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Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation.

Zunich SM, Douglas T, Valdovinos M, Chang T, Bushman W, Walterhouse D, Iannaccone P, Lamm ML - Mol. Cancer (2009)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Northwestern University Feinberg School of Medicine, Children's Memorial Research Center, Chicago, IL 60614, USA. SZunich@childrensmemorial.org

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.

Conclusion: 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.

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Morphologic pattern of LNShh human prostate cancer cells and MC3T3 mouse pre-osteoblasts in mixed culture. MC3T3 cells were mixed with Shh-expressing LNShh cells then seeded onto chamber slides and maintained for 14 days. (Panels a, b and c) GFP-expressing LNShh cells (stably transfected with pIRES2-hShh-EGFP vector) formed clusters surrounded by a stroma of MC3T3 cells which expressed intense phalloidin staining. DAPI was used as counterstain. Cells were visualized by fluorescent microscopy. (Panel d) LNShh cells in mixed culture were further identified by positive immunocytochemical staining for human cytokeratin 8 which was not detected in surrounding MC3T3 cells. Hematoxylin was used as counterstain. Scale bars: 50 μm.
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Figure 1: Morphologic pattern of LNShh human prostate cancer cells and MC3T3 mouse pre-osteoblasts in mixed culture. MC3T3 cells were mixed with Shh-expressing LNShh cells then seeded onto chamber slides and maintained for 14 days. (Panels a, b and c) GFP-expressing LNShh cells (stably transfected with pIRES2-hShh-EGFP vector) formed clusters surrounded by a stroma of MC3T3 cells which expressed intense phalloidin staining. DAPI was used as counterstain. Cells were visualized by fluorescent microscopy. (Panel d) LNShh cells in mixed culture were further identified by positive immunocytochemical staining for human cytokeratin 8 which was not detected in surrounding MC3T3 cells. Hematoxylin was used as counterstain. Scale bars: 50 μm.

Mentions: Shh-expressing LNCaP human prostate cancer cells obtained via stable transfection with hShh cDNA cloned into a pIRES2-EGFP vector, designated as LNShh cells, or vector-transfected control LNCaP cells were cultured within the same culture chamber with MC3T3 mouse pre-osteoblasts. The cells in this mixed culture system established, in a time-dependent manner, a distinctive morphologic pattern. There were no apparent differences in the spatial organization of the mixed cultures of MC3T3 cells and either control LNCaP or LNShh cells, and only images of mixed cultures of MC3T3 and LNShh cells are shown in Figure 1. Beginning about two weeks of mixed culture, clusters of LNShh cells, identified by their GFP fluorescence, were surrounded by a compact "stroma" of MC3T3 cells which exhibited more intense phalloidin staining of their F-actin filaments (Figure 1, panels a, b and c). Identification of the epithelial-derived LNShh cells in mixed cultures was further confirmed by their positive immunostaining for human cytokeratin 8 (Figure 1, panel d). When cultured alone, LNShh cells also formed colonies which became interconnected over time forming a meshwork instead of remaining discrete as shown in mixed cultures. On the other hand, single cultures of MC3T3 cells which reached confluence at about two weeks formed a sheet of cells instead of the reticulate network evident in mixed cultures (data not shown).


Paracrine sonic hedgehog signalling by prostate cancer cells induces osteoblast differentiation.

Zunich SM, Douglas T, Valdovinos M, Chang T, Bushman W, Walterhouse D, Iannaccone P, Lamm ML - Mol. Cancer (2009)

Morphologic pattern of LNShh human prostate cancer cells and MC3T3 mouse pre-osteoblasts in mixed culture. MC3T3 cells were mixed with Shh-expressing LNShh cells then seeded onto chamber slides and maintained for 14 days. (Panels a, b and c) GFP-expressing LNShh cells (stably transfected with pIRES2-hShh-EGFP vector) formed clusters surrounded by a stroma of MC3T3 cells which expressed intense phalloidin staining. DAPI was used as counterstain. Cells were visualized by fluorescent microscopy. (Panel d) LNShh cells in mixed culture were further identified by positive immunocytochemical staining for human cytokeratin 8 which was not detected in surrounding MC3T3 cells. Hematoxylin was used as counterstain. Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654862&req=5

Figure 1: Morphologic pattern of LNShh human prostate cancer cells and MC3T3 mouse pre-osteoblasts in mixed culture. MC3T3 cells were mixed with Shh-expressing LNShh cells then seeded onto chamber slides and maintained for 14 days. (Panels a, b and c) GFP-expressing LNShh cells (stably transfected with pIRES2-hShh-EGFP vector) formed clusters surrounded by a stroma of MC3T3 cells which expressed intense phalloidin staining. DAPI was used as counterstain. Cells were visualized by fluorescent microscopy. (Panel d) LNShh cells in mixed culture were further identified by positive immunocytochemical staining for human cytokeratin 8 which was not detected in surrounding MC3T3 cells. Hematoxylin was used as counterstain. Scale bars: 50 μm.
Mentions: Shh-expressing LNCaP human prostate cancer cells obtained via stable transfection with hShh cDNA cloned into a pIRES2-EGFP vector, designated as LNShh cells, or vector-transfected control LNCaP cells were cultured within the same culture chamber with MC3T3 mouse pre-osteoblasts. The cells in this mixed culture system established, in a time-dependent manner, a distinctive morphologic pattern. There were no apparent differences in the spatial organization of the mixed cultures of MC3T3 cells and either control LNCaP or LNShh cells, and only images of mixed cultures of MC3T3 and LNShh cells are shown in Figure 1. Beginning about two weeks of mixed culture, clusters of LNShh cells, identified by their GFP fluorescence, were surrounded by a compact "stroma" of MC3T3 cells which exhibited more intense phalloidin staining of their F-actin filaments (Figure 1, panels a, b and c). Identification of the epithelial-derived LNShh cells in mixed cultures was further confirmed by their positive immunostaining for human cytokeratin 8 (Figure 1, panel d). When cultured alone, LNShh cells also formed colonies which became interconnected over time forming a meshwork instead of remaining discrete as shown in mixed cultures. On the other hand, single cultures of MC3T3 cells which reached confluence at about two weeks formed a sheet of cells instead of the reticulate network evident in mixed cultures (data not shown).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Northwestern University Feinberg School of Medicine, Children's Memorial Research Center, Chicago, IL 60614, USA. SZunich@childrensmemorial.org

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus