A p53 Super-tumor Suppressor Reveals a Tumor Suppressive p53-Ptpn14-Yap Axis in Pancreatic Cancer

Abstract

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p53^53,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

Keywords: Hippo pathway; Ptpn14; YAP; mouse model; p53; pancreas cancer; transactivation domain; tumor suppressor.

Figure 1. Analysis of pancreatic cancer suppression potential of p53 TAD mutants

(A) Schematic of p53 transcriptional activation domain (TAD) mutants analyzed in this study. The p53 DNA binding domain (DBD), tetramerization domain (T) and C-terminal domain (C) are also shown. (B) Schematic for pancreatic cancer study. Kras^+/LSL-G12D;Pdx1-Cre;Trp53^+/+ (n=16), Kras^+/LSL-G12D;Pdx1-Cre;Trp53^+/LSL-WT (n=14), Kras^+/LSL-G12D;Pdx1-Cre;Trp53^+/LSL-53,54 (n=10), Kras^+/LSL-G12D;Pdx1-Cre;Trp53^{+/LSL-25,26,53,54} (n=12), and Kras^+/LSL-G12D;Pdx1-Cre;Trp53^+/− (n=8) mouse cohorts were analyzed for pancreatic cancer-free survival. (C) Kaplan-Meier analysis of PDAC-free survival of cohorts listed in (B). Labels indicate the p53 status of each cohort. (D) Representative histological images of the most advanced lesions found in each of the cohorts studied. The same field was analyzed in consecutive sections by H&E, Muc5ac, and Ck19 immunostaining. (E) Table summarizing the cohort genotypes and percentage of mice with PanINs (pancreatic intraepithelial neoplasias), IPMNs (intraductal papillary mucinous neoplasms), PDACs (pancreatic ductal adenocarcinomas), and metastatic lesions. Differences in pancreatic cancer incidence in Kras^+/LSL-G12D;Pdx1-Cre;Trp53^+/LSL-53,54 (p=0.019, n=10), Kras^+/LSLG12D;Pdx1-Cre;Trp53^{+/LSL-25,26,53,54} (p=0.0003, n=12) and Kras^+/LSLG12D;Pdx1-Cre;Trp53^+/− (p=0.042, n=8) mice are relative to p53 wild-type mice (Kras^+/LSLG12D;Pdx1-Cre;Trp53^+/+, n=16 and Kras^+/LSLG12D;Pdx1-Cre;Trp53^+/LSL-WT, n=14), using the two-tailed Fisher’s exact test. Each mouse was scored for the most advanced lesions found. (F) Representative H&E and Ck19 staining on organs with metastases from Kras^+/LSLG12D;Pdx1-Cre;Trp53^{+/LSL-25,26,53,54} and Kras^+/LSLG12D;Pdx1-Cre;Trp53^+/− mice. The scale bar in each panel applies to all images in that panel.

Figure 2. p53 transcriptomics and ChIP-seq analysis reveal genes hyperactivated by p53^53,54

(A) Genes activated in both Hras^V12;Trp53^53,54/53,54 and Hras^V12;Trp53^+/+ MEFs relative to Hras^V12;Trp53^−/− MEFs were further filtered based on fold change (Hras^V12;Trp53^53,54/53,54 vs. Hras^V12;Trp53^+/+ ≥ 1.3 fold) and on direct binding by p53, as established by p53 ChIP-seq data. The heat map shows the expression of 103 genes that satisfy these criteria in MEFs of these genotypes as well as in Hras^V12;Trp53^{25,26,53,54/25,26,53,54} MEFs. (B) qRT-PCR analysis of p53 target gene expression in Kras^G12D-expressing lung cancer cells derived from tumors in Kras^+/LA2;Trp53^{LSL-WT/LSL-WT}, Kras^+/LA2;Trp53^{LSL-25,26/LSL-25,26}, Kras^+/LA2;Trp53^{LSL-53,54/LSL-53,54} or Kras^+/LA2;Trp53^{LSL-25,26,53,54/LSL-25,26,53,54} mice and transduced with Ad-Cre to allow for the recombination of the lox-stop-lox cassette. Expression is relative to Actb (n=1, triplicate). Data are presented as mean ± SD. (C) Western blot analysis of p53 in Hras^V12-expressing MEFs homozygous for the different p53 variants. Cells were transduced with Ad-Cre (Adenovirus-Cre) to allow for the recombination of the lox-stop-lox cassette present in the different Trp53 alleles. Gapdh is a loading control. (D) Immunoprecipitation (IP) of p53 in MEFs homozygous for different Trp53 alleles, followed by immunoblotting for Mdm2 and p53. HC denotes the immunoglobulin heavy chain. Input is 2.5% of the total amount immunoprecipitated. Actin serves as a loading control. The fraction of Mdm2 bound to each p53 variant is indicated at the bottom and is relative to wild-type p53. (E) In silico structural modeling showing how the 53,54 residues in human and mouse p53 interact with HMGB1, a representative p53 TAD2-interacting protein, and how this interaction is affected with the QS mutations. (F) Heat map showing the subset of the 103 genes identified in Hras^V12-expressing MEFs whose expression is p53-dependent in PDAC, based on the comparison of PDAC cells from Kras^+/LSL-G12D;Ptf1a-Cre;Trp53^+/fl and Kras^+/LSL-G12D;Ptf1a-Cre;Cdkn2a^+/fl mice, where tumors undergo LOH for Trp53 or Cdkn2a (Collisson et al., 2011).

Figure 3. Ptpn14 is a bona fide p53 target gene

(A–B) p53 ChIP-seq profiles showing peaks in the Ptpn14 locus in doxorubicin-treated MEFs (A) and the PTPN14 locus in human fetal fibroblasts (B). Exons are represented by gray boxes and introns by dashed lines. The orientation of the vertices indicates whether the gene is on the sense (vertex down) or antisense (vertex up) strand. Transcription start sites (TSS) are marked by arrows. Inverted red triangles mark significant “called” peaks, and numbers denote the distance from the TSS. p53 response elements in each peak are indicated, with red denoting nucleotides in the conserved cores. Spacers between the two half-sites and number of mismatches relative to the consensus are indicated. (C) qRT-PCR for Ptpn14 expression in Hras^V12-expressing MEFs homozygous for different Trp53 alleles, relative to cells with wild-type Trp53 (n=1, triplicate). (D) qRT-PCR for Ptpn14 expression in Kras^G12D-expressing lung adenocarcinoma cells homozygous for each of the different Trp53 alleles (See Fig. 2B; n=1, triplicate). (E) qRT-PCR for Ptpn14 expression in Eμ-myc-driven B-cell lymphomas with and without p53-ER activation by 4-OHT (n=1, triplicate). (F) qRT-PCR for Ptpn14 expression in MEFs 6 hr after treatment with 8 Gy ionizing radiation, relative to untreated MEFs with wild-type p53 (n=3, triplicate). In all qRT-PCR experiments, Ptpn14 expression is relative to Actb. Data represent mean ± SD; * p ≤ 0.05; ** p ≤ 0.001, two-tailed unpaired Student’s t-test.

Figure 4. Ptpn14 overexpression drives growth arrest

(A) The effect of HA-GFP, HA-Ptpn14 or HA-p53 expression on cell cycle progression in Kras^+/G12D;Pdx1-Cre;Trp53^fl/fl mouse PDAC cells was examined by BrdU immunostaining cells expressing each antigen (detected by GFP or HA immunofluorescence). (Left) The average BrdU incorporation ± SD from (n=3), with 100 cells counted per experiment, is shown. (Right) Representative images are shown; arrows point to BrdU⁺ cells, while arrowheads point to BrdU⁻ cells. (B) (Left) Average colony number ± SD of KPC cells infected with an empty vector or a HA-Ptpn14 vector for low plating and soft agar assays (n=2, triplicate). (Right) Representative images of crystal violet-stained low plating (2 weeks after seeding), Giemsa-stained soft agar (4 weeks after seeding) and Ptpn14 localization by immunofluorescence. (C) The effect of HA-GFP, HA-Ptpn14 or HA-p53 expression on cell cycle progression in PANC-1 and MIA PaCa-2 human PDAC cells was examined by BrdU immunostaining. The average BrdU incorporation ± SD, with 100 cells counted per experiment, is shown (n=3). (D) (Left) Average colony number ± SD of MIA PaCa-2 cells infected with an empty vector or a HA-Ptpn14 vector for both low plating and soft agar assays (n=2, triplicate). (Right) Representative images of crystal violet-stained low plating (2 weeks after seeding), Giemsa-stained soft agar (4 weeks after seeding) and Ptpn14 localization by immunofluorescence. (E) The effect of HA-p53 expression on cell cycle progression in KPC PDAC cells expressing luciferase control shRNA (shCont) or shPtpn14-2 was examined by BrdU immunostaining. The average BrdU incorporation ± SD was assessed, with 100 cells counted per experiment (n=3). * p ≤ 0.05; ** p ≤ 0.001, two-tailed unpaired Student’s t-test. The scale bar in each panel applies to all images in that panel.

Figure 5. Ptpn14 is a pancreatic cancer suppressor

(A) Western blot for Ptpn14 and p53 in PDAC cells from Kras^+/LSL-G12D;Pdx1-Cre;Cdkn2a^fl/fl (KIC) and KPC mice after introducing a luciferase control shRNA (shCont), either of two Ptpn14 shRNAs (shPtpn14-1 and shPtpn14-2), or p53 shRNA (shp53). Ptpn14 quantification relative to Gapdh loading control is shown below the blot. (B) (Left) Low plating experiment quantification, showing the average colony number ± SD after introduction of the indicated shRNA into two lines of KIC cells (KIC1 and KIC2) and one line of KPC cells (n=3, triplicate). (Right) Representative images of crystal violet-stained low plating assays 2 weeks after seeding. (C) (Left) Average soft agar colony number ± SD after introduction of the indicated shRNA into KIC1 and KIC2 cells (n=2, triplicate). (Right) Representative images of soft agar experiment 4 weeks after seeding. (D) (Left) Tumor volume (average ±SD): KIC cells transduced with luciferase control shRNA, p53 shRNA, Ptpn14 shRNA-1 or Ptpn14 shRNA-2 were injected subcutaneously into ICR/Scid mice and tumor volume was measured as a function of time (n=3). (Right) Representative tumor images at the end of the experiment (22 days), with quantification of average tumor weight ± SD below. Each individual point represents a mouse. * p ≤ 0.05; ** p ≤ 0.001, two-tailed unpaired Student’s t-test.

Figure 6. Ptpn14 restrains Yap activity

(A) Schematic representation of wild-type Ptpn14 and a panel of mutants altered in key domains, including the FERM, PPxY and protein tyrosine phosphatase domains. PPxA1/PPxA2 denotes alanine mutations in both PPxY domains (Wilson et al., 2014). (B) HA-GFP or V5-tagged Ptpn14 variants were expressed in KPC mouse PDAC cells and BrdU-positivity of cells expressing each antigen (detected by GFP, HA or V5 immunofluorescence) was assessed. (Left) The average BrdU incorporation from 3 experiments ± SD, with 100 cells counted per experiment, is shown. (Right) Representative immunofluorescence images showing the localization pattern for each Ptpn14 variant. (C) Effects of HA-Ptpn14 on YAP localization in PANC-1 human PDAC cells, compared to HA-GFP. (Left) Graph showing the average percentage of cells with YAP nuclear exclusion from 3 experiments ± SD. (Right) Representative images with arrows pointing to HA-GFP-positive cells with nuclear YAP and arrowheads pointing to Ptpn14-expressing cells without nuclear Yap. (D) Colony forming ability of KIC cells treated with the Yap inhibitor verteporfin (10 μM) after introduction of control, p53, Ptpn14-1 or Ptpn14-2 shRNAs. (Left) Low plating experiment quantification, showing the average colony number ± SD of 3 experiments. (Right) Representative images show the crystal violet-stained colonies 2 weeks after seeding. (E) Sorting scheme to isolate PanIN cells from Kras^+/G12D;Pdx1-Cre;Trp53^+/+ and Kras^+/G12D;Pdx1-Cre;Trp53^−/− mice, based on CD133 positivity. Side-scatter area (SSC-A) and CD133 area (CD133-A) are used to define the PanIN population. (F) Yap GSEA signature (YAP1_UP) found enriched in Kras^+/G12D;Pdx1-Cre;Trp53^−/− cells relative to Kras^+/G12D;Pdx1-Cre;Trp53^+/+ cells. Nominal p value is indicated. The heat map represents the expression of the genes that contributed to the enrichment score. (G) Heat map showing the expression of an expanded list of 114 Yap-activated target genes based on 3 different signatures in Kras^+/G12D;Pdx1-Cre;Trp53^+/+ and Kras^+/G12D;Pdx1-Cre;Trp53^−/− PanIN cells. (H) (Left) Representative histological images of PanIN lesions from Kras^+/G12D;Ptf1a-Cre;Trp53^+/+ (n=4) and Kras^+/G12D;Ptf1a-Cre;Trp53^fl/fl (n=3) mice. The same field was analyzed by H&E and Yap staining, at 2 different magnifications, as indicated by the scale bars. Box indicates region of magnification. (Right) Average percentage of cells in PanINs with nuclear Yap ± SD. At least 300 nuclei were analyzed per mouse. * p ≤ 0.05; ** p ≤ 0.001, two-tailed unpaired Student’s t-test.

Figure 7. The p53-Ptpn14-Yap tumor suppressive axis is important in human cancers

(A) (Left) Representative images of Ptpn14 immunohistochemistry on PanIN (n=53) and PDAC (n=109) samples. (Right) Percentage of samples with Ptpn14 plasma membrane staining. (B) (Left) Representative images of Ptpn14 and Yap immunohistochemistry in PDAC samples with known p53 status (n=32 and n=64, for TP53 wild-type and mutant respectively; defined by sequencing of the TP53 gene). (Right) Percentage of samples with Ptpn14 plasma membrane staining and with Yap nuclear localization. (C) Heat map showing the fold-change in the expression of Yap-activated genes in tumor samples harboring TP53 mutations relative to tumors with wild-type p53. Only genes where p value for summary statistics across different tumors was ≤ 9.8E-17 are shown. Asterisks denote significant changes (p ≤ 0.05) within each gene/tumor type, based on a linear regression model. The black box highlights the results obtained in pancreatic cancer (PAAD). Ovarian cancer (OV), lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), breast cancer (BRCA), bladder cancer (BLCA), kidney cancer (KIRC), glioblastoma (GMB), uterine cancer (UCEC), acute leukemia (LAML), head and neck carcinoma (HNSC), colon (COAD) and rectal adenocarcinomas (READ). (D) Map depicting TP53 and PTPN14 mutations in gastrointestinal cancers. Mutual exclusivity was evaluated using the DISCOVER algorithm. (E) Proposed model incorporating Ptpn14 and Yap into the p53 tumor suppression program. For the TMA analysis, * p ≤ 0.05; ** p ≤ 0.001, Fisher test. For the heat map of the fold-change, * p ≤ 0.05, based on a linear model built for each Yap target gene.