Somatic MMR gene mutations as a cause for MSI-H sebaceous neoplasms in Muir-Torre syndrome-like patients.

Sebaceous neoplasms are a major clinical feature of Muir-Torre syndrome (MTS) associated with visceral malignancies, especially colorectal and endometrial tumors. The diagnosis of MTS relies largely on the microsatellite instability (MSI) phenotype in tumors, suggesting germline mutations in DNA mismatch repair (MMR) genes responsible for the inherited disease. We hypothesized that in some MSI-H sebaceous tumors, acquired rather than inherited mutations in MMR genes could be involved. Using next-generation sequencing, we screened MMR gene mutations in 18 MSI-H sebaceous tumors. We found mutations in 17 samples (94%). Indeed, 12/17 (71%) were shown to carry acquired somatic mutations and among 12 samples, seven were shown to be associated with additional somatic alterations like loss of heterozygosity or multiple mutations, suggesting somatic second hits. Our findings strongly suggest that somatic MMR deficiency is responsible for a proportion of MSI-H sebaceous tumors.

Intensity-dependent constitutional MLH1 promoter methylation leads to early onset of colorectal cancer by affecting both alleles.

Constitutional epimutation is one of the causes for MLH1 gene inactivation associated with hereditary non-polyposis colon cancer (HNPCC) syndrome. Here we investigate MLH1 promoter hypermethylation in 110 sporadic early-onset colorectal cancer patients. Variable levels of hypermethylation were detected in 55 patients (50%). Importantly a reduced MLH1 gene expression was found in patients with high-level methylation, with the association of microsatellite instability (MSI) in their tumor cells. Such high-level methylation accounts for 7.4% of all patients included in this study. Furthermore, we found that in one case constitutional methylation affected both alleles, indicating a post-zygotic methylation dysregulation. Our findings suggest that constitutional epimutation is a mechanism underlying early-onset colorectal cancer, although it is involved in only a small proportion of patients, who require appropriate surveillance. Our findings provide further insight into the role of aberrant constitutional methylation in colon carcinogenesis and raise the question of whether prevalent low-level methylation constitutes a potential risk factor for cancer development.

Epithelial-to-mesenchymal transition promotes immune escape by inducing CD70 in non-small cell lung cancer.

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. METHODS: We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). RESULTS: We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. CONCLUSION: Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

MLH1 promoter hypermethylation: are you absolutely sure about the absence of MLH1 germline mutation? About a new case.

Lynch syndrome accounts for 3-5% of colorectal cancers and is due to a germline mutation in one of the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. Somatic hypermethylation of the MLH1 promoter is commonly associated to sporadic cases. Strategies have been developed to identify patients with Lynch Syndrome based on clinical findings, tumoral phenotype, family history and immunohistochemistry analysis. However, there still are some pitfalls in this strategy, possibly responsible for an underdiagnosis of Lynch syndrome. Here we report the case of a 37 years-old man presenting with two concomitant tumors located in the rectosigmoid and in the ileocecal angle. Both tumors were microsatellites instability-high (MSI-H) and showed a loss of MLH1 and PMS2 protein expression, but only one had MLH1 promoter hypermethylation. Constitutional analysis of mismatch repair genes could not be performed from a blood sample, because of the early death of the patient. However, tumoral tissue analyses revealed in both tumors a pathogenic variant in the MLH1 gene. Further analysis of the surrounding tumor-free tissue also showed the presence of this alteration of the MHL1 gene. Finally, the same pathogenic variant was present constitutionally in one of the siblings of the patient, confirming its hereditary nature. This new case of concomitant presence of MLH1 promoter hypermethylation and MLH1 germline mutation demonstrates that the presence of MLH1 promoter hypermethylation should not rule out the diagnosis of Lynch Syndrome.

Novel biallelic mutations in MSH6 and PMS2 genes: gene conversion as a likely cause of PMS2 gene inactivation.

Since the first report by our group in 1999, more than 20 unrelated biallelic mutations in DNA mismatch repair genes (MMR) have been identified. In the present report, we describe two novel cases: one carrying compound heterozygous mutations in the MSH6 gene; and the other, compound heterozygous mutations in the PMS2 gene. Interestingly, the inactivation of one PMS2 allele was likely caused by gene conversion. Although gene conversion has been suggested to be a mutation mechanism underlying PMS2 inactivation, this is the first report of its involvement in a pathogenic mutation. The clinical features of biallelic mutation carriers were similar to other previously described patients, with the presence of café-au-lait spots (CALS), early onset of brain tumors, and colorectal neoplasia. Our data provide further evidence of the existence, although rare, of a distinct recessively inherited syndrome on the basis of MMR constitutional inactivation. The identification of this syndrome should be useful for genetic counseling, especially in families with atypical hereditary nonpolyposis colon cancer (HNPCC) associated with childhood cancers, and for the clinical surveillance of these mutation carriers.

Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing.

A substantial proportion of MLH1 and MSH2 gene mutations in hereditary nonpolyposis colon cancer syndrome (HNPCC) families are characterized by nucleotide substitutions, either within the coding sequence (missense or silent mutations) or in introns. The question of whether these mutations affect the normal function of encoding mismatch DNA repair proteins and thus lead to the predisposition to cancer is determinant in genetic testing. Recent studies have suggested that some nucleotide substitutions can induce aberrant splicing by disrupting cis-transcription elements such as exonic enhancers (ESEs). ESE disruption has been proposed to be the mechanism that underlies the presumed pathological missense mutations identified in HNPCC families. To investigate the prevalence of aberrant splicing resulting from nucleotide substitutions, and its relevance to predicted ESEs, we conducted a systematic RNA screening of a series of 60 patients who carried unrelated exonic or intronic mutations in MLH1 or MSH2 genes. Aberrant splicing was found in 15 cases, five of which were associated with exonic mutations. We evaluated the link between those splicing mutations and predicted putative ESEs by using the computational tools ESEfinder and RESCUE-ESE. Our study shows that the algorithm-based ESE prediction cannot be definitely correlated to experimental observations from RNA screening. By using minigene constructs and in vitro transcription assay, we demonstrated that nucleotide substitutions are the direct cause of the splicing defect. This is the first systematic screening for the effect of missense and silent mutations on splicing in HNPCC patients. The pathogenic splicing mutations identified in this study will contribute to the assessment of "unclassified variants" in genetic counseling. Our results also suggest that one must use caution when determining the pathogenic effect of a missense or silent mutation using ESE prediction algorithms. Analysis at the RNA level is therefore necessary.

Chfr inactivation is not associated to chromosomal instability in colon cancers.

Numerous observations suggest that chromosome instability is caused by mitotic abnormalities such as errors in the partitioning of chromosomes. Chfr was recently defined as a central component of a new mitotic checkpoint that delays chromosome condensation in response to mitotic stress. Chfr was shown to be frequently inactivated in several human neoplasms, including colon, lung and esophageal cancers. To test whether Chfr inactivation may lead or participate to chromosomal instability (CIN), we analysed the genetic and epigenetic status of the gene in a large panel of primary colon and breast cancers, as well as in colon and breast cancer cell lines displaying either a microsatellite instability or a CIN. Our results confirm that Chfr is frequently inactivated in colon cancers, through a mechanism of hypermethylation of the promoter sequences. In contrast, the loss of Chfr expression appears to be a rare event in breast cancers. Furthermore, our data demonstrate that Chfr inactivation is not associated with CIN in these frequent types of human cancers.

IkappaB kinase beta phosphorylates Dok1 serines in response to TNF, IL-1, or gamma radiation.

Dok1 is an abundant Ras-GTPase-activating protein-associated tyrosine kinase substrate that negatively regulates cell growth and promotes migration. We now find that IkappaB kinase beta (IKKbeta) associated with and phosphorylated Dok1 in human epithelial cells and B lymphocytes. IKKbeta phosphorylation of Dok1 depended on Dok1 S(439), S(443), S(446), and S(450). Recombinant IKKbeta also phosphorylated Dok1 or Dok1 amino acids 430-481 in vitro. TNF-alpha, IL-1, gamma radiation, or IKKbeta overexpression phosphorylated Dok1 S(443), S(446), and S(450) in vivo, as detected with Dok1 phospho-S site-specific antisera. Moreover, Dok1 with S(439), S(443), S(446), and S(450) mutated to A was not phosphorylated by IKKbeta in vivo. Surprisingly, mutant Dok1 A(439), A(443), A(446), and A(450) differed from wild-type Dok1 in not inhibiting platelet-derived growth factor-induced extracellular signal-regulated kinase 1/2 phosphorylation or cell growth. Mutant Dok1 A(439), A(443), A(446), and A(450) also did not promote cell motility, whereas wild-type Dok1 promoted cell motility, and Dok1 E(439), E(443), E(446), and E(450) further enhanced cell motility. These data indicate that IKKbeta phosphorylates Dok1 S(439)S(443) and S(446)S(450) after TNF-alpha, IL-1, or gamma-radiation and implicate the critical Dok1 serines in Dok1 effects after tyrosine kinase activation.