Involvement of the membrane-bound mucin MUC1 in renal-clear cell carcinoma progression and identification of new therapeutic targets

Le carcinome rénal représente 5% des tumeurs de l’adulte et se développe au niveau des tubules rénaux. Le sous-type histologique majeur des cancers du rein est le carcinome rénal à cellules claires (cRCC). 90% des cRCC présentent une inactivation biallélique du gène suppresseur de tumeur de Von Hippel Lindau (VHL) induisant une activation constitutive de la voie de l’hypoxie via le facteur de transcription HIF1-α (Hypoxia Inducible Factor) qui contribue à la physiologie des tumeurs. Les cRCC sont des tumeurs à la fois radio- et chimiorésistantes, rendant la prise en charge thérapeutique des patients très difficile.Nos recherches consistaient en l’étude des rôles de la mucine membranaire MUC1, dont la queue cytoplasmique (MUC1 CT) peut interagir avec différentes voies de signalisation et agir en tant que co-activateur transcriptionnel de nombreux gènes impliqués dans la progression tumorale et la diffusion métastatique. Des travaux antérieurs réalisés au laboratoire montraient que la surexpression de MUC1 observée dans les cRCC était associée au statut métastatique des patients et marquait un mauvais pronostic. Cette surexpression de MUC1 est également impliquée dans la voie de l’hypoxie, voie majeure de la carcinogenèse rénale. Le premier objectif de l’étude était donc de déterminer les effets de la surexpression de MUC1 sur les propriétés des cellules de cRCC. Nous montrons ainsi que le domaine extracellulaire de MUC1 ainsi que sa partie cytoplasmique sont impliqués dans l’augmentation des capacités migratoires et de la viabilité des cellules cancéreuses rénales et qu’elle leur confère une résistance à l’anoïkis, programme de mort cellulaire déclenché lorsque la cellule perd ses contacts avec les cellules voisines ou avec la matrice extra-cellulaire et diminuent les propriétés d’agrégation des cellules tumorales. Nous montrons également que MUC1 est impliquée dans la chimiorésistance des cRCC en induisant l’expression de genes de chimiorésistance comme ABCG2 et GSTO2. Nous montrons par ailleurs que les propriétés invasives des cellules de cRCC sont exclusivement liées à MUC1 CT. Le deuxième objectif de l’étude était d’identifier les mécanismes moléculaires à l’origine du clivage de MUC1 CT. En utilisant différentes stratégies (siARN, inhibiteurs pharmacologiques et peptides), nous montrons pour la première fois que deux sheddases, ADAM10 et ADAM17 et la gamma secrétase sont nécessaires au clivage de MUC1 C, permettant ainsi sa délocalisation nucléaire et l’augmentation des propriétés invasives des cellules de cRCC. Enfin, nous montrons que la surexpression de MUC1 augmente l’expression protéique d’ADAM10/17, suggérant une boucle de régulation positive existant en conditions pathologiques.En conclusion, notre étude souligne le rôle de MUC1 dans la progression tumorale rénale et montre que la localisation nucléaire de MUC1-C est à l’origine de l’acquisition d’un phénotype invasif et chimiorésistant via l’action des sheddases ADAM10/17 et de la gamma secrétase. MUC1 apparait alors comme une cible thérapeutique potentielle intéressante dans la prise en charge des cRCC.Renal cell carcinoma corresponds to 5% of all adult malignancies and originates from renal tubules. The main histologic subtype is represented by clear renal cell carcinoma. Ninety percent of cRCC present a biallelic inactivation of the von Hippel Lindau (VHL) tumor suppressor gene resulting in constitutive activation of hypoxia signaling pathway via the Hypoxia Inducible Factor (HIF) -1 transcription factor that contributes to the physiology of tumours. cRCC is typically highly resistant to conventional systemic therapies. MUC1 is a membrane-anchored mucin and its cytoplasmic tail (CT) can interact with many signaling pathways and act as a co-transcription factor to activate genes involved in tumor progression and metastasis. Previous studies have shown that MUC1 is diffusely overexpressed in cRCC and MUC1 overexpression has been found to be associated with metastatic disease and a worse prognosis.MUC1 is overexpressed in renal cell carcinoma with correlation to prognosis and has been implicated in the hypoxic pathway, the main renal carcinogenetic pathway. In this context, we assessed the effects of MUC1 overexpression on renal cancer cells properties. Using shRNA strategy and/or different MUC1 constructs, we found that MUC1-extracellular domain and MUC1 CT are both involved in increase of migration, cell viability, resistance to anoikis and to decrease of cell aggregation in cancer cells. We also showed that MUC1 is involved in cRCC chemoresistance by inducing chemoresistance genes expression like ABCG2 and GSTO2. Invasiveness depends only on MUC1 CT. Then, by using siRNA strategy and/or pharmacological inhibitors or peptides, we showed that sheddases ADAM10, ADAM17 and gamma-secretase are necessary for MUC1 C-terminal subunit (MUC1-C) nuclear location and in increase of invasion property. Finally, MUC1 overexpression increases ADAM10/17 protein expression suggesting a positive regulatory loop. In conclusion, we report that MUC1 acts in renal cancer progression and MUC1-C nuclear localization is driving invasiveness of renal cancer cells through a sheddase/gamma secretase dependent pathway. MUC1 appears as a therapeutic target by blocking MUC1 cleavage or nuclear translocation by using pharmacological approach and peptide strategies

Implication de la mucine membranaire MUC1 dans la progression tumorale rénale et identification de nouvelles cibles thérapeutiques

Renal cell carcinoma corresponds to 5% of all adult malignancies and originates from renal tubules. The main histologic subtype is represented by clear renal cell carcinoma. Ninety percent of cRCC present a biallelic inactivation of the von Hippel Lindau (VHL) tumor suppressor gene resulting in constitutive activation of hypoxia signaling pathway via the Hypoxia Inducible Factor (HIF) -1 transcription factor that contributes to the physiology of tumours. cRCC is typically highly resistant to conventional systemic therapies. MUC1 is a membrane-anchored mucin and its cytoplasmic tail (CT) can interact with many signaling pathways and act as a co-transcription factor to activate genes involved in tumor progression and metastasis. Previous studies have shown that MUC1 is diffusely overexpressed in cRCC and MUC1 overexpression has been found to be associated with metastatic disease and a worse prognosis.MUC1 is overexpressed in renal cell carcinoma with correlation to prognosis and has been implicated in the hypoxic pathway, the main renal carcinogenetic pathway. In this context, we assessed the effects of MUC1 overexpression on renal cancer cells properties. Using shRNA strategy and/or different MUC1 constructs, we found that MUC1-extracellular domain and MUC1 CT are both involved in increase of migration, cell viability, resistance to anoikis and to decrease of cell aggregation in cancer cells. We also showed that MUC1 is involved in cRCC chemoresistance by inducing chemoresistance genes expression like ABCG2 and GSTO2. Invasiveness depends only on MUC1 CT. Then, by using siRNA strategy and/or pharmacological inhibitors or peptides, we showed that sheddases ADAM10, ADAM17 and gamma-secretase are necessary for MUC1 C-terminal subunit (MUC1-C) nuclear location and in increase of invasion property. Finally, MUC1 overexpression increases ADAM10/17 protein expression suggesting a positive regulatory loop. In conclusion, we report that MUC1 acts in renal cancer progression and MUC1-C nuclear localization is driving invasiveness of renal cancer cells through a sheddase/gamma secretase dependent pathway. MUC1 appears as a therapeutic target by blocking MUC1 cleavage or nuclear translocation by using pharmacological approach and peptide strategies.Le carcinome rénal représente 5% des tumeurs de l’adulte et se développe au niveau des tubules rénaux. Le sous-type histologique majeur des cancers du rein est le carcinome rénal à cellules claires (cRCC). 90% des cRCC présentent une inactivation biallélique du gène suppresseur de tumeur de Von Hippel Lindau (VHL) induisant une activation constitutive de la voie de l’hypoxie via le facteur de transcription HIF1-α (Hypoxia Inducible Factor) qui contribue à la physiologie des tumeurs. Les cRCC sont des tumeurs à la fois radio- et chimiorésistantes, rendant la prise en charge thérapeutique des patients très difficile.Nos recherches consistaient en l’étude des rôles de la mucine membranaire MUC1, dont la queue cytoplasmique (MUC1 CT) peut interagir avec différentes voies de signalisation et agir en tant que co-activateur transcriptionnel de nombreux gènes impliqués dans la progression tumorale et la diffusion métastatique. Des travaux antérieurs réalisés au laboratoire montraient que la surexpression de MUC1 observée dans les cRCC était associée au statut métastatique des patients et marquait un mauvais pronostic. Cette surexpression de MUC1 est également impliquée dans la voie de l’hypoxie, voie majeure de la carcinogenèse rénale. Le premier objectif de l’étude était donc de déterminer les effets de la surexpression de MUC1 sur les propriétés des cellules de cRCC. Nous montrons ainsi que le domaine extracellulaire de MUC1 ainsi que sa partie cytoplasmique sont impliqués dans l’augmentation des capacités migratoires et de la viabilité des cellules cancéreuses rénales et qu’elle leur confère une résistance à l’anoïkis, programme de mort cellulaire déclenché lorsque la cellule perd ses contacts avec les cellules voisines ou avec la matrice extra-cellulaire et diminuent les propriétés d’agrégation des cellules tumorales. Nous montrons également que MUC1 est impliquée dans la chimiorésistance des cRCC en induisant l’expression de genes de chimiorésistance comme ABCG2 et GSTO2. Nous montrons par ailleurs que les propriétés invasives des cellules de cRCC sont exclusivement liées à MUC1 CT. Le deuxième objectif de l’étude était d’identifier les mécanismes moléculaires à l’origine du clivage de MUC1 CT. En utilisant différentes stratégies (siARN, inhibiteurs pharmacologiques et peptides), nous montrons pour la première fois que deux sheddases, ADAM10 et ADAM17 et la gamma secrétase sont nécessaires au clivage de MUC1 C, permettant ainsi sa délocalisation nucléaire et l’augmentation des propriétés invasives des cellules de cRCC. Enfin, nous montrons que la surexpression de MUC1 augmente l’expression protéique d’ADAM10/17, suggérant une boucle de régulation positive existant en conditions pathologiques.En conclusion, notre étude souligne le rôle de MUC1 dans la progression tumorale rénale et montre que la localisation nucléaire de MUC1-C est à l’origine de l’acquisition d’un phénotype invasif et chimiorésistant via l’action des sheddases ADAM10/17 et de la gamma secrétase. MUC1 apparait alors comme une cible thérapeutique potentielle intéressante dans la prise en charge des cRCC

Enantioselective synthesis of thioesters as substrates for high-through put screening assays of Penicillin Binding Proteins

Excessive utilization of beta-lactam antibiotics like penicillin has created drug-resistant strains in bacteria. One of the main mechanisms of resistance is the production of drug resistant Penicillin Binding Proteins (PBPs) and the over expression of these proteins. The transglycosidase and transpeptidase activities of PBPs catalyze the last two steps of peptidoglycan biosynthesis, which is unique to bacteria, and lies outside the cytoplasmic membrane. PBPs are interesting targets and efforts are still done to find new inhibitors. A thioesterase activity has been described for various PBPs. For example, the thioester S2d is a substrate of PBP R39 of Actinomadura and of PBP2x of Streptococcus pneumoniae. The utilization of thioesters allows a rapid screening of active compounds in high-through put screening assays. Furthermore detailed kinetic studies using thioesters as reporter substrates are also possible. Here we will present the enantioselective synthesis of the thioesters and their application as substrates in high through put screening assays

DNA METHYLATION BY DNMT1 AND DNMT3b METHYLTRANSFERASES IS DRIVEN BY THE MUC1-C ONCOPROTEIN IN HUMAN CARCINOMA CELLS

Aberrant expression of the DNA methyltransferases (DNMTs) and disruption of DNA methylation patterns are associated with carcinogenesis and cancer cell survival. The oncogenic MUC1-C protein is aberrantly overexpressed in diverse carcinomas; however, there is no known link between MUC1-C and DNA methylation. Our results demonstrate that MUC1-C induces expression of DNMT1 and DNMT3b, but not DNMT3a, in breast and other carcinoma cell types. We show that MUC1-C occupies the DNMT1 and DNMT3b promoters in complexes with NF-κB p65 and drives DNMT1 and DNMT3b transcription. In this way, MUC1-C controls global DNA methylation as determined by analysis of LINE-1 repeat elements. The results further demonstrate that targeting MUC1-C downregulates DNA methylation of the CDH1 tumor suppressor gene in association with induction of E-cadherin expression. These findings provide compelling evidence that MUC1-C is of functional importance to induction of DNMT1 and DNMT3b and, in turn, changes in DNA methylation patterns in cancer cells

Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C.

In response to the widespread use of beta-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent beta-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3muM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50)=26muM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50)=138muM) and a class C PBP (R39 from Actinomadura sp., IC(50)=0.6muM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species

Targeting MUC1-C suppresses polycomb repressive complex 1 in multiple myeloma

The polycomb repressive complex 1 (PRC1) includes the BMI1, RING1 and RING2 proteins. BMI1 is required for survival of multiple myeloma (MM) cells. The MUC1-C oncoprotein is aberrantly expressed by MM cells, activates MYC and is also necessary for MM cell survival. The present studies show that targeting MUC1-C with (i) stable and inducible silencing and CRISPR/Cas9 editing and (ii) the pharmacologic inhibitor GO-203, which blocks MUC1-C function, downregulates BMI1, RING1 and RING2 expression. The results demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism. MUC1-C thus promotes MYC occupancy on the BMI1 promoter and thereby activates BMI1 expression. We also show that the MUC1-C→MYC pathway induces RING2 expression. Moreover, in contrast to BMI1 and RING2, we found that MUC1-C drives RING1 by an NF-κB p65-dependent mechanism. Targeting MUC1-C and thereby the suppression of these key PRC1 proteins was associated with downregulation of the PRC1 E3 ligase activity as evidenced by decreases in ubiquitylation of histone H2A. Targeting MUC1-C also resulted in activation of the PRC1-repressed tumor suppressor genes, PTEN, CDNK2A and BIM. These findings identify a heretofore unrecognized role for MUC1-C in the epigenetic regulation of MM cells

MUC1-C activates EZH2 expression and function in human cancer cells

The EZH2 histone methyltransferase is a member of the polycomb repressive complex 2 (PRC2) that is highly expressed in diverse human cancers and is associated with a poor prognosis. MUC1-C is an oncoprotein that is similarly overexpressed in carcinomas and has been linked to epigenetic regulation. A role for MUC1-C in regulating EZH2 and histone methylation is not known. Here, we demonstrate that targeting MUC1-C in diverse human carcinoma cells downregulates EZH2 and other PRC2 components. MUC1-C activates (i) the EZH2 promoter through induction of the pRB→E2F pathway, and (ii) an NF-κB p65 driven enhancer in exon 1. We also show that MUC1-C binds directly to the EZH2 CXC region adjacent to the catalytic SET domain and associates with EZH2 on the CDH1 and BRCA1 promoters. In concert with these results, targeting MUC1-C downregulates EZH2 function as evidenced by (i) global and promoter-specific decreases in H3K27 trimethylation (H3K27me3), and (ii) activation of tumor suppressor genes, including BRCA1. These findings highlight a previously unreported role for MUC1-C in activating EZH2 expression and function in cancer cells

MUC1-C induces DNA methyltransferase 1 and represses tumor suppressor genes in acute myeloid leukemia

Aberrant DNA methylation is a hallmark of acute myeloid leukemia (AML); however, the regulation of DNA methyltransferase 1 (DNMT1), which is responsible for maintenance of DNA methylation patterns, has largely remained elusive. MUC1-C is a transmembrane oncoprotein that is aberrantly expressed in AML stem-like cells. The present studies demonstrate that targeting MUC1-C with silencing or a pharmacologic inhibitor GO-203 suppresses DNMT1 expression. In addition, MUC1 expression positively correlates with that of DNMT1 in primary AML cells, particularly the CD34+/CD38− population. The mechanistic basis for this relationship is supported by the demonstration that MUC1-C activates the NF-κB p65 pathway, promotes occupancy of the MUC1-C/NF-κB complex on the DNMT1 promoter and drives DNMT1 transcription. We also show that targeting MUC1-C substantially reduces gene promoter-specific DNA methylation, and derepresses expression of tumor suppressor genes, including CDH1, PTEN and BRCA1. In support of these results, we demonstrate that combining GO-203 with the DNMT1 inhibitor decitabine is highly effective in reducing DNMT1 levels and decreasing AML cell survival. These findings indicate that (i) MUC1-C is an attractive target for the epigentic reprogramming of AML cells, and (ii) targeting MUC1-C in combination with decitabine is a potentially effective clinical approach for the treatment of AML

MUC1-C ACTIVATES BMI1 IN HUMAN CANCER CELLS

BMI1 is a component of the PRC1 complex that is overexpressed in breast and other cancers, and promotes self-renewal of cancer stem-like cells. The oncogenic mucin 1 (MUC1) C-terminal (MUC1-C) subunit is similarly overexpressed in human carcinoma cells and has been linked to their self-renewal. There is no known relationship between MUC1-C and BMI1 in cancer. The present studies demonstrate that MUC1-C drives BMI1 transcription by a MYC-dependent mechanism in breast and other cancer cells. In addition, we show that MUC1-C blocks miR-200c-mediated downregulation of BMI1 expression. The functional significance of this MUC1-C→BMI1 pathway is supported by the demonstration that targeting MUC1-C suppresses BMI1-induced ubiquitylation of H2A and thereby derepresses homeobox HOXC5 and HOXC13 gene expression. Notably, our results further show that MUC1-C binds directly to BMI1 and promotes occupancy of BMI1 on the CDKN2A promoter. In concert with BMI1-induced repression of the p16INK4a tumor suppressor, we found that targeting MUC1-C is associated with induction of p16INK4a expression. In support of these results, analysis of three gene expresssion datasets demonstrated highly significant correlations between MUC1-C and BMI1 in breast cancers. These findings uncover a previously unrecognized role for MUC1-C in driving BMI1 expression and in directly interacting with this stem cell factor, linking MUC1-C with function of the PRC1 in epigenetic gene silencing