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DDIT4 Novel Mutations in Pancreatic Cancer.

ABSTRACT: Pancreatic cancer is one of the most common malignancies worldwide. This study is aimed at searching the possible genetic mutations and the value of novel gene mutation in the DNA damage-inducible transcript 4 (DDIT4) and signaling pathway in pancreatic cancer. Polymerase chain reaction (PCR) was performed to amplify the DNA sequences of DDIT4 from patients with pancreatic ductal adenocarcinoma. In addition, we used IHC to detect the expression level of DDIT4 in patients with pancreatic cancer in different types of gene mutation. Double-labeled immunofluorescence was employed to explore the expression levels of DDIT4/LC3 and their potential correlation. Our work indicated the two novel stable gene mutations in DDIT4 mRNA 3'-untranslated region (m.990 U>A and m.1246 C>U). Thirteen samples were found to have mutation in the DDIT4 3'-untranslated regions (UTR). To further verify the influence of gene mutation on protein expression, we performed immunohistochemistry on different gene mutation types, and we found a correlation between DDIT4 expression and gene mutation, which is accompanied by nuclear staining deepening. In order to further discuss the clinical value of DDIT4 gene mutation, immunofluorescence suggested that the expression of DDIT4 colocated with LC3; thus, we speculated that DDIT4 mutation may be involved in autophagy in pancreatic cancer cell. In this study, we found mutation in the 3'-UTR region of DDIT4, which may be associated with DDIT4 expression and tumor autophagy in pancreatic cancer tissues.

SUBMITTER: Ding F

PROVIDER: S-EPMC8110378 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Pancreatic cancer has a five-year survival rate of ~8%, with characteristic molecular heterogeneity and restricted treatment options. Targeting metabolism has emerged as a potentially effective therapeutic strategy for cancers such as pancreatic cancer, which are driven by genetic alterations that are not tractable drug targets. Although somatic mitochondrial genome (mtDNA) mutations have been observed in various tumors types, understanding of metabolic genotype-phenotype relationships is limited. METHODS:We deployed an integrated approach combining genomics, metabolomics, and phenotypic analysis on a unique cohort of patient-derived pancreatic cancer cell lines (PDCLs). Genome analysis was performed via targeted sequencing of the mitochondrial genome (mtDNA) and nuclear genes encoding mitochondrial components and metabolic genes. Phenotypic characterization of PDCLs included measurement of cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a Seahorse XF extracellular flux analyser, targeted metabolomics and pathway profiling, and radiolabelled glutamine tracing. RESULTS:We identified 24 somatic mutations in the mtDNA of 12 patient-derived pancreatic cancer cell lines (PDCLs). A further 18 mutations were identified in a targeted study of ~1000 nuclear genes important for mitochondrial function and metabolism. Comparison with reference datasets indicated a strong selection bias for non-synonymous mutants with predicted functional effects. Phenotypic analysis showed metabolic changes consistent with mitochondrial dysfunction, including reduced oxygen consumption and increased glycolysis. Metabolomics and radiolabeled substrate tracing indicated the initiation of reductive glutamine metabolism and lipid synthesis in tumours. CONCLUSIONS:The heterogeneous genomic landscape of pancreatic tumours may converge on a common metabolic phenotype, with individual tumours adapting to increased anabolic demands via different genetic mechanisms. Targeting resulting metabolic phenotypes may be a productive therapeutic strategy.

Project description:BackgroundChemoresistance is a major factor contributing to the poor prognosis of patients with pancreatic cancer, and cancer stemness is one of the most crucial factors associated with chemoresistance and a very promising direction for cancer treatment. However, the exact molecular mechanisms of cancer stemness have not been completely elucidated.Methodsm6A-RNA immunoprecipitation and sequencing were used to screen m6A-related mRNAs and lncRNAs. qRT-PCR and FISH were utilized to analyse DDIT4-AS1 expression. Spheroid formation, colony formation, Western blot and flow cytometry assays were performed to analyse the cancer stemness and chemosensitivity of PDAC cells. Xenograft experiments were conducted to analyse the tumour formation ratio and growth in vivo. RNA sequencing, Western blot and bioinformatics analyses were used to identify the downstream pathway of DDIT4-AS1. IP, RIP and RNA pulldown assays were performed to test the interaction between DDIT4-AS1, DDIT4 and UPF1. Patient-derived xenograft (PDX) mouse models were generated to evaluate chemosensitivities to GEM.ResultsDDIT4-AS1 was identified as one of the downstream targets of ALKBH5, and recruitment of HuR onto m6A-modified sites is essential for DDIT4-AS1 stabilization. DDIT4-AS1 was upregulated in PDAC and positively correlated with a poor prognosis. DDIT4-AS1 silencing inhibited stemness and enhanced chemosensitivity to GEM (Gemcitabine). Mechanistically, DDIT4-AS1 promoted the phosphorylation of UPF1 by preventing the binding of SMG5 and PP2A to UPF1, which decreased the stability of the DDIT4 mRNA and activated the mTOR pathway. Furthermore, suppression of DDIT4-AS1 in a PDX-derived model enhanced the antitumour effects of GEM on PDAC.ConclusionsThe ALKBH5-mediated m6A modification led to DDIT4-AS1 overexpression in PDAC, and DDIT-AS1 increased cancer stemness and suppressed chemosensitivity to GEM by destabilizing DDIT4 and activating the mTOR pathway. Approaches targeting DDIT4-AS1 and its pathway may be an effective strategy for the treatment of chemoresistance in PDAC.

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DDIT4 Novel Mutations in Pancreatic Cancer.

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