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TGF-? 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-? 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-? signaling. In TNBC cell lines and mouse xenografts, the chemotherapeutic drug paclitaxel increased autocrine TGF-? signaling and IL-8 expression and enriched for CSCs, as indicated by mammosphere formation and CSC markers. The TGF-? type I receptor kinase inhibitor LY2157299, a neutralizing TGF-? 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-? signaling enhances tumor recurrence through IL-8-dependent expansion of CSCs and that TGF-? pathway inhibitors prevent the development of drug-resistant CSCs. These findings support testing a combination of TGF-? inhibitors and anticancer chemotherapy in patients with TNBC.

SUBMITTER: Bhola NE

PROVIDER: S-EPMC3582135 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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

Bhola Neil E NE Balko Justin M JM Dugger Teresa C TC Kuba María Gabriela MG Sánchez Violeta V Sanders Melinda M Stanford Jamie J Cook Rebecca S RS Arteaga Carlos L CL

The Journal of clinical investigation 20130208 3

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 bi ...[more]

PMID: 23391723

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Project description:Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored. Methods: Here, we re-purposed the TGF-β inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors. Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4. Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

Project description:TGFβ signaling plays crucial role during development and cancer, however the role for TGFβ signaling in regulating the noncoding part of the human genome in triple negative breast cancer (TNBC) is still being unraveled. Herein, we provide the transcriptional landscape of TNBC in response to TGFβ activation and subsequent inhibition employing SB431542, selective TGFβ1 Receptor ALK5 Inhibitor. Our data revealed 72 commonly upregulated [fold change (FC) ≥ 2.0], including PLAU, TPM1, TAGLN, COL1A1, TGFBI, and SNAI1, and 53 downregulated (FC ≤ 2.0) protein coding genes in BT-549 and MDA-MB-231 models in response to TGFβ1 activation. Alignment to the geocode (V33) identified 41 upregulated (FC ≥ 2.0) and 22 downregulated (FC ≤ 2.0) long non-coding RNA (lncRNA) in response to TGFβ1 activation, which were inhibited by concurrent treatment with SB431542. To place our data from the in vitro models into their clinical context, we identified AC015909.1, AC013451.1, CYP1B1-AS1, AC004862.1, LINC01824, AL138828.1, B4GALT1-AS1, AL353751.1, AC090826.3, AC104695.4, ADORA2A-AS1, PTPRG-AS1, LINC01943, AC026954.3, TPM1-AS, ZFPM2-AS1, AC007362.1, AC112721.2, MALAT1, AL513314.2, AC112721.1, AC010343.3, LINC01711, and MAP3K2-DT lncRNA expression to positively correlate with TGFβ1 expression in a cohort of 360 TNBC patients. To provide mechanistic insight into lncRNA regulation by TGFβ signaling, SMAD2/3 ChIp-Seq data from BT-549 TNBC model retrieved from Gene Expression Omnibus (GEO) revealed direct binding of SMAD2/SMAD3 to the promoter of AC112721.1, AC112721.2, MALAT1, HHIP-AS1, LINC00472, and SLC7A11, suggesting their direct regulation by TGFβ1/SMAD2/SMAD3 pathway. Interestingly, AC112721.1, AC112721.2 exhibited higher expression in TNBC compared to normal breast tissue suggesting a possible role for those lncRNA in TNBC biology. Our miRNA analysis in the BT-549 model in response to exogenous TGFB1 revealed several affected miRNAs (2.0 ≤ FC ≤ 2.0), whose expression pattern was reversed in the presence of SB431542, suggesting those miRNA as plausible targets for TGFβ regulation. In particular, we observed hsa-miR-1275 to be downregulated in response to TGFB1 which was highly predicted to regulate PCDH1, FIBCD1, FXYD7, GDNF, STC1, EDN1, ZSWIM4, FGF1, PPP1R9B, NUAK1, PALM2AKAP2, IGFL3, and SPOCK1 whose expression were upregulated in response to TGFβ1 stimulus. On the other hand, hsa-miR-181b-5p was among the top upregulated miRNAs in response to TGFB1, which is also predicted to regulate CDKN1B, TNFRSF11B, SIM1, and ARSJ in the BT-549 model. Taken together, our data is the first to provide such in depth analysis of lncRNA and miRNA epigenetic changes in response to TGFβ signaling in TNBC.

Project description:BACKGROUND:Immunotherapy with chimeric antigen receptor (CAR)-engineered T-cells is effective in some hematologic tumors. In solid tumors, however, sustained antitumor responses after CAR T-cell therapy remain to be demonstrated both in the pre-clinical and clinical setting. A perceived barrier to the efficacy of CAR T-cell therapy in solid tumors is the hostile tumor microenvironment where immunosuppressive soluble factors like transforming growth factor (TGF)-? are thought to inhibit the cellular immune response. Here, we analyzed whether CAR T-cells specific for the receptor tyrosine kinase-like orphan receptor 1 (ROR1) antigen, that is frequently expressed in triple-negative breast cancer (TNBC), are susceptible to inhibition by TGF-? and evaluated TGF-?-receptor signaling blockade as a way of neutralizing the inhibitory effect of this cytokine. METHODS:CD8+ and CD4+ ROR1-CAR T-cells were prepared from healthy donors and their antitumor function analyzed using the TNBC cell line MDA-MB-231 in vitro and in a microphysiologic 3D tumor model. Analyses were performed in co-culture assays of ROR1-CAR T-cells and MDA-MB-231 cells with addition of exogenous TGF-?. RESULTS:The data show that exposure to TGF-? engages TGF-?-receptor signaling in CD8+ and CD4+ ROR1-CAR T-cells as evidenced by phosphorylation of small mothers against decapentaplegic homolog 2. In the presence of TGF-?, the cytolytic activity, cytokine production and proliferation of ROR1-CAR T-cells in co-culture with MDA-MB-231 TNBC cells were markedly impaired, and the viability of ROR1-CAR T-cells reduced. Blockade of TGF-?-receptor signaling with the specific kinase inhibitor SD-208 was able to protect CD8+ and CD4+ ROR1-CAR T-cells from the inhibitory effect of TGF-?, and sustained their antitumor function in vitro and in the microphysiologic 3D tumor model. Combination treatment with SD-208 also led to increased viability and lower expression of PD-1 on ROR1-CAR T-cells at the end of the antitumor response. CONCLUSION:We demonstrate the TGF-? suppresses the antitumor function of ROR1-CAR T-cells against TNBC in preclinical models. Our study supports the continued preclinical development and the clinical evaluation of combination treatments that shield CAR T-cells from TGF-?, as exemplified by the TGF-?-receptor kinase inhibitor SD-208 in this study.

Project description:Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-β cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-β, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-β and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.

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TGF-? inhibition enhances chemotherapy action against triple-negative breast cancer.

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

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