The G Protein Estrogen Receptor (GPER) is involved in the resistance to the CDK4/6 inhibitor palbociclib in breast cancer

Abstract

Background: The cyclin D1-cyclin dependent kinases (CDK)4/6 inhibitor palbociclib in combination with endocrine therapy shows remarkable efficacy in the management of estrogen receptor (ER)-positive and HER2-negative advanced breast cancer (BC). Nevertheless, resistance to palbociclib frequently arises, highlighting the need to identify new targets toward more comprehensive therapeutic strategies in BC patients.

Methods: BC cell lines resistant to palbociclib were generated and used as a model system. Gene silencing techniques and overexpression experiments, real-time PCR, immunoblotting and chromatin immunoprecipitation studies as well as cell viability, colony and 3D spheroid formation assays served to evaluate the involvement of the G protein-coupled estrogen receptor (GPER) in the resistance to palbociclib in BC cells. Molecular docking simulations were also performed to investigate the potential interaction of palbociclib with GPER. Furthermore, BC cells co-cultured with cancer-associated fibroblasts (CAFs) isolated from mammary carcinoma, were used to investigate whether GPER signaling may contribute to functional cell interactions within the tumor microenvironment toward palbociclib resistance. Finally, by bioinformatics analyses and k-means clustering on clinical and expression data of large cohorts of BC patients, the clinical significance of novel mediators of palbociclib resistance was explored.

Results: Dissecting the molecular events that characterize ER-positive BC cells resistant to palbociclib, the down-regulation of ERα along with the up-regulation of GPER were found. To evaluate the molecular events involved in the up-regulation of GPER, we determined that the epidermal growth factor receptor (EGFR) interacts with the promoter region of GPER and stimulates its expression toward BC cells resistance to palbociclib treatment. Adding further cues to these data, we ascertained that palbociclib does induce pro-inflammatory transcriptional events via GPER signaling in CAFs. Of note, by performing co-culture assays we demonstrated that GPER contributes to the reduced sensitivity to palbociclib also facilitating the functional interaction between BC cells and main components of the tumor microenvironment named CAFs.

Conclusions: Overall, our results provide novel insights on the molecular events through which GPER may contribute to palbociclib resistance in BC cells. Additional investigations are warranted in order to assess whether targeting the GPER-mediated interactions between BC cells and CAFs may be useful in more comprehensive therapeutic approaches of BC resistant to palbociclib.

Keywords: Breast cancer; Cancer-associated fibroblasts (CAFs); Estrogen receptor; G protein-coupled estrogen receptor (GPER); Palbociclib; Resistance.

Fig. 1

Establishment of palbociclib-resistant MCF7 (MCF7/PalbR) BC cells. A Cell cycle analysis performed by flow cytometry in MCF7 and MCF7/PalbR cells treated with vehicle or 1 μM palbociclib (Palb) for 12 h. B Percentage of cells in G0/G1, S and G2/M phases of cell cycle. C Proliferation of MCF7 and MCF7/PalbR cells after 3 days treatment with vehicle or 1 μM palbociclib (Palb). Values of vehicle-treated MCF7 cells were set as 100% upon which cell viability was determined. D Representative pictures of spheroids (a single spheroid/well) from the MCF7 and MCF7/PalbR spheroid cultures grown on agar-coated plates and exposed for 6 days to vehicle or 1 μM palbociclib (Palb), as indicated. Scale bar: 500 μm. E Quantification of spheroid growth. Values of vehicle-treated MCF7 cells were set as 100% upon which spheroid growth was determined. F Colony formation assay in MCF7 and MCF7/PalbR cells exposed to vehicle or 1 μM palbociclib (Palb). Plates were stained with Crystal Violet and colonies were counted following 10 days of incubation (G). Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05

Fig. 2

EGFR mediates the increase of GPER expression in MCF7/PalbR cells. mRNA (A) and protein (B) expression of ERα in MCF7 and MCF7/PalbR cells, as evaluated by real-time PCR and immunoblotting assays, respectively. mRNA (C, E) and protein (D, F) levels of GPER and EGFR in MCF7 and MCF7/PalbR cells, as evaluated by real-time PCR and immunoblotting, respectively. In RNA experiments, values are normalized to the actin beta (ACTB) expression and shown as fold changes of mRNA expression in MCF7/PalbR respect to MCF7 cells. G GPER protein expression in MCF7 and MCF7/PalbR cells transiently transfected with a control shRNA or a shEGFR plasmid, as indicated. H Efficacy of EGFR silencing in MCF7/PalbR cells. Side panels show densitometric analyses of the blots normalized to β-actin, which served as loading control. I Recruitment of EGFR to the AT-rich sequence located within the GPER promoter, as ascertained by ChIP assay in MCF7/PalbR cells transiently transfected with a control shRNA or a shEGFR plasmid. In control samples, nonspecific IgGs were used instead of the primary antibody. The amplified sequences were evaluated by real-time PCR. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05. Created with BioRender.com

Fig. 3

The up-regulation of c-Fos, EGR1 and Cyr61 in MCF7/PalbR cells relies on both EGFR and GPER. A Immunoblots of c-Fos, EGR1 and Cyr61 in MCF7 and MCF7/PalbR cells transiently transfected with a control shRNA or a shEGFR plasmid, as indicated. B Efficacy of EGFR silencing in MCF7/PalbR cells. C Protein levels of c-Fos, EGR1 and Cyr61 in MCF7 and MCF7/PalbR cells transiently transfected with a control shRNA or a shGPER plasmid, as indicated. D Efficacy of GPER silencing in MCF7/PalbR cells. Side panels show densitometric analyses of the blots normalized to β-actin, which served as loading control. c-Fos (E), EGR1 (F) and Cyr61 (G) mRNA levels in METABRIC ER-positive BC patients with elevated expression of both EGFR and GPER (median values were used as threshold). (*) indicates p < 0.05. (****) indicates p < 0.0001

Fig. 4

EGFR or GPER silencing restore palbociclib sensitivity in MCF7/PalbR cells. A Representative pictures of spheroids (a single spheroid/well) from MCF7/PalbR/shRNA and MCF7/PalbR/shEGFR spheroid cultures grown for 6 days on agar-coated plates. B Quantification of spheroid growth; values of MCF7/PalbR/shRNA cells were set as 100% upon which the number of MCF7/PalbR/shEGFR cells was determined. C Efficacy of EGFR silencing in MCF7/PalbR/shEGFR cells. D Representative pictures of spheroids (a single spheroid/well) from the MCF7/PalbR/shRNA and MCF7/PalbR/shGPER spheroid cultures grown for 6 days on agar-coated plates. Scale bar 500 μm. E Quantification of spheroid growth; values of MCF7/PalbR/shRNA cells were set as 100% upon which the number of MCF7/PalbR/shGPER cells was determined. F Efficacy of GPER silencing in MCF7/PalbR/shGPER cells. G Colony formation assay in in MCF7/PalbR/shRNA and MCF7/PalbR/shGPER cells. Plates were stained with Crystal Violet and colonies were counted following 10 days of incubation. (H). I Protein levels of cyclin D1, cyclin E1 and GPER in MCF7/PalbR/shRNA and MCF7/PalbR/shGPER cells. Side panels show densitometric analyses of the blots normalized to β-actin, which served as loading control. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05. Kaplan-Meier survival curves representing the overall survival (J) and relapse-free survival (K) in ER-positive BC patients of the METABRIC database, based on low vs high EGFR and GPER mRNA levels (median values were used as threshold)

Fig. 5

Palbociclib triggers the activation of GPER signaling in CAFs. A Superimposed binding modes of palbociclib (orange), G-1 (green) and E2 (yellow) in a GPER model, and details of the binding site. Protein backbone is represented as a ribbon and the key protein residues Tyr55, Thr66, Tyr123, Arg299 and His300 are in cyan. The ligands are also shown separately: palbociclib (B), G-1 (C) and E2 (D). E ERK1/2 phosphorylation in CAFs transiently transfected with a control shRNA or a shGPER plasmid and then exposed for 15 min to vehicle (–) or 1 μM palbociclib (Palb). (F, H) Efficacy of GPER silencing in CAFs. G c-Fos protein levels in CAFs transiently transfected with a control shRNA or a shGPER plasmid and thereafter exposed for 4 h to vehicle (–) or 1 μM palbociclib (Palb). I Immunoblot of c-Fos in CAFs treated with vehicle (-) or 1 μM palbociclib (Palb) in the presence or absence of 100 nM trametinib (Tram). Side panels show densitometric analyses of the blots normalized to ERK2 and β-actin that served as loading controls, as indicated. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05

Fig. 6

Palbociclib stimulates a pro-inflammatory gene expression profile through GPER in CAFs. A CAFs were transiently transfected with a control shRNA or a shGPER plasmid and then treated with vehicle or 1 μM palbociclib (Palb) for 8 h. Values were normalized to the 18S gene expression; the colors indicate the log₂ fold changes of gene expression upon palbociclib respect to vehicle-treated cells, as indicated. B Multiple boxplot showing the differential expression of GPER-dependent pro-inflammatory genes in ER-positive BC samples of the TCGA dataset characterized by a low or high stromal score. C Kaplan Meier survival curves in METABRIC ER-positive BC patients exhibiting high expression levels of the GPER-regulated inflammatory genes, according to k-means clustering analysis. D ER-positive BC samples characterized by elevated expression of GPER-dependent pro-inflammatory genes display worse clinical features in terms of tumor grade, tumor stage and NPI (Nottingham Prognostic Index), as indicated. (***) indicates p < 0.001; (****) indicates p < 0.0001

Fig. 7

Palbociclib-treated BC cells acquire an increased survival capacity following GPER activation in CAFs. A Workflow of the 2D cell co-cultures and 3D co-culture spheroid assays. BC patient-derived CAFs, which were previously transfected with control shRNA or shGPER plasmids and stained with CellTracker™ Green CMFDA dye, were co-cultured with MCF7 cells previously stained with CellTracker™CM-DiI dye. Co-cultures were exposed for 3 days to vehicle or 1 μM palbociclib (Palb), then MCF7 cell number and spheroid areas were analyzed on day 4. Created with BioRender.com. B Viability of MCF7 cells after 3 days treatment with vehicle or 1 μM palbociclib (Palb) and 2D co-cultured with CAFs that were previously transfected with control shRNA or shGPER plasmids. Values of vehicle-treated MCF7 cells were set as 100% upon which cell viability was determined. C Representative pictures of MCF7 and CAFs (previously transfected with control shRNA or shGPER plasmids) 3D co-culture spheroids (a single spheroid/well) grown for 3 days on agar-coated plates in the presence or absence of palbociclib (Palb). Scale bar 1000 μm. D Quantification of spheroid area; values of vehicle-treated spheroids were set as 100% upon which the area of palbociclib-treated spheroids was determined. Values represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p < 0.05