Perlecan, a candidate gene for the CAPB locus, regulates prostate cancer cell growth via the Sonic Hedgehog pathway

Abstract

Background: Genetic studies associated the CAPB locus with familial risk of brain and prostate cancers. We have identified HSPG2 (Perlecan) as a candidate gene for CAPB. Previously we have linked Perlecan to Hedgehog signaling in Drosophila. More recently, we have demonstrated the importance of Hedgehog signaling in humans for advanced prostate cancer.

Results: Here we demonstrate Perlecan expression in prostate cancer, and its function in prostate cancer cell growth through interaction and modulation of Sonic Hedgehog (SHH) signaling. Perlecan expression in prostate cancer tissues correlates with a high Gleason score and rapid cell proliferation. Perlecan is highly expressed in prostate cancer cell lines, including androgen insensitive cell lines and cell lines selected for metastatic properties. Inhibition of Perlecan expression in these cell lines decreases cell growth. Simultaneous blockade of Perlecan expression and androgen signaling in the androgen-sensitive cell line LNCaP was additive, indicating the independence of these two pathways. Perlecan expression correlates with SHH in tumor tissue microarrays and increased tumor cell proliferation based on Ki-67 immunohistochemistry. Inhibition of Perlecan expression by siRNA in prostate cancer cell lines decreases SHH signaling while expression of the downstream SHH effector GLI1 rescues the proliferation defect. Perlecan forms complexes with increasing amounts of SHH that correlate with increasing metastatic potential of the prostate cancer cell line. SHH signaling also increases in the more metastatic cell lines. Metastatic prostate cancer cell lines grown under serum-starved conditions (low androgen and growth factors) resulted in maintenance of Perlecan expression. Under low androgen, low growth factor conditions, Perlecan expression level correlates with the ability of the cells to maintain SHH signaling.

Conclusion: We have demonstrated that Perlecan, a candidate gene for the CAPB locus, is a new component of the SHH pathway in prostate tumors and works independently of androgen signaling. In metastatic tumor cells increased SHH signaling correlates with the maintenance of Perlecan expression and more Perlecan-SHH complexes. Perlecan is a proteoglycan that regulates extracellular and stromal accessibility to growth factors such as SHH, thus allowing for the maintenance of SHH signaling under growth factor limiting conditions. This proteoglycan represents an important central regulator of SHH activity and presents an ideal drug target for blocking SHH effects.

Figure 1

Perlecan protein levels in human prostate tumors. Immunohistochemistry of Perlecan protein in prostate cancer (A) and normal prostate (B). Perlecan is present as a secreted protein in the tumor gland lumens (C) but not in the lumens of benign glands or benign corpora amylacea secretions (D). Staining is also seen in metastatic prostate cancer specimens (E). Secondary antibody alone control fails to demonstrate staining (F). All images originally photographed at 400 × magnification. Quantitation of Perlecan mRNA expression by Real Time PCR (G) or protein by digitized dot blot (H) in normal prostate and tumor samples from individual patients presented as fold change in tumor versus normal. Gleason scores for the tumors are listed. Red numbers or columns indicate patients previously shown to have increased expression of SHH, PTCH1 and GLI1 [7].

Figure 2

Perlecan expression and functional analysis in cell lines. A. Relative Perlecan mRNA levels from Realtime PCR (LNCaP series) and spotted cDNA microarray data (LNCaP, DU145, PC3). All samples presented normalized to LNCaP at 100%. Androgen sensitive: LNCaP. Androgen insensitive: C4, C4-2, C4-2B, PC3, DU145. B. Inhibition by Perlecan siRNA decreases prostate cancer cell proliferation. BrdU incorporation in the LNCaP, C4, C4-2 and C4-2B cell lines. All samples were normalized to control (scrambled siRNA treated) cells at 100%. Black bars represent control samples transfected with scrambled siRNA. Grey bars represent samples transfected with Perlecan siRNA. Error bars represent n = 3 independent samples. C. Additive effect of Perlecan siRNA and androgen blockade on cell proliferation. BrdU incorporation in LNCaP cells after Perlecan siRNA and/or bicalutimide (Casodex) treatment. Control and Casodex alone samples were treated with a scrambled siRNA. p < 0.0001 for comparisons between groups. Error bars represent n = 6 for independent transfections.

Figure 3

Co-localization of Shh and Perlecan, and correlation with Ki-67 staining. Immunohistochemistry for Sonic Hedgehog (A), demonstrating both weak cytoplasmic staining in prostate cancer epithelial cells and stronger intraluminal staining of secreted SHH. Co-localization of Perlecan (B) and Sonic Hedgehog (C) in consecutive sections of prostate carcinoma. Examples of co-localization of the secreted proteins in gland lumens are highlighted (red asterisks). All histologic images originally photographed at 400 × magnification. Significant co-localization of Perlecan and SHH staining was associated with higher cellular proliferation rates as indicated by Ki-67 nuclear staining by immunohistochemistry (D).

Figure 4

Perlecan and the SHH-GLI1 pathway. A. Decreased Perlecan and SHH signaling in Perlecan RNAi treated LNCaP cells. Expression of Perlecan, and the SHH signaling molecules PTCH1 and GLI1 as determined by Real Time PCR. Black columns represent control samples, Grey columns represent Perlecan RNAi treated cells. All expression normalized to β-actin levels. Real Time PCR studies were run with an n = 9. Error bars indicate standard deviation. B. Gli-1 transfection restores BrdU Proliferation in Perlecan RNAi treated cells. Percent BrdU incorporation normalized to levels of BrdU incorporation in control (scrambled RNAi treated) cells. BrdU analysis was done with n = 6. Error bars indicate standard deviation. C. Immunoprecipitation with anti-Perlecan antibody pulls down SHH. Co-immunoprecipitation of SHH and Perlecan from equal amounts of medium conditioned by 80% confluent cells. Size marker is indicated. Due to modifications, mature SHH runs as an approximately 22 kD band. Note the increased amount of bound SHH in the C4-2 and C4-2B cell lines. D. Relative expression of the SHH pathway components in LNCaP series cells. Black columns represent SHH mRNA, grey columns represent PTCH mRNA, with expression presented as ratios with respect to expression in LNCaP cells. While SHH is lower, PTCH is higher in the androgen insensitive metastatic cell lines C4-2 and C4-2B compared to LNCaP. All mRNAs by QRT-PCR were normalized to Beta-actin.

Figure 5

Perlecan function under androgen and growth factor limitation. A. Minimal changes in Perlecan mRNA levels in LNCaP-derived cell lines upon serum starvation. RealTime PCR analysis of Perlecan mRNA levels presented as fold increase in Perlecan under normal (black bars) or starved (grey bars) growth conditions. While Perlecan mRNA is decreased in LNCaP, all other cell lines demonstrate no change in Perlecan mRNA levels. B. Top Panel: No change in Perlecan protein levels upon serum starvation. Agarose based western blots from protein extracts derived from exponentially growing or serum starved LNCaP, C4, C4-2, and C4-2B cells. No significant differences are noted in protein levels between the cell lines or under the differing conditions. Bottom Panel: Equivalent amounts of the same samples loaded on traditional SDS-PAGE and probed for GAPDH as a loading control. C. Increases in expression of SHH and Gli-1 mRNA upon serum starvation. RealTime PCR analysis of SHH (black bars) and GLI1 (grey bars) as increased fold change compared to normal growth conditions. Gene expression determined by All Real Time PCR with an n = 9 and normalized to Beta-actin. Error bars indicate standard deviation.

Figure 6

Modulation of androgen and Perlecan regulated Sonic Hedgehog signaling. As changes occur to the tumor microenvironment, prostate cancer cells modulate their use of both androgen and Perlecan mediated Sonic Hedgehog signaling. The use of androgen (T) occurs via the androgen receptor (AR). Perlecan (P) is produced, binds Sonic Hedgehog (S) and signals through the Gli (G) proteins. The heaviness of each arrow indicates relative signaling strength (gene expression, complex formation). Androgen sensitive cells (LNCaP) utilize both androgen and Perlecan-SHH signaling under normal conditions, but decrease Perlecan-SHH signaling under poor growth conditions. In contrast aggressive androgen insensitive cells (C4, C4-2, C4-2B) utilize both pathways, and upregulate the Perlecan-SHH signaling under poor growth conditions. This may occur through increased SHH binding affinity to Perlecan.