Institute of Chemical Biology and Fundamental Medicine Siberian Branch of the Russian Academy of Sciences, Akad. Lavrentiev Ave. 8, 630090 Novosibirsk, Russia.
Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, Akad. Lavrentiev Ave. 8/2, 630090 Novosibirsk, Russia.
Int J Mol Sci. 2023 Jan 27;24(3):2494. doi: 10.3390/ijms24032494.
Hypoxia arises in most growing solid tumors and can lead to pleotropic effects that potentially increase tumor aggressiveness and resistance to therapy through regulation of the expression of genes associated with the epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). The main goal of the current work was to obtain and investigate the intermediate phenotype of tumor cells undergoing the hypoxia-dependent transition from fibroblast to epithelial morphology. Primary breast cancer fibroblasts BrC4f, being cancer-associated fibroblasts, were subjected to one or two rounds of "pulsed hypoxia" (PH). PH induced transformation of fibroblast-shaped cells to semi-epithelial cells. Western blot analysis, fluorescent microscopy and flow cytometry of transformed cells demonstrated the decrease in the mesenchymal markers vimentin and N-cad and an increase in the epithelial marker E-cad. These cells kept mesenchymal markers αSMA and S100A4 and high ALDH activity. Real-time PCR data of the cells after one (BrC4f_Hyp1) and two (BrC4f_Hyp2) rounds of PH showed consistent up-regulation of TWIST1 gene as an early response and ZEB1/2 and SLUG transcriptional activity as a subsequent response. Reversion of BrC4f_Hyp2 cells to normoxia conditions converted them to epithelial-like cells (BrC4e) with decreased expression of EMT genes and up-regulation of MET-related OVOL2 and c-MYC genes. Transplantation of BrC4f and BrC4f_Hyp2 cells into SCID mice showed the acceleration of tumor growth up to 61.6% for BrC4f_Hyp2 cells. To summarize, rounds of PH imitate the MET process of tumorigenesis in which cancer-associated fibroblasts pass through intermediate stages and become more aggressive epithelial-like tumor cells.
缺氧发生在大多数生长中的实体肿瘤中，并通过调节与上皮间质转化（EMT）和间质上皮转化（MET）相关的基因表达，导致潜在的肿瘤侵袭性和治疗抵抗的多效性效应。当前工作的主要目标是获得和研究经历缺氧依赖性从成纤维细胞向上皮形态转变的肿瘤细胞的中间表型。作为癌相关成纤维细胞的原发性乳腺癌成纤维细胞 BrC4f 经历了一轮或两轮“脉冲缺氧”（PH）。PH 诱导成纤维细胞样细胞向半上皮细胞的转化。转化细胞的 Western blot 分析、荧光显微镜和流式细胞术显示，间充质标志物波形蛋白和 N-钙黏蛋白减少，上皮标志物 E-钙黏蛋白增加。这些细胞保持间充质标志物 αSMA 和 S100A4 和高 ALDH 活性。经过一轮（BrC4f_Hyp1）和两轮（BrC4f_Hyp2）PH 处理的细胞的实时 PCR 数据显示，TWIST1 基因作为早期反应一致上调，ZEB1/2 和 SLUG 转录活性作为随后的反应上调。将 BrC4f_Hyp2 细胞返回到常氧条件下，将其转化为上皮样细胞（BrC4e），降低 EMT 基因的表达，并上调 MET 相关的 OVOL2 和 c-MYC 基因。将 BrC4f 和 BrC4f_Hyp2 细胞移植到 SCID 小鼠中，显示 BrC4f_Hyp2 细胞的肿瘤生长加速高达 61.6%。总之，多轮 PH 模拟了肿瘤发生的 MET 过程，其中癌相关成纤维细胞经历中间阶段并成为更具侵袭性的上皮样肿瘤细胞。
