Loss of the wild-type KRAS allele promotes pancreatic cancer progression through functional activation of YAP1.

Human pancreatic ductal adenocarcinoma (PDAC) harboring one KRAS mutant allele often displays increasing genomic loss of the remaining wild-type (WT) allele (known as LOH at KRAS) as tumors progress to metastasis, yet the molecular ramification of this WT allelic loss is unknown. In this study, we showed that the restoration of WT KRAS expression in human PDAC cell lines with LOH at KRAS significantly attenuated the malignancy of PDAC cells both in vitro and in vivo, demonstrating a tumor-suppressive role of the WT KRAS allele. Through RNA-Seq, we identified the HIPPO signaling pathway to be positively regulated by WT KRAS in PDAC cells. In accordance with this observation, PDAC cells with LOH at KRAS exhibited increased nuclear localization and activation of transcriptional co-activator YAP1. Mechanistically, we discovered that WT KRAS expression sequestered YAP1 from the nucleus, through enhanced 14-3-3zeta interaction with phosphorylated YAP1 at S127. Consistently, expression of a constitutively-active YAP1 mutant in PDAC cells bypassed the growth inhibitory effects of WT KRAS. In patient samples, we found that the YAP1-activation genes were significantly upregulated in tumors with LOH at KRAS, and YAP1 nuclear localization predicted poor survival for PDAC patients. Collectively, our results reveal that the WT allelic loss leads to functional activation of YAP1 and enhanced tumor malignancy, which explains the selection advantage of the tumor cells with LOH at KRAS during pancreatic cancer clonal evolution and progression to metastasis, and should be taken into consideration in future therapeutic strategies targeting KRAS.

Smad4 loss synergizes with TGFα overexpression in promoting pancreatic metaplasia, PanIN development, and fibrosis.

AIMS: While overexpression of TGFα has been reported in human pancreatic ductal adenocarcinoma (PDAC), mice with overexpressed TGFα develop premalignant pancreatic acinar-to-ductal metaplasia (ADM) but not PDAC. TGF-ß signaling pathway is pivotal to the development of PDAC and tissue fibrosis. Here we sought to investigate the interplay between TGFα and TGF-ß signaling in pancreatic tumorigenesis and fibrosis, namely via Smad4 inactivation. METHODS: The MT-TGFα mouse was crossed with a new Smad4 conditional knock-out mouse (Smad4flox/flox;p48-Cre or S4) to generate Smad4flox/flox;MT-TGFα;p48-Cre (STP). After TGFα overexpression was induced with zinc sulfate water for eight months, the pancreata of the STP, MT-TGFα, and S4 mice were examined for tumor development and fibrotic responses. PanIN lesions and number of ducts were counted, and proliferation was measured by Ki67 immunohistochemistry (IHC). Qualitative analysis of fibrosis was analyzed by Trichrome Masson and Sirius Red staining, while vimentin was used for quantification. Expression analyses of fibrosis, pancreatitis, or desmoplasia associated markers (α-SMA, Shh, COX-2, Muc6, Col1a1, and Ctgf) were performed by IHC and/or qRT-PCR. RESULTS: Our STP mice exhibited advanced ADM, increased fibrosis, increased numbers of PanIN lesions, overexpression of chronic pancreatitis-related marker Muc6, and elevated expression of desmoplasia-associated marker Col1A1, compared to the MT-TGFα mice. The inactivation of Smad4 in the exocrine compartment was responsible for both the enhanced PanIN formation and fibrosis in the pancreas. The phenotype of the STP mice represents a transient state from ADMs to PanINs, closely mimicking the interface area seen in human chronic pancreatitis associated with PDAC. CONCLUSION: We have documented a novel mouse model, the STP mice, which displayed histologic presentations reminiscent to those of human chronic pancreatitis with signs of early tumorigenesis. The STP mice could be a suitable animal model for interrogating the transition of chronic pancreatitis to pancreatic cancer.