Genetic polymorphisms and protein expression of P53 and BRCA1 in preneoplastic and neoplastic rat mammary glands

Al-Dhaheri,Wafa; Hassouna,Imam; Karam,Sherif  M.

doi:10.3892/or.2018.6284

Genetic polymorphisms and protein expression of P53 and BRCA1 in preneoplastic and neoplastic rat mammary glands

Authors:
- Wafa Al-Dhaheri
- Imam Hassouna
- Sherif M. Karam
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Affiliations: Department of Anatomy, College of Medicine and Health Sciences, UAE University, Al-Ain, United Arab Emirates, Department of Biology, College of Science, UAE University, Al-Ain, United Arab Emirates
Published online on: February 28, 2018 https://doi.org/10.3892/or.2018.6284
Pages: 2193-2200

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Abstract

Breast cancer is the most common type of cancer and the leading cause of cancer-related deaths among women in the United Arab Emirates and worldwide. Although many factors contribute to the high incidence of breast cancer, a considerable number of cases are related to environmental factors. In the present study, breast cancer was induced in female rats using a single dose, 80 mg/kg body wt, of the environmental carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The aim of the present study, was to characterize some of the molecular changes that occur during breast cancer development in the DMBA-treated rat model. Mammary gland tissues of control and DMBA-treated rats were processed for: i) immunohistochemical probing using anti-BRCA1 antibody to characterize and correlate the localization of this cell cycle protein during progression to cancer, ii) western blotting to analyze the alteration of p53 protein expression in preneoplastic and neoplastic lesions of the mammary glands, and iii) polymerase chain reactions using primers specific for BRCA1 and P53 genes followed by single stranded conformational polymorphism (SSCP) or restriction fragment length polymorphism (RFLP) assays to detect possible mutations in these genes during development of breast cancer. Microscopic examination revealed a wide range of preneoplastic and neoplastic lesions providing a sequence representing the multistep process of breast cancer formation in DMBA-treated rats. Probing for BRCA1 protein revealed a gradual defect in its translocation from the cytoplasm to the nucleus during breast cancer progression. In control rats, BRCA1 was present in the nuclei of terminal duct epithelial cells. However, in the preneoplastic lesions, BRCA1 was localized in both the cytoplasm and nuclei of the epithelial duct cells. In all malignant lesions, BRCA1 was mostly found in the cytoplasm. Western blotting revealed initial downregulation in the expression of p53 protein during breast cancer development. However, with progression towards malignancy, upregulation of p53 was observed. These changes were associated with polymorphism in p53 gene, which was detected in exon 5 using SSCP assay. However, using RFLP and BamHI to digest the PCR products of exon 11 of BRCA1 gene revealed no detectable polymorphisms. In conclusion, molecular characterization of the early changes that occur during development of breast cancer provides some clues for better understanding of its pathogenesis.

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1	Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D and Bray F: Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 136:E359–E386. 2015. View Article : Google Scholar : PubMed/NCBI
2	Dieterich M, Stubert J, Reimer T, Erickson N and Berling A: Influence of lifestyle factors on breast cancer risk. Breast Care. 9:407–414. 2014. View Article : Google Scholar : PubMed/NCBI
3	Seltenrich N: Institutes in the Lead: Identifying environmental factors in breast cancer. Environ Health Perspect. 124:A199–A205. 2016. View Article : Google Scholar : PubMed/NCBI
4	Rennó AL, Alves-Júnior MJ, Rocha RM, De Souza PC, de Souza VB, Jampietro J, Vassallo J, Hyslop S, Anhê GF, de Moraes Schenka NG, et al: Decreased expression of stem cell markers by simvastatin in 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer. Toxicol Pathol. 43:400–410. 2015. View Article : Google Scholar : PubMed/NCBI
5	Russo J: Significance of rat mammary tumors for human risk assessment. Toxicol Pathol. 43:145–170. 2015. View Article : Google Scholar : PubMed/NCBI
6	Al-Dhaheri WS, Hassouna I, Al-Salam S and Karam SM: Characterization of breast cancer progression in the rat. Ann NY Acad Sci. 1138:121–131. 2008. View Article : Google Scholar : PubMed/NCBI
7	Ciriello G, Miller ML, Aksoy BA, Senbabaoglu Y, Schultz N and Sander C: Emerging landscape of oncogenic signatures across human cancers. Nat Genet. 45:1127–1133. 2013. View Article : Google Scholar : PubMed/NCBI
8	Peng L, Xu T, Long T and Zuo H: Association between BRCA status and P53 status in breast cancer: A meta-analysis. Med Sci Monit. 22:1939–1945. 2016. View Article : Google Scholar : PubMed/NCBI
9	Rivlin N, Brosh R, Oren M and Rotter V: Mutations in the p53 tumor suppressor gene: Important milestones at the various steps of tumorigenesis. Genes Cancer. 2:466–474. 2011. View Article : Google Scholar : PubMed/NCBI
10	Elledge RM and Allred DC: The P53 tumor suppressor gene in breast cancer. Breast Cancer Res Treat. 32:39–47. 1994. View Article : Google Scholar : PubMed/NCBI
11	Maslon MM and Hupp TR: Drug discovery and mutant p53. Trends Cell Biol. 20:542–555. 2010. View Article : Google Scholar : PubMed/NCBI
12	Jiang Q and Greenberg RA: Deciphering the BRCA1 tumor suppressor network. J Biol Chem. 290:17724–17732. 2015. View Article : Google Scholar : PubMed/NCBI
13	Rosen EM, Fan S, Pestell RG and Goldberg ID: BRCA1 gene in breast cancer. J Cell Physiol. 196:19–41. 2003. View Article : Google Scholar : PubMed/NCBI
14	Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al: A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 266:66–71. 1994. View Article : Google Scholar : PubMed/NCBI
15	Easton DF, Bishop DT, Ford D and Crockford GP: Genetic linkage analysis in familial breast and ovarian cancer: Results from 214 families. The breast cancer linkage consortium. Am J Hum Genet. 52:678–701. 1993.PubMed/NCBI
16	Keshavarzi F, Javadi GR and Zeinali S: BRCA1 and BRCA2 germline mutations in 85 Iranian breast cancer patients. Fam Cancer. 11:57–67. 2011. View Article : Google Scholar
17	Shen SX, Weaver Z, Xu X, Li C, Weinstein M, Chen L, Guan XY, Ried T and Deng CX: A targeted disruption of the murine Brca1 gene causes gamma-irradiation hypersensitivity and genetic instability. Oncogene. 17:3115–3124. 1998. View Article : Google Scholar : PubMed/NCBI
18	Ottini L, D'Amico C, Noviello C, Lauro S, Lalle M, Fornarini G, Colantuoni OA, Pizzi C, Cortesi E, Carlini S, et al: BRCA1 and BRCA2 mutations in central and southern Italian patients. Breast Cancer Res. 2:307–310. 2000. View Article : Google Scholar : PubMed/NCBI
19	Fraser JA, Reeves JR, Stanton PD, Black DM, Going JJ, Cooke TG and Bartlett JM: A role for BRCA1 in sporadic breast cancer. Br J Cancer. 88:1263–1270. 2003. View Article : Google Scholar : PubMed/NCBI
20	Feng Z, Kachnic L, Zhang J, Powell SN and Xia F: DNA damage induces p53-dependent BRCA1 nuclear export. J Biol Chem. 279:28574–28584. 2004. View Article : Google Scholar : PubMed/NCBI
21	Yang ES and Xia F: BRCA1 16 years later: DNA damage-induced BRCA1 shuttling. FEBS J. 277:3079–3085. 2010. View Article : Google Scholar : PubMed/NCBI
22	Hollstein M, Sidransky D, Vogelstein B and Harris CC: p53 mutation in human cancers. Science. 253:49–53. 1991. View Article : Google Scholar : PubMed/NCBI
23	Zurer I, Hofseth LJ, Cohen Y, Xu-Welliver M, Hussain SP, Harris CC and Rotter V: The role of p53 in base excision repair following genotoxic stress. Carcinogenesis. 25:11–19. 2004. View Article : Google Scholar : PubMed/NCBI
24	Gannon JV, Greaves R, Iggo R and Lane DP: Activating mutation in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J. 9:1595–1602. 1990.PubMed/NCBI
25	Andersen TI, Holm R, Nesland JM, Heimdal KR, Ottestad L and Børresen AL: Prognostic significance of TP53 alterations in breast carcinoma. Br J Cancer. 68:540–548. 1993. View Article : Google Scholar : PubMed/NCBI
26	Lee CH, Kim WH, Lim JH, Kang MS, Kim DY and Kweon OK: Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors. J Vet Sci. 5:63–69. 2004. View Article : Google Scholar : PubMed/NCBI
27	Rossner P Jr, Gammon MD, Zhang YJ, Terry MB, Hibshoosh H, Memeo L, Mansukhani M, Long CM, Garbowski G, Agrawal M, et al: Mutations in p53, p53 protein overexpression and breast cancer survival. J Cell Mol Med. 13:3847–3857. 2009. View Article : Google Scholar : PubMed/NCBI
28	Kalemi TG, Lambropoulos AF, Gueorguiev M, Chrisafi S, Papazisis KT and Kotsis A: The association of p53 mutations and p53 codon 72, Her 2 codon 655 and MTHFR C677T polymorphisms with breast cancer in Northern Greece. Cancer Lett. 222:57–65. 2004. View Article : Google Scholar
29	Jerry DJ, Kittrell FS, Kuperwasser C, Laucirica R, Dickinson ES, Bonilla PJ, Butel JS and Medina DA: Mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development. Oncogene. 19:1052–1058. 2000. View Article : Google Scholar : PubMed/NCBI
30	Malisic E, Jankovic R, Slavkovic D, Milovic-Kovacevic M and Radulovic S: P53 gene mutations and codon 72 polymorphism in ovarian carcinoma patients from Serbia. J BUON. 15:101–106. 2010.PubMed/NCBI
31	Keohavong P, Gao WM, Mady HH, Kanbour-Shakir A and Melhem MF: Analysis of p53 mutations in cells taken from paraffin-embedded tissue sections of ductal carcinoma in situ and atypical ductal hyperplasia of the breast. Cancer Lett. 212:121–130. 2004. View Article : Google Scholar : PubMed/NCBI
32	Zhou W, Muggerud AA, Vu P, Due EU, Sørlie T, Børresen-Dale AL, Wärnberg F and Langerød A: Full sequencing of TP53 identifies identical mutations within in situ and invasive components in breast cancer suggesting clonal evolution. Mol Oncol. 3:214–219. 2009. View Article : Google Scholar : PubMed/NCBI
33	Jerry DJ, Ozbun MA, Kittrell FS, Lane DP, Medina D and Butel JS: Mutations in p53 are frequent in the preneolpastic stage of mouse mammary tumor development. Cancer Res. 53:3374–3381. 1993.PubMed/NCBI
34	Manjanatha MG, Chen JB, Shaddock JG Jr, Harris AJ, Shelton SD and Casciano DA: Molecular analysis of lacI mutations in Rat2™ cells exposed to 7,12-dimethylbenz[a]anthracene: Evidence for DNA sequence and DNA strand biases for mutation. Mutat Res. 372:53–64. 1996. View Article : Google Scholar : PubMed/NCBI
35	Chatterjee M, Janarthan M, Manivannan R, Rana A and Chatterjee M: Combinatorial effect of fish oil (Maxepa) and 1α,25-dihydroxyvitamin D₃ in the chemoprevention of DMBA-induced mammary carcinogenesis in rats. Chem Biol Interact. 188:102–110. 2010. View Article : Google Scholar : PubMed/NCBI
36	Chakravarti D, Pelling JC, Cavalieri EL and Rogan EG: Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: The role of apurinic sites. Proc Natl Acad Sci USA. 92:10422–10426. 1995. View Article : Google Scholar : PubMed/NCBI