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.S ONIC HEDGEHOG (SHH) signaling has been implicated in different aspects of animal development, acting through several components, including the transmembrane proteins PATCHED1 (PTCH1) and SMOOTHENED (SMOH), to activate the GLI zinc-finger transcription factors (1, 2). In addition, we and others have shown that SHH signaling is implicated in a number of tumors (reviewed in refs. 2 and 3), such as basal cell carcinomas (4-6), medulloblastomas (7,8), gliomas (7), sarcomas (9, 10), tumors of the digestive tract (11), small cell lung cancers (12,) and pancreatic carcinomas (13). To date there is no direct evidence linking SHH signaling to prostate cancer, the most common solid cancer in men (14), although we have found that sporadic prostate tumors express GLI1 (7), a reliable marker of SHH signaling (15,16). This observation allowed us to propose the hypothesis that the SHH-GLI pathway participates in prostate cancer (7). Consistently, Shh signaling has been found to be essential for prostate patterning and development (17)(18)(19)(20)(21)(22), and genetic mapping data has revealed that at least two key components of the SHH-GLI pathway [SMOH and SUPPRESSOR OF FUSED (SUFUH)] are located in chromosomal regions implicated in familial human prostate cancer (23,24). Here we have tested the involvement of SHH-GLI signaling in prostate cancer. MethodsCell Lines and Primary Cultures. The PC3, LNCaP, and DU145 cell lines (25-27) were purchased from American Type Culture Collection and grown as specified. All primary prostate tumors were obtained following approved protocols. Tumors in PBS were chopped with a razor blade and incubated with Papain for 1 h at 37°C, they were then dissociated by passing them through a fire-polished pipette and washed several times in serum containing media. All dissociated primary tumors were plated in polyornithinand laminin-treated p16 plates in DMEM-F12 with 10% FBS at Ϸ30,000 cells per p16 well. Primary cultures were ...

SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development

“…Given the relevant role of Fbxl17 in regulating Hh signaling and that Hh signaling activity has been implicated in the proliferation of prostate cancer cell lines (Karhadkar et al , 2004; Sanchez et al , 2004), we further tested the effect of Fbxl17 on Hh activation by quantifying Gli1 mRNA levels upon either depletion or expression of Fbxl17 using PC3 cells. Fbxl17 silencing resulted in a decrease in Gli1 mRNA levels (Appendix Fig S2A), and contrarily, Fbxl17 expression led to an increase in Gli1 mRNA levels (Appendix Fig S2B).…”

Distinct roles of first exon variants of the tumor-suppressor Patched1 in Hedgehog signaling

“…Deregulation of HH signaling causes malformation and tumor formation, with the first example being basal cell carcinomas (BCC) (Johnson et al, 1996;Hahn et al, 1996b). In a number of tumors that were recently found to include prostatic and pancreatic cancers, HH signaling is abnormally upregulated, and effective signaling inhibition can repress tumor growth and induce apoptosis (Sanchez et al, 2004). Among core components of the HH pathway, the secreted ligand HH, the membrane receptor Patched (PTCH), the membrane signal transductor Smoothened (SMO) and the transcription factor Ci/glioma (GLI) are evolutionarily conserved from insects to mammals.…”

Targeting the CB2 cannabinoid receptor in osteoporosis

“…This concept received substantial impetus from two early reports of cyclopamine- or Shh antibody-mediated suppression of prostate cancer cell growth in vitro and in vivo [66,96], and the outcomes of these experimental studies were viewed as evidence for an active auto-crine-like hedgehog signaling process in these cell lines. This conclusion should now be reconsidered, especially in light of the concerns discussed previously.…”

“…There are general concerns that the so-called ‘normal’ regions of human prostate specimens that are available for study might be affected by the common prostate benign disease states that might also invoke abnormal hedgehog responses [100] and this raises questions regarding the establishment of normal prostate basal expression levels for any of these genes. Approaches that assess RNA levels by in situ hybridization are complicated by the uneven cellular architecture of a prostate tumor (in which the cellularity of the stroma can appear sparse compared with the adjacent epithelium) and this might account for the conflicting findings of Gli1 RNAs localized to benign and malignant prostate epithelium in one study [96] versus selective expression in the stroma around tumors in another [100]. Likewise, quantitative reverse-transcriptase PCR approaches that involve bulk extraction from tumor tissues are complicated by the comixtures of tumor and benign stromal cells in the specimens that complicate analysis, so it is difficult to comment on observations based on this approach.…”