PARP-1 Functional Deficiency With Breast Cancer Carcinogenesis

PARP-1 functional deficiency with breast cancer carcinogenesisSpeciality:Biochemistry and Molecular Biology,School of Basic Medicine, Peking Union Medical CollegePhD Candidate:Cao,Wen-HuiSupervisor:Professor Shen,YanBreast cancer is one of the most common malignancies among females worldwide and its incidence is increasing with every year.Although there have been improvements in early diagnosis and therapy,this disease is still the second leading cause of cancer mortality in women.It is urgent to clarify its molecular mechanism.It has been shown that germ line mutations in many genes,such as BRCA1,BRCA2,ATM,CHK2 and p53,which involve in DNA damage response,contribute to familial breast cancer,implying that combinational deficiency in both DNA repair and cell cycle checkpoint plays an important role in breast cancer carcinogenesis.For～40%familial breast cancer cases,their breast cancer-related genes are still not clear.PARP-1 encodes a multifunctional protein,studies showed that loss of PARP-1 function lead to deficiencies in DNA-damage repair and chromosome instability,implying that PARP-1 deficiency may play a role in carcinogenisis.After more than ten years' studies on PARP-1 knockout mice,it is clear that loss of PARP-1 function would give rise to late-onset spontaneous tumors in mice,and carcinogenic agents could accelerate tumor formation in PARP-1^-/-mice.Tong's studies indicate that PARP-1 deficiency changed tumor types in p53^+/- mice,and gave rise to many cancers,including breast cancer.Further studies showed that PARP-1 deficiency caused late onset breast cancer in 19.5%female mice,and p53 deficiency could promote the incidence of breast cancer,indicating that PARP-1 and p53 have synergistic functions in suppressing breast cancer carcinogenesis. 1.Our studies in this workBased on the above evidences,our work mainly focused on two sides.First,we study the molecular and cellular changes existed in primary epithelial cells,which were separated from the PARP-1^-/- mice model.Second,we also analyzed PARP-1 mutations and single nucleotide polymorphisms(SNP) in sporadic French breast cancer cases to evaluate the correlation between PARP-1 mutation/polymorphism and human sporadic breast cancer.2.Results1) In this study,twenty rare heterozygous variants were found in nine(10.8%) breast cancer cases,including novel variants in promoter g.2772371T＞C(n=1),at 5'UTR c.-62C＞T(n=1),at introns(n=9),and at exon c.2819A＞G(Arg452Arg,n=1).Although two exon variants c.1148C＞A(Tyr383Ser) and c.2819A＞G(Arg940Lys),two 3'UTR variants c.3140G＞A and c.3415T＞C,and 4 intron variants of PARP-1 are registered in SNPs database,none of these rare variants was detected in the 100 controls.Mutational analysis indicated that functional mutation are rare in sporadic breast cancer.In this study,we detected a total of Twenty-five common SNPs(minor allele frequency＞10%) in 100 controls.We next divided them into 5 groups according to their genotype distribution.The genotype distribution for group A was significantly different between cases and controls(P=0.035),suggesting that their genotypes may be associated with susceptibility for breast cancer.We further performed association analysis between expression of ER,PR and PARP-1 SNPs.Interestingly,gtSNP of PARP-1 c.852T＞C CC-carriers had a significantly increased frequency of loss of ER expression compared with homozygote TT carriers(OR=38,95%CI 4.6-316).In addition,CC-carriers also showed a strong association of loss of PR expression compared with homozygote TT carriers(OR=5.17,95%CI 1.2-22.7).In summary,the results imply that functional PARP-lmutations are rare in sporadic breast cancer cases,but PARP-1 polymorphisms may confer susceptibility to breast cancer. 2) In this work,we mainly analyzed the early molecular and cellular changes in PARP-1^-/- PME cell,which may lead to carcinogenesis,and found that(1) PARP-1 deficiency causes aneupoidy,chromosome aberration and centrosome amplification in PME cells;PARP-1 and p53 play a synergistic role in the maintenance of centrosome function and chromosomal stability in PME cells.(2) PARP-1 deficiency compromises p53 function upon DNA damage response(DDR). Although Western blotting analysis revealed a similar basal level of p53 phosphorylation at serl5 in PARP-1-proficient and-deficient PME cells,upon DNA damage induced by adriamycin,phosphorylation of p53 in wild-type PME cells was significantly increased at 4 hrs.Although phosphorylation of p53 at ser15 could occur in response to DNA damage,PARP-1 deficiency reduced level of p53 activation.In addition,PARP-1 deficiency compromised activation of p21.(3) Upon DNA DSB damage induced by adriamycin treatment,PARP-1 deficiency didn't affectγH2AX foci formation.But compared to wild-type cells,BRCA1 foci were dramatically reduced in PARP-1^-/- PME cells,suggesting that PARP-1 deficiency compromises BRCA1 foci formation after DSB induction.Immunoprecipitation analysis showed that PARP-1 and BRCA1 has direct interaction.In summary,this study indicates that,(1) PARP-1 functional mutations are rare in sporadic breast cancer cases;polymorphisms existed in PARP-1 gene may contribute to breast cancer susceptibility.(2) PARP-1 and Poly(ADP-ribosyl)ation play an important role in DNA damage response;PARP-1 functional deficiency could result in genomic instability in PME cell,promoting breast cancer carcinogenesis.