Significance of the Fanconi Anemia FANCD2 Protein in Sporadic and Metastatic Human Breast Cancer

FANCD2, a pivotal protein in the Fanconi anemia and BRCA pathway/network, is monoubiquitylated in the nucleus in response to DNA damage. This study examines the subcellular location and relationship with prognostic factors and patient survival of FANCD2 in breast cancer. Antibodies to FANCD2 were used to immunocytochemically stain 16 benign and 20 malignant breast specimens as well as 314 primary breast carcinomas to assess its association with subcellular compartment and prognostic factors using Fisher’s Exact test or with patient survival over 20 years using Wilcoxon-Gehan statistics. Immunoreactive FANCD2 was found in the nucleus and cytoplasm of all 16 benign tissues, but nuclear staining was lost from a significant 19/20 malignant carcinomas (P < 0.0001). Antibodies to FANCD2 stained the cytoplasm of 196 primary carcinomas, leaving 118 as negatively stained. Negative cytoplasmic staining was significantly associated with positive staining for the metastasis-inducing proteins S100A4, S100P, osteopontin, and AGR2 (P ≤ 0.002). Survival of patients with FANCD2-negative carcinomas was significantly worse (P < 0.0001) than those with positively stained carcinomas, and only 4% were alive at the census date. Multivariate regression analysis identified negative staining for cytoplasmic FANCD2 as the most significant indicator of patient death (P = 0.001). Thus FANCD2’s cytoplasmic loss in the primary carcinomas may allow the selection of cells overexpressing proteins that can induce metastases before surgery.Previous reports have shown that the expression of four proteins that can induce metastasis in experimental rats are highly significantly correlated with each other and separately with early patient death in human breast cancer. These four proteins are S100A4, osteopontin (OPN), AGR2, and S100P.^1,2,3,4 If their expression is coordinated, then markers of the underlying mechanism should be even more highly correlated with patient demise. One possible coordination mechanism is the generation of an unstable genome by failure of a DNA repair pathway or some other protective mechanism. The majority of familial breast cancer is associated with individuals heterozygous for the BRCA1 or BRCA2 genes that encode proteins important for the repair of DNA double strand breaks and interstrand crosslinks by homologous recombination.^5,6 Biallelic inactivation of BRCA2 results in one form of the cancer-prone syndrome Fanconi anemia (complementation group FA-D1) and the BRCA2/FANCD1 protein operates with 12 other Fanconi anemia (FA) proteins and BRCA1 in a multifaceted response to DNA damage known as the FA/BRCA tumor suppressor pathway/network.^7,8,9,10 Eight of the 13 proteins, together with two FA-associated-proteins, participate in a nuclear core-complex that is required for the monoubiquitylation of FANCD2 and FANCI.⁸ FANCD2 is pivotal in the FA pathway translocating to chromatin and on monoubiquitylation, co-localizing with BRCA1, BRCA2 and the recombinase RAD51 in DNA damage inducible foci.^{11,12,13,14,15} FANCD2 is primarily regarded as being active within the nucleus and specifically in chromatin, although a cytoplasmic function has recently been proposed.¹⁶ The FA proteins BRCA2/FANCD1 and FANCN/PALB2 are believed to function downstream of, or in parallel with, FANCD2 monoubiquitylation and FANCN and FANCJ/BRIP1, like BRCA2, are now considered to be breast cancer susceptibility genes.^17,18 While FANCD2 directly interacts with BRCA2,¹⁹ and is found in complex with BRCA2 in mammalian cells,^13,20 there is no direct evidence for changes in FANCD2 in the development of familial breast cancer. However, FancD2 knockout mice exhibit an increased incidence of epithelial cancers including hepatic, ovarian, and mammary tumors,²¹ reduced FANCD2 expression is associated with familial ovarian cancer²² and several FA core-complex genes have now been implicated with several types of sporadic cancer, including ovarian and pancreatic cancer.²³ This suggests that a failure to express or post-translationally modify FANCD2 may play a role in the development of sporadic breast cancer. In contrast, a previous report in human breast cancer over a relatively short 6-year follow-up has suggested that high nuclear FANCD2 expression is correlated with poor patient survival.²⁴ To resolve this apparent contradiction we now show that breast carcinomas contain mainly cytoplasmic FANCD2 and that its loss is strongly correlated with the expression of the four metastasis-inducing proteins and particularly with premature death of patients with sporadic breast cancer over a much longer follow-up period of 20 years.