Palmitoylation of GNAQ/11 is critical for tumor cell proliferation and survival in GNAQ/11-mutant uveal melanoma

Abstract

More than 85% of patients with uveal melanoma (UM) carry a GNAQ or GNA11 mutation at a hotspot codon (Q209) that encodes G protein α subunit q/11 polypeptides (Gα_q/11). GNAQ/11 relies on palmitoylation for membrane association and signal transduction. Despite the palmitoylation of GNAQ/11 was discovered long before, its implication in UM remains unclear. Here, results of palmitoylation-targeted mutagenesis and chemical interference approaches revealed that the loss of GNAQ/11 palmitoylation substantially affected tumor cell proliferation and survival in UM cells. Palmitoylation inhibition through the mutation of palmitoylation sites suppressed GNAQ/11^Q209L-induced malignant transformation in NIH3T3 cells. Importantly, the palmitoylation-deficient oncogenic GNAQ/11 failed to rescue the cell death initiated by the knock down of endogenous GNAQ/11 oncogenes in UM cells, which are much more dependent on Gα_q/11 signaling for cell survival and proliferation than other melanoma cells without GNAQ/11 mutations. Furthermore, the palmitoylation inhibitor, 2-bromopalmitate, also specifically disrupted Gα_q/11 downstream signaling by interfering with the MAPK pathway and BCL2 survival pathway in GNAQ/11-mutant UM cells and showed a notable synergistic effect when applied in combination with the BCL2 inhibitor, ABT-199, in vitro. The findings validate that GNAQ/11 palmitoylation plays a critical role in UM and may serve as a promising therapeutic target for GNAQ/11-driven UM.

Keywords

uveal melanoma / mutant GNAQ/11 / palmitoylation / BCL2 / combination target therapy

References

[1] Simanshu DK, Nissley DV, McCormick F. RAS proteins and their regulators in human disease. Cell 2017; 170( 1): 17– 33

[2] Dorsam RT, Gutkind JS. G-protein-coupled receptors and cancer. Nat Rev Cancer 2007; 7( 2): 79– 94

[3] O’Hayre M, Vázquez-Prado J, Kufareva I, Stawiski EW, Handel TM, Seshagiri S, Gutkind JS. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nat Rev Cancer 2013; 13( 6): 412– 424

[4] Jager MJ, Shields CL, Cebulla CM, Abdel-Rahman MH, Grossniklaus HE, Stern MH, Carvajal RD, Belfort RN, Jia R, Shields JA, Damato BE. Uveal melanoma. Nat Rev Dis Primers 2020; 6( 1): 24

[5] Jovanovic P, Mihajlovic M, Djordjevic-Jocic J, Vlajkovic S, Cekic S, Stefanovic V. Ocular melanoma: an overview of the current status. Int J Clin Exp Pathol 2013; 6( 7): 1230– 1244

[6] Seth R, Messersmith H, Kaur V, Kirkwood JM, Kudchadkar R, McQuade JL, Provenzano A, Swami U, Weber J, Alluri KC, Agarwala S, Ascierto PA, Atkins MB, Davis N, Ernstoff MS, Faries MB, Gold JS, Guild S, Gyorki DE, Khushalani NI, Meyers MO, Robert C, Santinami M, Sehdev A, Sondak VK, Spurrier G, Tsai KK, van Akkooi A, Funchain P. Systemic therapy for melanoma: ASCO Guideline. J Clin Oncol 2020; 38( 33): 3947– 3970

[7] Hu Q, Shokat KM. Disease-causing mutations in the G protein Gαs subvert the roles of GDP and GTP. Cell 2018; 173( 5): 1254– 1264.e11

[8] Chen X, Wu Q, Depeille P, Chen P, Thornton S, Kalirai H, Coupland SE, Roose JP, Bastian BC. RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma. Cancer Cell 2017; 31( 5): 685– 696.e6

[9] Van Raamsdonk CD, Griewank KG, Crosby MB, Garrido MC, Vemula S, Wiesner T, Obenauf AC, Wackernagel W, Green G, Bouvier N, Sozen MM, Baimukanova G, Roy R, Heguy A, Dolgalev I, Khanin R, Busam K, Speicher MR, O’Brien J, Bastian BC. Mutations in GNA11 in uveal melanoma. N Engl J Med 2010; 363( 23): 2191– 2199

[10] Feng X, Arang N, Rigiracciolo DC, Lee JS, Yeerna H, Wang Z, Lubrano S, Kishore A, Pachter JA, König GM, Maggiolini M, Kostenis E, Schlaepfer DD, Tamayo P, Chen Q, Ruppin E, Gutkind JS. A platform of synthetic lethal gene interaction networks reveals that the GNAQ uveal melanoma oncogene controls the Hippo pathway through FAK. Cancer Cell 2019; 35( 3): 457– 472.e5

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

[12] Yu FX, Luo J, Mo JS, Liu G, Kim YC, Meng Z, Zhao L, Peyman G, Ouyang H, Jiang W, Zhao J, Chen X, Zhang L, Wang CY, Bastian BC, Zhang K, Guan KL. Mutant Gq/11 promote uveal melanoma tumorigenesis by activating YAP. Cancer Cell 2014; 25( 6): 822– 830

[13] Vader MJC, Madigan MC, Versluis M, Suleiman HM, Gezgin G, Gruis NA, Out-Luiting JJ, Bergman W, Verdijk RM, Jager MJ, van der Velden PA. GNAQ and GNA11 mutations and downstream YAP activation in choroidal nevi. Br J Cancer 2017; 117( 6): 884– 887

[14] Chua V, Lapadula D, Randolph C, Benovic JL, Wedegaertner PB, Aplin AE. Dysregulated GPCR signaling and therapeutic options in uveal melanoma. Mol Cancer Res 2017; 15( 5): 501– 506

[15] Steeb T, Wessely A, Ruzicka T, Heppt MV, Berking C. How to MEK the best of uveal melanoma: a systematic review on the efficacy and safety of MEK inhibitors in metastatic or unresectable uveal melanoma. Eur J Cancer 2018; 103 : 41– 51

[16] Annala S, Feng X, Shridhar N, Eryilmaz F, Patt J, Yang J, Pfeil EM, Cervantes-Villagrana RD, Inoue A, Häberlein F, Slodczyk T, Reher R, Kehraus S, Monteleone S, Schrage R, Heycke N, Rick U, Engel S, Pfeifer A, Kolb P, König G, Bünemann M, Tüting T, Vázquez-Prado J, Gutkind JS, Gaffal E, Kostenis E. Direct targeting of Gα_q and Gα₁₁ oncoproteins in cancer cells. Sci Signal 2019; 12( 573): eaau5948

[17] Singh AD, Turell ME, Topham AK. Uveal melanoma: trends in incidence, treatment, and survival. Ophthalmology 2011; 118( 9): 1881– 1885

[18] Arnold JJ, Blinder KJ, Bressler NM, Bressler SB, Burdan A, Haynes L, Lim JI, Miller JW, Potter MJ, Reaves A, Rosenfeld PJ, Sickenberg M, Slakter JS, Soubrane G, Strong HA, Stur M; Treatment of Age-Related Macular Degeneration with Photodynamic Therapy Study Group; Verteporfin in Photodynamic Therapy Study Group. Acute severe visual acuity decrease after photodynamic therapy with verteporfin: case reports from randomized clinical trials—TAP and VIP report no. 3. Am J Ophthalmol 2004; 137( 4): 683– 696

[19] Khalili JS, Yu X, Wang J, Hayes BC, Davies MA, Lizee G, Esmaeli B, Woodman SE. Combination small molecule MEK and PI3K inhibition enhances uveal melanoma cell death in a mutant GNAQ- and GNA11-dependent manner. Clin Cancer Res 2012; 18( 16): 4345– 4355

[20] Chen X, Wu Q, Tan L, Porter D, Jager MJ, Emery C, Bastian BC. Combined PKC and MEK inhibition in uveal melanoma with GNAQ and GNA11 mutations. Oncogene 2014; 33( 39): 4724– 4734

[21] Paradis JS, Acosta M, Saddawi-Konefka R, Kishore A, Gomes F, Arang N, Tiago M, Coma S, Lubrano S, Wu X, Ford K, Day CP, Merlino G, Mali P, Pachter JA, Sato T, Aplin AE, Gutkind JS. Synthetic lethal screens reveal cotargeting FAK and MEK as a multimodal precision therapy for GNAQ-driven uveal melanoma. Clin Cancer Res 2021; 27( 11): 3190– 3200

[22] Sagoo MS, Harbour JW, Stebbing J, Bowcock AM. Combined PKC and MEK inhibition for treating metastatic uveal melanoma. Oncogene 2014; 33( 39): 4722– 4723

[23] Takasaki J, Saito T, Taniguchi M, Kawasaki T, Moritani Y, Hayashi K, Kobori M. A novel Gα_q/11-selective inhibitor. J Biol Chem 2004; 279( 46): 47438– 47445

[24] Nishimura A, Kitano K, Takasaki J, Taniguchi M, Mizuno N, Tago K, Hakoshima T, Itoh H. Structural basis for the specific inhibition of heterotrimeric G_q protein by a small molecule. Proc Natl Acad Sci USA 2010; 107( 31): 13666– 13671

[25] Zaima K, Deguchi J, Matsuno Y, Kaneda T, Hirasawa Y, Morita H. Vasorelaxant effect of FR900359 from Ardisia crenata on rat aortic artery. J Nat Med 2013; 67( 1): 196– 201

[26] Schrage R, Schmitz AL, Gaffal E, Annala S, Kehraus S, Wenzel D, Büllesbach KM, Bald T, Inoue A, Shinjo Y, Galandrin S, Shridhar N, Hesse M, Grundmann M, Merten N, Charpentier TH, Martz M, Butcher AJ, Slodczyk T, Armando S, Effern M, Namkung Y, Jenkins L, Horn V, Stößel A, Dargatz H, Tietze D, Imhof D, Galés C, Drewke C, Müller CE, Hölzel M, Milligan G, Tobin AB, Gomeza J, Dohlman HG, Sondek J, Harden TK, Bouvier M, Laporte SA, Aoki J, Fleischmann BK, Mohr K, König GM, Tüting T, Kostenis E. The experimental power of FR900359 to study Gq-regulated biological processes. Nat Commun 2015; 6( 1): 10156

[27] Ahearn IM, Haigis K, Bar-Sagi D, Philips MR. Regulating the regulator: post-translational modification of RAS. Nat Rev Mol Cell Biol 2012; 13 : 39– 51

[28] Linder ME, Middleton P, Hepler JR, Taussig R, Gilman AG, Mumby SM. Lipid modifications of G proteins: alpha subunits are palmitoylated. Proc Natl Acad Sci USA 1993; 90( 8): 3675– 3679

[29] De I, Sadhukhan S. Emerging roles of DHHC-mediated protein S-palmitoylation in physiological and pathophysiological context. Eur J Cell Biol 2018; 97( 5): 319– 338

[30] Greaves J, Chamberlain LH. DHHC palmitoyl transferases: substrate interactions and (patho)physiology. Trends Biochem Sci 2011; 36( 5): 245– 253

[31] Cuiffo B, Ren R. Palmitoylation of oncogenic NRAS is essential for leukemogenesis. Blood 2010; 115( 17): 3598– 3605

[32] Liu P, Jiao B, Zhang R, Zhao H, Zhang C, Wu M, Li D, Zhao X, Qiu Q, Li J, Ren R. Palmitoylacyltransferase Zdhhc9 inactivation mitigates leukemogenic potential of oncogenic Nras. Leukemia 2016; 30( 5): 1225– 1228

[33] Xia Z, Zhang X, Liu P, Zhang R, Huang Z, Li D, Xiao X, Wu M, Ning N, Zhang Q, Zhang J, Liu M, Jiao B, Ren R. GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner. Cell Death Dis 2021; 12( 1): 54

[34] Duncan JA, Gilman AG. Autoacylation of G protein alpha subunits. J Biol Chem 1996; 271( 38): 23594– 23600

[35] Grassie MA, McCallum JF, Guzzi F, Magee AI, Milligan G, Parenti M. The palmitoylation status of the G-protein G(o)1 alpha regulates its activity of interaction with the plasma membrane. Biochem J 1994; 302( 3): 913– 920

[36] Sikarwar AS, Hinton M, Santhosh KT, Chelikani P, Dakshinamurti S. Palmitoylation of Gαq determines its association with the thromboxane receptor in hypoxic pulmonary hypertension. Am J Respir Cell Mol Biol 2014; 50( 1): 135– 143

[37] Evanko DS, Thiyagarajan MM, Siderovski DP, Wedegaertner PB. Gβγ isoforms selectively rescue plasma membrane localization and palmitoylation of mutant Gα_s and Gα_q. J Biol Chem 2001; 276( 26): 23945– 23953

[38] Tsutsumi R, Fukata Y, Noritake J, Iwanaga T, Perez F, Fukata M. Identification of G protein alpha subunit-palmitoylating enzyme. Mol Cell Biol 2009; 29( 2): 435– 447

[39] Wu M, Huang J, Zhang J, Benes C, Jiao B, Ren R. N-Arachidonoyl dopamine inhibits NRAS neoplastic transformation by suppressing its plasma membrane translocation. Mol Cancer Ther 2017; 16( 1): 57– 67

[40] Ning N, Yu Y, Wu M, Zhang R, Zhang T, Zhu C, Huang L, Yun CH, Benes CH, Zhang J, Deng X, Chen Q, Ren R. A novel microtubule inhibitor overcomes multidrug resistance in tumors. Cancer Res 2018; 78( 20): 5949– 5957

[41] He F, Yu J, Yang J, Wang S, Zhuang A, Shi H, Gu X, Xu X, Chai P, Jia R. m⁶A RNA hypermethylation-induced BACE2 boosts intracellular calcium release and accelerates tumorigenesis of ocular melanoma. Mol Ther 2021; 29( 6): 2121– 2133

[42] Slater K, Heeran AB, Garcia-Mulero S, Kalirai H, Sanz-Pamplona R, Rahman A, Al-Attar N, Helmi M, O’Connell F, Bosch R, Portela A, Villanueva A, Gallagher WM, Jensen LD, Piulats JM, Coupland SE, O’Sullivan J, Kennedy BN. High cysteinyl leukotriene receptor 1 expression correlates with poor survival of uveal melanoma patients and cognate antagonist drugs modulate the growth, cancer secretome, and metabolism of uveal melanoma cells. Cancers (Basel) 2020; 12( 10): 2950

[43] Tan S, Yang H, Xue S, Qiao J, Salarian M, Hekmatyar K, Meng Y, Mukkavilli R, Pu F, Odubade OY, Harris W, Hai Y, Yushak ML, Morales-Tirado VM, Mittal P, Sun PZ, Lawson D, Grossniklaus HE, Yang JJ. Chemokine receptor 4 targeted protein MRI contrast agent for early detection of liver metastases. Sci Adv 2020; 6( 6): eaav7504

[44] Jager MJ, Magner JAB, Ksander BR, Dubovy SR. Uveal melanoma cell lines: where do they come from? (An American Ophthalmological Society Thesis). Trans Am Ophthalmol Soc 2016; 114 : T5

[45] Yu X, Ambrosini G, Roszik J, Eterovic AK, Stempke-Hale K, Seftor EA, Chattopadhyay C, Grimm E, Carvajal RD, Hendrix MJ, Hodi FS, Schwartz GK, Woodman SE. Genetic analysis of the ‘uveal melanoma’ C918 cell line reveals atypical BRAF and common KRAS mutations and single tandem repeat profile identical to the cutaneous melanoma C8161 cell line. Pigment Cell Melanoma Res 2015; 28( 3): 357– 359

[46] Li Y, He J, Qiu C, Shang Q, Qian G, Fan X, Ge S, Jia R. The oncolytic virus H101 combined with GNAQ siRNA-mediated knockdown reduces uveal melanoma cell viability. J Cell Biochem 2019; 120( 4): 5766– 5776

[47] Wu X, Zhu M, Fletcher JA, Giobbie-Hurder A, Hodi FS. The protein kinase C inhibitor enzastaurin exhibits antitumor activity against uveal melanoma. PLoS One 2012; 7( 1): e29622

[48] O’Hayre M, Degese MS, Gutkind JS. Novel insights into G protein and G protein-coupled receptor signaling in cancer. Curr Opin Cell Biol 2014; 27 : 126– 135

[49] Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011; 11( 11): 761– 774

[50] Wang M, Casey PJ. Protein prenylation: unique fats make their mark on biology. Nat Rev Mol Cell Biol 2016; 17( 2): 110– 122

[51] Linder ME, Deschenes RJ. Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 2007; 8( 1): 74– 84

[52] Yoo JH, Shi DS, Grossmann AH, Sorensen LK, Tong Z, Mleynek TM, Rogers A, Zhu W, Richards JR, Winter JM, Zhu J, Dunn C, Bajji A, Shenderovich M, Mueller AL, Woodman SE, Harbour JW, Thomas KR, Odelberg SJ, Ostanin K, Li DY. ARF6 is an actionable node that orchestrates oncogenic GNAQ signaling in uveal melanoma. Cancer Cell 2016; 29( 6): 889– 904

[53] Yu FX, Zhang K, Guan KL. YAP as oncotarget in uveal melanoma. Oncoscience 2014; 1( 7): 480– 481

[54] Onken MD, Makepeace CM, Kaltenbronn KM, Kanai SM, Todd TD, Wang S, Broekelmann TJ, Rao PK, Cooper JA, Blumer KJ. Targeting nucleotide exchange to inhibit constitutively active G protein α subunits in cancer cells. Sci Signal 2018; 11( 546): eaao6852

[55] Yao H, Lan J, Li C, Shi H, Brosseau JP, Wang H, Lu H, Fang C, Zhang Y, Liang L, Zhou X, Wang C, Xue Y, Cui Y, Xu J. Inhibiting PD-L1 palmitoylation enhances T-cell immune responses against tumours. Nat Biomed Eng 2019; 3( 4): 306– 317

[56] Lu Y, Yan JS, Xia L, Qin K, Yin QQ, Xu HT, Gao MQ, Qu XN, Sun YT, Chen GQ. 2-Bromopalmitate targets retinoic acid receptor alpha and overcomes all-trans retinoic acid resistance of acute promyelocytic leukemia. Haematologica 2019; 104( 1): 102– 112

[57] Némati F, de Montrion C, Lang G, Kraus-Berthier L, Carita G, Sastre-Garau X, Berniard A, Vallerand D, Geneste O, de Plater L, Pierré A, Lockhart B, Desjardins L, Piperno-Neumann S, Depil S, Decaudin D. Targeting Bcl-2/Bcl-XL induces antitumor activity in uveal melanoma patient-derived xenografts. PLoS One 2014; 9( 1): e80836

Acknowledgements

We thank Professors Renbing Jia and Xianqun Fan (Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine) for their gift of UM cells. We also thank Dr. Jiale Wu, Dr. Yun Tan, Dr. Hezhou Guo, Dr. Na Liu, Dr. Shanhe Yu, Dr. Jinyan Huang, Changzhou Feng, and Ran Li (Shanghai Institute of Hematology) for insightful discussions. This work was supported by the Key Project of National Natural Science Foundation of China (No. 81530006 to Ruibao Ren), Shanghai Collaborative Innovation Program on Regenerative Medicine and Stem Cell Research (No. 2019CXJQ01 to Ruibao Ren), National Natural Science Foundation of China (No. 81870112 to Ruibao Ren, No. 81770171 to Bo Jiao, and No. 81970134 to Ping Liu), Samuel Waxman Cancer Research Foundation (to Ruibao Ren), and the Innovative Research Team of High-level Local Universities in Shanghai.

Compliance with ethics guidelines

Yan Zhang, Baoyuan Zhang, Yongyun Li, Yuting Dai, Jiaoyang Li, Donghe Li, Zhizhou Xia, Jianming Zhang, Ping Liu, Ming Chen, Bo Jiao, and Ruibao Ren declare that they have no conflict of interests. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000 (5).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11684-021-0911-0 and is accessible for authorized users.

RIGHTS & PERMISSIONS

2022 Higher Education Press