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

Abstract

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.1-4 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. 7-10 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.

CCAAT/enhancer binding protein delta (C/EBPδ, CEBPD)-mediated nuclear import of FANCD2 by IPO4 augments cellular response to DNA damage

“…Interestingly, a very recent immunohistochemical study showed that a significant proportion of malignant breast tumors had lost expression of nuclear FANCD2 but retained cytoplasmic expression, whereas benign lesions retained both nuclear and cytoplasmic staining (32). These observations are consistent with FANCD2 nuclear import serving as a tumor-suppressing mechanism.…”

FANCD2 re-expression is associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents

“…Indeed, analyses of the proliferation marker Ki67 in GBM tissue micro-arrays agree with this hypothesis (Figures 1C and S1C). It is interesting to note that in contrast to these findings, a previous study showed that a cohort of breast tumours exhibiting reduced cytoplasmic FANCD2 expression were correlated with poor survival outcomes [26]. However, our findings are also consistent with previous reports showing that highly proliferative tissues such as testis, tonsils and spleen exhibit high expression of FANCD2 and FANCA, yet non-proliferative tissues such as lung, liver, muscle and the brain exhibit minimal expression of these proteins [9, 10].…”

Patient-derived C-terminal mutation of FANCI causes protein mislocalization and reveals putative EDGE motif function in DNA repair

“…In a recent study, FANCD2 was found to be largely cytoplasmic in more malignant human breast tumor tissues. 47 Cytoplasmic localization of FANCD2 in cells, similar to that of FANCI, also probably prevents FA pathway activation, leading to genome instability. It is still unclear what is the precise mechanism that causes the FANCD2 protein mislocalization in these human breast cancer tissues.…”

“…It is still unclear what is the precise mechanism that causes the FANCD2 protein mislocalization in these human breast cancer tissues. 47 It will be interesting to determine whether FANCI may also be abnormally expressed and cytoplasmically localized in human breast cancers and whether this is the result of sporadic mutations that target the C-terminal NLS function of FANCI. It is known that defects in protein NLSs have been associated with human disease conditions, such as numerous cancers and developmental disorders.…”