STUDY OF P53 EXPRESSION IN CARCINOMA CERVIX AND NORMAL CERVICAL EPITHELIUM WITH CLINICAL CORRELATION

Cervical cancer is a world- wide public health problem with an incidence of 530,232 new cases and 275,008 deaths every year. Across the world, carcinoma of the uterine cervix is the second most common malignancy in women, and is a major cause of morbidity and mortality. Cervical carcinoma is the leading cancer in India, common in the females between 15 and 44 years of age group [1]. India accounts for one-fth of the world's burden of cervical cancer and the incidence of cervical carcinoma has increased from 0.11 million in the year 2000 to 0.16 million cases in 2012 [2].

TP73 is a credible biomarker for predicting clinical progression and prognosis in cervical cancer patients

Tumor protein p73 (TP73) has been reported to be dysregulated in various types of human cancer and associated with clinical progression and outcome. Owing to the lack of reports on the correlation between TP73 protein expression and clinicopathologic features of cervical cancer, the aim of our research was to explore the clinical and prognostic significance of TP73 protein expression in cervical cancer patients. In our study, TP73 protein expression was detected by immunochemistry in 118 paraffin-embedded cervical cancer tissue specimens and 40 paraffin-embedded normal cervical epithelium tissue specimens. In the results, we found cervical cancer tissues exhibited high TP73 expression in comparison with normal cervical epithelium tissues, which was consistent with the expression status of TP73 in The Cancer Genome Atlas (TCGA) database. Furthermore, we analyzed the relationships between TP73 expression and clinicopathologic features through using the chi-square test or Fisher’s exact test, and found high expression of TP73 was markedly associated with early clinical stage, less lymph node metastasis, absent distant metastasis, squamous cell carcinoma and favorable histological grade. The Kaplan–Meier method and log-rank test were performed based on the expression level of TP73 in a cervical cancer cohort from the TCGA database, and showed that TP73 expression was positively correlated with overall survival time in cervical cancer patients. Moreover, univariate and multivariate Cox proportional hazards regression model indicated that high TP73 expression was identified as an independent factor for predicting favorable overall survival in cervical cancer patients. In conclusion, TP73 expression is increased in cervical cancer tissues and cells, and acts as a credible biomarker for predicting favorable overall survival in cervical cancer patients.

Deregulation of LIMD1–VHL–HIF-1α–VEGF pathway is associated with different stages of cervical cancer

To understand the mechanism of cellular stress in basal–parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal–parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal–parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal–parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2′-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal–parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

Association of P16-RBSP3 inactivation with phosphorylated RB1 overexpression in basal–parabasal layers of normal cervix unchanged during CACX development

To understand the molecular mechanism of RB1 phosphorylation in basal–parabasal layers of normal cervix and during cervical cancer (CACX) development, we analyzed the alterations (expression/methylation/deletion/mutation) of RB1/phosphorylated RB1 (p-RB1) (ser807/811 and ser567) and two RB1 phosphorylation inhibitors, P16 and RBSP3, in disease-free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 28), CACX (n = 102) samples and two CACX cell lines. Immunohistochemical analysis revealed high/medium expression of RB1/p-RB1 (ser807/811 and ser567) and low expression of P16 and RBSP3 in proliferating basal–parabasal layers of majority of normal cervical epitheliums, irrespective of HPV16 infection. Interestingly, 35–52% samples showed high/medium expression of P16 in basal–parabasal layers of normal and had significant association with deleterious non-synonimous SNPs of P16. Methylation of P16 and RBSP3 in basal–parabasal layers of normal cervix (32 and 62%, respectively) showed concordance with their respective expressions in basal–parabasal layers. The methylation frequency of P16 and RBSP3 in basal–parabasal layers of normal did not change significantly in CIN and CACX. The deletion frequency of P16 and RB1 increased significantly with CACX progression. While, deletion of RBSP3 was high in CIN and comparable during CACX progression. P16 showed scattered and infrequent mutation in CACX. The alteration of P16 and RBSP3 was synergistic and showed association with overexpression of p-RB1 in tumors and associated with poor prognosis of patients. Thus, our data suggest that overexpression of p-RB1 in basal–parabasal layers of normal cervical epithelium was due to methylation/low functional-linked non-synonimous SNPs of P16 and RBSP3. This pattern was maintained during cervical carcinogenesis by additional deletion/mutation.

S100A7 in the Fallopian tube: a comparative study

SummaryThe oviduct is a dynamic organ in which final gamete maturation, fertilization and early embryo development take place. It is considered to be a sterile site; however the mechanism for sterility maintenance is still unknown. S100A7 is an anti-microbial peptide that has been reported in human reproductive tissues such as prostate, testicle, ovary, normal cervical epithelium and sperm. The current work reports the presence of S100A7 in the Fallopian tube and its localization at the apical surface of epithelial cells. For comparison, porcine S100A7 was used for antibody development and search for peptide in reproductive tissues. Although present in boar seminal vesicles and seminal plasma, S100A7 was not detected on female porcine organs. Also, in contrast with the human protein, porcine S100A7 did not show anti-microbial activity under the conditions tested. Phylogenetic analyses showed high divergence of porcine S100A7 from human, primate, bovine, ovine and equine sequences, being the murine sequence at a most distant branch. The differences in sequence homology, Escherichia coli-cidal activity, detectable presence and localization of S100A7 from human and pig, suggest that there are possible different functions in each organism.