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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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Cyclopamine treatment decreases the expression of the SHH ligand, Smo receptor, Gli1 factors, and increases the expression of the Ptch1. (A) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Shown are western blot analysis of the SHH ligand in cell lysates from cells treated in each condition and incubated with antibodies against human SHH ligand and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Quantitative gene expression of Ptch1, Smo receptors, Gli1, Gli2 and Gli3. Results are shown as mean ± SEM, n = 6 *, P < 0.05 from cells treated in Ctl at the same time point.
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Figure 5: Cyclopamine treatment decreases the expression of the SHH ligand, Smo receptor, Gli1 factors, and increases the expression of the Ptch1. (A) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Shown are western blot analysis of the SHH ligand in cell lysates from cells treated in each condition and incubated with antibodies against human SHH ligand and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Quantitative gene expression of Ptch1, Smo receptors, Gli1, Gli2 and Gli3. Results are shown as mean ± SEM, n = 6 *, P < 0.05 from cells treated in Ctl at the same time point.

Mentions: To check further the specificity of the inhibitor towards the SHH signaling pathway, we measured the expression of all the molecular components of the pathway by western blot or quantitative analysis of mRNAs expression in 786-0 cells. The expression of the SHH ligand was surprisingly, but interestingly, decreased as a function of time by cyclopamine, suggesting that the SHH ligand may itself be a target of the SHH pathway (Figure 5A).


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)

Cyclopamine treatment decreases the expression of the SHH ligand, Smo receptor, Gli1 factors, and increases the expression of the Ptch1. (A) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Shown are western blot analysis of the SHH ligand in cell lysates from cells treated in each condition and incubated with antibodies against human SHH ligand and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Quantitative gene expression of Ptch1, Smo receptors, Gli1, Gli2 and Gli3. Results are shown as mean ± SEM, n = 6 *, P < 0.05 from cells treated in Ctl at the same time point.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: Cyclopamine treatment decreases the expression of the SHH ligand, Smo receptor, Gli1 factors, and increases the expression of the Ptch1. (A) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Shown are western blot analysis of the SHH ligand in cell lysates from cells treated in each condition and incubated with antibodies against human SHH ligand and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Human 786-0 CRCC cells were seeded and treated for the indicated periods of time in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM). Quantitative gene expression of Ptch1, Smo receptors, Gli1, Gli2 and Gli3. Results are shown as mean ± SEM, n = 6 *, P < 0.05 from cells treated in Ctl at the same time point.
Mentions: To check further the specificity of the inhibitor towards the SHH signaling pathway, we measured the expression of all the molecular components of the pathway by western blot or quantitative analysis of mRNAs expression in 786-0 cells. The expression of the SHH ligand was surprisingly, but interestingly, decreased as a function of time by cyclopamine, suggesting that the SHH ligand may itself be a target of the SHH pathway (Figure 5A).

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