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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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Related in: MedlinePlus

The SHH signaling pathway plays a pivotal and orchestral role in the constitutive activation of oncogenic pathways in human CRCC. (A) Western blots analysis of human CRCC 786-0 cell lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against non-phosphorylated GSK-3 (GSK-3), phospho-GSK-3 (P-GSK3), non-phosphorylated Akt (Akt), phospho-Akt (P-Akt), non-phosphorylated NF-κB (NF-κB), phospho-NF-κB (P-NF-κB), non-phosphorylated Erk1/2 (Erk1/2), phospho-Erk1/2 (P-Erk1/2) and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Western blots analysis of human CRCC 786-0 cells lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against Gli1, cyclin D1, Pax2, Lim1, VEGF, TGF-β1 and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (C) Western blot analysis in 9 human tumors (T1 ...) and normal corresponding tissues (N1 ...) lysates incubated with antibodies against Gli1, cyclin D1, Pax2, Lim1, and corresponding β-actin. The gels shown are representative for at least 3 independent experiments.
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Figure 7: The SHH signaling pathway plays a pivotal and orchestral role in the constitutive activation of oncogenic pathways in human CRCC. (A) Western blots analysis of human CRCC 786-0 cell lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against non-phosphorylated GSK-3 (GSK-3), phospho-GSK-3 (P-GSK3), non-phosphorylated Akt (Akt), phospho-Akt (P-Akt), non-phosphorylated NF-κB (NF-κB), phospho-NF-κB (P-NF-κB), non-phosphorylated Erk1/2 (Erk1/2), phospho-Erk1/2 (P-Erk1/2) and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Western blots analysis of human CRCC 786-0 cells lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against Gli1, cyclin D1, Pax2, Lim1, VEGF, TGF-β1 and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (C) Western blot analysis in 9 human tumors (T1 ...) and normal corresponding tissues (N1 ...) lysates incubated with antibodies against Gli1, cyclin D1, Pax2, Lim1, and corresponding β-actin. The gels shown are representative for at least 3 independent experiments.

Mentions: We next evaluated the effect of cyclopamine and of Smo and Gli1 silencing through transient transfection on GSK-3 activation and of all of the above-mentioned signaling pathways by western blot in 786-0 cells. The non-phosphorylated states of GSK-3, Akt, NF-κB and Erk1/2 remain unchanged after cyclopamine treatments (Figure 7A). However, cyclopamine treatments induced a decrease in the phosphorylation state of Akt, NF-κB and Erk1/2, and an increase in the phosphorylated state of GSK-3 (Figure 7A), thus inhibiting their biological activities. Again, similar results were obtained after Smo or Gli1 silencing (Additional file 8).


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)

The SHH signaling pathway plays a pivotal and orchestral role in the constitutive activation of oncogenic pathways in human CRCC. (A) Western blots analysis of human CRCC 786-0 cell lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against non-phosphorylated GSK-3 (GSK-3), phospho-GSK-3 (P-GSK3), non-phosphorylated Akt (Akt), phospho-Akt (P-Akt), non-phosphorylated NF-κB (NF-κB), phospho-NF-κB (P-NF-κB), non-phosphorylated Erk1/2 (Erk1/2), phospho-Erk1/2 (P-Erk1/2) and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Western blots analysis of human CRCC 786-0 cells lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against Gli1, cyclin D1, Pax2, Lim1, VEGF, TGF-β1 and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (C) Western blot analysis in 9 human tumors (T1 ...) and normal corresponding tissues (N1 ...) lysates incubated with antibodies against Gli1, cyclin D1, Pax2, Lim1, and corresponding β-actin. The gels shown are representative for at least 3 independent experiments.
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Figure 7: The SHH signaling pathway plays a pivotal and orchestral role in the constitutive activation of oncogenic pathways in human CRCC. (A) Western blots analysis of human CRCC 786-0 cell lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against non-phosphorylated GSK-3 (GSK-3), phospho-GSK-3 (P-GSK3), non-phosphorylated Akt (Akt), phospho-Akt (P-Akt), non-phosphorylated NF-κB (NF-κB), phospho-NF-κB (P-NF-κB), non-phosphorylated Erk1/2 (Erk1/2), phospho-Erk1/2 (P-Erk1/2) and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Western blots analysis of human CRCC 786-0 cells lysates treated for 2 days in control (Ctl) or with cyclopamine (Cy) at 20 μM and incubated with the antibodies against Gli1, cyclin D1, Pax2, Lim1, VEGF, TGF-β1 and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (C) Western blot analysis in 9 human tumors (T1 ...) and normal corresponding tissues (N1 ...) lysates incubated with antibodies against Gli1, cyclin D1, Pax2, Lim1, and corresponding β-actin. The gels shown are representative for at least 3 independent experiments.
Mentions: We next evaluated the effect of cyclopamine and of Smo and Gli1 silencing through transient transfection on GSK-3 activation and of all of the above-mentioned signaling pathways by western blot in 786-0 cells. The non-phosphorylated states of GSK-3, Akt, NF-κB and Erk1/2 remain unchanged after cyclopamine treatments (Figure 7A). However, cyclopamine treatments induced a decrease in the phosphorylation state of Akt, NF-κB and Erk1/2, and an increase in the phosphorylated state of GSK-3 (Figure 7A), thus inhibiting their biological activities. Again, similar results were obtained after Smo or Gli1 silencing (Additional file 8).

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