title: Association of P16-RBSP3 inactivation with phosphorylated RB1 overexpression in basal-parabasal layers of normal cervix unchanged during CACX development.
abstract: 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.
authors.affiliation: Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, West Bengal, India.
authors.forename: Chandraditya
authors.initials: C
authors.lastname: Chakraborty
authors.affiliation: Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, West Bengal, India.
authors.forename: Anirban
authors.initials: A
authors.lastname: Roychowdhury
authors.affiliation: Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, West Bengal, India.
authors.forename: Sudip
authors.initials: S
authors.lastname: Samadder
authors.affiliation: North Bengal Medical College and Hospital, Siliguri, West Bengal, India.
authors.forename: Anup
authors.initials: A
authors.lastname: Roy
authors.affiliation: Department of Gynecologic Oncology, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, West Bengal, India.
authors.forename: Ranajit Kumar
authors.initials: RK
authors.lastname: Mandal
authors.affiliation: Department of Gynecologic Oncology, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, West Bengal, India.
authors.forename: Partha
authors.initials: P
authors.lastname: Basu
authors.affiliation: Saroj Gupta Cancer Centre & Research Institute, MG Road, Thakurpukur, Kolkata, India.
authors.forename: Susanta
authors.initials: S
authors.lastname: Roychoudhury
authors.affiliation: Department of Oncogene Regulation, Chittaranjan National Cancer Institute, 37, S.P. Mukherjee Road, Kolkata 700026, West Bengal, India.
authors.forename: Chinmay Kumar
authors.initials: CK
authors.lastname: Panda
mesh_terms: D052685:Alphapapillomavirus / Q000302:isolation & purification; D045744:Cell Line, Tumor; D002584:Cervix Uteri / Q000378:metabolism*; D019175:DNA Methylation; D005260:Female; D019942:Genes, p16; D006801:Humans; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D056286:Retinoblastoma Binding Proteins / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D002583:Uterine Cervical Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000821:virology
chemical_list: C486483:CTDSPL protein, human; C000606393:RB1 protein, human; D056286:Retinoblastoma Binding Proteins; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases
keywords: CACX progression; HPV; RB1 phosphorylation; basal–parabasal layers