Jacques Robert

Protein arginine (N)-methyl transferase 7 (PRMT7) as a potential target for the sensitization of tumor cells to camptothecins

PRMT7 belongs to the protein arginine methyl‐transferases family. We show that downregulation of PRMT7α and β isoforms in DC‐3F hamster cells was associated with increased sensitivity to the Top1 inhibitor camptothecin (CPT). This effect was not due to a change in Top1 contents or catalytic activity, or to a difference in the reversal of DNA breaks. Overexpression of PRMT7α and β in DC‐3F cells had no effect on CPT sensitivity, whereas it conferred a resistance to DC‐3F/9‐OH‐E cells for which both isoforms are reduced by two‐ to three‐fold as compared to DC‐3F parental cells. Finally, downregulation of the human PRMT7 could also sensitize HeLa cells to CPT, suggesting that it could be used as a target to potentiate CPT derivatives.

Short curcumin treatment modulates oxidative stress, arginase activity, aberrant crypt foci, and TGF-β1 and HES-1 transcripts in 1,2-dimethylhydrazine-colon carcinogenesis in mice

This study investigated the effect of short curcumin treatment, a natural antioxidant on 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in mice. The incidence of aberrant crypt foci (ACF) was 100%, with 54 ± 6 per colon, 10 weeks after the first DMH injection and reached 67 ± 12 per colon after 12 weeks. A high level of undifferentiated goblet cells and a weak apoptotic activity were shown in dysplastic ACF. The morphological alterations of colonic mucosa were associated to severe oxidative stress ratio with 43% increase in malondialdehyde vs. 36% decrease in GSH. DMH also increased inducible nitric synthase (iNOS) mRNA transcripts (250%), nitrites level (240%) and arginase activity (296%), leading to nitrosative stress and cell proliferation. Curcumin treatment, starting at week 10 post-DMH injection for 14 days, reduced the number of ACF (40%), iNOS expression (25%) and arginase activity (73%), and improved redox status by approximately 46%, compared to DMH-treated mice. Moreover, curcumin induced apoptosis of dysplastic ACF cells without restoring goblet cells differentiation. Interestingly, curcumin induced a parallel increase in TGF-β1 and HES-1 transcripts (42% and 26%, respectively). In conclusion, the protective effect of curcumin was driven by the reduction of arginase activity and nitrosative stress. The up regulation of TGF-β1 and HES-1 expression by curcumin suggests for the first time, a potential interplay between these signalling pathways in the chemoprotective mechanism of curcumin.

FOLFIRI® and Bevacizumab in first-line treatment for colorectal cancer patients: safety, efficacy and genetic polymorphisms

BackgroundOver 50% of colorectal cancer (CRC) patients develop metastases. The aim of this study was to evaluate efficacy and tolerance of first-line FOLFIRI®u2009+u2009bevacizumab (B) treatment for metastatic CRC, and to assess genetic polymorphisms as potential markers.MethodsAdult patients with histologically-proven, non-resectable metastatic CRC and ECOGu2009≤u20092 were included. 14-day cycles consisted of bevacizumab (5xa0mg/kg), irinotecan (180xa0mg/m2), bolus FU (400xa0mg/m2) and leucovorin (400xa0mg/m2), followed by 46-hour FU infusions (2400xa0mg/m2). Primary endpoint was response rate according to RECIST criteria. Secondary endpoints were overall (OS) and progression-free (PFS) survivals, response duration, and toxicity. Associations between clinical data, UGT1A1, thymidylate synthase, VEGFA polymorphisms and PFS, OS and toxicity were analyzed.ResultsSixty-two patients were enrolled (median age 68y). 59/62 patients were eligible and evaluable for response at 6xa0months: 28 showed partial response (47.5%; 95% CI; 34.3-60.9), 20 stable disease (33.9%) and 11 progression (18.6%). Grade 3/4 toxicities were as follows: neutropenia 16.1%; diarrhea 11.3%; nausea-vomiting 1.6%. Median response duration was 9.5xa0months (range 2.7-20); median PFS 10.3xa0months (range 8.8-11.7); and median OS 25.7xa0months (range 20.2-29.7). 11/59 initially unresectable patients were resectable after treatment. VEGFA polymorphism (rs25648) was associated with better OS (HR: 3.61; 95% CI: 1.57-8.30).ConclusionsFOLFIRI®u2009+u2009bevacizumab is active with good response rate, long median OS, and a good safety profile. A VEGFA polymorphism might have a prognostic value in this malignancy.Trial registrationClinicaltrials.gov: NCT00467142 (registration date: April 25, 2007)

Anti‐Inflammatory and Antiproliferative Activities of Organic Fractions from the Mediterranean Brown Seaweed, Cystoseira Compressa

Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I‐III Regulatory, Quality, Manufacturing Postmarketing Phase IV

Identification of methylguanine methyltransferase polymorphisms as genetic markers of individual susceptibility to therapy-related myeloid neoplasms

PURPOSEnMyelodysplastic syndromes (MDS) are pre-leukaemic haematopoietic stem cell disorders. Among them, 10-20% occur after chemotherapy and/or radiotherapy, and are called therapy-related MDS (t-MDS). The aim of this study was to identify genetic markers in t-MDS.nnnMETHODSnA prospective cohort of 59 MDS patients (39 de novo MDS, 20 t-MDS) was studied. A total of 384 single nucleotide polymorphisms (SNP) selected among genes involved in DNA repair, drug metabolism and transport, signal transduction and oncogenesis, were genotyped using a custom-made SNP chip.nnnRESULTSnTwo non-synonymous SNPs present in the methylguanine methyltransferase (MGMT) gene, in complete linkage disequilibrium, were significantly associated with t-MDS: rs2308321 and rs2308327, with a raw p value of 7.4 × 10(-5) and a corrected p value after Benjamini-Hochberg correction of 0.014. Other associations tested between clinical and cytogenetic features and SNP chip gene variants gave corrected p values above 0.05. A validation cohort was separately constituted of 43 patients (24 de novo MDS, 19 t-MDS) and the two MGMT SNPs were genotyped; it confirmed a significant association between the variant allele of MGMT and t-MDS (p=0.038).nnnCONCLUSIONnWe thus identified a putative marker of the risk to develop MDS after cancer treatment.

Abstract 4478: SHMT2 modulates DNA methylation and differentially affects prostate cancer cell response to platinum derivatives.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnProstate cancer (PCa) is one of the leading causes of death from cancer in men. Several prognostic factors allow differentiating low-grade from high-grade PCa that are often refractory to chemical castration but are still treated with hormone therapy to which docetaxel or cabazitaxel are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low, pointing towards the need for new alternative therapies to treat these aggressive tumors. Using a rational in silico approach based on the NCI60-cell line panel, we recently identified a signature of 6 genes, the expression of which could predict at the functional level, sensitivity to oxaliplatin but not to cisplatin in DU145, LNCaP and C42B prostate cancer cell lines (Puyo et al. Mol. Pharmacol, 2012). Among them, we focused on SHMT2, the mitochondrial isoform of serine hydroxymethyl transferase involved in the biosynthesis of purines. Downregulation of SHMT2 or absence of SHMT2 catalytic activity was associated with a resistance to oxaliplatin, whereas it had no effect on cisplatin sensitivity, a selectivity that was attributed to the DACH moiety of platinum derivatives. Here, we investigated the role of DNA methylation in this selective response to platinum compounds since SHMTs can indirectly regulate the methylation status of DNA. Variations in global level of methylation as measured by the methylation status of LINE-1 retrotransposon, was associated with differences in sensitivity of PCa cells to platinum compounds. Using our in silico approach, we found significant correlations between SHMT2 expression and cell sensitivity to both demethylating agents azacytidine and decitabine. We also found that treatment of DU145 cells, with high level of global DNA methylation, sensitized cells to platinum compounds. In order to evaluate whether methylation could impair the formation of Pt-adducts in vitro, we used purified oligonucleotides containing a unique site of platination. We show that methylation at specific CpG in the vicinity of the platination site could reduce the kinetics of DNA adducts formation. This inhibition was more pronounced for DACH platin than for cisplatin. We also assessed the effect of transient repression of SHMT2 in LNCaP cells on the methylation status of ∼450,000 CpG sites using the Infinium Meth450K beadchip from Illumina. Preliminary results show that significant changes in methylation status was only observed in a reduced number of CpGs which were not located in genes that are known to be involved in cell response to Pt adducts formation. Together our results demonstrate that SHMT2-mediated specific response to oxaliplatin can be due to both methylation in the vicinity of the platination site and the modulation of the expression of genes that are not directly linked to the processing of DNA-adducts.nnCitation Format: Stephane Puyo, Nadine Houede, Marina Hamant, Pierre Richaud, Jacques Robert, Philippe Pourquier. SHMT2 modulates DNA methylation and differentially affects prostate cancer cell response to platinum derivatives. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2013-4478

Abstract 5064: A gene expression signature which could predict high grade prostate cancer response to oxaliplatin

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnProstate cancer is one of the leading causes of death from cancer in men. The prognosis of patients is defined according to staging, PSA levels and Gleason score, which differentiates low grade (Gleason < 4) and high grade (Gleason ≥ 4) cancers. High-grade tumors are associated with high metastatic potential and poor prognosis. They are treated with hormone therapy, to which microtubule poisons are added when they become resistant to the anti-androgen. Despite many clinical trials with other chemotherapeutic agents, response rates remain low. Moreover, none of these trials took into account the tumor grade. We therefore envisaged a new rational in silico approach to screen for drug candidates that could be used as an alternative to docetaxel, based on an expression signature of 86 genes that could distinguish low-grade and high-grade tumors, with a reliability of 81% (PNAS 2006 103:10991-6). We explored the NCI databases, which allow access to both gene expression profiles of 60 human tumor cell lines and their in vitro sensitivity to thousands of anticancer drugs and extracted the expression profiles of the 86 genes’ signature. We calculated for each gene the Pearson coefficients of correlation (r) between their expression level in the 60 cell lines and cell sensitivity to 152 core anticancer compounds. We found that the expression of 11 genes was associated with sensitivity to oxaliplatin. They include DPM1, PCCB, ATP5G3, and SHMT2 genes involved in metabolism; RHOT2, CD59 and JUN genes involved in signal transduction, the CDKN2C cyclin-dependent kinase inhibitor gene, RPL13 and EIF4A1 genes involved in translation, and the unknown [FLJ35093][1] gene. This signature seems specific to DACH platinum derivatives since no correlation was found with the sensitivity to cisplatin. Functional validation of this signature was performed in vitro using the prostate cancer cell lines DU145 and LNCaP and the benign prostate hyperplasia (BPH) cells as “normal” cells. We measured the effect of siRNA-mediated downregulation of each of the 11 genes on the sensitivity to oxaliplatin or cisplatin by clonogenic or MTT assays. Preliminary data confirmed our in silico results for 7 out of the 11 genes, for which donwregulation induced a significant change in IC50 for oxaliplatin (but not cisplatin) in DU145 and/or LNCaP cells. For EIF4A1 we found a correlation opposite to that obtained from in silico results. Also, inhibition of CDKN2C could affect the sensitivity to cisplatin in LNCaP cells. Conversely, no effect of each siRNA tested could be observed in BPH cells, suggesting that functional validation should be tested in a tumoral background. Our new rational approach allowed the identification of oxaliplatin as an alternative therapy for high grade prostate cancers. It also identified a potential gene expression signature that could be used to predict tumor response to oxaliplatin (and potentially DACH platinum derivatives) in the clinic.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5064. doi:10.1158/1538-7445.AM2011-5064nn [1]: /lookup/external-ref?link_type=GENPEPT&access_num=FLJ35093&atom=%2Fcanres%2F71%2F8_Supplement%2F5064.atom

Abstract 2699: ERCC5 (XPG) status and clinical activity of trabectedin in patients with advanced soft-tissue sarcoma

Trabectedin is approved in Europe for the treatment of advanced soft tissue sarcoma (STS) and provides objective response and disease stabilisation rates ranging from 5%-10% and 30-40%, respectively. Although the precise mechanism of action of trabectedin is not fully elucidated, various in vitro studies have shown that its activity depends, at least in part, on the nucleotide excision repair (NER) status of the cells. Indeed, NER-deficient cell lines are less sensitive to trabectedin than their wild-type counterparts. Our aim was to determine whether the status of ERCC5 (XPG), a 3′-endonuclease which plays a crucial role in the excision of the damaged DNA, was associated with the clinical activity of trabectedin in advanced STS patients. We analyzed both, the single nucleotide polymorphism (Asp1104His, G>C, exon 15) and the expression status (real-time quantitative RT-PCR) of ERCC5 in tumors from a cohort of 119 patients with advanced STS (median age at the start of treatment: 49 years old). All patients included in this retrospective analysis were treated between 1999 and 2007 in phase I-II clinical trials or in the context of a compassionate-use program. Trabectedin was given at different doses (0.5-3 mg/m2) with either a 3-h infusion or a 24-h continuous infusion schedule. The two most frequent histological subtypes were leiomyosarcoma (non-uterine: 21, uterine: 11) and liposarcoma (myxoid round cell: 24, other: 15). Overall, tumour control (CR, PR, and SD ≥ 6 months) was achieved in 42 patients (35%, 95% CI 26-44). The median progression-free survival (PFS) and overall survival (OS) of the entire patient group were 3.3 months (95% CI 2.5-4.1) and 12 months (95% CI 7.8-16.1), respectively. Variant allele carriers for ERCC5 were found in 58 cases: G/C (40; 33%), C/C (17; 15%). ERCC5 mRNA was found to be overexpressed in 48% of analyzed cases. In patients with tumors homozygous for the wild-type allele (Asp), ERRC5 mRNA overexpression was associated with significantly higher median PFS: 17.1 months (95% CI 4.7-29.4) versus 1.8 months (95% CI 1.4-2.2), p=0.002. In the group of patients with tumors heterozygous or homozygous for the variant allele (His), median PFS was poor regardless to the expression status of ERRC5 mRNA: 2.8 months (95% CI 1.3-4.2) versus 3.4 months (95% CI 1.4-5.5), p=0.50. We also compared the effect of wild-type and variant ERCC5 expression in human 94RD27 XPG-deficient fibroblasts (deletion of the last 261 aa) on the sensitivity to trabectedin. Preliminary results show that cells stably expressing wild-type ERCC5 are more sensitive to trabectedin than cells expressing the variant allele of ERCC5. Altogether, our data suggest that overexpression of wild-type ERCC5 might be predictive of clinical benefit from trabectedin in advanced STS patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2699.

Abstract 2692: Overexpression of serine hydroxy methyl transferase 2 (SHMT2) in high grade prostate cancers as a marker of tumor response to oxaliplatin

Prostate cancer is one of the leading causes of death from cancer in men and its incidence is constantly rising. In most cases, the prognosis of patients is defined according to staging, PSA levels and Gleason score, which differentiates low grade (Gleason Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2692.