Hippo pathway在許多生物性調控上為一重要角色，包含細胞生長、細胞凋亡和器官發育。Yes-associated protein 1 (YAP)功能屬於轉錄輔因子，可決定hippo路徑的訊息傳遞及功能。YAP 於腫瘤生中的角色可成為癌蛋白或在細胞去氧核醣核甘酸受損反應時做為腫瘤抑制因子。 YAP於細胞中確切的角色取決於和不同轉錄因子的結合。雖然YAP 和腫瘤抑制因子RUNX3於試管中的交互作用以被報導,但此一交互作用於細胞內的功能性調控仍然未知。 在我們的先前研究中顯示RUNX3可藉由轉錄調控，於胃癌細胞中抑制Akt的基因表現。為了探討YAP 和RUNX3的交互作用是否參與Akt 的表現調控,我們於細胞株偵測RUNX3和YAP的蛋白表現並發現RUNX3和YAP普遍表現於於非小細胞肺癌細胞中。利用穀胱胺酸沉降試驗和免疫共沉澱實驗中確認RUNX3 和YAP透過脯氨酸motif和雙色胺酸區域於試管中和細胞中交互作用。將RUNX3的脯氨酸motif 點突變則中斷和 YAP的交互作用。RUNX3和YAP於細胞內的為致主要在核周邊區域,暗示著YAP可能影響RUNX3的入核作用。於非小細胞肺癌細胞中過表現 YAP 會減少Akt及其下遊β-catenin 和 Cyclin D1的蛋白表現。於YAP穩定過表現的非小細胞肺癌細胞株H1299中Akt蛋白表現同為減少。於H1299細胞中穩定減弱YAP基因表現則增加Akt, β-catenin 和Cyclin D1的蛋白表現，顯示 YAP可能為一負向調控 Akt基因表現的調節因子 我們並進行Akt基因啟動子報導試驗驗證YAP對Akt啟動子的調控。如同先前於胃癌細胞的研究，RUNX3可在H1299細胞中抑制Akt啟動子的活性.細胞中過表現 YAP則降低Akt啟動子活性， 減弱YAP基因表現則增加Akt啟動子活性。將Akt啟動子上的RUNX3結合位做突變則阻止YAP抑制Akt promoter的活性。減弱YAP基因表現可減少非小細胞肺癌細胞株的抗放敏感性。在RUNX3和YAP交互作用的研究裡提供些證據來支持YAP於去氧核醣核酸受損反應中的腫瘤抑制因子的角色。

Hippo pathway plays an essential role in many biological regulations, including cell proliferation, apoptosis and organ development. Yes-associated protein 1 (YAP) is a transcriptional cofactor which determines the signaling and function of Hippo pathway. The role of YAP in could be a oncoprotein in tumorigenesis or tumor suppressor in cell DNA damage response. The definite role of YAP in cell is dependent on the binding of different transcriptional factors. Although the binding of YAP and the tumor suppressor, RUNX3 had been reported in vitro, the functional regulation in cell is still unclear. In our unpublished study show that RUNX3 represses Akt protein expression through transcriptional regulation in gastric cancer cells. To investigate if the interaction of YAP and RUNX3 is involved in the regulation of Akt gene expression, we detect the RUNX3 and YAP protein expression in cell lines and find that RUNX3 and YAP prevalently expression in non-small cell lung cancer cell lines. The GST pull down and Co-immunoprecipitation assay are performed to confirm the interaction. The interaction of YAP and RUNX3 in vitro and vivo are dependent on their WW domain of YAP and proline-rich motif of RUNX3. Point mutation of proline-rich motif of RUNX3 disrupts the interaction with YAP. The subcellular localization of RUNX3 and YAP is around nucleus, implies the YAP could influence the nuclear localization of RUNX3. Over-expression of YAP in non-small cell lung cancer reduces Akt and downstream effectors including β-catenin and Cyclin D1 protein expression. The Akt protein expression is also down-regulated in the YAP stably over-expressed non-small cell lung cancer cell line, H1299. Stable knockdown of YAP in H1299 cells enhance Akt, β-catenin and Cyclin D1 protein expression, indicating YAP is a negative regulator to Akt gene expression probably. Akt promoter reporter assay is performed to demonstrate the regulation of YAP on the Akt promoter. As previous studies in gastric cancer cells, RUNX3 also represses Akt promoter activity in H1299 cells. Over-expression of YAP represses promoter activity and knockdown of YAP increase promoter activity. Mutation of two RUNX3-binding sites on Akt promoter abolishes the YAP-mediated repression on Akt promoter. Knockdown of YAP reduces irradiation sensitivity of no-small cell lung cancers. This study on interaction of RUNX3 and YAP may provide evidences to support the tumor suppressor role of YAP in DNA damage response.

[1] Hanahan D, Weinberg RA. The hallmarks of cancers Cell. 100(1):57-70 (2000).
[2] Hanahan D, Weinberg RA.The hallmarks of cancer: The next generation. Cell. 144(5):646-74 (2011).
[3] Harvey KF, Pfleger CM, Hariharan IK. The Drosophila Mst Ortholog, hippo, Restricts Growth and Cell Proliferation and Promotes Apoptosis Cell. 114(4):457-67 (2003).
[4] Xu T, Wang W, Zhang S, Stewart RA, Yu W. Identifying tumor suppressors in genetic mosaics: the Drosophila lats gene encodes a putative protein kinase Development. 121(4):1053-63 (1995).
[5] Pantalacci S, Tapon N, Léopold P. The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila Nat Cell Biol. 5(10):921-7 (2003).
[6] Bennett FC, Harvey KF. Fat Cadherin Modulates Organ Size in Drosophila via the Salvador/Warts/Hippo Signaling Pathway. Curr Biol. 16(21):2101-10 (2006).
[7] Justice RW, Zilian O, Woods DF, Noll M, Bryant PJ. The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation Genes Dev. 9(5):534-46 (1995).
[8] Huang J, Wu S, Barrera J, Matthews K, Pan D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila Homolog of YAP Cell. 122(3):421-34 (2005).
[9]. Zhao B, Wei X, Li W, Udan RS, Yang Q, Kim J, Xie J, Ikenoue T, Yu J, Li L, Zheng P, Ye K, Chinnaiyan A, Halder G, Lai ZC, Guan KL. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control Genes Dev. 21(21):2747-61 (2007).
[10] Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA, Gayyed MF, Anders RA, Maitra A, Pan D. Elucidation of a universal size-control mechanism in Drosophila and mammals Cell. 130(6):1120-33 (2007).
[11] Wu S, Huang J, Dong J, Pan D. hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts Cell. 114(4):445-56 (2003).
[12] Tapon N, Harvey KF, Bell DW, Wahrer DC, Schiripo TA, Haber DA, Hariharan IK. salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines. Cell. 110(4):467-78 (2002).
[13] Thompson BJ, Cohen SM. The Hippo pathway regulates the bantam microRNA to control cell proliferation and apoptosis in Drosophila Cell. 126(4):767-74 (2006).
[14] Nolo R, Morrison CM, Tao C, Zhang X, Halder G. The bantam microRNA is a target of the hippo tumor-suppressor pathway. Curr Biol. 16(19):1895-904 (2006).
[15] Cinar B, Collak FK, Lopez D, Akgul S, Mukhopadhyay NK, Kilicarslan M, Gioeli DG, Freeman MR. MST1 Is a Multifunctional Caspase-Independent Inhibitor of androgenic signaling Cancer Res. 71(12):4303-4313 (2011).
[16] Oh S, Lee D, Kim T, Kim TS, Oh HJ, Hwang CY, Kong YY, Kwon KS, Lim DS. Crucial Role for Mst1 and Mst2 Kinases in Early Embryonic Development of the Mouse Mol Cell Biol. 29(23):6309-20 (2009).
[17] Zhou D, Conrad C, Xia F, Park JS, Payer B, Yin Y, Lauwers GY, Thasler W, Lee JT, Avruch J, Bardeesy N. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene Cancer Cell. 16(5):425-38 (2009).
[18] Lee KP, Lee JH, Kim TS, Kim TH, Park HD, Byun JS, Kim MC, Jeong WI, Calvisi DF, Kim JM, Lim DS. The Hippo–Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis Proc Natl Acad Sci U S A. 107(18):8248-53 (2010).
[19] Hao Y, Chun A, Cheung K, Rashidi B, Yang X. Tumor Suppressor LATS1 Is a Negative Regulator of Oncogene YAP J Biol Chem. 283(9):5496-509 (2008).
[20] St John MA, Tao W, Fei X, Fukumoto R, Carcangiu ML, Brownstein DG, Parlow AF, McGrath J, Xu T. Mice deficient of Lats1 develop soft-tissue sarcomas, ovarian tumours and pituitary dysfunction Nat Genet. 21(2):182-6 (1999).
[21] Takahashi Y, Miyoshi Y, Takahata C, Irahara N, Taguchi T, Tamaki Y, Noguchi S. Down-regulation of LATS1 and LATS2 mRNA expression by promoter hypermethylation and its association with biologically aggressive phenotype in human breast cancers. Clin Cancer Res. 11(4):1380-5 (2005).
[22] Morin-Kensicki EM, Boone BN, Howell M, Stonebraker JR, Teed J, Alb JG, Magnuson TR, O'Neal W, Milgram SL. Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65 Mol Cell Biol. 26(1):77-87 (2006).
[23] kita R, Uchijima Y, Nishiyama K, Amano T, Chen Q, Takeuchi T, Mitani A, Nagase T, Yatomi Y, Aburatani H, Nakagawa O, Small EV, Cobo-Stark P, Igarashi P, Murakami M, Tominaga J, Sato T, Asano T, Kurihara Y, Kurihara H. Multiple renal cysts, urinary concentration defects, and pulmonary emphysematous changes in mice lacking TAZ. Am J Physiol Renal Physiol. 294(3):F542-53 (2008).
[24] Kang W, Tong JH, Chan AW, Lee TL, Lung RW, Leung PP, So KK, Wu K, Fan D, Yu J, Sung JJ, To KF. Yes-associated protein 1 exhibits oncogenic property in gastric cancer and its nuclear accumulation associates with poor prognosis. Clin Cancer Res. 17(8):2130-9 (2011).
[25] Zhou Z, Hao Y, Liu N, Raptis L, Tsao MS, Yang X. TAZ is a novel oncogene in non-small cell lung cancer. Oncogene. 30(18):2181-6 (2011)
[26] Xu Y, Stamenkovic I, Yu Q. CD44 attenuates activation of the hippo signaling pathway and is a prime therapeutic target for glioblastoma. Cancer Res. 70(6):2455-64(2010).
[27] Salah Z, Aqeilan RI. Cell Death Dis. 2011 Jun WW domain interactions regulate the Hippo tumor suppressor pathway. Cell Death Dis. 2:e172 (2011).
[28] Sudol M. Yes-associated protein (YAP65) is a proline-rich phosphoprotein that binds to the SH3 domain of the Yes proto-oncogene product. Oncogene. 9(8):2145-52( 1994).
[29] Levy D, Adamovich Y, Reuven N, Shaul Y Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage. Mol Cell. 29(3):350-61 (2008).
[30] Zhao B, Li L, Tumaneng K, Wang CY, Guan KL. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(beta-TRCP). Genes Dev. Jan 24(1):72-85 (2010).
[31] Basu S, Totty NF, Irwin MS, Sudol M, Downward J. Akt phosphorylates the Yes-associated protein, YAP, to induce interaction with 14-3-3 and attenuation of p73-mediated apoptosis. Mol Cell. 11(1):11-23.(2003)
[32] Hao Y, Chun A, Cheung K, Rashidi B, Yang X. Tumor suppressor LATS1 is a negative regulator of oncogene YAP. J Biol Chem. 283(9):5496-509.(2008)
[33] Chen L, Chan SW, Zhang X, Walsh M, Lim CJ, Hong W, Song H. Structural basis of YAP recognition by TEAD4 in the hippo pathway. Genes Dev. 24(3):290-300. (2010)
[34] Zhao B, Li L, Lu Q, Wang LH, Liu CY, Lei Q, Guan KL. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein. Genes Dev. 25(1):51-63. (2011)
[35] Schlegelmilch K, Mohseni M, Kirak O, Pruszak J, Rodriguez JR, Zhou D, Kreger BT, Vasioukhin V, Avruch J, Brummelkamp TR, Camargo FD. Yap1 acts downstream of α-catenin to control epidermal proliferation. Cell. 144(5):782-95 (2011).
[36] Lian I, Kim J, Okazawa H, Zhao J, Zhao B, Yu J, Chinnaiyan A, Israel MA, Goldstein LS, Abujarour R, Ding S, Guan KL The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation. Genes Dev. 24(11):1106-18 (2010)
[37]. Cao X, Pfaff SL, Gage FH. YAP regulates neural progenitor cell number via the TEA domain transcription factor. Genes Dev. 22(23):3320-34 (20080
[38].Zhang H, Pasolli HA, Fuchs E. Yes-associated protein (YAP) transcriptional coactivator functions in balancing growth and differentiation in skin. Proc Natl Acad Sci U S A. 108(6):2270-5 (2011)
[39] Cai J, Zhang N, Zheng Y, de Wilde RF, Maitra A, Pan D. The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev. 24(21):2383-8. (2010)
[40] Lee KP, Lee JH, Kim TS, Kim TH, Park HD, Byun JS, Kim MC, Jeong WI, Calvisi DF, Kim JM, Lim DS. The Hippo-Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc Natl Acad Sci U S A. 107(18):8248-53 (2010)
[41] Overholtzer M, Zhang J, Smolen GA, Muir B, Li W, Sgroi DC, Deng CX, Brugge JS, Haber DA. Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon Proc Natl Acad Sci U S A. 103(33):12405-10 (2006)
[42] Zhao B, Ye X, Yu J, Li L, Li W, Li S, Yu J, Lin JD, Wang CY, Chinnaiyan AM, Lai ZC, Guan KL. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22(14):1962-71 (2008).
[43] Wang Y, Dong Q, Zhang Q, Li Z, Wang E, Qiu X. Overexpression of yes-associated protein contributes to progression and poor prognosis of non-small-cell lung cancer. Cancer Sci. 101(5):1279-85 (2010).
[44] Steinhardt AA, Gayyed MF, Klein AP, Dong J, Maitra A, Pan D, Montgomery EA, Anders RA. Expression of Yes-associated protein in common solid tumors. Hum Pathol. 39(11):1582-9 (2008).
[45] Levy D, Adamovich Y, Reuven N, Shaul Y. The Yes-associated protein 1 stabilizes p73 by preventing Itch-mediated ubiquitination of p73. Cell Death Differ. 14(4):743-51 (2007).
[46] Lapi E, Di Agostino S, Donzelli S, Gal H, Domany E, Rechavi G, Pandolfi PP, Givol D, Strano S, Lu X, Blandino G. PML, YAP, and p73 are components of a proapoptotic autoregulatory feedback loop. Mol Cell. 32(6):803-14 (2008).
[47] Zagurovskaya M, Shareef MM, Das A, Reeves A, Gupta S, Sudol M, Bedford MT, Prichard J, Mohiuddin M, Ahmed MM. EGR-1 forms a complex with YAP-1 and upregulates Bax expression in irradiated prostate carcinoma cells. Oncogene. Feb 28(8):1121-31 (2009).
[48] Wu C, Xu B, Yuan P, Miao X, Liu Y, Guan Y, Yu D, Xu J, Zhang T, Shen H, Wu T, Lin D. Genome-wide interrogation identifies YAP1 variants associated with survival of small-cell lung cancer patients. Cancer Res. 70(23):9721-9 (2010).
[49].Pan D The hippo signaling pathway in development and cancer. Dev Cell. 19(4):491-505 (2010).
[50] Levanon D, Negreanu V, Bernstein Y, Bar-Am I, Avivi L, Groner Y. AML1, AML2, and AML3, the human members of the runt domain gene-family: cDNA structure, expression, and chromosomal localization. Genomics. 23(2):425-32 (1994).
[51] Kagoshima H, Shigesada K, Satake M, Ito Y, Miyoshi H, Ohki M, Pepling M, Gergen P. The Runt domain identifies a new family of heteromeric transcriptional regulators. Trends Genet. 9(10):338-41 (1993).
[52] Levanon D, Goldstein RE, Bernstein Y, Tang H, Goldenberg D, Stifani S, Paroush Z, Groner Y. Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors. Proc Natl Acad Sci U S A. 95(20):11590-5 (1998).
[53] Levanon D, Bettoun D, Harris-Cerruti C, Woolf E, Negreanu V, Eilam R, Bernstein Y, Goldenberg D, Xiao C, Fliegauf M, Kremer E, Otto F, Brenner O, Lev-Tov A, Groner Y. The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons. EMBO J. 21(13):3454-63 (2002).
[54] Blyth K, Cameron ER, Neil JC. The RUNX genes: gain or loss of function in cancer. Nat Rev Cancer. 5(5):376-87 (2005).
[55] Woolf E, Xiao C, Fainaru O, Lotem J, Rosen D, Negreanu V, Bernstein Y, Goldenberg D, Brenner O, Berke G, Levanon D, Groner Y. Runx3 and Runx1 are required for CD8 T cell development during thymopoiesis. Proc Natl Acad Sci U S A. 100(13):7731-6 (2003).
[56] Li QL, Ito K, Sakakura C, Fukamachi H, Inoue K, Chi XZ, Lee KY, Nomura S, Lee CW, Han SB, Kim HM, Kim WJ, Yamamoto H, Yamashita N, Yano T, Ikeda T, Itohara S, Inazawa J, Abe T, Hagiwara A, Yamagishi H, Ooe A, Kaneda A, Sugimura T, Ushijima T, Bae SC, Ito Y. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell. 109(1):113-24 (2002).
[57] Yano T, Ito K, Fukamachi H, Chi XZ, Wee HJ, Inoue K, Ida H, Bouillet P, Strasser A, Bae SC, Ito Y. The RUNX3 tumor suppressor upregulates Bim in gastric epithelial cells undergoing transforming growth factor beta-induced apoptosis. Mol Cell Biol. 26(12):4474-88 (2006).
[58]. Kim JH, Choi JK, Cinghu S, Jang JW, Lee YS, Li YH, Goh YM, Chi XZ, Lee KS, Wee H, Bae SC. Jab1/CSN5 induces the cytoplasmic localization and degradation of RUNX3. J Cell Biochem. 107(3):557-65 (2009).
[59] Fujii S, Ito K, Ito Y, Ochiai A. Enhancer of zeste homologue 2 (EZH2) down-regulates RUNX3 by increasing histone H3 methylation. J Biol Chem. 283(25):17324-32 (2008).
[60] Lee SH, Kim J, Kim WH, Lee YM. Hypoxic silencing of tumor suppressor RUNX3 by histone modification in gastric cancer cells. Oncogene. 28(2):184-94 (2009)
[61] Ito K, Liu Q, Salto-Tellez M, Yano T, Tada K, Ida H, Huang C, Shah N, Inoue M, Rajnakova A, Hiong KC, Peh BK, Han HC, Ito T, Teh M, Yeoh KG, Ito Y. RUNX3, a novel tumor suppressor, is frequently inactivated in gastric cancer by protein mislocalization. Cancer Res. 65(17):7743-50 (2005).
[62] Yanada M, Yaoi T, Shimada J, Sakakura C, Nishimura M, Ito K, Terauchi K, Nishiyama K, Itoh K, Fushiki S. Frequent hemizygous deletion at 1p36 and hypermethylation downregulate RUNX3 expression in human lung cancer cell lines. Oncol Rep. 14(4):817-22 (2005).
[63] Ito K, Lim AC, Salto-Tellez M, Motoda L, Osato M, Chuang LS, Lee CW, Voon DC, Koo JK, Wang H, Fukamachi H, Ito Y. RUNX3 attenuates beta-catenin/T cell factors in intestinal tumorigenesis. Cancer Cell. 14(3):226-37 (2008).
[64] Wei D, Gong W, Oh SC, Li Q, Kim WD, Wang L, Le X, Yao J, Wu TT, Huang S, Xie K. Loss of RUNX3 expression significantly affects the clinical outcome of gastric cancer patients and its restoration causes drastic suppression of tumor growth and metastasis. Cancer Res. 65(11):4809-16 (2005).
[65] Yagi R, Chen LF, Shigesada K, Murakami Y, Ito Y. A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator. EMBO J. 18(9):2551-62. (1999)
[66] Zaidi SK, Sullivan AJ, Medina R, Ito Y, van Wijnen AJ, Stein JL, Lian JB, Stein GS. Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. EMBO J. 23(4):790-9 (2004).
[67] Herbst RS, Heymach JV, Lippman SM. Lung cancer. N Engl J Med 359(13):1367-80 (2008)
[68] Sekido Y, Fong KM, Minna JD. Molecular genetics of lung cancer Annu Rev Med. 54:73-87. (2003)
[69] da Cunha Santos G, Shepherd FA, Tsao MS. EGFR mutations and lung cancer. Annu Rev Pathol. 28;6:49-69. (2011)