Abstract 3: TIP60 modulates colorectal cancer stemness through cd44 expression

Abstract Objective: To identify the functional role of TIP60 in regulating colorectal cancer (CRC) stemness. Materials and Methods: The HCT116 CRC cell lines were cultured in DMEM (high glucose) supplemented with 10% FBS. ShRNA-mediated depletion of TIP60 was generated using lentiviral vectors. To confirm TIP60 knockdown efficiency and CD44 expression levels, qPCR analyses were performed. For the colony formation assay, 1000 cells of HCT116 ShLuc control and ShTIP60 were seeded in 6-well plates and incubated for 11 days. For immunofluorescence staining, cells were grown on cover slips, fixed with 100% methanol for 20 minutes, and blocked with 3% BSA. They were then incubated with E-cadherin primary antibody at a 1:500 dilution and secondary antibody Alexa Fluor-488 at a 1:1000 dilution. Cells were mounted with Vectashield mounting medium containing DAPI. Spheroid culture was conducted in 96-well ultra-low attachment plates, with 100 cells of HCT116 ShLuc control and ShTIP60 seeded in each well and incubated for 7 days. The 3D invasion assay was performed in 96-well ultra-low round-bottom plates supplemented with 3D basement membrane extract (BME) invasion matrix. Findings: This study uncovered the pivotal role of HIV Tat-interactive protein (TIP60) in regulating CRC stemness. TIP60 depletion resulted in a significant decrease in cellular proliferation, highlighting the importance of TIP60 in CRC growth. Furthermore, TIP60 depletion altered colony formation, transitioning from densely packed formations to scattered spindle networks implying its involvement in the epithelial-mesenchymal transition (EMT). 3D culture models demonstrated that TIP60 is essential for spheroids formation, emphasizing its significance in promoting cancer stemness. TIP60-depleted cells exhibited enhanced invasion in a 3D BME invasion matrix, accentuating its crucial contribution to cellular invasiveness. Furthermore, we have identified that TIP60 undergoes liquid-liquid phase separation (LLPS). TIP60 harbors an intrinsically disordered region (IDR), which is essential for inducing LLPS in CRC. We have also discovered that TIP60 IDR regulates CRC stemness and EMT in CRC. In addition, the reduction of TIP60 was linked to a decrease in the expression of CD44, which is a crucial indicator for cancer stem cells (CSCs). Significantly, CD44 overexpression reinstated the efficiency of spheroid formation and reversed the EMT phenotype. Conclusions and Clinical Implications: Our findings suggest that TIP60 has CSC characteristics in CRC, and targeting TIP60 will offer a novel therapeutic approach against colorectal cancer stem cells. Citation Format: Mohammad Asad, Sudhakar Jha. TIP60 modulates colorectal cancer stemness through cd44 expression [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 3.