RESUMO
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). In the present study, decreased intratumoral MLH1 and SPTAN1 expression in CRCs could be significantly correlated with enhanced serum IL-8. Furthermore, using stably reduced SPTAN1-expressing SW480, SW620 or HT-29 cell lines, the RAS-mediated RAF/MEK/ERK pathway was analyzed. Here, a close connection between low SPTAN1 expression, increased IL-8 secretion, enhanced extracellular-signal-regulated kinase (ERK) phosphorylation and a mesenchymal phenotype were detected. The inhibition of ERK by U0126 led to a significant reduction in IL-8 secretion, and the combination therapy of U0126 with FOLFOX optimizes the response of corresponding cancer cell lines. Therefore, we hypothesize that the combination therapy of FOLFOX and U0126 may have great potential to improve drug efficacy on this subgroup of CRCs, showing decreased MLH1 and SPTAN1 accompanied with high serum IL-8 in affected patients.
Assuntos
Butadienos , Neoplasias Colorretais , Fluoruracila , Interleucina-8 , Nitrilas , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Interleucina-8/metabolismo , Interleucina-8/genética , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Butadienos/farmacologia , Nitrilas/farmacologia , Linhagem Celular Tumoral , Compostos Organoplatínicos/farmacologia , Compostos Organoplatínicos/uso terapêutico , Leucovorina/uso terapêutico , Leucovorina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Feminino , Masculino , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HT29 , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína 1 Homóloga a MutL/metabolismo , Proteína 1 Homóloga a MutL/genética , Pessoa de Meia-Idade , Idoso , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fosforilação/efeitos dos fármacosRESUMO
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.(His727Profs*57) and MSH2 p.(Thr788Asnfs*11)), but also two missense variants (MLH1 p.(Asn338Ser) and p.(Gly181Ser)) and two small, in-frame deletion variants (p.(Val647-Leu650del) and p.(Lys678_Cys680del)). For such small coding genetic variants, it is unclear if they are inactivating or not. We here provide clinical description of the variant carriers and their families, and we performed biochemical laboratory testing on the variant proteins to test if their stability or their MMR activity are compromised. Subsequently, we compared the results to in-silico predictions on structure and conservation. We demonstrate that neither missense alteration affected function, while both deletion variants caused a dramatic instability of the MLH1 protein, resulting in MMR deficiency. These results were consistent with the structural analyses that were performed. The study shows that knowledge of protein function may provide molecular explanations of results obtained with functional biochemical testing and can thereby, in conjunction with clinical information, elevate the evidential value and facilitate clinical management in affected families.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Reparo de Erro de Pareamento de DNA , Proteína 1 Homóloga a MutL , Neoplasias Colorretais Hereditárias sem Polipose/genética , Humanos , Masculino , Proteína 1 Homóloga a MutL/genética , Feminino , Reparo de Erro de Pareamento de DNA/genética , Pessoa de Meia-Idade , Proteína 2 Homóloga a MutS/genética , Adulto , Tunísia , Linhagem , Turquia , Idoso , Mutação de Sentido IncorretoRESUMO
Acute-on-chronic liver failure (ACLF) is associated with increased mortality. Specific therapy options are limited. Hypoxia-inducible factor 1 alpha (HIF-1α) has been linked to the pathogenesis of chronic liver disease (CLD), but the role of HIF-1α in ACLF is poorly understood. In the current study, different etiologies of CLD and precipitating events triggering ACLF were used in four rodent models. HIF-1α expression and the intracellular pathway of HIF-1α induction were investigated using real-time quantitative PCR. The results were verified by Western blotting and immunohistochemistry for extrahepatic HIF-1α expression using transcriptome analysis. Exploratory immunohistochemical staining was performed to assess HIF-1α in human liver tissue. Intrahepatic HIF-1α expression was significantly increased in all animals with ACLF, regardless of the underlying etiology of CLD or the precipitating event. The induction of HIF-1α was accompanied by the increased mRNA expression of NFkB1 and STAT3 and resulted in a marked elevation of mRNA levels of its downstream genes. Extrahepatic HIF-1α expression was not elevated. In human liver tissue samples, HIF-1α expression was elevated in CLD and ACLF. Increased intrahepatic HIF-1α expression seems to play an important role in the pathogenesis of ACLF, and future studies are pending to investigate the role of therapeutic HIF inhibitors in ACLF.
Assuntos
Insuficiência Hepática Crônica Agudizada , Subunidade alfa do Fator 1 Induzível por Hipóxia , Animais , Humanos , Insuficiência Hepática Crônica Agudizada/etiologia , Insuficiência Hepática Crônica Agudizada/metabolismo , Previsões , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , RNA Mensageiro/metabolismoRESUMO
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. In the current study, we focused on S87 located in the ATPase domain of MLH1 and on S446, S456 and S477 located in its linker region. We analysed the phosphorylation-dependent impact of these amino acids on DNA binding, MMR ability and thermal stability of MutLα. We were able to demonstrate that phosphorylation at S87 of MLH1 inhibits DNA binding of MutLα. In addition, we detected that its MMR function seems to be regulated predominantly via phosphorylation of serines in the linker domain, which are also partially involved in the regulation of DNA binding. Furthermore, we found that the thermal stability of MutLα decreased in relation to its phosphorylation status implying that complete phosphorylation might lead to instability and degradation of MLH1. In summary, we showed here, for the first time, a phosphorylation-dependent regulation of DNA binding of MutLα and hypothesized that this might significantly impact its functional regulation during MMR in vivo.
Assuntos
Reparo de Erro de Pareamento de DNA , DNA , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Fosforilação , Domínios Proteicos , DNA/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismoRESUMO
Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.
Assuntos
Insuficiência Hepática Crônica Agudizada , Doença Hepática Terminal , Varizes Esofágicas e Gástricas , Humanos , Doença Hepática Terminal/complicações , Varizes Esofágicas e Gástricas/complicações , Hemorragia Gastrointestinal/complicações , Cirrose Hepática/complicações , Cirrose Hepática/terapia , Insuficiência Hepática Crônica Agudizada/terapia , Insuficiência Hepática Crônica Agudizada/etiologiaRESUMO
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. This region has as yet been regarded as a flexible spacer, and missense alterations in this region have been considered non-pathogenic. However, we have identified and investigated a small motif (ConMot) in this linker which is conserved in eukaryotes. Deletion of the ConMot or scrambling of the motif abolished mismatch repair activity. A mutation from a cancer family within the motif (p.Arg385Pro) also inactivated MMR, suggesting that ConMot alterations can be causative for Lynch syndrome. Intriguingly, the mismatch repair defect of the ConMot variants could be restored by addition of a ConMot peptide containing the deleted sequence. This is the first instance of a DNA mismatch repair defect conferred by a mutation that can be overcome by addition of a small molecule. Based on the experimental data and AlphaFold2 predictions, we suggest that the ConMot may bind close to the C-terminal MLH1-PMS2 endonuclease and modulate its activation during the MMR process.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Reparo de Erro de Pareamento de DNA , Proteína 1 Homóloga a MutL , Humanos , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Mutação , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismoRESUMO
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.(Glu736Lys), p.(Pro640Thr) and p.(Leu73Pro)) in six individuals from large Tunisian families. For none of these alterations, a classification of pathogenicity was available, consequently diagnosis, predictive testing and targeted surveillance in affected families was impossible. We therefore performed functional laboratory testing using a system testing stability as well as catalytic activity that includes clinically validated reference variants. Both p.(Leu73Pro) and p.(Pro640Thr) were found to be non-functional due to severe defects in protein stability and catalytic activity. In contrast, p.(Glu736Lys) was comparable to the wildtype protein and therefore considered a neutral substitution. Analysis of residue conservation and of the structural roles of the substituted residues corroborated these findings. In conjunction with the available clinical data, two variants fulfil classification criteria for class 4 "likely pathogenic". The findings of this work clarify the mechanism of pathogenicity of two unclear MLH1 variants and enables predictive testing and targeted surveillance in members of carrier families worldwide.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Mutação de Sentido Incorreto , Humanos , Virulência , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA , Mutação em Linhagem Germinativa , Proteína 1 Homóloga a MutL/genéticaRESUMO
Cancer is a leading cause of death worldwide. Casein kinase 2 (CK2) is commonly dysregulated in cancer, impacting diverse molecular pathways. CK2 is a highly conserved serine/threonine kinase, constitutively active and ubiquitously expressed in eukaryotes. With over 500 known substrates and being estimated to be responsible for up to 10% of the human phosphoproteome, it is of significant importance. A broad spectrum of diverse types of cancer cells has been already shown to rely on disturbed CK2 levels for their survival. The hallmarks of cancer provide a rationale for understanding cancer's common traits. They constitute the maintenance of proliferative signaling, evasion of growth suppressors, resisting cell death, enabling of replicative immortality, induction of angiogenesis, the activation of invasion and metastasis, as well as avoidance of immune destruction and dysregulation of cellular energetics. In this work, we have compiled evidence from the literature suggesting that CK2 modulates all hallmarks of cancer, thereby promoting oncogenesis and operating as a cancer driver by creating a cellular environment favorable to neoplasia.
RESUMO
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. Using a cohort of 165 patients, we identified 88 CRCs showing significantly increased nuclear/cytoplasmic CK2α expression, 28 tumors with high nuclear CK2α expression and 49 cases showing a general low CK2α expression. Patients with high nuclear/cytoplasmic CK2α expression demonstrated significantly reduced 5-year survival outcome. By immunoprecipitation and Western blot analysis, we showed that high nuclear/cytoplasmic CK2α expression significantly correlates with increased MLH1 phosphorylation and enriched somatic tumor mutation rates. The CK2α mRNA levels tended to be enhanced in high nuclear/cytoplasmic and high nuclear CK2α-expressing tumors. Furthermore, we identified various SNPs in the promotor region of CK2α, which might cause differential CK2α expression. In summary, we demonstrated that high nuclear/cytoplasmic CK2α expression in CRCs correlates with enhanced MLH1 phosphorylation in vivo and seems to be causative for increased mutation rates, presumably induced by reduced MMR. These observations could provide important new therapeutic targets.
RESUMO
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. The analysis was based on histologic assessment of SPTAN1 protein levels in our own CRC cohort, and transcriptome data of 573 CRC cases from The Cancer Genome Atlas (TCGA). We first establish that high intratumoural levels of SPTAN1 protein and mRNA associate with favourable survival outcomes in patients with CRC. Next, a response prediction signature applied to the TCGA data reveals a possible link between high SPTAN1 transcript levels and improved patient responses to FOLFOX chemotherapy. Complementary in vitro experiments confirm that SPTAN1 knockdown strains of the colon cancer cell lines HT-29, HCT116 mlh1-2 and Caco-2 are less responsive to FOLFOX chemotherapy compared with SPTAN1-proficient control strains. Taken together, we identify SPTAN1 as a novel prognostic biomarker in CRC and show that SPTAN1 expression levels may predict patient responses to chemotherapy. These investigations illustrate how an affordable, histology-based diagnostic test could directly impact therapeutic decision-making at the bedside.
RESUMO
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. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.
Assuntos
ATPases Transportadoras de Cobre/genética , Cobre/sangue , Degeneração Hepatolenticular/genética , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Ceruloplasmina/metabolismo , Voluntários Saudáveis , Células Hep G2 , Humanos , Polimorfismo GenéticoRESUMO
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. AKT and other AGC kinases possess a regulatory mechanism that relies on a conserved hydrophobic motif (HM) C-terminal to the catalytic core. In AKT, the HM is contiguous to the serine 473 and two other newly discovered (serine 477 and tyrosine 479) regulatory phosphorylation sites. In AKT genes, this regulatory HM region is encoded in the final exon. We identified a splice variant of AKT2 (AKT2-13a), which contains an alternative final exon and lacks the HM regulatory site. We validated the presence of mRNA for this AKT2-13a splice variant in different tissues, and the presence of AKT2-13a protein in extracts from HEK293 cells. When overexpressed in HEK293 cells, AKT2-13a is phosphorylated at the activation loop and at the zipper/turn motif phosphorylation sites but has reduced specific activity. Analysis of the human transcriptome corresponding to other AGC kinases revealed that all three AKT isoforms express alternative transcripts lacking the HM regulatory motif, which was not the case for SGK1-3, S6K1-2, and classical, novel and atypical PKC isoforms. The transcripts of splice variants of Akt1-3 excluding the HM regulatory region could lead to expression of deregulated forms of AKT.
Assuntos
Processamento Alternativo/genética , Proteínas Proto-Oncogênicas c-akt/genética , Sequências Reguladoras de Ácido Nucleico/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Motivos de Nucleotídeos/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/genética , Isoformas de Proteínas/genética , Transdução de Sinais/genéticaRESUMO
Nonerythroid spectrin αII (SPTAN1) expression is decreased in ~40% of cases of MLH1deficient colorectal cancer (CRC). SPTAN1 knockdown reduces cell viability, cellular mobility and cellcell 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. Therefore, in the present study, proteome arrays were used to analyse the expression of 119 different chemokines and soluble receptors in CRC cell lines in which mutL homologue 1 (MLH1) or SPTAN1 were knocked down. The levels of interleukin (IL)8 were significantly increased in the cells in which SPTAN1 was knocked down, both at the mRNA and protein level. ELISA demonstrated that the cells in which SPTAN1 was knocked down secreted increased quantities of IL8, and chemotaxis assays revealed the enhanced trafficking of neutrophils, which was induced by media containing higher levels of IL8. The IL8 receptors, CRCX1 and CRCX2, were expressed in all the cell lines examined; however, their expression was not directly associated with IL8 expression. The results of the present study thus demonstrated that CRC cells in which SPTAN1 was knocked down secreted significantly higher levels of IL8, which inturn increased the migration of neutrophilic granulocytes. As MLH1deficient CRC exhibits an increased infiltration of cytotoxic Tcells and is associated with a decreased SPTAN1 expression, it can thus be hypothesized that CRC with a low SPTAN1 expression may release increased quantities of IL8, resulting in increased immune cell infiltration.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais/patologia , Interleucina-8/genética , Interleucina-8/metabolismo , Proteínas dos Microfilamentos/genética , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Células HT29 , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de NeoplasiasRESUMO
Nonerythroid spectrin αII (SPTAN1) is an important cytoskeletal protein that ensures vital cellular properties including polarity and cell stabilization. In addition, it is involved in cell adhesion, cell-cell contact, and apoptosis. The detection of altered expression of SPTAN1 in tumors indicates that SPTAN1 might be involved in the development and progression of cancer. SPTAN1 has been described in cancer and therapy response and proposed as a potential marker protein for neoplasia, tumor aggressiveness, and therapeutic efficiency. On one hand, the existing data suggest that overexpression of SPTAN1 in tumor cells reflects neoplastic and tumor promoting activity. On the other hand, nuclear SPTAN1 can have tumor suppressing effects by enabling DNA repair through interaction with DNA repair proteins. Moreover, SPTAN1 cleavage products occur during apoptosis and could serve as markers for the efficacy of cancer therapy. Due to SPTAN1's multifaceted functions and its role in adhesion and migration, SPTAN1 can influence tumor growth and progression in both positive and negative directions depending on its specific regulation. This review summarizes the current knowledge on SPTAN1 in cancer and depicts several mechanisms by which SPTAN1 could impact tumor development and aggressiveness.
RESUMO
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. METHODS AND RESULTS: To investigate the link between MLH1 and SPTAN1 in cancer progression, a cohort of 189 patients with CRC was analyzed by immunohistochemistry. Compared with the surrounding normal mucosa, SPTAN1 expression was reduced in MLH1-deficient CRCs, whereas MLH1-proficient CRCs showed a significant upregulation of SPTAN1. Overall, we identified a strong correlation between MLH1 status and SPTAN1 expression. When comparing TNM classification and SPTAN1 levels, we found higher SPTAN1 levels in stage I CRCs, while stages II to IV showed a gradual reduction of SPTAN1 expression. In addition, SPTAN1 expression was lower in metastatic compared with non-metastatic CRCs. Knockdown of SPTAN1 in CRC cell lines demonstrated decreased cell viability, impaired cellular mobility and reduced cell-cell contact formation, indicating that SPTAN1 plays an important role in cell growth and cell attachment. The observed weakened cell-cell contact of SPTAN1 knockdown cells might indicate that tumor cells expressing low levels of SPTAN1 detach from their primary tumor and metastasize more easily. CONCLUSION: Taken together, we demonstrate that MLH1 deficiency, low SPTAN1 expression, and tumor progression and metastasis are in close relation. We conclude that SPTAN1 is a candidate molecule explaining the tumor progression and metastasis of MLH1-deficient CRCs. The detailed analysis of SPTAN1 is now mandatory to substantiate its relevance and its potential value as a candidate protein for targeted therapy, and as a predictive marker of cancer aggressiveness.
Assuntos
Proteínas de Transporte/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas dos Microfilamentos/metabolismo , Proteína 1 Homóloga a MutL/deficiência , Idoso , Idoso de 80 Anos ou mais , Células CACO-2 , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Estudos de Coortes , Neoplasias Colorretais/genética , Progressão da Doença , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Masculino , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Pessoa de Meia-Idade , Metástase Neoplásica/genética , Metástase Neoplásica/patologiaRESUMO
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. Moreover, the potential contribution of CpG island methylation and specific transcription factors on MUTYH transcription was addressed. The findings allowed to attribute regulatory roles to three conserved motifs in the promoter: an M4 motif, a transcription factor IIB recognition element, and a GC box. Moreover, the data showed that three patient variants compromised MUTYH expression and therefore have the potential to cause pathogenic effects. We did not find evidence for a biologically relevant contribution of CpG island methylation or a direct transcriptional activation by DNA damage. Besides insight into the regulation of MUTYH transcription, the work therefore provides a functional MUTYH minigene experimental system suitable as a diagnostic tool for analyzing patient variants, and a functional map of the promotor that also can facilitate pathogenicity classifications of human variants.
Assuntos
Processamento Alternativo , DNA Glicosilases/genética , Regulação da Expressão Gênica , Variação Genética , Regiões Promotoras Genéticas , Linhagem Celular , Biologia Computacional/métodos , Ilhas de CpG , Metilação de DNA , Análise Mutacional de DNA , Éxons , Perfilação da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Leucócitos Mononucleares , Mutação , Estresse OxidativoRESUMO
DNA mismatch repair (MMR) is a highly conserved process and ensures the removal of mispaired DNA bases and insertion-deletion loops right after replication. For this, a MutSα or MutSß protein complex recognizes the DNA damage, MutLα nicks the erroneous strand, exonuclease 1 removes the wrong nucleotides, DNA polymerase δ refills the gap and DNA ligase I joins the fragments to seal the nicks and complete the repair process. The failure to accomplish these functions is associated with higher mutation rates and may lead to cancer, which highlights the importance of MMR by the maintenance of genomic stability. The post-replicative MMR implies that involved proteins are regulated at several levels, including posttranslational modifications (PTMs). Phosphorylation is one of the most common and major PTMs. Suitable with its regulatory force phosphorylation was shown to influence MMR factors thereby adjusting eukaryotic MMR activity. In this review, we summarized the current knowledge of the role of phosphorylation of MMR process involved proteins and their functional relevance.
Assuntos
Reparo de Erro de Pareamento de DNA , Animais , Ciclo Celular , Humanos , Fosforilação , ProteóliseRESUMO
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. Pharmacological treatment indicates that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.
Assuntos
Caseína Quinase II/metabolismo , Proteína 1 Homóloga a MutL/química , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL/metabolismo , Animais , Ciclo Celular , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Espectrometria de Massas , Endonuclease PMS2 de Reparo de Erro de Pareamento/química , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Modelos Moleculares , Proteínas MutL/química , Fosforilação , Processamento de Proteína Pós-Traducional , Serina/metabolismo , Células Sf9RESUMO
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.His112Gln, p.Pro141Ala, p.Arg265Pro, p.Asn338Ser, p.Ile501del, p.Arg575Lys, p.Lys618del, p.Leu676Pro), and evidence of pathogenicity or neutrality was not available for the majority of these variants. We therefore performed biochemical laboratory testing of the variant proteins and compared the results to protein in silico predictions on structure and conservation. Additionally, we collected all available clinical information of the families to come to a conclusion concerning their pathogenic potential and facilitate clinical diagnosis in the affected families. We provide evidence that four of the alterations are causative for Lynch syndrome, four are likely neutral and one shows compromised activity which can currently not be classified with respect to its pathogenic potential. The work demonstrates that biochemical testing, corroborated by congruent evolutionary and structural information, can serve to reliably classify uncertain variants when other data are insufficient.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Predisposição Genética para Doença , Proteína 1 Homóloga a MutL/genética , Mutação , Neoplasias Colorretais Hereditárias sem Polipose/etnologia , Simulação por Computador , Células HEK293 , Humanos , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/química , Conformação Proteica , América do SulRESUMO
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. The stability of MutLα subunits was investigated by transiently overexpression of wild type and mutant forms of MLH1 and PMS2 using immunoblotting for measuring the protein levels after treatment. We found that treatment with the cell-permeable serine/threonine phosphatase inhibitor, Calyculin, leads to degradation of PMS2 when MLH1 or its C-terminal domain is missing or if amino acids of MLH1 essential for PMS2 interaction are mutated. In addition, we discovered that the C-terminal tail of PMS2 is relevant for this Calyculin-dependent degradation. A direct involvement of AKT, which was previously described to be responsible for PMS2 degradation, could not be detected. The multi-kinase inhibitor Sorafenib, in contrast, was able to avoid the degradation of PMS2 which postulates that cellular phosphorylation is involved in this process. Together, we show that pharmacologically induced phosphorylation by Calyculin can induce the selective proteasome-dependent degradation of PMS2 but not of MLH1 and that the PMS2 degradation could be blocked by Sorafenib treatment. Curiously, the C-terminal Lynch Syndrome-variants MLH1L749P and MLH1Y750X make PMS2 prone to Calyculin induced degradation. Therefore, we conclude that the specific degradation of PMS2 may represent a new mechanism to regulate MutLα.