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 Apr 15;21(8):

Rescue mammalian Hippo Wu S, Huang J, Dong J, Pan D. Cell Aug 22;114(4): Harvey KF, Pfleger CM, Hariharan IK. Cell Aug 22;114(4):

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 & Warts kinases Salvador & Mats co-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 constitutively active

Dong J, et al. Cell Sep 21;130(6):

Dong J, et al. Cell Sep 21;130(6):

Dong J, et al. Cell Sep 21;130(6):

(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 Dong J, et al. Cell Sep 21;130(6):

(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) Dong J, et al. Cell Sep 21;130(6):

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

5 days post-induction Rosa26 rtTA - ubiquitous loss of differentiated cells periodic acid-Schiff- lose goblet cells Hes - only in progenitors Camargo FD, et al. Curr Biol Dec 4;17(23):

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

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 - Met 2 - Yap 3 - many genes Overholtzer M, et al. Proc Natl Acad Sci U S A Aug 15;103(33):

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

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

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

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

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

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

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

Hippo pathway Regulated by neighboring cells - mediates contact inhibition Inputs still obscure Other means of regulation…

Fat proto-cadherin Expanded & Merlin FERM domain mammalian homologs for all components

Cell Density Regulation Zhao B, et al. Genes Dev Nov 1;21(21):

Merlin Zhao B, et al. Genes Dev Nov 1;21(21):

Katagiri, et al. Nat Immunol Sep;7(9):

Katagiri, et al. Nat Immunol Sep;7(9):

Katagiri, et al. Nat Immunol Sep;7(9):

ICAM coated surface, CXCL12 stimulation Katagiri, et al. Nat Immunol Sep;7(9):

ICAM coated surface, CXCL12 stimulation Katagiri, et al. Nat Immunol Sep;7(9):

Matallanas, et al. Mol Cell Sep 21;27(6):

Lei, et al. Mol Cell Biol Apr;28(7):

Lei, et al. Mol Cell Biol Apr;28(7):

Lei, et al. Mol Cell Biol Apr;28(7):