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Comparative Study
, 70 (21), 8517-25

Hippo Pathway Effector Yap Is an Ovarian Cancer Oncogene

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Comparative Study

Hippo Pathway Effector Yap Is an Ovarian Cancer Oncogene

Chad A Hall et al. Cancer Res.

Abstract

The Hippo pathway regulates organ size and tumorigenesis in Drosophila and mammals and is altered in a variety of human cancers, yet it remains unclear if the Hippo pathway is of prognostic significance to cancer patients. Here we show that the key targets of Hippo signaling, transcriptional coactivators Yki and Yap, play a conserved role in promoting ovarian cancer in flies and humans, respectively. Whereas studies linking Yap to cancer in other tissues have focused on overall Yap levels, we show for the first time that subcellular levels of Yap show an exceptionally strong association with poor patient survival. Specifically, high levels of nuclear Yap (nYap), or low levels of cytoplasmic phosphorylated Yap (cpYap), associated with poor survival from ovarian cancer. Patients with both high nYap and low cpYap had ∼50% lower 5-year survival, and this combination is an independent prognostic marker for survival, with an exceptionally high hazard ratio of 7.8. We find that Yap2 is the predominantly expressed Yap isoform in both the ovarian surface epithelium (OSE) and epithelial ovarian cancers. Overexpression of Yap2 and phosphorylation-defective Yap2-5SA in immortalized OSE cells resulted in increased cell proliferation, resistance to cisplatin-induced apoptosis, faster cell migration, and anchorage-independent growth, whereas Yap knockdown resulted in increased sensitivity to cisplatin-induced apoptosis. Findings argue that the Hippo signaling pathway defines an important pathway in progression of ovarian cancer.

Figures

Figure 1
Figure 1
Drosophila Yki-S168A or human Yap-S127A overexpression induces tumorigenesis in the Drosophila ovary. A. Confocal section through wildtype Drosophila egg chamber (upper) stained with propidium iodide to reveal nuclei and with phalloidin to reveal cell cortices. Follicle cells (small nuclei, arrow heads) form a monolayer epithelium around the germ cells (large nuclei, arrows). In egg chambers in which all follicle cells overexpress fly Yki-S168A (middle) or human Yap-S127A (lower) the follicular epithelium becomes multilayered and has supernumerary follicle cells.
Figure 2
Figure 2
Yap and pYap associate with disease-specific survival. A. OSE (left) and serous ovarian tumor (right) stained with Haematoxylin (blue) to reveal cell nuclei, and for Yap (top; brown in OSE, red in tumor) or pYap (bottom, brown). In OSE and tumors, Yap localizes primarily to the nucleus (arrows) and pYap localizes primarily to the cytoplasm (arrowheads). B. Kaplan-Meier estimates of disease-specific survival (truncated at 5 years) of patients with high verses low nYap (left), high versus low cpYap (middle), and nY/cpY Category 3 (high nYap and low cpYap) vs other nY/cpY Categories (0, 1, 2)(right)(see table 1). P-values from Log-rank tests are shown.
Figure 3
Figure 3
Yap overexpression promotes proliferation and resistance to apoptosis. A. Western analysis (left) of Yap and pYap from 80T cells expressing nontargeting shRNA, shRNAs targeting Yap (shYap7,8,9), empty vector (overexpression control), Yap2-myc, and Yap2-5SA-myc. The blot on the right was exposed for shorter time. Immunofluorescence staining (right) for myc and DNA in 80T cells expressing Yap2-myc and Yap2-5SA-myc. For clarity, the fields were selected to contain fewer overexpressing cells than typically observed. A higher portion of overexpressed Yap2-5SA localizes to the nucleus (arrowheads) compared to overexpression of Yap2 (arrows). B. Quantification of cell number (left) in Yap knockdown and overexpressing cells after six days of growth. Control and Yap2-5SA cells stained for DNA (middle) to reveal condensed DNA of mitotic cells (asterics). Quantification of mitotic index (right). C. Light micrographs of cells treated with 30 μM cisplatin for 48 hours (left) to show morphology of adherent cells (arrows) and nonadherent cells (arrowheads). Western analysis (right) of full length Caspase-8 (C8-FL), cleaved Caspase-8 (cC8), and cleaved Caspase-3 (cC3) from cells treated with 30 μM cisplatin for 48 hours.*p<.05, **p<.01, and ***p<.001.
Figure 4
Figure 4
Yap overexpression reduces contact inhibition and promotes wound closure and anchorage independent growth. A. Micrographs of confluent cultures stained for DNA to reveal cell density. Greater fluorescence intensity reveals multilayering of cells. B. Representative bright field micrographs (left) of control and Yap2-5SA cells 0.5 and 10 hours after wounding. Quantification of wound closure (right) after 10 hours. C. Representative micrograph of cell colonies visualized with MTT (left) after 14 days of growth in soft agar. Quantification of the number of colonies per well (right).*p<.05 and ***p<.001.

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