Wild-type KRAS Allele Exerts Its Tumor-suppressive Function Through Cytoplasmic Retention Of YAP1 In Pancreatic Ductal Adenocarcinoma

Background:KRAS is the most frequently mutated oncogene(~95%)in pancreatic ductal adenocarcinoma(PDAC)and it has been shown to be essential for pancreatic tumor initiation and maintenance in both humans and mice.We have previously reported that the wild-type Kras allele was selectively lost in both primary pancreatic tumors and metastases developed in a mouse model of PDAC,and the frequency of the wild-type loss increased from primary tumors to metastases in this mouse model and human pancreatic cancer cells.However,the mechanisms underlying the loss of heterozygous at KRAS locus and the function of wild-type KRAS allele in PDAC remain unclear.Objectives:1.To understand the function of wild-type KRAS allele in PDAC.2.To understand the mechanisms of signaling pathways underlying the regulation of wild-type KRAS.Methods:1.Construction of the inducible wild-type KRAS expression pancreatic cancer cell-line in MIA PacA-2 and AsPC-1 cells,which have undergone LOH(loss of heterozygosity)at KRAS locus.2.Examine the phenotype changes after restoring the wild-type KRAS allele through MTT assay,colony formation assay,and scratch assay in vitro.3.Xenograft model were used to observe the impact of restoration of wild-type KRAS on tumorigenesis and tumor growth in vivo.4.Examine the canonical downstream signaling pathways of KRAS after restoration of wild-type KRAS by Western Blot.5.Immunofluorescent,immunocytochemistry labeling were used to verify the translocation of YAP1 after restoration of wild-type KRAS.6.Western blot analysis to detect the YAP1 translocation level after restoration of wild-type KRAS by separately extract protein lysis from cytoplasm and nucleus.7.Dual-Glo luciferase assay were used to access to endogenous YAP1-TEADs binding level in situations with/without the restoration of wild-type KRAS.8.RNA sequencing was used to identify the differentially transcription genes after the restoration of wild-type KRAS.9.Differentially expressed genes were overlapped with YAP1-TEADs downstream target genes.Heatmap and cluster diagrams were generated to show the intersection of statistically differentially expressed genes with YAP1-TEADs targets genes.Some of these intersected genes were further validated through qRT-PCR.10.Cytoplasmic/nuclear proteomic analysis to verify protein level change within the restoration of wild-type KRAS allele.11.Western Blot analysis to show the YAP1,p-YAP1 protein level changes within the restoration of wild-type KRAS.Immunoprecipitation was performed to show the endogenous YAP1 and 14-3-3zeta interaction level changes within the restoration of wild-type KRAS.12.Mutant-type YAP1(activated YAP1)as well as wild-type YAP1 were transiently transfected to iWT KRAS stable transfected cell-lines to observe the phenotype changes such as proliferation activity and motility changes.12.Immunohistochemistry staining were utilized to label YAP1 in human post-surgical PDAC samples on Tissue Microarray(TMA).Clinicopathological and prognostic significance of YAP 1 were analyzed.Results:1.Restored wild-type KRAS expressing pancreatic cancer cell-lines were featured with inhibited malignancies.2.Expression of wild-type KRAS significantly decreased the tumor volume in xenograft model.3.Induction of wild-type KRAS expression caused Yapl cytoplasmic retention in iWT KRAS MIA PaCa-2 and iWT KRAS AsPC-1 cell-lines.4.RNA sequencing identified 5276 differentially expressed genes within the restoration of wild-type KRAS.45.74%(172/376)YAP1-TEADs downstream target genes were overlapped with the RNA Seq data.Overlapped genes were further validated by qRT-PCR,by restoration of the wild-type KRAS,YAP1-TEADs downstream target genes were transcriptionally downregulated.5.By restoration of the wild-type KRAS,phosphorylation of YAP 1 at S127 site was increased.Immunoprecipitation showed that the interactions between p-YAP1 and 14-3-3zeta increased in the cytoplasm.6.Overexpressed mutant-type YAP1 completely off-set the wild-type KRAS induced tumor-suppressive function while overexpressed wild-type YAP 1 didn't fully antagonize the wild-type KRAS inhibitory effect in proliferation and motility.7.IHC labeling of YAP1 on TMAs showed the overexpression of YAP1,and overexpressed YAP1 was significantly associated with poor prognosis in post-surgical PDAC patients.Conclusions:1.Wild-type KRAS allele presented tumor-suppressive function counterpart with mutant KRAS in human PD AC cell-lines.2.Wild-type KRAS sequestered YAP1 from nuclear translocation through increasing YAP1 phosphorylation at S127 and binding with 14-3-3zeta,and YAP1-TEADs downstream targets were transcriptionally downregulated.3.YAP1 was overexpressed in PD AC and associated with poor prognosis.