CDKN2A promoter methylation enhances self-renewal of glioblastoma stem cells and confers resistance to carmustine

Abstract

Abstract Background Glioblastoma, a highly aggressive form of brain cancer, poses significant challenges due to its resistance to therapy and high recurrence rates. This study aimed to investigate the expression and functional implications of CDKN2A, a key tumor suppressor gene, in glioblastoma cells, building upon the existing background of knowledge in this field. Method Quantitative reverse transcription PCR (qRT-PCR) analysis was performed to evaluate CDKN2A expression in U87 glioblastoma cells compared to normal human astrocytes (NHA). CDKN2A expression levels were manipulated using small interfering RNA (siRNA) and CDKN2A overexpression vector. Cell viability assays and carmustine sensitivity tests were conducted to assess the impact of CDKN2A modulation on glioblastoma cell viability and drug response. Sphere formation assays and western blot analysis were performed to investigate the role of CDKN2A in glioblastoma stem cell (GSC) self-renewal and pluripotency marker expression. Additionally, methylation-specific PCR (MSP) assays and demethylation treatment were employed to elucidate the mechanism of CDKN2A downregulation in U87 cells. Result CDKN2A expression was significantly reduced in glioblastoma cells compared to NHA. CDKN2A overexpression resulted in decreased cell viability and enhanced sensitivity to carmustine treatment. CDKN2A inhibition promoted self-renewal capacity and increased pluripotency marker expression in U87 cells. CDKN2A upregulation led to elevated protein levels of p16INK4a, p14ARF, P53, and P21, which are involved in cell cycle regulation. CDKN2A downregulation in U87 cells was associated with high promoter methylation, which was reversed by treatment with a demethylating agent. Conclusion Our findings demonstrate that CDKN2A downregulation in glioblastoma cells is associated with decreased cell viability, enhanced drug resistance, increased self-renewal capacity, and altered expression of pluripotency markers. The observed CDKN2A expression changes are mediated by promoter methylation. These results highlight the potential role of CDKN2A as a therapeutic target and prognostic marker in glioblastoma.