Reprogramming pancreatic stellate cells via p53 activation: A putative target for pancreatic cancer therapy

<div><p>Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely dense fibrotic stroma, which contributes to tumor growth, metastasis, and drug resistance. During tumorigenesis, quiescent pancreatic stellate cells (PSCs) are activated and become major contributors to fibrosis, by increasing growth factor signaling and extracellular matrix deposition. The p53 tumor suppressor is known to restrict tumor initiation and progression through cell autonomous mechanisms including apoptosis, cell cycle arrest, and senescence. There is growing evidence that stromal p53 also exerts anti-tumor activity by paracrine mechanisms, though a role for stromal p53 in PDAC has not yet been described. Here, we demonstrate that activation of stromal p53 exerts anti-tumor effects in PDAC. We show that primary cancer-associated PSCs (caPSCs) isolated from human PDAC express wild-type p53, which can be activated by the Mdm2 antagonist Nutlin-3a. Our work reveals that p53 acts as a major regulator of PSC activation and as a modulator of PDAC fibrosis. In vitro, p53 activation by Nutlin-3a induces profound transcriptional changes, which reprogram activated PSCs to quiescence. Using immunofluorescence and lipidomics, we have also found that p53 activation induces lipid droplet accumulation in both normal and tumor-associated fibroblasts, revealing a previously undescribed role for p53 in lipid storage. <i>In vivo</i>, treatment of tumor-bearing mice with the clinical form of Nutlin-3a induces stromal p53 activation, reverses caPSCs activation, and decreases fibrosis. All together our work uncovers new functions for stromal p53 in PDAC.</p></div

Stromal p53 activation reverses caPSC activation and reduces pancreatic desmoplasia in vivo.

<p>(A) Tumor bearing mice were treated with vehicle or RG7112 (200 mg/kg) and were sacrificed. Epithelial cells (EPCAM+) and fibroblasts (PDGFRα+) were isolated from dissociated tumors by FACS. p21 and Mdm2 mRNA levels were quantified by RT-qPCR and normalized to Rplp0 mRNA. Bars represent mean +SD of 3 different mice. *, p<0.05 by two-way ANOVA. (B-C) Mice transplanted with KPC cells were treated for 15 days with Vehicle or RG7112 (200 mg/kg) starting day 8 post-transplantation. Tumors were harvested and fixed in formalin. FFPE sections were subjected to (B) IHC using an αSMA antibody and (C) Masson’s Trichrome staining. Representative images are shown on the left and quantification on the right. At least eight 15X fields were quantified using Inform 2.1 software and mean values for each tumor are plotted on the graph. *, p<0.05 by Student’s test. Scale bar, 50 μM. (D) Metascape analysis was performed on RG7112 downregulated and upregulated genes (fold change >1.4 or <0.7, adjusted p<0.05). Six of the twenty most significant canonical pathways are shown and -log(pval) are indicated. (E) Heatmap representing selected genes from the RNA-seq analysis. Data are represented as log2 fold change, RG7112 versus Vehicle.</p

p53 reprograms human caPSCs towards quiescence.

<p>caPSCs were treated with Nutlin-3a, Nutlin-3b (control) or PD332991 for 48h (C, G) or 72h (A,B, F). (A) Cells were immunostained for Ki67 and nuclei were stained with DAPI. Bar graph represents the percentage of nuclei positive for the Ki67 antigen. At least 100 cells per condition were counted. Values are plotted as mean +SD of at least 2 experiments. ***, p<0.001; **, p<0.01; *, p<0.05 by two-way ANOVA. (B) Representative images of caPSCs stained with BODIPY 493/503 for detection of neutral lipids. (C) Acta2 mRNA levels were quantified by RT-qPCR. Values were normalized to Rplp0 mRNA levels and are represented as fold change relative to Nutlin-3b treated cells. Bars indicate mean +SD of at least 3 experiments. ***, p<0.001; **, p<0.01; *, p<0.05 by one-way ANOVA. (D-E) caPSCs were treated with Nutlin-3a or Nutlin-3b for 72h. Nutlin-3a was removed from treated caPSCs and cells were cultured for an additional 72h with (+) or without (-) Nutlin-3a. (D) Representative images of cells stained with BODIPY 493/503 (E) Acta2 mRNA levels were quantified and represented as described in C. (F) Representative images of cells stained with BODIPY 493/503. (G) Acta2 mRNA levels were quantified and represented as described in C. Scale bars, 25 μM.</p

p53-induced lipid droplet accumulation is associated with an increase in triacylglycerols and cholesterol esters.

<p>(A) Relative abundance of selected lipids from mass spectrometry-based lipidomics analysis of caPSC-82 treated for 72h with Nutlin-3a or Nutlin-3b (control). Bar graphs represent mean +SD of triplicates. #, p<0.0001; ***, p<0.001; **, p<0.01; *, p<0.05 by two-way ANOVA. (B) Representative images of cells treated with Nutlin-3a or Nutlin-3b for 72h and stained with BODIPY 493/503. Scale bar, 10 μM. (C) Relative abundance of selected lipids from mass spectrometry-based lipidomic analysis of the skin fibroblast line HF treated for 72h with Nutlin-3a or Nutlin-3b. Data are represented as in A. (D) Heatmap representing selected genes from the RNA-seq analysis of caPSCs and skin fibroblasts (SkinF). Data are represented as log2 fold change, Nutlin-3a versus Nutlin-3b.</p

p53 transcriptionally regulates the PSC activation network.

<p>(A-B) Four primary human caPSCs were treated for 48h with Nutlin-3a or Nutlin-3b (control) and analyzed by RNA-seq. (A) Ingenuity Pathway Analysis (IPA) was performed on p53 downregulated and upregulated genes from RNA-seq analysis (fold change >1.5 or <0.67, adjusted p <0.05). The 5 most significant canonical pathways are shown and–log(pval) are indicated. (B) Heatmap representing selected genes from the RNA-seq analysis. Data are represented as log2 fold change, Nutlin-3a versus Nutlin-3b. (C-D) Primary mouse PSCs isolated from pancreata of wild-type C57B6/J mice were treated with Nutlin-3a or Nutlin-3b and harvested on day 3 (pre-activated) or on day 7 (activated) of culture. (C) Heatmap shows the relative abundance of selected genes from the RNA-seq analysis of Nutlin-3b treated mPSC (day 3 and 7) and Nutlin-3a treated mPSC (day 7). (D) IPA analysis was performed on p53 downregulated and upregulated genes at day 7 (fold change>1.5 or <0.67, adjpval<0.05). The 5 most significant canonical pathways are shown and -log(pval) are indicated.</p

Cancer-associated pancreatic stellate cells express functional p53.

<p>(A) Immunoblot for p53 and p21 from primary caPSCs treated with Nutlin-3a (+) or its inactive enantiomer Nutlin-3b (-) for 48h. β-Actin serves as a loading control. (B) p21 and Mdm2 mRNA levels were quantified by real-time qPCR (RT-qPCR) in caPSCs treated with Nutlin-3a or Nutlin-3b for 48h. Values were normalized to Rplp0 mRNA levels and are represented as fold change relative to Nutlin-3b treated cells. Bars indicate mean +SD of at least 3 experiments. ***, p<0.001; **, p<0.01; *, p<0.05 by One-way ANOVA.</p