Cross-talk between mutant p53 and p62/SQSTM1 augments cancer cell migration by promoting the degradation of cell adhesion proteins

Abstract

Missense mutations in the p53 tumor suppressor abound in human cancer. Common (“hotspot”) mutations endow mutant p53 (mutp53) proteins with oncogenic gain of function (GOF), including enhanced cell migration and invasiveness, favoring cancer progression. GOF is usually attributed to transcriptional effects of mutp53. To elucidate transcription-independent effects of mutp53, we characterized the protein interactome of the p53R273H mutant in cells derived from pancreatic ductal adenocarcinoma (PDAC), where p53R273H is the most frequent p53 mutant. We now report that p53R273H, but not the p53R175H hotspot mutant, interacts with SQSTM1/p62 and promotes cancer cell migration and invasion in a p62-dependent manner. Mechanistically, the p53R273H-p62 axis drives the proteasomal degradation of several cell junction–associated proteins, including the gap junction protein Connexin 43, facilitating scattered cell migration. Concordantly, down-regulation of Connexin 43 augments PDAC cell migration, while its forced overexpression blunts the promigratory effect of the p53R273H-p62 axis. These findings define a mechanism of mutp53 GOF.

Keywords: cell adhesion; migration; mutant p53; p62; protein–protein interaction.

Fig. 1.

p53^R273H interacts with p62 via its CTD. (A) Volcano plot of the top hits (red) in immunoprecipitation and mass spectrometry analysis of p53^R273H IPs from CTRL PANC-1 cells, compared to p53KO PANC-1 cells. Top hits were defined as having absolute fold change ≥1.4 (log2 fold-change [FC] ≥ 0.49), P ≤ 0.05, and unique peptides ≥2. (B) PANC-1 cell lysates were IP with p53-specific antibodies (Santa Cruz FL-393) or IgG CTRL, followed by WB analysis with antibodies against p62 (Abcam 56416); inputs are shown on the left. (C) PANC-1 cell lysates underwent IP with p62-specific antibodies (Santa Cruz D-3) or IgG CTRL, followed by WB analysis with antibodies against p53 (p53-HRP). (D) PLA between p62 and p53, performed on CTRL and shp53 cells using the Duolink PLA kit (Sigma) with tetramethylrhodamine-5-isothiocyanate (TRITC) as a probe (red). Counterstaining was with DAPI (blue) and phalloidin-FITC (green) to visualize nuclear DNA and F-actin, respectively. Quantification of PLA foci is shown below, ****P < 0.0001 (Student’s t test), based on counting of 40 cells. (Scale bar, 10 μm.) (E) PANC-1 cells stably expressing shRNA directed against the 3′UTR of the endogenous p53 mRNA (3′UTR shp53) were transiently transfected to express wtp53, p53^R175H, p53^R273H or p53^R248W. Cell lysates were subjected to IP with antibodies against p62 (Santa Cruz D-3), followed by WB analysis with antibodies against p53 (p53-HRP) or p62 (Santa Cruz D-3). (F) PANC-1 cells stably expressing shRNA directed against the 3′UTR of the endogenous p53 mRNA (3′UTR shp53) were stably transduced to overexpress V5-tagged intact p53^R273H (FL) or a p53^R273H mutant in which residues 331–335 have been replaced by Ala (5A). Cell lysates underwent IP with antibodies against p62 (Santa Cruz D-3), followed by WB analysis with antibodies against p62 (Santa Cruz D-3) or p53 (anti rabbit V5 CST #13202). RASGAP was used as a loading CTRL. (G) PANC-1 cells as in F were subjected to PLA with antibodies against the V5 tag (reactive with the V5-tagged overexpressed mutp53) and p62. Nuclei were visualized by DAPI staining. (Scale bar, 10 μm.)

Fig. 2.

p62 promotes migration and invasion of cancer cells harboring p53^R273H. (A and B) (Top) Representative images of transwell migration (A) and invasion (B) assays (t = 18 h) of PANC-1 cells stably transfected with CTRL shRNA, p53 shRNA (shp53), or p62 shRNA (shp62). (Bottom) Quantification of area covered by migrating/invading cells, from three independent biological repeats, normalized to CTRL. Error bars indicate SEM. Statistical significance was determined using one-way ANOVA and Tukey’s post hoc test of the indicated comparisons, *P < 0.05, **P < 0.01, ***P < 0.001. (C) Quantification of transwell migration (t = 18 h) of 3′UTR shp53 PANC-1 cells stably transfected with p53^R273H (FL) or p53^R273H-5A, area covered by migrating cells, from three independent biological repeats, normalized to nontransfected cells (−). Statistical analysis as in A and B. *P < 0.05, NS, not significant. (D) Quantification of gap closure assays of CTRL and shp53 PANC-1 cells without or with stable overexpression of GFP-tagged p62, from three independent biological repeats. Statistical analysis as in A and B. *P < 0.05, **P < 0.01. (E) PANC-1 cells were subjected to a gap closure assay for 8 h. Cells were then fixed and subjected to PLA analysis of p62-p53^R273H interaction. Shown are representative sections from the migrating front (yellow box) and the center of the monolayer (red box). Yellow arrow indicates direction of migration. Right: Quantification of PLA foci, based on counting the average numbers of foci/cell in four different fields in each indicated region. ***P < 0.001 (Student’s t test). (Scale bar, 10 μm.) (F) CTRL HCT116 cells (expressing endogenous wtp53; wt), p53KO HCT116 cells, or KO cells ectopically expressing p53^R273H or p53^R175H were stably transduced with either CTRL shRNA or shRNA directed against p62. Cells were seeded in 12-well plates containing ibidi culture inserts. Gaps were created by removing the insert at t = 0, and cells were allowed to migrate for 24 h. Shown is quantification of percent of gap covered, from two independent biological repeats, for three fields in each condition. Error bars represent SEM. Statistical analysis as in A and B. *P < 0.05. (G) GFP tagged PANC-1 cells expressing CTRL shRNA, p53 shRNA, or p62 shRNA were injected into the tail vein of NSG mice (0.1 million cells/mouse). Lung metastases were evaluated 2 wk postinjection. (Top) Representative images of lung metastases. (Bottom) Total area of metastases at the lung surface (calibrated units), as quantified with ImageJ (n = 5 mice per group). Error bars represent SEM. Statistical analysis as in A and B. **P < 0.01, ***P < 0.001. (Scale bar, 0.5 cm.)

Fig. 3.

The p53^R273H-p62 axis down-regulates proteins associated with cell adhesion. (A) Total proteome analysis by LC-MS/MS was performed on PANC-1 cells stably expressing p53 shRNA, p62 shRNA, or CTRL shRNA. The Venn diagrams show the numbers of proteins increased upon knockdown of either p53 (beige) or p62 (gray) compared to CTRL (absolute fold change ≥ 1.4, P ≤ 0.05), and likewise for proteins decreased upon knockdown of p53 or p62, from two biological repeats. Numbers of overlapping proteins are indicated by arrows. (B) WB analysis of Connexin 43 in CTRL, shp62, and shp53 PANC-1 cells. Actin was used as loading CTRL. (C) IF analysis of Connexin 43 (red) in CTRL, shp62, and shp53 PANC-1 cells. Cells were counterstained with DAPI (blue) to visualize nuclei. (Scale bar, 10 μm.) (D) 3′UTR shp53 PANC-1 cells (see SI Appendix, Fig. S1F) were transiently transfected with DNA encoding p53^R273H. Then, 72 h later, cells were fixed and stained for Connexin 43 (red) and p53 (green) and counterstained with DAPI. Shown is a field in which some cells overexpress ectopic p53^R273H while the rest have only residual endogenous p53^R273H. (Scale bar, 10 μm.) (E) IF imaging of Connexin 43 (red) and V5 (green) in 3′UTR shp53 PANC-1 cells stably expressing V5-tagged p53^R273H (FL) or p53^R273H-5A (5A). (Scale bar, 10 μm.) (F) IF imaging of the migrating front (indicated by yellow arrow) of CTRL, shp62, and shp53 PANC-1 cells, stained for Connexin 43 (red) and phalloidin-FITC to visualize F-actin (green). (Scale bar, 10 μm.) (G) CTRL, shp62, and shp53 PANC-1 cells, either nontransfected or 48 h after transfection with a Connexin 43 expression plasmid, were subjected to a transwell migration assay for 18 h. Area covered by migrating cells was calculated from three independent biological repeats, normalized to CTRL. Error bars indicate SEM. One-way ANOVA and Tukey’s post hoc test of the indicated comparisons are shown. **P < 0.01, ****P < 0.0001. (H) CTRL, shp62, and shp53 PANC-1 cell cultures were transfected with the indicated siRNAs. Then, 36 h later, cells were subjected to a transwell migration assay for 18 h. Area covered by migrating cells was calculated from two independent biological repeats and normalized to CTRL. Error bars indicate SEM. One-way ANOVA and Tukey’s post hoc test of the indicated comparisons are shown in the table (right). *P < 0.05; **P < 0.01.

Fig. 4.

p53^R273H and p62 promote proteasomal degradation of Connexin 43. (A) CTRL, shp62, and shp53 PANC-1 cells were treated for 4 h with either MG132 (10 µM) or chloroquine (25 µM) or were left untreated. (Top) Cell lysates were subjected to WB analysis for Connexin 43 and GAPDH as loading CTRL. (Bottom) Quantification of change in Connexin 43 abundance, relative to untreated cells, from three biological repeats. (B) Extracts of CTRL and shp53 PANC-1 cells were IP with FK2 antibody, which binds mono- and polyubiquitinated proteins, followed by SDS-PAGE (7% polyacrylamide) and WB analysis with the indicated antibodies. Separate aliquots of the same IPs were resolved on another 7% polyacrylamide gel, followed by WB analysis with FK2, anti-p53 (p53-HRP), anti-p62 (Santa Cruz D-3), and anti-beta actin as loading CTRL. (C) CTRL PANC-1 cells were treated for 4 h with MG132 (10 µM) or were left untreated. Cell lysates underwent IP with p62-specific antibodies (Santa Cruz D-3) or IgG CTRL, followed by WB analysis with antibodies against Connexin 43 and p53 (p53-HRP). (D) PLA between p62 and Connexin 43 in 3′UTR shp53 PANC-1 cells stably expressing p53^R273H (FL) or p53^R273H-5A (5A). Counterstaining with DAPI (blue) and phalloidin-FITC (green) to visualize F-actin. (Scale bar, 10 μm.) (E) PANC-1 cells with stable p62 knockdown (using 3′UTR targeted shRNA) were transiently transfected with FLAG-tagged FL p62 or p62ΔUBA. Then, 48 h later, cells were fixed and stained with anti-FLAG (green) and anti-Connexin 43 (red) antibodies. Yellow borders indicate p62-overexpressing cells. (Scale bar, 10 μm.) (F) PANC-1 cells were treated with 1,6-HD or 1,6-HD (3.5% solution) for 5 min or left untreated (NT), fixed, and stained for Connexin 43 (red) and p62 (green). (Scale bar, 10 μm.)