High nuclear expression of HIF1α, synergizing with inactivation of LIMD1 and VHL, portray worst prognosis among the bladder cancer patients: association with arsenic prevalence.

ABSTRACT

PURPOSE: Our study was aimed to understand the importance of LIMD1-VHL-HIF1α pathway in development of bladder carcinoma (BlCa) in association with arsenic prevalence. METHODS: At first, the mRNA expression pattern of the genes of this pathway (LIMD1, VHL and HIF1α) was checked in GEO datasets and in our samples. Next, genetic and epigenetic profiling of LIMD1 and VHL was done in our sample pool, validated in T24 BlCa cell line. The results were next correlated with various clinico-pathological parameters. RESULTS: Differential under-expression of LIMD1 and VHL genes was found in muscle-invasive BlCa (MIBC) in comparison to non-muscle-invasive BlCa (NMIBC). However, HIF1α protein, but mRNA, was found to be overexpressed among the MIBC samples; depicting the probability of HIF1α protein stabilization. Analysis of genetic and epigenetic profiles of LIMD1 and VHL exposed a frequent promoter methylation of LIMD1 gene in MIBC samples. Further, in-depth look into the results unveiled that the high nuclear expression of HIF1α was significantly correlated with genetic alterations of LIMD1, alone or in combination with VHL. Moreover, treating the T24 cells with a de-methylating agent (5-aza-2'-deoxycytidine) re-expressed the methylated LIMD1 and VHL genes, which in turn, reduced the HIF1α protein level significantly. Additionally, patients with high arsenic content (> 112 ng/g, AsH) seemed to have recurrent promoter methylation in LIMD1, as well as co-methylation/alteration of LIMD1 and VHL gene. Lastly, high nuclear expression of HIF1α in association with co-alteration of VHL and LIMD1 showed the worst overall survival (OS) among the patients. CONCLUSION: To conclude, MIBC samples portrayed higher alterations in VHL and LIMD1, thereby, stabilizing HIF1α protein and lowering the OS of patients.