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

Peyton Rous at The Rockefeller Institute of Medical Research. in 1911 Rous reported on a virus-induced sarcomas in chickens

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.

SH3 KINASE SH2

Modular and X-Ray Structure of Src - CLOSED CONFORMATION

M

SRC

Normal Transformed

Light

Microscopy

Scanning

Microscopy

Reiko Imai, Toru Akiyama, Akira Kawai

Hidesaburao Hanafusa The Rockefeller University

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.

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.

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

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

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/

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

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

Michael Sheetz

MBI, Singapore

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/

Mechanical stretch of MKN28 cells causes

fast translocation of YAP effector

Verification of CRISPR-Cas9 KO

of YAP in MKN28 cells

YAP- KO cells show prominent stress fibers

F-Actin is increased in YAP KO cells

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

***

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

YAP KO cells have more active RhoA, leading to increased phosphorylation of Cofilin (Ser3)

YAP KO cells have more active RhoA, leading to increased phosphorylation of Cofilin (Ser3)

YAP KO cells have more active RhoA, leading to increased phosphorylation of Cofilin (Ser3)

Atomic Force Microscopy was used to measure stiffness of adherent YAP KO MKN28 cells

**

Young’s Modulus of single adherent YAP KO MKN28 cells and YAP rescued cells

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

CONTROL YAP+/+

KNOCK-OUT YAP-/-

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

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)

YAP knock down in ERMS (RD) cells reduces

transformation and xenotransplant size

A B

Tremblay et al Cancer Cell (2010)

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

My Laboratory

Yiting QIAO Ashwini

KARANATH

Megan FINCH EDMONDSON

Bowen ZHU Pearlyn TOH Max HOSELBARTH

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

Alberta Innovates –Health Solutions

Acknowledgements 2

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