Metformin attenuates transforming growth factor beta (TGF-β) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer

Abstract

Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-β (TGF-β) genes. We examined the biological, molecular and prognostic impact of TGF-β upregulation and/or inhibition using in vivo and in vitro methods. Using publically available breast cancer gene expression databases, we show that upregulation and enrichment of a TGF-β gene signature is most frequent in MSL/CL breast cancers and is associated with a worse outcome. Using several MSL/CL breast cancer cell lines, we show that TGF-β elicits significant increases in cellular proliferation, migration, invasion, and motility, whereas these effects can be abrogated by a specific inhibitor against TGF-β receptor I and the anti-diabetic agent metformin, alone or in combination. Prior reports from our lab show that TNBC is exquisitely sensitive to metformin treatment. Mechanistically, metformin blocks endogenous activation of Smad2 and Smad3 and dampens TGF-β-mediated activation of Smad2, Smad3, and ID1 both at the transcriptional and translational level. We report the use of ID1 and ID3 as clinical surrogate markers, where high expression of these TGF-β target genes was correlated to poor prognosis in claudin-low patients. Given TGF-β's role in tumorigenesis and immunomodulation, blockade of this pathway using direct kinase inhibitors or more broadly acting inhibitors may dampen or abolish pro-carcinogenic and metastatic signaling in patients with MCL/CL TNBC. Metformin therapy (with or without other agents) may be a heretofore unrecognized approach to reduce the oncogenic activities associated with TGF-β mediated oncogenesis.

Keywords: Breast cancer; TGF-β; claudin-low; mesenchymal; mesenchymal stem-like; metformin; triple negative breast cancer (TNBC).

Figure 1.

TGF-β Gene Expression Signature Upregulated in MSL/CL Subtype of TNBC. A. Heat maps showing relative gene expression of the TGF-β differentially expressed genes (P < 0.05) in each intrinsic subtype of breast cancer using UNC337 data set. Colored squares in the heat map are the relative mean transcript abundance (log₂, –3 to 3) for each subtype with highest expression in red, average expression in black, and lowest expression in green. B. Box-and-whisker plots are representative of the average expression of the TGF-β upregulated gene signatures across the intrinsic breast cancer subtypes. C. Box-and-whisker plots are representative of the average expression TGFB1 in the different breast cancer subtypes (P = 5.48e-13). D. Average probe intensity for TGFB1 in each of the defined intrinsic subtypes of breast cancer as was extrapolated from. Bar graph is representative of luminal A/B and HER2 (LumA/B/HER2+), Basal, and Mesenchymal/Mesenchymal Stem-like/Claudin-low (M/MSL) cell lines. Standard deviations between examined cell lines are identified. E. Kaplan-Meier plot for relapse free survival (RFS) and log-rank test P values. Tumors were independently ranked from low to high signature score for TGF-β expression utilizing the UNC254 tumors with survival data. The Kaplan-Meier plot and log rank test P value compares the tumors with the lowest TGF-β signature (TGF-β downregulated genes) expression relative to TGF-β-high (TGF-β upregulated genes) expression in all intrinsic breast cancer subtypes, P = 0.014. Statistics were performed using a two-tailed t-test using excel. P in box-whisker plots were calculated by comparing gene expression means across all subtypes.

Figure 2.

Prognostic Significance of ID1 and ID3 TGF-β Gene Expression in Relapse Free Survival. Box-and-whisker plots are representative of the expression ID1 gene expression (A, P = 5.89e-23), ID3 gene expression (B, P = 1.07e-20), and the average expression of ID1and ID3 genes (C, P = 2.17e-27) signatures were defined in the intrinsic breast cancer subtypes from UNC855 dataset. D. Kaplan-Meier plot for relapse free survival in claudin-low tumors with log-rank test p-values. The P value compared high ID1 and ID3 expression to low ID1 and ID3 gene expression within claudin-low tumors using the UNC855 data set (P = 0.0472).

Figure 3.

TGF-β Increases Proliferation, Signal Transduction, and Gene Expression of Downstream Target Genes in MSL/CL Cell Lines. A. SUM159PT-Nuc-GFP and BT-549 parental cells were treated with increasing concentrations of TGF-β1 (0, 0.16, 0.32, 0.64, 1.25 ng/ml) monitored for proliferation using IncuCyte Zoom. Images are representative to 72 hr, n = 12 of three independent experiments. GFP transposed to black and phase for visualization. B. Bar graph quantitation of green object count (1/mm²) of live proliferation over time at 72 hr (*P < 0.001, #P < 0.01). C. SUM159PT and BT-549 cells were treated with increasing concentrations of TGF-β1 (0, 0.16, 0.32, 0.64, 1.25, 2.5, 5, 10 ng/ml) for 4–6 hours then harvested for WB and probed with TGF-β signaling pathway proteins. D. SUM159PT cells were treated TGF-β1 (T;1.25 ng/ml) or vehicle control (C) for 4–6 hours then harvested for mRNA analysis of TGF-β gene targets (***P < 0.0001, **P < 0.001, *P = 0.01). Experiments are representative triplicate experiments.

Figure 4.

Metformin Alone or in Combination with TGF-β-KI Attenuates TGF-β-induced Proliferation and Activation of TGF-β Signaling Proteins and mRNA. A. SUM159PT-Nuc-GFP cells were treated with increasing concentrations of metformin (0–40 mM) in the presence of increasing concentrations of TGF-β-KI (LY2157299; 0, 1.25, 2.5, and 5.0 µM) and monitored for proliferation over time using IncuCyte Zoom™ for 6 days, (n = 4, *P < 0.001). Bar graph represents metformin dose-response (0–40 mM) mediated inhibition of proliferation at 72 hrs in SUM159PT cells (**P < 0.0001, *P < 0.001, #P < 0.01). B. SUM159PT cells were treated with metformin dose response (0, 0.64, 1.25, 2.5, 5, 10, 20, 40mM) for 24 h then harvested for WB analysis of TGF-β-protein targets. C. SUM159PT or BT-549 cells were treated with metformin (0, 0.64, 1.25, 2.5, 5, 10 mM) for 20 h prior to TGF-β1 (1 ng/ml) stimulation for 4 hours then harvested for WB examination of TGF-β signaling proteins. D. SUM159PT were treated with metformin (10 mM) for 20 hours prior to TGF-β1 (1ng/ml) stimulation for 4 hours then mRNA was isolated and purified for qRT-PCR examination of TGF-β-specific gene targets (n = 3, *P < 0.0001, #P < 0.001). Experiments are representative at least three experiments.

Figure 5.

Metformin blocks TGF-β-mediated Motility in MSL/CL Cells. SUM159PT expressing ZsG-Luc and BT-549 parental cells were seeded in a monolayer prior to infliction of wound then treated with metformin (10 mM), TβRI-KI (5 µM), TGF-β1 (1 ng/ml), vehicle control or defined combinations. Cells were monitored for motility indicated by relative wound closure percentage for 6 days using IncuCyte Zoom™ in SUM159PT (A), BT-549 (B) cell lines. Representative images were taken at 24 hr time point, with black time indicating t = 0. Bar graph is represents relative wound closure at 24 hr time point for each cell line treated as indicated. Experiments were done n = 8 for each assay. Each assay was repeated at least three times for each cell line. Statistics were performed using a two-tailed t-test using excel and GraphPad Prism 6® to generate comparisons in bar graphs quantitation of relative wound closure (*P < 0.001 and #P < 0.01).

Figure 6.

Metformin Retards TGF-β-induced Invasion in MSL/CL Cells. SUM159PT-Luc-ZsG (A) and BT-549-Luc-ZsG (B) cells were seeded in a monolayer prior to infliction of wound followed by filling the void with Matrigel® to mimic an invasive boundary for cells to intravasate through. Cells were treated with metformin (5 or 10 mM), TβRI-KI (5 µM), TGF-β1 (1 ng/ml), vehicle control or defined combinations. Cells were monitored for relative wound closure using IncuCyte Zoom™ for 6 days. Images are representative of 48 hr time point, with t = 0 represented as a black line. Relative scratch wound mask is indicated in blue. Bar graph quantitation of relative wound closure is shown to the right of the images for each cell line at 48 hr time point. Each experiment is representative of n = 8 and performed as three independent experiments, (*P < 0.0001, #P < 0.001). C. MSL/CL cell lines were treated with 5 mM or 10 mM metformin for TβRI-KI (5 µM) for 20 hrs prior to TGF-β1 (1 ng/ml) stimulation for 4 hrs. Cells were harvested for WB and probed for TGF-β protein targets.