Abstract
Purpose
Uveal melanoma (UM) is the most common intraocular malignant tumor in adults. Due to the lack of effective treatments for metastatic UM, the survival of UM has not changed over the past 3 decades. Therefore, it is important to identify essential genes regulating the metastasis of UM.
Methods
In this study, a genome-wide CRISPR knockout screen in an orthotopic mouse model of UM was performed to identify the regulatory genes conferring the metastatic phenotype. Loss-of-function analyses were performed to explore the function of G protein pathway suppressor 2 (GPS2) in UM metastasis in vitro and in vivo. RNA sequencing was performed to investigate the molecular mechanism underlying the function of GPS2 as a tumor suppressor in UM.
Results
Among the highest-ranking genes, we found several validated tumor suppressors, such as SHPRH, GPS2, PRPH2, and hsa-mir-1229; GPS2 was chosen as the candidate gene for further studies. GPS2 was lower expressed in the tumor tissues of UM patients. Furthermore, knocking-down GPS2 promoted the proliferation and metastatic abilities of UM cells both in vivo and in vitro. Finally, analysis of the transcriptome data revealed that silencing GPS2 upregulates oncogenic signaling pathways MAPK and PI3K-Akt, and in the meantime downregulates tumor suppressor signaling pathway Slit/Robo in UM cells.
Conclusion
Altogether, our study proved that the GPS2 gene functions as a tumor suppressor and might be a novel potential therapeutic target for UM treatment.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Amodeo V, Deli A, Betts J et al (2017) A PML/slit axis controls physiological cell migration and cancer invasion in the CNS. Cell Rep 20(2):411–426. https://doi.org/10.1016/j.celrep.2017.06.0
Bartha Á, Győrffy B (2021) TNMplot.com: a web tool for the comparison of gene expression in normal, tumor and metastatic tissues. Int J Mol Sci 22(5):2622. https://doi.org/10.3390/ijms22052622
Basile MS, Mazzon E, Russo A et al (2019) Differential modulation and prognostic values of immune-escape genes in uveal melanoma. PLoS ONE 14(1):e0210276. https://doi.org/10.1371/journal.pone.0210276
Broggi G, Russo A, Reibaldi M et al (2020) Histopathology and genetic biomarkers of choroidal melanoma. Appl Sci. https://doi.org/10.3390/app10228081
Cao Q, Shi Y, Wang X et al (2019) Circular METRN RNA hsa_circ_0037251 promotes glioma progression by sponging miR-1229–3p and regulating mTOR expression. Sci Rep 9(1):19791. https://doi.org/10.1038/s41598-019-56417-8
Chan S, Smith E, Gao Y et al (2021) Loss of G-protein pathway suppressor 2 promotes tumor growth through activation of AKT signaling. Front Cell Dev Biol. 8:608044. https://doi.org/10.3389/fcell.2020.608044
Chandrashekar DS, Bashel B, Balasubramanya SAH et al (2017) UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses. Neoplasia 19(8):649–658. https://doi.org/10.1016/j.neo.2017.05.0022
Chang A, Chen Y, Shen W et al (2015) Ifit1 protects against lipopolysaccharide and D-galactosamine-induced fatal hepatitis by inhibiting activation of the JNK pathway. J Infect Dis 212(9):1509–1520. https://doi.org/10.1093/infdis/jiv221
Cheng X, Kao HY (2009) G protein pathway suppressor 2 (GPS2) is a transcriptional corepressor important for estrogen receptor alpha-mediated transcriptional regulation. J Biol Chem 284(52):36395–36404. https://doi.org/10.1074/jbc.M109.062109
Dong K, Xue H, Cheng J et al (2019) PRPH2 activates hippo signalling and suppresses the invasion and anoikis inhibition of laryngeal cancer. Cancer Manag Res 11:10107–10115. https://doi.org/10.2147/CMAR.S222527
Fagone P, Caltabiano R, Russo A et al (2017) Identification of novel chemotherapeutic strategies for metastatic uveal melanoma. Sci Rep 7:44564. https://doi.org/10.1038/srep44564
Fang J, Ji WH, Wang FZ et al (2021) Circular RNA hsa_circ_0000700 promotes cell proliferation and migration in esophageal squamous cell carcinoma by sponging miR-1229. J Cancer 12(9):2610–2623. https://doi.org/10.7150/jca.47112
Han X, Zhang MC (2010) Potential anti-angiogenic role of Slit2 in corneal neovascularization. Exp Eye Res 90(6):742–749. https://doi.org/10.1016/j.exer.2010.03.007
Han S, Cao C, Tang T et al (2015) ROBO3 promotes growth and metastasis of pancreatic carcinoma. Cancer Lett 366(1):61–70. https://doi.org/10.1016/j.canlet.2015.06.004
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144(5):646–674. https://doi.org/10.1016/j.cell.2011.02.013
Huang XD, Xiao FJ, Wang SX et al (2016) G protein pathway suppressor 2 (GPS2) acts as a tumor suppressor in liposarcoma. Tumour Biol 37(10):13333–13343. https://doi.org/10.1007/s13277-016-5220-x
Huang K, Liu X, Li Y et al (2019) Genome-wide CRISPR-Cas9 screening identifies NF-κB/E2F6 responsible for EGFRvIII-associated temozolomide resistance in glioblastoma. Adv Sci (weinh). 6(17):1900782. https://doi.org/10.1002/advs.201900782
Hui K, Gao Y, Huang J et al (2017) RASAL2, a RAS GTPase-activating protein, inhibits stemness and epithelial-mesenchymal transition via MAPK/SOX2 pathway in bladder cancer. Cell Death Dis 8(2):e2600. https://doi.org/10.1038/cddis.2017.9
Jarmalavicius S, Trefzer U, Walden P (2010) Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma. FASEB J 24(3):937–946. https://doi.org/10.1096/fj.09-136283
Ji W, Qiu C, Wang M, Mao N, Wu S, Dai Y (2018) Hsa_circ_0001649: a circular RNA and potential novel biomarker for colorectal cancer. Biochem Biophys Res Commun 497(1):122–126. https://doi.org/10.1016/j.bbrc.2018.02.036
Jiang Y, Wang T, Yan L, Qu L (2018) A novel prognostic biomarker for pancreatic ductal adenocarcinoma: hsa_circ_0001649. Gene 675:88–93. https://doi.org/10.1016/j.gene.2018.06.099
Jin DY, Teramoto H, Giam CZ, Chun RF, Gutkind JS, Jeang KT (1997) A human suppressor of c-Jun N-terminal kinase 1 activation by tumor necrosis factor alpha. J Biol Chem 272(41):25816–25823. https://doi.org/10.1074/jbc.272.41.25816
Kivela T, Simpson ER, Grossniklaus HE, Jager MJ, Sough AD, Caminal JM (2002) Uveal melanoma. AJCC cancer staging manual, 8th edn. Springer, New York, pp 805–817
Kolde R, Laur S, Adler P, Vilo J (2012) Robust rank aggregation for gene list integration and meta-analysis. Bioinformatics 28(4):573–580. https://doi.org/10.1093/bioinformatics/btr709
Kong R, Yi F, Wen P et al (2015) Myo9b is a key player in SLIT/ROBO-mediated lung tumor suppression. J Clin Invest 125(12):4407–4420. https://doi.org/10.1172/JCI81673
Lánczky A, Győrffy B (2021) Web-based survival analysis tool tailored for medical research (KMplot): development and Implementation. J Med Internet Res 23(7):e27633. https://doi.org/10.2196/27633
Lee TH, Yi W, Griswold MD, Zhu F, Her C (2006) Formation of hMSH4-hMSH5 heterocomplex is a prerequisite for subsequent GPS2 recruitment. DNA Repair (amst) 5(1):32–42. https://doi.org/10.1016/j.dnarep.2005.07.004
Li W, Xu H, Xiao T et al (2014) MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 15(12):554. https://doi.org/10.1186/s130.59-014-0554-4
Li WH, Song YC, Zhang H et al (2017) Decreased expression of Hsa_circ_00001649 in gastric cancer and its clinical significance. Dis Markers 2017:4587698. https://doi.org/10.1155/2017/4587698
Li J, Qin X, Shi J et al (2021a) A systematic CRISPR screen reveals an IL-20/IL20RA-mediated immune crosstalk to prevent the ovarian cancer metastasis. Elife 10:e66222. https://doi.org/10.7554/eLife.66222
Li J, Wang J, Xie D et al (2021b) Characteristics of the PI3K/AKT and MAPK/ERK pathways involved in the maintenance of self-renewal in lung cancer stem-like cells. Int J Biol Sci 17(5):1191–1202. https://doi.org/10.7150/ijbs.57871
Liu T, Song Z, Gai Y (2018) Circular RNA circ_0001649 acts as a prognostic biomarker and inhibits NSCLC progression via sponging miR-331-3p and miR-338-5p. Biochem Biophys Res Commun 503(3):1503–1509. https://doi.org/10.1016/j.bbrc.2018.07.070
Lv Y, Wang X, Li X et al (2020) Nucleotide de novo synthesis increases breast cancer stemness and metastasis via cGMP-PKG-MAPK signaling pathway. PLoS Biol 18(11):e3000872. https://doi.org/10.1371/journal.pbio.3000872
Madsen RR, Erickson EC, Rueda OM et al (2021) Positive correlation between transcriptomic stemness and PI3K/AKT/mTOR signaling scores in breast cancer, and a counterintuitive relationship with PIK3CA genotype. PLoS Genet 17(11):e1009876. https://doi.org/10.1371/journal.pgen.1009876
Mohankumar KM, Currle DS, White E et al (2015) An in vivo screen identifies ependymoma oncogenes and tumor-suppressor genes. Nat Genet 47(8):878–887. https://doi.org/10.1038/ng.3323
Ng SR, Rideout WM 3rd, Akama-Garren EH et al (2020) CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer. Proc Natl Acad Sci U S A 117(1):513–521. https://doi.org/10.1073/pnas.1821893117
Niederkorn J, Streilein JW, Shadduck JA (1981) Deviant immune responses to allogeneic tumors injected intracamerally and subcutaneously in mice. Invest Ophthalmol vis Sci 20(3):355–363
Paul EV, Parnell BL, Fraker M (1962) Prognosis of malignant melanaomas of the choroid and ciliary body. Int Ophthalmol Clin 2(2):387–402
Peng YC, Kuo F, Breiding DE, Wang YF, Mansur CP, Androphy EJ (2001) AMF1 (GPS2) modulates p53 transactivation. Mol Cell Biol 21(17):5913–5924. https://doi.org/10.1128/MCB.21.17.5913-5924.2001
Qin M, Liu G, Huo X et al (2016) Hsa_circ_0001649: a circular RNA and potential novel biomarker for hepatocellular carcinoma. Cancer Biomark 16(1):161–169. https://doi.org/10.3233/CBM-150552
Rama N, Dubrac A, Mathivet T et al (2015) Slit2 signaling through Robo1 and Robo2 is required for retinal neovascularization. Nat Med 21(5):483–491. https://doi.org/10.1038/nm.3849
Rietschel P, Panageas KS, Hanlon C, Patel A, Abramson DH, Chapman PB (2005) Variates of survival in metastatic uveal melanoma. J Clin Oncol 23(31):8076–8080. https://doi.org/10.1200/JCO.2005.02.6534
Riverso M, Montagnani V, Stecca B (2017) KLF4 is regulated by RAS/RAF/MEK/ERK signaling through E2F1 and promotes melanoma cell growth. Oncogene 36(23):3322–3333. https://doi.org/10.1038/onc.2016.481
Russo D, Di Crescenzo RM, Broggi G et al (2020) Expression of P16INK4a in uveal melanoma: new perspectives. Front Oncol 10:562074. https://doi.org/10.3389/fonc.2020.562074
Shalem O, Sanjana NE, Hartenian E et al (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343(6166):84–87. https://doi.org/10.1126/science.1247005
Si Y, Zhang H, Peng P et al (2021) G protein pathway suppressor 2 suppresses gastric cancer by destabilizing epidermal growth factor receptor. Cancer Sci 112(12):4867–4882. https://doi.org/10.1111/cas.15151
Spain BH, Bowdish KS, Pacal AR et al (1996) Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells. Mol Cell Biol 16(12):6698–6706. https://doi.org/10.1128/MCB.16.12.6698
Stine RR, Greenspan LJ, Ramachandran KV, Matunis EL (2014) Coordinate regulation of stem cell competition by Slit-Robo and JAK-STAT signaling in the Drosophila testis. PLoS Genet 10(11):e1004713. https://doi.org/10.1371/journal.pgen.1004713
Su Y, Xu C, Liu Y, Hu Y, Wu H (2019) Circular RNA hsa_circ_0001649 inhibits hepatocellular carcinoma progression via multiple miRNAs sponge. Aging (albany NY) 11(10):3362–3375. https://doi.org/10.18632/aging.101988
Sun D, Zhu D (2020) Circular RNA hsa_circ_0001649 suppresses the growth of osteosarcoma cells via sponging multiple miRNAs. Cell Cycle 19(20):2631–2643. https://doi.org/10.1080/1538.4101.2020.1814026
Sun H, Wang Q, Yuan G et al (2020) Hsa_circ_0001649 restrains gastric carcinoma growth and metastasis by downregulation of miR-20a. J Clin Lab Anal 34(6):e23235. https://doi.org/10.1002/jcla.23235
Tie J, Pan Y, Zhao L et al (2010) MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor. PLoS Genet 6(3):e1000879. https://doi.org/10.1371/journal.pgen.1000879
Valenti MT, Dalle Carbonare L, Mottes M (2018) Ectopic expression of the osteogenic master gene RUNX2 in melanoma. World J Stem Cells 10(7):78–81. https://doi.org/10.4252/wjsc.v10.i7.78
Wang SM, Tie J, Wang WL et al (2016) POU2F2-oriented network promotes human gastric cancer metastasis. Gut 65(9):1427–1438. https://doi.org/10.1136/gutjnl-2014-308932
Wang Y, Sui X, Zhao H et al (2018) Decreased circular RNA hsa_circ_0001649 predicts unfavorable prognosis in glioma and exerts oncogenic properties in vitro and in vivo. Gene 676:117–122. https://doi.org/10.1016/j.gene.2018.07.037
Wang Z, Kang L, Zhang H et al (2019) AKT drives SOX2 overexpression and cancer cell stemness in esophageal cancer by protecting SOX2 from UBR5-mediated degradation. Oncogene 38(26):5250–5264. https://doi.org/10.1038/s41388-019-0790-x
Wang Y, Lou N, Zuo M et al (2021) STAT3-induced ZBED3-AS1 promotes the malignant phenotypes of melanoma cells by activating PI3K/AKT signaling pathway. RNA Biol 18(sup1):355–368. https://doi.org/10.1080/15476286.2021.1950463
Xing L, Zhang L, Feng Y, Cui Z, Ding L (2018) Downregulation of circular RNA hsa_circ_0001649 indicates poor prognosis for retinoblastoma and regulates cell proliferation and apoptosis via AKT/mTOR signaling pathway. Biomed Pharmacother 105:326–333. https://doi.org/10.1016/j.biopha.2018.05.141
Xu Y, Yao Y, Zhong X et al (2018) Downregulated circular RNA hsa_circ_0001649 regulates proliferation, migration and invasion in cholangiocarcinoma cells. Biochem Biophys Res Commun 496(2):455–461. https://doi.org/10.1016/j.bbrc.2018.01.077
Yang J, Manson DK, Marr BP, Carvajal RD (2018a) Treatment of uveal melanoma: where are we now? Ther Adv Med Oncol. 10:1758834018757175. https://doi.org/10.1177/1758834018757175
Yang T, Huang H, Shao Q, Yee S, Majumder T, Liu G (2018b) miR-92 suppresses Robo1 translation to modulate slit sensitivity in commissural axon guidance. Cell Rep 24(10):2694-2708.e6. https://doi.org/10.1016/j.celrep.2018.08.021
Yu J, Cao Q, Yu J et al (2010) The neuronal repellent SLIT2 is a target for repression by EZH2 in prostate cancer. Oncogene 29(39):5370–5380. https://doi.org/10.1038/onc.2010.269
Zhang X, Dong J, He Y et al (2017) miR-218 inhibited tumor angiogenesis by targeting ROBO1 in gastric cancer. Gene 615:42–49. https://doi.org/10.1016/j.gene.2017.03.022
Zhang X, Qiu S, Luo P et al (2018) Down-regulation of hsa_circ_0001649 in hepatocellular carcinoma predicts a poor prognosis. Cancer Biomark 22(1):135–142. https://doi.org/10.3233/CBM-171109
Zhou WJ, Geng ZH, Chi S et al (2011) Slit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis. Cell Res 21(4):609–626. https://doi.org/10.1038/cr.2011.17
Zhou X, Li R, Jing R, Zuo B, Zheng Q (2020) Genome-wide CRISPR knockout screens identify ADAMTSL3 and PTEN genes as suppressors of HCC proliferation and metastasis, respectively. J Cancer Res Clin Oncol 146(6):1509–1521. https://doi.org/10.1007/s00432-020-03207-9
Acknowledgements
We would like to thank the professors of the School of Medicine, Nankai University, for their kind support in writing this research paper.
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This work was supported by the Science and Technology Project of the Health Committee of Binhai New Area (2019BWKZ009).
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All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by SS and HC. TW contributed to the design of this study. HW and JW were involved in the animal experiments. The first draft of the manuscript was written by FS and LW, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Supplementary file1: Representative H&E-stained sections of the liver, brain, and spleen of BALB/C nude mice orthotopically transplanted with the indicated OCM1 cells. (TIF 2982 KB)
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Shi, S., Chen, H., Wang, H. et al. Genome-wide CRISPR knockout screening identified G protein pathway suppressor 2 as a novel tumor suppressor for uveal melanoma metastasis. J Cancer Res Clin Oncol 149, 3349–3360 (2023). https://doi.org/10.1007/s00432-022-04160-5
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DOI: https://doi.org/10.1007/s00432-022-04160-5