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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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LNShh cells induce osteoblast differentiation. (A) Effect on cell proliferation. MC3T3 cells were grown in 24-well plates with culture inserts containing either control LNCaP cells (squares), LNShh cells (triangles), or no cells (circles). Absorbance measurements are in direct proportion to the number of living cells. Each point is the mean ± SD of 3 replicate wells, and results are representative of two independent experiments. *P < 0.05 and **P < 0.001, compared to MC3T3 cells co-cultured with LNShh cells at indicated time points. (B) Effect on ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates. Quantitative ALP activity was determined in cell lysates at indicated time points. ALP activity in mixed cultures of MC3T3 and LNShh cells (filled bars) were compared to those in mixed cultures of MC3T3 and control LNCaP cells (open bars). Data are means ± SD of 6 replicate determinations from 3 independent samples for each group. *, P < 0.05. (C) Localization of ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates and stained for ALP activity at indicated time points. Magnification, 10×. (D) Effect on Akp2 expression. MC3T3 cells were grown in mixed culture with either LNCaP or LNShh cells. Expression of Akp2 in MC3T3 cells cultured with LNShh cells (filled bars) relative to those cultured with control LNCaP cells (open bars) were compared. (E) MC3T3 cells were cultured alone in the absence (open bar) or presence (filled bar) of 1 μg/ml Shh-N for 24 h, and their relative Akp2 expression was compared. In (D) and (E), values are means ± SD of 2–5 assays. *, P < 0.05.
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Figure 3: LNShh cells induce osteoblast differentiation. (A) Effect on cell proliferation. MC3T3 cells were grown in 24-well plates with culture inserts containing either control LNCaP cells (squares), LNShh cells (triangles), or no cells (circles). Absorbance measurements are in direct proportion to the number of living cells. Each point is the mean ± SD of 3 replicate wells, and results are representative of two independent experiments. *P < 0.05 and **P < 0.001, compared to MC3T3 cells co-cultured with LNShh cells at indicated time points. (B) Effect on ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates. Quantitative ALP activity was determined in cell lysates at indicated time points. ALP activity in mixed cultures of MC3T3 and LNShh cells (filled bars) were compared to those in mixed cultures of MC3T3 and control LNCaP cells (open bars). Data are means ± SD of 6 replicate determinations from 3 independent samples for each group. *, P < 0.05. (C) Localization of ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates and stained for ALP activity at indicated time points. Magnification, 10×. (D) Effect on Akp2 expression. MC3T3 cells were grown in mixed culture with either LNCaP or LNShh cells. Expression of Akp2 in MC3T3 cells cultured with LNShh cells (filled bars) relative to those cultured with control LNCaP cells (open bars) were compared. (E) MC3T3 cells were cultured alone in the absence (open bar) or presence (filled bar) of 1 μg/ml Shh-N for 24 h, and their relative Akp2 expression was compared. In (D) and (E), values are means ± SD of 2–5 assays. *, P < 0.05.

Mentions: The ability of LNShh cells to induce osteoblast differentiation was examined. The initial phase of osteoblast differentiation is characterized by cell proliferation followed by growth arrest [15]. As shown in Figure 3A, proliferation of MC3T3 cells co-cultured with LNShh cells was significantly decreased by days 5 and 7 of co-culture compared to that of MC3T3 cells co-cultured with control LNCaP cells or cultured alone. These data suggest that Shh-expressing prostate cancer cells can inhibit cell proliferation in pre-osteoblasts.


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)

LNShh cells induce osteoblast differentiation. (A) Effect on cell proliferation. MC3T3 cells were grown in 24-well plates with culture inserts containing either control LNCaP cells (squares), LNShh cells (triangles), or no cells (circles). Absorbance measurements are in direct proportion to the number of living cells. Each point is the mean ± SD of 3 replicate wells, and results are representative of two independent experiments. *P < 0.05 and **P < 0.001, compared to MC3T3 cells co-cultured with LNShh cells at indicated time points. (B) Effect on ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates. Quantitative ALP activity was determined in cell lysates at indicated time points. ALP activity in mixed cultures of MC3T3 and LNShh cells (filled bars) were compared to those in mixed cultures of MC3T3 and control LNCaP cells (open bars). Data are means ± SD of 6 replicate determinations from 3 independent samples for each group. *, P < 0.05. (C) Localization of ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates and stained for ALP activity at indicated time points. Magnification, 10×. (D) Effect on Akp2 expression. MC3T3 cells were grown in mixed culture with either LNCaP or LNShh cells. Expression of Akp2 in MC3T3 cells cultured with LNShh cells (filled bars) relative to those cultured with control LNCaP cells (open bars) were compared. (E) MC3T3 cells were cultured alone in the absence (open bar) or presence (filled bar) of 1 μg/ml Shh-N for 24 h, and their relative Akp2 expression was compared. In (D) and (E), values are means ± SD of 2–5 assays. *, P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: LNShh cells induce osteoblast differentiation. (A) Effect on cell proliferation. MC3T3 cells were grown in 24-well plates with culture inserts containing either control LNCaP cells (squares), LNShh cells (triangles), or no cells (circles). Absorbance measurements are in direct proportion to the number of living cells. Each point is the mean ± SD of 3 replicate wells, and results are representative of two independent experiments. *P < 0.05 and **P < 0.001, compared to MC3T3 cells co-cultured with LNShh cells at indicated time points. (B) Effect on ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates. Quantitative ALP activity was determined in cell lysates at indicated time points. ALP activity in mixed cultures of MC3T3 and LNShh cells (filled bars) were compared to those in mixed cultures of MC3T3 and control LNCaP cells (open bars). Data are means ± SD of 6 replicate determinations from 3 independent samples for each group. *, P < 0.05. (C) Localization of ALP activity. Mixed cultures of MC3T3 cells with either control LNCaP or LNShh cells were grown in 6-well plates and stained for ALP activity at indicated time points. Magnification, 10×. (D) Effect on Akp2 expression. MC3T3 cells were grown in mixed culture with either LNCaP or LNShh cells. Expression of Akp2 in MC3T3 cells cultured with LNShh cells (filled bars) relative to those cultured with control LNCaP cells (open bars) were compared. (E) MC3T3 cells were cultured alone in the absence (open bar) or presence (filled bar) of 1 μg/ml Shh-N for 24 h, and their relative Akp2 expression was compared. In (D) and (E), values are means ± SD of 2–5 assays. *, P < 0.05.
Mentions: The ability of LNShh cells to induce osteoblast differentiation was examined. The initial phase of osteoblast differentiation is characterized by cell proliferation followed by growth arrest [15]. As shown in Figure 3A, proliferation of MC3T3 cells co-cultured with LNShh cells was significantly decreased by days 5 and 7 of co-culture compared to that of MC3T3 cells co-cultured with control LNCaP cells or cultured alone. These data suggest that Shh-expressing prostate cancer cells can inhibit cell proliferation in pre-osteoblasts.

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