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Mutant p53^R175H promotes cancer initiation in the pancreas by stabilizing HSP70.

ABSTRACT: Pancreatic cancer remains a highly lethal malignancy. We have recently shown that simultaneous expression of Kras and mutant Tp53^R175H promotes invasive ductal adenocarcinoma from pancreatic ductal cells. We hypothesized specific mutations in TP53 have divergent mechanisms of transforming ductal cells. In order to understand the role of mutant TP53 in transforming pancreatic ductal cells, we used a lentiviral system to express mutant TP53^R175H and TP53^R273H, two of the most frequently mutated TP53 alleles in pancreatic cancer patients, in immortalized, but not transformed, pancreatic ductal epithelial cells carrying a KRAS mutation (HPNE:KRAS^G12D). Mutant TP53 expression enhanced colony formation and an RPPA assay results revealed TP53^R175H uniquely induced HSP70 expression in HPNE:KRAS^G12D cells. In the context of TP53^R175H expression; we observed nuclear localization of HSP70. We performed immunoprecipitation experiments to show mutant p53^R175H binds to HSP70. We also provide evidence mutant p53^R175H is important for HSP70 stability and, more importantly, HSP70 is required for mutant p53 stability. These data are critical in the context of events leading to cellular transformation in the pancreas.

SUBMITTER: Polireddy K

PROVIDER: S-EPMC9753970 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Mutant p53R175H promotes cancer initiation in the pancreas by stabilizing HSP70.

Polireddy Kishore K Singh Kanchan K Pruski Melissa M Jones Neal C NC Manisundaram Naveen V NV Ponnela Pavani P Ouellette Michel M Van Buren George G Younes Mamoun M Bynon John S JS Dar Wasim A WA Bailey Jennifer M JM

Cancer letters 20190401

Pancreatic cancer remains a highly lethal malignancy. We have recently shown that simultaneous expression of Kras and mutant Tp53R175H promotes invasive ductal adenocarcinoma from pancreatic ductal cells. We hypothesized specific mutations in TP53 have divergent mechanisms of transforming ductal cells. In order to understand the role of mutant TP53 in transforming pancreatic ductal cells, we used a lentiviral system to express mutant TP53R175H and TP53R273H, two ...[more]

PMID: 30946870

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Project description:BackgroundThe mutation rate of the tumor protein P53 (TP53) has been reported to be greater than 50% in non-small cell lung cancer (NSCLC), and gain-of-function (GOF) mutations in unfolded P53 (TP53R175H and TP53Y220C) have been associated with poor prognosis. However, the best treatment for patients with NSCLC harboring unfolded mutant P53 (mutp53) remains unclear. Triptolide is a natural compound derived from Tripterygium wilfordii that has shown a strong antitumor effect in a variety of cancers. Our study aimed to explore the GOF mutations in unfolded mutp53 (TP53R175H and TP53Y220C) and to clarify the molecular mechanisms by which triptolide regulates the degradation of unfolded mutp53 proteins in NSCLC.MethodsTwo unfolded proteins harboring TP53R175H and TP53Y220C mutations were selected to explore their functions in NSCLC progression. NCI-H1299 cells (TP53-null) were transfected with wild-type TP53 (TP53WT), TP53R175H, or TP53Y220C genes and treated with triptolide or a vehicle. Wound healing and transwell assays were performed to measure cell migration and invasion in vitro. Lung metastasis models were constructed through tail vein injection of mutant cells into BALB/c nude mice to evaluate the effect of triptolide on metastasis in vivo. Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunoprecipitation, and dual-luciferase reporter assays were performed to explore the relevant molecular mechanisms.ResultsOur study revealed that triptolide treatment reduced TP53R175H levels and that the TP53Y220C mutation enhanced the invasion and migration of NCI-H1299 cells. Mechanistically, triptolide promoted TP53R175H and TP53Y220C protein proteasomal degradation mediated through the E3 ligase murine double minute 2 (MDM2) by directly interacting with heat shock protein 70 (HSP70). Moreover, by upregulating HSP70 transcription, triptolide contributed to the protein degradation of the GOF mutp53.ConclusionsOur study reports, for the first time, the mechanism underlying triptolide-regulated protein degradation of TP53R175H or TP53Y220C, which offers new insight into developing a better therapeutic strategy for patients with NSCLC who express the unfolded mutp53 GOF protein.

Dataset Information

Mutant p53^R175H promotes cancer initiation in the pancreas by stabilizing HSP70.

Publications

Mutant p53<sup>R175H</sup> promotes cancer initiation in the pancreas by stabilizing HSP70.

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