Tumor suppressor BRCA1 is expressed in prostate cancer and controls insulin-like growth factor I receptor (IGF-IR) gene transcription in an androgen receptor-dependent manner

Abstract

Purpose: The insulin-like growth factor (IGF) system plays an important role in prostate cancer. The BRCA1 gene encodes a transcription factor with tumor suppressor activity. The involvement of BRCA1 in prostate cancer, however, has not yet been elucidated. The purpose of the present study was to examine the functional correlations between BRCA1 and the IGF system in prostate cancer.

Experimental design: An immunohistochemical analysis of BRCA1 was done on tissue microarrays comprising 203 primary prostate cancer specimens. In addition, BRCA1 levels were measured in prostate cancer xenografts and in cell lines representing early stages (P69 cells) and advanced stages (M12 cells) of the disease. The ability of BRCA1 to regulate IGF-I receptor (IGF-IR) expression was studied by coexpression experiments using a BRCA1 expression vector along with an IGF-IR promoter-luciferase reporter.

Results: We found significantly elevated BRCA1 levels in prostate cancer in comparison with histologically normal prostate tissue (P<0.001). In addition, an inverse correlation between BRCA1 and IGF-IR levels was observed in the androgen receptor (AR)-negative prostate cancer-derived P69 and M12 cell lines. Coexpression experiments in M12 cells revealed that BRCA1 was able to suppress IGF-IR promoter activity and endogenous IGF-IR levels. On the other hand, BRCA1 enhanced IGF-IR levels in LNCaP C4-2 cells expressing an endogenous AR.

Conclusions: We provide evidence that BRCA1 differentially regulates IGF-IR expression in AR-positive and AR-negative prostate cancer cells. The mechanism of action of BRCA1 involves modulation of IGF-IR gene transcription. In addition, immunohistochemical data are consistent with a potential survival role of BRCA1 in prostate cancer.

Figure 1. Expression of BRCA1 in prostate cancer

Two tissue microarrays including 203 specimens were immunostained with BRCA1 antibody C20 as described in Materials and methods. (A) Gleason score 6 cancer glands (red arrows) expressing intense immunoreactivity and a benign gland (green arrow) expressing faint immunoreactivity. (B) Gleason score 6 cancer glands expressing faint immunoreactivity (red arrow) and adjacent normal gland (green arrow) with intraluminal crystalloid lacking immunoreactivity. (C) Statistical analysis of BRCA1 staining in prostate cancer versus normal adjacent prostate epithelium (average of the respective scores with standard deviations).

Figure 1. Expression of BRCA1 in prostate cancer

Two tissue microarrays including 203 specimens were immunostained with BRCA1 antibody C20 as described in Materials and methods. (A) Gleason score 6 cancer glands (red arrows) expressing intense immunoreactivity and a benign gland (green arrow) expressing faint immunoreactivity. (B) Gleason score 6 cancer glands expressing faint immunoreactivity (red arrow) and adjacent normal gland (green arrow) with intraluminal crystalloid lacking immunoreactivity. (C) Statistical analysis of BRCA1 staining in prostate cancer versus normal adjacent prostate epithelium (average of the respective scores with standard deviations).

Figure 2. Nuclear BRCA1 staining in prostate cancer

Nuclear staining by anti-BRCA1 of both benign epithelial (green arrow) and stromal (black arrow) cells in a section of prostate (A) was abolished when the primary anti-BRCA1 antibody was preincubated with a BRCA1 blocking peptide (B). (C) Tissue lysate from LuCap 35 human prostate cancer xenograft was electrophoresed through SDS-PAGE and immunoblotted with anti-BRCA1 antibody in the absence or presence of a BRCA1 blocking peptide. Note loss of BRCA1 band with ten-fold molar excess of the blocking peptide.

Figure 3. Western immunoblots with BRCA1, IGF-IR, and ERK antibodies of cell extracts of 27 individual human prostate cancer xenografts grown in SCID mice

Xenografts with a (v) or (ai) after the number are androgen-independent lines grown in castrate mice. All of the other lines were from intact mice. Tissue was kindly supplied by Dr. Robert Vessella.

Figure 4. Expression of endogenous IGF-IR and BRCA1 in P69 and M12 prostate cancer cells

(A) Western blot analysis of IGF-IR and BRCA1 expression in prostate cancer cells. Untransfected M12 and P69 cells were lysed in the presence of protease inhibitors, as indicated in Materials and Methods. Equal amounts of protein (100 μg) were separated by 8% SDS-PAGE, transferred to nitrocellulose filters, and blotted with anti-BRCA1 (upper panel), anti-IGF-IR (medium panel), and anti-tubulin (lower panel) antibodies. The positions of the ~220-kDa BRCA1, ~97-kDa IGF-IR β-subunit, and ~50-kDa tubulin proteins are indicated. The figure shows a typical Western blot repeated at least three times with similar results. (B) Quantitative Real Time-PCR of BRCA1 mRNA levels in prostate cancer cells. Total RNA was prepared from P69 and M12 cells and BRCA1 mRNA and GAPDH mRNA values were measured using the TaqMan^® Real-time PCR system. Analysis of the data was performed as described under Materials and Methods. *, p < 0.01 versus M12 cells.

Figure 4. Expression of endogenous IGF-IR and BRCA1 in P69 and M12 prostate cancer cells

(A) Western blot analysis of IGF-IR and BRCA1 expression in prostate cancer cells. Untransfected M12 and P69 cells were lysed in the presence of protease inhibitors, as indicated in Materials and Methods. Equal amounts of protein (100 μg) were separated by 8% SDS-PAGE, transferred to nitrocellulose filters, and blotted with anti-BRCA1 (upper panel), anti-IGF-IR (medium panel), and anti-tubulin (lower panel) antibodies. The positions of the ~220-kDa BRCA1, ~97-kDa IGF-IR β-subunit, and ~50-kDa tubulin proteins are indicated. The figure shows a typical Western blot repeated at least three times with similar results. (B) Quantitative Real Time-PCR of BRCA1 mRNA levels in prostate cancer cells. Total RNA was prepared from P69 and M12 cells and BRCA1 mRNA and GAPDH mRNA values were measured using the TaqMan^® Real-time PCR system. Analysis of the data was performed as described under Materials and Methods. *, p < 0.01 versus M12 cells.

Figure 5. Regulation of IGF-IR gene expression by BRCA1 in prostate cancer cells

(A) Regulation of IGF-IR promoter activity by BRCA1. M12 cells were transiently transfected with 1 μg of the p(−476/+640)LUC IGF-IR promoter-luciferase reporter construct, along with 1 μg of the BRCA1 expression vector (or empty pcDNA3) and 0.3 μg of the pCMVβ plasmid, using the Jet-PEI^™ reagent. Forty hours after transfection cells were harvested and the levels of luciferase and β-galactosidase were measured. Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. Results are mean ± S.E.M. of three independent experiments, performed in duplicate dishes. *, p < 0.01 versus pcDNA3-transfected cells. (B) Quantitative Real Time-PCR of BRCA1 mRNA in P69-derived and M12-derived BRCA1 stable overexpressing clones. BRCA1 mRNA levels were normalized to GAPDH mRNA levels and expressed in arbitrary units. Analyses were done as indicated in the Legend to Figure 4. Control, pcDNA3-transfected clones; BRCA1, full length BRCA1-transfected clones. (C) Regulation of endogenous IGF-IR levels by BRCA1. Stable BRCA1-overexpressing (or pcDNA-3 transfected) P69 and M12 cells were lysed and endogenous IGF-IR levels were measured by Western blots. Blots were re-probed with anti-tubulin as a loading control. (D) Regulation of total and phosphorylated IGF-IR levels and downstream mediators by BRCA1. M12 cells were transfected with a BRCA1 expression vector (or empty pcDN3 vector) and after 48 h cells were lysed and Western blots were performed using antibodies against BRCA1, tubulin, and total and phospho-IGF-IR, Akt, and Erk.

Figure 5. Regulation of IGF-IR gene expression by BRCA1 in prostate cancer cells

(A) Regulation of IGF-IR promoter activity by BRCA1. M12 cells were transiently transfected with 1 μg of the p(−476/+640)LUC IGF-IR promoter-luciferase reporter construct, along with 1 μg of the BRCA1 expression vector (or empty pcDNA3) and 0.3 μg of the pCMVβ plasmid, using the Jet-PEI^™ reagent. Forty hours after transfection cells were harvested and the levels of luciferase and β-galactosidase were measured. Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. Results are mean ± S.E.M. of three independent experiments, performed in duplicate dishes. *, p < 0.01 versus pcDNA3-transfected cells. (B) Quantitative Real Time-PCR of BRCA1 mRNA in P69-derived and M12-derived BRCA1 stable overexpressing clones. BRCA1 mRNA levels were normalized to GAPDH mRNA levels and expressed in arbitrary units. Analyses were done as indicated in the Legend to Figure 4. Control, pcDNA3-transfected clones; BRCA1, full length BRCA1-transfected clones. (C) Regulation of endogenous IGF-IR levels by BRCA1. Stable BRCA1-overexpressing (or pcDNA-3 transfected) P69 and M12 cells were lysed and endogenous IGF-IR levels were measured by Western blots. Blots were re-probed with anti-tubulin as a loading control. (D) Regulation of total and phosphorylated IGF-IR levels and downstream mediators by BRCA1. M12 cells were transfected with a BRCA1 expression vector (or empty pcDN3 vector) and after 48 h cells were lysed and Western blots were performed using antibodies against BRCA1, tubulin, and total and phospho-IGF-IR, Akt, and Erk.

Figure 5. Regulation of IGF-IR gene expression by BRCA1 in prostate cancer cells

(A) Regulation of IGF-IR promoter activity by BRCA1. M12 cells were transiently transfected with 1 μg of the p(−476/+640)LUC IGF-IR promoter-luciferase reporter construct, along with 1 μg of the BRCA1 expression vector (or empty pcDNA3) and 0.3 μg of the pCMVβ plasmid, using the Jet-PEI^™ reagent. Forty hours after transfection cells were harvested and the levels of luciferase and β-galactosidase were measured. Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. Results are mean ± S.E.M. of three independent experiments, performed in duplicate dishes. *, p < 0.01 versus pcDNA3-transfected cells. (B) Quantitative Real Time-PCR of BRCA1 mRNA in P69-derived and M12-derived BRCA1 stable overexpressing clones. BRCA1 mRNA levels were normalized to GAPDH mRNA levels and expressed in arbitrary units. Analyses were done as indicated in the Legend to Figure 4. Control, pcDNA3-transfected clones; BRCA1, full length BRCA1-transfected clones. (C) Regulation of endogenous IGF-IR levels by BRCA1. Stable BRCA1-overexpressing (or pcDNA-3 transfected) P69 and M12 cells were lysed and endogenous IGF-IR levels were measured by Western blots. Blots were re-probed with anti-tubulin as a loading control. (D) Regulation of total and phosphorylated IGF-IR levels and downstream mediators by BRCA1. M12 cells were transfected with a BRCA1 expression vector (or empty pcDN3 vector) and after 48 h cells were lysed and Western blots were performed using antibodies against BRCA1, tubulin, and total and phospho-IGF-IR, Akt, and Erk.

Figure 5. Regulation of IGF-IR gene expression by BRCA1 in prostate cancer cells

(A) Regulation of IGF-IR promoter activity by BRCA1. M12 cells were transiently transfected with 1 μg of the p(−476/+640)LUC IGF-IR promoter-luciferase reporter construct, along with 1 μg of the BRCA1 expression vector (or empty pcDNA3) and 0.3 μg of the pCMVβ plasmid, using the Jet-PEI^™ reagent. Forty hours after transfection cells were harvested and the levels of luciferase and β-galactosidase were measured. Promoter activities are expressed as luciferase values normalized for β-galactosidase levels. Results are mean ± S.E.M. of three independent experiments, performed in duplicate dishes. *, p < 0.01 versus pcDNA3-transfected cells. (B) Quantitative Real Time-PCR of BRCA1 mRNA in P69-derived and M12-derived BRCA1 stable overexpressing clones. BRCA1 mRNA levels were normalized to GAPDH mRNA levels and expressed in arbitrary units. Analyses were done as indicated in the Legend to Figure 4. Control, pcDNA3-transfected clones; BRCA1, full length BRCA1-transfected clones. (C) Regulation of endogenous IGF-IR levels by BRCA1. Stable BRCA1-overexpressing (or pcDNA-3 transfected) P69 and M12 cells were lysed and endogenous IGF-IR levels were measured by Western blots. Blots were re-probed with anti-tubulin as a loading control. (D) Regulation of total and phosphorylated IGF-IR levels and downstream mediators by BRCA1. M12 cells were transfected with a BRCA1 expression vector (or empty pcDN3 vector) and after 48 h cells were lysed and Western blots were performed using antibodies against BRCA1, tubulin, and total and phospho-IGF-IR, Akt, and Erk.

Figure 6. Effect of AR status on BRCA1 action

(A) LnCaP C4-2 cells were transfected with a BRCA1 expression vector (hatched columns) or an empty vector (solid columns) as described in Materials and Methods. Twenty-four hours after transfection dihydrotestosterone (DHT) 10⁻⁹M was added to the medium and total RNA was collected after an additional 3 h. Quantitative RT-PCR was run for the androgen regulated gene TSC22 and IGF-IR mRNAs. *, p < 0.01 versus control. (B) The AAR3 luciferase reporter construct was cotransfected onto LnCaP C4-2 cells along with a BRCA1 expression vector (solid columns) or an empty vector (open columns). Twenty-four hours after transfection DHT 10⁻⁹M (or diluent) was added to the cells for an additional 3 h, after which luciferase activity was measured. Note the significant increase in reporter activity in the BRCA1-containing cells compared to control. RLU, relative luciferase units. (C) Effect of BRCA1 on cellular proliferation. BRCA1-expressing and control P69 and M12 cells were plated in 6-well plates at a density of 2×10⁵ cells/well in complete medium. Cells were trypsinized every 24 h, stained with Trypan Blue, and counted with a hemocytometer. The number of cells at time 0 was assigned a value of 100%. The y-axis denotes cell numbers (percentage of cells at time 0). Bars are mean ± S.D. (n=3 independent experiments). Proliferation rates of BRCA1-expressing P69 and M12 cells at 72 h were significantly higher than control, pcDNA3-transfected cells (p < 0.05).

Figure 6. Effect of AR status on BRCA1 action

(A) LnCaP C4-2 cells were transfected with a BRCA1 expression vector (hatched columns) or an empty vector (solid columns) as described in Materials and Methods. Twenty-four hours after transfection dihydrotestosterone (DHT) 10⁻⁹M was added to the medium and total RNA was collected after an additional 3 h. Quantitative RT-PCR was run for the androgen regulated gene TSC22 and IGF-IR mRNAs. *, p < 0.01 versus control. (B) The AAR3 luciferase reporter construct was cotransfected onto LnCaP C4-2 cells along with a BRCA1 expression vector (solid columns) or an empty vector (open columns). Twenty-four hours after transfection DHT 10⁻⁹M (or diluent) was added to the cells for an additional 3 h, after which luciferase activity was measured. Note the significant increase in reporter activity in the BRCA1-containing cells compared to control. RLU, relative luciferase units. (C) Effect of BRCA1 on cellular proliferation. BRCA1-expressing and control P69 and M12 cells were plated in 6-well plates at a density of 2×10⁵ cells/well in complete medium. Cells were trypsinized every 24 h, stained with Trypan Blue, and counted with a hemocytometer. The number of cells at time 0 was assigned a value of 100%. The y-axis denotes cell numbers (percentage of cells at time 0). Bars are mean ± S.D. (n=3 independent experiments). Proliferation rates of BRCA1-expressing P69 and M12 cells at 72 h were significantly higher than control, pcDNA3-transfected cells (p < 0.05).