CHD4 and NOX4 expression in thyroid tumor tissues

Abstract

Aim: Chromodomain-helicase-DNA-binding protein 4 (CHD4) is a core NURD remodeling complex ATPase that plays a crucial role as a gene repressor. Its overexpression has been reported in several cancers. In papillary thyroid carcinomas (PTCs), CHD4 is overexpressed and associated with aggressive features of the tumor, such as proliferation, migration, and epithelial-mesenchymal transition (EMT). We previously showed in PTCs that NADPH oxidase NOX4 expression is positively regulated by BRAF^V600E mutation, which is the most aggressive alteration in PTCs. In this retrospective study, we wondered whether there is a link between CHD4 and NOX4 protein expression in malignant thyroid tissues.

Methods: We explored CHD4 protein expression by immunostaining analysis in 86 human thyroid tissues: 44 thyroid tumor tissues [28 classical forms of PTCs (C-PTCs), 13 follicular variants of PTCs (F-PTCs), and three anaplastic thyroid carcinomas (ATCs)] and 42 of their normal adjacent tissues (NATs). The detection ofBRAF^V600E mutation was performed using Sanger sequencing and digital droplet PCR. Statistical analyses were conducted using GraphPad Prism 8 software. Various tests were used to assess the statistical relevance of different correlations, such as the chi-square test, Fisher’s exact test, and the Pearson correlation coefficient. Ap-value of less than 0.05 indicates statistical significance.

Results: The CHD4 protein expression analysis with already published data from our group BRAF^V600E status and NOX4 expression) reveals a highly significant level of CHD4 protein expression in C-PTCs compared to F-PTCs and ATC. Importantly, 70% of C-PTCs-BRAF^V600E overexpress CHD4 at the protein level, confirming the positive correlation between the CHD4 expression and BRAF^V600E mutation. Furthermore, a high level of CHD4 is associated with the presence of capsular breach and vascular emboli, affirming the involvement of CHD4 in thyroid tumor aggressiveness. Interestingly, we showed for the first time, to our knowledge, a positive correlation between CHD4 and NOX4 protein expression in malignant thyroid tissues.

Conclusions: The results of this study suggest that CHD4 could be used as a complementary molecular marker to improve the diagnosis and the management of PTCs-BRAF^V600E.

Keywords

chromodomain-helicase-DNA-binding protein 4 (CHD4) / papillary thyroid carcinomas / NADPH oxidase 4 / BRAFV600E mutation / molecular marker / epigenetic

References

[1] Nikiforov YE, Nikiforova MN. Molecular genetics and diagnosis of thyroid cancer. Nat Rev Endocrinol. 2011; 7: 569-80.

[2] Newfield RS, Jiang W, Sugganth DX, Hantash FM, Lee E, Newbury RO. Mutational analysis using next generation sequencing in pediatric thyroid cancer reveals BRAF and fusion oncogenes are common. Int J Pediatr Otorhinolaryngol. 2022; 157: 111121.

[3] Kebebew E, Weng J, Bauer J, Ranvier G, Clark OH, Duh QY, et al. The prevalence and prognostic value of BRAF mutation in thyroid cancer. Ann Surg. 2007; 246: 466-70.

[4] Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res. 2003; 63: 1454-7.

[5] Xing M, Alzahrani AS, Carson KA, Viola D, Elisei R, Bendlova B, et al. Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA. 2013; 309: 1493-501.

[6] Galrão AL, Camargo RY, Friguglietti CU, Moraes L, Cerutti JM, Serrano-Nascimento C, et al. Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS expression in thyroid tumors. J Clin Endocrinol Metab. 2014; 99: E944-52.

[7] Yi JW, Ha SY, Jee HG, Kim K, Kim SJ, Chai YJ, et al. Induction of the BRAFV600E Mutation in Thyroid Cells Leads to Frequent Hypermethylation. Clin Exp Otorhinolaryngol. 2022; 15: 273-82.

[8] Azouzi N, Cailloux J, Cazarin JM, Knauf JA, Cracchiolo J, Ghuzlan AA, et al. NADPH Oxidase NOX4 Is a Critical Mediator of BRAFV600E-Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas. Antioxid Redox Signal. 2017; 26: 864-77.

[9] Riesco-Eizaguirre G, Santisteban P, De la Vieja A. The complex regulation of NIS expression and activity in thyroid and extrathyroidal tissues. Endocr Relat Cancer. 2021; 28: T141-65.

[10] Fenniche S, Oukabli M, Oubaddou Y, Chahdi H, Damiri A, Alghuzlan A, et al. A Comparative Analysis of NOX4 Protein Expression in Malignant and Non-Malignant Thyroid Tumors. Curr Issues Mol Biol. 2023; 45: 5811-23.

[11] Xue Y, Wong J, Moreno GT, Young MK, Côté J, Wang W. NURD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities. Mol Cell. 1998; 2: 851-61.

[12] McKenzie LD, LeClair JW, Miller KN, Strong AD, Chan HL, Oates EL, et al. CHD4 regulates the DNA damage response and RAD51 expression in glioblastoma. Sci Rep. 2019; 9: 4444.

[13] Wang Y, Chen Y, Bao L, Zhang B, Wang JE, Kumar A, et al. CHD4 Promotes Breast Cancer Progression as a Coactivator of Hypoxia-Inducible Factors. Cancer Res. 2020; 80: 3880-91.

[14] Novillo A, Fernández-Santander A, Gaibar M, Galán M, Romero-Lorca A, Abdellaoui-Soussi FE, et al. Role of Chromodomain-Helicase-DNA-Binding Protein 4 (CHD4) in Breast Cancer. Front Oncol. 2021; 11: 633233.

[15] D’Alesio C, Bellese G, Gagliani MC, Lechiara A, Dameri M, Grasselli E, et al. The chromodomain helicase CHD4 regulates ERBB2 signaling pathway and autophagy in ERBB2+ breast cancer cells. Biol Open. 2019; 8: bio038323.

[16] Xia L, Huang W, Bellani M, Seidman MM, Wu K, Fan D, et al. CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes. Cancer Cell. 2017; 31: 653-68.e7.

[17] Xu N, Liu F, Wu S, Ye M, Ge H, Zhang M, et al. CHD4 mediates proliferation and migration of non-small cell lung cancer via the RhoA/ROCK pathway by regulating PHF5A. BMC Cancer. 2020; 20: 262.

[18] Wang HC, Chou CL, Yang CC, Huang WL, Hsu YC, Luo CW, et al. Over-Expression of CHD4 Is an Independent Biomarker of Poor Prognosis in Patients with Rectal Cancers Receiving Concurrent Chemoradiotherapy. Int J Mol Sci. 2019; 20: 4087.

[19] Pratheeshkumar P, Siraj AK, Divya SP, Parvathareddy SK, Alobaisi K, Al-Sobhi SS, et al. CHD4 Predicts Aggressiveness in PTC Patients and Promotes Cancer Stemness and EMT in PTC Cells. Int J Mol Sci. 2021; 22: 504.

[20] Polo SE, Kaidi A, Baskcomb L, Galanty Y, Jackson SP. Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4. EMBO J. 2010; 29: 3130-9.

[21] Zhang J, Shih DJH, Lin SY. The Tale of CHD4 in DNA Damage Response and Chemotherapeutic Response. J Cancer Res Cell Ther. 2019; 3: 052.

[22] Lloyd RV, Osamura RY, Klöppel G, Rosai J, editors. WHO classification of tumours of endocrine organs. 4th ed. Lyon: International Agency for Research on Cancer; 2017.

[23] Li P, Ding Y, Liu M, Wang W, Li X. Sex disparities in thyroid cancer: a SEER population study. Gland Surg. 2021; 10: 3200- 10.

[24] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68: 394-424.

[25] Kaabouch M, Chahdi H, Azouzi N, Oukabli M, Rharrassi I, Boudhas A, et al. BRAFV600E hot spot mutation in thyroid carcinomas: first Moroccan experience from a single-institution retrospective study. Afr Health Sci. 2020; 20: 1849-56.

[26] Shi X, Liu R, Basolo F, Giannini R, Shen X, Teng D, et al. Differential Clinicopathological Risk and Prognosis of Major Papillary Thyroid Cancer Variants. J Clin Endocrinol Metab. 2016; 101: 264-74.

[27] Shafique K, Baloch Z. Risk stratification of papillary thyroid carcinoma and its variants; from clinicopathologic features to molecular profiling. Diagn Histopathol. 2019; 25: 143-53.

[28] Hu J, Yuan IJ, Mirshahidi S, Simental A, Lee SC, Yuan X. Thyroid Carcinoma: Phenotypic Features, Underlying Biology and Potential Relevance for Targeting Therapy. Int J Mol Sci. 2021; 22: 1950.

[29] Kim S, Lee KE, Myong JP, Park JH, Jeon YK, Min HS, et al. BRAF V600E mutation is associated with tumor aggressiveness in papillary thyroid cancer. World J Surg. 2012; 36: 310-7.

[30] Choi YW, Kim HJ, Kim YH, Park SH, Chwae YJ, Lee J, et al. B-RafV600E inhibits sodium iodide symporter expression via regulation of DNA methyltransferase 1. Exp Mol Med. 2014; 46: e120.

[31] Masoodi T, Siraj AK, Siraj S, Azam S, Qadri Z, Albalawy WN, et al. Whole-Exome Sequencing of Matched Primary and Metastatic Papillary Thyroid Cancer. Thyroid. 2020; 30: 42-56.

[32] Li S, Hu G, Chen Y, Sang Y, Tang Q, Liu R. TERT upstream promoter methylation regulates TERT expression and acts as a therapeutic target in TERT promoter mutation-negative thyroid cancer. Cancer Cell Int. 2024; 24: 271.

[33] Oskouie AA, Ahmadi MS, Taherkhani A. Identification of Prognostic Biomarkers in Papillary Thyroid Cancer and Developing Non-Invasive Diagnostic Models Through Integrated Bioinformatics Analysis. Microrna. 2022; 11: 73-87.

[34] Larsen DH, Poinsignon C, Gudjonsson T, Dinant C, Payne MR, Hari FJ, et al. The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage. J Cell Biol. 2010; 190: 731-40.