Suppressive cancer nonstop extension mutations increase C-terminal hydrophobicity and disrupt evolutionarily conserved amino acid patterns.

Nonstop extension mutations, a.k.a. stop-lost or stop-loss mutations, convert a stop codon into a sense codon resulting in translation into the 3' untranslated region until the next in-frame stop codon, thereby extending the C-terminus of a protein. In cancer, only nonstop mutations in SMAD4 have been functionally characterized, while the impact of other nonstop mutations remain unknown. Here, we exploit our pan-cancer NonStopDB dataset and test all 2335 C-terminal extensions arising from somatic nonstop mutations in cancer for their impact on protein expression. In a high-throughput screen, 56.1% of the extensions effectively reduce protein abundance. Extensions of multiple tumor suppressor genes like PTEN, APC, B2M, CASP8, CDKN1B and MLH1 are effective and validated for their suppressive impact. Importantly, the effective extensions possess a higher hydrophobicity than the neutral extensions linking C-terminal hydrophobicity with protein destabilization. Analyzing the proteomes of eleven different species reveals conserved patterns of amino acid distribution in the C-terminal regions of all proteins compared to the proteomes like an enrichment of lysine and arginine and a depletion of glycine, leucine, valine and isoleucine across species and kingdoms. These evolutionary selection patterns are disrupted in the cancer-derived effective nonstop extensions.

MLH1 Gene Expression in Peripheral Blood in Colon Cancer Patients and Their Association with Colon Cancer.

MutL homolog 1 (MLH1) is a component of the heterodimeric complex MutLα that detects and fixes base-base mismatches and insertion/deletion loops caused by nucleotide misincorporation. In the absence of MLH1 protein, the frequency of non-repaired mismatches increases, resulting in organ cancer. The current study sought to quantify MLH1 gene expression and its relationship with tumor invasion (T) and lymph node invasion (N) in blood samples from patients with colorectal cancer (CRC). Blood samples were obtained from 36 CRC patients. RNA was extracted, and cDNA was synthesized using a kit. The primers were built using the exon-exon junction approach, and MLH1 and ß-actin genes were tested 3x using real-time polymerase chain reaction (Real-Time PCR). Gene expression analysis software was used to analyze the data, and a t-test was used to examine the expression of MLH1 and its connection with T and N variables. In this study, 36 patients with colorectal cancer, including 15 (41.6%) women and 21 (58.4%) men, with a mean age of 57.35 ± 4.22 years and in the age range of 26-87 years, were included. The results showed that the ratio of MLH1 gene expression in patients decreased compared to that in healthy individuals, and the decrease in gene expression at different stages of the disease was significant. The results of this study showed that the reduction of MLH1 gene expression has an effective role in the development of CRC.

Bladder Cancer detection by urinary methylation markers GHSR/MAL: a validation study.

PURPOSE: Although cystoscopy is a reliable tool for detecting bladder cancer, it poses a high burden on patients and entails high costs. This highlights the need for non-invasive and cost-effective alternatives. This study aimed to validate a previously developed urinary methylation marker panel containing GHSR and MAL. METHODS: We enrolled 134 patients who underwent cystoscopy because of hematuria, including 63 individuals with primary bladder cancer and 71 with non-malignant findings. Urine samples were self-collected at home and sent via regular mail. Subsequently, DNA was extracted and the hypermethylation of GHSR and MAL was evaluated using quantitative methylation-specific polymerase chain reaction. The performance of methylation markers was assessed using area-under-the-curve (AUC) analysis and sensitivity and specificity based on pre-established cut-off values. RESULTS: Validation of the marker panel GHSR/MAL resulted in an AUC of 0.87 at 79% sensitivity and 80% specificity. Sensitivity was comparable to the previous investigation (P > 0.9), though specificity was significantly lower (P = 0.026). Sensitivity was higher for high-grade tumors compared to low-grade tumors (94% vs. 60%, P = 0.002). CONCLUSION: Validation of the GHSR/MAL methylation marker panel on at home collected urine samples confirms its robust performance for bladder cancer detection in a hematuria population, and underscores the diagnostic potential for future clinical application.

Germline DNA Damage Repair Gene Alterations in Patients with Metachronous Breast and Colorectal Cancer.

A hereditary component of breast (BC) and colorectal cancer (CRC) has been described in approximately one-third of these tumor types. BC patients have an increased risk of developing CRC as a second primary tumor and vice versa. Germline genomic variants (NextSeq550, Illumina) were investigated in 24 unrelated BC and/or CRC patients and 7 relatives from 3 index patients. Fifty-six pathogenic or likely pathogenic variants were identified in 19 of 24 patients. We detected single-nucleotide variants (SNVs) in CRC predisposition genes (MLH1 and MUTYH) and other promising candidates (CDK5RAP3, MAD1L1, NOS3, and POLM). Eighteen patients presented SNVs or copy number variants (CNVs) in DNA damage repair genes. We also identified SNVs recently associated with BC or CRC predisposition (PABPC1, TYRO3, MAP3K1, SLC15A4, and LAMA1). The PABPC1c.1255C>T variant was detected in nine unrelated patients. Each patient presented at least one SNV/CNV in a candidate gene, and most had alterations in more than one gene, reinforcing a polygenic model for BC/CRC predisposition. A significant fraction of BC/CRC patients with a family history of these tumors harbored deleterious germline variants in DNA repair genes. Our findings can lead to strategies to improve the diagnosis, genetic counseling, and treatment of patients and their relatives.

Resistance to 5-fluorouracil: The molecular mechanisms of development in colon cancer cells.

Colon cancer is a significant health problem worldwide as it is one of the most common and deadliest cancers. The standard approach for the treatment of colon cancer is 5-fluorouracil (5-FU) based chemotherapy, which is limited by the development of resistance to this drug. Therefore, our study aimed to establish 5-FU resistance in SW-480 and HT-29 colon cancer cells and to precisely determine the molecular mechanisms and biomarkers that contribute to its development, both after short-term exposure and in cells with already developed resistance (SW-480-5FUR and HT-29-5FUR). The expression of various molecules involved in the different mechanisms of resistance development was monitored at the gene (qPCR) and protein (immunocytochemistry) levels. Based on the obtained results, alterations in the 5-FU anabolic pathway, biotransformation, drug efflux, mismatch repair, and apoptosis process together contributed to the development of 5-FU resistance in SW-480 and HT-29 colon cancer cells. In addition, UMPS, ABCC1, ABCC5, and MLH1, as well as the disturbed ratio of pro-apoptotic BAX and anti-apoptotic BCL2, should be taken into consideration as potential targets for the discovery of 5-FU resistance-related biomarkers in colon cancer cells. We suggest that future investigations focus on further validation of these findings by additional in vitro and in vivo testing, which is a limitation of our study.

Risk of Gastric and Small Intestinal Cancer in Patients With Lynch Syndrome: Data From a Large, Community-Based US Population.

INTRODUCTION: Risk of gastric and small intestinal cancer in Lynch syndrome (LS) remains poorly understood. We investigated the risk of gastric and small intestinal cancer in patients with LS in a large, community-based population. METHODS: This retrospective cohort study included all patients diagnosed with LS between January 1, 1997, and December 31, 2020, at Kaiser Permanente Northern California. Cumulative incidence of gastric cancer and small intestinal cancer was calculated using competing risk methodology. RESULTS: Among 1,106 patients with LS with a median follow-up of 19.3 years (interquartile range [IQR] 9.4-24.0 years), 11 developed gastric cancer (8 MSH2 , 2 MLH1 and 1 PMS2 ) with a median diagnosis age of 56 years (IQR 42-63 years) and 11 developed small intestinal cancer (6 MSH2 , 3 MLH1 , 1 MSH6 and 1 PMS2 ) with a median diagnosis age of 57 years (IQR 50-66 years). Cumulative incidence by age 80 years was 7.26% (95% confidence internal [CI], 1.80-18.03%) for men and 3.43% (95% CI, 0.50-11.71%) for women for gastric cancer and 7.28% (95% CI, 3.19-13.63%) for men and 2.21% (95% CI, 0.23-9.19%) for women for small intestinal cancer. Pathogenic variant carriers of MSH2 and MLH1 had the highest risk of gastric and small intestinal cancer. History of Helicobacter pylori infection was associated with increased risk of gastric cancer (adjusted odds ratio 5.52; 95% CI, 1.72-17.75). DISCUSSION: Patients with LS, particularly MSH2 and MLH1 pathogenic variant carriers, had significantly increased lifetime risk of gastric and small intestinal cancer. Testing and treatment of H. pylori infection should be considered for all patients with LS.

Somatic and germline mutations in endometrial cancer.

Endometrial cancer is a complex disease influenced by both somatic and germline mutations. While individual mutations in genes such as PTEN, PIK3CA, and members of the DNA mismatch repair (MMR) system have been extensively studied, comprehensive analyses comparing somatic and germline mutations within the same cohort are limited. This study compares these mutations using whole exome sequencing (WES) data from tumor and blood samples in patients with endometrial cancer. Thirteen female patients with histologically confirmed endometrial cancer were selected. Tumor tissues and matched blood samples were collected and subjected to WES at the CeGaT laboratory, followed by bioinformatics analysis and annotation using the Geneyx platform. WES revealed significant somatic and germline DNA mutations, with key pathogenic variants identified in genes such as PTEN, PIK3CA, TP53, MLH1, and MSH2. Comparative analysis showed distinct and overlapping mutation profiles, highlighting the importance of integrating somatic and germline data in endometrial cancer research.

The Relationship Between DNA Mismatch Repair Status and Clinicopathologic Characteristics in Colon Cancer.

DNA mismatch repair (MMR) proteins are essential for repairing genetic mutations that occur during DNA replication. Deficiency of MMR proteins results in a phenotype called microsatellite instability (MSI), which occurs in Lynch syndrome as well as sporadic colorectal cancers (CRC), and it is associated with several clinicopathological features. We aimed to investigate the association of the loss of MMR proteins with clinicopathologic considerations in our CRC series. In this retrospective study, DNA MMR protein status in CRC is evaluated in a total of 200 colorectal resection specimens by immunohistochemistry (IHC) for MLH1, MSH2, MSH6 and PMS2 protein expression. The BRAF mutation was investigated by the real-time PCR in cases with loss of MLH1 protein expression. The relationship between MMR status and clinicopathological parameters was investigated statistically. Loss of MMR protein expression was detected in 26 of 200 CRC cases. The BRAFV600E mutation was detected in 2 of the cases with MLH1 loss and accepted as sporadic. The remaining 24 cases (12%) were identified as Lynch syndrome candidates. There were statistical differences observed regarding the presence of tumor-infiltrating lymphocytes (P < .001), Crohn's-like reaction (P = .001), expansile growth (P < .001), tumor heterogeneity (P < .001), mucinous differentiation (P < .001), and presence of metastatic lymph nodes (P = .045) between sporadic cases with preserved MMR and Lynch candidates. However, difference in the survival rates between sporadic cases and Lynch candidates was not significant. Immunohistochemical staining for MMR is a practical method for predicting MSI phenotype as well as Lynch candidates. MMR expression status was found to be associated with certain clinicopathological features some of which also have prognostic significance.

Lynch Syndrome-associated Genomic Variants.

BACKGROUND AND AIMS: Lynch Syndrome, a hereditary disorder characterized by germline mutations in mismatch repair (MMR) genes, is a major contributor to colorectal cancers. It has also been identified in endometrial cancer. Despite the established role of MMR deficiency in tumorigenesis, the specific genomic alterations driving Lynch syndrome-associated endometrial cancer, and their overlap with colorectal cancer, remain incompletely understood. This study aims to fill this gap by performing a detailed comparative analysis of germline and somatic mutations in endometrial cancer within the context of Lynch syndrome. METHODS: We conducted whole exome sequencing on matched germline and somatic DNA from 13 patients diagnosed with Lynch syndrome-associated endometrial cancer. High-depth sequencing was performed, followed by rigorous bioinformatics analysis to identify and annotate variants, focusing on their potential pathogenicity and relevance to both endometrial and colorectal cancer. RESULTS: Our analysis revealed 1,118 germline and 14,051 somatic variants, with 493 variants common to both. Recurrent pathogenic mutations in MLH1, MSH2, and MSH6 were confirmed, highlighting their critical role in Lynch syndrome. Notably, frequent somatic mutations in the PIK3CA and PTEN genes were identified, implicating the PI3K/AKT/mTOR pathway as a key oncogenic driver in these cancers. Additionally, novel somatic mutations in genes related to the extracellular matrix such as FBN1 and SPARC were uncovered, suggesting a possible unique role in endometrial tumor progression. CONCLUSIONS: This study provides new insights into the molecular basis of Lynch syndrome-associated endometrial cancer, emphasizing the overlap in oncogenic pathways with colorectal cancer. The discovery of shared and unique genetic mutations highlights the importance of developing combined treatment strategies and suggests that targeting these specific mutations could improve therapy for patients with Lynch syndrome-associated cancers.

Lynch syndrome screening in patients with young-onset extra-colorectal Lynch syndrome-associated cancers.

BACKGROUND: Lynch syndrome (LS) is a hereditary cancer syndrome caused by pathogenic germline variants in mismatch repair (MMR) genes, which predisposes to various types of cancers showing deficient MMR (dMMR). Identification of LS probands is crucial to reduce cancer-related deaths in affected families. Although universal screening is recommended for colorectal and endometrial cancers, and age-restricted screening is proposed as an alternative, LS screening covering a broader spectrum of cancer types is needed. In the current study, we elucidated the rate of dMMR tumors and evaluated the outcome of LS screening in young-onset extra-colorectal LS-associated cancers. METHODS: Immunohistochemistry for MMR proteins were retrospectively performed in a total of 309 tissue samples of endometrial, non-mucinous ovarian, gastric, urothelial, pancreatic, biliary tract, and adrenal cancers in patients < 50 years of age. Clinicopathological information and the results of genetic testing were obtained from medical charts. RESULTS: There were 24 dMMR tumors (7.8%) including 18 endometrial, three ovarian, two urothelial, and one gastric cancer. Co-occurrence of colorectal cancer and family history of LS-associated cancers was significantly enriched in patients with dMMR tumors. Among the 16 patients with dMMR tumors who were informed of the immunohistochemistry results, five with endometrial and one with urothelial cancer were diagnosed as LS with positive pathogenic variants in MMR genes. CONCLUSIONS: We report the outcome of immunohistochemistry for MMR proteins performed in multiple types of young-onset extra-colorectal LS-associated cancers. Our study demonstrates the feasibility of a comprehensive LS screening program incorporating young-onset patients with various types of extra-colorectal LS-associated cancers.

Constitutional mismatch repair deficiency: a case on a commonly misinterpreted mutation in colon cancer.

It is estimated that 153,020 cases of CRC per year, with an increase in diagnoses in younger patients. We present a case of a female with an early presentation of Lynch Syndrome and CRC, who, on her third malignant presentation, was re-diagnosed as a constitutional mismatch repair deficiency.

Universal Lynch Syndrome Screening in Colorectal Cancer: A 5-Year Experience of a Portuguese Pathology Department.

Lynch syndrome (LS) is a prevalent genetic condition associated with colorectal cancer (CRC). Accurate identification of LS patients is challenging, and a universal tumor screening approach has been recommended. We present the methodology and results of universal LS screening in our hospital's Pathology Department. This retrospective study analyzed CRC tumors from a 5-year period (2017-2021). Immunohistochemistry was used to assess MMR protein expression, followed by BRAF V600E analysis and MLH1 promoter methylation. Statistical analysis examined associations between clinicopathologic variables MMR status and LS-suspected tumors. The study analyzed 939 colorectal carcinomas, with 8.7% exhibiting mismatch repair (MMR) deficiency, significantly lower than previous research. After applying the algorithm, 24 LS-suspected cases were identified, accounting for 2.6% of tested patients and 29.3% of MMR-deficient tumors. Our study establishes the feasibility of universal testing for all new cases of CRC in detecting individuals at risk for LS, even in the absence of clinical information. To gain a comprehensive understanding of the MMR status in our population, further investigations are warranted.

Ochratoxin A-induced DNA damage triggers G2 phase arrest via hMLH1-p53-p21 signaling pathway in human gastric epithelium immortalized cells in vitro.

Ochratoxin A (OTA), as one of the most important and harmful mycotoxins, is classed as possible human carcinogen (group 2B). As we all know, DNA damage may cause genomic instability, cell cycle disorder, activation of DNA damage pathway, and stimulation of DNA repair system. To explore the roles of DNA damage repair protein (hMLH1) on OTA-induced G2 arrest, the DNA damage, chromosome aberration, cell cycle distribution and p53-p21 signaling pathway were evaluatd after different time OTA exposure (6, 12, 24, 48 h) in immortalized human gastric epithelial cells (GES-1). Our results demonstrated that OTA exposure could trigger genomic instability, DNA damage and G2 phase arrest of GES-1 cells. At the same time, OTA treatment could increase the expression of hMLH1, and induce phosphorylation of the p53 protein, as well as p21, in response to DNA damage. Finally, inhibition of hMLH1 by siRNA effectively prevented the activation of p53-p21 signaling pathway and rescued the G2 arrest elicited by OTA. This study demonstrated that hMLH1-p53-p21 signaling pathway played an important role in DNA damage and G2 cell cycle arrest the mediated by OTA in GES-1 cells.

Adherence to national Lynch syndrome testing guidelines for colorectal cancer in an Aotearoa New Zealand hospital-based population.

AIM: Lynch syndrome (LS) is estimated to affect 1-3.9% of patients with colorectal cancer (CRC). Testing for LS is important in determining management and establishing surveillance for "Lynch families". Previous studies have identified poor rates of testing for LS in CRC patients. This study aimed to describe adherence to guidelines for testing of newly diagnosed CRC for LS. METHODS: A single institution cohort study of patients over 18 years with colorectal adenocarcinoma from 2018-2022 in Te Tai Tokerau, Aotearoa New Zealand was conducted. Rates of baseline immunohistochemistry (IHC) testing for mismatch repair (MMR) deficiency, further testing for MLH1-deficient cases and rates of germline mutational analysis were audited to determine adherence to national guidelines. The rate of LS in newly diagnosed CRC was estimated. RESULTS: Six hundred and sixty patients were eligible for universal testing for LS, of which 84% (n=553) completed initial IHC testing. MMR deficiency was reported in 20% (n=114) cases. Eighty-nine percent (n=101) was attributable to MLH1 deficiency, of which 99% (n=100) were appropriately tested for BRAF-V600E mutation. Sixty-four percent (4/11) patients indicated for hypermethylation testing were appropriately tested. Seventeen patients had an indication for germline mutational analysis, of which only 29% (n=5) were tested. The estimated incidence of LS in newly diagnosed CRC was 0.7-3.8%. CONCLUSION: Compliance with initial IHC testing was good. However, there is a need to improve rates of confirmation genetic testing. The incidence of confirmed LS in this study is 0.7%, however this may be as high as 3.9%.

[Analysis of gene mutation in a family with Muir-Torre syndrome accompanied with extraorbital cystic sebaceous carcinoma].

This study reported a family of MLH1 mutation-induced Muir-Torre syndrome (MTS) and evaluated it's clinical and genetic characteristics. A 51 year-old patient with extraorbital cystic sebaceous and colon adenocarcinoma diagnosed in November 2021 in Zhongshan Hospital of Xiamen University was included. The clinical data of the family were collected and a pedigree chart was drawn, which was in line with the Chinese Lynch syndrome diagnostic criteria and was a typical MTS family. NM_000249.4:c.298C>T(p.R100*) of MLH1 gene in exon 3 was detected by whole exome sequencing and multiplex ligation dependent amplification, which is a pathogenic mutation. After the pathogenic mutation was identified, Sanger sequencing was performed on 4 direct members of the family for MLH1 gene, and 3 family members were found to have detected the mutation and included in MTS risk control. Until December 25 2023, follow-up showed the proband patients were not suffered from recurrence or new occurrence of skin or gastrointestinal tumors. The study reported a typical MTS family and found a possible pathogenic nonsense mutation in the MLH1 gene, which provides new evidence for the pathogenicity of this mutation.

Modulation of miR-155-5p signalling via 5-ASA for the prevention of high microsatellite instability: an in vitro study using human epithelial cell lines.

5-aminosalicylic acid (5-ASA) is a first-line treatment for maintaining colitis remission. It is a highly effective, safe, and well-tolerated drug with anti-inflammatory and chemo-preventive properties. While patients with primary sclerosing cholangitis (PSC) with concomitant ulcerative colitis are treated with 5-ASA, the molecular mechanisms underlying the drug's chemo-preventive effects are not entirely understood. We previously reported that bile acids and lipopolysaccharide-induced miR-155 expression was associated with downregulating mismatch repair (MMR) proteins in CACO-2 cell lines. Therefore, in this investigation, a set of in vitro functional studies was performed to show the possible mechanisms behind the epigenetic relationship between miR-155 and 5-ASA's prevention of high microsatellite instability (MSI-H). In transient transfection with miR-155Mimic, which behaves like endogenous miRNA, we confirmed the relationships between miR-155 and its target MMR in three human intestinal epithelial cell lines: CACO-2, NCM460D and HT-29. We have shown, for the first time, that 5-ASA modulates MLH1, MSH2, MSH6 in miR-155 transfected cells. These findings underline that chemoprotective 5-ASA therapy can effectively attenuate the expression of miR-155 and potentially prevent a development of MSI-H in a subset of colorectal cancers associated with PSC.

Estimating cancer risk in carriers of Lynch syndrome variants in UK Biobank.

BackgroundLynch syndrome (LS) is an inherited cancer predisposition syndrome caused by genetic variants affecting DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 Cancer risk in LS is estimated from cohorts of individuals ascertained by individual or family history of cancer, which may upwardly bias estimates. METHODS: 830 carriers of pathogenic or likely pathogenic (path_MMR) MMR gene variants classified by InSiGHT were identified in 454 756 UK Biobank (UKB) participants using whole-exome sequence. Nelson-Aalen survival analysis was used to estimate cumulative incidence of colorectal, endometrial and breast cancer (BC). RESULTS: Cumulative incidence of colorectal and endometrial cancer (EC) by age 70 years was elevated in path_MMR carriers compared with non-carriers (colorectal: 11.8% (95% confidence interval (CI): 9.5% to 14.6%) vs 1.7% (95% CI: 1.6% to 1.7%), endometrial: 13.4% (95% CI: 10.2% to 17.6%) vs 1.0% (95% CI: 0.9% to 1.0%)), but the magnitude of this increase differed between genes. Cumulative BC incidence by age 70 years was not elevated in path_MMR carriers compared with non-carriers (8.9% (95% CI: 6.3% to 12.4%) vs 7.5% (95% CI: 7.4% to 7.6%)). Cumulative cancer incidence estimates in UKB were similar to estimates from the Prospective Lynch Syndrome Database for all genes and cancers, except there was no evidence for elevated EC risk in carriers of pathogenic PMS2 variants in UKB. CONCLUSION: These results support offering incidentally identified carriers of any path_MMR surveillance to manage colorectal cancer risk. Incidentally identified carriers of pathogenic variants in MLH1, MSH2 and MSH6 would also benefit from interventions to reduce EC risk. The results suggest that BC is not an LS-related cancer.

The ubiquitin ligase UBR4 and the deubiquitylase USP5 modulate the stability of DNA mismatch repair protein MLH1.

MLH1 plays a critical role in DNA mismatch repair and genome maintenance. MLH1 deficiency promotes cancer development and progression, but the mechanism underlying MLH1 regulation remains enigmatic. In this study, we demonstrated that MLH1 protein is degraded by the ubiquitin-proteasome system and have identified vital cis-elements and trans-factors involved in MLH1 turnover. We found that the region encompassing the amino acids 516 to 650 is crucial for MLH1 degradation. The mismatch repair protein PMS2 may shield MLH1 from degradation as it binds to the MLH1 segment key to its turnover. Furthermore, we have identified the E3 ubiquitin ligase UBR4 and the deubiquitylase USP5, which oppositely modulate MLH1 stability. In consistence, UBR4 or USP5 deficiency affects the cellular response to nucleotide analog 6-TG, supporting their roles in regulating mismatch repair. Our study has revealed important insights into the regulatory mechanisms underlying MLH1 proteolysis, critical to DNA mismatch repair related diseases.

Report of the sixth meeting of the European Consortium 'Care for CMMRD' (C4CMMRD), Paris, France, November 16th 2022.

Biallelic germline pathogenic variants in one of the four mismatch repair genes (MSH2, MSH6, MLH1 and PMS2) cause a very rare, highly penetrant, childhood-onset cancer syndrome, called constitutional mismatch repair deficiency (CMMRD). The European consortium "Care for CMMRD" (C4CMMRD) was founded in Paris in 2013 to facilitate international collaboration and improve our knowledge of this rare cancer predisposition syndrome. Following initial publications on diagnostic criteria and surveillance guidelines for CMMRD, several partners collaborating within the C4CMMRD consortium have worked on and published numerous CMMRD-related clinical and biological projects. Since its formation, the C4CMMRD consortium held meetings every 1-2 years (except in 2020 and 2021 due to the Covid 19 pandemic). The sixth C4CMMRD meeting was held in Paris in November 2022, and brought together 42 participants from nine countries involved in various fields of CMMRD healthcare. The aim was to update members on the latest results and developments from ongoing research, and to discuss and initiate new study proposals. As previously done for the fifth meeting of the C4CMMRD group, this report summarizes data presented at this meeting.

MLH1 Inhibits Metastatic Potential of Pancreatic Ductal Adenocarcinoma via Downregulation of GPRC5C.

DNA mismatch repair gene MutL homolog-1 (MLH1) has divergent effects in many cancers; however, its impact on the metastasis of pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, MLH1 stably overexpressed (OE) and knockdowned (KD) sublines were established. Wound healing and transwell assays were used to evaluate cell migration/invasion. In vivo metastasis was investigated in orthotopic implantation models (severe combined immunodeficiency mice). RT-qPCR and western blotting were adopted to show gene/protein expression. MLH1 downstream genes were screened by transcriptome sequencing. Tissue microarray-based immunohistochemistry was applied to determine protein expression in human specimens. In successfully generated sublines, OE cells presented weaker migration/invasion abilities, compared with controls, whereas in KD cells, these abilities were significantly stronger. The metastasis-inhibitory effect of MLH1 was also observed in mice. Mechanistically, G protein-coupled receptor, family C, group 5, member C (GPRC5C) was a key downstream gene of MLH1 in PDAC cells. Subsequently, transient GPRC5C silencing effectively inhibited cell migration/invasion and remarkably reversed the proinvasive effect of MLH1 knockdown in KD cells. In animal models and human PDAC tissues, tumoral GPRC5C expression, negatively associated with MLH1 expressions, was positively correlated with histologic grade, vessel invasion, and poor cancer-specific survival. In conclusion, MLH1 inhibits the metastatic potential of PDAC via downregulation of GPRC5C.