Study of expression alterations in genes associated with epithelial-mesenchymal transition in lung cancer

Abstract

Background. Lung cancer is the most common malignancy worldwide, with a high rate of adverse outcomes. One of the key processes contributing to lung cancer progression and metastasis is epithelial-mesenchymal transition (EMT). During it, epithelial cancer cells lose their characteristic features and acquire the properties of mesenchymal cells, which grants them motility, the ability to penetrate tissue, and metastasize. This also ensures resistance to therapy and stem cell properties, contributing to relapse. Proteins associated with the maintenance of the epithelial cell phenotype (CDH1, E-cadherin)—which are responsible for cell attachment to the basement membrane—as well as transcriptional repressors (SNAI1, SNAI2, ZEB1, ZEB2) and others, are involved in the regulation of EMT. Analysis of EMT gene expression can serve as the basis for the development of new prognostic biomarkers to assess tumor aggressiveness and the likelihood of disease recurrence, as well as facilitate the search for new targets for targeted therapy. The Aim was to study changes in the expression level of genes BMI1, CDH1, SNAI1, SNAI2, ZEB1, ZEB2 associated with EMT in lung cancer.

Methods. The study used 50 paired RNA samples isolated from non-small cell lung cancer (NSCLC) tissues (lung adenocarcinoma (LUAD) – 24 pairs, squamous cell lung cancer (LUSC) – 26 pairs) and adjacent histologically normal lung tissues. RNA was isolated from the tissue using a standard method. RNA integrity was RIN≥6. Expression levels were assessed by RT-PCR on a Real-Time CFX 96 Touch thermocycler (Bio-Rad, USA) using the ∆∆Cq method use of the BioMaster RT-PCR SYBR Blue (2×) kit (Biolabmix, Russia). B2M and ACTB served as reference genes. The significance of differences in gene expression was confirmed by multivariate analysis of variance (ANOVA) after assessing the normality of distribution in the Shapiro-Wilk test.

Results. The differences in the expression level of genes involved in EMT were revealed between LUAD and LUSC (p<0.05). In LUAD, changes atypical for classical EMT were demonstrated: a 3.47-fold increase in CDH1 expression, which contradicts the expected suppression of this epithelial marker, along with a global decrease in the expression level of mesenchymal factors: SNAI1, SNAI2, ZEB1 and ZEB2 by 1.42, 3.27, 2.41, 2.26 times, respectively, and a 1.83-fold increase in BMI1 expression. BMI1 is involved in maintaining the proliferation and self-renewal of tumor stem cells and is associated with therapy resistance. In LUSC, a simultaneous increase in CDH1, SNAI1, and SNAI2 mRNA expression was observed (3.02-, 1.02-, and 1.62-fold, respectively); this pattern deviates from the expected downregulation of CDH1 that typically accompanies the upregulation of its transcriptional repressors, SNAI1 and SNAI2. The 2.04-fold increase in BMI1 mRNA level significantly (p<0.05) correlates with tumor aggressiveness. Meanwhile, a 1.65- and 2.17-fold decrease in ZEB1 and ZEB2 expression is compensated for by an increase in SNAI1 and SNAI2 expression.

Conclusion. Complex heterogeneity in the mechanisms underlying the epithelial phenotype and EMT in lung cancer has been identified, demonstrating the importance of considering tumor histological features, molecular heterogeneity, and the ability to utilize alternative progression pathways when developing diagnostic criteria. The observed differences in the expression profiles of EMT-associated genes — specifically, the combination of elevated CDH1, BMI1, and SNAI2 expression in LUSC and elevated CDH1 and BMI1 expression in LUAD — may be of interest for further study regarding their biological and potential clinical significance in NSCLC.