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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 β-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-β 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.</p> <p>Conclusions</p> <p>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.</p

Implication de la protéine apparentée à l'hormone parathyroïdienne (PTHrP) dans la prolifération des cellules musculaires lisses vasculaires et identification d'une cible nucléaire de la PTHrP

In vivo, les cellules musculaires lisses vasculaires sont continuellement soumises au stretch mécanique. Dans des conditions pathologiques, ce stretch mécanique est augmenté et participe au remodelage vasculaire. Notre laboratoire avait montré que la PTHrP exerce des effets paradoxaux sur la prolifération des CMLV, à savoir une inhibition auto/paracrine et une stimulation intracrine. Dans cette étude, des CMLV issus de vaisseaux de résistance (rein) et de conductance (aorte) ont été utilisées. Le rôle de la PTHrP sur la prolifération des CMLV stretchées a été appréhendé. Nous montrons que l'expression de PTHrP est augmentée en réponse au stretch mécanique et qu'elle s'oppose par un mécanisme intracrine à l'effet inhibiteur du stretch sur la prolifération. De plus, nous montrons que l'expression de la PTHrP est contrôlée de façon post transcriptionnelle par le stretch mécanique. Ces résultats suggèrent que la PTHrP pourrait jouer un important rôle dans la modulation de la prolifération des CMLV en réponse au stretch mécanique.L'élucidation des voies intracrines de la PTHrP est un enjeu de taille. Les précédents modèles n'ont pu être utilisés pour étudier les effets de la PTHrP endogène. Pour cela, des cultures de CMLV ont été obtenues à partir de souris transgéniques dont l'exon 4 de la PTHrP a été "floxé". Ces cellules ont été transféctées par la Cre recombinase pour exciser le gène de la PTHrP et étudier les effets de la PTHrP endogène ainsi que les effets de la PTHrP auto/paracrine et intracrine par la transfection de différentes constructions de PTHrP. Une des cibles nucléaires identifiées est la culline 3, impliquée dans la dégradation de la cycline E, Nrf2 et la Topoisomérase I. Son expression est stimulée dans les CMLV en réponse à la PTHrP comprenant la SLN et le stretch mécanique, ainsi que dans les carcinomes rénaux à cellules claires surexprimant la PTHrP, suggérant une possible implication de la PTHrP et la culline 3 dans la cancérogénèse.In vivo, vascular smooth muscle cells (VSMC) are continuously exposed to mechanical cyclic stretch due to the pulsatile blood flow from the cardiac contractile cycle. Stretch is altered in pathological conditions and contribute to vascular remodeling by modulating VSMC proliferation and death. We have previously shown that PTHrP inhibits VSMC proliferation through the auto/paracrine pathway but stimulates VSMC proliferation through the intracrine pathway by translocating into the nucleus. Here we used VSMC isolated from both resistance and compliance vessels to study the role of PTHrP in VSMC proliferation under experimental stretch. We show that PTHrP gene expression is up-regulated by stretch and that PTHrP opposes the inhibitory effect induced by stretch on VSMC proliferation through the intracrine pathway. In addition, we demonstrate that PTHrP expression is controlled at the post-transcriptional level by stretch. These results strongly suggest that PTHrP plays a critical role in the modulation of VSMC proliferation in response to stretch. Elucidation of intracrine pathway of PTHrP is a reel challenge, nothing is known for years. Previous models are unable to define effect of endogenous PTHrP. Here, I used primary VSMC derived from transgenic mice in which exon 4 of the PTHrP was floxed. These cells were transfected with a Cre recombiase to obtain PTHrP null cells. Then it was possible to study endogenous PTHrP and intracrine or paracrine effect of PTHrP with transfection of different mutant construction. One of the nuclear target identified was the cullin-3 which is implicated in degradation of the cycle E, Nrf2 and the topoisomerase I. Cullin 3 expression is upregulated in VSMC both in response of transfection with PTHrP and mechanical stretch but not with transfection with a mutant PTHrP without the nuclear localization signal and in clear cell renal carcinoma which strongly expressed PTHrP indicating a possible implication of PTHrP and cullin 3 in cancer.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF