Interactions between cancer-associated fibroblasts and tumor cells promote MCL-1 dependency in estrogen receptor-positive breast cancers

Abstract

Selective inhibition of BCL-2 is expected to enhance therapeutic vulnerability in luminal estrogen receptor-positive breast cancers. We show here that the BCL-2 dependency of luminal tumor cells is nevertheless mitigated by breast cancer-associated fibroblasts (bCAFs) in a manner that defines MCL-1 as another critical therapeutic target. bCAFs favor MCL-1 expression and apoptotic resistance in luminal cancer cells in a IL-6 dependent manner while their own, robust, survival also relies on MCL-1. Studies based on ex vivo cultures of human luminal breast cancer tissues further argue that the contribution of stroma-derived signals to MCL-1 expression shapes BCL-2 dependency. Thus, MCL-1 inhibitors are beneficial for targeted apoptosis of breast tumor ecosystems, even in a subtype where MCL-1 dependency is not intrinsically driven by oncogenic pathways.

Fig. 1

bCAFs reduce BCL-2 dependency in luminal breast cancers. a Outgrowth of CAFs from partially enzymatically digested tissue of human breast tumor resections (left panel) and the resulting in vitro primo-culture (right panel) showing a homogenous fibroblastic phenotype. b Immunofluorescence of Pan Cytokeratin (green) in bCAFs and ZR-75-1 cell line, nuclei were stained in blue (4′,6-diamidino-2-phenylindole, DAPI). c Immunofluorescence of α-smooth muscle actin (α-SMA) and Fibroblast activation protein (FAP) (green) in NF and CAF (left panel) and in NHLF+ /- TGF-β (right panel), nuclei were stained in blue (4′,6-diamidino-2-phenylindole, DAPI). Images of fibroblasts (NF, CAF, left) or (NHLF+ /- TGF-β, right) contraction of collagen gels after 3 h. d-i Protective effects of media conditioned by bCAFs on ZR-75-1 cells (e, f, h, i) or T-47D cells (g). The indicated tumor cells were treated for 48 h with (e) Doxorubicin (2.5 µM)/5-Fluorouracil (27.5 µM)/Cisplatin (55 µM) or (f, g, h, i) ABT-737 (1 µM or 5 µM, as indicated) in presence of non-conditioned media (Control) or media conditioned for 48 h by normal fibroblast (NF), normal human lung fibroblast (NHLF) (pre-treated or not by TGFβ, overnight), or cancer-associated fibroblasts (CAFs) as indicated. Percentage of positive Annexin-V-FITC apoptotic cells was measured by flow cytometry in e, f, g; percentage of cytochrome C negative cells was measured by flow cytometry in h; Caspase 9 activity was measured by caspase Glo assay and expressed as fold change relative to the control (untreated w/o CAFs conditioned media) in i. Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 2

bCAFs exert their protective effects by paracrinely favoring MCL-1 expression. a Anti-apoptotic (BCL-2, MCL-1, BCL-xL) proteins expression levels in ZR-75-1 (top) or T-47D (bottom) cells, treated or not for 24 h with ABT-737 (1 µM) in presence of non-CM or CAFs-CM, were evaluated using western-blot analysis. b qRT-PCR of MCL-1 mRNA in ZR-75-1 cells grown in presence of non-CM or different CAFs CM for six and 12 h. Mean and SEM of three independent experiments are represented as relative quantity of mRNA normalized to the mean of RPLP0, B2M and GAPDH relative expression. c MCL-1 proteins expression levels in ZR-75-1 cells grown in presence of non-CM or CAFs-CM for 24 h and treated by cycloheximide for the indicated time, were evaluated using western-blot analysis. d ZR-75-1 cells (left) or T-47D cells (right) were treated for 48 h with ABT-737 1 µM and/or A-1210477 5 µM in presence of non-CM or Fibroblasts-CM. e ZR-75-1 cells infected by empty vector, MCL-1 or BCL-X_L shRNA, were treated for 48 h with ABT-737 1 µM in presence of non-CM or fibroblasts-CM. Percentage of positive Annexin-V-FITC or –APC apoptotic cells was measured by flow cytometry (d, e). Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 3

bCAFs produced IL-6 contributes to their protective effects. a ZR-75-1 cells were treated for 48 h with ABT-737 (1 µM) in presence of non-CM or CAFs-CM with or without IL-6 neutralizing antibody (anti IL-6). b ZR-75-1 or T-47D cells were treated for 48 h with ABT-737 (1 µM) in presence of non-CM, control (empty vector) or IL-6 shRNA CAFs-CM. For rescue, recombinant IL-6 (rIL-6; 500 pg/ml) was added on cancer cells within the CM (IL-6 shRNA + rIL-6) or previously during the media conditioning (IL-6 shRNA in presence of rIL-6) (as schematized in the bottom right panel). c ZR-75-1 or T-47D cells were treated for 48 h with ABT-737 1 µM in presence of non-CM, CAFs-CM (untreated), CAFs-CM co-treated with Stattic 8 µM (Stattic 8 µM) or CAFs-CM pre-incubated with Stattic 8 µM during CAFs media conditioning (Stattic pre-incubated). d Top: MCL-1, P-ERK, and ERK expression levels were evaluated by western-blot in ZR-75-1 cells treated for 24 h with ABT-737 (1 µM) alone or in combination with U0126 (5 µM) in presence of non-conditioned media or CAF-conditioned media. MCL-1, P-STAT-3 and STAT-3 expression levels were evaluated by western-blot in ZR-75-1 cells treated for 24 h with ABT-737 (1 µM) in presence of non-conditioned media or CAF-conditioned media. Bottom: ZR-75-1 cells were treated for 48 h with ABT-737 (1 µM) alone or in combination with U0126 (5 µM) or Stattic (8 µM) in presence of non-conditioned media or fibroblasts-conditioned media (NHLF, NF, CAFs). Percentage of positive Annexin-V-FITC cells was measured by flow cytometry. e In a co-culture model, ZR-75-1 cells (1/3) and CAFs (2/3) were treated with ABT-737 1 µM and U0126 5 µM alone or in combination, in DMEM 0.5% FBS for 48 h. CAFs were stained with anti CD-90 PE (bottom). Percentage of positive Annexin-V-APC apoptotic cells was measured by flow cytometry in each of the two cell populations (ZR-75 or CAF) (top). Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 4

MCL-1 is highly expressed by bCAFs. CAFs (CAF42, CAF46, CAF65) were treated for 48 h with U0126 (5 µM) or Stattic (8 µM) alone or in combination with ABT-737 (1 µM). Percentage of positive Annexin-V-FITC apoptotic cells was measured by flow cytometry. b Anti-apoptotic (BCL-xL, BCL-2, MCL-1) and pro-apoptotic (BAX, BAK) proteins expression levels in normal human lung fibroblast (NHFL) and primary culture of breast cancer-associated fibroblasts (CAF) were evaluated using western blots analysis. c MCL-1, P-ERK, and ERK expression levels were evaluated by western-blot in CAFs (CAF42, CAF46) treated for 24 h with Stattic (8 µM) or U0126 (5 µM). d–f MCL-1 expression in luminal-B breast cancer fibroblasts. d Proportion of MCL-1–positive cancer-associated fibroblasts in 20 luminal breast cancers. e Chromogenic detection (DAB) of Mcl-1 by immunohistochemical analysis of luminal breast cancers, representative cases. MCL-1–positive fibroblasts (arrows) with extended cytoplasm and hypochromatic nuclei, morphology of activated fibroblasts are shown on the left (left). Fibroblasts with undetectable MCL-1 expression (arrowheads) with smaller size and hyperchromatic nuclei, morphology of fibrocytes are shown on the right. Original magnification ×1000, bar = 20 micrometers. f MCL-1 (green) and a-SMA (red) fluorescent co-staining. Arrows and arrowheads indicate high and low MCL-1 expression in α-SMA positive cells respectively. Left: Green and Blue overlay, right, Green blue and red overlay

Fig. 5

bCAFs rely on MCL-1 for their survival. a BH3 profiling. Values indicate the percentage of cytochrome c loss and are representative of one experiment. b NHLF, NF, and CAFs cells were treated with A-1210477 (2.5 or 5 µM) or ABT-737 (1, 5, or 10 µM) for 48 h in DMEM containing 0.5% FBS, apoptosis was measured by Annexin-V flow cytometry. c Top: NHLF pre-treated or not by TGFβ for 24 h were treated with 10 µM ABT-737 or 5 µM A-1210477 for 48 h in DMEM containing 0.5% FBS, apoptosis was measured by Annexin-V flow cytometry. Bottom: MCL-1 proteins expression levels in NHLF + /-TGF-β cells treated by cycloheximide for the indicated time, were evaluated using western-blot analysis. d CAFs were treated with 5 µM A-1210477 + /- ABT-737 1 µM, Wehi-539 1 µM or ABT-199 1 µM for 48 h in DMEM containing 0.5% FBS, apoptosis was measured by Annexin-V flow cytometry. e Percentage of apoptotic cells estimated in ABT-737-treated CAFs cells previously infected with the control vector or the MCL-1 sh-RNA for 72 h. MCL-1 protein expression in CAFs cells infected with the control vector or the MCL-1 sh-RNA was evaluated by western blotting (insert). f Co-culture experiments. ZR-75-1 cells (1/3) and CAFs (2/3) were treated for 48 h with ABT-737 1 µM and A-1210477 5 µM alone or in combination in DMEM 0.5% FBS. MCL-1 protein expression levels in ZR-75-1 cells isolated by fluorescence-activated cell sorting after their co-culture with CAF is shown on the left. Representative FACS profiles of cell death analyzes (using Annexin V-APC as a marker) in co-culture of ZR-75-1 cells and CAFs (marked by anti CD-90 PE) are shown in the middle. Percentage of apoptotic (Annexin-V–APC positive) tumor cells (CD9O negative) and CAFs (CD 90 positive) grown alone or in co-culture and treated with the indicated combination of BH3 mimetic, measured by flow cytometry are shown on the right. Data are means ± SEM from three independent experiments. P-value was determined by two-way ANOVA. *P < 0.05, **P < 0.01,, ****P < 0.0001

Fig. 6

Co-variations between stromal score and MCL-1 expression characterize luminal breast cancers resistant to BCL-2 inhibition. a 169 ER⁺ primary human tumor samples were cultured 48 h with 1 μM ABT-737 or left untreated and then analyzed for active caspase-3 by immunohistochemistry. Left: data are represented as percentage of tumoral cells positive for active caspase-3 immunostaining in each treated and corresponding untreated specimen. Middle: percentage of tumoral cells positive for active caspase-3 immunostaining induced by ABT-737 above control (ABT-737 treated- untreated slice) is represented for all samples. Right: percentage of tumoral cells positive for active caspase-3 immunostaining induced by ABT-737 above control (ABT-737 treated- untreated slice) depending on the percentage of KI67 labelling as indicated. P-value were calculated using Wilcoxon test. b Left and Right: Data from the 71 samples used for molecular analysis are shown as in Fig. 5a Left and Right respectively. c Stromal score (z-score, Left) and MCL1 expression (z-score, Right) in “resistant” and “sensitive” groups (see Text for further details). P-value was calculated using Wilcoxon test. d Left: Plot of the 71 affymetrix samples with respect to their MCL1 mRNA expression and Stromal score (z-score) to which was added the TCGA regression line inferred from data used in Table 1. “Resistant” and “sensitive” groups are colored in gray and blue respectively. Right: Differences between MCL1 expression expected from the TCGA regression line and MCL1 expression samples from each group are presented as boxplots