Therapeutically activating RB: reestablishing cell cycle control in endocrine therapy-resistant breast cancer

Abstract

The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.

Figure 1

Differential RB-loss signature expression and survival among Luminal A and Luminal B breast cancer subtypes. (A) Gene expression heatmap depicting Luminal A and Luminal B breast cancer samples arranged by RB-loss signature expression level. (B) Boxplots depicting differential expression of the RB-loss signature in Luminal A and Luminal B subtypes. (C) Kaplan–Meier curves for relapse-free survival in Luminal A and Luminal B breast cancer subpopulations. (D) Stratification of Luminal B breast cancers based on an optimized RB-signature threshold and associated Kaplan–Meier curves for relapse-free survival.

Figure 2

Response to anti-estrogen therapy requires RB-mediated transcriptional repression. (A) Quantification of percent BrdU incorporation in MCF7 cells exposed to FBS, CDT, or CDT/ICI 182 780 (1.0 μM). (B) Immunoblot analysis of ERα, cyclin D1, p27^KIP1, pRB, ppRB (ser 780), cyclin A, and Lamin B in MCF7 cells exposed to FBS or CDT/ICI. (C) Chromatin immunoprecipitation experiments to examine the presence of RB at E2F target gene promoters in MCF7 cells treated with FBS or CDT/ICI. α-GFP IP and albumin promoters served as negative control and input lanes served as positive control. (D) Chromatin immunoprecipitation analysis to examine localization of Sin3B co-repressor at E2F target gene promoters in response to anti-estrogen exposure. α-Dbf4 IP and albumin promoters served as negative control and input lanes served as positive control. (E). Immunoblot analysis of pRB in miNS- and miRB-expressing MCF7 cells. (F) Sin3B ChIP assay on miRB-expressing MCF7 cells in response to FBS, CDT or CDT/ICI (1.0 μM) with CDT/ICI-treated wild-type MCF7 cells as a positive control. (G) Quantification of relative percent BrdU incorporation in miNS- and miRB-expressing MCF7 cells exposed to FBS, CDT, or CDT/ICI. ***, P<0.0005.

Figure 3

Inability of RB to mediate repression of gene transcription in models of anti-estrogen resistance. (A) Schematic diagram demonstrating the generation of multiple models of anti-estrogen resistance. (B) Immunoblot analysis of ERα, cyclin D1, pRB, ppRB (ser 780), cyclin A, RNR II, and Lamin B in ICI-resistant LCC9 cells postexposure to FBS, CDT, or CDT/ICI for 72 h. (C) Detection of RB protein on E2F target gene promoters cyclin A by chromatin immunoprecipitation assay in ICI-resistant LCC9 cells with CDT/ICI-treated wild-type MCF7 cells as a positive control. (D) Percent BrdU incorporation of ICI 182 780-resistant LCC9 cells postexposure to FBS, CDT, or CDT/ICI for 48 h. (E) BrdU incorporation, protein expression, and RB chromatin immunoprecipitation to examine occupancy on cyclin A promoter in MCF7/CSS/Tam^R cells exposed to FBS, CDT, or CDT/Tamoxifen for 72 h. (F) Analyses of cyclin D1 mRNA levels by qRT-PCR and concurrent analyses of protein levels from MCF7 and LCC9 models. ***, P<0.0005.

Figure 4

CDK4/6 inhibition promotes RB-mediated transcriptional repression and decreases cellular proliferation in models of antiestrogen resistance. (A) Immunoblot analysis of pRB, ppRB (Ser 780), cyclin A, RNRII, and Lamin B (loading control) in LCC9 cells in response to DMSO or PD-0332991 (500 nM) for 24 h. (B) Identification of pRB on E2F target gene promoter cyclin A by ChIP in LCC9 cells treated with 500 nM PD-0332991 for 24 h (E2F target gene cyclin A as experimental gene, α-GFP IP and albumin promoter as negative controls and input served as positive control). (C) Percent BrdU incorporation in ICI 182 780-resistant LCC9 cells in response to 500 nM PD-0332991 for 24 h. (D) Immunoblot analysis of pRB, ppRB (ser 780), E2F target genes cyclin A, RNRII, PCNA, and Lamin B (loading control) in Tamoxifen-resistant MCF7/CSS/Tam^R cells postexposure to DMSO or PD-0332991 (500 nM) for 24 h. (E) Identification of pRB on E2F target gene promoter cyclin A by ChIP in Tamoxifen-resistant MCF7/CSS/Tam^R cells in response to DMSO or PD-0332991 (500 nM) exposed for 24 h (E2F target gene cyclin A as experimental gene, α-GFP IP and albumin promoter as negative controls, and input served as positive control). (F) Percentage BrdU incorporation in Tamoxifen-resistant MCF7/CSS/Tam^R cells in response to DMSO and PD-0332991 (500 nM) exposed for 24 h. ***, P<0.0005.

Figure 5

Long-term proliferation control and pRB-mediated cellular senescence in MCF7 and LCC9 cells in response to ICI 182 780 and PD-0332991. (A) Growth of MCF7 cells in response to DMSO, PD-0332991 (500 nM), FBS, CDT, or CDT/ICI (1.0 μM) treated for 10 days. (B) Representative bright field microscopy images of crystal violet-stained MCF7 cells treated with PD-0332991 and vehicle (DMSO) for 10 days. (C) Representative images of β-galactosidase-positive MCF7 cells treated with DMSO, PD-0332991, FBS, and CDT/ICI. (D) Graphic representation of percentage (%) of β-galactosidase-positive MCF7 cells treated with DMSO, PD-0332991, FBS, or CDT/ICI. (E) Growth curve of ICI-resistant LCC9 cells in response to DMSO, PD-0332991 (500 nM), FBS, CDT, or CDT/ICI (1.0 μM) exposed for 10 days. (F) Representative microscopy images of β-galactosidase-positive LCC9 cells treated with DMSO, PD-0332991 (500 nM), FBS, or CDT/ICI (1.0 μM). (G) Graphic representation of percentage (%) of β-galactosidase-positive LCC9 cells treated with DMSO, PD-0332991 (500 nM), FBS, or CDT/ICI (1.0 μM). ***, P<0.0005.