title: Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation.
abstract: BACKGROUND
  Sonic hedgehog (Shh) and components of its signalling pathway have been identified in human prostate carcinoma and increased levels of their expression appear to correlate with disease progression and metastasis. The mechanism through which Shh signalling could promote metastasis in bone, the most common site for prostate carcinoma metastasis, has not yet been investigated. The present study determined the effect of Shh signalling between prostate cancer cells and pre-osteoblasts on osteoblast differentiation, a requisite process for new bone formation that characterizes prostate carcinoma metastasis.
  
  
  RESULTS
  LNCaP human prostate cancer cells modified to overexpress Shh (designated LNShh cells) and MC3T3 mouse pre-osteoblasts were maintained as mixed populations within the same culture chamber. In this non-conventional mixed culture system, LNShh cells upregulated the expression of Shh target genes Gli1 and Patched 1 (Ptc1) in MC3T3 cells and this was inhibited by cyclopamine, a specific chemical inhibitor of hedgehog signalling. Concomitantly, MC3T3 cells exhibited time-dependent decreased cell proliferation, upregulated alkaline phosphatase Akp2 gene expression, and increased alkaline phosphatase activity indicative of early phase osteoblast differentiation. LNShh cell-induced differentiation was inhibited in MC3T3 cells stably transfected with a dominant negative form of Gli1, a transcription factor that mediates Shh signalling. Interestingly, LNShh cells did not significantly increase the endogenous expression of the osteoblast differentiation transcription factor Runx2 and its target genes osteocalcin and osteopontin. Consistent with these results, exogenous Shh peptide did not upregulate Runx2 expression in MC3T3 cells. However, Runx2 levels were increased in MC3T3 cells by ascorbic acid, a known stimulator of osteoblast differentiation.
  
  
  CONCLUSIONS
  Altogether, these data demonstrate that Shh-expressing prostate cancer cells can directly and specifically induce differentiation in pre-osteoblasts via a Gli1-dependent mechanism that does not require transcriptional upregulation of Runx2. Paracrine activation of the Shh pathway in osteoblast progenitors and subsequent induction of osteoblast differentiation could be a mechanism through which high levels of Shh expression in prostate carcinoma contribute to bone metastasis. Targeting of paracrine Shh signalling may provide an effective therapeutic strategy against prostate carcinoma metastasis in bone.
authors.affiliation: Department of Pediatrics, Northwestern University Feinberg School of Medicine, Children's Memorial Research Center, Chicago, IL 60614, USA. SZunich@childrensmemorial.org
authors.forename: Samantha M
authors.initials: SM
authors.lastname: Zunich
authors.forename: Taneka
authors.initials: T
authors.lastname: Douglas
authors.forename: Maria
authors.initials: M
authors.lastname: Valdovinos
authors.forename: Tiffany
authors.initials: T
authors.lastname: Chang
authors.forename: Wade
authors.initials: W
authors.lastname: Bushman
authors.forename: David
authors.initials: D
authors.lastname: Walterhouse
authors.forename: Philip
authors.initials: P
authors.lastname: Iannaccone
authors.forename: Marilyn L G
authors.initials: ML
authors.lastname: Lamm
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics; D005799:Genes, Dominant; D053823:Hedgehog Proteins / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D010006:Osteoblasts / Q000166:cytology*; D019899:Paracrine Communication; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D011956:Receptors, Cell Surface / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000235:genetics; D000071676:Zinc Finger Protein GLI1
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; C400551:GLI1 protein, human; D053823:Hedgehog Proteins; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; C000606240:Ptch1 protein, mouse; D011956:Receptors, Cell Surface; C509239:SHH protein, human; D014157:Transcription Factors; D000071676:Zinc Finger Protein GLI1