Genetic and epigenetic alterations affecting PARK-2 expression in cervical neoplasm among North Indian patients

Abstract

The recent investigation on PARK-2, a putative tumor suppressor gene, has found that it has been altered in multiple human malignancies. However, the clinical impact of PARK-2 alteration in uterine cervix carcinoma has not yet been studied. Therefore, we aimed to examine mutations, promoter hypermethylation, and protein expression of PARK-2 among the North Indian patients and their association with clinical parameters to evaluate the implication of PARK-2 in the genesis of cervical cancer. A total of 168 patient samples were processed for mutational analysis by single-strand conformation polymorphism, sequencing, and further in silico analysis of the identified mutations. Promoter hypermethylation by methylation-specific polymerase chain reaction and expression of PARK-2 were performed using immunohistochemistry. Statistical correlation between molecular findings and the clinicopathological parameters was taken to figure out the meaningful outcome. As per our findings, 3.5% (6/168) tumors showed novel missense mutations in exon 11 of PARK-2. In silico analysis showed high structural deviations manifested by mutations, A398D and Y391N, in both mutant proteins as compared to wild type. Promoter hypermethylation was observed in total of 29% of (48/168) tumor samples. Furthermore, 46.43% tumors (78/168) exhibited loss of PARK-2 expression in cervical carcinoma. The loss of expression of PARK-2 when correlated with clinical parameters resulted in significant association with tumor stage (p = 0.002) and with histological grade (p = 0.025). However, only clinical stage remained significant after Bonferroni correction (p < 0.007). A trend was observed between PARK-2 promoter hypermethylation and its protein expression. Our study provided sufficient information and insight for investigation of PARK-2 and highlighted its role as a tumor suppressor gene in cervical cancer in North Indian population.

Parkin gene mutations are not common, but its epigenetic inactivation is a frequent event and predicts poor survival in advanced breast cancer patients

“…A total of 12 exons were amplified to reveal any somatic mutation of Parkin by single-stranded conformational polymorphism (SSCP). Extracted DNA was used for PCR amplification using primers (Additional file 1: Table S2) and amplification conditions as described earlier [9]. The amplified products were visualized by electrophoresis using 2% agarose gel and stained with ethidium bromide.…”

“…The amplified products were visualized by electrophoresis using 2% agarose gel and stained with ethidium bromide. SSCP protocol was followed as mentioned earlier [9]. Samples that demonstrated differences in band-shifts with respect to the wild-type bands were categorized as mutants.…”

“…The 3–4 μm thin tissue sections were then taken on Poly-L-lysine coated slides. The protocol was followed as described previously [9]. The slides were incubated in a humidified chamber with 1:100 dilution of anti- PARK-2 antibody (cat #ab15954, Abcam) at 4 °C for 24 h. After washing with PBS thrice, slides were next incubated with biotinylated secondary antibody and with an avidin-horseradish peroxidase for 25–30 min.…”

“…It is an emerging molecular marker which raises the hopes for the development of novel therapeutics in combating cancer [20, 21]. Recent studies have reported aberrant methylation at Parkin promoter among acute lymphoid leukemia (ALL), chronic granulocytic leukemia (CGL) [15], nasopharyngeal carcinoma [22] and cervical cancer [9]. Although the precise genetic and epigenetic mechanisms contributing to Parkin loss in breast cancer remain elusive, this prompted us to investigate the possible mechanisms as well as the potential role of Parkin gene in breast cancer.…”

The Progress of Methylation Regulation in Gene Expression of Cervical Cancer

“…In the North Indian population, methylation of the p16 gene promoter which induces loss of tumor-suppressing activity and promotes the development of cervical cancer is observed significantly in FIGO stage III [ 44 , 45 ]. Meanwhile, correlated with clinical parameters, promoter hypermethylation and expression loss of PARK-2, RAR β , and FHIT are significantly higher in cervical cancer than in CINs and normal tissues, resulting in a significant association with tumor stage and histological grade [ 46 , 47 ]. In Uighur women, increased methylation was detected at 13 CpG sites, and a high methylation level was associated with the risk of CIN2 + ; the strongest related site was 6650 [ 48 ].…”