Mutant p53R175H 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.

Keywords: Pancreatic cancer; Proteomics.

Figure 1:. Mutant p53 promotes survival and neoplastic growth of normal pancreatic ductal cells.

A. An MTT assay reveals mutant p53^R175H promotes proliferation in 10% Serum, 2% Serum and Serum Free Conditions. Mutant p53^R273H also promotes proliferation, but in serum free conditions at 72 hours, the increase is not significant. A 2-way Anova using Prism Graphpad software was used to test for significance. B. Western blot for extracellular-regulated kinase p-Erk^T202/Y204 and Akt^S473 phosphorylation. In conditions of serum deprivation, expression of mutant 53 sustained p-Erk^T202/Y204 C. Images of colonies in soft agar assay. D. Ability of mutant p53 to induce anchorage independent growth was measured using soft agar assay. A Students t-test using Prism software was used to test for significance.

Figure 2.. A functional proteomics screen reveals mutant p53^R175H increases expression of HSP70

A. Heat map showing differential expression of proteins upon either vector control or mutant p53 over expression in HPNE:KRAS^G12D cells. B. RPPA assay samples were used to validate the expression of HSP70 using western blotting. C,D. Normalized RPPA data in linear values was used to draw the bar graphs showing the expression of p53 and HSP70. E. Western blot analysis to detect HSP70 expression in total cell lysate from stable cell lines in culture. F. HPNE:KRAS ^G12D cells expressing either vector control or mutant p53 exposed either 37 ⁰C or 42 ⁰C for 1hr and then recovered at 37 ⁰C for 6 hrs. Total cell lysate was prepared for western blot analysis and then HSP70 was detected. A students t-test was used to evaluate significance.

Figure 3.. Mutant p53^R175H increases mRNA levels of multiple HSP70 isoforms

A. Real time PCR was carried out to detect gene expression changes in different HSP70 isoforms with mutant p53 over expression. Mutant p53^R175H significantly increased expression of HSPA-1b, HSPA-1L and HSPA-8. B. Real time PCR was used to confirm mutant p53^R157H increases mRNA levels of HSP70 in HPNE cells, HPNE:KRAS^G12D and T-HPNE cells, which indicated mutant p53^R175H exerts an effect on HSP70 independent of mutant KRAS C. Western blot analysis to detect HSP27, 40, 90 and HSF1, 2, 4 in HPNE:KRAS^G12D cells expressing either vector control or mutant p53. Mutant p53^R175H increased expression of HSP40 and HSF2. A student’s t test was used to calculate statistics.

Figure 4:. mutant p53^R175H promotes nuclear accumulation of and stabilizes HSP70.

A. Western blot analysis was performed to detect HSP70 expression in isolated cytosolic and nuclear fractions from HPNE:KRAS^G12D cells that are stably expressing either vector control or different p53 mutants. Histone 3 was used as a nuclear marker and α-tubulin was used as a cytosolic markers. B. HPNE:KRAS^G12D cells that are stably expressing either vector control or different p53 mutants subjected to heat shock treatment and then HSP70 detected in cytosolic and nuclear fractions. C. Representative IHC showing intensity scores of staining used to define the levels of HSP70 in each human PDA sample analyzed. D. A p53^R175H specific antibody was used to evaluate mutant p53 status in a human PDA array. Representative HSP70 IHC in p53R175H mutant positive versus negative samples are also shown. E. Pie graph showing the percentage of human PDA samples that stained positive for mutant p53^R175H (47%). F. Quantification of HSP70 Intensity Score (1, 2 or 3) as a function of mutant p53^R175H status. IHC analysis revealed a significant increase in the intensity of HSP70 in patients with mutant p53^R175H. G. Quantification of percentage of cells per field with positive nuclear localization of HSP70. We observed a significant increase in the percentage of tumors cells with positive accumulation of HSP70 in patients with mutant p53. A student’s t-test using Prism software was used for all analysis.

Figure 5.. HSP70 interacts with and stabilizes mutant p53.

A. Western blot analysis of immunoprecipitated samples for HSP70 antibody. HPNE:KRAS^G12D cells stably expressing either vector control or mutant p53 were immunoprecipitated with V5 antibody. B, D. Western blot for p53 and HSP70 in the cytosolic and nuclear fractions after Triptolide treatment in HPNE:KRAS^G12D cells stably expressing either vector control or mutant p53. C. Immunoblot analysis of HSP70 and p53 expression after scrambled siRNA or HSP70 specific siRNA transfection, followed by heat shock treatment in HPNE:KRAS^G12D cells stably expressing either vector control or mutant p53.

Figure 6.. HSP70 is required for mutant p53 mediated cellular transformation:

A. Bar graph showing the number of colonies from HPNE:KRAS ^G12D cells that are stably expressing either vector control or mutant p53^R175H treated with DMSO or Triptolide in a soft agar assay. B. Western blot analysis of wild type and mutant p53 expression levels in HPNE:KRAS^G12D cells treated with a dose response C. Bar graph showing the number of colonies from HPNE:KRAS^G12D cells that are stably expressing either vector control or mutant p53 transfected with scrambled siRNA or HSP70 specific siRNA in soft agar assay. D. Western blot analysis showing siRNA guided deletion of HSP70 decreases the stability of mutant p53.