Abstract B19: MET and IL6 signaling in triple-negative breast cancer
Authors: Tovar Elizabeth A. , Sameni Mansoureh , Essenburg Curt J. , Chalasani Anita , Linklater Erik S. , Cherba David M. , Anbalagan Aruselvi , Winn Mary E. , Sloane Bonnie F. , Graveel Carrie R. 
Published: May 2016

Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancers and is associated with advanced stage at diagnosis and poorer outcome compared to other breast cancer subtypes. There is an unmet need for targeted therapeutic strategies for TNBC patients since current treatment options are restricted to standard chemotherapy. Both receptor tyrosine kinase (RTK) and inflammatory signaling have been shown to promote cancer progression and are promising therapeutic targets. Our laboratory was the first to demonstrate that the MET receptor tyrosine kinase is highly expressed in TNBC. Hepatocyte growth factor (HGF), the MET ligand, is highly expressed in breast carcinoma and breast carcinoma-associated fibroblasts (CAFs) and is able to induce paracrine or autocrine MET signaling. MET/HGF signaling is also connected with the pro-inflammatory cytokine interleukin 6 (IL6). HGF and IL6 have been shown to interact to enhance invasion of lung cancer cells and progression of multiple myeloma. In breast cancer patients, high serum expression of HGF and IL6 distinguishes metastatic breast cancers. Nonetheless, there is a gap in knowledge as to whether MET and IL6 signaling pathways directly or indirectly interact and how MET/IL6 activation promotes TNBC progression.

We are examining the novel concept that MET and IL6 signaling pathways act through a positive signaling feedback loop to drive TNBC progression. By understanding the interactions between these signaling networks, we will be able to design therapeutic strategies that target critical signaling nodes in TNBC. Analysis of gene expression profiles in the four molecular TNBC subtypes defined by Burstein et al. revealed that MET, HGF, and IL6 are expressed in each of the TNBC subtypes. Immunohistochemical analysis of HGF and IL6 expression in breast cancer tissues revealed significantly higher HGF and IL6 expression in TNBC compared to ER+ breast cancers. To determine the effect of MET and IL6 inhibition, we established Mammary Architecture and Microenvironment Engineering (MAME) 3D co-culture models of TNBC cells ± fibroblasts. In these models, TNBC cells have high MET expression, moderate to high IL6 expression, and minimal IL6 receptor (IL6R) expression; whereas the CAF cells have high HGF expression and moderate IL6R expression. Our preliminary studies revealed that an IL6 neutralizing antibody (siltuximab) reduced TNBC structure volumes relative to IL6 expression in the TNBC cells, whereas an IL6 receptor (IL6R) neutralizing antibody (tocilizumab) had no effect. These results correlate with IL6 and IL6R expression levels in TNBC cell lines. We evaluated the efficacy of MET inhibition using XL184 (cabozantinib) and observed that XL184 significantly inhibited TNBC growth, proliferation, and invasion of diverse TNBC cell lines, yet was ineffective against MET-negative breast cancer cells. We are currently evaluating the effect of HGF-mediated MET activation on IL6 signaling and inhibition in our 3D TNBC models.

To evaluate the effect of MET and/or IL6 inhibition in vivo we utilized a novel xenograft mouse model that expresses human HGF (hHGFtg SCID). In TNBC cell lines MDA-MB-231 and HCC70, IL6R inhibition slowed tumor progression marginally, whereas MET inhibition with XL184 and the combination of XL184 + anti-IL6R drastically inhibited tumor growth. In a model of established tumor growth, we started treatment when tumor reached 500 mm3. Again we observed a significant decrease in tumor growth with XL184 treatment (p&lt;0.005), but also observed a decrease in tumor growth with anti-IL6R treatment (p&lt;0.03). In our current studies we are evaluating the effects of XL184 and siltuximab treatment in TNBC + CAF tumors. These studies will identify the RTK and inflammatory signaling networks that are critical for TNBC progression and are potential targets for combination therapy.

Citation Format: Elizabeth A. Tovar, Mansoureh Sameni, Curt J. Essenburg, Anita Chalasani, Erik S. Linklater, David M. Cherba, Aruselvi Anbalagan, Mary E. Winn, Bonnie F. Sloane, Carrie R. Graveel. MET and IL6 signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B19.