FGFR1 abrogates inhibitory effect of androgen receptor concurrent with induction of androgen-receptor variants in androgen receptor-negative prostate tumor epithelial cells

Abstract

Background: Despite dramatic positive effects, there is evidence that the androgen receptor (AR) may negatively influence prostate tumor progression. Understanding the AR repressor function and how it is subverted is of particular importance in anti-androgen and AR intervention strategies.

Methods: AR, resident FGFR2IIIb, and ectopic FGFR1 were expressed by transfection in the AR-negative epithelial cell line DTE that predominates in cell culture of AR-positive androgen-responsive model Dunning R3327 rat prostate tumors. Androgen-responsiveness at transcription was measured by a luciferase reporter. Cell population growth rates were assessed by cell counts, DNA synthesis, and expression of cell cycle genes. AR variants (ARVs) were assessed by immunochemistry and nuclease protection of mRNA.

Results: Expression of AR inhibited cell population growth of AR-negative DTE cells at the G1-S phase of the cell cycle. Ectopic FGFR1, but not resident FGFR2IIIb abrogated the growth inhibitory effects of AR. Appearance of ARVs was coincident with co-expression of FGFR1 and AR and abrogation of the AR-dependent inhibition of cell growth.

Conclusions: DTE cells may represent non-malignant AR-negative progenitors whose population is restricted by activation of AR in vivo. Ectopic expression of epithelial FGFR1, a common observation in tumors, overrides the inhibition of AR and thus may contribute to evolution of androgen and AR independent tumors. These results are consistent with the notion that some tumor cells are negatively restricted by AR and are unleased by androgen-deprivation or ectopic expression of FGFR1. ARV's may play a role in the bypass of the negative restrictions of AR.

Fig. 1

DTE cell cultures display basal and luminal epithelial cell markers, but no synaptophysin-positive neuroendocrine cells. DTE and DTE/R1 cells on cover slides were immunostained with the indicated antibodies. The nuclei were counterstained with To-Pro 3. Inserts are prostate tissue sections stained with synaptophysin for positive controls. Images were representative of the entire cultures collected with a Zeiss LSM 510 Confocal Microscope. CK, cytokeratin; Syn, synaptophysin.

Fig. 2

AR expression in Dunning tumor tissue and cells. AR mRNA expression in the indicated tissues and cells were analyzed by RT-PCR (A) and nuclease protection of total RNA (40 ug) extracted from the indicated sources (B) as described in Materials and Methods. mRNA was protected with the 57-164 probe described in Fig. 4. C. Cell lysates were subjected to immunoblot analysis with anti-AR antibody. C, control; NP, normal rat prostate tissue; DT, Dunning R3327PAP tumor tissue; AT3, Dunning R3327AT3 tumor tissue; DTE, nonmalignant epithelial cells that emerge in culture from the Dunning R3327PAP tumor.

Fig. 3

Expression of AR in DTE cells suppresses cell proliferation. A. Luciferase activity was assessed in control DTE and AR-expressing DTE/AR cells after transient transfection with pGL3-ARR₂PB-LUC and pcDNA3.1-Zeo-β-gal in the presence or absence of DHT. Luciferase activity induced by DHT was normalized and expressed as the relative increase over DTE cells in absence of DHT. Expression of AR assessed by immunoblot is shown in the lower panels. B. DTE and DTE/AR cells were grown in 5 percent charcoal-stripped serum with or without 10 nM DHT. The population doubling times were calculated from time points described in Materials and Methods. C. Cells were grown in 5 percent charcoal-stripped serum with DHT added at the indicated concentrations and ³H-thymidine measured by the sequence described in Materials and Methods. D. Cells were treated with 10 nM DHT or vehicle for 24 hr and lysates then subjected to analyses by immunoblot with the indicated antibodies.

Fig. 4

Ectopic FGFR1 abrogates androgen response and AR-dependent inhibition of cell growth concurrent with C-terminal truncation of AR. A. The indicated permanently transfected variants of DTE cells were transiently transfected with pGL3-ARR₂PB-LUC and pcDNA3.1-Zeo/β-gal and relative luciferase activity assess in presence or absence of DHT as described in Fig. 2A. A-E were five different clones of DTE/R1AR cells. B. Cell population doubling rates were assessed in the variant DTE cells in (A) as described in Fig. 2B. C, D. The indicated cell lysates were subjected to immunoblot analyses with anti-AR N-20 against the AR N terminus (upper panel) and anti-AR C-19 against the AR C-terminus (middle panel).

Fig. 5

Androgen receptor variants (ARVs) in cells expressing ectopic FGFR1 and AR. A. Schematic of AR mRNA protection probes. Probes are numbered in respect to amino acids (aa) coded by probes overlapping the N-terminal transactivating domain (TAD), the DNA binding domain (DBD) and the C-terminal ligand binding domain (LBD) (47). B. Total RNA from the indicated DTE variants and clonal lines cells (Fig. 3) was analyzed by RPA with the indicated probes. Arrows indicate the band corresponding to the full length probe.