Abstract 4636: Keratin 6 and SOX2 regulate growth, stemness, basal/squamous expression, and chemotherapy response in muscle-invasive urothelial carcinoma

Abstract Background and purpose: Urothelial carcinoma (UC) is prevalent globally and it is well-established that arsenic exposure is associated with development of UC. Muscle-invasive UC (MIUC) is aggressive and accounts for 25% of all diagnosed UC cases. Cisplatin-based treatment is the standard treatment for MIUC. Patients with MIUC often have a dismal prognosis and frequently acquire chemoresistance, especially if the tumors exhibit squamous differentiation (SD). UC can be further subtyped as being either basal or luminal based on gene expression signatures of the tumors. The basal tumors are considered more aggressive and are enriched with expression of genes associated with SD (KRT1, KRT5, KRT6, KRT14, and KRT16). The UROtsa cell line is derived from normal human urothelium and our lab has malignantly transformed these cells using arsenite (As3+). The tumors derived from As3+-transformed cells resemble clinical cases of basal MIUC displaying focal areas of SD and expression of basal keratins (KRT1, 5, 6, 14, and 16). Additionally, SOX2 was found to be overexpressed in these As3+-transformed UROtsa cells. SOX2 expression in UC is correlated with reduced survival and promotes malignant stemness and proliferation. SOX2 is also associated with the development of cisplatin resistance in other cancers. The goal of this study was to define the role of KRT6 and SOX2 in regulating basal/squamous expression, regulating growth, and regulating sensitivity to cisplatin. Methods: We performed stable lentiviral knockdown of KRT6 or SOX2 in the As3+-transformed UROtsa cells (basal MIUC with SD), along with the commercially available UC cell lines 5637 (basal, invasive) and RT4 (luminal, non-invasive). After successful knockdown KRT6 or SOX2 in these cells, we assessed cellular proliferation and colony formation. Additionally, the gene/protein expression of the basal keratins along with other genes/proteins involved with growth and stemness were analyzed. Migration was evaluated using a wound healing assay and drug sensitivity was tested after KRT6 or SOX2 knockdown. Results: Knockdown of KRT6 or SOX2 in the As3+-transformed cells attenuated basal keratin expression, reduced cell proliferation, decreased colony forming abilities, and increased sensitivity to cisplatin compared to control cells. Furthermore, cell migration was reduced in SOX2 knockdown cells. Conclusion: These results indicate that both KRT6 and SOX2 play critical roles in basal/squamous expression, growth, migration, and cisplatin sensitivity in MIUC. However, further in vivo studies are required to confirm the impact of KRT6 and SOX2 expression on tumor growth, SD, invasion, and chemotherapy response. Citation Format: Nelofar Nargis, Aaron Mehus, Donald A. Sens. Keratin 6 and SOX2 regulate growth, stemness, basal/squamous expression, and chemotherapy response in muscle-invasive urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4636.