G0S2 represses PI3K/mTOR signaling and increases sensitivity to PI3K/mTOR pathway inhibitors in breast cancer

Abstract

G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target implicated in cancer biology and therapy based on frequent methylation-mediated silencing in diverse solid tumors. We recently reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, correlates with increased rates of recurrence, indicating that G0S2 plays a role in breast cancer progression. However, the function(s) and mechanism(s) of G0S2 tumor suppression remain unclear. In order to determine potential mechanisms of G0S2 anti-oncogenic activity, we performed genome-wide expression analysis that revealed an enrichment of gene signatures related to PI3K/mTOR pathway activation in G0S2 null cells as compared to G0S2 wild-type cells. G0S2 null cells also exhibited a dramatic decreased sensitivity to PI3K/mTOR pathway inhibitors. Conversely, restoring G0S2 expression in human ER+ breast cancer cells decreased basal mTOR signaling and sensitized the cells to pharmacologic mTOR pathway inhibitors. Notably, we provide evidence here that the increase in recurrence seen with low G0S2 expression is especially prominent in patients who have undergone antiestrogen therapy. Further, ER+ breast cancer cells with restored G0S2 expression had a relative increased sensitivity to tamoxifen. These findings reveal that in breast cancer G0S2 functions as a tumor suppressor in part by repressing PI3K/mTOR activity, and that G0S2 enhances therapeutic responses to PI3K/mTOR inhibitors. Recent studies implicate hyperactivation of PI3K/mTOR signaling as promoting resistance to antiestrogen therapies in ER+ breast cancer. Our data establishes G0S2 as opposing this form of antiestrogen resistance. This promotes further investigation of the role of G0S2 as an antineoplastic breast cancer target and a biomarker for recurrence and therapy response.

Keywords: Breast cancer; PI3K; estrogen receptor; mTOR; recurrence.

Figure 1.

G0S2 null cells have upregulated gene signatures associated with PI3K/mTOR activation. A, Gene Set Enrichment Analysis (GSEA) of gene expression profiles of G0S2 null MEFs and wild-type MEFs in biological triplicate. G0S2 null cells are enriched for gene signatures associated with MYC activation and PI3K/mTOR signaling. B, Representative gene set enrichment plots. NES, normalized enrichment score.

Figure 2.

G0S2 null cells have enhanced basal mTOR signaling. A, Real-time PCR of select mTOR targets (from HALLMARK_MTORC1_SIGNALING) in G0S2 null cells as compared with wild-type cells. Samples were independent from those used in microarray analysis. Bars are the average of biological triplicates and the error bars are SD. *, P < 0.05. Experiment was repeated with similar results. B, G0S2 null cells have increased levels of the mTOR downstream effector, phospho-p70S6K. G0S2 wild-type and G0S2 null cells were treated with vehicle control, 20 nM rapamycin (rap) or 20 nM everolimus (eve) for 24 hours before cells were harvested for Western analysis with anti-P-p70S6K antibody. Experiment was repeated twice with similar results.

Figure 3.

G0S2 null cells exhibit decreased sensitivity to inhibitors of PI3K/mTOR signaling as compared with wild-type cells. Wild-type and G0S2 null MEFs were treated with the mTOR inhibitors rapamycin and everolimus and the dual PI3K/mTOR inhibitor, BEZ235. Left, Cell proliferation was assessed by CellTiter-Glo assay after 72 hours of drug treatment. Data points are the average of biological triplicates. Error bars, SD. *, P < 0.05; **, P < 0.01. Right, representative long-term clonogenic assay. Cells were stained 9 to 11 days after initial drug treatment. Experiments were repeated three times with similar results.

Figure 4.

G0S2 overexpression represses basal mTOR signaling in ER+ breast cancer cell lines. Engineered overexpression of G0S2 in BT474, MCF7 and T47D cells results in decreased basal mTOR signaling as indicated by decreased levels of P-p70S6K as compared with empty vector control cells. Each immunoblot blot represents independent lysates harvested from newly generated stable cell lines.

Figure 5.

G0S2 overexpression increases sensitivity of breast cancer cells to pharmacological inhibitors targeting the PI3K/mTOR pathway. BT474 and MCF7 cells stably expressing V5-tagged G0S2 or empty-vector control were treated with rapamycin, everolimus or BEZ235 for 7 to 10 days and assessed for changes in cell proliferation as compared to vehicles. Experiments were repeated three times using independently generated stable cell lines, and similar results were obtained. Data points are biological triplicates. Error bars, SD. *, P < 0.05; **, P < 0.01; ***, P < 0.005. Similar results were seen with engineered T47D cells (Figure S2).

Figure 6.

G0S2 expression in breast cancer is associated with decreased recurrence after endocrine or tamoxifen therapy. Kaplan-Meier log-rank test and univariant Cox proportion analysis was performed using the SurvExpress database with cases divided into two groups above and below the median G0S2 expression value. Results are from the 10 data set meta-analysis constructed by SurvExpress and the independent Sotiriou 2006 dataset. For list of other studies supporting an association between recurrence free survival and G0S2 level after antiestrogen therapy see Supplementary Table S1.

Figure 7.

G0S2 overexpressing breast cancer cells exhibit enhanced response to tamoxifen. T47D and MCF7 cells stably expressing V5-tagged, Flag-tagged G0S2 or empty-vector control were treated with tamoxifen in phenol free media containing 0.1 nM estradiol for 8 to 10 days and assessed for changes in cell proliferation as compared to vehicle. Data points are biological triplicates. Error bars, SEM. *, P < 0.05; **, P < 0.01. Results in MCF7 cells trended similar to T47D but did not reach statistical significance.