Abstract 120: Paracrine TGF-β1 induces tamoxifen resistance in cancer-associated fibroblsts by activation of p44/42 MAPK signaling pathway

Abstract

Abstract Introduction: Biochemical crosstalk between tumor and stroma by several secretory factors has evolved as a significant contributor for the development of therapeutic resistance in breast cancer, where cancer-associated fibroblasts (CAFs) in the tumor stroma play a critical role. However, the role of secretory factors of cancer cells in assisting CAF chemoresistance is least apparent. TGF-β is the major cytokine secreted by the cancer cells to the tumor stroma that further imparts resistance in CAFs to the chemotherapy. Here, we hypothesize that tamoxifen-resistant breast cancer cells stimulate surrounding CAFs towards chemoresistant phenotype through TGF-β1 activated p44/42 MAPK signaling pathway. Methodology: The tamoxifen-resistant (TAM-R) clones of parental MCF-7 cell line were established by treating with sub-lethal doses over several cycles and characterized by MTT assay. Western blot (WB) was performed to quantify the TGF-β1 expression in the parental MCF-7 and the resistant MCF-7 (MCF-7/TAM-R) cells. ELISA was carried out to quantify TGF-β1 in the conditioned media (CM) from MCF-7 and MCF-7/TAM-R. CM from both the MCF-7 and MCF-7/TAM-R was collected and treated to the fibroblasts in dose and time-dependent manner. Initial screening for the expression of CAF markers α-SMA and caveolin-1 was carried out by qPCR and confirmed by WB. Cell cycle analysis was performed by flow cytometry and WB. Scratch assay was performed to evaluate migration and proliferation activity of CAFs. Downstream signaling pathway analysis was carried out by WB. Expression of TGF-β1 and p44/42 was downregulated by appropriate inhibitors. Results: Tamoxifen treatment to the MCF-7 cells showed a marked increase in TGF- β1 expression both in time and dose-dependent manner. α-SMA was upregulated, and Caveolin-1 was downregulated in CAFs after treatment of CM from MCF-7/ TAM-R cells. MTT result showed a higher IC50 value of TAM in CAFs treated with CM compared to untreated one. WB analysis confirmed the elevated anti-apoptotic and reduced pro-apoptotic protein expressions in CAFs post-treatment. Flow cytometry analysis depicts less number of cells in the sub G0/G1 phage after TAM treatment in CM treated CAFs compared to untreated one. WB results showed increased cyclin D1, and cyclin E in CM treated CAFs. Further, inhibiting TGF- β1 in MCF-7/TAM-R cells and p44/42 MAPK signaling cascade in CM treated CAFs found a strong correlation between the elevated expressions of TGF- β1 in MCF-7/TAM-R cells and p44/42 signaling pathway in CM treated CAFs. Conclusion: Here we report that chemoresistant breast cancer cells with increased TGF- β1 expression exhibit a greater potential in inducing TAM resistance in CAFs compared to parental cells, with an up-regulated p44/42 MAPK signaling cascade. The mechanism underscores the p44/42 MAPK signaling pathway as an important therapeutic target in tumor stroma. Note: This abstract was not presented at the meeting. Citation Format: Bikash Chandra Jena, Chandan Kanta Das, Deblina Bharadwaj, Subhayan Das, Indranil Banerjee, Santosh Gupta, Mahitosh Mandal. Paracrine TGF-β1 induces tamoxifen resistance in cancer-associated fibroblsts by activation of p44/42 MAPK signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 120.