SHANK2 is a frequently amplified oncogene with evolutionarily conserved roles in regulating Hippo signaling

Abstract

Dysfunction of the Hippo pathway enables cells to evade contact inhibition and provides advantages for cancerous overgrowth. However, for a significant portion of human cancer, how Hippo signaling is perturbed remains unknown. To answer this question, we performed a genome-wide screening for genes that affect the Hippo pathway in Drosophila and cross-referenced the hit genes with human cancer genome. In our screen, Prosap was identified as a novel regulator of the Hippo pathway that potently affects tissue growth. Interestingly, a mammalian homolog of Prosap, SHANK2, is the most frequently amplified gene on 11q13, a major tumor amplicon in human cancer. Gene amplification profile in this 11q13 amplicon clearly indicates selective pressure for SHANK2 amplification. More importantly, across the human cancer genome, SHANK2 is the most frequently amplified gene that is not located within the Myc amplicon. Further studies in multiple human cell lines confirmed that SHANK2 overexpression causes deregulation of Hippo signaling through competitive binding for a LATS1 activator, and as a potential oncogene, SHANK2 promotes cellular transformation and tumor formation in vivo. In cancer cell lines with deregulated Hippo pathway, depletion of SHANK2 restores Hippo signaling and ceases cellular proliferation. Taken together, these results suggest that SHANK2 is an evolutionarily conserved Hippo pathway regulator, commonly amplified in human cancer and potently promotes cancer. Our study for the first time illustrated oncogenic function of SHANK2, one of the most frequently amplified gene in human cancer. Furthermore, given that in normal adult tissues, SHANK2’s expression is largely restricted to the nervous system, SHANK2 may represent an interesting target for anticancer therapy.

Key words： SHANK2; oncogene; Hippo signaling; cancer

References

1 Atkins M, Potier D, Romanelli L, Jacobs J, Mach J, Hamaratoglu F, Aerts S, Halder G(2016) An ectopic network of transcription factors regulated by hippo signaling drives growth and invasion of a malignant tumor model. Curr Biol 26(16):2101–2113

2 Barros-Filho MC, Reis-Rosa LA, Hatakeyama M, Marchi FA, Chulam T, Scapulatempo-Neto C, Nicolau UR, Carvalho AL, Pinto CAL, Drigo SA (2018) Oncogenic drivers in 11q13 associated with prognosis and response to therapy in advanced oropharyngeal carcinomas. Oral Oncol 83:81–90

3 Berkel S, Marshall CR, Weiss B, Howe J, Roeth R, Moog U, Endris V, Roberts W, Szatmari P, Pinto D (2010) Mutations in the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation. Nat Genet 42(6):489–491

4 Brown J, Stepien AJ, Willem P (2020) Landscape of copy number aberrations in esophageal squamous cell carcinoma from a high endemic region of South Africa. BMC Cancer 20(1):281

5 Carneiro A, Isinger A, Karlsson A, Johansson J, Jönsson G, Bendahl PO, Falkenback D, Halvarsson B, Nilbert M (2008) Prognostic impact of array-based genomic profiles in esophageal squamous cell cancer. BMC Cancer 8:98

6 Chakraborty S, Njah K, Pobbati AV, Lim YB, Raju A, Lakshmanan M, Tergaonkar V, Lim CT, Hong W (2017) Agrin as a mechanotransduction signal regulating YAP through the Hippo pathway. Cell Rep 18(10):2464–2479

7 Chang L, Azzolin L, Di Biagio D, Zanconato F, Battilana G, Lucon Xiccato R, Aragona M, Giulitti S,Panciera T, Gandin A (2018) The SWI/SNF complex is a mechanoregulated inhibitor of YAP and TAZ. Nature 563(7730):265–269

8 Dent LG, Poon CLC, Zhang X, Degoutin JL, Tipping M, Veraksa A, Harvey KF (2015) The GTPase regulatory proteins pix and git control tissue growth via the hippo pathway. Curr Biol 25(1):124–130

9 Dong J, Feldmann G, Huang J, Wu S, Zhang N, Comerford SA, Gayyed MF, Anders RA, Maitra A, Pan D (2007) Elucidation of a universal size-control mechanism in drosophila and mammals. Cell 130(6):1120–1133

10 Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S (2011) Role of YAP/TAZ in mechanotransduction. Nature 474 (7350):179–183 Engels WR (1996) P elements in Drosophila. Curr Top Microbiol Immunol 204:103–123

11 Feng X, Degese MS, Iglesias-Bartolome R, Vaque JP, Molinolo AA, Rodrigues M, Zaidi MR, Ksander BR, Merlino G, Sodhi A (2014) Hippo-independent activation of YAP by the GNAQ uveal melanoma oncogene through a Trio-regulated Rho GTPase signaling circuitry. Cancer Cell 25(6):831–845

12 Fernandez-L A, Northcott PA, Dalton J, Fraga C, Ellison D, Angers S,Taylor MD, Kenney AM (2009) YAP1 is amplified and upregulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation. Genes Dev 23(23):2729–2741

13 Gadd MS, Testa A, Lucas X, Chan KH, Chen W,Lamont DJ, Zengerle M, Ciulli A (2017) Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol 13(5):514–521

14 Gumbiner BM, Kim NG (2014) The Hippo-YAP signaling pathway and contact inhibition of growth. J Cell Sci 127(Pt 4):709–717

15 Guo T, Lu Y, Li P, Yin MX, Lv D, Zhang W, Wang H, Zhou Z,Ji H, Zhao Y (2013) A novel partner of Scalloped regulates Hippo signaling via antagonizing Scalloped-Yorkie activity. Cell Res 23 (10):1201–1214

16 Halder G, Johnson RL (2011) Hippo signaling: growth control and beyond. Development 138(1):9–22

17 Halder G, Dupont S, Piccolo S (2012) Transduction of mechanical and cytoskeletal cues by YAP and TAZ. Nat Rev Mol Cell Biol 13 (9):591–600

18 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674

19 Harvey KF, Pfleger CM, Hariharan IK (2003) The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell 114(4):457–467

20 Hayashi MK, Tang C, Verpelli C,Narayanan R, Stearns MH, Xu RM, Li H, Sala C, Hayashi Y (2009) The postsynaptic density proteins Homer and Shank form a polymeric network structure. Cell 137 (1):159–171

21 Heidary Arash E, Song KM, Song S, Shiban A, Attisano L (2014) Arhgef7 promotes activation of the Hippo pathway core kinase Lats. EMBO J 33(24):2997–3011

22 Hu L, Xu J, Yin MX, Lu Y, Wu W, Xue Z, Ho MS, Gao G, Zhao Y, Zhang L (2016) Ack promotes tissue growth via phosphorylation and suppression of the Hippo pathway component Expanded. Cell Discov 2:15047

23 Huang HL, Wang S, Yin MX, Dong L, Wang C, Wu W, Lu Y, Feng M, Dai C, Guo X(2013) Par-1 regulates tissue growth by influencing hippo phosphorylation status and Hippo-Salvador association. PLoS Biol 11(8):e1001620

24 Huang J, Wu S, Barrera J, Matthews K, Pan D (2005) The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP. Cell 122(3):421–434

25 Hung AY, Futai K, Sala C,Valtschanoff JG, Ryu J, Woodworth MA, Kidd FL, Sung CC, Miyakawa T, Bear MF (2008) Smaller dendritic spines, weaker synaptic transmission, but enhanced spatial learning in mice lacking Shank1. J Neurosci 28(7):1697–1708

26 Jia J, Zhang W, Wang B, Trinko R, Jiang J (2003) The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis. Genes Dev 17(20):2514–2519

27 Jiao S, Wang H, Shi Z, Dong A, Zhang W, Song X, He F, Wang Y, Zhang Z, Wang W et al (2014) A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer. Cancer Cell 25(2):166–180

28 Lee T, Luo L (2001) Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development. Trends Neurosci 24(5):251–254

29 Li J, Belogortseva N, Porter D, Park M (2008) Chmp1A functions as a novel tumor suppressor gene in human embryonic kidney and ductal pancreatic tumor cells. Cell Cycle 7(18):2886–2893

30 Li Z, Razavi P, Li Q, Toy W, Liu B, Ping C,Hsieh W, Sanchez-Vega F, Brown DN, Da Cruz Paula AF (2018) Loss of the FAT1 tumor suppressor promotes resistance to CDK4/6 inhibitors via the Hippo pathway. Cancer Cell 34(6):893–905

31 Lim SK, Lu SY, Kang SA, Tan HJ, Li Z, Wee ZNA, Guan JS, Chichili VPR, Sivaraman J, Putti T (2016) Wnt signaling promotes breast cancer by blocking ITCH-mediated degradation of YAP/ TAZ transcriptional coactivator WBP2. Cancer Res 76(21):6278–6289

32 Liu CY, Zha ZY, Zhou X, Zhang H, Huang W, Zhao D, Li T, Chan SW, Lim CJ, Hong Wet al (2010) The hippo tumor pathway promotes TAZ degradation by phosphorylating a phosphodegron and recruiting the SCFβ-TrCP E3 ligase. J Biol Chem 285 (48):37159–37169

33 Maille E, Brosseau S, Hanoux V, Creveuil C, Danel C, Bergot E, Scherpereel A, Mazières J, Margery J, Greillier L (2019) MST1/Hippo promoter gene methylation predicts poor survival in patients with malignant pleural mesothelioma in the IFCT-GFPC-0701 MAPS Phase 3 trial. Br J Cancer 120(4):387–397

34 Malik SA, Khan MS, Dar M, Hussain MU, Shah MA, Shafi SM, Mudassar S (2018) Molecular alterations and expression dynamics of LATS1 and LATS2 genes in non-small-cell lung carcinoma. Pathol Oncol Res 24(2):207–214

35 Merritt NM, Fullenkamp CA, Hall SL, Qian Q, Desai C, Thomason J, Lambertz AM, Dupuy AJ, Darbro B,Tanas MR (2018) A comprehensive evaluation of Hippo pathway silencing in sarcomas. Oncotarget 9(60):31620–31636

36 Naisbitt S, Eunjoon K, Tu JC, Xiao B, Sala C, Valtschanoff J, Weinberg RJ, Worley PF, Sheng M (1999) Shank, a novel family of postsynaptic density proteins that binds to the NMDA receptor/ PSD-95/GKAP complex and cortactin. Neuron 23(3):569–582

37 Overholtzer M, Zhang J, Smolen GA, Muir B, Li W, Sgroi DC, Deng C-X, Brugge JS, Haber DA (2006) Transforming properties of YAP, a candidate oncogene on the chromosome 11q22 amplicon. Proc Natl Acad Sci USA 103(33):12405–12410

38 Pantalacci S, Tapon N, Léopold P (2003) The salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila. Nat Cell Biol 5(10):921–927

39 Park E,Na M, Choi J, Kim S, Lee JR, Yoon J,Park D, Sheng M, Kim E (2003) The Shank family of postsynaptic density proteins interacts with and promotes synaptic accumulation of the βPIX guanine nucleotide exchange factor for Rac1 and Cdc42. J Biol Chem 278(21):19220–19229

40 Peça J, Feliciano C, Ting JT, Wang W, Wells MF, Venkatraman TN, Lascola CD, Fu Z, Feng G(2011) Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472 (7344):437–442

41 Pontén FK, Schwenk JM, Asplund A, Edqvist PHD (2011) The Human Protein Atlas as a proteomic resource for biomarker discovery. J Intern Med 270(5):428–446

42 Qin H De, Liao XY, Chen Y Bin, Huang SY, Xue WQ, Li FF, Ge XS, Liu DQ, Cai Q, Long J (2016) Genomic characterization of esophageal squamous cell carcinoma reveals critical genes underlying tumorigenesis and poor prognosis. Am J Hum Genet 98(4):709–727

43 Rørth P (1996) A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci USA 93 (22):12418–12422

44 Sanchez-Vega F, Mina M, Armenia J, Chatila WK, Luna A, La KC, Dimitriadoy S,Liu DL, Kantheti HS, Saghafinia S (2018) Oncogenic signaling pathways in the cancer genome atlas. Cell 173(2):321–337

45 Sansores-Garcia L, Atkins M, Moya IM, Shahmoradgoli M, Tao C, Mills GB, Halder G (2013) Mask is required for the activity of the hippo pathway effector Yki/YAP. Curr Biol 23(3):229–235

46 Schmeisser MJ, Ey E, Wegener S,Bockmann J, Stempel AV, Kuebler A, Janssen AL, Udvardi PT, Shiban E, Spilker C (2012) Autistic-like behaviours and hyperactivity in mice lacking ProSAP1/Shank2. Nature 486(7402):256–260

47 Schneider K, Seemann E, Liebmann L, Ahuja R, Koch D, Westermann M, Hübner CA, Kessels MM, Qualmann B (2014) ProSAP1 and membrane nanodomain-associated syndapin i promote postsynapse formation and function. J Cell Biol 205(2):197–215

48 Seidel C, Schagdarsurengin U, Blümke K, Würl P, Pfeifer GP, Hauptmann S, Taubert H, Dammann R (2007) Frequent hypermethylation of MST1 and MST2 in soft tissue sarcoma. Mol Carcinog 46(10):865–871

49 Setten RL, Rossi JJ, Han S (2019) The current state and future directions of RNAi-based therapeutics. Nat Rev Drug Discov 18 (6):421–446

50 Siew WC, Chun JL, Guo K, Chee PN, Lee I, Hunziker W, Zeng Q, Hong W (2008) A role for TAZ in migration, invasion, and tumorigenesis of breast cancer cells. Cancer Res 68(8):2592–2598

51 Song H, Mak KK, Topol L, Yun K, Hu J, Garrett L, Chen Y, Park O, Chang J, Simpson RM (2010) Mammalian Mst1 and Mst2 kinases play essential roles in organ size control and tumor suppression. Proc Natl Acad Sci USA 107(4):1431–1436

52 Song Y, Li L, Ou Y, Gao Z, Li E, Li X, Zhang W, Wang J, Xu L, Zhou Y (2014) Identification of genomic alterations in oesophageal squamous cell cancer. Nature 509(7498):91–95

53 Stein CA, Castanotto D (2017) FDA-approved oligonucleotide therapies in 2017. Mol Ther 25(5):1069–1075

54 Stepanenko AA, Dmitrenko VV (2015) HEK293 in cell biology and cancer research: Phenotype, karyotype, tumorigenicity, and stress-induced genome-phenotype evolution. Gene 569(2):182–190

55 Tang Z, Li C, Kang B, Gao G, Li C,Zhang Z (2017) GEPIA: A web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45(W1):W98–W102

56 Udan RS,Kango-Singh M,Nolo R, Tao C, Halder G (2003) Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway. Nat Cell Biol 5(10):914–920

57 Wang W, Xiao ZD, Li X, Aziz KE, Gan B, Johnson RL, Chen J (2015) AMPK modulates Hippo pathway activity to regulate energy homeostasis. Nat Cell Biol 17(4):490–499

58 Wang X, McCoy PA, Rodriguiz RM, Pan Y, Je HS, Roberts AC, Kim CJ, Berrios J, Colvin JS, Bousquet-Moore D (2011) Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum Mol Genet 20(15):3093–3108

59 Wehr MC, Holder MV, Gailite I, Saunders RE, Maile TM, Ciirdaeva E, Instrell R, Jiang M, Howell M, Rossner MJ (2013) Saltinducible kinases regulate growth through the Hippo signalling pathway in Drosophila. Nat Cell Biol 15(1):61–71

60 Wierzbicki PM, Adrych K, Kartanowicz D, Stanislawowski M, Kowalczyk A, Godlewski J, Skwierz-Bogdanska I,Celinski K, Gach T, Kulig J (2013) Underexpression of LATS1 TSG in colorectal cancer is associated with promoter hypermethylation. World J Gastroenterol 19(27):4363–4373

61 Won H, Lee HR, Gee HY, Mah W, Kim JI, Lee J, Ha S, Chung C, Jung ES, Cho YS(2012) Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function. Nature 486(7402):261–265

62 Wu C,Jin X, Tsueng G, Afrasiabi C, Su AI (2016) BioGPS: Building your own mash-up of gene annotations and expression profiles. Nucleic Acids Res 44(D1):D313–D316

63 Wu S, Huang J, Dong J, Pan D (2003) hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell 114 (4):445–456

64 Xiao GH, Chernoff J, Testa JR (2003) NF2: The Wizardry of Merlin. Genes Chromosom Cancer 38(4):389–399

65 Yang C-C, Graves HK, Moya IM, Tao C, Hamaratoglu F, Gladden AB, Halder G (2015) Differential regulation of the Hippo pathway by adherens junctions and apical–basal cell polarity modules. Proc Natl Acad Sci USA 112(6):1785–1790

66 Yant SR, Park J, Huang Y, Mikkelsen JG, Kay MA (2004) Mutational analysis of the N-terminal DNA-binding domain of sleeping beauty transposase: critical residues for DNA binding and hyperactivity in mammalian cells. Mol Cell Biol 24(20):9239–9247

67 Yin F, Yu J, Zheng Y, Chen Q, Zhang N, Pan D (2013) XSpatial organization of hippo signaling at the plasma membrane mediated by the tumor suppressor merlin/NF2. Cell 154(6):1342–1355

68 Yu FX, Zhao B, Panupinthu N, Jewell JL, Lian I, Wang LH, Zhao J, Yuan H, Tumaneng K, Li H (2012) Regulation of the Hippo- YAP pathway by G-protein-coupled receptor signaling. Cell 150 (4):780–791

69 Yu FX, Luo J, Mo JS, Liu G,Kim YC, Meng Z, Zhao L, Peyman G,Ouyang H, Jiang W(2014) Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP. Cancer Cell 25 (6):822–830

70 Yu FX, Zhao B, Guan KL (2015) Hippo pathway in organ size control, tissue homeostasis, and cancer. Cell 163(4):811–828

71 Yu T, Bachman J, Lai ZC (2013) Evidence for a tumor suppressor role for the Large tumor suppressor genes LATS1 and LATS2 in human cancer. Genetics 195(3):1193–1196

72 Yu Y, Cao J, Wu W, Zhu Q, Tang Y, Zhu C, Dai J, Li Z, Wang J, Xue L et al (2019) Genome-wide copy number variation analysis identified ANO1 as a novel oncogene and prognostic biomarker in esophageal squamous cell cancer. Carcinogenesis 40 (10):1198–1208

73 Zack TI, Schumacher SE, Carter SL, Cherniack AD, Saksena G, Tabak B, Lawrence MS, Zhang CZ, Wala J, Mermel CH (2013) Pan-cancer patterns of somatic copy number alteration. Nat Genet 45(10):1134–1140

74 Zender L, Spector MS, Xue W, Flemming P, Cordon-Cardo C, Silke J, Fan ST, Luk JM, Wigler M, Hannon GJ(2006) Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 125(7):1253–1267

75 Zhang L, Ren F, Zhang Q, Chen Y,Wang B, Jiang J (2008) The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. Dev Cell 14(3):377–387

76 Zhang N, Bai H, David KK, Dong J, Zheng Y, Cai J, Giovannini M, Liu P,Anders RA, Pan D (2010) The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals. Dev Cell 19(1):27–38

77 Zhang W, Gao Y, Li P, Shi Z, Guo T, Li F, Han X, Feng Y, Zheng C, Wang Z(2014) VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex. Cell Res 24(3):331–343

78 Zhao B, Li L, Tumaneng K, Wang CY, Guan KL (2010) A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP. Genes Dev 24(1):72–85

79 Zhao B, Li L, Wang L, Wang CY, Yu J, Guan KL (2012) Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev 26(1):54–68

80 Zhou D, Conrad C, Xia F, Park JS, Payer B, Yin Y, Lauwers GY, Thasler W, Lee JT, Avruch J (2009) Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell 16(5):425–438