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 ...

In mammalian cells, Id proteins coordinate proliferation and differentiation. Id2 is a dominant-negative antagonist of basic helix-loop-helix transcription factors and proteins of the retinoblastoma (Rb) family. Here we show that Id2-Rb double knockout embryos survive to term with minimal or no defects in neurogenesis and haematopoiesis, but they die at birth from severe reduction of muscle tissue. In neuroblastoma, an embryonal tumour derived from the neural crest, Id2 is overexpressed in cells carrying extra copies of the N-myc gene. In these cells, Id2 is in molar excess of the active form of Rb. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell-cycle progression induced by Myc oncoproteins requires inactivation of Rb by Id2. Thus, a dual connection links Id2 and Rb: during normal cell-cycle, Rb prohibits the action of Id2 on its natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumour-suppressor function of Rb.

Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.