Cyclin G2 promotes cell cycle arrest in breast cancer cells responding to fulvestrant and metformin and correlates with patient survival

Abstract

Definition of cell cycle control proteins that modify tumor cell resistance to estrogen (E2) signaling antagonists could inform clinical choice for estrogen receptor positive (ER+) breast cancer (BC) therapy. Cyclin G2 (CycG2) is upregulated during cell cycle arrest responses to cellular stresses and growth inhibitory signals and its gene, CCNG2, is directly repressed by E2-bound ER complexes. Our previous studies showed that blockade of HER2, PI3K and mTOR signaling upregulates CycG2 expression in HER2+ BC cells, and that CycG2 overexpression induces cell cycle arrest. Moreover, insulin and insulin-like growth factor-1 (IGF-1) receptor signaling strongly represses CycG2. Here we show that blockade of ER-signaling in MCF7 and T47D BC cell lines enhances the expression and nuclear localization of CycG2. Knockdown of CycG2 attenuated the cell cycle arrest response of E2-depleted and fulvestrant treated MCF7 cells. These muted responses were accompanied by sustained inhibitory phosphorylation of retinoblastoma (RB) protein, expression of cyclin D1, phospho-activation of ERK1/2 and MEK1/2 and expression of cRaf. Our work indicates that CycG2 can form complexes with CDK10, a CDK linked to modulation of RAF/MEK/MAPK signaling and tamoxifen resistance. We determined that metformin upregulates CycG2 and potentiates fulvestrant-induced CycG2 expression and cell cycle arrest. CycG2 knockdown blunts the enhanced anti-proliferative effect of metformin on fulvestrant treated cells. Meta-analysis of BC tumor microarrays indicates that CCNG2 expression is low in aggressive, poor-prognosis BC and that high CCNG2 expression correlates with longer periods of patient survival. Together these findings indicate that CycG2 contributes to signaling networks that limit BC.

Keywords: CCNG2; CDK10; Cell Cycle Arrest; Estrogen Deprivation; Fulvestrant; IGF-1R; Insulin; Metformin; RAF/MEK/MAPK pathway; Tamoxifen-Resistant.

Figure 1.

CycG2 expression is modulated by estrogen signaling in breast cancer cells. (A) Left, Histogram overlays of DNA content in MCF7 cells grown in E2-depleted medium (DM, dashed line) for 4 d vs. those cultured in non-treated medium (NT, light gray fill) and E2-depleted cultures re-stimulated with 10 nM E2 for an additional 48 h (DM+E2, dark gray fill). Right, One-way ANOVA statistical analysis of cumulative DNA flow cytometry data arising from experimental conditions shown at left. (B) Immunoblot analysis of CycG2 expression in MCF7 and T47D cells depleted of estrogen (DM) for 4 d relative to control protein (GAPDH or α-tubulin and Ponceau S stain). (C) Immunoblot analysis of CycG2 expression following 4 h re-stimulation of 3 day E2-depleted MCF7 cells with (+) 10 nM E2 compared to non-treated control (-). (D) Confocal immunofluorescence micrographs of expression and localization of CycG2 in MCF7 cells cultured in E2 containing (NT) or in E2-depleted (DM) medium for 6 d. Cells were stained with antibodies to CycG2 (shown in green channel) and α-tubulin (αTub, red). (E)  Confocal micrographs of CycG2 expression and localization following 72 h re-stimulation of E2-starved (DM) MCF7 cells with 10 nM E2 (DM+E2).  Micrographs at top show immunosignals for CycG2 (green), lamin B (Lam B, blue; nuclear marker) and γ-tubulin (γTub, red; centrosome marker); micrographs at bottom show single channel anti-CycG2 immunosignal from above images in black and white for better contrast.

Figure 2.

Inhibition of estrogen signaling with fulvestrant upregulates CycG2 expression in ER positive breast cancer cell lines. (A, B) MCF7 and T47D cells cultured in the presence or absence of fulvestrant (ICI, 100 nM) for 24 (A) or 48h (A, B) or absence of E2 (DM, 6 days) were assessed for changes in CycG2 expression by immunoblot analysis. (C) Confocal immunofluorescence microscopy images showing CycG2 expression (green) in fulvestrant-treated (ICI, bottom panel) and non-treated control (NT, top) MCF7 cells. Cells were stained with antibodies against CycG2 (green), and α-tubulin (α-Tub, red). (D) Immunoblot of CycG2 in MCF7 cells cultured for 48h in the absence (NT) or presence of the E2-signaling antagonists fulvestrant (ICI, 100 nM) or tamoxifen (TAM, 100 nM). (E) Immunoblot of CycG2 in E2-depleted (DM, 5 days) MCF7 cultures compared to E2-depleted cultures re-stimulated for an additional 48h with E2 alone or E2 plus ICI or TAM as indicated.

Figure 3.

Stable silencing of CycG2 expression attenuates the cell cycle arrest response of MCF7 cells to estrogen withdrawal. (A) Immunoblot of endogenous CycG2 in MCF7 clones expressing CCNG2-targeting shRNAs (1-B and ID3) or a non-silencing control shRNA (NSC) cultured with (+) or without (-) estrogen depletion (DM). Numbers underneath shRNA denominators refer to clone number for the respective stably transfected cell line. (B) One-way ANOVA statistical analysis of CycG2 expression data from > 3 replicate experiments quantified by immunoblotting as in A. (C) Histogram overlays of DNA content in MCF7 control and CycG2 KD clones cultured in E2-depleted (black line, DM days) or normal media (gray area, NT). (D) One-way ANOVA statistical analysis of G₁- and S-phase cell cycle distribution of single parameter DNA flow cytometry data analyses similar to that shown in C. (E) Two-parameter flow cytometry analysis of BrdU-labeled DNA in E2-depleted (DM) compared to non-treated (NT) CycG2 KD clones and controls. (F) One-way ANOVA analysis of BrdU incorporation and cell cycle distribution data as shown in E. ***p = < 0.001; **p = < 0.01; *p = < 0.05.

Figure 4.

Silencing CycG2 expression reduces the cell cycle inhibitory effects of fulvestrant-mediated ER signaling blockade. (A) Immunoblots of CycG2 levels in indicated MCF7 CycG2 KD clones and controls (NSC, WT) following 48 h culture in the presence (+) or absence (-) of fulvestrant (ICI, 100 nM). (B) One-way ANOVA statistical analysis of CycG2 expression data from >3 replicate experiments quantified by immunoblotting as in A. (C) Representative histogram overlays of DNA content in parental MCF7 control (WT, NSC) and CycG2 KD clones cultured in normal (gray area, NT) or ICI (black line) containing medium for 48h). (D) Statistical analysis (one-way ANOVA) of G₁- and S-phase DNA distribution data of flow cytometry analyses as shown in C. (E) Two-parameter flow cytometry analysis of BrdU-labeled DNA in CycG2 KD clones and WT and NSC controls cultured with (ICI) or without (NT) fulvestrant. (F) One way ANOVA analysis of BrdU data from >3 repeats of experiments shown in E. ***p = < 0.001; **p = < 0.01; *p = < 0.05.

Figure 5.

Silencing CycG2 expression blunts fulvestrant-induced dephosphorylation of Rb, inhibition of MEK/ERK signaling, and downregulation of Raf1. Immunoblot analysis of the indicated cell cycle regulatory and MAP kinase pathway proteins in MCF7 control (WT, NSC) and CycG2 KD (1-B, ID3) clones cultured with (+) or without (-) fulvestrant (ICI, 100 nM) for 48 h (A, C, E-H) or (B,D) E2-depletion (DM). Immunoblots for (A, B) S780 pRb and total Rb, (C, D) CycD1, E) T202/204 pERK 1/2, total ERK, (F) S217/221 pMEK1/2 and total MEK, (G) total cRaf, and (G) S338 p-cRaf are shown atop the indicated loading controls (GAPDH, α-tubulin, or β-actin). Numbers below lanes in blot panels indicate relative expression levels normalized to level in non-treated no-knockdown control (MCF7 WT or NSC control set at 1, marked with an *). Band intensity quantification is relative to amount of either loading control (GAPDH (GDH), α-tubulin (Tub) or β-actin (β−Act)) or total Rb, ERK, or MEK as indicated.

Figure 6.

CycG2 associates with CDK10. (A) Schema showing the sequence predicted α-helical regions in the conserved Cyclin Box domain and c-terminal bundle repeat of full length (FL) CycG2 and the relative extent of the regions remaining in each expression construct used in B. The # symbol depicts the glutamate residue in CycG2 equivalent to the well conserved Glu295 of cyclin A critical for the cyclin fold and CDK interactions; * indicates the CycG2 residues conserved with the cyclin A CDK-interacting residues Asp305 and Leu306. (B) Top, Immunoblots of whole lysates from the indicated transfected U2OS cultures used for the co-immunoprecipitation studies of CycG2-GFP and HA-CDK10 complexes. Below, Immunoblots of indicated immunoprecipitates isolated from lysates of transfected U2OS cells. (C) Confocal immunofluorescence micrographs of CycG2-GFP (green) and HA-CDK10 (red) co-expressed in transfected U2OS cells and cytoskeleton marker α-tubulin (blue). Single channel micrographs of the individual fluorescence signals are shown in black and white for better contrast while the merged 3 channel image at the bottom shows the areas of colocalization (yellow). (D) Immunoblots of endogenous CycG2 coimmunoprecipitated from HA-CDK10 transfected U2OS cells by antibodies against HA (E, F) Immunoprecipitation of endogenous CycG2 or CDK10 from lysates of MCF7 cultures depleted of E2 for 5 d (DM), with or without E2 re-stimulation (E2) in the presence or absence fulvestrant (ICI,) or tamoxifen (TAM) for an additional 48h. NT = non-treated control.

Figure 7.

CycG2 contributes the cell cycle inhibitory effects induced by metformin. (A) Two parameter DNA flow cytometry analysis of DNA synthesis (BrdU incorporation) and cell cycle status of metformin treated cultures of MCF7 WT cells. (B) Immunoblot of CycG2 vs. loading control signals (α-tubulin, β-actin) in MCF7 WT cells cultured in normal medium (NT) or in the presence of metformin (Met,1 mM), or fulvestrant (ICI, 100 nM) or in the absence of insulin (-Ins) (C) Top, representative immunoblots show metformin (Met) enhancement of fulvestrant (ICI)-induced CycG2 expression in MCF7 control (WT, NSC) cultures I + M = fulvestrant + metformin; AG = 10 μM IGF-1R inhibitor AG1024. Bottom, statistical analysis of CycG2 expression in MCF7 WT cells treated for 24 hours with either metformin, fulvestrant or both agents. (D) CycG2 expression in CycG2 KD clones (1-B, ID3) treated for 24 hours with either metformin, fulvestrant or both agents compared to similarly treated MCF7 controls. (E) Statistical analysis of (one-way ANOVA) bar graphs of G₁- and S-phase DNA distribution data. MCF7 control and CycG2 KD cultures that were treated for 24 h with metformin or fulvestrant alone or in combination. ***p = < 0.001; **p = < 0.01; *p = < 0.05.

Figure 8.

Meta-analysis of CCNG2 expression in breast cancer (BC) tumor types and its correlation with relapse free (RFS) and distant metastasis free (DMFS) survival in breast cancer patients. (A, B) Box-plots assessing CCNG2 expression in BC subtype (A) or grade classified BC tumor tissues (B) from all or tamoxifen-treated (B, right) patients (numbers of sample size at top of box-plot panels). (C, D) Kaplan-Meier survival curves estimating probability of survival for patients with tumors expressing high or low levels of CCNG2 (stratified on basis of tumor characteristics and therapy). HR = Hazard ratio. Data were assembled from online microarray databases and analyzed using the GOBO - Gene expression-based Outcome for Breast cancer Online software (http://co.bmc.lu.se/gobo) and the KM plotter tool for breast cancer (http://kmplot.com/analysis/). PR = progesterone receptor, LN = lymph node.