TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer
Neil E. Bhola, Justin M. Balko, Teresa C. Dugger, María Gabriela Kuba, Violeta Sánchez, Melinda Sanders, Jamie Stanford, Rebecca S. Cook, Carlos L. Arteaga
Neil E. Bhola, Justin M. Balko, Teresa C. Dugger, María Gabriela Kuba, Violeta Sánchez, Melinda Sanders, Jamie Stanford, Rebecca S. Cook, Carlos L. Arteaga
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Research Article Oncology

TGF-β inhibition enhances chemotherapy action against triple-negative breast cancer

Abstract

After an initial response to chemotherapy, many patients with triple-negative breast cancer (TNBC) have recurrence of drug-resistant metastatic disease. Studies with TNBC cells suggest that chemotherapy-resistant populations of cancer stem-like cells (CSCs) with self-renewing and tumor-initiating capacities are responsible for these relapses. TGF-β has been shown to increase stem-like properties in human breast cancer cells. We analyzed RNA expression in matched pairs of primary breast cancer biopsies before and after chemotherapy. Biopsies after chemotherapy displayed increased RNA transcripts of genes associated with CSCs and TGF-β signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-β signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-β type I receptor kinase inhibitor LY2157299, a neutralizing TGF-β type II receptor antibody, and SMAD4 siRNA all blocked paclitaxel-induced IL8 transcription and CSC expansion. Moreover, treatment of TNBC xenografts with LY2157299 prevented reestablishment of tumors after paclitaxel treatment. These data suggest that chemotherapy-induced TGF-β signaling enhances tumor recurrence through IL-8–dependent expansion of CSCs and that TGF-β pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-β inhibitors and anticancer chemotherapy in patients with TNBC.

Authors

Neil E. Bhola, Justin M. Balko, Teresa C. Dugger, María Gabriela Kuba, Violeta Sánchez, Melinda Sanders, Jamie Stanford, Rebecca S. Cook, Carlos L. Arteaga

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Figure 3

TGF-β increases the CSC population in a SMAD4-dependent manner.

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TGF-β increases the CSC population in a SMAD4-dependent manner. (A) SUM1...
(A) SUM159 and BT549 cells were preincubated with 1 to 5 μM LY2157299 for 24 hours, followed by treatment with 2.5 ng/ml TGF-β1 for 2 hours in media supplemented with 0.5% FBS. Cell lysates were prepared and subjected to immunoblot analysis with total SMAD2/3 and P-SMAD2 antibodies. (B) SUM159 and BT549 cells were treated with 2.5 ng/ml TGF-β1 with or without 5 μM LY2157299 for 6 days. TGF-β1 and inhibitor were replenished every 3 days. ALDEFLUOR assay was performed in SUM159 cells as described in Methods (n = 3; *P < 0.01). BT549 cells were stained with CD44 and PROCR antibodies, followed by FACS analysis (n = 3; #P < 0.002). Error bars indicate SEM. (C) SUM159 and BT549 cells were seeded in low-adherent dishes and treated with 2.5 ng/ml TGF-β1 with or without 5 μM LY2157299 for 6 days. TGF-β and inhibitor were replenished every 3 days. Mammosphere number was calculated using a GelCount reader and software. Each bar represents the mean number ± SEM (*P < 0.02). (D) SUM159 and BT549 cells were transfected with nontargeting control (CTL) or 2 different SMAD4 siRNA oligonucleotides for 72 hours; lysates of these cells were separated by SDS-PAGE and subjected to immunoblot analysis with SMAD4 and actin antibodies. (E) Control and SMAD4 siRNA-transfected SUM159 and BT549 cells were cultured as mammospheres in the presence or absence of 2.5 ng/ml TGF-β1. Mammosphere number was calculated using a Gel Count reader and software after 6 days. Each bar represents the mammosphere number ± SEM (n = 3; *P < 0.04).