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. 2013 Mar;123(3):1348-58.
doi: 10.1172/JCI65416. Epub 2013 Feb 8.

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

Neil E Bhola  1 , Justin M Balko, Teresa C Dugger, María Gabriela Kuba, Violeta Sánchez, Melinda Sanders, Jamie Stanford, Rebecca S Cook, Carlos L Arteaga
Affiliations

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

Neil E Bhola et al. J Clin Invest. 2013 Mar.

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.

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Figures

Figure 1
Figure 1. Chemotherapy-treated breast cancers display increased markers of TGF-β signaling and CSCs.
(A) NanoString analysis of TGF-β pathway genes in RNA extracted from breast cancer biopsies before chemotherapy (Pre-Tx) and after chemotherapy (Post-Tx). A TGF-β gene expression score was generated in both groups, as described in Methods (n = 17; paired Student’s t test, P = 0.002). (B) Box plots of TGFBR2, TGFBR3, and SMAD4 gene expression between biopsies before and after chemotherapy (TGFBR2: P = 0.007, TGFBR3: P = 0.0026, SMAD4: P = 0.012). Symbols indicate individual tumors; horizontal bars represent the mean. (C) Box plots of gene expression of CSC genes CD44 and ALDH1A1 (CD44: P = 0.013; ALDH1A1: P = 0.0067). (D) Box plots of FOS, JUNB, JUN, CDKN1A gene expression in the 17 paired tumors (paired Student’s t test, P < 0.0008 for all 4 comparisons). (B–D) In box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate 10th and 90th percentiles. (E) TGFB1 and TGFBR2 mRNA from SUM159 cells treated with or without 10 nM paclitaxel for 6 days was measured by quantitative RT-PCR (*P = 0.03). Using the TGF-β–specific PCR array described in Methods, the expression of JUNB, JUN, FOS, and CDKN1A were assessed and quantified in both control (C) and paclitaxel-treated (P) cells. Error bars indicate SEM.
Figure 2
Figure 2. Paclitaxel enriches a CSC population with increased TGF-β signaling.
(A and B) SUM159 and BT549 cells were treated with vehicle (control) or 10 nM paclitaxel for 4 days and allowed to recover in fresh media for another 4 days. Cells were trypsinized and analyzed by FACS for (A) ALDH activity and CD44hi/PROCR+ expression (*P < 0.01) or (B) assessed for their ability to form mammospheres (*P = 0.04). (C) SUM159 xenografts were treated with vehicle or 10 and 20 mg/kg/d paclitaxel for 5 consecutive days (treatment started at day 1 and ended at day 6 of x axis). Tumor diameters were measured every 3 days using calipers, and volume in mm3 was calculated as described in Methods. Xenografts were harvested on day 15, dissociated into single cells, and grown as mammospheres. Each bar represents the mean mammosphere number ± SEM (n = 3; *P < 0.05). Original magnification, ×100. (D) Whole lysates from the same SUM159 xenografts as in C were subjected to P-SMAD2, total SMAD2/3, and actin (control) immunoblot analysis. exp, exposure. (E) SUM159 and BT549 cells were treated with vehicle or 5 nM or 10 nM paclitaxel for 4 days and seeded in 24-well plates. Cells were transfected with pCAGA-Luc and Renilla plasmids; luciferase activity was determined 24 hours later using the Dual Luciferase Kit, as described in Methods (n = 3; *P < 0.05). Error bars indicate SEM.
Figure 3
Figure 3. TGF-β increases the CSC population in a SMAD4-dependent manner.
(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).
Figure 4
Figure 4. Paclitaxel and TGF-β induce SMAD4-dependent expression of IL-8.
(A) SUM159 and BT549 cells were treated with 5 to 10 nM paclitaxel for 4 days. RT-qPCR analysis was performed to assess IL8 and GAPDH RNA levels (*P < 0.05, **P < 0.003). (B) SUM159 cells were treated with 2.5 ng/ml TGF-β with or without 5 μM LY2157299 and grown as mammospheres. Media was collected, and IL-8 protein levels were measured by ELISA; IL-8 levels were normalized to total protein (*P = 0.02). (C) SUM159 cells were transfected with control or SMAD4 siRNA and plated as mammospheres with or without TGF-β1. After 6 days, mammospheres and media were collected and analyzed for IL-8 levels by ELISA. IL-8 levels were normalized to total protein (*P < 0.001). (D) SUM159 cells were transfected with control or both CXCR1 and CXCR2 siRNA and plated as mammospheres with or without 2.5 ng/ml TGF-β1 for 6 days. Mammosphere number was then quantitated, as described in Methods (*P = 0.016). (E) SUM159 cells were transfected with control or SMAD4 siRNA. Forty-eight hours later, 10 nM paclitaxel was added for 24 hours before mRNA extraction and RT-qPCR using IL-8–specific primers (*P < 0.002, **P = 0.002). (F) RT-qPCR analysis of IL8 mRNA levels in SUM159 and BT549 cells treated with 5 μM LY2157299 and 5 nM paclitaxel (BT549 cells) or 10 nM paclitaxel (SUM159 cells) as indicated for 6 days (*P < 0.007, **P < 0.001). (G) Media from cells treated with paclitaxel with or without LY2157299 was collected and subjected to IL-8 ELISA assay. Raw IL-8 levels were normalized to cell number (*P < 0.001). Error bars indicate SEM.
Figure 5
Figure 5. Paclitaxel requires TGF-β signaling to expand CSCs.
(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.
Figure 6
Figure 6. TGF-β inhibition in vivo abrogates tumor-initiating potential after chemotherapy.
(A–C) Female athymic mice were injected with SUM159 cells in the number 4 mammary fat pad. (A) Once tumors reached a volume of ≥75 mm3, mice were randomized to 4 treatment groups (n = 10 mice per group): (a) vehicle (control), (b) LY2157299 (100 mg/kg/p.o. twice daily), (c) paclitaxel (20 mg/kg/d i.p. 5 times), and (d) both drugs. The arrow at day 5 represents the cessation of paclitaxel treatment. The arrow and star at day 15 indicate the cessation of LY2157299 treatment and the procurement of xenografts, respectively. The arrow at day 23 represents the cessation of monitoring for tumor growth. Lysates from xenografts harvested after (B) 5 days and (C) 14 days were assessed for total SMAD2/3 and P-SMAD2 levels by immunoblot analysis. (D) Tumor volumes calculated on day 14 (*P = 0.035; Mann-Whitney). Symbols indicate individual tumors; horizontal bars represent the mean. (E) Single cell suspensions derived from xenografts harvested on day 14 were analyzed for ALDH activity using the ALDEFLUOR assay (*P = 0.002). (F) Serum was collected from mice at the time of tumor harvesting and sacrifice and analyzed for IL-8 protein levels by ELISA. Each bar represents mean IL-8 levels over control ± SEM (n = 3). ns, not significant. (G) Progression of tumor volumes during days 14–23 in mice treated with paclitaxel with or without LY2157299 during days 1–14 (n = 5). Tumors treated with vehicle (controls) or LY2157299 alone are not included, as they had reached a volume of ≥1 cm3 on day 14 (start of x axis). Because of the large tumor burden, these mice had to be sacrificed as per institutional guidelines. Error bars indicate SEM.
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