PDGFRβ is an essential therapeutic target for BRCA1-deficient mammary tumors

Brca1^f/f and Tg (MMTV-Cre) 4Mam mice were obtained from the NCI Mouse Repository and JAX lab, respectively [46, 47]. The generation of p16^−/−, p18^−/−, Brca1^+/−, Brca1^MGKO (Brca1^f/f;MMTV-Cre or Brca1^f/−;MMTV-Cre) mice was described previously [31, 48‐50]. The Institutional Animal Care and Use Committee at the University of Miami and Shenzhen University approved all animal procedures. Histopathology and immunohistochemistry (IHC) were performed as described previously [11, 28, 31]. The primary antibodies used were Ck14, cleaved caspase 3 (Thermal Scientific), PDGFRβ, phosphorylated PKCα (p-PKCα), phosphorylated FRA1 (p-FRA1), FRA1 (Cell signaling), ERα, BRCA1 (Santa Cruz), E-cadherin (E-cad), PKCα (BD Biosciences), and Vimentin (Vim) (Abcam). Immunocomplexes were detected by using the Vectastain ABC DAB kit according to the manufacturer’s instructions (Vector Laboratories) or by using FITC- or rhodamine-conjugated secondary antibodies (Jackson Immunoresearch).

Mammary tumors were dissected and tumor cell suspensions were prepared as previously described [11, 28, 31]. For the primary and secondary tumorsphere formation assays, primary p18^−/−;Brca1^MGKO mammary tumor cells were plated onto six-well, ultra-low attachment plates, in serum-free DMEM-F12 supplemented with B27, EGF, and bFGF as described [28, 31]. Primary tumorspheres formed were collected and dissociated after 9 days of culture. 10⁴ cells dissociated from the primary tumorsphere were plated in triplicate with or without treatment. Secondary tumorspheres that formed after 6 days of culture were counted under the microscope.

T47D, HCC1937, MCF7, SUM149, MDA-MB231, and BT20 cells were cultured per ATCC recommendations. To determine cell viability, 50,000 cells were plated in 24-well plates and treated with DMSO, PDGFR tyrosine kinase inhibitor III (PDGFR inh III, EMD Biosciences) [44, 51], or RO31-8220, a specific PKCα inhibitor (Cayman Chemical) [44, 52], at the indicated concentrations. Viable cell numbers were determined by an automatic cell counter (Bio-rad). Dead cells were determined by trypan blue or propidium iodide (PI) staining, and the percentage of dead cells was calculated from at least 1000 cells. For ectopic expression of BRCA1, cells were transfected with pBabe-empty, pBabe-HA-BRCA1, or pBabe-Myc-BRCA1 as previously described [31]. For CRISPR-mediated Pdgfrβ knockout (KO) in primary tumor cells, Pdgfrβ CRISPR/Cas9 KO plasmids (mouse) (Santa Cruz, SC-422171) were transfected into p18^−/−;Brca1^MGKO primary tumor cells following the manufacturer’s protocol. GFP-positive and GFP-negative cells were sorted 2 days after transfection on a BD FACS SORP Aria-IIu machine for further analysis. For annexin V analysis, p18^−/−;Brca1^MGKO tumor cells were transfected with the Pdgfrβ CRISPR/Cas9 KO plasmids, and 48 h after transfection, cells were stained with annexin V-pacific blue (Biolegend) and analyzed by the LSR–Fortessa machine (BD Pharmingen). Data analysis was performed using Kaluza software (Beckman Coulter).

For in vivo transplantation, p18^−/−;Brca1^MGKO primary tumor cells transfected with the Pdgfrβ CRISPR/Cas9 KO plasmids were FACS sorted. Six thousand GFP^neg and GFP^pos live cells (trypan blue negative) were suspended in a 50% solution of Matrigel (BD) and then inoculated into the left and right inguinal mammary fat pads (MFPs) of 6-week-old female NSG mice (Jackson Laboratory), respectively. Four weeks after transplantation, animals were euthanized and mammary tumors were dissected for histopathological and immunohistochemical analyses. For ex vivo transplantation, primary p18^−/−;Brca1^MGKO tumor cells were cultured to generate primary tumorsphere. 10⁴ cells dissociated from primary tumorspheres were treated with DMSO, Ro-31-8220, or PDGFR inh III for 6 days. One thousand live cells were transplanted into MFPs of NSG mice. Four weeks after transplantation, animals were euthanized and mammary tumors were analyzed. For treatment of established mammary tumors, p18^−/−;Brca1^MGKO tumor cells were transplanted into MFPs of NSG mice and allowed to reach ~ 200 mm³ in size. Mice were then treated with daily i.p. injection of the agents, and the tumor size was determined.

RNA was extracted and purified from tumors using an RNeasy kit (Qiagen). Tumor RNA was reverse transcribed, amplified, and labeled with Cy5, and WT mammary tissue reference RNA was reverse transcribed, amplified, and labeled with Cy3. The amplified sample and reference were co-hybridized to Agilent 4x180k custom mouse microarrays and were analyzed as described previously [14, 53]. Murine tumor gene expression data was deposited at the Gene Expression Omnibus under accession number GSE155239. Single sample gene set enrichment analysis (ssGSEA) was performed using GenePattern [54]. The gene signature for PDGF signaling was taken from Reactome [55], while EMT and mammary stem cell signatures were published [56, 57].

For the western blot, tissue and cell lysates were prepared as previously reported [28, 31]. The primary antibodies used were BRCA1 (Santa Cruz), E-cad, Vim, PDGFRβ, p-PDGFRβ, p-PKCα, p-Fra1 and Snail (Cell signaling), Gapdh (Ambion), and Twist (Abcam). ChIP assays were carried out as previously described [28, 31]. Briefly, T47D and HCC1937 cells were treated with 1.5% formaldehyde and sonicated. Anti-BRCA1 antibody (D-9, Santa Cruz) or control mouse IgG was used to precipitate chromatin associated with BRCA1. Q-PCR was performed to determine the relative abundance of target DNA. Specific primers for the analysis of BRCA1 binding to PDGFRβ are listed in Additional file 1.

Formalin-fixed paraffin-embedded (FFPE) human breast cancer samples lacking patient-identifying information were obtained with IRB approval from the Tissue Banks at the University of Miami and the Department of Pathology at Shenzhen University. All samples obtained were non-treated invasive breast cancers with known ER status. The MetaBric human breast cancer dataset [58] was analyzed for correlation between BRCA1 with PDGFRβ and PKCα mRNA levels.

All data are presented as the mean ± SD for at least three repeated individual experiments for each group. Quantitative results were analyzed by the two-tailed Fisher exact test or two-tailed Student’s t test. p < 0.05 was considered statistically significant.

We previously discovered that the majority of p18^−/− mice developed CK8⁺ and ER⁺ luminal type mammary tumors while most p18^−/−;Brca1^+/− mice formed mammary tumors that were ER⁻ and CK5⁺ basal-like tumors expressing mesenchymal markers as well as EMT-TFs [11, 28, 31]. In addition, p18^−/−;Brca1^+/− mammary tumors were enriched with cancer stem cells [31, 59] and significantly more metastatic than p18^−/− tumors (Fig. 1a). These data suggest that heterozygous germline deletion of Brca1 induces basal-like tumors with activation of EMT and promotes metastasis. To further explore the role of PDGFRβ signaling in activating EMT and promoting tumor initiation and progression [40, 44, 45], we performed a microarray analysis of mammary tumors from p18^−/− and p18^−/−;Brca1^+/− mutant mice. Analysis of differentially expressed genes identified Pdgfrβ mRNA to be significantly higher in p18^−/−;Brca1^+/− tumors than in p18^−/− tumors (Fig. 1b). We then performed IHC analysis and detected that 75% (n = 16) of p18^−/−;Brca1^+/− tumors expressed Pdgfrβ, whereas only 16% (n = 19) of p18^−/− tumors yielded similar results. Strong Pdgfrβ expression ranged from 2 to 60% of p18^−/−;Brca1^+/− tumor cells, while Pdgfrβ expression was found in 2–5% of p18^−/− tumor cells and was much weaker in intensity. All the EMT-positive p18^−/−;Brca1^+/− tumors were also positive for Pdgfrβ (Fig. 1a, c). We also noted p18^−/−;Brca1^+/− tumor cells that had invaded into muscles were marked by high levels of Pdgfrβ expression (Fig. 1c). Taking advantage of the majority of p18^−/−;Brca1^+/− tumors expressing a high level of Pdgfrβ and a few of these tumors expressing a low level of Pdgfrβ, we performed ssGSEA and found that the lowest Pdgfrβ samples had lower Pdgf signaling as expected (Fig. 1d, Additional file 2A). Furthermore, Pdgfrβ mRNA levels in p18^−/−;Brca1^+/− mammary tumors strongly correlated with EMT and stem cell signatures (Fig. 1e, f, and Additional file 2B, C, D), in agreement with the data derived from IHC and published elsewhere [31, 59]. Together, these results suggest that germline deletion of Brca1 in a p18^−/− background activates EMT in mammary tumorigenesis, which is associated with an increase of Pdgfrβ and Pdgfrβ-activated signaling pathway.

Pdgfrβ is abundantly expressed in stromal fibroblasts which also play an important role in facilitating breast cancer growth and progression [35, 38, 39]. To confirm the role of Brca1 loss in regulating Pdgfrβ in mammary epithelial and carcinoma cells, and to rule out the effect of stromal Pdgfrβ regulated by Brca1 loss in mammary tumorigenesis, we generated Brca1^MGKO (Brca1^f/f;MMTV-Cre or Brca1^f/−;MMTV-Cre), p18^−/−;Brca1^MGKO, and p16^−/−;Brca1^MGKO mice in the Balb/c-B6 mixed background in which Brca1 was specifically deleted in mammary epithelia. We previously reported the successful deletion of Brca1 and the activation of EMT in mammary epithelia of p18^−/−;Brca1^MGKO and p16^−/−;Brca1^MGKO mice [31]. We found that 47% (n = 15) of p18^−/−, 73% (n = 15) of p18^−/−;Brca1^MGKO, and 60% (n = 10) of p16^−/−;Brca1^MGKO mice in the Balb/c-B6 mixed background developed mammary tumors, whereas only 8% (n = 13) of Brca1^MGKO and no p16^−/− mice did so at similar ages (Table 1). Notably, 36% (n = 11) of p18^−/−;Brca1^MGKO and 50% (n = 6) of p16^−/−;Brca1^MGKO tumors, but no p18^−/− tumors, metastasized to the lung (Table 1), consolidating the role of loss-of-function of Brca1 in promoting tumor metastasis.

Though the mammary tumor incidence of p18^−/− mice in the Balb/c-B6 background was lower than that of the Balb/c background, p18^−/− tumors in the Balb/c-B6 background were also predominantly luminal (Fig. 1a and Table 1) [28, 31]. Eighty-two percent (n = 11) of p18^−/−;Brca1^MGKO and all of p16^−/−;Brca1^MGKO tumors were positive for Vim, EMT-TFs, and Pdgfrβ, whereas only 29% (n = 7) of p18^−/− tumors were positive for these features. Further, comparing these p18^−/−;Brca1^MGKO and p16^−/−;Brca1^MGKO tumors with p18^−/− tumors, we identified more tumors with high p-Pkcα and p-Fra1 which together indicated Pdgfrβ pathway activity in Brca1-deficient tumors (Table 1, Fig. 2a–c).

Implying epithelial origin, most Pdgfrβ-positive p18^−/−;Brca1^MGKO and p16^−/−;Brca1^MGKO mammary tumor cells were also positive for Ck14, an epithelial marker. Notably, Pdgfrβ and Ck14 doubly positive tumor cells were also detected in lung metastases of p18^−/−;Brca1^MGKO and p16^−/−;Brca1^MGKO mammary tumors (Fig. 2a, b, and Additional file 3), which demonstrated that the metastases were derived from Brca1-deficient basal-like mammary tumors with an increased Pdgfrβ expression. Using western blots to assess protein, we found that the expression of Pdgfrβ, p-Pkcα, and p-Fra1 was clearly increased in p18^−/−;Brca1^MGKO tumors relative to that of adjacent tumor-free mammary tissues in the same mice or to p18^−/− tumors (Fig. 2d, Additional file 4), and in p18^−/−;Brca1^MGKO tumor cells relative to that in p18^−/− tumor cells (Fig. 2e). As a whole, these data suggest that loss of Brca1 induces EMT and metastatic basal-like tumors in an epithelium-autonomous manner and that Brca1 loss activates Pdgfrβ-Pkcα signaling in epithelial tumor cells thereby enriching the number of tumor cells with EMT-like features.

To confirm the role of BRCA1 in controlling Pdgfrβ-Pkcα signaling, we overexpressed WT BRCA1 in two BRCA1-mutant breast cancer cell lines, HCC1937 and SUM149, and a basal-like cell line, MDA-MB231. BRCA1 inhibited the mRNA and protein levels of PDGFRβ indicating BRCA1 repressed transcription of PDGFRβ (Fig. 3a, b, and Additional file 5). Consistently, the level of phosphorylation of PKCα was also reduced in BRCA1-overexressed cells confirming that BRCA1 suppressed PDGFRβ-PKCα signaling (Fig. 3a, b, and Additional file 5). Furthermore, we found that overexpression of BRCA1 in these cells induced expression of CDH1 (encoding E-cadherin) while inhibiting expression of FOSL1 (encoding FRA1) and VIM, consistent with our finding that deficiency of Brca1 induced EMT in tumor cells. Importantly, these results confirm the role of Brca1 in suppressing EMT in a mammary tumor cell autonomous manner.

We and others have shown that BRCA1 binds to FOXC1/2 and TWIST loci to repress their transcription [31, 60]. Our bioinformatic analysis revealed that there exist at least four putative BRCA1 binding sites in the PDGFRβ locus (Fig. 3c). We performed chromatin-immunoprecipitation (ChIP) assays and found that endogenous and exogenous BRCA1 bound to the PDGFRβ locus in T47D and HCC1937 cells, respectively (Fig. 3d, e). Together with the inhibitory effect of BRCA1 for the expression of PDGFRβ, these data suggest that BRCA1 binds to the PDGFRβ locus to repress its transcription.

To determine the role of Pdgrfβ in Brca1-deficient tumorigenesis, we knocked out Pdgfrβ in p18^−/−;Brca1^MGKO primary tumor cells by using the CRISPR/Cas9 system. We transfected Pdgfrβ CRISPR/Cas9 KO plasmids (encoding GFP) into p18^−/−;Brca1^MGKO primary tumor cells. Two days after transfection, GFP-negative (GFP^neg) and GFP-positive (GFP^pos) cells were FACS sorted for further analysis. We found that GFP^pos tumor cells expressed nearly no detectable Pdgfrβ and drastically reduced p-Pkcα, p-Fra1, and Vim, when compared with GFP^neg tumor cells (Fig. 4a). These data confirmed successful depletion of Pdgfrβ protein in the GFP^pos p18^−/−;Brca1^MGKO mammary tumor cells. Additionally, these data also indicate that activation of Pkcα and its downstream target Fra1 is dependent on Pdgfrβ signaling, and Pdgfrβ signaling is required for maintaining the mesenchymal traits in Brca1-deficient tumor cells.

To test the impact of Pdgfrβ CRISPR/Cas9 KO on tumor cell viability, we performed annexin V staining. We found that 8.8% of GFP^pos (Pdgfrβ KO) tumor cells were positive for annexin V compared to 4.3% of the GFP^neg (Pdgfrβ WT) cells (Fig. 4b). These data indicate that acute deletion of Pdgfrβ slightly enhances apoptosis of Brca1-deficient tumor cells. We further analyzed FACS-sorted Pdgfrβ WT and Pdgfrβ KO p18^−/−;Brca1^MGKO tumor cells in dishes and observed that the population with characteristic morphological features of apoptosis (i.e., cell shrinkage, pyknosis, dense cytoplasm with tightly packed organelles) or dead cells was significantly increased in Pdgfrβ KO cells relative to that in Pdgfrβ WT cells after 5–7 days of culture (Fig. 4c). We found that the viable cells were significantly less and the dead cells were drastically more in Pdgfrβ KO population than in Pdgfrβ WT population (Fig. 4d, e). These data indicate that long-term depletion of Pdgfrβ in p18^−/−;Brca1^MGKO tumor cells in vitro increases tumor cell death.

We next transplanted freshly FACS-sorted Pdgfrβ WT and Pdgfrβ KO p18^−/−;Brca1^MGKO tumor cells that were viable into MFPs of NSG mice. We found that Pdgfrβ WT p18^−/−;Brca1^MGKO tumor cells generated palpable tumors in 10–14 days whereas Pdgfrβ KO cells did not generate palpable tumors in the same time period. Four weeks after transplantation, tumors generated by Pdgfrβ KO cells were significantly smaller than tumors generated by Pdgfrβ WT cells (Fig. 4f). We performed western blot in conjunction with IHC analysis and observed that tumors generated by Pdgfrβ KO cells expressed clearly reduced levels of Pdgfrβ, p-Pkcα, p-Fra1, Vim, Snail, and Twist, but increased levels of E-cad, when compared with tumors generated by Pdgfrβ WT cells (Fig. 4g, h, and data not shown). Notably, we detected that some cells in tumors generated by Pdgfrβ KO p18^−/−;Brca1^MGKO cells expressed high levels of E-cad, whereas E-cad-positive cells in tumors generated by Pdgfrβ WT p18^−/−;Brca1^MGKO tumor cells were rarely observed (Fig. 4h). These results demonstrate that deletion of Pdgfrβ in Brca1-deficient tumor cells promotes MET and suppresses tumorigenesis.

To determine whether Pdgfrβ and Pkcα activity represented therapeutic target in BRCA1-deficient tumors, we treated p18^−/−;Brca1^MGKO tumor cells with a PDGFR Inh III and PKCα inhibitor, Ro-31-8220. The activity and specificity of these drugs in inhibiting phosphorylation of PDGFRβ and PKCα have been well confirmed by multiple groups [44, 51, 52, 61]. Illustrating these inhibitors were having the intended effects, we observed reduced Pdgfrβ and Pkcα phosphorylation following treatment (Fig. 5a, and Additional file 6A, B). In addition, we examined the impact of dosage on tumor cells. Treatment of p18^−/−;Brca1^MGKO tumor cells with PDGFR Inh III and Ro-31-8220 significantly reduced cell number and increased cell death, particularly with high dosages (200 nM for PDGFR Inh III and 350 nM for Ro-31-8220) (Fig. 5b, c). At a low dosage (20 nM for PDGFR Inh III and 35 nM for Ro-31-8220), these drugs converted spindle-shaped, mesenchymal-like cells into epithelial-like cells (Fig. 5d). This is consistent with the data derived from targeted deletion of Pdgfrβ, and these results further confirm that inhibition of Pdgfrβ-Pkcα signaling promotes Brca1-deficient tumor cell death and MET.

We have previously reported that deletion of Brca1 in p18-deficient mice activates EMT and enhances TICs [31]. We detected that p18^−/−;Brca1^MGKO tumor cells formed significantly more and larger spheres than p18^−/− cells (data not shown) confirming the role of loss-of-function of Brca1 in stimulating TICs. We treated p18^−/−;Brca1^MGKO tumorsphere-dissociated cells with PDGFR Inh III or Ro-31-8220 and found that inhibition of Pdgfrβ or Pkcα activity significantly reduced secondary tumorsphere forming potential (Fig. 5e, g). Moreover, we transplanted p18^−/−;Brca1^MGKO tumorsphere-dissociated cells into NSG mice and found that cells pretreated with low-dose PDGFR Inh III or Ro-31-8220 produced significantly smaller tumors than control cells (Fig. 5f, h). Western blot and IHC analysis revealed that tumors generated by PDGFR Inh III or Ro-31-8220 pretreated cells expressed higher levels of E-cad and lower levels of Vim (Fig. 5i, j) than DMSO-pretreated cells. Again, these results confirm that the inhibition of Pdgfrβ and Pkcα activity promotes MET and reduces Brca1-deficient tumor initiating potential.

We then determined if pharmaceutical inhibition of Pdgfrβ and Pkcα activity had any effect on the progression of established Brca1-deficient tumors. Transplanted p18^−/−;Brca1^MGKO tumors were allowed to reach 150–200 mm³ in size and then mice were treated with DMSO or inhibitors daily. Three days after treatment, tumors from PDGFR Inh III- or Ro-31-8220-treated mice began to show a significant size reduction in comparison with the tumors from DMSO-treated animals (Fig. 6a, b). After a 9-day treatment, tumors from DMSO-treated mice reached 578 ± 265 mm³ in size, whereas those from PDGFR Inh III-treated mice only reached 131 ± 22 mm³, which was even smaller than that of the tumor size at the start of treatment (194 ± 48 mm³) (Fig. 6a), indicating that treatment with PDGFR Inh III induced regression of Brca1-deficient tumors. Consistently, animals that received Ro-31-8220 treatment for 9 days had a significant reduction in tumor size compared with DMSO treatment controls (772 ± 366 mm³ vs. 313 ± 182 mm³, Fig. 6b). These data demonstrate that pharmaceutical inhibition of Pdgfrβ and Pkcα activity has therapeutic effects on established Brca1-deficient tumors.

To determine the mechanisms associated with inhibition of tumor progression by Pdgfrβ and Pkcα inhibitors, we analyzed Brca1-deficient tumors by western blot. This revealed that all tumors treated with PDGFR Inh III or Ro-31-8220 expressed more cleaved caspase 3 and less Vim (Fig. 6c, and data not shown), and one expressed higher levels of E-cad when compared with those with DMSO treatment (tumor 5 in Fig. 6c, and data not shown). IHC analysis confirmed that all tumors treated with PDGFR Inh III or Ro-31-8220 displayed more cleaved caspase 3-positive cells than control tumors, though the level of increase varied among tumors and different areas of an individual tumor. All control tumors widely expressed high levels of Vim while PDGFR Inh- or Ro-31-8220-treated tumors showed faint or undetectable Vim (Fig. 6d, and data not shown). In sum, these results suggest that inhibition of Pdgfrβ or Pkcα activity suppresses tumor progression by reducing mesenchymal features and inducing apoptosis.

To confirm the cytotoxic effects of inhibition of PDGFR and PKCα on BRCA1-deficient human breast cancer cells, we screened six widely used cell lines including two BRCA1 WT luminal lines, T47D and MCF7; two BRCA1 WT basal lines, MDA-MB231 and BT20; and two BRCA1 mutated lines, HCC1937 and SUM149. Importantly, all these cell lines carry defective INK4-RB pathway (undetectable p16 in T47D, MCF7, MDA-MB231, BT20, SUM149, and undetectable RB in HCC1937) [62‐64], which are similar with our p18^−/−, p18^−/−;Brca1^MGKO, and p16^−/−;Brca1^MGKO mouse tumor cell system. We observed that the expression of PDGFRβ and PKCα was drastically increased in two BRCA1 mutant cell lines (HCC1937 and SUM149) and a BRCA1 lowly expressing MDA-MB231 line, whereas PDGFRβ and PKCα levels were extremely low in BRCA1 WT luminal lines (T47D and MCF7) and slightly increased in a BRCA1 WT basal line (BT20) (Fig. 7a, b). Western blot analysis revealed that BRCA1 mutant HCC1937 cells expressed significantly higher amounts of PDGFRβ, p-PKCα, and p-FRA1 than BRCA1-WT T47D cells (Fig. 7b) which suggested the activation of PDGFRβ-PKCα signaling by BRCA1 loss, consistent with our finding in mice and the findings from others [44, 65]. We confirmed that PDGFR inh III and Ro-31-8220 suppressed PDGFRβ and PKCα activities in HCC1937 and T47D cells, though the latter of which exhibited extremely low level of PDGFRβ and PKCα activities (Additional file 6A, B). We treated these cells with the inhibitors and found that PDGFR Inh III and Ro-31-8220 significantly reduced the number of viable cells and promoted cell death in BRCA1-mutated HCC1937 and SUM149 cells. In BRCA1 lowly expressing MDA-MB231 cells, these effects were moderate and even more slight in BRCA1 WT luminal cells (Fig. 7c, d, Additional file 6C). Importantly, BRCA1 in BT20 cells was lower than that in luminal cells (MCF7 and T47D), but not as low as that in MDA-MB231 cells. Thus, the slightly increased level of PDGFRβ and PKCα in BT20 cells relative to that in luminal cells suggested that other factors were involved in inactivation of PDGFRβ and PKCα and may explain the poor response to PDGFRβ and PKCα inhibitors in BT20 cells. Together, these data suggest that inhibition of PDGFR or PKCα activity efficiently kills BRCA1-deficient human breast cancer cells.

To extend these results to human breast tumors, we performed immunostaining analysis of 8 ER⁺ and 10 ER⁻ primary human breast cancers. We found that PDGFRβ, PKCα, FRA1, and VIM were readily detected in ER⁻ and BRCA1 weak or non-detectable tumor cells, whereas these proteins were barely detectable in ER⁺ and BRCA1⁺ tumor cells (Fig. 7e and Additional file 7A, B). Further analysis revealed that PDGFRβ and PKCα detected by immunostaining were significantly inversely correlated with BRCA1 expression (Fig. 7f). We then analyzed PDGFRβ and PKCα mRNA expression in TCGA breast cancer dataset and did not find significant differences of the expression between BRCA1 mutant and WT TNBCs (data not shown). This may be partly resulted from the small sample size of BRCA1 mutant TNBCs in TCGA dataset (PDGFRβ and PKCα mRNA data are available in only five BRCA1 mutant TNBCs). Moreover, we queried the mRNA expression of genes in the METABRIC 1584 breast cancer sample sets and found a significant inverse correlation between BRCA1 with PDGFRβ and PKCα mRNA levels (Fig. 7g). These clinical findings, consistent with our results in mice, further confirm that BRCA1 suppresses the PDGFRβ-PKCα signaling pathway in breast basal-like cancer development and progression.