The cancer-related Runx2 protein enhances cell growth and responses to androgen and TGFbeta in prostate cancer cells

Abstract

Prostate cancer cells often metastasize to bone where osteolytic lesions are formed. Runx2 is an essential transcription factor for bone formation and suppresses cell growth in normal osteoblasts but may function as an oncogenic factor in solid tumors (e.g., breast, prostate). Here, we addressed whether Runx2 is linked to steroid hormone and growth factor signaling, which controls prostate cancer cell growth. Protein expression profiling of prostate cell lines (i.e., PC3, LNCaP, RWPE) treated with 5alpha-dihydrotestosterone (DHT) or tumor growth factor beta (TGFbeta) revealed modulations in selected cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors that are generally consistent with mitogenic responses. Endogenous elevation of Runx2 and diminished p57 protein levels in PC3 cells are associated with faster proliferation in vitro and development of larger tumors upon xenografting these cells in bone in vivo. To examine whether TGFbeta or DHT signaling modulates the transcriptional activity of Runx2 and vice versa, we performed luciferase reporter assays. In PC3 cells that express TGFbetaRII, TGFbeta and Runx2 synergize to increase transcription of synthetic promoters. In LNCaP cells that are DHT responsive, Runx2 stimulates the androgen receptor (AR) responsive expression of the prostate-specific marker PSA, perhaps facilitated by formation of a complex with AR. Our data suggest that Runx2 is mechanistically linked to TGFbeta and androgen responsive pathways that support prostate cancer cell growth.

Figure 1. Cell cycle responses of prostate cancer cells to androgen and TGFβ

Prostate cancer cell lines PC-3-b and LNCaP, and immortalized prostate cells RWPE were treated for 24 h with DHT (10 nM) or TGFβ (10 ng/ml) for 24 h in 2% CCS medium. (A) Cell lines respond differently to treatment dependent on the presence or absence of a functional androgen or TGFβ pathway. Equal amounts of protein were subjected to western blot analysis and stained for cell cycle markers. Dotted boxes indicate proteins that show more than 2 fold increase or decrease compared to control treatments. (B) mRNA detection under the same treatment conditions. Grey boxes indicate no effect upon treatment, colored boxes indicate, respectively, more than 2 fold increase (red) or decrease (blue) in mRNA levels. Transcript levels were normalized to GAPDH mRNA levels.

Figure 2. Endogenous levels of Runx2, cell cycle proteins, and AR in prostate cancer cells

(A) Prostate cancer cells were analyzed for protein expression with western blot for Runx2, p57, p27, and p21, Cyclin D1 and AR. Equal amounts of protein were loaded for all cell lines, with tubulin as a loading control. HeLa cells were included as a control cell line. Dotted boxes indicate interesting differences in Runx2 and p57 expression in two PC-3 sublines (PC-3-a and PC-3-b). For comparison, mRNA levels for Runx2 (B) and p57 (C) are shown in the lower panels. The graphs show data from representative and reproducible experiments.

Figure 3. Runx2 expression in PC-3 cells is related to higher cell growth rate and larger tumor volume

(A) Upper panel depicts tumor size resulting from PC-3-a and PC-3-b cells. Cells were injected in tibiae of SCID mice (n=3), and photographs were taken 6 weeks after injection. Representative images are shown. (B) Cell growth studies were performed with PC-3 sublines a and b. PC-3-a cells with higher Runx2 expression grow faster than PC-3-b cells that express higher p57 levels (see Figure 2). Data shown are from averages of two independent growth curves counted in quadruplicate. (C) Growth curves in which PC-3-a cells were subjected to Runx2 siRNA. At day 4 there is less cell growth compared to control treated cells (non-silencing, NS) and mock-treated cells. Data shown are from averages of cells counted in triplicate.

Figure 4. Runx2 and TGFβ responses cooperate in PC-3 cells

(A) Treatment of PC-3 (subline a) cells with TGFβ (10 ng/ml) for 24 h increased p21 levels but did not significantly affect Runx2 protein levels. (B) In functional luciferase reporter assays, TGFβ (10 or 50 ng/ml) stimulated Runx2 activity in a dose-dependent manner. Empty vector plasmid (control) was transfected or Runx2-HA plasmid (250 ng). (C) Runx2 enhances the TGFβ response in PC-3 cells. Averages were taken from duplicate experiments and firefly luciferase activity was normalized to Renilla values.

Figure 5. Runx2 interacts with AR and increases androgen activity and PSA production in LNCaP cells

(A) Treatment of LNCaP cells with DHT (10 nM) and/or TGFβ (10 ng/ml) for 24 h did not affect AR or Runx2 expression at the protein level. (B) DHT (20 nM) does not significantly increase Runx2 activity in LNCaP cells. Cells were transfected with 50 ng, 100 ng and 200 ng Runx2-HA plasmid DNA or empty factor (EV) as a negative control. Luciferase activity was normalized to Renilla values. A representative experiment is shown from 3 independent experiments performed in triplicate. (C) Runx2 enhances ARE-activity in LNCaP cells in a dose-dependent manner. A representative experiment is shown from 3 independent experiments performed in triplicate. (D) Pulldown with Runx2 antibody in immunoprecipitation assay using LNCaP cell lysate demonstrates that Runx2 and AR physically interact. (E) DHT incubation (10 nM) for 24 h stimulates PSA protein production in LNCaP cells as measured by ELISA. (F) Forced Runx2-HA expression (250 ng, 500 ng, 1000 ng) in LNCaP cells strongly increases PSA production after 24 h of DHT (20 nM) treatment. A representative experiment is shown performed in triplicate.