CellBio  Vol.6 No.3 , September 2017
N-Myc Inhibition: Advances in Neuroblastoma Treatment
Abstract: Neuroblastoma (NBL) is one of the most common solid tumors and around 15% of cancer mortality in children. Amplification of the N-Myc proto-oncogene is strongly correlated with advanced disease and poor clinical outcome in NBL. Recent studies described that ubiquitin-specific protease 7 (USP7; also known as HAUSP) interacts with N-Myc, induces deubiquitination and subsequent stabilization of N-Myc that in-turn potentiates N-Myc function, and treatment with the HAUSP inhibitor (P22077) blocked such effects.

1. Introduction

Neuroblastoma (NBL), the most common solid tumor of childhood, is originated from the neural crest cells of the developing sympathetic nervous system [1] . It represents 8% - 10% of pediatric tumors and accounts for 15% of all pediatric cancer deaths [2] [3] [4] . In the recent years, considerable progress has been made in the treatment effect of NBL including chemotherapy, radiotherapy, surgical resection and hematopoietic stem cell transplantation; however the 5-year overall survival (OS) is still less than 50% in high-risk NBL [4] [5] .

The genetic feature most consistently associated with treatment failure is an amplification of the N-Myc proto-oncogene, which is strongly correlated with advanced disease and poor prognosis [6] [7] [8] [9] [10] . Generally, amplification of N-Myc occurred in neuroblastoma based on the mechanisms involving double minutes (dmin) or homogeneously staining regions (hsr) [11] . Expression of N-Myc is associated with accelerated proliferation, migration, invasion and metastasis [3] [12] [13] [14] [15] . Consistence with these evidences, N-Myc transgene can induce tumor formation in transgenic mice [16] , and N-Myc-knockout mouse shows embryonic lethality [17] [18] [19] , whereas the Nes-Cre-driven conditional knockout of N-Myc has a decrease in cerebellar and cerebral cortex mass due to defective cellular proliferation [20] .

At the early stage of NBL, it is often clinically unrecognized [21] . The primary tumor usually occurs in the abdomen (60%), but neuroblastoma children present with metastasis more than 50% at diagnosis [2] . NBL metastasis is usually present in the bone marrow (70.5%) or the skeleton (55.7%); patients may also present with metastasis in the lymph nodes (30.9%), liver (29.6%), or intracranial and orbital sites (18.2%) [22] . Recently, Yue Z-X. et al. demonstrated that clinical outcome was poorer in NBL patients metastases to bone marrow with N-Myc amplification than in those without amplification [23] .

2. Results

2.1. PI3K/mTOR/N-Myc Inhibition

Both C-Myc and N-Myc contribute to the regulation of VEGF and angiogenesis. Like C-Myc, N-Myc is stabilized by activation of phosphatidylinositol 3-kinase (PI3K) [24] , and inhibition of PI3K and mTOR (mammalian target of rapamycin) leading to reduced secretion of VEGF and decreased levels of N-Myc protein [25] [26] [27] . Moreover, Chanthery Y.H. et al. demonstrated that a clinical PI3K/mTOR inhibitor, NVP-BEZ235, decreased angiogenesis and improved survival on N-Myc dependent mechanism in both primary human (highly pretreated recurrent N-Myc-amplified orthotopic xenograft) and transgenic mouse models for N-Myc-driven neuroblastoma, suggesting that NVP-BEZ235 should be tested in children with high-risk, N-Myc-amplified neuroblastoma [28] .

2.2. PARP1/N-Myc Inhibition

Poly (ADP-ribose) polymerase (PARP) is involved in a number of cellular processes such as DNA repair, genomic stability, and programmed cell death in the response to numerous endogenous and environmental genotoxic agents [29] [30] [31] . Survival studies of PARP knockout (PARP-/-) mice after γ-irradiation showed that, PARP-/- mice died within 10 days post-irradiation compared to wild-type controls, those remained apparently healthy [29] . Recently, Colicchia V. et al. described that higher expression of PARP1 was associated with poor clinical outcome in NBL patients [32] . Moreover, PARP1 is highly expressed in N-Myc amplified and advanced stages compared to N-Myc non-amplified and lower stages in primary NBL or NBL cell lines; supporting N-Myc inhibition might be a promising developmental therapy in NBL.

2.3. PD-L1/N-Myc Inhibition

Cancer immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies [33] . Cancer cells frequently produced factors such

Figure 1. Schematic diagram of model shows the association between HAUSP and N-MYC, and how HAUSP inhibitor (P22077) can facilitate the degradation of N-MYC protein that in-turn suppressed Neuroblastoma (NBL) cell proliferation & tumor growth. Courtesy of Tavana O. et al. (modified by M.K. Hasan).

as PD-L1, adenosine, IL-10 and TGF-β that bind negative regulatory surface receptors expressed on cytotoxic T cells [34] - [39] . Tumor cells expressed PD-L1 on the surface and prevent binding of its inhibitory receptor PD-1 on T cells [40] . Targeting PD-L1 or PD-1 by mAb immunotherapies was shown to have pronounced anti-tumor activity in clinical trials [34] [35] [41] [42] . Recently, Melaiu O. et al. described that higher level of PD-L1 expression was correlated to unfavorable prognosis in NBL patients [43] . PD-L1 expression was observed higher with N-Myc amplification in NBL patients and cell lines. Moreover, N-Myc blockade causes suppression of PD-L1 expression in NBL; suggesting N-Myc-inhibition therapy could restore an efficient anti-tumor immunity in high-risk neuroblastoma.

2.4. HAUSP/N-Myc Inhibition

Ubiquitin-specific protease 7 (USP7; also known as HAUSP) is a ubiquitin specific protease or a deubiquitylating enzyme that cleaves ubiquitin from its substrates [44] . As ubiquitylation process (polyubiquitination) is most commonly associated with the stability and degradation of cellular proteins, HAUSP activity generally stabilizes its substrate proteins. In cancer biology, HAUSP have important role for the modulation of the stability and activity of several cellular proteins [45] - [50] .

Recently, Tavana O. et al. found that ubiquitin-specific protease 7 (USP7 or HAUSP) directly interacts with N-Myc, and HAUSP expression induces deubiquitination and subsequent stabilization of N-Myc [51] . RNA interference (RNAi)-mediated knockdown of USP7 in neuroblastoma cancer cell lines, or genetic ablation of Usp7 in the mouse brain, inhibits stabilization of N-Myc protein, which leads to suppression of N-Myc function. Structural analysis revealed that amino acids from 281 to 345 region of N-Myc protein are necessary for this interaction.

Moreover, high expression of HAUSP in patients with neuroblastoma is associated with poor prognosis, and significantly correlates with N-Myc transcriptional activity. Treatment with the small-molecule inhibitor of HAUSP (P22077) suppressed cell proliferation, and the growth of xenograft tumor models in mouse derived from N-Myc-amplified human neuroblastoma cell lines. (See Figure 1).

3. Conclusion

Overall results suggesting that inhibition of N-Myc might have important applications for the treatment of NBL patients with N-Myc amplification.

Cite this paper: Hasan, M. (2017) N-Myc Inhibition: Advances in Neuroblastoma Treatment. CellBio, 6, 27-34. doi: 10.4236/cellbio.2017.63003.
References

[1]   Davidoff, A.M. (2012) Neuroblastoma. Seminars in Pediatric Surgery, 21, 2-14.
https://doi.org/10.1053/j.sempedsurg.2011.10.009

[2]   Morandi, F., Corrias, M.V. and Pistoia, V. (2015) Evaluation of Bone Marrow as a Metastatic Site of Human Neuroblastoma. Annals of the New York Academy of Sciences, 1335, 23-31.
https://doi.org/10.1111/nyas.12554

[3]   Hasan, M.K., Nafady, A., Takatori, A., Kishida, S., Ohira, M., Suenaga, Y., et al. (2013) ALK is a MYCN Target Gene and Regulates Cell Migration and Invasion in Neuroblastoma. Scientific Reports, 3, 3450.
https://doi.org/10.1038/srep03450

[4]   Domingo-Fernandez, R., Watters, K., Piskareva, O., Stallings, R.L. and Bray, I. (2013) The Role of Genetic and Epigenetic Alterations in Neuroblastoma Disease Pathogenesis. Pediatric Surgery International, 29, 101-119.
https://doi.org/10.1007/s00383-012-3239-7

[5]   Chaturvedi, N.K., McGuire, T.R., Coulter, D.W., Shukla, A., McIntyre, E.M., Sharp, J.G., et al. (2016) Improved Therapy for Neuroblastoma Using a Combination Approach: Superior Efficacy with Vismodegib and Topotecan. Oncotarget, 7, 15215-15229.
https://doi.org/10.18632/oncotarget.7714

[6]   Brodeur, G.M., Seeger, R.C. and Schwab, M., Varmus, H.E., Bishop, J.M. (1984) Amplification of N-myc in Untreated Human Neuroblastomas Correlates with Advanced Disease Stage. Science, 224, 1121-1124.
https://doi.org/10.1126/science.6719137

[7]   Seeger, R.C., Brodeur, G.M., Sather, H., Dalton, A., Siegel, S.E., Wong, K.Y., et al. (1985) Association of Multiple Copies of the N-myc Oncogene with Rapid Progression of Neuroblastomas. The New England Journal of Medicine, 313, 1111-1116.
https://doi.org/10.1056/NEJM198510313131802

[8]   Kohl, N.E., Gee, C.E. and Alt, F.W. (1984) Activated Expression of the N-myc Gene in Human Neuroblastomas and Related Tumors. Science, 226, 1335-1337.
https://doi.org/10.1126/science.6505694

[9]   Riley, R.D., Heney, D., Jones, D.R., Sutton, A.J., Lambert, P.C., Abrams, K.R., et al. (2004) A Systematic Review of Molecular and Biological Tumor Markers in Neuroblastoma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research, 10, 4-12.

[10]   Huang, M. and Weiss, W.A. (2013) Neuroblastoma and MYCN. Cold Spring Harbor Perspectives in Medicine, 3, a014415.
https://doi.org/10.1101/cshperspect.a014415

[11]   Storlazzi, C.T., Lonoce, A., Guastadisegni, M.C., Trombetta, D., D’Addabbo, P., Daniele, G., et al. (2010) Gene Amplification as Double Minutes or Homogeneously Staining Regions in Solid Tumors: Origin and Structure. Genome Research, 20, 1198-1206.
https://doi.org/10.1101/gr.106252.110

[12]   Lutz, W., Stohr, M., Schurmann, J., Wenzel, A., Lohr, A. and Schwab, M. (1996) Conditional Expression of N-Myc in Human Neuroblastoma Cells Increases Expression of Alpha-Prothymosin and Ornithine Decarboxylase and Accelerates Progression into S-Phase Early after Mitogenic Stimulation of Quiescent Cells. Oncogene, 13, 803-812.

[13]   Bernards, R., Dessain, S.K. and Weinberg, R.A. (1986) N-myc Amplification Causes Down-Modulation of MHC Class I Antigen Expression in Neuroblastoma. Cell, 47, 667-674.
https://doi.org/10.1016/0092-8674(86)90509-X

[14]   Goodman, L.A., Liu, B.C., Thiele, C.J., Schmidt, M.L., Cohn, S.L., Yamashiro, J.M., et al. (1997) Modulation of N-Myc Expression Alters the Invasiveness of Neuroblastoma. Clinical & Experimental Metastasis, 15, 130-139.
https://doi.org/10.1023/A:1018448710006

[15]   Tanaka, N. and Fukuzawa, M. (2008) MYCN Downregulates Integrin Alpha1 to Promote Invasion of Human Neuroblastoma Cells. International Journal of Oncology, 33, 815-821.

[16]   Weiss, W.A., Aldape, K., Mohapatra, G., Feuerstein, B.G. and Bishop, J.M. (1997) Targeted Expression of MYCN Causes Neuroblastoma in Transgenic Mice. The EMBO Journal, 16, 2985-2995.
https://doi.org/10.1093/emboj/16.11.2985

[17]   Charron, J., Malynn, B.A., Fisher, P., Stewart, V., Jeannotte, L., Goff, S.P., et al. (1992) Embryonic Lethality in Mice Homozygous for a Targeted Disruption of the N-Myc Gene. Genes & Development, 6, 2248-2257.
https://doi.org/10.1101/gad.6.12a.2248

[18]   Stanton, B.R., Perkins, A.S., Tessarollo, L., Sassoon, D.A. and Parada, L.F. (1992) Loss of N-myc Function Results in Embryonic Lethality and Failure of the Epithelial Component of the Embryo to Develop. Genes & Development, 6, 2235-2247.
https://doi.org/10.1101/gad.6.12a.2235

[19]   Sawai, S., Shimono, A., Wakamatsu, Y., Palmes, C., Hanaoka, K. and Kondoh, H. (1993) Defects of Embryonic Organogenesis Resulting from Targeted Disruption of the N-Myc Gene in the Mouse. Development, 117, 1445-1455.

[20]   Knoepfler, P.S., Cheng, P.F. and Eisenman, R.N. (2002) N-myc Is Essential during Neurogenesis for the Rapid Expansion of Progenitor Cell Populations and the Inhibition of Neuronal Differentiation. Genes & Development, 16, 2699-2712.
https://doi.org/10.1101/gad.1021202

[21]   Brodeur, G.M. and Bagatell, R. (2014) Mechanisms of Neuroblastoma Regression. Nature Reviews Clinical Oncology, 11, 704-713.
https://doi.org/10.1038/nrclinonc.2014.168

[22]   DuBois, S.G., Kalika, Y., Lukens, J.N., Brodeur, G.M., Seeger, R.C., Atkinson, J.B., et al. (1999) Metastatic Sites in Stage IV and IVS Neuroblastoma Correlate with Age, Tumor Biology, and Survival. Journal of Pediatric Hematology/Oncology, 21, 181-189.
https://doi.org/10.1097/00043426-199905000-00005

[23]   Yue, Z.X., Huang, C., Gao, C., Xing, T.Y., Liu, S.G., Li, X.J., et al. (2017) MYCN Amplification Predicts Poor Prognosis Based on Interphase Fluorescence in Situ Hybridization Analysis of Bone Marrow Cells in Bone Marrow Metastases of Neuroblastoma. Cancer Cell International, 17.

[24]   Kenney, A.M., Widlund, H.R. and Rowitch, D.H. (2004) Hedgehog and PI-3 Kinase Signaling Converge on Nmyc1 to Promote Cell Cycle Progression in Cerebellar Neuronal Precursors. Development, 131, 217-228.
https://doi.org/10.1242/dev.00891

[25]   Kang, J., Rychahou, P.G., Ishola, T.A., Mourot, J.M., Evers, B.M. and Chung, D.H. (2008) N-myc Is a Novel Regulator of PI3K-Mediated VEGF Expression in Neuroblastoma. Oncogene, 27, 3999-4007.
https://doi.org/10.1038/onc.2008.15

[26]   Chesler, L., Schlieve, C., Goldenberg, D.D., Kenney, A., Kim, G., McMillan, A., et al. (2006) Inhibition of Phosphatidylinositol 3-Kinase Destabilizes Mycn Protein and Blocks Malignant Progression in Neuroblastoma. Cancer Research, 66, 8139-8146.
https://doi.org/10.1158/0008-5472.CAN-05-2769

[27]   Johnsen, J.I., Segerstrom, L., Orrego, A., Elfman, L., Henriksson, M., Kagedal, B., et al. (2008) Inhibitors of Mammalian Target of Rapamycin Downregulate MYCN Protein Expression and Inhibit Neuroblastoma Growth in Vitro and in Vivo. Oncogene, 27, 2910-2922.
https://doi.org/10.1038/sj.onc.1210938

[28]   Chanthery, Y.H., Gustafson, W.C., Itsara, M., Persson, A., Hackett, C.S., Grimmer, M., et al. (2012) Paracrine Signaling through MYCN Enhances Tumor-Vascular Interactions in Neuroblastoma. Science Translational Medicine, 4, 115ra113.
https://doi.org/10.1126/scitranslmed.3002977

[29]   Herceg, Z. and Wang, Z.Q. (2001) Functions of poly(ADP-ribose) Polymerase (PARP) in DNA Repair, Genomic Integrity and Cell Death. Mutation Research, 477, 97-110.
https://doi.org/10.1016/S0027-5107(01)00111-7

[30]   Jubin, T., Kadam, A., Jariwala, M., Bhatt, S., Sutariya, S., Gani, A.R., et al. (2016) The PARP Family: Insights into Functional Aspects of Poly (ADP-ribose) Polymerase-1 in Cell Growth and Survival. Cell Proliferation, 49, 421-437.
https://doi.org/10.1111/cpr.12268

[31]   De la Lastra, C.A., Villegas, I. and Sanchez-Fidalgo, S. (2007) Poly(ADP-ribose) Polymerase Inhibitors: New Pharmacological Functions and Potential Clinical Implications. Current Pharmaceutical Design, 13, 933-962.
https://doi.org/10.2174/138161207780414241

[32]   Colicchia, V., Petroni, M., Guarguaglini, G., Sardina, F., Sahun-Roncero, M., Carbonari, M., et al. (2017) PARP Inhibitors Enhance Replication Stress and Cause Mitotic Catastrophe in MYCN-Dependent Neuroblastoma. Oncogene.
https://doi.org/10.1038/onc.2017.40

[33]   Vinay, D.S., Ryan, E.P., Pawelec, G., Talib, W.H., Stagg, J., Elkord, E., et al. (2015) Immune Evasion in Cancer: Mechanistic Basis and Therapeutic Strategies. Seminars in Cancer Biology, 35, S185-S198.
https://doi.org/10.1016/j.semcancer.2015.03.004

[34]   Brahmer, J.R., Tykodi, S.S., Chow, L.Q., Hwu, W.J., Topalian, S.L., Hwu, P., et al. (2012) Safety and Activity of Anti-PD-L1 Antibody in Patients with Advanced Cancer. The New England Journal of Medicine, 366, 2455-2465.
https://doi.org/10.1056/NEJMoa1200694

[35]   Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., McDermott, D.F., et al. (2012) Safety, Activity, and Immune Correlates of anti-PD-1 Antibody in Cancer. The New England Journal of Medicine, 366, 2443-2454.
https://doi.org/10.1056/NEJMoa1200690

[36]   Zhang, B. (2010) CD73: A Novel Target for Cancer Immunotherapy. Cancer Research, 70, 6407-6411.
https://doi.org/10.1158/0008-5472.CAN-10-1544

[37]   Hoskin, D.W., Mader, J.S., Furlong, S.J., Conrad, D.M. and Blay, J. (2008) Inhibition of T Cell and Natural Killer Cell Function by Adenosine and Its Contribution to Immune Evasion by Tumor Cells (Review). International Journal of Oncology, 32, 527-535.
https://doi.org/10.3892/ijo.32.3.527

[38]   Mosser, D.M. and Zhang, X. (2008) Interleukin-10: New Perspectives on an Old Cytokine. Immunological Reviews, 226, 205-218.
https://doi.org/10.1111/j.1600-065X.2008.00706.x

[39]   Thomas, D.A. and Massague, J. (2005) TGF-Beta Directly Targets Cytotoxic T Cell Functions during Tumor Evasion of Immune Surveillance. Cancer Cell, 8, 369-380.
https://doi.org/10.1016/j.ccr.2005.10.012

[40]   Dondero, A., Pastorino, F., Della Chiesa, M., Corrias, M.V., Morandi, F., Pistoia, V., et al. (2016) PD-L1 Expression in Metastatic Neuroblastoma as an Additional Mechanism for Limiting Immune Surveillance. Oncoimmunology, 5, e1064578.
https://doi.org/10.1080/2162402X.2015.1064578

[41]   Ansell, S.M., Lesokhin, A.M., Borrello, I., Halwani, A., Scott, E.C., Gutierrez, M., et al. (2015) PD-1 Blockade with Nivolumab in Relapsed or Refractory Hodgkin’s Lymphoma. The New England Journal of Medicine, 372, 311-319.
https://doi.org/10.1056/NEJMoa1411087

[42]   Kopp, L.M. and Katsanis, E. (2016) Targeted Immunotherapy for Pediatric Solid Tumors. Oncoimmunology, 5, e1087637.

[43]   Melaiu, O., Mina, M., Chierici, M., Boldrini, R., Jurman, G., Romania, P., et al. (2017) PD-L1 Is a Therapeutic Target of the Bromodomain Inhibitor JQ1 and, Combined with HLA Class I, a Promising Prognostic Biomarker in Neuroblastoma. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research.

[44]   Holowaty, M.N., Sheng, Y., Nguyen, T., Arrowsmith, C. and Frappier, L. (2003) Protein Interaction Domains of the Ubiquitin-Specific Protease, USP7/HAUSP. The Journal of Biological Chemistry, 278, 47753-47761.
https://doi.org/10.1074/jbc.M307200200

[45]   Nicholson, B. and Suresh Kumar, K.G. (2011) The Multifaceted Roles of USP7: New Therapeutic Opportunities. Cell Biochemistry and Biophysics, 60, 61-68.
https://doi.org/10.1007/s12013-011-9185-5

[46]   Van der Horst, A., de Vries-Smits, A.M., Brenkman, A.B., van Triest, M.H., van den Broek, N., Colland, F., et al. (2006) FOXO4 Transcriptional Activity Is Regulated by Monoubiquitination and USP7/HAUSP. Nature Cell Biology, 8, 1064-1073.
https://doi.org/10.1038/ncb1469

[47]   Song, M.S., Salmena, L., Carracedo, A., Egia, A., Lo-Coco, F., Teruya-Feldstein, J., et al. (2008) The Deubiquitinylation and Localization of PTEN Are Regulated by a HAUSP-PML Network. Nature, 455, 813-817.
https://doi.org/10.1038/nature07290

[48]   Du, Z., Song, J., Wang, Y., Zhao, Y., Guda, K., Yang, S., et al. (2010) DNMT1 Stability Is Regulated by Proteins Coordinating Deubiquitination and Acetylation-Driven Ubiquitination. Science Signaling, 3, ra80.
https://doi.org/10.1126/scisignal.2001462

[49]   Faesen, A.C., Dirac, A.M., Shanmugham, A., Ovaa, H., Perrakis, A. and Sixma, T.K. (2011) Mechanism of USP7/HAUSP Activation by Its C-Terminal Ubiquitin-Like Domain and Allosteric Regulation by GMP-Synthetase. Molecular Cell, 44, 147-159.
https://doi.org/10.1016/j.molcel.2011.06.034

[50]   Pfoh, R., Lacdao, I.K. and Saridakis, V. (2015) Deubiquitinases and the New Therapeutic Opportunities Offered to Cancer. Endocrine-Related Cancer, 22, T35-T54.
https://doi.org/10.1530/ERC-14-0516

[51]   Tavana, O., Li, D., Dai, C., Lopez, G., Banerjee, D., Kon, N., et al. (2016) HAUSP Deubiquitinates and Stabilizes N-Myc in Neuroblastoma. Nature Medicine.
https://doi.org/10.1038/nm.4180

 
 
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