Complex interplay at the epigenomic, genomic, transcriptomic, and proteomic levels have earlier been reported in GBC. BNIP3 belong to Bcl-2 protein family member which contain only Bcl-2 homology 3 (BH3) domain. Unlike BH3 only proteins, BNIP3 pro-death activity is controlled by both the BH3 domain and transmembrane (TM) domain of BNIP3.It has been reported during hypoxic and ischemic condition BNIP3 can play different roles including apoptosis, programmed necrosis, autophagy and mitophagy. In uveal melanoma BNIP3 expression detection by IHC reveals downregulation in 68.1% (32/47) samples while 31.9% (15/47) were upregulated. BNIP3 is reported as upregulated in breast cancer[18, 19], prostate cancer, lung cancer[21, 22], cervical cancer and endometrial cancer whereas studies demonstrated its reduced expression in colorectal cancer, pancreatic cancer, gastric carcinoma, ccRCC and haematopoietic tumors. However, BNIP3 expression profile has not been correlated with its promoter methylation status in GBC. In the present study we have demonstrated through semi-quantitative RT-PCR that 56% (58/84) of the GBC samples show down regulation of BNIP3 at transcript level. Interestingly, the lower expression in GBCshow significant correlation between BNIP3 mRNA expression and clinico-pathological parameters of GBC patients including pathological late stage and nodal spread. Similar correlation has also been demonstrated in invasive breast cancer and pancreatic cancer[5, 28]. Significant reduced expression in late stage and GBC with presence of nodal metastasis suggests its role in GBC initiation and progression. Although not significant, the patients with reduced BNIP3 expression have lower survival. Significant correlation of reduced expression of BNIP3 with low survival has been demonstrated in pancreatic cancer[5, 29]. We have also found similar correlation of BNIP3 expression with patient overall survival, however, it did not reach the significance level. In the present study, we examined sensitivity and specificity of BNIP3 for GBC. Our diagnostic utility analysis indicates high diagnostic potential of BNIP3 at mRNA level.
It has been reported from Indian population that there is increased promoter methylation of tumor suppressor genes in late stages of GBC. Promoter hypermethylation may be directly related to high incidence and poor prognosis of GBC in Asian countries. Thus, we further investigated whether the promoter hypermethylation of BNIP3 is a possible mechanism of its reduced expression. BNIP3 promoter hypermethylation is reported in colorectal cancer[25, 26, 31], pancreatic cancer[5, 12, 32, 33], gastric cancer[26, 34]whereas hypomethylation of BNIP3 in prostate cancer is also observed. Shao et al. (2019) demonstrated that inactivation of BNIP3 is through histone deacetylation but not promoter methylation in clear cell renal cell carcinoma while a few reports depicts that DNA methylation as well as histone deacetylation play key role in silencing of BNIP3 in hematopoietic tumors and colorectal cancer. In our study 69% of GBC samples show promoter hypermethylation. Further, correlation of methylation status of BNIP3 with late stage and lymph node metastasis reveals promoter hypermethylation may play a critical role in initiation of GBC. Sugita et al. (2011) and Shimizuet et al. (2010) demonstrated association of BNIP3 methylation with poor overall survival in gastric and colorectal cancer respectively [25, 34]. Similarly, our study also demonstrates that hypermethylation of BNIP3 promoter is associated with reduced overall survival and is correlated with its transcription suggesting its prognostic utility in GBC.
In conclusion our study suggests that promoter hypermethylation is a major mechanism of BNIP3 downregulation in GBC, suggesting its role in initiation and progression of the disease. Since the promoter hypermethylation of BNIP3 is more frequent in late stage tumors and there is significant decreasing expression of its transcripts from gallstone to late stage of GBC, promoter hypermethylation and down regulation of BNIP3 are early events in GBC progression. This study also suggests that BNIP3 may serve as diagnostic and prognostic marker in GBC. Further studies are needed to explore the functional role of BNIP3 for its therapeutic potential in GBC.