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 5

Paclitaxel requires TGF-β signaling to expand CSCs.

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Paclitaxel requires TGF-β signaling to expand CSCs. (A) SUM159 and BT549...
(A) SUM159 and BT549 cells were treated with 5 μM LY2157299 and 5 nM paclitaxel (BT549 cells) or 10 nM paclitaxel (SUM159 cells) for 6 days. The proportion of ALDH+ and CD44hi/PROCR+ cells was determined by flow cytometry (SUM159: *P < 0.03; BT549: #P < 0.001; n = 3). (B) P-SMAD2 levels were assessed by immunoblot analysis of cell lysates. (C) SUM159 and BT549 cells were treated with 5 μM LY2157299 and 5 nM paclitaxel for 6 days. Cells were then seeded in either 6- or 24-well ultra-low adherent plates for mammosphere formation. Mammosphere number was calculated using the GelCount reader and software (BT549: *P < 0.002; n = 3). (D) SUM159 cells were treated with 10 nM paclitaxel for 3 days, followed by transfection with either CTL or SMAD4 siRNA. Transfected cells were seeded as mammospheres for 6 days. Mammospheres were imaged and their total number was quantitated (*P < 0.001). Original magnification, ×100. (E) SUM159 ALDH+ and ALDH– cells were flow sorted; an equal number of ALDH+ and ALDH– cells were seeded in a 3D Matrigel morphogenesis assay, as described in Methods. Three days later, cells were treated with LY2157299 (5 μM) or paclitaxel (5 nM) or both for 9 days. Fresh medium and inhibitors were added every 3 days. Original magnification, ×100. Error bars indicate SEM.