Xin Hong1,2,*, Hung Thanh Nguyen3,*, Qingfeng Chen1,4, Rui Zhang5, Zandra Hagman6, P. Mathijs Voorhoeve3,# & Stephen M. Cohen1,2,#
1: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore, 138673
2: Department of Biological Sciences, National University of Singapore, 117543
3: Duke-NUS Graduate Medical School, 8 College Rd, Singapore,169857
4: Singapore-MIT Alliance for Research and Technology
5: Department of Genetics, Stanford University
6: Department of Cellular and Molecular Medicine, University of Copenhagen
*# Equal contribution
Published online in EMBOJ on 5 September 2014.
Ras antagonises Hippo tumor suppressor activity by direct regulation of YAP-stability. Ras controls SOCS-protein expression, channeling YAP into ubiquitin-dependent degradation.
- YAP expression is sufficient to bypass the role of oncogenic Ras in transformation of primary human cells, and acts via a feedback loop involving Amphiregulin and EGFR.
- Ras acts via downregulation of SOCS5/6 to reduce YAP protein turnover.
- By reducing YAP turnover, the Ras pathway acts in opposition to the Hippo tumor suppressor pathway, which promotes YAP turnover.
- The levels of SOCS6 mRNA and the YAP target AREG were inversely correlated in colorectal cancer with a wild-type K-Ras gene, but not in K-Ras mutant cancers.
The Ras/SOCS/YAP/AREG feedback loop.
A Upregulation of AREG and survivin in BJT/p53KD/p16KD/ST cells transduced to express RasV12, two independent shRNAs targeting SOCS5/6, or YAPS127A. RNA was isolated from a subset of the cells prepared for the colony formation assay in Fig 3A. The immunoblot control for SOCS6 depletion is shown in Fig 3A. Transcript levels were measured by quantitative PCR. Data: average of three independent experiments ± SD. GAPDH was used as a control.
B Immunoblot showing the effect of YAP depletion on AREG protein levels. BJT/p53KD/p16KD/ST cells were transduced to express two independent shRNAs targeting YAP or TEAD-DN. Blots were probed with antibody to YAP and AREG. Actin served as a loading control.
C Immunoblot showing the effect of AREG depletion on EGFR activity in BJT/p53KD/p16KD/ST/Rasv12 cells. Cells were transduced to express two independent shRNAs targeting AREG. Blots were probed with antibody to Y1068 phosphorylated EGFR to monitor EGFR activity, and with antibody to EGFR and AREG. Actin served as a loading control.
D Effect of AREG depletion on soft agar colony formation by fully transformation-competent BJT/p53KD/p16KD/S/RasV12 cells. Cells were transduced to express two independent shRNAs targeting AREG or with empty vector as a control. Lower left: average colony number from three independent experiments ± SD shown as % of vector control. P < 0.05 comparing each siRNA to control. Lower right: AREG mRNA levels were measured by quantitative PCR to monitor shRNA efficacy.
E Effect of AREG depletion on soft agar colony formation by YAP1S127A-expressing cells. BJT/p53KD/p16KD/ST/ER-Rasv12 cells were transduced to express YAP1S127A and with shRNA targeting AREG or with empty vector as a control. Below: average colony number from three independent experiments ± SD, normalized to YAP1S127A-expressing cells. P < 0.05 comparing each siRNA to it respective control.
Source data are available online for this figure.
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