Genetic Inactivation of Peroxiredoxin-I Impairs the Growth of Human Pancreatic Cancer Cells.
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ABSTRACT:
SUBMITTER: Dahou H
PROVIDER: S-EPMC8068151 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundOur previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells.MethodsCRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples.ResultsPRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate.ConclusionsOur study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.
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Project description:Epigenetic gene silencing by aberrant DNA methylation is one of the important mechanisms leading to loss of key cellular pathways in tumorigenesis. Methyl-CpG-targeted transcriptional activation (MeTA) reactivates hypermethylation-mediated silenced genes in a different way from DNA-demethylating agents. Microarray coupled with MeTA (MeTA-array) identified seven commonly hypermethylation-mediated silenced genes in 12 pancreatic ductal adenocarcinoma (PDAC) cell lines. Among these, we focused on IRX4 (Iroquois homeobox 4) because IRX4 is located at chromosome 5p15.33 where PDAC susceptibility loci have been identified through genome-wide association study. IRX4 was greatly downregulated in all of the analyzed 12 PDAC cell lines by promoter hypermethylation. In addition, the IRX4 promoter region was found to be frequently and specifically hypermethylated in primary resected PDACs (18/28: 64%). Reexpression of IRX4 inhibited colony formation and proliferation in two PDAC cell lines, PK-1 and PK-9. In contrast, knockdown of IRX4 accelerated cell proliferation in an IRX4-expressing normal pancreatic ductal epithelial cell line, HPDE-1. Because IRX4 is a sequence-specific transcription factor, downstream molecules of IRX4 were pursued by microarray analyses utilizing tetracycline-mediated IRX4 inducible PK-1 and PK-9 cells; CRYAB, CD69, and IL32 were identified as IRX4 downstream candidate genes. Forced expression of these genes suppressed colony formation abilities for both PK-1 and PK-9. These results suggest that DNA methylation-mediated silencing of IRX4 contributes to pancreatic tumorigenesis through aberrant transcriptional regulation of several cancer-related genes.
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Project description:We have previously shown that L-methionine inhibits proliferation of breast, prostate, and colon cancer cells. This study extends these findings to BXPC-3 (mutated p53) and HPAC (wild-type p53) pancreatic cancer cells and explores the reversibility of these effects. Cells were exposed to L-methionine (5 mg/ml) for 7 days or for 3 days, followed by 4 days of culture without L-methionine (recovery). Cell proliferation, apoptosis, and cell cycle effects were assessed by flow cytometry after staining for Ki-67 or annexin V/propidium iodide. Cell proliferation was reduced by 31-35% after 7 days of methionine exposure; the effect persisted in BXPC-3 and HPAC cells after 4 days of recovery. Methionine increased apoptosis by 40-75% in HPAC cells, but not in BXPC-3 cells. Continuous exposure to methionine caused accumulation of BXPC-3 cells in the S phase and HPAC cells in both the G0/G1 and S phases; however, after 4 days of recovery, these effects disappeared. In conclusion, L-methionine inhibits proliferation and interferes with the cell cycle of BXPC-3 and HPAC pancreatic cancer cells; the effects on apoptosis remarkably persisted after methionine withdrawal. Apoptosis was induced only in BXPC-3 cells. Some of the differences in the effects of methionine between cell lines may be related to disparate p53 status. These findings warrant further studies on the potential therapeutic benefit of L-methionine against pancreatic cancer.
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Project description:BackgroundThe vertebrate pancreas contains islet, acinar and ductal cells. These cells derive from a transient pool of multipotent pancreatic progenitors during embryonic development. Insight into the genetic determinants regulating pancreatic organogenesis will help the development of cell-based therapies for the treatment of diabetes mellitus. Suppressor enhancer lin12/Notch 1 like (Sel1l) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by Sel1l remain elusive.ResultsWe have characterized the pancreatic phenotype of mice carrying a gene trap mutation in Sel1l. We show that Sel1l expression in the developing pancreas coincides with differentiation of the endocrine and exocrine lineages. Mice homozygous for the gene trap mutation die prenatally and display an impaired pancreatic epithelial morphology and cell differentiation. The pancreatic epithelial cells of Sel1l mutant embryos are confined to the progenitor cell state throughout the secondary transition. Pharmacological inhibition of Notch signaling partially rescues the pancreatic phenotype of Sel1l mutant embryos.ConclusionsTogether, these data suggest that Sel1l is essential for the growth and differentiation of endoderm-derived pancreatic epithelial cells during mouse embryonic development.
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Project description:Pancreatic ductal adenocarcinoma, which accounts for the majority of pancreatic cancers, is a lethal disease with few therapeutic options. Genomic profiling of pancreatic ductal adenocarcinoma has identified a complex and heterogeneous landscape. Understanding the molecular characteristics of pancreatic ductal adenocarcinoma will facilitate the identification of potential therapeutic strategies. We analyzed the gene expression profiles of primary tumors from patients compared to normal pancreas and identified high co-overexpression of core components of the spindle assembly checkpoint, including the protein kinase TTK (also known as MPS-1). We found overexpression of TTK protein in a subset of pancreatic ductal adenocarcinoma primary tumors and cell lines. siRNA-mediated depletion or catalytic inhibition of TTK resulted in an aberrant cell cycle profile, multi- and micro-nucleation, induction of apoptosis, and decreased cell proliferation and transformed growth. Selective catalytic inhibition of TTK caused override of the spindle assembly checkpoint-induced cell cycle arrest. Interestingly, we identified ubiquitin specific peptidase 16 (Usp16), an ubiquitin hydrolase, as a phosphorylation substrate of TTK. Usp16 regulates chromosomal condensation and G2/M progression by deubiquitinating histone H2A and polo-like kinase 1. Phosphomimetic mutants of Usp16 show enhanced proteosomal degradation and may prolong the G2/M transition allowing for correction of replication errors. Taken together, our results suggest a critical role for TTK in preventing aneuploidy-induced cell death in pancreatic cancer.
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Project description:Phosphotidylinositol-3-kinase (PI3K) signaling is altered in the majority of human cancers. To gain insight into the roles of members of this pathway in growth regulation, we inactivated AKT1, AKT2, or PDPK1 genes by targeted homologous recombination in human colon cancer cell lines. Knockout of either AKT1 or AKT2 had minimum effects on cell growth or downstream signaling. In contrast, knockout of both AKT1 and AKT2 resulted in markedly reduced proliferation in vitro when growth factors were limiting and severely affected experimental metastasis in mice. Unexpectedly, AKT1 and AKT2 appeared to regulate growth through FOXO proteins, but not through either GSK3beta or mTOR. In contrast, inactivation of PDPK1 affected GSK3beta and mTOR activation. These findings show that the PI3K signaling pathway is wired differently in human cancer cells than in other cell types or organisms, which has important implications for the design and testing of drugs that target this pathway.
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Project description:Although dietary fructose is associated with an elevated risk for pancreatic cancer, the underlying mechanisms remain elusive. Here, we report that ketohexokinase (KHK), the rate-limiting enzyme of fructose metabolism, is a driver of PDAC development. We demonstrate that fructose triggers KHK and induces fructolytic gene expression in mouse and human PDAC. Genetic inactivation of KhkC enhances the survival of KPC-driven PDAC even in the absence of high fructose diet. Furthermore, it decreases the viability, migratory capability, and growth of KPC cells in a cell autonomous manner. Mechanistically, we demonstrate that genetic ablation of KHKC strongly impairs the activation of KRAS-MAPK pathway and of rpS6, a downstream target of mTORC signaling. Moreover, overexpression of KHKC in KPC cells enhances the downstream KRAS pathway and cell viability. Our data provide new insights into the role of KHK in PDAC progression and imply that inhibiting KHK could have profound implications for pancreatic cancer therapy.
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Project description:Fluorescent protein reporter genes are widely used to identify and sort murine pancreatic ?-cells. In this study, we compared use of the MIP-GFP transgene, which exhibits aberrant expression of human growth hormone (hGH), with a newly derived Ins2Apple allele that lacks hGH expression on the expression of sex-specific genes. ?-Cells from MIP-GFP transgenic mice exhibit changes in the expression of 7,733 genes, or greater than half of their transcriptome, compared with ?-cells from Ins2Apple/+ mice. To determine how these differences might affect a typical differential gene expression study, we analyzed the effect of sex on gene expression using both reporter lines. Six hundred fifty-seven differentially expressed genes were identified between male and female ?-cells containing the Ins2Apple allele. Female ?-cells exhibit higher expression of Xist, Tmed9, Arpc3, Eml2, and several islet-enriched transcription factors, including Nkx2-2 and Hnf4a, whereas male ?-cells exhibited a generally higher expression of genes involved in cell cycle regulation. In marked contrast, the same male vs. female comparison of ?-cells containing the MIP-GFP transgene revealed only 115 differentially expressed genes, and comparison of the 2 lists of differentially expressed genes revealed only 17 that were common to both analyses. These results indicate that 1) male and female ?-cells differ in their expression of key transcription factors and cell cycle regulators and 2) the MIP-GFP transgene may attenuate sex-specific differences that distinguish male and female ?-cells, thereby impairing the identification of sex-specific variations.
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Project description:A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G(0) tumor cells. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G(0) state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase.
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Project description:Mammalian Target of Rapamycin complex 2 (mTORC2) and its regulatory component Rapamycin-insensitive companion of mTOR (RICTOR) are increasingly recognized as important players in human cancer development and progression. However, the role of RICTOR in human pancreatic ductal adenocarcinoma (PDAC) is unclear so far. Here, we sought to analyze the effects of RICTOR inhibition in human pancreatic cancer cell lines in vitro and in vivo. Furthermore, RICTOR expression was determined in human PDAC samples. Results demonstrate that depletion of RICTOR with siRNA (transient knock-down) or shRNA (stable knock-down) has an inhibitory effect on tumor growth in vitro. Moreover, RICTOR inhibition led to impaired phosphorylation/activity of AGC kinases (AKT, SGK1). Interestingly, hypoxia-induced expression of hypoxia-induced factor-1α (HIF-1α) was diminished and secretion of vascular-endothelial growth factor-A (VEGF-A) was impaired upon targeting RICTOR. Stable RICTOR knock-down led to significant inhibition of tumor growth in subcutaneous and orthotopic tumor models which was accompanied by significant reduction of tumor cell proliferation. Finally, immunohistochemical analyses of 85 human PDAC samples revealed significantly poorer survival in patients with higher RICTOR expression. In conclusion, these findings provide first evidence for mTORC2/RICTOR as an attractive novel target for treatment of human PDAC.
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