Nicotinamide N-methyltransferase is related to MELF pattern invasion in endometrioid carcinoma [RNA-seq]

In endometrioid carcinoma (EC), grade 1 (G1) EC is generally associated with a good prognosis. However, in a minority of G1, we often find a more aggressive histological pattern: MELF (microcystic, elongated, and fragmented) pattern. We previously revealed that EC with high expression of S100A4 and serum deprivation-response protein (SDPR) was related to MELF pattern invasion. However, the molecular feature of the invasive front area of MELF pattern has not been investigated. In the current study, we searched for genes preferentially expressed in the invasive front area of EC with MELF pattern by using laser microdissection and RNA sequencing, and revealed that Nicotinamide N-methyltransferase (NNMT) is related to invasiveness of MELF pattern. We confirmed that NNMT expression was high in the invasive front area of MELF pattern in immunohistochemical analysis. Moreover, using endometrioid carcinoma cell lines, we showed that NNMT promotes migration, invasion, colony formation, epithelial-mesenchymal transition (EMT), and chemoresistance. We speculated that depletion of NNMT promotes histone methylation and leads to tumor suppression because NNMT consumes S-adenosyl methionine (SAM), which is an essential methylation cofactor. We showed that NNMT-knockout cells the expression of H3K9me2 was enhanced. We speculate that methylation of H3K9 lead to repress the transcription of various oncogenic genes by performing RNA sequencing using NNMT-knockout cell lines. Our findings showed the possibility that NNMT inhibitors, which are expected to be used for the treatment of metabolic disorders, is effective for the treatment of an aggressive EC. This is the first report of the gene analysis focused on the morphological changes of MELF pattern invasion of EC. Overall design: To compare the levels of gene expression profiles between NNMT-knockout AN3CA cells and control cells, total RNA was extracted from KO1, KO2, and EV cells.

子宫内膜样癌（endometrioid carcinoma, EC）中，1级（G1）EC通常预后良好。然而，少数G1病例中常可见更具侵袭性的组织学表型：MELF（微囊、细长及碎裂）型。我们既往研究证实，高表达S100A4与血清剥夺反应蛋白（SDPR）的EC与MELF型侵袭相关，但目前针对MELF型侵袭前沿区域的分子特征尚未得到系统研究。
本研究通过激光显微切割与RNA测序技术，筛选出MELF型EC侵袭前沿区域优先表达的基因，发现烟酰胺N-甲基转移酶（NNMT）与MELF型的侵袭性密切相关。免疫组化分析进一步证实，MELF型侵袭前沿区域的NNMT表达水平显著升高。
此外，利用子宫内膜样癌细胞系，我们证实NNMT可促进细胞迁移、侵袭、集落形成、上皮间质转化（EMT）以及化疗耐药。鉴于NNMT会消耗作为核心甲基化辅因子的S-腺苷甲硫氨酸（SAM），我们推测敲除NNMT可促进组蛋白甲基化并发挥肿瘤抑制作用。实验结果显示，NNMT敲除细胞中H3K9me2的表达水平显著上调。通过对NNMT敲除细胞系开展RNA测序分析，我们推测H3K9甲基化可通过抑制多种致癌基因的转录实现肿瘤抑制效应。
本研究结果表明，原本用于代谢性疾病治疗的NNMT抑制剂，有望用于侵袭性子宫内膜样癌的临床治疗。这是首个针对EC的MELF型侵袭形态学改变开展基因分析的研究。
总体实验设计：为比较NNMT敲除AN3CA细胞与对照细胞的基因表达谱差异，我们从KO1、KO2及空载体（EV）细胞中提取总RNA。