PELP1/SRC-3-dependent regulation of metabolic PFKFB kinases drives therapy resistant ER+ breast cancer

Abstract

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER⁺) breast cancer. Disseminated ER⁺ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the maintenance and expansion of breast cancer stem cells (CSCs). Breast CSCs frequently exist as a minority population in therapy resistant tumors. In this study, we show that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. Seahorse metabolic assays demonstrated that cytoplasmic PELP1 influences cellular metabolism by increasing both glycolysis and mitochondrial respiration. PELP1 interacts with PFKFB3 and PFKFB4 proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB4 knockdown inhibited in vivo emergence of circulating tumor cell (CTC) populations in mammary intraductal (MIND) models. Application of PFKFB inhibitors in combination with ER targeted therapies blocked tumorsphere formation in multiple models of advanced breast cancer including tamoxifen (TamR) and paclitaxel (TaxR) resistant models, murine tumor cells, and ER⁺ patient-derived organoids (PDxO). Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER⁺ tumor cell populations that include CSCs and CTCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance.

Figure 1.

PELP1-induced gene expression is altered in 3D conditions. (A) Quantification of endogenous cytoplasmic PELP1 in ER+ and ER− cell lines (MCF-7, SUM225, and CCH1). (B) ALDH+ and (C) CD44^hi/CD24^lo populations in MCF-7 PELP1 cells. (D) Venn diagrams showing unique genes up or downregulated >2-fold in MCF-7 PELP1 cells (3D vs. 2D). IPA analysis of (E) upstream regulators and (F) diseases or functions. (G) Heat-map showing log2(FPKM) values of cyto PELP1 gene signature. (H) Volcano plots of 3D vs. 2D comparison of MCF-7 PELP1 cells. (I) S-plot showing differentially expressed genes in 3D comparison of MCF-7 WT vs. cyto PELP1 cells from integration of RNA-seq and ATAC-seq analysis. Kaplan-Meier curves for upper and lower 50^th percentile of cyto PELP1 gene signature in the METABRIC (J) all subtypes (n=1904), (K) ER+ only (n=1222), (L) Her+ (n=188), and (M) TNBC (n=290) patient cohorts. Graphed data represent the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2.

PELP1 cytoplasmic signaling upregulates HIF-activated metabolic pathways. (A) mRNA levels of EPAS1, PFKFB3, PFKFB4, NDRG1, and SOX9 in MCF-7 PELP1 cells. (B) OCR and ECAR measured in MCF-7 PELP1 cells by Seahorse Cell Energy Phenotype test. (C) OCR measured in MCF-7 PELP1 cells by Seahorse Mito Stress test. (D) Glucose uptake in cells treated with 2-NBDG. Graphed data represent the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3.

PFKFB inhibition blocks PELP1/SRC-3 signaling. Co-immunoprecipitation of (A) PELP1 and PFKFB3 or (B) PFKFB4 in MCF-7 PELP1 cells. Co-immunoprecipitation of PELP1 and SRC-3 in MCF-7 PELP1 cells treated with vehicle (DMSO), (C) PFK158 (100 nM), or (D) 5MPN (5 μM). Cell lysate controls (right). Proximity ligand assay (PLA) in MCF-7 PELP1 cells treated with vehicle, (E) PFK158, or (F) 5MPN. (G) Secondary tumorspheres in MCF-7 PELP1 shGFP control and shPFKFB4 knockdown cells. Secondary tumorspheres in MCF-7 PELP1 cells treated with vehicle, (H) PFK158 or (I) 5MPN. (J) Secondary tumorspheres in J110 cells treated with vehicle, PFK158, or 5MPN. Western blot shows PFKFB3 and PFKFB4 levels. Graphed data represent the mean ± SD (n = 3). PLA data represent the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4.

PFKFB4 knockdown abrogates cyto PELP1 CTCs in MIND xenografts. (A) Representative H&E stains from MIND glands (WT and cyto PELP1). (B) Tumor area (%) calculated from H&E sections from (A). (C) Tumor area (%) calculated from H&E sections from WT and cyto PELP1 (shGFP, shPFKFB4) MIND glands. (D) Representative images of CTCs from blood samples collected from mice injected with WT or cyto PELP1 (shGFP, shPFKFB4) cells. (E) Average size of soft agar colonies (CTCs) from (D). (F) Average number of colonies/well (CTCs). Graphed data represent the mean ± SD (n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5.

Therapy resistant models phenocopy cyto PELP1 cancer biology. (A) Taxol dose response in MCF-7 PELP1 cells (0-50 nM Taxol). (B) Quantification of endogenous cytoplasmic PELP1 in MCF-7 TaxR and TamR cells. (C) Co-immunoprecipitation of PELP1 and SRC-3 in MCF-7 TaxR (top) or TamR (bottom) cells. (D) PELP1/SRC-3 interaction measured by PLA in MCF-7 TaxR cells; quantification of PLA signal (cytoplasmic and nuclear interactions). (E) mRNA levels of EPAS1, PFKFB3, and PFKFB4 in MCF-7 TaxR (top) or TamR (bottom) cells cultured in 2D or 3D conditions. (F) OCR and ECAR measured in MCF-7 TaxR cells by Seahorse Cell Energy Phenotype test. (G) OCR measured in MCF-7 TaxR cells by Seahorse Mito Stress test. (H) Glucose uptake in cells treated with 2-NBDG. Secondary tumorspheres in (I) MCF-7 TaxR and (J) MCF-7 TamR cells treated with vehicle (DMSO), PFK158 (100 nM), 5MPN (5 μM), or SI-2 (100 nM). Graphed data represent the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6.

Endocrine therapies exhibit combinatorial effects with PELP1 complex inhibitors. Tumorsphere assays in MCF-7 PELP1 cells treated with: (A) tam/SI-2, (B) tam/5MPN, or (C) SI-2/5MPN. Tumorsphere assays in MCF-7 TaxR cells treated with: (D) tam/SI-2, (E) tam/5MPN, or (F) SI-2/5MPN. Concentrations: tam (100 nM), 5MPN (5 μM), SI-2 (100 nM). Graphed data represent the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7.

Co-treatments in preclinical ER+ PDxO models target CSCs. CellTiter Glo assays in HCI-003 and −017 co-treated with (A) tam/SI-2, (B) tam/5MPN, or (C) SI-2/5MPN. (D) Tables summarizing most synergistic area scores from 7A-C and SFigure 20A-20C. (E) Western blot of PELP1, SRC-3, PFKFB3, PFKFB4, and ER protein in HCI-003, −007, −011, and −017. Tumorsphere assays in HCI-003 and HCI-017 co-treated with (F) tam/SI-2, (G) tam/5MPN, or (H) SI-2/5MPN. Prior to assay, PDxO models were pre-treated with the indicated compounds for 3 days and continued treatment during the assay. Concentrations: tam (100 nM), 5MPN (5 μM), SI-2 (100 nM). Graphed data represent the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.