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The sonic hedgehog signaling pathway is reactivated in human renal cell carcinoma and plays orchestral role in tumor growth.

Dormoy V, Danilin S, Lindner V, Thomas L, Rothhut S, Coquard C, Helwig JJ, Jacqmin D, Lang H, Massfelder T - Mol. Cancer (2009)

Bottom Line: The sonic hedgehog (SHH) signaling pathway is crucial to normal development.Gli1, cyclin D1, Pax2, Lim1, VEGF, and TGF-beta were exclusively expressed in tumors and were shown to be regulated by SHH, as evidenced by immunoblot after SHH inhibition.Using specific inhibitors and immunoblot, the activation of the oncogenic PI3K/Akt, NF-kB and MAPK pathways was decreased by SHH inhibition.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U682, Section of Renal Cancer and Renal Physiopathology, University of Strasbourg, School of Medicine, Strasbourg, 67085 France. valerian.dormoy@medecine.u-strasbg.fr

ABSTRACT

Background: Human clear cell renal cell carcinoma (CRCC) remains resistant to therapies. Recent advances in Hypoxia Inducible Factors (HIF) molecular network led to targeted therapies, but unfortunately with only limited clinical significance. Elucidating the molecular processes involved in kidney tumorigenesis and resistance is central to the development of improved therapies, not only for kidney cancer but for many, if not all, cancer types. The oncogenic PI3K/Akt, NF-kB and MAPK pathways are critical for tumorigenesis. The sonic hedgehog (SHH) signaling pathway is crucial to normal development.

Results: By quantitative RT-PCR and immunoblot, we report that the SHH signaling pathway is constitutively reactivated in tumors independently of the von Hippel-Lindau (VHL) tumor suppressor gene expression which is inactivated in the majority of CRCC. The inhibition of the SHH signaling pathway by the specific inhibitor cyclopamine abolished CRCC cell growth as assessed by cell counting, BrdU incorporation studies, fluorescence-activated cell sorting and beta-galactosidase staining. Importantly, inhibition of the SHH pathway induced tumor regression in nude mice through inhibition of cell proliferation and neo-vascularization, and induction of apoptosis but not senescence assessed by in vivo studies, immunoblot and immunohistochemistry. Gli1, cyclin D1, Pax2, Lim1, VEGF, and TGF-beta were exclusively expressed in tumors and were shown to be regulated by SHH, as evidenced by immunoblot after SHH inhibition. Using specific inhibitors and immunoblot, the activation of the oncogenic PI3K/Akt, NF-kB and MAPK pathways was decreased by SHH inhibition.

Conclusions: These findings support targeting SHH for the treatment of CRCC and pave the way for innovative and additional investigations in a broad range of cancers.

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Inhibition of the SHH signaling pathway induces tumor regression in nude mice. (A) Tumor growth in mice treated according to the first experimental protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. Immunoblotting experiments to measure expression of Gli1 in tumors lysates. The gels shown are representative for at least 3 independent experiments. Photographs show the implanted tumors in representative mice at day 1 of drug injection, at day 19 in Ctl-treated group or cyclopamine-treated group. (B) Tumor growth in mice treated according to the second protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. (C) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained with an antibody against Ki67 (magnification ×400). Right, proliferative index. Results are shown as mean ± SEM, n = 7. **, P < 0.01 from Ctl-treated mice. (D) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained for CD31 (magnification ×400). Right, quantification of neovascularization. Results are shown as mean ± SEM, n = 7. *, P < 0.05 from Ctl-treated mice.
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Figure 6: Inhibition of the SHH signaling pathway induces tumor regression in nude mice. (A) Tumor growth in mice treated according to the first experimental protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. Immunoblotting experiments to measure expression of Gli1 in tumors lysates. The gels shown are representative for at least 3 independent experiments. Photographs show the implanted tumors in representative mice at day 1 of drug injection, at day 19 in Ctl-treated group or cyclopamine-treated group. (B) Tumor growth in mice treated according to the second protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. (C) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained with an antibody against Ki67 (magnification ×400). Right, proliferative index. Results are shown as mean ± SEM, n = 7. **, P < 0.01 from Ctl-treated mice. (D) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained for CD31 (magnification ×400). Right, quantification of neovascularization. Results are shown as mean ± SEM, n = 7. *, P < 0.05 from Ctl-treated mice.

Mentions: We next analyzed the effect of cyclopamine in vivo in the tumor xenografted nude mice model. In the first protocol (injections every other day), tumor growth was completely abolished by cyclopamine treatment (Figure 6A and Additional file 5A). The expression of Gli1 was decreased by 80% in tumors harvested from cyclopamine-treated mice compared to tumors from control mice showing adequate targeting of the drug (Figure 6A, top gels).


The sonic hedgehog signaling pathway is reactivated in human renal cell carcinoma and plays orchestral role in tumor growth.

Dormoy V, Danilin S, Lindner V, Thomas L, Rothhut S, Coquard C, Helwig JJ, Jacqmin D, Lang H, Massfelder T - Mol. Cancer (2009)

Inhibition of the SHH signaling pathway induces tumor regression in nude mice. (A) Tumor growth in mice treated according to the first experimental protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. Immunoblotting experiments to measure expression of Gli1 in tumors lysates. The gels shown are representative for at least 3 independent experiments. Photographs show the implanted tumors in representative mice at day 1 of drug injection, at day 19 in Ctl-treated group or cyclopamine-treated group. (B) Tumor growth in mice treated according to the second protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. (C) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained with an antibody against Ki67 (magnification ×400). Right, proliferative index. Results are shown as mean ± SEM, n = 7. **, P < 0.01 from Ctl-treated mice. (D) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained for CD31 (magnification ×400). Right, quantification of neovascularization. Results are shown as mean ± SEM, n = 7. *, P < 0.05 from Ctl-treated mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Inhibition of the SHH signaling pathway induces tumor regression in nude mice. (A) Tumor growth in mice treated according to the first experimental protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. Immunoblotting experiments to measure expression of Gli1 in tumors lysates. The gels shown are representative for at least 3 independent experiments. Photographs show the implanted tumors in representative mice at day 1 of drug injection, at day 19 in Ctl-treated group or cyclopamine-treated group. (B) Tumor growth in mice treated according to the second protocol. Results are shown as mean ± SEM, n = 7 for both groups; **, P < 0.01 cyclopamine-treated mice vs Ctl-treated mice. (C) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained with an antibody against Ki67 (magnification ×400). Right, proliferative index. Results are shown as mean ± SEM, n = 7. **, P < 0.01 from Ctl-treated mice. (D) Left, tumor sections of control- (Ctl) or cyclopamine (Cyclopamine)-treated mice immunostained for CD31 (magnification ×400). Right, quantification of neovascularization. Results are shown as mean ± SEM, n = 7. *, P < 0.05 from Ctl-treated mice.
Mentions: We next analyzed the effect of cyclopamine in vivo in the tumor xenografted nude mice model. In the first protocol (injections every other day), tumor growth was completely abolished by cyclopamine treatment (Figure 6A and Additional file 5A). The expression of Gli1 was decreased by 80% in tumors harvested from cyclopamine-treated mice compared to tumors from control mice showing adequate targeting of the drug (Figure 6A, top gels).

Bottom Line: The sonic hedgehog (SHH) signaling pathway is crucial to normal development.Gli1, cyclin D1, Pax2, Lim1, VEGF, and TGF-beta were exclusively expressed in tumors and were shown to be regulated by SHH, as evidenced by immunoblot after SHH inhibition.Using specific inhibitors and immunoblot, the activation of the oncogenic PI3K/Akt, NF-kB and MAPK pathways was decreased by SHH inhibition.

View Article: PubMed Central - HTML - PubMed

Affiliation: INSERM U682, Section of Renal Cancer and Renal Physiopathology, University of Strasbourg, School of Medicine, Strasbourg, 67085 France. valerian.dormoy@medecine.u-strasbg.fr

ABSTRACT

Background: Human clear cell renal cell carcinoma (CRCC) remains resistant to therapies. Recent advances in Hypoxia Inducible Factors (HIF) molecular network led to targeted therapies, but unfortunately with only limited clinical significance. Elucidating the molecular processes involved in kidney tumorigenesis and resistance is central to the development of improved therapies, not only for kidney cancer but for many, if not all, cancer types. The oncogenic PI3K/Akt, NF-kB and MAPK pathways are critical for tumorigenesis. The sonic hedgehog (SHH) signaling pathway is crucial to normal development.

Results: By quantitative RT-PCR and immunoblot, we report that the SHH signaling pathway is constitutively reactivated in tumors independently of the von Hippel-Lindau (VHL) tumor suppressor gene expression which is inactivated in the majority of CRCC. The inhibition of the SHH signaling pathway by the specific inhibitor cyclopamine abolished CRCC cell growth as assessed by cell counting, BrdU incorporation studies, fluorescence-activated cell sorting and beta-galactosidase staining. Importantly, inhibition of the SHH pathway induced tumor regression in nude mice through inhibition of cell proliferation and neo-vascularization, and induction of apoptosis but not senescence assessed by in vivo studies, immunoblot and immunohistochemistry. Gli1, cyclin D1, Pax2, Lim1, VEGF, and TGF-beta were exclusively expressed in tumors and were shown to be regulated by SHH, as evidenced by immunoblot after SHH inhibition. Using specific inhibitors and immunoblot, the activation of the oncogenic PI3K/Akt, NF-kB and MAPK pathways was decreased by SHH inhibition.

Conclusions: These findings support targeting SHH for the treatment of CRCC and pave the way for innovative and additional investigations in a broad range of cancers.

Show MeSH
Related in: MedlinePlus