KRAS and YAP1 converge to regulate EMT and tumor survival

Cell (Impact Factor: 33.12). 06/2014; 158(1). DOI: 10.1016/j.cell.2014.06.004
Source: PubMed

ABSTRACT Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.

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    • "nuclear enrichment in Er/Er keratinocytes led to elevated expression of its target genes, we performed qPCR to assay the mRNA levels of the genes Cyr61, Ctgf, Zeb1, and Snai2, which were previously shown to be Yap1 transcriptional targets (Heallen et al., 2011; Shao et al., 2014). They exhibited reduced expression in differentiating WT keratinocytes (Fig. 3a), which was correlated with the Yap1 cytoplasmic retention pattern shown above. "
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    ABSTRACT: The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting DMBA/TPA induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In the present study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in DMBA/TPA-induced tumors from Er/+ skin. Furthermore, shRNA knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length WT or the mutant form found in Er/Er mice. However Er 14-3-3σ does not interact with Yap1, as demonstrated by co-immunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in Er/Er epidermis.Journal of Investigative Dermatology accepted article preview online, 10 February 2015. doi:10.1038/jid.2015.42.
    Journal of Investigative Dermatology 02/2015; 135(6). DOI:10.1038/jid.2015.42 · 6.37 Impact Factor
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    • "In agreement with this supposition, Shao et al. (2014) identified Yap1 in a gain-of-function screen to identify genes that can substitute for Ras signaling in KRAS-dependent human cancer cells. Consistent with the pleiotropic effects of Yap1, both studies converge on overlapping networks, such as ATF and E2Fs, and diverge on distinct transcriptional programs , such as Tead2 (this study) and Fos (Shao et al., 2014). Our convergent and contrasting findings are consistent with the established fact that the Yap1-mediated gene expression program is largely dictated by the cellular context and its interacting transcription factors (Pobbati and Hong, 2013; Zhao et al., 2011). "
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    ABSTRACT: Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.
    Cell 06/2014; 158(1). DOI:10.1016/j.cell.2014.06.003 · 33.12 Impact Factor
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    ABSTRACT: It is of great therapeutic importance to understand why tumors relapse after the failure of therapies targeting oncogenes to which cancer cells are addicted. In this issue, Kapoor et al. and Shao et al. identify the transcriptional coactivator YAP1 as a central driver of compensation for the loss of K-Ras signaling in K-Ras-dependent cancers.
    Cell 07/2014; 158(1):11-2. DOI:10.1016/j.cell.2014.06.021 · 33.12 Impact Factor
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Questions & Answers about this publication

  • Andrei Nikonov added an answer in Cell Signaling:
    Does anyone has tried a good antibody for YAP in mouse cell lines samples?

    I have tried the YAP antibody from Cell Signaling, but I am using it 1:500 and I get a very weak signal. 

    Andrei Nikonov · University of Tartu

    According to the recent paper on YAP1 and KRAS pathways convergence, the antibody you mentioned is used either at a dilution 1:50 to 1:10 (in co-immunoprecipitation). So if you are doing IP, just increase the Ab concentration.

    In the same paper ChIP is also performed but with antibody from Santa.

    If you are doing western blot, then simply increase the concentration of primary-YAP Ab and after incubation save it and conserve by adding sodium azide to 0.04% storing it at +4C. It should be stable for more than a month. I typically performed some 7-12 westerns with such a solution (add azide only once, after first incubation).

    Increasing the concentration of Ab often also means decreasing the volume of your solution. You can easily do a western in 5-10 ml final volume on orbital shaker inside a 50-ml falcon (hence avoiding evaporation). However, to avoid background perform blocking and all washes in ordinary vessels with plenty on solution.