A new natural killer cell-specific gene signature predicting recurrence in colorectal cancer patients

The protective role of Natural Killer (NK) cell tumour immunosurveillance has long been recognised in colorectal cancer (CRC). However, as most patients show limited intra-tumoral NK cell infiltration, improving our ability to identify those with high NK cell activity might aid in dissecting the molecular features which underlie NK cell sensitivity. Here, a novel CRC-specific NK cell gene signature that infers NK cell load in primary tissue samples was derived and validated in multiple patient CRC cohorts. In contrast with other NK cell gene signatures that have several overlapping genes across different immune cell types, our NK cell signature has been extensively refined to be specific for CRC-infiltrating NK cells. The specificity of the signature is substantiated in tumour-infiltrating NK cells from primary CRC tumours at the single cell level, and the signature includes genes representative of NK cells of different maturation states, activation status and anatomical origin. Our signature also accurately discriminates murine NK cells, demonstrating the applicability of this geneset when mining datasets generated from preclinical studies. Differential gene expression analysis revealed tumour-intrinsic features associated with NK cell inclusion versus exclusion in CRC patients, with those tumours with predicted high NK activity showing strong evidence of enhanced chemotactic and cytotoxic transcriptional programs. Furthermore, survival modelling indicated that NK signature expression is associated with improved survival outcomes in CRC patients. Thus, scoring CRC samples with this refined NK cell signature might aid in identifying patients with high NK cell activity who could be prime candidates for NK cell directed immunotherapies

Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium

The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active β-catenin–Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis

Pregnane × Receptor (PXR) expression in colorectal cancer cells restricts irinotecan chemosensitivity through enhanced SN-38 glucuronidation

<p>Abstract</p> <p>Background</p> <p>Clinical efficacy of chemotherapy in colorectal cancer is subjected to broad inter-individual variations leading to the inability to predict outcome and toxicity. The topoisomerase I inhibitor irinotecan (CPT-11) is worldwide approved for the treatment of metastatic colorectal cancer and undergoes extensive peripheral and tumoral metabolism. PXR is a xenoreceptor activated by many drugs and environmental compounds regulating the expression of drug metabolism and transport genes in detoxification organs such as liver and gastrointestinal tract. Considering the metabolic pathway of irinotecan and the tissue distribution of Pregnane × Receptor (PXR), we hypothesized that PXR could play a key role in colon cancer cell response to irinotecan.</p> <p>Results</p> <p>PXR mRNA expression was quantified by RT-quantitative PCR in a panel of 14 colon tumor samples and their matched normal tissues. PXR expression was modulated in human colorectal cancer cells LS174T, SW480 and SW620 by transfection and siRNA strategies. Cellular response to irinotecan and its active metabolic SN38 was assessed by cell viability assays, HPLC metabolic profiles and mRNA quantification of PXR target genes. We showed that PXR was strongly expressed in colon tumor samples and displayed a great variability of expression. Expression of hPXR in human colorectal cancer cells led to a marked chemoresistance to the active metabolite SN38 correlated with PXR expression level. Metabolic profiles of SN38 showed a strong enhancement of SN38 glucuronidation to the inactive SN38G metabolite in PXR-expressing cells, correlated with an increase of UDPglucuronosyl transferases UGT1A1, UGT1A9 and UGT1A10 mRNAs. Inhibition of PXR expression by lentivirus-mediated shRNA, led to SN38 chemoresistance reversion concomitantly to a decrease of UGT1A1 expression and SN38 glucuronidation. Similarly, PXR mRNA expression levels correlated to UGT1A subfamily expression in human colon tumor biopsies.</p> <p>Conclusion</p> <p>Our results demonstrate that tumoral metabolism of SN38 is affected by PXR and point to potential therapeutic significance of PXR quantification in the prediction of irinotecan response. Furthermore, our observations are pharmacologically relevant since many patients suffering from cancer diseases are often exposed to co-medications, food additives or herbal supplements able to activate PXR. A substantial part of the variability observed among patients might be caused by such interactions</p

Reference gene selection for head and neck squamous cell carcinoma gene expression studies

<p>Abstract</p> <p>Background</p> <p>It is no longer adequate to choose reference genes blindly. We present the first study that defines the suitability of 12 reference genes commonly used in cancer studies (<it>ACT, ALAS, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPL27, RPS29, SHAD </it>and <it>TBP</it>) for the normalization of quantitative expression data in the field of head and neck squamous cell carcinoma (HNSCC).</p> <p>Results</p> <p>Raw expression levels were measured by RT-qPCR in HNSCC and normal matched mucosa of 46 patients. We analyzed the expression stability using geNorm and NormFinder and compared the expression levels between subgroups. In HNSCC and/or normal mucosa, the four best normalization genes were <it>ALAS, GAPDH, RPS18 </it>and <it>SHAD </it>and the most stable combination of two genes was <it>GAPDH-SHAD</it>. We recommend using <it>KALPHA-TBP </it>for the study of T1-T2 tumors, <it>RPL27-SHAD </it>for T3-T4 tumors, <it>KALPHA-SHAD </it>for N0 tumors, and <it>ALAS-TBP </it>for N+ tumors. <it>ACT, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPS29, SHAD </it>and <it>TBP </it>were slightly misregulated (<1.7-fold) between tumor and normal mucosa but can be used for normalization, depending on the resolution required for the assay.</p> <p>Conclusion</p> <p>In the field of HNSCC, this study will guide researchers in selecting the most appropriate reference genes from among 12 potentially suitable reference genes, depending on the specific setting of their experiments.</p

Clinical relevance of nine transcriptional molecular markers for the diagnosis of head and neck squamous cell carcinoma in tissue and saliva rinse

<p>Abstract</p> <p>Background</p> <p>Analysis of 23 published transcriptome studies allowed us to identify nine genes displaying frequent alterations in HNSCC (<it>FN1, MMP1, PLAU, SPARC</it>, <it>IL1RN, KRT4, KRT13, MAL</it>, and <it>TGM3</it>). We aimed to independently confirm these dysregulations and to identify potential relationships with clinical data for diagnostic, staging and prognostic purposes either at the tissue level or in saliva rinse.</p> <p>Methods</p> <p>For a period of two years, we systematically collected tumor tissue, normal matched mucosa and saliva of patients diagnosed with primary untreated HNSCC. Expression levels of the nine genes of interest were measured by RT-qPCR in tumor and healthy matched mucosa from 46 patients. <it>MMP1 </it>expression level was measured by RT-qPCR in the salivary rinse of 51 HNSCC patients and 18 control cases.</p> <p>Results</p> <p>Dysregulation of the nine genes was confirmed by the Wilcoxon test. <it>IL1RN, MAL </it>and <it>MMP1 </it>were the most efficient diagnostic markers of HNSCC, with ROC AUC > 0.95 and both sensitivity and specificity above 91%. No clinically relevant correlation was found between gene expression level in tumor and T stage, N stage, tumor grade, global survival or disease-free survival. Our preliminary results suggests that with 100% specificity, <it>MMP1 </it>detection in saliva rinse is potentially useful for non invasive diagnosis of HNSCC of the oral cavity or oropharynx, but technical improvement is needed since sensitivity was only 20%.</p> <p>Conclusion</p> <p><it>IL1RN, MAL </it>and <it>MMP1 </it>are prospective tumor diagnostic markers for HNSCC. <it>MMP1 </it>overexpression is the most promising marker, and its detection could help identify tumor cells in tissue or saliva.</p

Études de la compartimentation des Claudines 1, 2, 4 et 7 dans le côlon humain normal et tumoral (dialogue avec la voie Wnt/b-caténine/Tcf-4)

MONTPELLIER-BU Médecine UPM (341722108) / SudocMONTPELLIER-BU Médecine (341722104) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Régulation des Cellules Souches Cancéreuses du cancer colorectal par une protéine des jonctions serrées (la Claudine-2)

Les claudines sont des protéines des jonctions serrés (JS) dont l'expression est perturbée en cas de cancer. Dans ce travail, nous montrons d'abord que la surexpression de la claudine-2 agit comme intermédiaire de l'effet pro-tumorigène de la symplékine en coopération avec le facteur de transcription ZONAB dans le cancer colorectal (CCR) (Buchert, Papin et al 2010). Nous avons alors étudié le rôle de la claudine-2 sur les cellules souches cancéreuses (CSC), considérées comme responsables du maintien à longue durée de la tumorigénèse, en analysant les conséquences de sa sous-expression expérimentale dans les CSC coliques putatives exprimant le marqueur LGR5. Sur la lignée de CCR humain DLD1, l'inhibition de claudine-2 par shRNA ou siRNA diminue le nombre de cellules LGR5(+) et augmente l'expression du marqueur de différenciation CK20. La sous-expression de la claudine-2 dans les cellules LGR5(+) diminue leur capacité à survivre en dilution clonale en suspension dans un milieu sans sérum, réduit le nombre et la croissance de tumeurs qu'elles induisent après xénogreffe, et augmente l'expression des clusters de microRNA miR-182/183/203, miR-141/200a et miR-200b/c/429, inhibiteurs du phénotype CSC. Ces résultats suggèrent que la claudine-2 participe au maintien du phénotype CSC des cellules LGR5(+). Nous montrons de plus que le génotype des cellules LGR5(-) isolées est plastique, et que cette population contient aussi une sous-population de type CSC. Celle-ci pourrait exprimer une activité ALDH, et l'inhibition de claudine-2 induit une augmentation paradoxale de la formation de sphéroïdes par les cellules ALDH(+) isolées. La claudine-2 peut donc moduler différemment la capacité d'initiation tumorale des cellules LGR5(+) et ALDH(+). L'inhibition de tumorigénicité induite par la sous-expression de la claudine-2 prédomine dans la population tumorale globale, au sein de laquelle les cellules LGR5(+)/(-) et ALDH(+)/(-) coexistent.Claudins are tight junction proteins frequently displaying abnormal expression patterns in cancer. In this work, we first showed that claudin-2 overexpression acts as an essential intermediate in the tumor-promoting role of the Symplekin/ZONAB complex in colorectal cancer (CRC). We then set out to analyze the function of Claudin-2 in cancer stem cells (CSC), which are strongly suspected to drive long-term tumorigenicity. To this effect, we experimentally down-regulated claudin-2 expression in LGR5-expressing cells, putative CRC cancer stem cells. We showed that claudin-2 inhibition by shRNA increased the expression of the epithelial differentiation marker CK20 and decreased the number of LGR5(+) cells, without inducing their apoptosis. Claudin-2-depleted LGR5(+) cells were no longer able to survive as single cells in suspension in serum free medium, and formed less and smaller tumors in a xenograft model. Moreover, the inhibition of claudin-2 increased the expression of the miRNA clusters miR-182/183/203, miR-141/200a and miR-200b/c/429, known as potent CSC phenotype inhibitors. Hence, our results suggest that claudin-2 promotes the CSC phenotype of LGR5(+) cells. In addition, we show that the genotype of isolated LGR5(-) cells is unstable and that this population also contains a CSC-like sub-population, potentially displaying a high ALDH activity. Interestingly, claudin-2 down-regulation in isolated ALDH(+) cells induces a paradoxical increase in their sphere-forming ability. Claudin-2 can thus differentially modulate the tumor-initiating capability of LGR5(+) and ALDH(+) cells. The inhibitory effect on tumorigenicitiy induced by claudin-2 depletion is clearly predominant in the global, heterogenous tumor cell population, where LGR5(+)/(-) and ALDH(+)/(-) cells coexist.MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

The long road to colorectal cancer therapy: Searching for the right signals

International audienceDuring the last 25 years, improvements in our understanding of signaling processes mediating tumor initiation, growth and dissemination have opened the door towards increasingly efficient, selective, but also complex therapeutic approaches. In parallel, the better characterization of altered signaling pathways in tumor cells, as well as the relationship between these alterations and the sensitivity of tumors to given compounds, have forced us to contemplate the use of biomarkers allowing patient selection and evaluation of treatment efficacy. Such biomarkers should become a corner stone for the success of a more rational and "personalized" clinical approach to colorectal cancer. Here we give an overview of the "signaling pathway-selective" compounds that are currently in use or under clinical development in the setting of colorectal cancer (CRC) and we discuss promising targets, based on our current knowledge of colorectal tumor cell biology, such as Src family, Wnt and Hegdehog signaling