Wesoly Joanna, Pstrąg Natalia, Derylo Kamil, Michalec-Wawiórka Barbara, Derebecka Natalia, Nowicka Hanna, Kajdasz Arkadiusz, Kluzek Katarzyna, Srebniak Malgorzata, Tchórzewski Marek, Kwias Zbigniew, Bluyssen Hans
Laboratory of High Throughput Technologies, Adam Mickiewicz University Poznan, Poland.
Department of Molecular Biology, Maria Curie-Sklodowska University Lublin, Poland.
Am J Cancer Res. 2023 May 15;13(5):1863-1883. eCollection 2023.
Due to their involvement in the development of various cancers Transmembrane Proteins (TMEMs) are the focus of many recent studies. Previously we reported TMEM de-regulation in clear cell Renal Cell Carcinoma (ccRCC) with TMEM213, 207, 116, 72 and 30B being among the most downregulated on mRNA level. TMEM down-regulation was also more pronounced in advanced ccRCC tumors and was potentially linked to clinical parameters such as: metastasis (TMEM72 and 116), Fuhrman grade (TMEM30B) and overall survival (TMEM30B). To further investigate these findings, first, we set off to prove experimentally that selected TMEMs are indeed membrane-bound as predicted in silico, we verified the presence of signaling peptides on their N-termini, orientation of TMEMs within the membrane and validated their predicted cellular localization. To investigate the potential role of selected TMEMs in cellular processes overexpression studies in HEK293 and HK-2 cell lines were carried out. Additionally, we tested TMEM isoform expression in ccRCC tumors, identified mutations in TMEM genes and examined chromosomal aberrations in their loci. We confirmed the membrane-bound status of all selected TMEMs, assigned TMEM213, and 207 to early endosomes, TMEM72 to early endosomes and plasma membrane, TMEM116 and 30B to the endoplasmic reticulum. The N-terminus of TMEM213 was found to be exposed to the cytoplasm, the C-terminus of TMEM207, 116 and 72 were directed toward the cytoplasm, and both termini of TMEM30B faced the cytoplasm. Interestingly, TMEM mutations and chromosomal aberrations were infrequent in ccRCC tumors, yet we identified potentially damaging mutations in TMEM213 and TMEM30B and found deletions in the TMEM30B locus in nearly 30% of the tumors. Overexpression studies suggested selected TMEMs may take part in carcinogenesis processes such as cell adhesion, regulation of epithelial cell proliferation, and regulation of adaptive immune response, which could indicate a link to the development and progression of ccRCC.
由于跨膜蛋白（TMEMs）参与了多种癌症的发展，它们是近期许多研究的重点。此前我们报道了透明细胞肾细胞癌（ccRCC）中TMEM的失调情况，其中TMEM213、207、116、72和30B在mRNA水平上是下调最为明显的。TMEM下调在晚期ccRCC肿瘤中也更为显著，并且可能与临床参数相关，如：转移（TMEM72和116）、Fuhrman分级（TMEM30B）和总生存期（TMEM30B）。为了进一步研究这些发现，首先，我们着手通过实验证明所选的TMEMs确实如计算机预测的那样是膜结合的，我们验证了它们N端信号肽的存在、TMEMs在膜内的方向，并验证了它们预测的细胞定位。为了研究所选TMEMs在细胞过程中的潜在作用，我们在HEK293和HK-2细胞系中进行了过表达研究。此外，我们检测了ccRCC肿瘤中TMEM异构体的表达，鉴定了TMEM基因中的突变，并检查了其基因座中的染色体畸变。我们证实了所有所选TMEMs的膜结合状态，将TMEM213和207定位于早期内体，TMEM72定位于早期内体和质膜，TMEM116和30B定位于内质网。发现TMEM213的N端暴露于细胞质中，TMEM207、116和72的C端指向细胞质，TMEM30B的两端都面向细胞质。有趣的是，TMEM突变和染色体畸变在ccRCC肿瘤中并不常见，但我们在TMEM213和TMEM30B中鉴定出了潜在的有害突变，并在近30%的肿瘤中发现了TMEM30B基因座的缺失。过表达研究表明，所选的TMEMs可能参与致癌过程，如细胞黏附、上皮细胞增殖的调节和适应性免疫反应的调节，这可能表明与ccRCC的发生和发展有关。
