Yes-associated protein mediates immune reprogramming in pancreatic ductal adenocarcinoma - PubMed (original) (raw)

Yes-associated protein mediates immune reprogramming in pancreatic ductal adenocarcinoma

S Murakami et al. Oncogene. 2017.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a high degree of inflammation and profound immune suppression. Here we identify Yes-associated protein (Yap) as a critical regulator of the immunosuppressive microenvironment in both mouse and human PDAC. Within Kras:p53 mutant pancreatic ductal cells, Yap drives the expression and secretion of multiple cytokines/chemokines, which in turn promote the differentiation and accumulation of myeloid-derived suppressor cells (MDSCs) both in vitro and in vivo. Pancreas-specific knockout of Yap or antibody-mediated depletion of MDSCs promoted macrophage reprogramming, reactivation of T cells, apoptosis of Kras mutant neoplastic ductal cells and pancreatic regeneration after acute pancreatitis. In primary human PDAC, YAP expression levels strongly correlate with an MDSC gene signature, and high expression of YAP or MDSC-related genes predicts decreased survival in PDAC patients. These results reveal multifaceted roles of YAP in PDAC pathogenesis and underscore its promise as a therapeutic target for this deadly disease.

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Figures

Figure 1

Figure 1. Deletion of Yap in Kras:p53 mutant pancreata results in tissue regeneration following acute pancreatitis

(a) Experimental design and summary of phenotypes at the indicated time points. (b) Representative images of H&E, CK19, Vimentin, αSMA, and CD45 IHC staining of pancreas sections from KPC and KPYC mice at 1 week or 5 weeks post caerulein injections. Scale bar indicates 200 μm.

Figure 2

Figure 2. Deletion of Yap reactivates T cells and promotes Kras:Trp53 mutant neoplastic ducts to undergo apoptosis following acute pancreatitis

(a) Representative images and quantification of the percentage of cleaved-Caspase 3 (cleaved Casp3) and CD3 IHC staining, and IF staining for CD3/CD44/CK19 and CD3/Gzmb/CK19 in the pancreas sections from KPC and KPYC mice at 1 week post caerulein injections. Error bars represent standard errors. Scale bar indicates 200 μm. *P<0.05; **P<0.005; NS: not significant. (b) Percentage of CD3+ T cells among total CD45+ leukocytes in pancreata from 9-week-old KPC (n = 6) and KPYC (n = 3) mice at 1 week post caerulein injections as determined by flow cytometry. Error bars indicate standard deviations. NS: not significant. (c) Ratio of CD4/CD8 positive cells in the pancreata of 9-week-old KPC (n = 7) and KPYC (n = 3) mice at 1 week post caerulein injections as determined by flow cytometry. Error bars indicate standard deviations. NS: not significant. (d) Percentage of CD4+FoxP3+ regulatory T cells among total CD4+ leukocytes in pancreata from 9-week-old KPC (n = 4) and KPYC (n = 3) mice at 1 week post caerulein injections as determined by flow cytometry. Error bars indicate standard deviations. NS: not significant. (e) Relative mRNA expression of Prf1, Gzmb, and Pcna in CD45+CD3+CD8+ cytotoxic T cells isolated from pancreata of 9-week-old KPC (n = 3) and KPYC ( n= 4) mice at 1 week post caerulein challenge as determined by qRT-PCR. Error bars indicate standard errors. ***P<0.0005. (f) Heatmap of relative mRNA expression of genes involved in TCR signaling in pancreata from 9-week-old KPC and KPYC mice at 1 week post caerulein injections as determined by microarray analysis.

Figure 3

Figure 3. Depletion of Yap enhances infiltration of MHCII+ macrophages in Kras:Trp53 mutant pancreata following acute pancreatitis

(a) Representative images and quantification of the percentage of MHCII and F4/80 IHC staining, and CD3, MHCII and CK19 IF signal intensity in pancreatic sections from 9-week-old KPC and KPYC mice at 1 week post caerulein injections. Error bars indicate standard errors. Scale bar indicates 200 μm. *P<0.05; **P<0.005; ***P<0.0005. (b) Percentage of CD11b+F4/80+ macrophages among total CD45+ leukocytes in pancreata from 9-week-old KPC (n = 6) and KPYC (n = 5) mice at 1 week post caerulein injections as determined by flow cytometry. Error bars indicate standard deviations. **P<0.005. (c) Percentage of MHC II+ cells among CD45+CD11b+F4/80+ macrophages in pancreata from 9-week-old KPC (n = 5) and KPYC (n = 4) mice at 1 week post caerulein injections as determined by flow cytometry. Error bars indicate standard deviations. **P<0.005. (d) Percentage of CD19+MHCII+ B-cells among total CD45+ leukocytes in pancreata from 9-week-old KPC (n = 6) and KPYC (n = 4) mice at 1 week post caerulein injections as determined by flow cytometry. Error bars indicate standard deviations.. NS: not significant. (e) Percentage of CD11c+MHCII+ dendritic cells among total CD45+ leukocytes in pancreata from 9-week-old KPC (n = 3) and KPYC (n = 5) mice at 1 week post caerulein injections as determined by flow cytometry. Error bars indicate standard deviations.. NS: not significant. (f) Relative mRNA expression of Nos2 and Arg in CD45+F4/80+ macrophages isolated from pancreata of 9-week-old KPC (n = 3) and KPYC (n = 7) animals at 1 week post caerulein challenge as determined by qRT-PCR. Error bars indicate standard errors. *P<0.05; **P<0.005.

Figure 4

Figure 4. Yap knockout blocks accumulation of MDSCs in the spleen and pancreata of Kras:Trp53 mutant mice following acute pancreatitis

(a) Percentage of of Gr1+CD11b+ MDSC cells among total CD45+ leukocytes in spleens and pancreata from 9-week-old KPC (spleen, n = 8; pancreas, n = 7) and KPYC (spleen, n = 6; pancreas, n = 5) mice at 1 week post caerulein injections as determined by flow cytometry. Symbols represent data from individual mice, and bars show the mean. Error bars indicate standard deviations. *P<0.05; ***P<0.0005. (b) Representative plots of flow cytometry analysis of Gr1+CD11b+ MDSC. The percentage of Gr1+CD11b+ cells among all CD45+ leukocytes is indicated in red. (c) Representative images and quantification of the percentage of Gr1, Ly6C, and Ly6G IHC staining in pancreatic sections from 9-week-old KPC and KPYC mice at 1 week post caerulein challenge. Error bars indicate standard errors. Scale bar indicates 200 μm. *P<0.05; ***P<0.0005. (d) Representative plots of flow cytometry analysis of Gr1+CD11b+ MDSC cells following 5 days of incubation of WT bone marrow cells with control medium or conditioned medium (CM) from KPYC cells transduced with vector control or Flag-Yap. The Gr1+CD11b+ population is gated and the percentage among all CD45+ leukocytes is indicated in red. Error bars indicate standard errors.

Figure 5

Figure 5. Silencing of Yap in Kras:Trp53 mutant PDAC cells inhibits the transcription and secretion of MDSC-polarizing cytokines

(a) Quantification of indicated cytokines in pancreata from 9-week-old WT (n = 3), KPC (n =6), and KPYC (n = 7) mice at 1 week post caerulein injection as determined by ELISA. Symbols represent data from individual mice, and bars show the mean. Error bars indicate standard deviation. *P<0.05; **P<0.005; ***P<0.0005. (b) Relative log2 fold changes (FC) of indicated cytokines in the conditioned media (CM) of KPC PDAC cells harboring pTRIPZ empty vector (Ctrl) or pTRIPZ-shYap (Yapsh) after addition of Dox. Error bars indicate standard deviation. *P<0.05; ***P<0.0005. (c) Relative log2 FC of indicated cytokines in the CM from KPYC cells re-expressing Yap compared to CM from KPYC control cells. Error bars indicate standard deviation. *P<0.05; **P<0.005; NS: not significant. (d) Relative mRNA expression of indicated genes in KPYC cells introduced with vector control or Flag-Yap as determined by qRT-PCR analysis. Error bars indicate standard error. *P<0.05; ***P<0.0005. (e) qRT-PCR analysis of ChIP with control IgG and Yap antibodies against promoter regions containing putative Tead-binding motifs (S1-3) of indicated genes in KPC PDAC cells. The 3’UTR sequences were used as negative controls. Error bars indicate standard error. *P<0.05; **P<0.005; ***P<0.0005; NS: not significant. (f) qRT-PCR analysis of ChIP with Flag antibodies in KPYC cells introduced with control vector or Flag-Yap using the same set of primers as in (e). Error bars indicate standard error. *P<0.05; **P<0.005; ***P<0.0005.

Figure 6

Figure 6. Depletion of MDSCs restores infiltration of MHCII+ cells and promotes pancreatic regeneration in KC mice

(a) Experimental design. (b) Representative images and quantification of H&E, Gr1 IHC, and MHCII/CD3 IF staining of pancreatic sections from 10-week-old KC mice that were subjected to caerulein and antibody injections as outlined in (a). Error bars represent standard errors. #1-3 indicate three different mice injected with αGr1 antibody. Scale bar indicates 200 μm. *P<0.05; **P<0.005; ***P<0.0005; NS: not significant.

Figure 7

Figure 7. Yap expression correlates with expression of MDSC-related genes and predicts survival in human PDAC

(a) Heatmap of unsupervised hierarchical clustering analysis of expression of 40 MDSC-related genes in TCGA primary PDAC tumor samples with RNAseq expression information (n=179). Three major clusters are identified as “Low” (n=19), “Medium” (n=63), or “High” (n=97) . (b) Relative YAP mRNA levels in TCGA primary PDAC tumors classified as either MDSC “High” or MDSC “Low” as in (a). (c) Kaplan-Meier overall survival curve of PDAC patients with either “High” (n=71) or “Low” (n=16) MDSC expression profiles. (d) Kaplan-Meier survival curve of PDAC patients stratified by Low (n=39), Medium (n=98), or High (n=10) relative YAP expression.

Figure 8

Figure 8. Yap orchestrates an immune suppressive microenvironment in Kras mutant pancreas by promoting the expression of MDSC-promoting cytokines

Schematic representation of the crucial function of Yap as a master switch of an ongenic Kras secretome that promotes the accumulation of immune suppressive MDSCs and TAMs and downregulates antigen-presenting macrophages, leading to suppression of CTLs.

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