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

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.

Figure 1

All the SHH signaling pathway components are expressed in human CRCC cells independently of VHL expression. (A) Western blot analysis of the SHH ligand in human CRCC cell lysates incubated with antibodies against human SHH ligand and corresponding β-actin. The expression of the ligand was assessed in 786-0 cells either untransfected (786-0 wt) or transfected with the vector alone (786-0 V), the full-length human VHL cDNA (786-0 VHL) or truncated inactive VHL cDNA sequence (786-0 ΔVHL), as well as in a panel of human CRCC cell lines either deficient in VHL expression (Deficient in VHL) or expressing VHL (Expressing VHL). The gels shown are representative for at least 3 independent experiments. (B) Quantitative gene expression of SHH ligand, of Ptch1 and Smo receptors, and of the Glis transcription factors (Gli1, Gli2 and Gli3) in the same panel of cells depicted in (A). Results are shown as mean ± SEM, n = 4.

Figure 2

All the SHH signaling pathway components are expressed in human CRCC tumors. (A) Western blot analysis of the SHH ligand in 9 human tumors (T1 ...) and normal corresponding tissues (N1 ...) lysates incubated with antibodies against human SHH ligand and corresponding β-actin. The gels shown are representative for at least 3 independent experiments. (B) Quantitative gene expression of Ptch1 and Smo receptors, and of the Gli1 transcription factor in the same normal/tumoral tissue pairs shown in (A). Results are representative of 4 independent experiments.

Figure 3

The inhibition of the SHH signaling pathway decreases human CRCC cell proliferation. (A) Human 786-0 cells were treated in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM) and cells were counted each day. Results are shown as mean ± SEM, n = 6; *, P < 0.05; **, P < 0.01 from Ctl. (B) Human 786-0 cells were treated for 5 days in control (Ctl) or with cyclopamine (Cyclopamine) and adherent cells were counted. Results are shown as mean ± SEM, n = 6; **, P < 0.01 from 0 (without cyclopamine). (C) Our panel of human CRCC cells either deficient or expressing VHL were treated treated for 5 days in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM) and adherent cells were counted. Results are shown as mean ± SEM, n = 6; **, P < 0.01 from Ctl (without cyclopamine) that was set to 100%. (D) Human 786-0 wt, 786-0 V, 786-0 VHL and 786-0 πVHL cells were analyzed for BrdU incorporation after treatment in control (Ctl) or with cyclopamine (Cyclopamine, 20 μM), for the indicated periods of time. Results are shown as mean ± SEM, n = 6; *, P < 0.05; **, P < 0.01 from corresponding Ctl.

Figure 4

The inhibition of the SHH signaling pathway induces human CRCC cells apoptosis. FACS analysis of 786-0 treated in control (Ctl) or with cyclopamine (Cyclopamine) at 20 μM. (A) Exemples of FACS analysis (day 4). No evidence of necrosis was observed in any cases. (B) Quantitative analysis of apoptotic cells as a function of the time of treatments. Results are shown as mean ± SEM, n = 6 *, P < 0.05 and **, P < 0.01 from Ctl apoptosis. Bars, 5 μm.

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.

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.

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.