Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG-GLI1 signaling

Abstract

Prostate cancer is the most common solid tumor in men, and it shares with all cancers the hallmark of elevated, nonhomeostatic cell proliferation. Here we have tested the hypothesis that the SONIC HEDGEHOG (SHH)-GLI signaling pathway is implicated in prostate cancer. We report expression of SHH-GLI pathway components in adult human prostate cancer, often with enhanced levels in tumors versus normal prostatic epithelia. Blocking the pathway with cyclopamine or anti-SHH antibodies inhibits the proliferation of GLI1+/PSA+ primary prostate tumor cultures. Inversely, SHH can potentiate tumor cell proliferation, suggesting that autocrine signaling may often sustain tumor growth. In addition, pathway blockade in three metastatic prostate cancer cell lines with cyclopamine or through GLI1 RNA interference leads to inhibition of cell proliferation, suggesting cell-autonomous pathway activation at different levels and showing an essential role for GLI1 in human cells. Our data demonstrate the dependence of prostate cancer on SHH-GLI function and suggest a novel therapeutic approach.

Fig. 1.

Expression of SHH–GLI pathway components in normal prostate tissue and prostate tumors. Sections of normal prostate tissue (A, C, E, G, I, L, and O) and prostate tumors (B, D, F, H, J, K, M, N, P, and Q) show hematoxylin and eosin (H&E) staining (A and B) or the expression of SHH (C, D, and I–K), PTCH1 (E, F, and L–N), and GLI1 (G, H, and O–Q). (G Inset) Sense GLI1 probe control showing no background. Prostate tumors have many small epithelial glandular structures. Black arrows point to expressing cells. White arrows point to nonexpressing cells. (R–T) Sections from the tissue microarrays of normal prostate tissue (R) and prostate tumors (S and T) showing expression of SHH protein with an anti-SHH antibody (αSHH Ab) (R–T) and a no-primary antibody control (T Inset). All sections were counterstained with hematoxylin to visualize nuclei and tissue structure. Arrow in T points to localization of SHH protein in the cytoplasm of epithelial cells. e, epithelium; l, lumen; s, stroma; t, tumor. (Scale bar in T is 150 μmin A–H, R, and S, 20 μmin J, M, P, and T, and 10 μmin I–L, N, O, and Q.)

Fig. 2.

Response of prostate tumor cell lines to alterations in the SHH–GLI pathway. (A) PCR analyses for the expression of SHH–GLI pathway components in three cell lines as indicated. In this and all other PCR assays, the expression of the ubiquitous gene GAPDH is measured as quantitative control. (B) Inhibition of prostate cell line proliferation as measured by BrdUrd incorporation in the three prostate cell lines used with cyclopamine. Tomatidine is used as control. (C and D) PCR analyses of the suppression of GLI1 expression in LNCaP cells by cyclopamine treatment at 36 h (C) or of the expression of prostate specific antigen (PSA), GLI1, SHH, and PTCH1 expression in whole prostate tumor tissue (T), primary culture (C), the glioblastoma cell line U87 (U), and LNCaP (L) cells (D). PSA is expressed in prostate but not in brain cells. All samples express GLI1 and SHH. The whole tissue and primary culture correspond to PT6. (E) Histogram of the inhibition of BrdUrd incorporation in primary cultures of prostate tumor (PT3-PT8) by cyclopamine treatment. (F–I) Immunocytochemistry for BrdUrd incorporation with secondary FITC-antibodies showing BrdUrd⁺ nuclei (green) in a field of primary prostate cells (PT6) in control cells (treated with ethanol as the carrier for cyclopamine, F), cyclopamine (G), SHH protein (H), or anti-SHH antibody (αSHH Ab, I). All nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). (J and K) Histograms of the increase in (J) or inhibition of (K) BrdUrd incorporation of primary prostate tumors after treatment with SHH (J) or anti-SHH antibody (αSHH Ab, K) for 48 h. Histogram error bars represent SEM in all panels.

Fig. 3.

Response of prostate cell lines to GLI1 RNA interference. (A–C) Immunocytochemisty of the three prostate cell lines indicated showing the efficiency of lipofection of an FITC-tagged control siRNA (green). Note the lower efficiency in PC3 cells. (D) Effect of GLI1 siRNA on gene expression. RNA interference reduces GLI1 and PTCH1 mRNA levels as seen at 24 and 48 h, respectively (E) Histogram of the inhibition of BrdUrd incorporation in prostate tumor cell lines by GLI1 siRNA. (F) Specificity of the effects of GLI1 siRNA on GLI1 mRNA levels in the three prostate cell lines, compared with those of a control unrelated siRNA, 8 h after transfection. The levels of GAPDH are shown below as controls.