from rsv oncogene src to yap oncogene of the hippo tumor ...€¦ · from rsv oncogene src to yap...
TRANSCRIPT
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From RSV Oncogene Src to YAP
Oncogene of the Hippo Tumor
Suppressor Pathway
4th Singapore Sarcoma Meeting Academia, SGH, Singapore
November 6th, 2016
Marius Sudol, Ph.D.
Thanks to Richard Quek, Mark Puhaindran,
Amos Loh and Kenneth Chang
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Peyton Rous at The Rockefeller Institute of Medical Research. in 1911 Rous reported on a virus-induced sarcomas in chickens
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One of the seminal experiments in biology, which led to identification of the first oncogene, Src
Rous P. (1911). A Sarcoma of the Fowl Transmissible by an
Agent Separable from the Tumor Cells. J Exp Med 13: 397–411.
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SH3 KINASE SH2
Modular and X-Ray Structure of Src - CLOSED CONFORMATION
M
SRC
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Normal Transformed
Light
Microscopy
Scanning
Microscopy
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Reiko Imai, Toru Akiyama, Akira Kawai
Hidesaburao Hanafusa The Rockefeller University
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WW TAD YAP (1)
WW WW TAD YAP (2)
WW domain was identified
as a differentially spliced-in coding exon
in cDNA of Yes- and Src-associated protein
SH3-BM
ASGPLPDGWEQAMTQDGEIYYINHKNKTTSWLDPRLDPR
Sudol, M. (1994) Oncogene 9, 2145-2152.; Bork, P., & Sudol, M. (1994) TiBS. 19, 531-533; Chen H.I., & Sudol, M. (1995) Proc. Natl. Acad. Sci. USA. 92,
7819-7823.
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W
NH2
COOH
P
Y P
P
S
Y
V WW domain is the smallest among protein modules
X-Ray Structure of WW Domain-Ligand Complex
Macias, M.J., et al., (1996). Nature, 382, 646-649 Huang, X., et al., (2000) Nature Struct. Biol. 7, 634-638.
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SH3 WW
pYxx PxxP PPxY
SH2
WW domain complexes share binding modes with those of SH3 and SH2 complexes
Chen H.I. & Sudol, M. (1995) Proc. Natl. Acad. Sci. USA. 92, 7819-7823 Sudol, M & Hunter, T. (2000) Cell 103, 1001-1004
PPxpY
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WW
WW WW WW WW
WW
WW WW
WW DOMAIN IS PRESENT IN ADAPTORS ENZYMES AND REGULATORS OF TRANSCRIPTION
ABD SPECTRIN Repeats
HECT C2
ISOMERASE
TAD
DR ER
Dystrophin
Nedd-4
PQBP1 Pin1
YAP 1-1
WW WW TAD YAP 1-2
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Hippo-YAP signaling pathway
– Loss of function
increased growth
• Well conserved
• Two effectors
– Paralogous proteins
– Yes-associated
protein (YAP)
– Transcriptional
coactivator with PDZ-
binding motif (TAZ)
• Tumor suppressor signaling pathway
• Originally identified in Drosophila
http://www.mechanobio.info/topics/mechanosignaling/signaling-pathways/hippo-signaling/
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Hippo-YAP signaling pathway
– Loss of function
increased growth
• Well conserved
• Two effectors
– Paralogous proteins
– Yes-associated
protein (YAP)
– Transcriptional
coactivator with PDZ-
binding motif (TAZ)
• Tumor suppressor signaling pathway
• Originally identified in Drosophila
http://www.mechanobio.info/topics/mechanosignaling/signaling-pathways/hippo-signaling/
W = WW domain
W W
W
W
W
W
W
W
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HIPPO-YAP is a main regulatory pathway for tissue growth and organ development
YAP-8
weeks
YAP-3
months
YAP Normal YAP Normal
Dong & DJ Pan, Cell, (2007) 122, 421-434
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Michael Sheetz
MBI, Singapore
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1. Dense vs. sparse cultured cells (Zhao et al. 2007)
1. Small vs. large surface (Wada et al. 2011)
1. Soft vs. stiff substrate (Dupont et al. 2011)
1. Stretching contact inhibited
cells (Aragona et al. 2013, Yidan et al.
2015)
Hippo-YAP signaling is regulated by
mechanical cues
http://www.mechanobio.info/topics/mechanosignaling/signaling-pathways/hippo-signaling/
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Mechanical stretch of MKN28 cells causes
fast translocation of YAP effector
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Verification of CRISPR-Cas9 KO
of YAP in MKN28 cells
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YAP- KO cells show prominent stress fibers
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F-Actin is increased in YAP KO cells
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YAP KO cells express less ARHGAP29, a suppressor of RhoA
Qiao, Y., and Sudol, M. – unpublished
Zhang, H., et al., (2009) J. Biol. Chem. 284, 13355-13362
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***
YAP-null cells express less ARHGAP29, a suppressor of RhoA. RNA was harvested from wild-type, YAP-null (YAP K/O) and YAP-rescue (YAP K/O+Flag-YAP) cells and reverse-transcribed into cDNA before real-time PCR analysis (n=4).
Promoter of ARHGAP29
Chromatin Immuno-Precipitation
EXON1 EXON2
-1356 -1351 Locus #1
-536 -531 Locus #2
GGAATT GGAATT
+1
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YAP KO cells have more active RhoA, leading to increased phosphorylation of Cofilin (Ser3)
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YAP KO cells have more active RhoA, leading to increased phosphorylation of Cofilin (Ser3)
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YAP KO cells have more active RhoA, leading to increased phosphorylation of Cofilin (Ser3)
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Atomic Force Microscopy was used to measure stiffness of adherent YAP KO MKN28 cells
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**
Young’s Modulus of single adherent YAP KO MKN28 cells and YAP rescued cells
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Aspiration flow rate (ml/hr) Volume aspirated ∆V(ml) Corresponding aspiration pressure (Pa) Pipette Radius (μm)
Wild-type YAP K/O
Micropipette aspiration assay of YAP KO in MKN28 cells
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CONTROL YAP+/+
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KNOCK-OUT YAP-/-
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YAP in Cancers Potent oncogene in HCC, Dj Pan, JHSM, Baltimore, USA PTPN14–YAP WW domain partner is mutated in relapsed neuroblastoma, Angelika Egger, Berlin-Charite Germany YAP is nuclear in pediatric liver cancers, Kashayar Vakili, Boston Children Hospital, USA YAP-TEF3 fusion in 10% of Epithelioid Hemangio- Endothelioms, Brian Rubin, Cleveland Clinic, Ohio, USA Activated YAP causes embryonic rhabdomyosarcoma in mice, Fernando Camargo, Boston Children Hospital, USA
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Persistent YAP hyperactivity in activated but
not quiescent satellite cells causes tumours in
vivo
Pax7-YAP1 S127A
Yap P S127A
Yap P S127A
Pax7, Myf5, MyoD-YAP1 S127A + activated satellite cells
No tumourigenesis apart from small tumour at injection site
Persistent Yap hyperactivity & activated satellite cells tumours in 100% of mice.
Tremblay et al Cancer Cell (2010)
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YAP knock down in ERMS (RD) cells reduces
transformation and xenotransplant size
A B
Tremblay et al Cancer Cell (2010)
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Src is the first oncogene, it causes spindle cell sarcoma in birds
YAP oncogene is amplified in liver cancer and may paly a role in
embryonic rhabdomyosarcoma
Cells without YAP proto-oncogene are more rigid than control cells
Changes in ARHGAP29 impinge on RhoA-LIMK1-Coflin to stabilize
F-actin
Mention a new modality for treating cancers
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My Laboratory
Yiting QIAO Ashwini
KARANATH
Megan FINCH EDMONDSON
Bowen ZHU Pearlyn TOH Max HOSELBARTH
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Acknowledgements 1
Yiting QIAO Megan FINCH EDMONDSON Ashwini KARANATH IMCB - Collaboratios and Advice: Walter Hunziker, Wanjin Hong, Ed Manser, Phil Kaldis, Uttam Surana, Haiwei Song, Vinay Tregaonkar, Nic Plachta
Collaborators: YAP-RIGIDITY - Himanshu Singh, Bena Lim and Chwee Teck Lim EBOLA - Prakash Amurugam, Fan Hao, Max Hoeslebarth, Gautam Sethi Ron Harty, Mark Bedford, Dev Sidhu, Amjad Farooq GIH-Francesco Gervasio, Yan Jie, Gene Yeo
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Alberta Innovates –Health Solutions
Acknowledgements 2
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?
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