Relationship between ZHX2 Expression and VHL Gene Alteration in VHL-associated and Sporadic Hemangioblastomas of the Central Nervous System

Main Article Content

Hiroshi Kanno
Kimihiro Nakahara
Sachiko Yamada
Satoshi Fujii
Hidetoshi Murata
Tetsuya Yamamoto
Hisashi Hasumi
Masahiro Yao

Keywords

central nervous system hemangioblastoma, gene alteration, VHL, ZHX2

Abstract

Central nervous system hemangioblastoma (CNS-HB) is the most common manifestation of von Hippel–Lindau disease (VHL). The main axis of the CNS-HB pathway is the VHL-HIF signaling pathway. Recently, we proposed an alternative VHL-JAK-STAT pathway in CNS-HB. In contrast, the VHL substrate transcription factor zinc fingers and homeoboxes 2 (ZHX2) have been identified as the oncogenic drivers in VHL-deficient clear cell renal cell carcinoma (RCC). However, ZHX2 expression in CNS-HB has not been previously reported. Furthermore, the VHL-ZXH2-NF-κB signaling pathway in CNS-HB remains unresolved. In this study, we aimed to investigate ZHX2 expression and VHL gene alteration in CNS-HB and propose the role of ZHX2 in CNS-HB. Using the MACS method, Scl+ hemangioblastoma-like cells were isolated from multipotent nestin-expressing stem cells. The ubiquitination of ZHX2 in these cells and the immunoprecipitation between ZHX2 and VHL were investigated. In addition, the VHL genes of patients with hemangioblastoma were analyzed. ZHX2 expression in CNS-HB tissues was examined by immunohistochemistry and western blotting. In addition, VHL gene mutations in CNS-HB were analyzed by sequencing. The association between ZHX2 expression and VHL gene mutation was analyzed. ZHX2 was ubiquitinated in Scl+hemangioblastoma-like cells after the transfer of the VHL expression vector into these cells. ZHX2 expression in these cells was well detected before transfer but disappeared after the transfer. ZHX2 expression was detected in 18 of the 21 CNS-HB tissues by immunoblotting and/or immunohistochemistry. Sporadic CNS-HB showed weak expression, whereas VHL-related CNS-HB showed moderate or strong expression. In particular, CNS-HB with severe VHL gene mutations, including large deletions, showed strong or moderate ZHX2 expression. The association between VHL gene mutation and ZHX2 expression revealed a significant correlation between VHL gene alteration severity and the level of immunoblotting (P < 0.05). In conclusion, the severity of VHL gene alteration correlates with the level of ZHX2 expression. ZHX2 is predominantly expressed in CNS-HB, especially in VHL-related cases with severe VHL gene alterations, suggesting a potential role in tumorigenesis and proliferation of CNS-HB.

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References

1. Tse JY, Wong JH, Lo KW, Poon WS, Huang DP, Ng HK. Molecular genetic analysis of the von Hippel–Lindau disease tumor suppressor gene in familial and sporadic cerebellar hemangioblastomas. Am J Clin Pathol. 1997;107(4):459–66. 10.1093/ajcp/107.4.459

2. Latif F, Tory K, Gnarra J, Yao M, Duh F, Orcutt ML, et al. Identification of the von Hippel–Lindau disease tumor suppressor gene. Science. 1993;260 (5112):1317–20. 10.1126/science.8493574

3. Takayanagi S, Mukasa A, Tanaka S, Nomura M, Omata M, Yanagisawa S, et al. Differences in genetic and epigenetic alterations between von Hippel–Lindau disease-related and sporadic hemangioblastomas of the central nervous system. Neuro Oncol. 2017;19(9):1228–36. 10.1093/neuonc/nox034

4. Stebbins CE, Kaelin WG, Pavletich NP. Structure of the VHL–elongin C–elongin B complex: Implications for VHL tumor suppressor function. Science. 1999;284(5413):455–61. 10.1126/science.284.5413.455

5. Maxwell P, Wiesener MS, Chang GW, Clifford SC, Vaux EC, Cockman ME, et al. The von Hippel–Lindau gene product is necessary for oxgyen-dependent proteolysis of hypoxia-inducible factor-β subunits. Nature. 1999;399(6733):271–5. 10.1038/20459

6. Flamme I, Krieg M, Plate KH. Up-regulation of vascular endothelial growth factor in stromal cells of hemangioblastomas is correlated with up-regulation of the transcription factor HRF/HIF-2alpha. Am J Pathol. 1998;153(1):25–9. 10.1016/s0002-9440(10)65541-1

7. Trimble M, Caro J, Talalla A, Brain M. Secondary erythrocytosis due to a cerebellar hemangioblastoma: Demonstration of erythropoietin mRNA in the tumor. Blood. 1991;78(3):599–601. 10.1182/blood.V78.3.599.599

8. Iliopoulos O, Levy AP, Jiang C, Kaelin WG Jr, Goldberg MA. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein. Proc Natl Acad Sci USA. 1996;93:10595–9. 10.1073/pnas.93.20.10595

9. Krieg M, Marti HH, Plate KH. Coexpression of erythropoietin and vascular endothelial growth factor in nervous system tumors associated with von Hippel–Lindau tumor suppressor gene loss of function. Blood. 1998;92(9):3388–93. 10.1182/blood.V92.9.3388

10. Feldman MJ, Sizdahkhani S, Edwards NA, Merrill MJ, Ray-Chaudhury A, Zhuang Z, et al. Loss of quiescence in von Hippel–Lindau hemangioblastomas is associated with erythropoietin signaling. Sci Rep. 2016;6:35486. 10.1038/srep35486

11. Kanno H, Yoshizumi T, Shinonaga M, Kubo A, Murata H, Yao M. Role of VHL-JAK-STAT signaling pathway in central nervous system hemangioblastoma associated with von Hippel–Lindau disease. J Neurooncol. 2020;148(1):29–38. 10.1007/s11060-020-03506-8

12. Yue X, Zhang Z, Liang X, Gao L, Zhang X, Zhao D, et al. Zinc fingers and homeoboxes 2 inhibits hepatocellular carcinoma cell proliferation and represses expression of Cyclins A and E. Gastroenterology. 2012;142(7):1559–70.e2. 10.1053/j.gastro.2012.02.049

13. Nagel S, Schneider B, Meyer C, Kaufmann M, Drexler HG, Macleod RA. Transcriptional deregulation of homeobox gene ZHX2 in Hodgkin lymphoma. Leuk Res. 2012;36(5):646–55. 10.1016/j.leukres.2011.10.019

14. Kwon RJ, Kim YH, Jeong DC, Han ME, Kim JY, Liu L, et al. Expression and prognostic significance of zinc fingers and homeoboxes family members in renal cell carcinoma. PLoS One. 2017;12(2):e0171036. 10.1371/journal.pone.0171036

15. Zhang J, Wu T, Simon J, Takada M, Saito R, Fan C, et al. VHL substrate transcription factor ZHX2 as an oncogenic driver in clear cell renal cell carcinoma. Science. 2018;361(6399):290–5. 10.1126/science.aap8411

16. Yoshida M, Ashida K, Kondo K, Kobayashi H, Kanno H, Shinohara N, et al. Shitara N, Kishida T, Kawakami S, Baba M, Yamamoto I, Hosaka M, Shuin T, Yao M Germ-line mutation analysis in patients with von Hippel-Lindau disease in Japan: An extended study of 77 families. Jpn J Cancer Res 2000; 91 (2):204–212. 10.1111/j.1349-7006.2000.tb00933.x

17. Hattori K, Teranishi J, Stolle C, Yoshida M, Kondo K, Kishida T, et al. Detection of germline deletions using real-time quantitative polymerase chain reaction in Japanese patients with von Hippel-Lindau disease. Cancer Sci. 2006;97(5):400–5. 10.1111/j.1349-7006.2006.00193.x

18. Kanno H, Kubo A, Yoshizumi T, Mikami T, Maegawa J. Isolation of multipotent nestin-expressing stem cells-derived from the epidermis of elderly humans and TAT-VHL peptide-mediated neuronal differentiation of these cells. Int J Mol Sci. 2013;14(5):9604–17. 10.3390/ijms14059604

19. Park DM, Zhuang Z, Chen L, Szerlip N, Maric I, Li J, Sohn T, et al. von Hippel–Lindau disease associated hemangioblastomas are derived from embryologic multipotent cells. PLoS Med. 2007;4(2):e60. 10.1371/journal.pmed.0040060

20. Takada S, Hojo M, Takebe N, Tanigaki K, Miyamoto S. Stromal cells of hemangioblastomas exhibit mesenchymal stem cell-derived vascular progenitor cell properties. Brain Tumor Pathol. 2018;35(4):193–201. 10.1007/s10014-018-0323-2

21. Li M, Song J, Pytel P. Expression of HIF-1 regulated proteins vascular endothelial growth factor, carbonic anhydrase IX and hypoxia inducible gene 2 in hemangioblastomas. Folia Neuropathol. 2014;52(3):234–42. 10.5114/fn.2014.45564

22. Mukherjee T, Kim WS, Mandal L, Banerjee U. Interaction between Notch and Hif-alpha in development and survival of drosophila blood cells. Science. 2011;332(6034):1210–3. 10.1126/scien ce.11996 43

23. Vihanto MM, Plock J, Erni D, Frey BM, Frey FJ, Huynh-Do U. Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin. FASEB J. 2005;19(12):1689–91. 10.1096/fj.04-3647fje

24. Zagzag D, Krishnamachary B, Yee H, Okuyama H, Chiriboga L, Ali MA, et al. Stromal cell-derived factor-1A and CXCR4 expression in hemangioblastoma and clear cell-renal cell carcinoma: Von Hippel–Lindau loss-of-function induces expression of a ligand and its receptor. Cancer Res. 2005;65(14):6178–88. 10.1158/0008-5472.CAN-04-4406

25. Lappin TR, Lee FS. Update on mutations in the HIF: EPO pathway and their role in erythrocytosis. Blood Rev. 2019;37:100590. 10.1016/j.blre.2019.100590

26. Kaelin WG Jr. The VHL tumor suppressor gene: Insights into oxygen sensing and cancer. Trans Am Clin Climatol Assoc. 2017;128:298–307.

27. Kaelin WG Jr. The von Hippel-Lindau tumour suppressor protein: O2 sensing and cancer. Nat Rev Cancer. 2008;8(11):865–73. 10.1038/nrc250223

28. Kanno H, Sato H, Yokoyama TA, Yoshizumi T, Yamada S. The VHL tumor suppressor protein regulates tumorigenicity of U87-derived glioma stem-like cells by inhibiting the JAK/STAT signaling pathway. Int J Oncol. 2013;42(3):881–6. 10.3892/ijo.2013.1773

29. Pierscianek D, Wolf S, Keyvani K, El Hindy N, Stein KP, Sandalcioglu IE, et al. Study of angiogenic signaling pathways in hemangioblastoma. Neuropathology. 2017;37(1):3–11. 10.1111/neup.12316

30. Tarade D, Ohh M. The HIF and other quandaries in VHL disease. Oncogene. 2018;37(2):139–47. 10.1038/onc.2017.338

31. Dai H, Wang C, Yu Z, He D, Yu K, Liu Y, et al. MiR-17 regulates prostate cancer cell proliferation and apoptosis through inhibiting JAK-STAT3 signaling pathway. Cancer Biother Radiopharm. 2018;33(3):103–9. 10.1089/cbr.2017.2386

32. Dolatabadi S, Jonasson E, Lindén M, Fereydouni B, Bäcksten K, Nilsson M, et al. JAK-STAT signalling controls cancer stem cell properties including chemotherapy resistance in myxoid liposarcoma. Int J Cancer. 2019;145:435–49. 10.1002/ijc.32123

33. Villarino AV, Kanno Y, O’Shea JJ. Mechanisms and consequences of Jak-STAT signaling in the immune system. Nat Immunol. 2017;18(4):374–84. 10.1038/ni.3691

34. Xu CH, Liu Y, Xiao LM, Chen LK, Zheng SY, Zeng EM, et al. Silencing microRNA-221/222 cluster suppresses glioblastoma angiogenesis by suppressor of cytokine signaling-3-dependent JAK/STAT pathway. J Cell Physiol. 2019;234(12):22272–84. 10.1002/jcp.28794

35. Zhang Y, Wang D, Xu J, Wang Y, Ma F, Li Z, et al. Stat3 activation is critical for pluripotency maintenance. J Cell Physiol. 2019;234(2):1044–51. 10.1002/jcp.27241

36. Fang W, Liao C, Shi R, Simon JM, Ptacek TS, Zurlo G, et al. ZHX2 promotes HIF1β oncogenic signaling in triple-negative breast cancer. Elife. 2021;10:e70412. 10.7554/eLife.70412