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

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: focus on the cancer hallmark of tumor angiogenesis

One of the important ‘hallmarks’ of cancer is angiogenesis, which is the process of formation of new blood vessels that are necessary for tumor expansion, invasion and metastasis. Under normal physiological conditions, angiogenesis is well balanced and controlled by endogenous proangiogenic factors and antiangiogenic factors. However, factors produced by cancer cells, cancer stem cells and other cell types in the tumor stroma can disrupt the balance so that the tumor microenvironment favors tumor angiogenesis. These factors include vascular endothelial growth factor, endothelial tissue factor and other membrane bound receptors that mediate multiple intracellular signaling pathways that contribute to tumor angiogenesis. Though environmental exposures to certain chemicals have been found to initiate and promote tumor development, the role of these exposures (particularly to low doses of multiple substances), is largely unknown in relation to tumor angiogenesis. This review summarizes the evidence of the role of environmental chemical bioactivity and exposure in tumor angiogenesis and carcinogenesis. We identify a number of ubiquitous (prototypical) chemicals with disruptive potential that may warrant further investigation given their selectivity for high-throughput screening assay targets associated with proangiogenic pathways. We also consider the cross-hallmark relationships of a number of important angiogenic pathway targets with other cancer hallmarks and we make recommendations for future research. Understanding of the role of low-dose exposure of chemicals with disruptive potential could help us refine our approach to cancer risk assessment, and may ultimately aid in preventing cancer by reducing or eliminating exposures to synergistic mixtures of chemicals with carcinogenic potential

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Assessing the carcinogenic potential of low-dose exposures to chemical mixtures in the environment: the challenge ahead

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/ mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology

Les protéines de stabilisation des ARN messagers de la protéine apparentée à l hormone parathyroïdienne (PTHrP) (Identification et impact sur la croissance du carcinome à cellules rénales)

Le carcinome à cellules rénales (CCR), dont le sous-type le plus fréquent est le CCR conventionnel (CCC), représente 90% des cancers du rein. Son incidence augmente de façon constante et il reste de mauvais pronostic. La protéine apparentée à l hormone parathyroïdienne (PTHrP) est exprimée sous forme de 3 isoformes d ARNm codant pour des protéines de 139, 141 et 173 acides aminés. Elle a récemment été identifiée comme un facteur de survie essentiel à la croissance du CCC et comme une nouvelle cible du gène suppresseur de tumeur von Hippel Lindau (VHL) qui régule négativement son expression au niveau post-transcriptionnel. Le VHL est muté dans la majorité des cas de CCC, et dont la principale fonction est de dégrader en conditions normoxiques les facteurs de transcription induits par l hypoxie HIF-a qui régulent au niveau transcriptionnel et/ou post-transcriptionnel de nombreux facteurs favorisant la cancérogénèse, dont la PTHrP. Ce travail de thèse, a permis d établir que dans les CCC, l isoforme 141 de l ARNm de la PTHrP est celle qui est majoritairement exprimée est qu elle est stabilisée par HuR, une protéine ubiquitaire qui comme nous l avons montré, stabilise également les ARNm d autres facteurs cibles du système VHL/HIF comme le VEGF ou le TGF-b1. Nous avons également montré que HuR est surexprimée dans les CCC et que son extinction par siARN in vitro et in vivo dans un modèle de souris nude xénogreffées inhibe substanciellement la croissance tumorale via une inhibition de la prolifération cellulaire et une augmentation de l apoptose. Cibler cette protéine pourrait donc s avérer être une nouvelle option thérapeutique pour le traitement du CCC humain.Conventional renal cell carcinoma (CCC) is the main sub-type of RCC representing around 80% of all RCC cases. His incidence is in constant progression and the prognostic is poor. Parathyroid hormone-related protein (PTHrP) is a polyprotein alternatively spliced into 3 mRNA isoforms coding for proteins containing 139, 141 and 173 amino acids. We have previously shown that PTHrP is a survival factor for human RCC and that its expression is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor gene at the level of mRNA stability, as observed for growth and angiogenic factors such as VEGF or TGF-b. Using a panel of CCC cell lines either deficient for the VHL or expressing wild-type VHL, VHL-deficient 786-0 cells transfected with active or inactive VHL and human RCC samples and corresponding normal tissues, we showed first that PTHrP 141 mRNA isoform is prédominantly express in human RCC and a target for the mRNA stabilizing protein HuR. Interestingly, we showed that this ubiquitously expressed protein involved in the mRNA stabilization of number of others VHL/HIF target genes are overexpressed in all CCC samples we used. Moreover, through HuR knockdown using specific siRNA in cultured cell or in the nude mice xenografted model, we showed that this protein is criticaly involved in CCC tumor growth and that this effect is obtained through an inibition of cell proliferation and an increase in cell apoptosis. These results identify HuR as a potent new therapeutic target for the treatment of CRCC which remains a refractory disease.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Du développement au cancer (Implication des voies néphrogéniques dans la croissance du carcinome à cellules rénales humain)

Le cancer du rein est résistant aux thérapies existantes. Des acteurs intervenant dans l homéostasie peuvent être réexprimés par des cellules cancéreuses. Nous avons recherché ici si la cancérogenèse et la progression tumorale font appel à des voies/marqueurs développementaux. Nos investigations se sont portées spécifiquement sur la voie développementale du sonic hedgehog et par la suite sur le facteur de transcription néphrogénique Lim1. L accès aux tumeurs humaines et l exploitation de lignées cellulaires nous a permis d analyser l expression de cette voie signalétique et de ce marqueur néphrogénique dans le carcinome à cellules rénales. Des inhibiteurs pharmacologiques ainsi que l utilisation de transfections de siRNA/cDNA ont été utilisés pour évaluer l effet in vitro de ces acteurs du développement sur la prolifération et l apoptose des cellules cancéreuses. La migration a été étudiée par l utilisation de chambres de Boyden et l activation/interaction d autres voies oncogéniques a été recherchée par Western blot. Finalement une approche in vivo a permis une validation pré-clinique du bloquage de ces voies. Nous avons montré que la voie HH et que Lim1 sont réexprimés dans les tumeurs et dans l ensemble des lignées humaines. Leur inhibition entraine une diminution importante de la prolifération ainsi que de la migration cellulaires. In vivo, une inhibition de la croissance tumorale a été observée en ciblant cette voie et ce marqueur. Le ciblage de voies impliquées dans le développement peut constituer une innovation thérapeutique importante dans le traitement des cancers du rein et pose une pièce supplémentaire dans le puzzle moléculaire des mécanismes cancéreux.Kidney cancer remains resistant to therapies. Several genes play essential roles in human development, particularly during nephrogenesis. The concept suggesting that these actors could be expressed by cancer cells has recently emerged. In our studies, we investigated if cancerogenesis and tumor growth in renal cell carcinoma are linked to the developmental pathways. For this purpose, we focussed particularly on the developmental sonic hedgehog pathway, and then on the nephrogenic transcription factor Lim1. The expression of the SHH pathway and of Lim1 in RCC were analysed on RCC cell lines and tumors from human RCC. The proliferative and apoptosis effects of the SHH pathway and of Lim1 on kidney cancer cells were evaluated in vitro. Their involvement in kidney cancer cells migration and invasion were also studied, as well as their interaction with oncogenic pathways by Western blot. Focussed on the description of therapeutical innovation, we used xenografted mice to analyze the effects of the inhibition of these developmental pathways/markers in vivo. Our results demonstrate that the SHH pathway and Lim1 are reexpressed in RCC cell lines and in human tumors. The inhibition of these actors led to a radical decrease of cancer cells proliferation and migration. In vivo, targeting these pathways/markers, that we showed to participate to the regulation of oncogenic pathways, induced a decrease of tumor growth and even marked tumor regression. The developmental pathways implicated in RCC growth could constitute an important therapeutical innovation in the treatment of this cancer, and allow us to put an additional piece in the molecular puzzle of molecular cancer mechanisms.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Implication des voies signalétiques de survie dans la croissance du carcinome à cellules rénales

Au cours de cette thèse, nous nous sommes intéressés aux voies de signalisation PI3K/Akt et NF B et plus particulièrement à leur implication dans la croissance du carcinome à cellules rénales humain (CCR). Ces voies, impliquées dans la prolifération ou la mort cellulaire, jouent un rôle crucial dans la croissance et la survie cellulaire. Nous avons montré que ces voies sont activées de manière constitutive dans le CCR humain et qu elles jouent un rôle important dans la croissance et la survie de ces tumeurs in vitro et in vivo, quel que soit le statut en gène suppresseur de tumeur von Hippel-Lindau (VHL). Nos résultats identifient la PI3K ainsi que l inhibiteur endogène de NF B, I Ba, comme de nouvelles cibles thérapeutiques du CCR humain. Nous avons également mis en évidence l existence d une boucle de régulation entre Akt, GSK3 et NF B, et bien que cette régulation soit dépendante du statut en VHL, elle converge cependant vers NF B. De plus, des cibles de NF B ont été identifiées.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Recherche de nouveaux facteurs pronostiques et thérapeutiques dans le carcinome à cellules rénales humain

RESUME : Le cancer du rein, correspondant dans 90% des cas à un carcinome à cellules rénales (CCR), est d incidence croissante et de mauvais pronostic. Malgré l amélioration récente de la prise en charge thérapeutique des CCR métastatiques (anti-angiogéniques), il convient de préciser les critères pronostiques et de développer de nouvelles thérapies combinées et plus spécifiques. Nous avons étudié le mécanisme d action de la protéine apparentée à l hormone parathyroïdienne (PTHrP) exprimée dans 90% des CCR et son interaction avec d autres voies de signalisation. Nos études in vitro et in vivo ont mis en évidence que la PTHrP a des propriétés anti-apoptotique via la stimulation de la voie PI3K/Akt, avec une activation spécifique du facteur de transcription Nuclear Factor kappaB (NF B). La voie NF B est impliquée dans la résistance intrinsèque du CCR à l apoptose, indépendamment du statut du gène suppresseur de tumeur von Hippel-Lindau (VHL), souvent inactivé dans des CCR. Sur une puce à tissus de 249 CCR humains (suivi de 12-22 ans), l expression immunohistochimique de pAkt et pNF B (phosphorylés) est corrélée au type histologique (cellules claires). L augmentation de l expression de pAkt est corrélée au grade de Fuhrman et à une diminution de la survie globale et sans récidive. Par contre, l expression nucléaire de pNF B est plus importante si la tumeur est de petite taille et de stade localisé avec une meilleure survie sans récidive. Cependant, ces 2 facteurs ne sont pas indépendants. Les voies PTHrP/PI3K/Akt et NF B sont impliquées dans la croissance et la survie du CCR humain, ouvrant de nouvelles perspectives pronostiques mais également thérapeutiques dans cette pathologie.SUMMARY: Incidence of renal cell carcinoma (RCC) is increasing with a pejorative prognosis. It accounts for more than 90% of adult renal neoplasms. Despite the recent major improvements in the treatment of metastatic RCC (anti-angiogenic drugs), ones needs to precise prognostic factors and to develop more specific and combined targeted therapies. The aim of our project was to determine the implication of parathyroid hormone-related protein (PTHrP), expressed in 90% of RCC and its interaction with the other known signaling pathways involved in carcinogenesis. We identified the role of PTHrP in controlling tumor cell survival in vitro and in vivo. PTHrP acts through the PI3K/Akt pathway leading to the specific activation of Nuclear Factor kappaB (NF B). The NF B pathway is involved in the intrinsic resistance of RCC to apoptosis, with no influence of von Hippel-Lindau (VHL) tumor suppressor gene expression., often inactivated in RCC. We built up a tissue-microarray containing 249 human RCC with 12 to 22 years of clinical follow-up. Clear cell RCC showed increased pAkt and pNF B (phosphorylated) immunoreactivity. Increased expression of pAkt was significantly associated with Fuhrman grade and reduced survival. Increased expression of pNF B was correlated with small and localized tumor with best recurrence free survival. These two prognostic factors were not found to be independent for patient survival. This report provides the strong implication of PTHrP/PI3K/Akt and NF B pathways in renal growth and survival of RCC, as promising prognostic and therapeutic.STRASBOURG-Medecine (674822101) / SudocSudocFranceF