The Hippo Pathway. Hippo pathway Signaling components Over-expression & knockout studies Role in...

The Hippo Pathway

Hippo pathway

Signaling components

Over-expression & knockout studies

Role in cancer

Regulation

A good review:Pan D.Hippo signaling in organ size control.Genes Dev. 2007 Apr 15;21(8):886-97.

Rescue mammalianHippo

Wu S, Huang J, Dong J, Pan D.Cell. 2003 Aug 22;114(4):445-56.

Harvey KF, Pfleger CM, Hariharan IK.Cell. 2003 Aug 22;114(4):457-67.

Hippo pathway

Described about 5 years ago in flies- various mammalian components identified previously

Regulates growth, differentiation, cell death

Kinase cascade regulates P-state of transcription factor(s)- negative regulation

Hippo & Wartskinases

Salvador & Matsco-factors

Yorkie

Focus on Transcription Factor

Transcriptionally activates various growth & anti-apoptosis genes

Mammalian homolog Yap- identified P-site Ser127- S127A no longer sequestered by 14-3-3- constitutively active

Dong J, et al.Cell. 2007 Sep 21;130(6):1120-33.

(B and C) YAP overexpression drives cell proliferation. Control (B) and ApoE/rtTA-YAP (C) livers after 1 week of Dox exposure were analyzed for BrdU incorporation (red), counterstained with DAPI (blue).(D–H) YAP overexpression confers potent resistance to apoptosis. Control and ApoE/rtTA-YAP littermates were kept on Dox for 1 week, injected with Jo-2, and analyzed 3 hr postinjection by H&E staining (D and E), TUNEL (F and G), and western blotting for cell death markers (H). Note the widespread hemorrhage (asterisk) and apoptotic nuclei (arrowhead) in the control (D), but not the transgenic livers (E). Also note the extensive TUNEL staining in the control (F), but not the transgenic livers (G). In (H), cleavage of caspase-3 (Casp3) and PARP was detected in the control, but not the transgenic, livers (arrowhead marks the cleaved PARP product). Three animals were analyzed for each genotype.(I) RNAi knockdown of BIRC5/survivin reduces the transforming activity of YAP. Left: western blots showing increased BIRC5/survivin expression in YAP-HPNE cells compared to mock transfected cells, and the knockdown of BIRC5/survivin expression in YAP-HPNE cells stably expressing BIRC5/survivin shRNA. Right: soft agar assay showing anchorage-independent growth of YAP-HPNE cells, a feature that is not observed in the mock-transfected HPNE cells. Note that YAP-HPNE cells stably expressing BIRC5/survivin shRNA formed reduced numbers of colonies.

1 week Dox

BrdU

Jo2 Ab

H&E Tunel


(A) Liver from an ApoE/rtTA-YAP mouse raised on Dox for 8 weeks, starting at 3 weeks after birth. Note the presence of discrete nodules scattered throughout the liver (arrowheads).

(B) Liver from an ApoE/rtTA-YAP mouse raised on Dox for 3 months, starting at birth. Note the widespread development of HCC throughout the liver.

(C–E) Histolopathologic examination of murine liver nodules reveals characteristics of hepatocellular carcinoma. Mice were fed Dox-water as in (A). (C) shows cellular pleiomorphism of YAP-induced HCC, with a large cell (arrow) surrounded by smaller cells. (D) shows loss of cytoplasmic staining (arrows), or the so called “clear cell change.” (E) shows expanded hepatic plates. A reticulin stain highlights the edges of the hepatic plates (indicated by parallel lines), which are wider in HCC than the typical 1 to 2 cells in a nonneoplastic liver.

(F) Survival curves of control (Non-Tg) and ApoE/rtTA-YAP (Tg) littermates raised on Dox, starting at 3 or 8 weeks of age as indicated.

8 weeks dox(at 3 weeks)

3 months dox(at birth)


Yap over-expression beyond liver

Thijn Brummelkamp

(A) Schematic representation of liver-specific doxycycline-inducible YAP1 allele. The tetracycline transactivator (tTA) expression is driven by the liver activator protein (LAP) promoter. A tetracycline regulatable allele of YAP1-S127A was integrated downstream of the Col1A1 locus.

(B) Gross morphology of the liver in control and YAP1 transgenic mice induced for 5 weeks (top) and mice induced for 5 weeks followed by reversal of YAP1 activation for another 5 weeks (bottom).

5 weeks

5 weeks

Camargo FD, et al.Curr Biol. 2007 Dec 4;17(23):2054-60

5 days post-induction

Rosa26 rtTA- ubiquitous

loss of differentiatedcells

periodic acid-Schiff-lose gobletcells

Hes - only inprogenitors


Relevant role in human cancer?

Screen CGH - hits on chromosome 9

systenic to human 11q22 amplicon- common in diverse human tumors

Brca flox/-, p53 +/-, MMTC-Cre

1 - Met2 - Yap3 - many genes

Overholtzer M, et al.Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12405-10.

Overholtzer M, et al.Proc Natl Acad Sci U S A. 2006 Aug 15;103(33):12405-10.

Zhang J, Smolen GA, Haber DA.Cancer Res. 2008 Apr 15;68(8):2789-94.

WW45 epidermis e17.5 Lee JH, et al.EMBO J. 2008 Apr 23;27(8):1231-42.

WW45 intestinal epithelium Lee JH, et al.EMBO J. 2008 Apr 23;27(8):1231-42.

Lee JH, et al.EMBO J. 2008 Apr 23;27(8):1231-42.

Hippo pathway

Regulated by neighboring cells - mediates contact inhibition

Inputs still obscure

Other means of regulation…

Fatproto-cadherin

Expanded&

MerlinFERM domain

mammalian homologsfor all components

Cell Density Regulation

Zhao B, et al.Genes Dev. 2007 Nov 1;21(21):2747-61.

Merlin

Zhao B, et al.Genes Dev. 2007 Nov 1;21(21):2747-61.

Katagiri, et al.Nat Immunol. 2006 Sep;7(9):919-28.

ICAM coated surface, CXCL12 stimulation

Katagiri, et al.Nat Immunol. 2006 Sep;7(9):919-28.

Matallanas, et al.Mol Cell. 2007 Sep 21;27(6):962-75.

Lei, et al.Mol Cell Biol. 2008 Apr;28(7):2426-36.

The Hippo Pathway. Hippo pathway Signaling components Over-expression & knockout studies Role in...

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