| Introduction: Familial breast cancer refers to the presence of two blood-related members of a family with breast cancer.Breast cancer with a clear genetic factor is called hereditary breast cancer,which accounts for 5% to 10% of the entire breast cancer population.Most hereditary breast cancers are associated with BRCA1/2.In addition to BRCA1/2,there are other known breast cancer susceptibility genes,such as p53 and PTEN.Breast cancer associated with these genetic mutations is classified as hereditary breast cancer[1,2,3].Fanconi anemia(FA)is an autosomal recessive genetic disease involving hematopoietic stem cells.Its clinical manifestations are complex and diverse.Genetic analysis found at least 15 subtypes(FANCA,FANCB,FANCC,FANCD1/BRCA2,FANCD2,FANCE,FANCF,FANCG,FANCI,FANCJ/BRIP1,FANCL,FANCM,FANCN/PALB2,FANCO/RAD51 C,FANCP/SLX4),of which 12 genes have been cloned,which can explain almost all cases of Fanconi anemia [4,5].The current study found that proteins encoded by their genes may be coordinated with each other to regulate DNA repair through a common "FANCONI anemia/BRCA pathway." FANCD2 and BRCA1 are important components of the FANCONI anemia/BRCA pathway [6,7].DNA mutations are the leading cause of many diseases in humans,including tumors,and the direct cause of DNA mutations is different types of DNA damage[8,9].DNA damage caused by different factors can be repaired by different repair pathways.Genetic defects in DNA repair are also the molecular basis of a variety of hereditary diseases,and these patients often exhibit extremely high tumor susceptibility[10].The current study found that breast cancer-associated oncogenes BRCA1 and BRCA2 can form complexes with other tumor suppressor genes such as Fanconi anemia gene to regulate gene repair by homologous chromosome recombination,thereby preventing tumorigenesis[11,12].Objective: To understand whether BRCA1 and FANCD2 genes are expressed in familial,sporadic breast and benign tissues relevantly.To investigate the interaction of the two genes in the FA/BRCA pathway,the role of FANCD2 ubiquitination in sporadic breast cancer and FANCD2 ubiquitination in disease progression.All malignant cases were followed up to analyze the pathological characteristics of breast cancer patients with differences in BRCA1,FANCD2 gene expression and Fancd2 ubiquitination.Materials and Methods: Immunohistochemical SP method was used to detect the expression of BRCA1 and FANCD2 protein in 335 cases of familial,sporadic breast cancer and benign breast cancer.The expression of BRCA1 and FANCD2 in different tissues were discussed.The protein level and ubiquitination level of FANCD2 in 56 cases of sporadic breast cancer were detected by Western Blot.The clinical and pathological data of all breast cancer patients were collected and the prognosis of patients was followed up.The expression of BRCA1 and FANCD2,and the relationship between the degree of ubiquitination of FANCD2 and the prognosis of familial and sporadic breast cancer were analyzed.The experiment was verified by TCGA database.Results: 1.Patient characteristics The 47 women in the benign tumor group had a median age at diagnosis of 46 years(range: 18–71 years),with the tumors including fibroadenoma(15 cases),cystic hyperplasia(24 cases),intraductal papilloma(7 cases),and sclerosing adenosis(1 case).A total of 288 women had primary malignant breast cancer(Table 1),with median ages at diagnosis of 52 years(range: 31–76 years)in the FBC group and 50 years(range: 25–79 years)in the SBC group.The malignant tumors included intraductal carcinoma(39 cases),invasive ductal carcinoma(172 cases),invasive lobular carcinoma(47 cases),and other rare types(30 cases).The histological types were predominantly type I(FBC: 30.5%,SBC: 21.1%)and type II(FBC: 47.5%,SBC: 53.1%).Large proportions of the tumors were considered T1(46.9%),T2(43.1%),or involved lymphatic invasion(41%).Based on the 2017 AJCC criteria,most patients had stage I disease(FBC: 43.3%,SBC: 37.4%)or stage II disease(FBC: 36.2%,SBC: 38.1%).The follow-up was terminated in January 2016 with a median follow-up time of 74.4 months(range: 4–155 months).The median DFS time was 91.8 months,with 1-year,3-year,and 5-year DFS rates of 97.9%,91.7%,and 83.3%,respectively.There were 24(8.33%)local recurrences and 29(10.08%)distant metastases that occurred within 5 years.2.Immunocytochemical staining for BRCA1 and FANCD2 Typical cytoplasmic and nuclear staining results for BRCA1 in benign and malignant breast tissues are shown in Figure 1.The negative and positive control was selected from the previously diagnosed section of the pathology department of our hospital.Positive expression of BRCA1 was associated with a significantly better 5-year DFS rate among FBC cases(99/141,70.2%)than among SBC cases(21/147,14.3%;P<0.001).The typical staining results for FANCD2 were predominantly nuclear in the benign and malignant tissues(Fig.1D,1H).Positive expression of FANCD2 was associated with a significantly better 5-year DFS rate among FBC cases(59/141,41.8%)than among SBC cases(42/147,28.6%;P=0.037).Table 2 shows the patients' clinical characteristics and the expression of BRCA1 and FANCD2.Expression of BRCA1 in FBC was positively correlated with tumor size(P=0.021),lymphatic invasion(P=0.004),TNM stage(P=0.01),ER status(P=0.014),and FANCD2 expression(P<0.001).However,these correlations were not observed among SBC cases.Nevertheless,expression of FANCD2 in SBC was positively correlated with tumor size(P=0.003),TNM stage(P<0.001),and Ki-67 index(P=0.015).The benign tissues were frequently positive for BRCA1(29/47,61.7%)and FANCD2(21/47,44.7%).There was no significant correlation between the expression of the two proteins(P=0.206).Benign tissues were more frequently positive for BRCA1 than SBC tissues(61.7% vs.14.3%,P<0.001)and more frequently positive for FANCD2 than SBC tissues(44.7% vs.27.9%,P=0.069).Further analysis of BRCA1 and FANCD2 expression in breast cancer based on TCGA data Immunohistochemistry of this experiment revealed that BRCA1 and FANCD2 were expressed in both breast cancer tissues and adjacent tissues.Based on the results derived from GEPIA,the expression of BRCA1 and FANCD2 in breast cancer tissues was higher than in adjacent tissues(Fig 2).The association between the expression of FANCD2 and BRCA1 and clinical parameters was analyzed using the TCGA dataset.Due to different groupings,our results were not exactly the same as those from the TCGA database(Table 3).Expression of BRCA1 and FANCD2 in the TCGA dataset was positively correlated with tumor size(BRCA1: P <0.001;FANCD2: P <0.001)and ER expression(BRCA1: P <0.001;FANCD2: P<0.001);additionally,there was a positive correlation between BRCA1 and FANCD2 expression.Expression of FANCD2 in the TCGA dataset was positively correlated with the TNM stage(P=0.039).The above results from the TCGA database were consistent with the results of this study.4.Prognostic value of BRCA1 and FANCD2 expression The Kaplan-Meier curves were compared based on the patients' BRCA1 and FANCD2 status and FBC and SBC groupings.In the FBC group,BRCA1 expression was associated with significantly decreased DFS when compared to no BRCA1 expression(P=0.001,Fig.3A);this was based on a lower 5-year DFS rate(77.8% vs.95.2%)and a shorter median DFS time(81.4 months vs.108.8 months).However,in the FBC group,there was no significant difference in prognosis according to expression or non-expression of FANCD2(P=0.328,Fig.3B),although the group with FANCD2 expression had a lower 5-year DFS(78% vs.86.6%)and a shorter median DFS time(79.7 months vs.96.7 months).In the SBC group,BRCA1 expression was not associated with a significant difference in DFS(P=0.22,Fig.3C),although the group with BRCA1 expression had a slightly lower 5-year DFS rate(76.2% vs.84.9%)and a shorter median DFS time(92 months vs.94.3 months).In the SBC group,FANCD2 expression was associated with significantly decreased DFS(P<0.001,Fig.3D),based on a lower 5-year DFS rate(63.4% vs.91.5%)and a shorter median DFS time(69.7 months vs.103.3 months).Therefore,BRCA1 expression had significant prognostic value in FBC cases,while FANCD2 expression had significant prognostic value in SBC cases.5.Univariate and multivariate analysis of breast cancer patients The univariate analyses revealed that DFS among patients with FBC was significantly correlated with TNM stage(P=0.001)and BRCA1 expression(P=0.001),while DFS among patients with SBC was significantly correlated with tumor size(P=0.001),lymphatic invasion(P=0.004),TNM stage(P<0.001),the Ki-67 index(P=0.025),and FANCD2 expression(P<0.001).The multivariate Cox proportional hazards model also revealed that DFS among patients with FBC was independently predicted by the TNM stage(III–IV vs.I-II,HR: 2.042,95% CI: 1.150–3.624,P=0.015)and BRCA1 expression(positive vs.negative,HR: 2.168,95% CI: 1.142–4.113,P=0.018).In the SBC group,DFS was independently predicted by TNM stage(III–IV vs.I–II,HR: 4.361,95% CI: 2.465–7.716,P<0.001)and FANCD2 expression(positive vs.negative,HR: 1.192,95% CI: 1.041–3.512,P=0.037)(Table 4).6.FANCD2 ubiquitination is an independent prognostic factor for SBC Ubiquitination of FANCD2 reflected functional activation of the FA pathway,and FANCD2 expression was an independent prognostic factor for SBC.Thus,we performed Western blotting of 56 randomly selected SBC tissues to examine whether FANCD2 ubiquitination was associated with prognosis.The expressions of FANCD2-L and FANCD2-S were used to calculate the L/S ratio(Fig.4)to quantify FANCD2 ubiquitination,with L/S ratios ranging from 0.13 to 1.36(median: 0.645).The 56 cases were subsequently divided based on the median value into groups with ubiquitination High(Ub High,?0.645)or ubiquitination Low(Ub Low,<0.645),and representative Western blot results are shown in Figure 5(16,17).Patients with Ub High had significantly better DFS than patients with Ub Low(P=0.036,Fig.6),and the results of the univariable and multivariable analyses are shown in Table 5.Among FBC cases,patients with Ub High had a significantly better 5-year DFS rate(85.7% vs.71.4%)and a significantly better median DFS time(101.2 months vs.73.4 months).Multivariable analyses revealed that,in addition to TNM stage(P=0.003)and FANCD2 expression(P=0.006),FANCD2 ubiquitination independently predicted DFS in the SBC group(Ub High vs.Ub Low,HR: 0.335,95% CI: 0.128–0.875,P=0.026).Conclusion:1.There is a relationship between BRCA1 and FANCD2 in breast cancer.The occurrence and development of breast cancer are related to the expression of FANCD2 and the inactivation of FA/BRCA pathway.Mutant BRCA1 may affect breast cancer by activating FANCD2 expression.The occurrence and development of FANCD2 may also affect the formation of breast cancer through the FA/BRCA pathway.2.The prognosis of BRCA1-positive patients in familial breast cancer is worse,and the relationship between FANCD2 expression and prognosis is not clear.3.High FANCD2 expression and low FANCD2 ubiquitination in sporadic breast cancer predict a relatively poor prognosis.The process of ubiquitination of FANCD2 was inhibited.It has been reported that this may be related to the abnormal activity of ubiquitinated ligase,and may also be related to the upstream dysfunction of FA/BRCA pathway,leading to the ubiquitination of downstream FANCD2,which ultimately affects the entire pathway,leading to the appearance of malignant tumors.4.In breast cancer,tumor size,lymph node infiltration,TNM staging,ER and BRCA1 expression are significant prognostic factors.The degree of FANCD2 expression and ubiquitination in sporadic breast cancer can also be used as an independent factor in evaluating prognosis. |
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