A Novel Gene Expression Profile and Combined Effects in the Pathogenesis of Adrenocorticotropic Hormone-Secreting Pituitary Neuroendocrine Tumors

Abstract

Aims

Cushing’s disease is a rare endocrine disorder characterized by excessive endogenous glucocorticoid production, primarily resulting from adrenocorticotropic hormone-secreting pituitary neuroendocrine tumors (ACTH-PitNETs). This study investigated the expression of several genes implicated in the development of ACTH-PitNETs, including EGFR, USP8, CABLES1, USP2, STAM2, VPS28, HDAC2, IL-6, SMARCA4, WEE1, CDKN2A, CCND1, NR4A1, NEUROD1, and RIPK1. The methylation levels of the USP8 and CDKN2A genes were also assessed for insights into their regulatory mechanisms.

Methods

Formalin-fixed paraffin-embedded pituitary tumor tissue samples from 32 patients diagnosed with ACTH-PitNET and 15 anterior pituitary tissue samples were analyzed. Gene expression was analyzed through quantitative reverse transcription polymerase chain reaction, while methylation was examined through methylation-specific polymerase chain reaction. All data were analyzed with IBM SPSS Statistics 21. The relationships among gene expressions were assessed using principal component analysis.

Results

The expression of CABLES1, NR4A1, CCND1, NEUROD1, USP2, and WEE1 differed significantly between the patient and control groups. Additionally, significant correlations were observed between the levels of RIPK1, SMARCA4, and USP2 and pre-operative cortisol levels; WEE1 expression and pre-operative ACTH levels; CDKN2A expression and urinary cortisol levels; CABLES1, NEUROD1, SMARCA4, and STAM2 expression and post-operative cortisol levels at 48 h. CCND1 expression was correlated with adenoma size, while WEE1 expression was linked to remission status. Notably, the CDKN2A gene displayed partial methylation, whereas the USP8 gene was fully unmethylated.

Conclusions

The altered expression levels of the USP2, CABLES1, CDKN2A, and WEE1 may be closely associated with the development of ACTH-PitNETs. Notably, WEE1 emerged as a target gene for predicting clinical remission in patients with Cushing’s disease.

Keywords

Cushing’s disease - methylation - USP2 - CABLES1 - CDKN2A - WEE1

Publikationsverlauf

Eingereicht: 22. Oktober 2024

Angenommen nach Revision: 02. Juni 2025

Artikel online veröffentlicht:
30. Juli 2025

• References

• 1 Laws ER, Pace Louise. Cushing's Disease: An Often Misdiagnosed and Not So Rare Disorder. Boston, Massachusetts, United States: Academic Press; 2016
• 2 Asa SL, Mete O, Perry A. et al. Overview of the 2022 WHO classification of pituitary tumors. Endocr Pathol 2022; 33: 6-26
• 3 Albani A, Theodoropoulou M, Reincke M. Genetics of Cushing’s disease. Clin Endocrinol 2018; 88: 3-12
• 4 Fukuoka H, Takahashi Y. The role of genetic and epigenetic changes in pituitary tumorigenesis. Neurol Med Chir 2014; 54: 943-957
• 5 Marrero-Rodríguez D, Vela-Patiño S, Martinez-Mendoza F. et al. Genomics, transcriptomics, and epigenetics of sporadic pituitary tumors. Arch Med Res 2023; 54: 102915
• 6 Chang M, Yang C, Bao X. et al. Genetic and epigenetic causes of pituitary adenomas. Front Endocrinol (Lausanne) 2021; 11: 596554
• 7 Liu X, Feng M, Dai C. et al. Expression of EGFR in pituitary corticotroph adenomas and its relationship with tumor behavior. Front Endocrinol (Lausanne) 2019; 14: 785
• 8 Ma ZY, Song ZJ, Chen JH. et al. Recurrent gain-of-function USP8 mutations in Cushing's disease. Cell Res 2015; 25: 306-317
• 9 Hashemi-Madani N, Cheraghi S, Emami Z. et al. Targeted analysis of ubiquitin-specific peptidase (USP8) in a population of Iranian people with Cushing's disease and a systematic review of the literature. BMC Endocr Disord 2024; 24: 86
• 10 Kakihara K, Asamizu K, Moritsugu K. et al. Molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain. Commun Biol 2021; 4: 1272
• 11 Reincke M, Sbiera S, Hayakawa A. et al. Mutations in the deubiquitinase gene USP8 cause Cushing's disease. Nat Genet 2015; 47: 31-38
• 12 Fukuoka H, Cooper O, Ben-Shlomo A. et al. EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas. J Clin Investig 2011; 121: 471221
• 13 Rebollar-Vega RG, Zuarth-Vázquez JM, Hernández-Ramírez LC. Clinical spectrum of USP8 pathogenic variants in Cushing's disease. Arch Med Res 2023; 54: 102899
• 14 Kageyama K, Asari Y, Sugimoto Y. et al. Ubiquitin-specific protease 8 inhibitor suppresses adrenocorticotropic hormone production and corticotroph tumor cell proliferation. Endocr J 2020; 67: 177-184
• 15 Islam MT, Chen F, Chen H. The oncogenic role of ubiquitin specific peptidase (USP8) and its signaling pathways targeting for cancer therapeutics. Arch Biochem Biophys 2021; 15 701 108811
• 16 Mizuno E, Iura T, Mukai A. et al. Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. Mol Biol Cell 2005; 1611 5163-5174
• 17 Crespo-Yàñez X, Aguilar-Gurrieri C, Jacomin AC. et al. CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis. PLoS Genet 2018; 146: e1007456
• 18 Araki T, Liu X, Kameda H. et al. EGFR Induces E2F1-mediated corticotroph tumorigenesis. J Endocr Soc 2017; 12: 127-143
• 19 Asari Y, Kageyama K, Sugiyama A. et al. Lapatinib decreases the ACTH production and proliferation of corticotroph tumor cells. Endocr J 2019; 666: 515-522
• 20 Huang JR, Tan GM, Li Y. et al. The emerging role of Cables1 in cancer and other diseases. Mol Pharmacol 2017; 92: 240-245
• 21 Knosp E, Steiner E, Kitz K. et al. Pituitary adenomas with invasion of the cavernous sinus space: A magnetic resonance imaging classification compared with surgical findings. Neurosurgery 1993; 33: 610-618
• 22 Storr HL, Alexandraki KI, Martin L. et al. Comparisons in the epidemiology, diagnostic features and cure rate by transsphenoidal surgery between paediatric and adult-onset Cushing's disease. Eur J Endocrinol 2011; 164: 667-674
• 23 Evans CO, Young AN, Brown MR. et al. Novel patterns of gene expression in pituitary adenomas identified by complementary deoxyribonucleic acid microarrays and quantitative reverse transcription-polymerase chain reaction. J Clin Endocrinol Metab 2001; 86: 3097-3107
• 24 Qian ZR, Sano T, Yoshimoto K. et al. Tumor-specific downregulation and methylation of the CDH13 (H-cadherin) and CDH1 (E-cadherin) genes correlate with aggressiveness of human pituitary adenomas. Mod Pathol 2007; 20: 1269-1277
• 25 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001; 25: 402-408
• 26 Herman JG, Graff JR, Myöhänen S. et al. Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: 9821-9826
• 27 The Jamovi Project. Jamovi, (Version 2.2) https://www.jamovi.org 2021 (accessed on 10 June 2022)
• 28 Perez-Rivas LG, Theodoropoulou M, Ferraù F. et al. The gene of the ubiquitin-specific protease 8 is frequently mutated in adenomas causing Cushing's disease. J Clin Endocrinol Metab 2015; 100: E997-E1004
• 29 Xiong Q, Ge W. Gene mutations in Cushing's disease. Biomed Rep 2016; 5: 277-282
• 30 Beard JA, Tenga A, Chen T. The interplay of NR4A receptors and the oncogene-tumor suppressor networks in cancer. Cell Signal 2015; 27: 257-266
• 31 Roussel-Gervais A, Couture C, Langlais D. et al. The Cables1 gene in glucocorticoid regulation of pituitary corticotrope growth and Cushing disease. J Clin Endocrinol Metab 2016; 101: 513-522
• 32 Kageyama K, Asari Y, Sugimoto Y. et al. Ubiquitin-specific protease 8 inhibitor suppresses adrenocorticotropic hormone production and corticotroph tumor cell proliferation. Endocr J 2020; 67: 177-184
• 33 Lewis CW, Jin Z, Macdonald D. et al. Prolonged mitotic arrest induced by Wee1 inhibition sensitizes breast cancer cells to paclitaxel. Oncotarget 2017; 8: 73705-73722
• 34 Ghelli Luserna di Rorà A, Cerchione C, Martinelli G. et al. WEE1 family business: Regulation of mitosis, cancer progression, and therapeutic target. J Hematol Oncol 2020; 13: 126
• 35 Chen D, Hong R, Cao Y. et al. Combined Wee1 and EGFR inhibition reveals synergistic antitumor effect in esophageal squamous cell carcinoma. Carcinogenesis 2023; 44: 451-462
• 36 Hirai H, Iwasawa Y, Okada M. et al. Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther 2009; 8: 2992-3000
• 37 Bridges KA, Hirai H, Buser CA. et al. MK-1775, a novel Wee1 kinase inhibitor, radiosensitizes p53-defective human tumor cells. Clin Cancer Res 2011; 17: 5638-5648
• 38 Van Linden AA, Baturin D, Ford JB. et al. Inhibition of Wee1 sensitizes cancer cells to antimetabolite chemotherapeutics in vitro and in vivo, independent of p53 functionality. Mol Cancer Ther 2013; 12: 2675-2684
• 39 Zhang S, Guo Y, Zhang S. et al. Targeting the deubiquitinase USP2 for malignant tumor therapy (Review). Oncol Rep 2023; 50: 176
• 40 Mahul-Mellier AL, Datler C, Pazarentzos E. et al. De-ubiquitinating proteases USP2a and USP2c cause apoptosis by stabilising RIP1. Biochim Biophys Acta 2012; 1823: 1353-1365
• 41 Serra S, Chetty R. p16. J Clin Pathol 2018; 71: 853-858
• 42 Li J, Poi MJ, Tsai MD. Regulatory mechanisms of tumor suppressor P16(INK4A) and their relevance to cancer. Biochemistry 2011; 50: 5566-5582
• 43 Quereda V, Malumbres M. Cell cycle control of pituitary development and disease. J Mol Endocrinol 2009; 42: 75-86
• 44 Yoshino A, Katayama Y, Ogino A. et al. Promoter hypermethylation profile of cell cycle regulator genes in pituitary adenomas. J Neurooncol 2007; 83: 153-162
• 45 Rayess H, Wang MB, Srivatsan ES. Cellular senescence and tumor suppressor gene p16. Int J Cancer 2012; 130: 1715-1725
• 46 Simpson DJ, McNicol AM, Murray DC. et al. Molecular pathology shows p16 methylation in nonadenomatous pituitaries from patients with Cushing's disease. Clin Cancer Res 2004; 10: 1780-1788
• 47 Fratticci A, Grieco FA, Spilioti C. et al. Differential expression of neurogenins and NeuroD1 in human pituitary tumours. J Endocrinol 2007; 194: 475-484
• 48 Ferretti E, Di Stefano D, Zazzeroni F. et al. Human pituitary tumours express the bHLH transcription factors NeuroD1 and ASH1. J Endocrinol Invest 2003; 26: 957-965
• 49 Poulin G, Turgeon B, Drouin J. NeuroD1/beta2 contributes to cell-specific transcription of the proopiomelanocortin gene. Mol Cell Biol 1997; 17: 6673-6682
• 50 Parvin R, Saito-Hakoda A, Shimada H. et al. Role of NeuroD1 on the negative regulation of Pomc expression by glucocorticoid. PLoS One 2017; 12: e0175435
• 51 Araki T, Tone Y, Yamamoto M. et al. Two distinctive POMC promoters modify gene expression in Cushing disease. J Clin Endocrinol Metab 2021; 106: e3346-e3363
• 52 Mitrofanova LB, Vorobeva OM, Gorshkov AN. Analysis of pituitary adenoma expression patterns suggests a potential role for the NeuroD1 transcription factor in neuroendocrine tumor-targeting therapies. Oncotarget 2019; 10: 289-312
• 53 Seltzer J, Ashton CE, Scotton TC. et al. Gene and protein expression in pituitary corticotroph adenomas: A systematic review of the literature. Neurosurg Focus 2015; 38: E17
• 54 Oyama K, Sanno N, Teramoto A. et al. Expression of neuro D1 in human normal pituitaries and pituitary adenomas. Mod Pathol 2001; 14: 892-899