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Smoothened as a new therapeutic target for human osteosarcoma.

Hirotsu M, Setoguchi T, Sasaki H, Matsunoshita Y, Gao H, Nagao H, Kunigou O, Komiya S - Mol. Cancer (2010)

Bottom Line: Cell cycle analysis revealed that cyclopamine promoted G1 arrest.Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb.On the other hand, p21(cip1) wprotein was up-regulated by cyclopamine treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT

Background: The Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition.

Results: To evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21(cip1) wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo.

Conclusions: These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.

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SMO shRNA prevents osteosarcoma xenograft growth in vivo and prolongs survival. A, SMO shRNA or control shRNA transfected 143B cells (1 × 106) were inoculated subcutaneously. Established 143B tumors were measured. The tumor volume at day 7 was set at 1, and tumor volumes at subsequent time points were calculated. SMO shRNA inhibited tumor growth at 8 weeks compared to control shRNA. B, Kaplan-Meier survival curves from SMO shRNA groups and control shRNA. Kaplan-Meier analysis showed that SMO shRNA conferred a significant survival benefit (n = 6, p < 0.05). C, Immunohistochemical examination of ki67 was performed in xenograft tumors. SMO shRNA decreased SMO RNA in vivo. Ki67 staining revealed that proliferation of osteosarcoma cells was decreased by GSI treatment. The number of Ki67-positive cells was decreased to 30% of control revel by SMO shRNA (error bar means S.D.) (*: P < 0.01).
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Figure 6: SMO shRNA prevents osteosarcoma xenograft growth in vivo and prolongs survival. A, SMO shRNA or control shRNA transfected 143B cells (1 × 106) were inoculated subcutaneously. Established 143B tumors were measured. The tumor volume at day 7 was set at 1, and tumor volumes at subsequent time points were calculated. SMO shRNA inhibited tumor growth at 8 weeks compared to control shRNA. B, Kaplan-Meier survival curves from SMO shRNA groups and control shRNA. Kaplan-Meier analysis showed that SMO shRNA conferred a significant survival benefit (n = 6, p < 0.05). C, Immunohistochemical examination of ki67 was performed in xenograft tumors. SMO shRNA decreased SMO RNA in vivo. Ki67 staining revealed that proliferation of osteosarcoma cells was decreased by GSI treatment. The number of Ki67-positive cells was decreased to 30% of control revel by SMO shRNA (error bar means S.D.) (*: P < 0.01).

Mentions: To confirm the effect of SMO suppression, we examined the effect of SMO shRNA. 143B was transfected with control shRNA or SMO shRNA. SMO shRNA reduced the expression of SMO mRNA (Fig. 5A). MTT assay revealed that knock-down of SMO prevented osteosarcoma growth in vitro (Fig. 5A). We next used a clonogenic assay to determine whether cells capable of forming anchorage-independent colonies were depleted by SMO shRNA. This assay revealed SMO shRNA reduced colony formation in soft agar (Fig. 5B). These findings show that suppression of SMO prevents osteosarcoma growth in vitro. We then examined the transcription of cell cycle-related genes. Real-time PCR revealed that SMO shRNA prevented the transcription of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and E2F1 (see additional file 2). To examine the in vivo effect of SMO shRNA, nude mice were inoculated with control shRNA or SMO shRNA transfected 143B osteosarcoma cells intradermally. Results demonstrated significant inhibition of tumor growth SMO shRNA versus control shRNA (Fig. 6A, B). Kaplan-Meier analysis showed that SMO shRNA conferred a significant survival benefit (Fig. 6B). Next, we performed real-time PCR using formed tumors. Real-time PCR revealed that transcription of GLI1, GLI2, and PTCH1 was decreased in tumors formed by SMO shRNA-transfected 143B. These findings showed that SMO shRNA prevented the transcription of Hh target genes in vivo. In addition, SMO shRNA prevented the transcription of accelerators of the cell cycle including cyclin E1, SKP2, and E2F1 (see additional file 3). Histological analysis indicated that SMO shRNA prevented cell proliferation. The control tumors exhibited a number of cells positive for Ki67, a marker of cell proliferation. In contrast, SMO shRNA transfected tumors exhibited little evidence of proliferation, as evidenced by lack of Ki67 positivity. The number of Ki67-positive cells was decreased to 30% of control revel by SMO shRNA (Fig. 6C). These findings suggest that inhibition of SMO prevents osteosarcoma growth by cell cycle regulation in vivo.


Smoothened as a new therapeutic target for human osteosarcoma.

Hirotsu M, Setoguchi T, Sasaki H, Matsunoshita Y, Gao H, Nagao H, Kunigou O, Komiya S - Mol. Cancer (2010)

SMO shRNA prevents osteosarcoma xenograft growth in vivo and prolongs survival. A, SMO shRNA or control shRNA transfected 143B cells (1 × 106) were inoculated subcutaneously. Established 143B tumors were measured. The tumor volume at day 7 was set at 1, and tumor volumes at subsequent time points were calculated. SMO shRNA inhibited tumor growth at 8 weeks compared to control shRNA. B, Kaplan-Meier survival curves from SMO shRNA groups and control shRNA. Kaplan-Meier analysis showed that SMO shRNA conferred a significant survival benefit (n = 6, p < 0.05). C, Immunohistochemical examination of ki67 was performed in xenograft tumors. SMO shRNA decreased SMO RNA in vivo. Ki67 staining revealed that proliferation of osteosarcoma cells was decreased by GSI treatment. The number of Ki67-positive cells was decreased to 30% of control revel by SMO shRNA (error bar means S.D.) (*: P < 0.01).
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Figure 6: SMO shRNA prevents osteosarcoma xenograft growth in vivo and prolongs survival. A, SMO shRNA or control shRNA transfected 143B cells (1 × 106) were inoculated subcutaneously. Established 143B tumors were measured. The tumor volume at day 7 was set at 1, and tumor volumes at subsequent time points were calculated. SMO shRNA inhibited tumor growth at 8 weeks compared to control shRNA. B, Kaplan-Meier survival curves from SMO shRNA groups and control shRNA. Kaplan-Meier analysis showed that SMO shRNA conferred a significant survival benefit (n = 6, p < 0.05). C, Immunohistochemical examination of ki67 was performed in xenograft tumors. SMO shRNA decreased SMO RNA in vivo. Ki67 staining revealed that proliferation of osteosarcoma cells was decreased by GSI treatment. The number of Ki67-positive cells was decreased to 30% of control revel by SMO shRNA (error bar means S.D.) (*: P < 0.01).
Mentions: To confirm the effect of SMO suppression, we examined the effect of SMO shRNA. 143B was transfected with control shRNA or SMO shRNA. SMO shRNA reduced the expression of SMO mRNA (Fig. 5A). MTT assay revealed that knock-down of SMO prevented osteosarcoma growth in vitro (Fig. 5A). We next used a clonogenic assay to determine whether cells capable of forming anchorage-independent colonies were depleted by SMO shRNA. This assay revealed SMO shRNA reduced colony formation in soft agar (Fig. 5B). These findings show that suppression of SMO prevents osteosarcoma growth in vitro. We then examined the transcription of cell cycle-related genes. Real-time PCR revealed that SMO shRNA prevented the transcription of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and E2F1 (see additional file 2). To examine the in vivo effect of SMO shRNA, nude mice were inoculated with control shRNA or SMO shRNA transfected 143B osteosarcoma cells intradermally. Results demonstrated significant inhibition of tumor growth SMO shRNA versus control shRNA (Fig. 6A, B). Kaplan-Meier analysis showed that SMO shRNA conferred a significant survival benefit (Fig. 6B). Next, we performed real-time PCR using formed tumors. Real-time PCR revealed that transcription of GLI1, GLI2, and PTCH1 was decreased in tumors formed by SMO shRNA-transfected 143B. These findings showed that SMO shRNA prevented the transcription of Hh target genes in vivo. In addition, SMO shRNA prevented the transcription of accelerators of the cell cycle including cyclin E1, SKP2, and E2F1 (see additional file 3). Histological analysis indicated that SMO shRNA prevented cell proliferation. The control tumors exhibited a number of cells positive for Ki67, a marker of cell proliferation. In contrast, SMO shRNA transfected tumors exhibited little evidence of proliferation, as evidenced by lack of Ki67 positivity. The number of Ki67-positive cells was decreased to 30% of control revel by SMO shRNA (Fig. 6C). These findings suggest that inhibition of SMO prevents osteosarcoma growth by cell cycle regulation in vivo.

Bottom Line: Cell cycle analysis revealed that cyclopamine promoted G1 arrest.Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb.On the other hand, p21(cip1) wprotein was up-regulated by cyclopamine treatment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedic Surgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan.

ABSTRACT

Background: The Hedgehog signaling pathway functions as an organizer in embryonic development. Recent studies have demonstrated constitutive activation of Hedgehog pathway in various types of malignancies. However, it remains unclear how Hedgehog pathway is involved in the pathogenesis of osteosarcoma. To explore the involvement of aberrant Hedgehog pathway in the pathogenesis of osteosarcoma, we investigated the expression and activation of Hedgehog pathway in osteosarcoma and examined the effect of SMOOTHENED (SMO) inhibition.

Results: To evaluate the expression of genes of Hedgehog pathway, we performed real-time PCR and immunohistochemistry using osteosarcoma cell lines and osteosarcoma biopsy specimens. To evaluate the effect of SMO inhibition, we did cell viability, colony formation, cell cycle in vitro and xenograft model in vivo. Real-time PCR revealed that osteosarcoma cell lines over-expressed Sonic hedgehog, Indian hedgehog, PTCH1, SMO, and GLI. Real-time PCR revealed over-expression of SMO, PTCH1, and GLI2 in osteosarcoma biopsy specimens. These findings showed that Hedgehog pathway is activated in osteosarcomas. Inhibition of SMO by cyclopamine, a specific inhibitor of SMO, slowed the growth of osteosarcoma in vitro. Cell cycle analysis revealed that cyclopamine promoted G1 arrest. Cyclopamine reduced the expression of accelerators of the cell cycle including cyclin D1, cyclin E1, SKP2, and pRb. On the other hand, p21(cip1) wprotein was up-regulated by cyclopamine treatment. In addition, knockdown of SMO by SMO shRNA prevents osteosarcoma growth in vitro and in vivo.

Conclusions: These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with osteosarcoma.

Show MeSH
Related in: MedlinePlus