PDF - 39K, The ER, PR, and HER2 (ERBB2) status was determined by standardized immunohistochemical staining and pathological analysis at Spectrum Health. ER, PR, and HER2 scores are based on IHC guidelines recommended by the ASCO/CAP.

Supplementary Methods; Figure S1: Immunoblot and immunofluorescence analysis of MET and HGF in TNBC and fibroblast cell lines. Figure S2: Met inhibition has minimal effect on ERK5 signaling in human TNBC cells. Figure S3: XL184 and U0126 significantly reduce volume and outgrowths of MDA-MB-231 structures. Figure S4: Minimal growth recovery is observed after XL184 removal Figure S5: MET inhibition in C3H-SCID mice.

There is a vital need for improved therapeutic strategies that are effective in both primary and metastatic triple-negative breast cancer (TNBC). Current treatment options for TNBC patients are restricted to chemotherapy; however tyrosine kinases are promising druggable targets due to their high expression in multiple TNBC subtypes. Since coexpression of receptor tyrosine kinases (RTKs) can promote signaling crosstalk and cell survival in the presence of kinase inhibitors, it is likely that multiple RTKs will need to be inhibited to enhance therapeutic benefit and prevent resistance. The MET and EGFR receptors are actionable targets due to their high expression in TNBC; however crosstalk between MET and EGFR has been implicated in therapeutic resistance to single agent use of MET or EGFR inhibitors in several cancer types. Therefore it is likely that dual inhibition of MET and EGFR is required to prevent crosstalk signaling and acquired resistance. In this study, we evaluated the heterogeneity of MET and EGFR expression and activation in primary and metastatic TNBC tumorgrafts and determined the efficacy of MET (MGCD265 or crizotinib) and/or EGFR (erlotinib) inhibition against TNBC progression. Here we demonstrate that combined MET and EGFR inhibition with either MGCD265 and erlotinib treatment or crizotinib and erlotinib treatment were highly effective at abrogating tumor growth and significantly decreased the variability in treatment response compared to monotherapy. These results advance our understanding of the RTK signaling architecture in TNBC and demonstrate that combined MET and EGFR inhibition may be a promising therapeutic strategy for TNBC patients.

Abstract Purpose: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is associated with poor clinical outcome. There is a vital need for effective targeted therapeutics for TNBC patients, yet treatment strategies are challenged by the significant intertumoral heterogeneity within the TNBC subtype and its surrounding microenvironment. Receptor tyrosine kinases (RTK) are highly expressed in several TNBC subtypes and are promising therapeutic targets. In this study, we targeted the MET receptor, which is highly expressed across several TNBC subtypes. Experimental Design: Using the small-molecule inhibitor cabozantinib (XL184), we examined the efficacy of MET inhibition in preclinical models that recapitulate human TNBC and its microenvironment. To analyze the dynamic interactions between TNBC cells and fibroblasts over time, we utilized a 3D model referred to as MAME (Mammary Architecture and Microenvironment Engineering) with quantitative image analysis. To investigate cabozantinib inhibition in vivo, we used a novel xenograft model that expresses human HGF and supports paracrine MET signaling. Results: XL184 treatment of MAME cultures of MDA-MB-231 and HCC70 cells (± HGF-expressing fibroblasts) was cytotoxic and significantly reduced multicellular invasive outgrowths, even in cultures with HGF-expressing fibroblasts. Treatment with XL184 had no significant effects on METneg breast cancer cell growth. In vivo assays demonstrated that cabozantinib treatment significantly inhibited TNBC growth and metastasis. Conclusions: Using preclinical TNBC models that recapitulate the breast tumor microenvironment, we demonstrate that cabozantinib inhibition is an effective therapeutic strategy in several TNBC subtypes. Clin Cancer Res; 22(4); 923–34. ©2015 AACR.

Breast cancers display a remarkable phenotypic diversity that is exploited to promote both tumor progression and therapeutic resistance. Recent studies in several types of cancer have highlighted the significance of intratumoral heterogeneity on both innate and acquired resistance to tyrosine kinase inhibitors (TKIs). Tumor plasticity is supported by the heterogeneous expression of receptor tyrosine kinases (RTKs) and the robustness that the overlapping signaling networks provide. Therefore a thorough understanding of the intratumoral heterogeneity is necessary for the development of effective therapeutic strategies. The receptor tyrosine kinase MET is overexpressed in 20-30% of breast cancers and correlates with poor patient outcome. Previously, we determined that high MET expression correlated with ER-/ERBB2- and basal like breast cancers. These results and the efficacy of MET inhibitors in other cancers suggest that MET may be an effective clinical target for aggressive breast cancer subtypes. Recent studies have exposed interactions between MET and the ERBB receptor family in the progression and therapeutic resistance of several cancers. Since MET, ERBB2, and EGFR are known to be highly expressed in aggressive breast cancer subtypes, it is critical that we understand the relationships between these receptors in order to develop effective treatment strategies. We are investigating the relationship between MET and ERBB receptor signaling in the progression and resistance of ERBB2+ and triple-negative breast cancer (TNBC). We observe that there is a large subset of ERBB2+ breast cancers that express MET and contain MET+/ERBB2+ subpopulations. In a MET+/ERBB2+ breast cancer cell line, MET depletion results in increased ERBB2 activation whereas, ERBB2 depletion results in increased MET activation. Therefore, ERBB2+ breast cancers with MET+ subpopulations may have an innate resistance to ERBB2 inhibition and may benefit from combined MET and ERBB2 inhibition. In TNBC, we observe heterogeneous expression of MET and EGFR. We have developed patient-derived xenografts (PDX) from primary and metastatic TNBCs that have diverse patterns of MET and EGFR expression/activation. In these TNBC PDX models we observe varied responses to monotherapy with MET inhibitors MGCD265 and crizotinib and the EGFR inhibitor erlotinib. Interestingly, therapeutic response to MET inhibition does not correlate with protein expression levels. In all studies, we observe significantly increased efficacy of combination therapy with MET and EGFR inhibition and a decrease in response variability. Examination of phospho-MET localization in treated tumorgrafts revealed that MET and EGFR inhibition induces distinct phospho-MET localization changes. We also observe that in the residual resistant cells phospho-MET is highly expressed in cells with mitotic bodies. We are currently performing phospho-proteomic analysis to determine the effect of MET and/or EGFR inhibition on RTK signaling networks including ERK and AKT pathways. These results will identify proteomic signatures that represent MET and/or EGFR activation/inhibition and sensitivity or resistance to monotherapy or combined MET and EGFR inhibition. Overall these studies give us a more comprehensive view of TNBC network signaling, the level of RTK heterogeneity within TNBC, and the efficacy of MET and/or EGFR inhibition on TNBC progression. Citation Format: Elizabeth A. Tovar, Erik S. Linklater, Curt J. Essenburg, Zach Madaj, David M. Cherba, Mary E. Winn, Hasan Korkaya, Julie L. Boerner, Carrie R. Graveel. Targeting the intratumoral heterogeneity of receptor tyrosine kinases in 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 B14.

Abstract 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<0.005), but also observed a decrease in tumor growth with anti-IL6R treatment (p<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.

Abstract Breast cancer displays significant intratumoral heterogeneity, which has been shown to have a substantial impact on both innate and acquired resistance to tyrosine kinase inhibitors. The heterogeneous expression of multiple receptor tyrosine kinases (RTK) in cancers supports tumor signaling robustness and plays a significant role in resistance to targeted inhibition. Recent studies have revealed interactions between the MET receptor and the ERBB receptor family in the therapeutic resistance of several cancers. In this study, the relationship between MET expression/activity and the expression/activity of the ERBB receptor family in human breast cancer was interrogated. Importantly, a significant percentage of ERBB2+ tumors coexpressing MET and ERBB2 were observed and displayed significant heterogeneity with subpopulations of cells that are MET−/ERBB2+, MET+/ERBB2−, and MET+/ERBB2+. In a MET+/ERBB2+ breast cancer cell line, MET depletion resulted in increased ERBB2 activation, and conversely, ERBB2 depletion resulted in increased MET activation. Neither EGFR nor ERBB3 compensated for MET or ERBB2 knockdown. The loss of either MET or ERBB2 led to a decrease in PI3K/AKT signaling and increased dependency on MAPK. These data show that a subset of ERBB2+ breast cancers express MET and contain MET+/ERBB2+ subpopulations. Moreover, analysis of RTK activation during ERBB2 knockdown indicated that MET signaling is a compensatory pathway of resistance. Implications: ERBB2+ breast cancers with MET+/ERBB2+ subpopulations may have an innate resistance to ERBB2 inhibition and may benefit from combined MET and ERBB2 inhibition. Mol Cancer Res; 11(9); 1112–21. ©2013 AACR.

Breast cancer displays significant intratumoral heterogeneity, which has been shown to have a substantial impact on both innate and acquired resistance to tyrosine kinase inhibitors. The heterogeneous expression of multiple receptor tyrosine kinases (RTK) in cancers supports tumor signaling robustness and plays a significant role in resistance to targeted inhibition. Recent studies have revealed interactions between the MET receptor and the ERBB receptor family in the therapeutic resistance of several cancers. In this study, the relationship between MET expression/activity and the expression/activity of the ERBB receptor family in human breast cancer was interrogated. Importantly, a significant percentage of ERBB2þ tumors coexpressing MET and ERBB2 were observed and displayed significant heterogeneity with subpopulations of cells that are MET / ERBB2þ, METþ/ERBB2 , and METþ/ERBB2þ. In a METþ/ERBB2þ breast cancer cell line, MET depletion resulted in increased ERBB2 activation, and conversely, ERBB2 depletion resulted in increased MET activation. Neither EGFR nor ERBB3 compensated for MET or ERBB2 knockdown. The loss of either MET or ERBB2 led to a decrease in PI3K/AKT signaling and increased dependency on MAPK. These data show that a subset of ERBB2þ breast cancers express MET and contain METþ/ERBB2þ subpopulations. Moreover, analysis of RTK activation during ERBB2 knockdown indicated that MET signaling is a compensatory pathway of resistance. Implications: ERBB2þ breast cancers with METþ/ERBB2þ subpopulations may have an innate resistance to ERBB2 inhibition andmay benefit from combinedMET and ERBB2 inhibition.Mol Cancer Res; 11(9); 1112–21.