In this study, our research strategy encompassed several key objectives. Firstly, we aimed to identify differentially expressed genes of KRT8 between invasive and non-invasive NFPAs using qRT-PCR. Secondly, we sought to ascertain differentially expressed proteins of KRT8 between invasive and non-invasive NFPAs via Western blotting. Thirdly, we endeavored to investigate the invasive impact of KRT8 on GH3 cells utilizing the RNA interference technique. The primary goal of our study was to explore the correlation between KRT8 and NFPAs. Additionally, our study aimed to elucidate novel genes that could play a pivotal role in the pathogenesis of these enigmatic invasive pituitary NFPAs.
Cytokeratin 8 (KRT8) is an important member of the type II intermediate filament protein family, comprised of 483 amino acids and structured into three functional parts: the head structure area (N-terminal), rod structure area, and tail structure area (C-terminal).Notably, KRT8 expression levels increase during the differentiation of mammalian cells. In normal tissues, KRT8 and its complementary subunit KRT18 are co-expressed in various cell types, including glandular epithelial cells, transitional cell epithelial cells, and liver cells, while they are notably absent in squamous laminar epithelium.However, in tumor tissues, the expression profile of KRT8 exhibits significant divergence. For instance, KRT8 expression is found to be decreased in breast and colon cancer, while it is increased in head and neck cancer. Both of these patterns of expression have been shown to play crucial roles in tumor progression. Further understanding of these differential expressions may provide valuable insights into the pathogenesis of these cancers and potentially uncover novel therapeutic targets.
To the best of our knowledge, there is a dearth of research investigating the role of KRT8 in the invasion of NFPAs (non-functioning pituitary adenomas). Our experimental findings have demonstrated a significant up-regulation of KRT8 mRNA and protein in the invasion of NFPAs in vivo. In order to further elucidate the influence of KRT8 on invasiveness in vitro, we established a KRT8 silencing cell line by infecting GH3 cells with lentiviral shKRT8. Subsequent analysis revealed that the downregulation of KRT8 effectively inhibited cell invasion in GH3 cells. These observations not only underscore the potential role of KRT8 in promoting invasion in NFPAs but also provide valuable insights into the mechanisms underlying tumor invasiveness, thereby paving the way for the development of targeted therapeutic strategies.
KRT8 serves as an upstream regulatory gene in numerous signaling pathways associated with tumorigenesis, exhibiting abnormal expression in various epithelial-origin tumors such as breast cancer, gastric cancer, and pancreatic cancer15–17.The regulation of KRT8 is mediated by the MEK-ERK signaling pathway18. Busch et al have reported that the recombination process of cytokeratin and the metastasis of epithelial tumor cells are influenced by KRT8 phosphorylation19. Further mechanistic studies have confirmed that in epithelial cells of pancreatic and gastric cancer, the migratory capacity of these cells is enhanced through the regulation of neurosphingosine choline-mediated KRT8 phosphorylation and keratin recombination via the MEK-ERK signaling pathway20. These findings not only shed light on the pivotal role of KRT8 in tumor progression but also offer potential therapeutic targets for the management of these malignancies.
Cimpean AM et al discovered that KRT8 binds to tumor necrosis factor receptor 2 (TNFR2) and lymphocyte cytotoxic beta receptors within the cytoplasm of breast cancer cells, providing resistance to TNF-induced apoptosis21. Moreover, increased expression of KRT8 has been shown to alter the phenotypic characteristics of epithelial cells, potentially contributing to malignant transformation22. Furthermore, some researchers propose that KRT8 serves as the primary plasminogen receptor on the surface of breast cancer cells, thereby facilitating the rapid activation of plasminogen by plasminogen activators23, 24. This activation subsequently promotes invasion and the acquisition of a malignant phenotype. These findings collectively underscore the multifaceted roles of KRT8 in breast cancer pathobiology, providing critical insights into its potential as a therapeutic target for combating malignancy.
RNA interference (RNAi) was discovered and named by Fire et al during their investigations of the homologous gene to the justine RNA inhibition gene in Caenorhabditis elegans25. This phenomenon involves the introduction of exogenous double-stranded RNA (dsRNA) into organismal cells, leading to the degradation of mRNA homologous to the dsRNA and consequent inhibition of the RNA expression level of corresponding genes26. RNA interference has emerged as a powerful tool for silencing specific genes, with small interfering RNA (siRNA) technology being the most commonly used method27. In the context of this study, the silencing of the target gene KRT8 in GH3 pituitary tumor cell lines using siRNA technology resulted in a significant reduction in the invasiveness of these cells28. These findings not only underscore the utility of RNAi as a method for targeted gene silencing but also highlight the potential therapeutic implications of targeting KRT8 in the context of pituitary tumors.
Iyer SV et al demonstrated the silencing of KRT8 expression in human mammary MDA-MB-68 cells using RNAi technology, resulting in a significant increase in the transformative potential, motility, and invasive capabilities of these cells29. Similarly, Fortier et al found that specific KRT8 siRNA targeting epithelial tumor cells significantly enhances their invasiveness and sensitivity to cisplatin30. Additionally, Alam et al reported that the application of KRT8 siRNA to oral squamous cell carcinoma cells leads to a notable reduction in their tumorigenic potential and cellular invasive capacity31.These collective findings not only underscore the critical role of KRT8 in mediating cellular invasiveness and sensitivity to therapeutic agents but also highlight the potential of KRT8-targeted RNAi as a promising strategy for modulating malignant phenotypes in various cancer types.
While our study provides valuable insights, it is important to acknowledge its limitations. As a retrospective study, the data were selected from follow-up information, potentially leading to a loose grouping of cases and controls. Consequently, there is a need for larger multi-center randomized controlled trials to confirm and validate these findings in the future. Such trials will offer a more comprehensive and robust evaluation of the results, ensuring greater reliability and generalizability of the conclusions drawn from this research.