Combinatorial targeting of FGF and ErbB receptors blocks growth and metastatic spread of breast cancer models.
Breast Cancer Res. 2013 Jan 23;15(1):R8
Authors: Issa A, Gill JW, Heideman MR, Sahin O, Wiemann S, Dey JH, Hynes NE
Abstract
ABSTRACT: INTRODUCTION: Targeting receptor tyrosine kinases (RTKs) with kinase inhibitors is a clinically validated anti-cancer approach, however, the effectiveness of individual inhibitors is often short-lived and resistance emerges. Experimental approaches have revealed numerous feed-back loops in tumor cells and that blocking one signaling pathway, be it the receptor or downstream targets, is often not sufficient to cause tumor regression. Alterations in fibroblast growth factor receptor (FGFR) activity have been implicated in breast cancer. Using breast cancer models with autocrine activation of fibroblast growth factor receptors (FGFR), we have examined the impact of targeting FGFRs in vivo with a selective kinase inhibitor in combination with an inhibitor of PI3K/mTOR or with a pan-ErbB inhibitor. METHODS: The 4T1 or 67NR mammary cancer cells are models for basal-like breast cancer and display constitutive FGFR activity. Upon their injection into fat pads of female Balb/c mice both tumor cell lines form tumors; 4T1 tumors, but not 67NR tumors metastasize to lungs. Tumor growth was measured in mice treated with an FGFR inhibitor (dovitinib/TKI258), a PI3K/ mTOR inhibitor (NVP-BEZ235) or with a pan-ErbB inhibitor (AEE788). Inhibitors were administered individually or in combination and lung metastases were quantified in the 4T1 model. To uncover mechanisms underlying inhibitor activity, tumor lysates were examined by western analyses for signaling activity of FGFR/FRS2, ErbB2, the Erk and the PI3K/Akt/mTOR pathways. Tumor sections were examined for proliferation, apoptosis and vessel density using antibodies for P-Histone H3, cleaved Caspase-3 and CD31, respectively. A transcriptome analysis was carried out on tumors treated with dovitinib for different times to identify pathways upregulated by FGFR inhibition. Anti-phosphotyrosine receptor antibody arrays (P-Tyr RTK) were used to screen in an unbiased manner for active receptors in 4T1 tumors. RESULTS: Treatment of 4T1 and 67NR tumor-bearing mice with the combination of dovitinib + NVP-BEZ235 causes tumor stasis. Western analysis of tumor lysates shows strong down-regulation of the FRS2/Erk and PI3K/Akt/mTOR signaling pathways. Examination of tumor sections revealed that the combination treatment results in a significant decrease in proliferation and high numbers of apoptotic cells, in comparison to tumors treated with individual inhibitors. Using P-Tyr RTK arrays, we identified high levels of P-EGFR and P-ErbB2 in 4T1 tumors. Testing AEE788 in the 4T1 and 67NR models revealed that only the combination of dovitinib + AEE788 resulted in blockade of the PI3K/Akt/mTOR pathway, prolonged tumor stasis and in the 4T1 model, a highly significant decrease in lung metastasis. Analyses of the tumor sections revealed that the combination of dovitinib + AEE788 caused a significant decrease in proliferation and high levels of apoptosis. The results show that in vivo these breast cancer models become dependent upon co-activation of FGFR and ErbB receptors for PI3K pathway activity. CONCLUSIONS: The work presented here shows that in the 4T1 and 67NR breast cancer models the combination of dovitinib + NVP-BEZ235 or dovitinib + AEE788 leads to a strong inhibition of tumor growth and, for the 4T1 model, a block in metastatic spread. Only these combinations strongly down-regulate the FGFR/FRS2/Erk and the PI3K/Akt/mTOR signaling pathways and cause high levels of apoptosis. Interestingly, the decrease in mitosis and increase in apoptosis was consistently stronger in the dovitinib + AEE788 treatment-group suggesting that targeting ErbB receptors has broader downstream effects compared to targeting only PI3K/mTOR. In experiments aimed at testing the durability of treatment-response, this combination was also more effective. Considering that sub-classes of human breast tumors co-express ErbB receptors and FGFRs, these results might have implications for targeted therapy.
PMID: 23343422 [PubMed - as supplied by publisher]
