Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody,Journal of Cancer Research and Clinical Oncology

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Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody
Journal of Cancer Research and Clinical Oncology ( IF 2.7 ) Pub Date : 2023-10-07 , DOI: 10.1007/s00432-023-05433-3
Yan Wang ₁ , Xianqin Song ₁ , Yu Song ₁ , Kehua Fang ₂ , Xiaotian Chang ₁

Affiliation

Background

Peptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer.

Methods

The subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes.

Results

PADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated.

Conclusions

PADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.

Graphical abstract

中文翻译：

乳腺癌细胞中 PADI4 的细胞膜定位及抗 PADI4 单克隆抗体的抑制作用研究

背景

肽基精氨酸脱亚胺酶 4 (PADI4) 是一种翻译后修饰酶，可在钙离子存在下将蛋白质中的精氨酸转化为瓜氨酸，这称为瓜氨酸化。据报道PADI4在多种恶性肿瘤的细胞质和细胞核中表达。根据GeneCards数据库和我们之前的研究，推测PADI4也可能在细胞膜上表达。本研究旨在证实PADI4的膜表达以及抗PADI4抗体对细胞膜PADI4的影响。这可能是抗PADI4单克隆抗体治疗乳腺癌的另一种作用机制。

方法

通过免疫荧光、免疫电子显微镜和蛋白质印迹分析确定 PADI4 在 MDA-MB-231 和 MCF-7 乳腺癌细胞中的亚细胞定位。用PADI4抗体处理肿瘤细胞，测量细胞增殖、迁移、集落形成、凋亡、糖酵解和上皮间质转化（EMT）以及一些重要肿瘤基因的表达。

结果

PADI4不仅定位于乳腺癌细胞的细胞核和细胞质，还在细胞膜上检测到。 PADI4 抗体处理后，细胞增殖、迁移、集落形成、EMT 和糖酵解产生的 ATP 均减少，并且MYC原癌基因 ( MYC )、FAT 非典型钙粘蛋白 1 ( FAT1 )、核因子 kappa B 亚基 1 的 mRNA 表达降低乳腺癌细胞中NFκB（ NFκB ）和肿瘤坏死因子（ TNF-α ）的表达下调，而肿瘤蛋白p63 （ TP63 ）的mRNA表达上调。