Myc confers androgen-independent prostate cancer cell growth

Abstract

Prostate cancer is one of the most diagnosed and mortal cancers in western countries. A major clinical problem is the development of androgen-independent prostate cancer (AIPC) during antihormonal treatment. The molecular mechanisms underlying the change from androgen dependence to independence of these tumors are poorly understood and represent a challenge to develop new therapies. Based on genetic data showing amplification of the c-myc gene in AIPC, we studied the ability of c-myc to confer AIPC cell growth. Human androgen-dependent prostate cancer cells overexpressing c-myc grew independently of androgens and presented tumorigenic properties in androgen-depleted conditions. Analysis of signalling pathways by pharmacological inhibitors of the androgen receptor (AR) or by RNA interference directed against AR or c-myc showed that c-myc acted downstream of AR through multiple growth effectors. Thus c-myc is required for androgen-dependent growth and following ectopic expression can induce androgen-independent growth. Moreover, RNA interference directed against c-myc showed that growth of human AIPC cells, AR-positive or -negative, required c-myc expression. Furthermore, we showed that c-myc-overexpressing cells retain a functional p53 pathway and thus respond to etoposide.

Figure 1

c-myc expression renders LNCaP cells androgen-independent. LNCaP cells were infected with GFP or c-myc retroviral vectors and selected with hygromycin. One day after seeding, bicalutamide treatment was started and renewed every 2 days. (a) Growth curve assays. Cells were seeded in triplicate in 24-well plates. At 4-day intervals, the cell number was analyzed. The black lines represent untreated cells, and the gray lines show bicalutamide-treated cells. (b) Colony-formation assays. Cells (200,000) were seeded onto plates, and after 15 days of treatment with bicalutamide, the cells were stained using crystal violet. (c) DNA content analysis. After 11 days of bicalutamide treatment, cells were fixed, stained using propidium iodide, and analyzed. (d) Cell division analysis. After infection and selection, the cells were stained with CFDA SE dye and seeded in the presence of bicalutamide. Flow cytometry analysis on 10,000 cells was performed at day 9 and the number of cell divisions is presented. (e) Soft agar assays. Cells were plated in soft agar media containing or not containing androgen. Analysis of the appearance of the foci was performed after 2 weeks.

Figure 2

c-myc does not act through AR signalling. (a) c-myc does not induce PSA expression. Cells were treated with or without bicalutamide during 3 days. Cell extracts were resolved on SDS-PAGE gel, transferred, and analyzed for c-myc, PSA, and PSMA expression. β-Actin was used as loading control. (b) Cells were cultured with or without bicalutamide for 12 days after seeding. Representative pictures are presented. (c) Cellular extracts were prepared before bicalutamide treatment or after 4 and 12 days of treatment, resolved by SDS-PAGE, and analyzed by immunoblotting against neuron-specific enolase (NSE) and β-actin as a loading control. (d) Silencing of AR expression. LNCaP cells were infected by pRS or pRS/AR and drug-selected for 3 days. Next, cellular extracts were prepared and analyzed by immunoblotting for AR expression. β-Actin was used as loading control. (e) LNCaP or LNCaP/myc cells were infected by pRS or pRS/AR and drug-selected. Ten days after seeding 500,000 cells, crystal violet staining was performed and relative cell numbers were calculated.

Figure 3

c-myc is an essential downstream growth effector of AR. (a) c-myc expression during AR inhibition. Media containing bicalutamide was added to inhibit AR activity for the indicated times. RNA and protein extracts were prepared and analyzed by RT-PCR and Western blot, respectively. (b) c-myc expression during AR activation. Cells were maintained in media with CDS and bicalutamide for 2 days. Next, normal media was added to restimulate AR activity for the indicated times. Cellular extracts were prepared and analyzed. (c) Silencing of c-myc expression. Cells were infected and selected for 2 days, then cellular extracts were prepared and analyzed for c-myc and β-actin expression. (d) Colony-formation assays. Cells (400,000) were infected with pRS and pRS/myc retroviral vectors, and after 11 days the cells were stained with crystal violet. (e) Expression of growth regulators during AR inhibition. Cellular extracts were prepared before bicalutamide treatment or after 4 days and 12 days of treatment, resolved by SDS-PAGE, and analyzed by immunoblotting against c-myc, p16, RB, E2F1, cyclin A, Skp2, p27, CDK4, cyclin D1, hTERT, ornithine decarboxylase (ODC), and β-actin as a loading control. (f) Growth curve assays. Cells were infected successively with the different retroviruses and treated with polyamine. The growth curves were performed as described above. Shown is percentage of growth compared with 100% for c-myc–expressing cells treated with bicalutamide at day 12.

Figure 4

c-myc is required for the growth of human AIPC cells. (a) Cells were treated with or without bicalutamide for 5 days. Cellular extracts were prepared, resolved by SDS-PAGE, and analyzed by immunoblotting against c-myc, AR, and β-actin as a loading control. (b) The cells were seeded at low density and treated with (gray bars) or without (black bars) bicalutamide. After 2 weeks of treatment for LAPC-4 cells or 10 days for the other cell lines, crystal violet staining was performed and relative cell numbers were calculated and normalized to 100% for untreated cells. (c) Cells were seeded at low density and infected with pRS (black bars) or pRS/myc (gray bars). After drug selection, the cells were split 1 to 8 and allowed to grow for 1–2 weeks. Crystal violet staining was performed and relative cell numbers were calculated and normalized to 100% for untreated cells.

Figure 5

c-myc cells are sensitive to the p53 pathway. (a) One million infected cells were seeded in 10-cm dishes. One day later, they were treated overnight with or without 25 μM (LNCaP) or 50 μM (22Rv1) etoposide. Cellular extracts were prepared, resolved, and transferred before analysis for p53, p21, and GADD45 expression. β-Actin was used as a loading control. (b) After infection, 750,000 cells were seeded in 10-cm dishes and treated with 2 μM etoposide at day 1 and day 7. The cells were stained with crystal violet at day 17, and relative cell numbers were calculated. (c) After infection, 250,000 LNCaP cells were seeded, treated with bicalutamide for 15 days, stained with crystal violet, and analyzed. (d) After infection, 750,000 cells were seeded in 10-cm dishes and treated with bicalutamide every 2 days and with etoposide at day 1 and day 7. The cells were stained with crystal violet at day 19 and analyzed.