Metformin targets c-MYC oncogene to prevent prostate cancer

Abstract

Prostate cancer (PCa) is the second leading cause of cancer-related death in American men and many PCa patients develop skeletal metastasis. Current treatment modalities for metastatic PCa are mostly palliative with poor prognosis. Epidemiological studies indicated that patients receiving the diabetic drug metformin have lower PCa risk and better prognosis, suggesting that metformin may have antineoplastic effects. The mechanism by which metformin acts as chemopreventive agent to impede PCa initiation and progression is unknown. The amplification of c-MYC oncogene plays a key role in early prostate epithelia cell transformation and PCa growth. The purpose of this study is to investigate the effect of metformin on c-myc expression and PCa progression. Our results demonstrated that (i) in Hi-Myc mice that display murine prostate neoplasia and highly resemble the progression of human prostate tumors, metformin attenuated the development of prostate intraepithelial neoplasia (PIN, the precancerous lesion of prostate) and PCa lesions. (ii) Metformin reduced c-myc protein levels in vivo and in vitro. In Myc-CaP mouse PCa cells, metformin decreased c-myc protein levels by at least 50%. (iii) Metformin selectively inhibited the growth of PCa cells by stimulating cell cycle arrest and apoptosis without affecting the growth of normal prostatic epithelial cells (RWPE-1). (iv) Reduced PIN formation by metformin was associated with reduced levels of androgen receptor and proliferation marker Ki-67 in Hi-Myc mouse prostate glands. Our novel findings suggest that by downregulating c-myc, metformin can act as a chemopreventive agent to restrict prostatic neoplasia initiation and transformation.

Summary: Metformin, an old antidiabetes drug, may inhibit prostate intraepithelial neoplasia transforming to cancer lesion via reducing c-MYC, an 'old' overexpressed oncogene. This study explores chemopreventive efficacy of metformin in prostate cancer and its link to cMYC in vitro and in vivo.

Fig. 1.

Metformin decreases PIN formation in prostrates of Hi-Myc mice. Prostate tissues were dissected from mice that were treated orally with metformin (200 mg/kg/day) from 5 weeks old. Representative hematoxylin and eosin staining of paraffin-embedded prostate tissues at lower magnification (×10) and higher magnification (×40) from vehicle-treated (A) and metformin-treated group (B). (C) Incidence of PIN lesions in dorsal or ventral/lateral lobes of mouse prostates with or without metformin treatment for 4 weeks. (D) Incidence of PCa lesions in ventral/lateral lobes of mouse prostates with or without metformin treatment for 24 weeks. n = 10, *P < 0.05 compare to same lobe in vehicle group by Fisher’s exact test.

Fig. 2.

Metformin reduces c-Myc protein levels in vivo and in vitro. (A) Expression of c-Myc in prostates (8–9 weeks, n = 5) from mice that were treated orally with metformin (200 mg/kg/day) for 4 weeks. Percentage of stained cells was measured using the ImageJ processing program. (B–D) PCa Myc-CaP, C4-2b, PC-3 and LNCaP cells were treated with 2 mM metformin or vehicle (PBS), for 24h. Following treatment, cells were harvested and the amount of phosphorylated c-myc (p-c-myc), total c-myc and actin protein in each sample was determined using western blot analysis. (E) Myc-CaP cells were treated with 2mM metformin or PBS for 24h, in the presence or absence of the proteasome inhibitor MG132. Western blot analysis was used to measure the levels of c-myc and actin protein in treated cells. (F) Myc-CaP cells were treated with PBS or 2 mM metformin for 24h. Posttreatment cells were treated with 20mg/ml CHX at the indicated time points (0–60min) or left untreated. Lysates were prepared and analyzed by western blotting for c-Myc and actin loading control. (G) Myc-CaP cells were treated with vehicle, metformin (2 mM), AICAR (2 mM) or compound C (10 µM) + metformin (2 mM) for 24h. c-myc and actin protein levels were measured by western blots. *P < 0.05, **P < 0.01, two-tailed Student's t-test.

Fig. 3.

Metformin inhibits growth of PCa cells. (A–C) Myc-CaP, C4-2b and RWPE-1 cells were 2mM metformin for up to 72 h or (D–F) at indicated concentrations for 48h or left untreated (control, PBS vehicle). Following treatment, cell density was measured using the Alamar-Blue assay as described in Materials and methods. Each bar represents the mean ± SD of three independent samples. *P < 0.05, **P < 0.01 when compared with vehicle. Representative photos of each cell type treated with metformin or vehicle are shown.

Fig. 4.

Metformin induces G₁ cell cycle arrest. (A) Myc-CaP and (B) C4-2b cells were treated with 2mM metformin or PBS for 24 h. The percentage of cells within the different phases of the cell cycle was determined using flow cytometry as described in Materials and methods. *P < 0.01, **P < 0.001, Bonferroni (Dunn) t-tests after ANOVA. Each bar represents the mean ± SD of three independent samples. (C–F) Myc-CaP cells were treated with PBS or 2mM metformin for 24h and proteins were harvested. (C) Cyclin D1, (E) p21 and (F) p27 protein levels were determined using western blot analysis. Membranes were stripped and reblotted for actin as a loading control.

Fig. 5.

Metformin increases apoptosis in PCa cells. (A) Myc-CaP and (B) C4-2b cells were treated with PBS or 2mM metformin for 48 h before analyzing apoptosis using an annexin V–fluorescein isothiocyanate apoptosis detection kit as described in Materials and methods. *P < 0.05 and **P < 0.01, Bonferroni (Dunn) t-tests after ANOVA. Representative experiments are shown. (C and D) Myc-CaP and/or C4-2b cells were treated with PBS or metformin at indicated concentration for 24h and proteins from whole cell lysate were harvested. (C) Bak and (D) Bcl-2 protein levels were determined using western blot analysis. Membranes were stripped and reblotted for actin as a loading control.

Fig. 6.

Metformin reduces proliferation and AR expression in vivo. Immunohistochemistry of prostates (8–9 weeks, n = 5) from mice that were treated orally with metformin (200 mg/kg/day) for 4 weeks on (A and B) cell proliferation marker Ki-67 and (C and D) AR. Percentage of stained cells was measured using the ImageJ processing program. *P < 0.0001, one-tailed Student's t-test.