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Downstream of Mutant KRAS, the Transcription Regulator YAP Is Essential for Neoplastic Progression to Pancreatic Ductal Adenocarcinoma

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Downstream of Mutant KRAS, the Transcription Regulator YAP Is Essential for Neoplastic Progression to Pancreatic Ductal Adenocarcinoma

Weiying Zhang et al. Sci Signal.

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered Kras(G12D) and Kras(G12D):Trp53(R172H) mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS-mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.

Figures

Fig. 1
Fig. 1. Deletion of Yap blocks PDAC development in Kras and Kras:Trp53 mutant pancreas
(A) Representative images of H&E and CK19 IHC staining of pancreatic sections from KC, KPC, KYC, and KPYC mice of indicated ages. Asterisks, normal ducts; arrowheads, ADM or early PanINs. Scale bar, 100 µm. (B) Quantification of the disease stages in KC (n = 11 mice), KPC (n = 10), KYC (n = 7), and KPYC (n = 11) mice older than 9 weeks. (C) Quantification of the percentage of KPC (n = 13) or KPYC (n = 26) mice that developed PDAC over time. (D) Quantification of the mean percentage of histologically normal areas in pancreata from KC (n = 14), KPC (n = 13), KYC (n = 9), and KPYC (n = 16) mice younger or older than 9 weeks of age. (E) PDAC-free survival analysis of KPC (n = 26), KPYC (n = 14), KC (n = 16), and KYC (n = 10) mice.
Fig. 2
Fig. 2. Deletion of Yap does not affect ADM induced by oncogenic Kras
(A) Representative images of IHC staining for Yap, phosphorylated ERK(pERK), EGFR, and CK19 in pancreatic sections from 7-week-old KPC and KPYC mice. Scale bar, 100 µm. (B) Number of ADM lesions per 20× microscopic field in pancreata from 7-week-old KPC (n = 20) and KPYC (n = 17) mice. Lines mark the median numbers of ADM lesions per field for each genotype. (C) Representative IHC images of CK19 (brown) and Yap (blue) double staining and staining of Yap and α-SMA in serial pancreatic sections from 7-week-old KPC and KPYC mice. Asterisks, positively stained normal ducts; arrows, positively stained stromal cells; yellow arrowheads, positively stained ADM lesions; white arrowheads, negatively stained ADM lesions. Scale bar, 50 µm.
Fig. 3
Fig. 3. Deletion of Yap blocks the proliferation of Kras and Kras:Trp53mutant pancreatic ductal cells in vivo and in vitro
(A) Representative IHC images of Ki-67 in pancreatic sections from KPC and KPYC mice. Yellow arrowheads, Ki-67+ proliferating pancreatic ductal cells in KPC mice; gray arrowheads, Ki-67 ADMs in KPYC mice; yellow arrows, Ki-67+ proliferating stromal cells in both KPC and KPYC mice. Scale bar, 100 µm. (B) Number of Ki-67+ PanIN, stromal, and acinar cells per field from KC (n = 10) and KYC (n = 7) pancreata. Each data point represents the sum of five randomly selected fields from the pancreatic section of a different mouse. (C) Representative images from EdU incorporation assays of KrasG12D:Yapflox/+ and KrasG12D:Yapflox/flox pancreatic epithelial cells infected with Ad-GFP (GFP) or Ad-Cre-GFP (Cre). EdU-incorporated cells are red; nuclei are blue. Scale bar, 100 µm. (D) Quantification of the percentage of EdU+ cells per field from (C). Lines indicate median percentages of EdU+ cells. KrasG12D:Yapflox/+ (GFP), n = 23; KrasG12D:Yapflox/+ (Cre), n = 16; KrasG12D:Yapflox/flox (GFP), n= 13; KrasG12D:Yapflox/flox (Cre), n = 14. NS, not significant; P values were calculated using two-tailed t test. (E to H) Cell proliferation assessed in (E) KP and KPY cells, (F) KPY cells reconstituted with Yap or control vector, (G) mPDAC-G9 cells transfected with control or two independent mYap shRNAs (#1 and #2), and (H) human Colo-357 PDAC cells transfected with control or two independent hYap shRNAs (#1 and #2). Data are means ± SD from three independent experiments.
Fig. 4
Fig. 4. Yap promotes the proliferation of Kras and Kras:Trp53 mutant pancreatic ductal cells by sustaining the expression of a group of secreted factors
(A) qRT-PCR analysis of the indicated mRNA amounts in human Colo-357 PDAC cells infected with control or Yap shRNAs. Data are means ± SD of three independent experiments. (B) Western blot analysis in KPY cells infected with control or Yap retroviral vector, representative of three independent experiments. (C) qRT-PCR analysis of the indicated mRNA abundance in the cells as from (B). Data are means ± SD from three independent experiments. (D) Fold proliferation in KPY cells after 2 days in conditioned medium (+CM) from Yap-reconstituted (Yap) or control (vector) KPY cells. Data are means ± SD from four independent experiments. (E) Fold proliferation in Yap-reconstituted or control KPY cells after 3 days of exposure to dimethyl sulfoxide (DMSO) or phosphate-buffered saline (PBS) (control), MMP7 (200 ng/ml), PGE2 (10 µM), II-6 (150 ng/ml), or ll-1α (10 ng/ml), singly or in combination as indicated. Data are means ± SD from three independent experiments. *P< 0.01, **P< 0.001, ***P < 0.0001, two-tailed t test.
Fig. 5
Fig. 5. Yap controls the expression of Cox2 and Mmp7 in vitro and in vivo
(A) Representative images of IHC staining for Cox2 and Mmp7 in pancreatic sections from KC, KPC, KYC, and KPYC mice. Scale bar, 100 µm. (B) qRT-PCR analysis of ChIP with antibodies to immunoglobulin G (IgG), polymerase II (Pol II), and Yap on Cox2 and Mmp7 promoter regions that contain (S1 and S2) or do not contain (neg) putative TEAD-binding sites in Yap-reconstituted KPY cells. Data are means ± SD from three independent experiments. (C) Fold proliferation in KPY cells expressing control or Yap vector 3 days after addition of DMSO (control), marimastat (MMP inhibitor, 5 µM), or Celebrex (Cox2 inhibitor, 10 µM). Data are means ± SD from three independent experiments. *P< 0.01, **P< 0.001, ***P< 0.0001, two-tailed t test.
Fig. 6
Fig. 6. Deletion of Yap dampens the stromal response in Kras and Kras:Trp53 mutant pancreata
Representative images of IHC staining for α-SMA, vimentin, collagen, and CD45 on pancreatic sections from 3-month-old KC, KPC, KYC, and KPYC mice and 7-week-old KPC and KPYC mice. Yellow arrowheads, positively stained stroma; white arrowheads, negatively stained stroma; asterisks, blood vessels. Scale bar, 100 µm.
Fig. 7
Fig. 7. Oncogenic Ras signals through the MAPK pathway to promote the transcriptional activity of Yap
(A and B) Dual luciferase reporter assays of HEK293T cells transfected with Yap alone or in combination with wild-type (WT), G12V, or S17N HRAS (A) or KRAS (B). Data are means ± SD of the relative luciferase activity from three independent experiments. (C) qRT-PCR analysis for CTGF and CYR61 in HEK293T cells transfected with control vector, HRASG12V, or wild-type YAP alone or in combination. Data are means ± SD from three independent experiments. (D) Western blot analysis of HEK293T cells transfected with Flag-YAPS127A alone or in combination with wild-type KRAS, KRASG12V, or KRASS17N. (E) Western blot analysis of HEK293T cells transfected with vector control, MEKDD, or Flag-YAPS127A, either alone or in combination. (F) Western blot analysis of HEK293T cells transfected with vector control, HRASG12V, Flag-YAP5SA, and ERK siRNA (si-ERK) alone or in combination. (G) Western blot analysis of lysates from HEK293T cells expressing Flag-YAP5SA in combination with a control vector, HRASG12V, or MEKDD after extended separation on SDS-PAGE. (H) Western blotting for total and phosphorylated YAP (Ser127) in cytoplasmic and nuclear fractions of HEK293T cells transfected with either control vector or HRASG12V. Tubulin, cytoplasmic marker; lamin A/C, nuclear marker. All blots are representative of at least three independent experiments. (I) Schematic of the function of YAP in PDAC cells as a master transcriptional switch of the KRAS secre-tome promoting PDAC cell proliferation.

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