Microenvironmental control of breast cancer subtype elicited through paracrine platelet-derived growth factor-CC signaling

Abstract

Breast tumors of the basal-like, hormone receptor-negative subtype remain an unmet clinical challenge, as there is high rate of recurrence and poor survival in patients following treatment. Coevolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to support most, if not all, hallmarks of cancer progression. Here we delineate a previously unappreciated role for CAFs as determinants of the molecular subtype of breast cancer. We identified paracrine crosstalk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and CAFs expressing the cognate receptors in human basal-like mammary carcinomas. Genetic or pharmacological intervention of PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that specification of breast cancer to the basal-like subtype is under microenvironmental control and is therapeutically actionable.

Conflict of interest statement

Competing Financial Interest

KP, UE and PR are named inventors on PCT application# PCT/EP2016/077295 relating to the findings of the current study. KP, UE and AMS are shareholders of Paracrine Therapeutics that develop inhibitory agents to PDGF-CC.

Figures

Figure 1. Epithelial expression of PDGF-CC is associated with poor outcome in human breast carcinoma.

a-d, Immunostaining of normal breast tissue (a-b) or breast carcinoma (c-d) for PDGF-CC. Appropriate validation on >10 independent samples was performed to ensure reproducible staining pattern on human tissue. Dotted line represents the epithelium-stroma boundary and arrows identify tumor cells with particularly high expression of PDGF-CC on the invasive border. Scale bar, 40 µm. e, Kaplan-Meier analysis of breast cancer-specific survival dichotomized according to absence (negative, n=438 samples) or presence (positive, defined as a score of 1+ to 3+, n=452 samples) of PDGF-CC in tumor cells (for scoring scheme, see Supplementary Fig. 1a) of breast carcinomas from the Zürich cohort. ** p=0.002, log-rank test f, Average volume (with SEM) of tumors from 14 weeks old MMTV-PyMT mice (n=10 animals in each group). *** p=0.0001, two-sided, unpaired, equal variance Student’s t-test. g, Grading of tumors from 14 weeks old MMTV-PyMT mice (n=5 animals in each group). * Distribution of Pdgfc-/- vs Pdgfc+/- p=0.036, χ2 test. *** Distribution of Pdgfc-/- vs Pdgfc+/+ p=3*10-11 and distribution of Pdgfc+/- vs Pdgfc+/+ p=0.00052, χ2 test. h-k, Hematoxylin and eosin staining of tumors from MMTV-PyMT mice at 14 weeks of age. Scale bar, 500 µm. l, Average volume (with SEM) of tumors derived from MMTV-PyMT mice transplanted into the mammary fat pad of wt mice (n=21 animals in each group, composed of n=7 animals each from 3 independent experiments). *** p=6*10-6, two-sided, unpaired, equal variance Student’s t-test. m-n, Masson’s tri-chrome staining of tissue sections from tumors from 14 weeks old MMTV-PyMT mice. Representative pictures from the analysis of n=10 animals from each group are shown. Scale bar, 500 µm. o, Quantitative real-time PCR analysis of the average expression (with SEM) of markers for cancer-associated fibroblasts in tumor lysates of tumors from 14 weeks old MMTV-PyMT mice (n=3 animals in each group, analysis performed independently 3 times). ** Pdgfra, p=0.007; S100a4, p=0.003; Acta2, p=0.009 two-sided, unpaired, equal variance Student’s t-test.

Figure 2. Expression of PDGF-CC in breast carcinomas is associated with the basal-like molecular subtype and hormone receptor negativity.

a, Quantitative RT-PCR analysis of the luminal subtype marker Foxa1 in tumors from 14 weeks old MMTV-PyMT mice (n=5 animals in each group, analysis performed independently 3 times). *** p<0.001, two-sided, unpaired, equal variance Student’s t-test. b-c, Expression of FOXA1 (b) and PDGFC (c) in a panel of 51 breast cancer cell lines divided according to basal-like or luminal-like molecular subtype. d, Correlation between expression of FOXA1 and PDGFC in a panel of 51 breast cancer cell lines divided according to basal-like or luminal-like molecular subtype. Correlation coefficient and p-value from Pearson correlation analysis of n=26 basal cell lines and n=25 luminal cell lines. e, Expression of all PDGF family members in n=26 basal cell lines and n=25 luminal cell lines . Box represents the interquartile range, line represents the average expression and whiskers depict range of expression with statistical outliers indicated (>2 standard deviations from the mean). *** p=1*10-6, two-sided, unpaired Welch’s unequal variances t-test. f-k, Enrichment of luminal-defining transcription factors ESR1, FOXA1, and GATA3 binding sites, as derived from public datasets of ChIP-seq analysis in MCF7 cells, in the 1000 most up/down-regulated genes in Pdgfc-deficient PeRo-Lum1 cells compared to Pdgfc-proficient PeRo-Bas1 cells. Red, query signature; blue, n=105 randomly sampled gene lists of equal size to the query signature; green, average of the randomly sampled gene lists. The rank of the query gene list divided by the total number of re-sample instances was then used as a p-value for the probability to acquire the level of enrichment by chance. l-n, Correlation between expression of ERα and PDGF-CC in primary breast carcinomas (l, n=470 independent samples, *** p=2.1*10-14 independent samples, two-sided Jonckheere-Terpstra test for ordered alternatives), synchronous lymph nodes (m, n=132 independent samples, *** p=1.5*10-8 independent samples, two-sided Jonckheere-Terpstra test for ordered alternatives) or asynchronous distant replapses (n, n=29 independent samples, ** p=0.0015 independent samples, two-sided Jonckheere-Terpstra test for ordered alternatives) included in the Lund cohort. Box represents the interquartile range, line represents the median expression and whiskers depict 1.5x the height of the box. Circles and stars represent statistical outliers that fall outside of the whiskers and extreme outliers that fall outside of 3x the height of the box, respectively.

Figure 3. CAF-derived factors induced by PDGF-CC reduce the sensitivity of breast tumor cells to endocrine therapy.

a, Viability of PeRo-Lum1 luminal breast cancer cells derived from MMTV-PyMT; Pdgfc-/- mice in the presence of increasing concentrations of 4-OH-tamoxifen in medium conditioned by tumor cells (TC) or CAFs (the average (with SEM) of n=6 independent experiments is shown). ** p<0.01, *** p<0.001, (p-values for control vs STC1+HGF+IGFBP3 for each tamoxifen concentration (1-5 µM=0.0001, 0.00001, 0.00001, 0.00001, 0.005, respectively), two-way ANOVA with p-values from Bonferroni post-hoc test. b, Viability of PeRo-Lum1 cells in the presence of increasing concentrations of 4-OH-tamoxifen in medium conditioned by nothing or STC1, HGF and IGFBP3 (the average (with SEM) of n=6 independent experiments is shown). ** p<0.01, *** p<0.001 (p-values for control vs STC1+HGF+IGFBP3 for each tamoxifen concentration (1-5 µM=0.003, 0.0003, 0.00008, 0.0002, 0.005, respectively), two-way ANOVA with p-values from Bonferroni post-hoc test. c-e, Quantitative RT-PCR analysis of the average expression (with SEM) of Foxa1 (c, n=3 independent experiments), Esr1 (d, n=3 independent experiments) or Gata3 (e, n=2 independent experiments) in PeRo-Lum1 cells in the presence of combinations of STC1, HGF and IGFBP3. Foxa1 * p=0.03, ** p=0.002, *** p=0.0003, one-way ANOVA with p-values from Bonferroni post-hoc test. Esr1 * p=0.01, ** p=0.005, *** p=0.001, one-way ANOVA with p-values from Bonferroni post-hoc test. Gata3 * p=0.02, ** p=0.007, *** p=0.001, one-way ANOVA with p-values from Bonferroni post-hoc test. f-h, Immunostaining of tissue sections from tumors from 14 weeks old MMTV-PyMT mice for STC1 (f), HGF (g) or IGFBP3 (h). Immunostaining was performed n=3 independent times and representative images from n=5 tumors are shown. Scale bar, 100 µm. i, Pearson’s correlation analysis of factors denoting luminal-like (FOXA1, ESR1, GATA3) or basal-like (FOXC1, PDGFC) molecular subtype in luminal subtype breast carcinomas included in the TCGA cohort (n=1086 independent tumors included in analysis).

Figure 4. Genetic or pharmacological targeting of PDGF-CC induces expression of ERα and sensitizes tumors to endocrine therapy.

a-b, Treatment of wt mice transplanted orthotopically with tumors derived from 14 weeks old MMTV-PyMT; Pdgfc+/+ mice (a) or MMTV-PyMT; Pdgfc-/- mice (b) with oil vehicle (Pdgfc+/+ n=7 animals, Pdgfc-/- n=6 animals) or tamoxifen (Pdgfc-+/+ n=6 animals, Pdgfc-/- n=7 animals). Average tumor volume (with SEM) depicted. ** p=0.002, two-sided, unpaired, equal variance Student’s t-test. X-axis shows time after start of therapy. c-d, Immunostaining for ERα (brown) of tissue sections from the triple-negative patient-derived xenograft 12.58 from mice treated with control IgG or 6B3. Immunostaining performed n=3 independent times and representative images are shown. Scale bar, 40 µm. e, Quantification of the average ERα expression (with SEM) in patient-derived xenograft 12.58 from mice treated with control IgG or 6B3 (IgG, n=7 animals; 6B3, n=8 animals). *** p=7*10-7, χ2 test. f, Quantification of the average number of ERα-expressing cells (with SEM) in MDA-MB-231 tumors from mice treated with control IgG or 6B3 in combination with oil vehicle (IgG, n=10 animals; 6B3, n=12 animals) or tamoxifen (IgG, n=12 animals; 6B3, n=12 animals). ** p=0.003, two-sided, unpaired, equal variance Student’s t-test. g, Treatment of mice carrying orthotopically transplanted triple-negative patient-derived xenograft 14.32 with control IgG in combination with vehicle (n=10 animals) or tamoxifen (n=10 animals). Average tumor volume (with SEM) depicted. X-axis shows time after start of therapy. h, Treatment of mice carrying orthotopically transplanted MDA-MB-231 tumors with control IgG in combination with oil vehicle (n=10 animals) or tamoxifen (n=12 animals). Average tumor volume (with SEM) depicted. X-axis shows time after start of therapy. i, Treatment of mice carrying orthotopically transplanted triple-negative patient-derived xenograft 14.32 with the neutralizing PDGF-CC antibody 6B3 in combination with vehicle (n=10 animals) or tamoxifen (n=10 animals). Average tumor volume (with SEM) depicted. * p=0.03, two-sided, unpaired, equal variance Student’s t-test. X-axis shows time after start of therapy. j, Treatment of mice carrying orthotopically transplanted MDA-MB-231 tumors with the neutralizing PDGF-CC antibody 6B3 in combination with oil vehicle (n=12 animals) or tamoxifen (n=13 animals). Average tumor volume (with SEM) depicted. * p=0.025, two-sided, unpaired, equal variance Student’s t-test. X-axis shows time after start of therapy. k, Treatment of mice carrying orthotopically transplanted MCF7 tumors infected with lentivirus carrying empty vector or Pdgfc vector with oil vehicle (Vector, n=9 animals; Pdgfc, n=10 animals) or tamoxifen (Vector, n=10 animals, Pdgfc, n=8 animals). Average tumor volume (with SEM) depicted. * Vector + Tamoxifen vs Pdgfc + Tamoxifen, p=0.029, two-sided, unpaired, equal variance, Student’s t-test. X-axis shows time after start of therapy. l, Schematic diagram of the paracrine action of PDGF-CC in the breast tumor microenvironment. An active crosstalk between malignant cells and CAFs mediated through PDGF-CC results in specification of the molecular subtype and regulation of sensitivity to endocrine therapy.