Erk Inhibition as a Promising Therapeutic Strategy for High IL-8-Secreting and Low SPTAN1-Expressing Colorectal Cancer.

Tumor recurrence and drug resistance are responsible for poor prognosis in colorectal cancer (CRC). DNA mismatch repair (MMR) deficiency or elevated interleukin-8 (IL-8) levels are characteristics of CRCs, which have been independently correlated with treatment resistance to common therapies. We recently demonstrated significantly impaired therapeutical response and increased IL-8 release of CRC cell lines with reduced expression of MMR protein MLH1 as well as cytoskeletal non-erythrocytic spectrin alpha II (SPTAN1).

Functional analysis of MMR gene VUS from potential Lynch syndrome patients.

Lynch syndrome is caused by inactivating variants in DNA mismatch repair genes, namely MLH1, MSH2, MSH6 and PMS2. We have investigated five MLH1 and one MSH2 variants that we have identified in Turkish and Tunisian colorectal cancer patients. These variants comprised two small deletions causing frameshifts resulting in premature stops which could be classified pathogenic (MLH1 p.

Key role of phosphorylation sites in ATPase domain and Linker region of MLH1 for DNA binding and functionality of MutLα.

MutLα is essential for human DNA mismatch repair (MMR). It harbors a latent endonuclease, is responsible for recruitment of process associated proteins and is relevant for strand discrimination. Recently, we demonstrated that the MMR function of MutLα is regulated by phosphorylation of MLH1 at serine (S) 477.

A conserved motif in the disordered linker of human MLH1 is vital for DNA mismatch repair and its function is diminished by a cancer family mutation.

DNA mismatch repair (MMR) is essential for correction of DNA replication errors. Germline mutations of the human MMR gene MLH1 are the major cause of Lynch syndrome, a heritable cancer predisposition. In the MLH1 protein, a non-conserved, intrinsically disordered region connects two conserved, catalytically active structured domains of MLH1.

Functional characterization of MLH1 missense variants unveils mechanisms of pathogenicity and clarifies role in cancer.

Lynch syndrome is a heritable condition caused by a heterozygous germline inactivating mutation of the DNA mismatch repair (MMR) genes, most commonly the MLH1 gene. However, one third of the identified alterations are missense variants, for which the clinical significance is unclear in many cases. We have identified three MLH1 missense alterations (p.

High Expression of Casein Kinase 2 Alpha Is Responsible for Enhanced Phosphorylation of DNA Mismatch Repair Protein MLH1 and Increased Tumor Mutation Rates in Colorectal Cancer.

DNA mismatch repair (MMR) deficiency plays an essential role in the development of colorectal cancer (CRC). We recently demonstrated in vitro that the serine/threonine casein kinase 2 alpha (CK2α) causes phosphorylation of the MMR protein MLH1 at position serine 477, which significantly inhibits the MMR. In the present study, CK2α-dependent MLH1 phosphorylation was analyzed in vivo.

SPTAN1 Expression Predicts Treatment and Survival Outcomes in Colorectal Cancer.

Colorectal cancer (CRC) is a leading cause of cancer-related morbidity and mortality. In a cohort of 189 patients with CRC, we recently showed that expression of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1) was lower in advanced metastatic tumours. The aim of the present study was to clarify the association of intratumoural SPTAN1 expression levels with treatment and survival outcomes in patients with CRC.

Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations.

Pathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start.

Alternative AKT2 splicing produces protein lacking the hydrophobic motif regulatory region.

Three AKT serine/threonine kinase isoforms (AKT1/AKT2/AKT3) mediate proliferation, metabolism, differentiation and anti-apoptotic signals. AKT isoforms are activated downstream of PI3-kinase and also by PI3-kinase independent mechanisms. Mutations in the lipid phosphatase PTEN and PI3-kinase that increase PIP3 levels increase AKT signaling in a large proportion of human cancers.

Expression and secretion of the pro‑inflammatory cytokine IL‑8 is increased in colorectal cancer cells following the knockdown of non‑erythroid spectrin αII.

Non‑erythroid spectrin αII (SPTAN1) expression is decreased in ~40% of cases of MLH1‑deficient colorectal cancer (CRC). SPTAN1 knockdown reduces cell viability, cellular mobility and cell‑cell contact formation, indicating that the SPTAN1 plays an important role in tumour growth, attachment and in regulating the tumour microenvironment. Changes in the tumour microenvironment can affect the immune response.

Validation of an in Vitro Mismatch Repair Assay Used in the Functional Characterization of Mismatch Repair Variants.

A significant proportion of DNA-mismatch repair (MMR) variants are classified as of unknown significance, precluding diagnosis. The in vitro MMR assay is used to assess their MMR capability, likely the most important function of an MMR protein. However, the robustness of the assay, crucial for its use in the clinical setting, has been rarely evaluated.

Downregulation of SPTAN1 is related to MLH1 deficiency and metastasis in colorectal cancer.

Introduction: Colorectal cancers (CRCs) deficient in the DNA mismatch repair protein MutL homolog 1 (MLH1) display distinct clinicopathological features and require a different therapeutic approach compared to CRCs with MLH1 proficiency. However, the molecular basis of this fundamental difference remains elusive. Here, we report that MLH1-deficient CRCs exhibit reduced levels of the cytoskeletal scaffolding protein non-erythroid spectrin αII (SPTAN1), and that tumor progression and metastasis of CRCs correlate with SPTAN1 levels.

Diagnostic yield and clinical utility of a comprehensive gene panel for hereditary tumor syndromes.

Background: In a considerable number of patients with a suspected hereditary tumor syndrome (HTS), no underlying germline mutation is detected in the most likely affected genes. The present study aimed to establish and validate a large gene panel for HTS, and determine its diagnostic yield and clinical utility.

Methods: The study cohort comprised 173 patients with suspected, but unexplained, HTS (group U) and 64 HTS patients with a broad spectrum of known germline mutations (group K).

Analysis of MUTYH alternative transcript expression, promoter function, and the effect of human genetic variants.

The human DNA repair gene MUTYH, whose mutational loss causes a colorectal polyposis and cancer predisposition, contains three alternative first exons. In order to analyze alternative transcription and the effect of genetic alterations found in humans, we established a cell-based minigene experimental model supporting transcription and splicing and thoroughly verified its functionality. We identified highly conserved promoter areas and inactivated them in the minigene, and also introduced six human variants.

DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477).

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1.

Evaluation of MLH1 variants of unclear significance.

Inactivating mutations in the MLH1 gene cause the cancer predisposition Lynch syndrome, but for small coding genetic variants it is mostly unclear if they are inactivating or not. Nine such MLH1 variants have been identified in South American colorectal cancer (CRC) patients (p.Tyr97Asp, p.

Phosphorylation-dependent signaling controls degradation of DNA mismatch repair protein PMS2.

MutLα, a heterodimer consisting of MLH1 and PMS2, plays an important role in DNA mismatch repair and has been shown to be additionally involved in several other important cellular mechanisms. Previous work indicated that AKT could modulate PMS2 stability by phosphorylation. Still, the mechanisms of regulation of MutLα remain unclear.

Elucidating the clinical significance of two PMS2 missense variants coexisting in a family fulfilling hereditary cancer criteria.

The clinical spectrum of germline mismatch repair (MMR) gene variants continues increasing, encompassing Lynch syndrome, Constitutional MMR Deficiency (CMMRD), and the recently reported MSH3-associated polyposis. Genetic diagnosis of these hereditary cancer syndromes is often hampered by the presence of variants of unknown significance (VUS) and overlapping phenotypes. Two PMS2 VUS, c.

Loss of MLH1 sensitizes colon cancer cells to DNA-PKcs inhibitor KU60648.

Germline mutations of MLH1 are responsible for tumor generation in nearly 50% of patients with Lynch Syndrome, and around 15% of sporadic colorectal cancers show MLH1-deficiency due to promotor hypermethylation. Although these tumors are of lower aggressiveness the benefit for these patients from standard chemotherapy is still under discussion. Recently, it was shown that the sensitivity to the DNA-PKcs inhibitor KU60648 is linked to loss of the MMR protein MSH3.

Frequency and phenotypic spectrum of germline mutations in POLE and seven other polymerase genes in 266 patients with colorectal adenomas and carcinomas.

In a number of families with colorectal adenomatous polyposis or suspected Lynch syndrome/HNPCC, no germline alteration in the APC, MUTYH, or mismatch repair (MMR) genes are found. Missense mutations in the polymerase genes POLE and POLD1 have recently been identified as rare cause of multiple colorectal adenomas and carcinomas, a condition termed polymerase proofreading-associated polyposis (PPAP). The aim of the present study was to evaluate the clinical relevance and phenotypic spectrum of polymerase germline mutations.

Functional testing strategy for coding genetic variants of unclear significance in MLH1 in Lynch syndrome diagnosis.

Lynch syndrome is caused by inactivating mutations in the MLH1 gene, but genetic variants of unclear significance frequently preclude diagnosis. Functional testing can reveal variant-conferred defects in gene or protein function. Based on functional defect frequencies and clinical applicability of test systems, we developed a functional testing strategy aimed at efficiently detecting pathogenic defects in coding MLH1 variants.

Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1.

Introduction: Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear.

Promoter methylation of MLH1, PMS2, MSH2 and p16 is a phenomenon of advanced-stage HCCs.

Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion.

Refining the role of PMS2 in Lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants.

Background And Aim: The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients.