several malignancies [13,[21], [22], [23], [24], [25], [26], [27], [28]]. Thus, squalamine, an aminosterol isolated originally from dogfish shark liver, has been shown to exhibit potent antiangiogenic activity due to the selective inhibition of new blood vessel formation [25,[29], [30], [31]]. As VEGF is integral to the pathogenesis of neovascular age-related macular degeneration, early phase clinical trials of squalamine for this condition are underway [[32], [33], [34]]. Further, squalamine has also been reported to be effective in blocking tumor progression in several preclinical xenograft models, including breast [28,31,35], ovarian [24,36], lung [23,26], brain [27] and prostate [37] cancers. Additive antitumor effects have been demonstrated with squalamine in combination with chemotherapeutic agents such as cisplatin, carboplatin, cyclophosphamide and 5-fluorouracil [24,26,28]. In Phase I trials, squalamine administered IV was determined to be well-tolerated by patients and not associated with major toxicities at recommended dose levels [21]. In more advanced clinical trials, squalamine in combination with chemotherapy was also reported to be well-tolerated and demonstrated significant clinical benefit for treatment of patients with either non-small cell lung or ovarian cancers [22,23,36].

This study evaluates whether the in vivo use of either squalamine alone or combined with trastuzumab provides additional antitumor efficacy against human breast cancer cell xenografts with or without HER-2/neu-overexpression, respectively. Further, we have investigated potential molecular mechanisms by which squalamine may exert antiangiogenic effects. Our results indicate that squalamine administered alone inhibits the progression of breast tumors lacking HER2-overexpression. Furthermore, squalamine, particulalry in combination with trastuzumab, significantly suppresses the growth of HER2-overexpressing tumors in vivo, exceeding the inhibition expected with trastuzumab treatment alone. This antitumor effect of squalamine appears to be due in part to its blockade of tumor-associated angiogenesis stimulated by vascular endothelial growth factors. This action may be mediated mainly by inhibition of VEGF-induced phosphorylation of p42/p44 MAP kinase [24] and focal adhesion kinase (FAK), which, in turn, disrupts the critical assembly of stress fibers in tumor-associated vascular endothelial cells [[38], [39], [40], [41]].