Fanconi Anemia Complementation Group D2 Research Articles

Identification and prognostic analysis of ferroptosis‑related gene HSPA5 to predict the progression of lung squamous cell carcinoma.

Ferroptosis, an iron-dependent form of regulated cell death driven by excessive lipid peroxidation, is implicated in the development and therapeutic responses of cancer. However, the role of ferroptosis-related gene profiles in lung squamous cell carcinoma (LSCC) remains largely unknown. The present study aimed to identify the prognostic roles of ferroptosis-related genes in LSCC. Sequencing data from the Cancer Genome Atlas were analyzed and ferroptosis-related gene expression between tumor and para-tumor tissue was identified. The prognostic role of these genes was also assessed using Kaplan-Meier analyses and univariate and multivariate Cox proportional hazards regression model analyses. Immunological correlation, tumor stemness, drug sensitivity and the transcriptional differences of heat shock protein (HSP)A5 in LSCC were also analyzed. Thereafter, the expression of HSPA5 in 100 patients with metastatic LSCC was evaluated using immunohistochemistry (IHC) and the clinical significance of these markers with different risk factors was assessed. Of the 22 ferroptosis-related genes, the expression of HSPA5, HSPB1, glutathione peroxidase 4, Fanconi anemia complementation group D2, CDGSH iron sulfur domain 1, farnesyl-diphosphate farnesyltransferase 1, nuclear factor erythroid 2 like 2, solute carrier (SLC)1A5, ribosomal protein L8, nuclear receptor coactivator 4, transferrin receptor and SLC7A11 was significantly increased in LSCC compared with adjacent tissues. However, only high expression of HSPA5 was able to predict progression-free survival (PFS) and disease-free survival in LSCC. Although HSPA5 was also significantly elevated in patients with lung adenocarcinoma, HSPA5 expression did not predict the prognosis of patients with lung adenocarcinoma. Of note, a higher expression of HSPA5 was related to higher responses to chemotherapy but not to immunotherapy. In addition, HSPA5 expression was positively correlated with 'ferroptosis', 'cellular responses to hypoxia', 'tumor proliferation signature', 'G2M checkpoint', 'MYC targets' and 'TGFB'. IHC analysis also demonstrated that a high expression of HSPA5 in patients with metastatic LSCC in the study cohort was associated with shorter PFS and overall survival. In conclusion, the present study demonstrated that the expression of the ferroptosis-related gene HSPA5 may be a negative prognostic marker for LSCC.

Fanconi anemia complementation group D2 promotes sensitivity of endometrial cancer cells to chemotherapeutic agents by inhibiting the ferroptosis pathway

BackgroundResistance can develop during treatment of advanced endometrial cancer (EC), leading to unsatisfactory results. Fanconi anemia complementation group D2 (Fancd2) has been shown to be closely related to drug resistance in cancer cells. Therefore, this study was designed to explore the correlation of Fancd2 with EC resistance and the mechanism of Fancd2.MethodsReal-time quantitative PCR (RT-qPCR) was used to detect the expression of Fancd2 in EC tissues and cells. EC cells (Ishikawa) and paclitaxel-resistant EC cells (Ishikawa/TAX) were transfected to knock down Fancd2. In addition, the ferroptosis inhibitor Ferrostatin-1 was adopted to treat Ishikawa/TAX cells. The sensitivity of cancer cells to chemotherapeutic agents was observed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and inhibitory concentration (IC)50 was calculated. Reactive oxygen species (ROS) levels were measured by flow cytometry, the activity of malondialdehyde (MDA) and the levels of glutathione (GSH) and Fe2+ in cells were detected by corresponding kits, and protein expression of solute farrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) was obtained through western blot.ResultsCompared with the normal tissues and endometrial epithelial cells, Fancd2 expression was significantly increased in EC tissues and Ishikawa cells, respectively. After knock-down of Fancd2, Ishikawa cells showed significantly increased sensitivity to chemotherapeutic agents. Besides, compared with Ishikawa cells, the levels of ROS, the activity of MDA, and the levels of GSH and Fe2+ were significantly decreased in Ishikawa/TAX cells, while the expression levels of SLC7A11 and GPX4 were significantly increased. Knock-down of Fancd2 significantly increased the ferroptosis levels in Ishikawa/TAX cells, but this effect could be reversed by Ferrostatin-1.ConclusionFancd2 increases drug resistance in EC cells by inhibiting the cellular ferroptosis pathway.

Pan-cancer analysis of the tumorigenic role of Fanconi anemia complementation group D2 (FANCD2) in human tumors

Monoubiquitination of FANCD2 is a central step in the activation of the Fanconi anemia (FA) pathway after DNA damage. Defects in the FA pathway centered around FANCD2 not only lead to genomic instability but also induce tumorigenesis. At present, few studies have investigated FANCD2 in tumors, and no pan-cancer research on FANCD2 has been conducted. We conducted a comprehensive analysis of the role of FANCD2 in cancer using public databases and other published studies. Moreover, we evaluated the role of FANCD2 in the proliferation, migration and invasion of lung adenocarcinoma cells through in vitro and in vivo experiments, and explored the role of FANCD2 in cisplatin chemoresistance. We investigated the regulatory effect of FANCD2 on the cell cycle of lung adenocarcinoma cells by flow cytometry, and verified this effect by western blotting. FANCD2 expression is elevated in most TCGA tumors and shows a strong positive correlation with poor prognosis in tumor patients. In addition, FANCD2 expression shows strong correlations with immune infiltration, immune checkpoints, the tumor mutation burden (TMB), and microsatellite instability (MSI), which are immune-related features, suggesting that it may be a potential target of tumor immunotherapy. We further found that FANCD2 significantly promotes the proliferation, invasion, and migration abilities of lung adenocarcinoma cells and that its ability to promote cancer cell proliferation may be achieved by modulating the cell cycle. The findings indicate that FANCD2 is a potential biomarker and therapeutic target in cancer treatment by analyzing the oncogenic role of FANCD2 in different tumors.

CPT1a Controls the Function of Long-Term Hematopoietic Stem Cells By Regulating the Balance between Fatty Acid Oxidation and Oxphos in Mitochondria

Hematopoietic stem cells (HSCs) rely on the balance in place of quiescence and self-renewal to sustain stem cell potential and differentiation to generate mature blood cells. Appropriate regulation of cellular and mitochondrial metabolic processes such as glycolysis, OXPHOS, fatty acid metabolism, and amino acid metabolism are required for HSC maintenance. However, the mechanisms by which cellular and mitochondrial metabolisms regulate HSC quiescence, proliferation, and differentiation remain to be fully understood. Fatty acid oxidation (FAO) is a metabolic process involving the breakdown of fatty acids into acetyl-CoA, which is then transported into mitochondria by carnitine palmitoyltransferase 1a (CPT1a). It has been reported that inhibiting FAO using non-specific inhibitors of CPT1a impairs HSC maintenance. Interestingly, CPT1a forms a complex with the Fanconi anemia complementation group D2 (FANCD2) protein (T Zhang et al., Scientific Reports, 2017). Given that Fanconi anemia is associated with HSC defects, this CPT1a complex may play a critical role in maintaining fatty acid dependent mitochondrial metabolism that mediates HSC dormancy and stemness. However, the exact role, in HSCs, of mitochondrial CPT1a, the rate-limiting enzyme in FAO, remains unknown. Here, we find that CPT1a is highly enriched in hematopoietic stem cells. Further, we investigated the role of CPT1a in hematopoiesis utilizing a CPT1aconditional knockout in mice driven by Vav1-Cre. The CPT1a knockout ( Cpt1aΔ/Δ) displays profound HSC defects, including loss of HSC self-renewal and accelerated differentiation. Mechanistically, we find that loss of Cpt1a results in increased ATP production, mitochondrial membrane potential (Δφm), and mitochondrial ROS accumulation in long-term HSCs (LT-HSCs), as well as defective LT-HSC function indicated by bone marrow transplantation and 5-FU genotoxic stress assays. Furthermore, utilizing mitochondrial complex formation assays and measurements of mitochondrial complex activity, BM cells of Cpt1aΔ/Δ mice display enhanced mitochondrial complex formation and up-regulation of mitochondrial complex activity. Our results suggest that Cpt1a regulates FAO transport and that loss of Cpt1a deregulates mitochondrial metabolisms including oxidative phosphorylation (OXPHOS). Taken together, our study suggests that CPT1a plays a key role in LT-HSC maintenance by controlling the balance between FAO and OXPHOS in mitochondria. Because of the noncanonical relationship between CPT1a and FANCD2 for the regulation of mitochondrial metabolism, correction of fatty acid metabolism and OXPHOS may present a novel therapeutic avenue for Fanconi anemia.

FANCD2 as a novel prognostic biomarker correlated with immune and drug therapy in Hepatitis B-related hepatocellular carcinoma

BackgroundFerroptosis is related to the immunosuppression of tumors and plays a critical role in cancer progression. Fanconi anemia complementation group D2 (FANCD2) is a vital gene that regulates ferroptosis. However, the mechanism of action of FANCD2 in Hepatitis B-related hepatocellular carcinoma (HCC) remains unknown. In this study, we investigated the prognostic significance and mechanism of action of FANCD2 in Hepatitis B-related HCC.MethodsThe expression of FANCD2 in Hepatitis B-related HCC was explored using The Cancer Genome Atlas (TCGA) and validated using the Gene Expression Omnibus (GEO) database. Univariate and multivariate Cox regression analyses and Kaplan–Meier survival curves were used to analyze the relationship between FANCD2 expression and the overall survival of patients with Hepatitis B-related HCC. Protein–protein interaction networks for FANCD2 were built using the STRING website. In addition, correlations between FANCD2 expression and the dryness index, tumor mutational burden, microsatellite instability (MSI), immune pathways, genes involved in iron metabolism, and sorafenib chemotherapeutic response were analyzed.ResultsOur results indicated that FANCD2 was significantly overexpressed in Hepatitis B-related HCC and demonstrated a strong predictive ability for diagnosis (Area Under Curve, 0.903) and prognosis of the disease. High FANCD2 expression was associated with poor prognosis, high-grade tumors, high expression of PDL-1, high MSI scores, and low sorafenib IC50 in Hepatitis B-related HCC. BRCA1, BRCA2, FAN1, and FANCC were vital proteins interacting with FANCD2. The expression level of FANCD2 significantly correlated with the infiltration levels of Treg cells, B cells, CD8 + T cells, CD4 + T cells, neutrophils, macrophages, myeloid dendritic cells, and NK cells in Hepatitis B-related HCC. FANCD2 was positively correlated with the tumor proliferation signature pathway, DNA repair, and cellular response to hypoxia.ConclusionOur study indicated that FANCD2 was a potential novel biomarker and immunotherapeutic target against Hepatitis B-related HCC, which might be related to the chemotherapeutic response to sorafenib.

LncRNA SNHG1 upregulates FANCD2 and G6PD to suppress ferroptosis by sponging miR-199a-5p/3p in hepatocellular carcinoma.

Ferroptosis is a form of regulated cell death (RCD) triggered by iron-dependent lipid peroxidation and is closely associated with the occurrence and progression of hepatocellular carcinoma (HCC). The lncRNA SNHG1 (small nucleolar RNA host gene 1) has been shown to play an oncogenic role in HCC, but its function in RCD other than autophagy and apoptosis is still unknown. Here, we investigated the correlation between SNHG1 and 156 typical markers of five RCD types based on RNA sequencing data from The Cancer Genome Atlas database and showed the negative regulators of ferroptosis FANCD2 (Fanconi anemia complementation group D2) and G6PD (glucose-6-phosphate dehydrogenase) to be the most highly and fifth most highly correlating factors with SNHG1, respectively. A competitive endogenous RNA network of SNHG1 - miR-199a-5p/3p - FANCD2/G6PD was constructed bioinformatically. In vitro experiments showed that overexpression of the miR-199a precursor led to a decrease in expression of SNHG1, FANCD2, and G6PD, whereas knockdown of SNHG1 decreased expression of FANCD2 and G6PD but increased levels of miR-199a-5p and miR-199a-3p in HCC cells (Huh7 and HepG2). In addition, knockdown of SNHG1 increased erastin-mediated ferroptosis, iron accumulation, and lipid peroxidation. These results suggest that SNHG1 upregulates FANCD2 and G6PD by sponging miR-199a, thereby inhibiting ferroptosis in HCC. Moreover, a signature based on expression of SNHG1, FANCD2, and G6PD was identified as being associated with overall survival and the immunological microenvironment in HCC. Collectively, this study identified the SNHG1-miR-199a-FANCD2/G6PD axis in HCC, which is a potential marker for the prognosis and therapy of this tumor.

FANCD2 inhibits ferroptosis by regulating the JAK2/STAT3 pathway in osteosarcoma

BackgroundThis research aimed to investigate the roles of fanconi anemia complementation group D2 (FANCD2) on the regulation of ferroptosis in osteosarcoma progression.MethodsThe function of FANCD2 on cell viability, invasion, migration, and tumor growth were explored. FANCD2 and pathway-related genes were determined by western blot. Ferroptosis-associated markers were determined, including lipid peroxidation, labile iron pool (LIP), ferrous iron (Fe2+), and ferroptosis-related genes.ResultsFANCD2 expression was increased in osteosarcoma cells. FANCD2 knockdown reduced cell viability, invasion, and migration of osteosarcoma cells. FANCD2 knockdown regulated ferroptosis-related gene expression, and distinctly increased the levels of LIP, Fe2+, and lipid peroxidation, and these effects were reversed by a ferroptosis inhibitor Fer-1. In addition, JAK2 and STAT3 expression were reduced by silencing of FANCD2, and STAT3 activator (colivelin) distinctly reversed tumor suppressor effects of FANCD2 silencing on osteosarcoma development.ConclusionThese findings suggested that FANCD2 silencing could suppress osteosarcoma cell viability, migration, invasion, and tumor growth, and induced ferroptosis by regulating the JAK2/STAT3 axis. These findings may provide novel therapeutic ideas for clinical treatment of osteosarcoma.

FANCD2 promotes the malignant behavior of endometrial cancer cells and its prognostic value

Defective DNA damage repair is a key mechanism affecting tumor susceptibility, treatment response, and survival outcome of endometrial cancer (EC). Fanconi anemia complementation group D2 (FANCD2) is the core component of the Fanconi anemia repair pathway. To explore the function of FANCD2 in EC, we examined the expression of FANCD2 in human specimens and databases, and discussed the possible mechanism of carcinogenesis by in vitro assays. Immunohistochemistry results showed overexpression of FANCD2 was detected in EC tissues compared to normal and atypical hyperplasia endometrium. Higher FANCD2 expression was correlated with deeper myometrial invasion (MI) and proficient mismatch repair status. The Cancer Genome Atlas (TCGA) database analysis showed FANCD2 was upregulated in EC compared with normal tissue. The high expression of FANCD2 was associated with poor overall survival in EC. Knockdown of FANCD2 expression in EC cell lines inhibited malignant proliferation and migration ability. We demonstrated that decreased FANCD2 expression results in increased DNA damage and decreased S-phase cells, leading to a decrease in proliferative capacity in EC cells. Down-regulated FANCD2 confers sensitivity of EC cells to interstrand crosslinking agents. This study provides evidence for the malignant progression and prognostic value of FANCD2 in EC.

Integrative analyses of prognosis, tumor immunity, and ceRNA network of the ferroptosis-associated gene FANCD2 in hepatocellular carcinoma.

Extensive evidence has revealed that ferroptosis plays a vital role in HCC development and progression. Fanconi anemia complementation group D2 (FANCD2) has been reported to serve as a ferroptosis-associated gene and has a close relationship with tumorigenesis and drug resistance. However, the impact of the FANCD2-related immune response and its mechanisms in HCC remains incompletely understood. In the current research, we evaluated the prognostic significance and immune-associated mechanism of FANCD2 based on multiple bioinformatics methods and databases. The results demonstrated that FANCD2 was commonly upregulated in 15/33 tumors, and only the high expression of FANCD2 in HCC was closely correlated with worse clinical outcomes by OS and DFS analyses. Moreover, ncRNAs, including two major types, miRNAs and lncRNAs, were closely involved in mediating FANCD2 upregulation in HCC and were established in a ceRNA network by performing various in silico analyses. The DUXAP8-miR-29c-FANCD2 and LINC00511-miR-29c-FANCD2 axes were identified as the most likely ncRNA-associated upstream regulatory axis of FANCD2 in HCC. Finally, FANCD2 expression was confirmed to be positively related to HCC immune cell infiltration, immune checkpoints, and IPS analysis, and GSEA results also revealed that this ferroptosis-associated gene was primarily involved in cancer-associated pathways in HCC. In conclusion, our investigations indicate that ncRNA-related modulatory overexpression of FANCD2 might act as a promising prognostic and immunotherapeutic target against HCC.

Abnormally Expressed Ferroptosis-Associated FANCD2 in Mediating the Temozolomide Resistance and Immune Response in Glioblastoma.

Ferroptosis-related genes (FRGs) have been identified as potential targets involved in oncogenesis and cancer therapeutic response. Nevertheless, the specific roles and underlying mechanisms of FRGs in GBM and temozolomide (TMZ) resistance remain unclear. Through comprehensive bioinformatics, we found that ferroptosis-related Fanconi anemia complementation group D2 (FANCD2) was significantly up-regulated in GBM tissues, and the high expression level of FANCD2 was related to the poor prognosis in primary and recurrent GBM patients. Furthermore, FANCD2 could promote TMZ resistance by attenuating ferroptosis in GBM cells. Knockdown of FANCD2 could increase reactive oxygen species (ROS) levels and inhibit cell survival. The two characteristics were associated with ferroptosis in TMZ-resistant GBM cells T98G-R and U118-R. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that aberrantly expressed FANCD2 was potentially linked with several cancer-associated signaling pathways, including chromosome segregation, DNA replication, and cell cycle transition. In addition, we demonstrated that FANCD2 expression was positively correlated with several tumor-infiltrating lymphocytes (TILs) and multiple immune-associated signatures in GBM. Therefore, up-regulated FANCD2 could protect GBM cells from ferroptosis and promote TMZ resistance. FANCD2 may be a novel therapeutic target in GBM.

Modeling injury and repair in kidney organoids reveals that homologous recombination governs tubular intrinsic repair.

Kidneys have the capacity for intrinsic repair, preserving kidney architecture with return to a basal state after tubular injury. When injury is overwhelming or repetitive, however, that capacity is exceeded and incomplete repair results in fibrotic tissue replacing normal kidney parenchyma. Loss of nephrons correlates with reduced kidney function, which defines chronic kidney disease (CKD) and confers substantial morbidity and mortality to the worldwide population. Despite the identification of pathways involved in intrinsic repair, limited treatments for CKD exist, partly because of the limited throughput and predictivity of animal studies. Here, we showed that kidney organoids can model the transition from intrinsic to incomplete repair. Single-nuclear RNA sequencing of kidney organoids after cisplatin exposure identified 159 differentially expressed genes and 29 signal pathways in tubular cells undergoing intrinsic repair. Homology-directed repair (HDR) genes including Fanconi anemia complementation group D2 (FANCD2) and RAD51 recombinase (RAD51) were transiently up-regulated during intrinsic repair but were down-regulated in incomplete repair. Single cellular transcriptomics in mouse models of obstructive and hemodynamic kidney injury and human kidney samples of immune-mediated injury validated HDR gene up-regulation during tubular repair. Kidney biopsy samples with tubular injury and varying degrees of fibrosis confirmed loss of FANCD2 during incomplete repair. Last, we performed targeted drug screening that identified the DNA ligase IV inhibitor, SCR7, as a therapeutic candidate that rescued FANCD2/RAD51-mediated repair to prevent the progression of CKD in the cisplatin-induced organoid injury model. Our findings demonstrate the translational utility of kidney organoids to identify pathologic pathways and potential therapies.

FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes.

Individuals with Fanconi anemia (FA), a rare genetic bone marrow failure syndrome, have an increased risk of young‐onset head and neck squamous cell carcinomas (SCCs) and esophageal SCC. The FA DNA repair pathway is activated upon DNA damage induced by acetaldehyde, a chief alcohol metabolite and one of the major carcinogens in humans. However, the molecular basis of acetaldehyde‐induced genomic instability in SCCs of the head and neck and of the esophagus in FA remains elusive. Here, we report the effects of acetaldehyde on replication stress response in esophageal epithelial cells (keratinocytes). Acetaldehyde‐exposed esophageal keratinocytes displayed accumulation of DNA damage foci consisting of 53BP1 and BRCA1. At physiologically relevant concentrations, acetaldehyde activated the ATR‐Chk1 pathway, leading to S‐ and G2/M‐phase delay with accumulation of the FA complementation group D2 protein (FANCD2) at the sites of DNA synthesis, suggesting that acetaldehyde impedes replication fork progression. Consistently, depletion of the replication fork protection protein Timeless led to elevated DNA damage upon acetaldehyde exposure. Furthermore, FANCD2 depletion exacerbated replication abnormalities, elevated DNA damage, and led to apoptotic cell death, indicating that FANCD2 prevents acetaldehyde‐induced genomic instability in esophageal keratinocytes. These observations contribute to our understanding of the mechanisms that drive genomic instability in FA patients and alcohol‐related carcinogenesis, thereby providing a translational implication in the development of more effective therapies for SCCs.

Exploiting synthetic lethality to target BRCA1/2-deficient tumors: where we stand.

The principle of synthetic lethality, which refers to the loss of viability resulting from the disruption of two genes, which, individually, do not cause lethality, has become an attractive target approach due to the development and clinical success of Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi). In this review, we present the most recent findings on the use of PARPi in the clinic, which are currently approved for second-line therapy for advanced ovarian and breast cancer associated with mutations of BRCA1 or BRCA2 (BRCA1/2) genes. PARPi efficacy, however, appears to be limited by acquired and inherent resistance, highlighting the need for alternative and synergistic targets to eliminate these tumors. Here, we explore other identified synthetic lethal interactors of BRCA1/2, including DNA polymerase theta (POLQ), Fanconi anemia complementation group D2 (FANDC2), radiation sensitive 52 (RAD52), Flap structure-specific endonuclease 1 (FEN1), and apurinic/apyrimidinic endodeoxyribonuclease 2 (APE2), as well as other protein and nonprotein targets, for BRCA1/2-mutated cancers and their implications for future therapies. A wealth of information now exists for phenotypic and functional characterization of these novel synthetic lethal interactors of BRCA1/2, and leveraging these findings can pave the way for the development of new targeted therapies for patients suffering from these cancers.

TFG-maintaining stability of overlooked FANCD2 confers early DNA-damage response.

Emerging Fanconi Anemia (FA) signaling in the field of cancer research annotates the extreme importance of its center player, Fanconi Anemia complementation group D2 (FANCD2) in protecting human cells from going awry. However, a previously-unrecognized form of FANCD2, namely FANCD2-V2, is understudied. We report TRK-Fused Gene (TFG) is critical for roles played by FANCD2-V2 in early responses to DNA damage, but not for FANCD2-V1, the long-known form of FANCD2. FANCD2-V2 forms nuclear foci upon DNA damage, and both its focus appearance and disappearance are earlier than FANCD2-V1. The amino acid/aa 5-100 of TFG and the aa1437-1442 of FANCD2-V2 were identified to contribute to their interaction, which maintains the steady-state level of FANCD2-V2 protein. TFGΔaa5-100 or FANCD2-V2Δaa1437-1442-carrying cells could not show timely focus formation of FANCD2-V2 upon DNA damage and gained carcinogenicity over time. This study provides a previously-unknown key to unlock in-depth insights into maintaining genome stability, fostering translational studies on preventing, diagnosing and/or treating related diseases.

The synergistic proapoptotic effect of PARP-1 and HDAC inhibition in cutaneous T-cell lymphoma is mediated via Blimp-1

AbstractThe therapy of advanced mycosis fungoides (MF) presents a therapeutic challenge, and the search for new therapeutic targets is ongoing. Poly(ADP-ribose) polymerase 1 was shown to be upregulated in patients with advanced MF and could be druggable by a new class of chemotherapeutic agents, PARP-1 inhibitors, which are already in clinical trials for other malignancies; however, the role of PARP-1 inhibitors in MF has never been established. We examined the efficacy of talazoparib in the murine model of cutaneous T-cell lymphoma. The cytotoxic effect of talazoparib on Moloney MuLV-induced T-cell lymphoma (MBL2) cells was a result of G2/M cell cycle arrest via the upregulation of p53. The in vivo experiments confirmed the clinical impact of talazoparib on MF tumors. When talazoparib was combined with the histone deacetylase (HDAC) inhibitor, romidepsin, the cytotoxic effect was synergized via downregulation of the DNA-repair genes Fanconianemia complementation group A (FANCA), Fanconi anemia complementation group D2 (FANCD2), and DNA topoisomerase II binding protein 1(TOPBP1)and stimulation of apoptosis via Blimp-1 (PRDM1)/Bax axis. Romidepsin increased the expression of IRF8and Bcl-6, leading to upregulation of Blimp1and Bax; whereas talazoparib upregulated Blimp-1 and Bax via upregulation of interferon regulatory factor 4 (IRF4), leading to cleavage of caspases 6 and 7. Thus, a combination of talazoparib with romidepsin demonstrated the synergistic antilymphoma effect and warranted further investigation in a clinical trial.

FANCD2 knockdown with shRNA interference enhances the ionizing radiation sensitivity of nasopharyngeal carcinoma CNE-2 cells.

Fanconi anemia complementation group D2 (FANCD2) has been associated with the sensitivity of tumor cells to DNA crosslinking damaging agents in certain solid tumors. However, its role in nasopharyngeal carcinoma (NPC) is still unclear. In the present study, the role of FANCD2 in the response of NPC CNE-2 cells to radiation was investigated. A CNE-2 cell model with stable FANCD2 silencing was constructed by lentiviral transfection. Fluorescence quantitative PCR and western blotting were used to evaluate FANCD2 expression in CNE-2 cells. The biological impact of FANCD2 silencing on the response of CNE-2 cells to radiation was investigated in vitro and in vivo. The microarray technology, western blotting, and immunohistochemistry were used to analyze the proteins involved in related pathways after irradiation to investigate the underlying mechanism. Lentivirus-mediated shRNA interference stably silenced the FANCD2 gene in CNE-2 cells. In vitro, in the FANCD2-silenced group, cell proliferation was significantly inhibited, apoptosis was increased, and the cell cycle was arrested at the G2/M phase after irradiation. In vivo, FANCD2 silencing slowed tumor growth, as the volume and weight of the xenograft tumors were significantly decreased. Both in vitro and in vivo, the differentially expressed genes NUPR1, FLI1, and FGF21 were downregulated in the FANCD2-silenced group. Our results show that FANCD2 silencing affected the sensitivity of CNE-2 cells to ionizing radiation by regulating cell proliferation, apoptosis, and cell cycle distribution. The mechanism might be associated with changes in NUPR1, FLI1, and FGF21 protein expression due to the FANCD2 silencing. This study provides a promising target for NPC radiotherapy.

The DNA-binding activity of USP1-associated factor 1 is required for efficient RAD51-mediated homologous DNA pairing and homology-directed DNA repair

USP1-associated factor 1 (UAF1) is an integral component of the RAD51-associated protein 1 (RAD51AP1)-UAF1-ubiquitin-specific peptidase 1 (USP1) trimeric deubiquitinase complex. This complex acts on DNA-bound, monoubiquitinated Fanconi anemia complementation group D2 (FANCD2) protein in the Fanconi anemia pathway of the DNA damage response. Moreover, RAD51AP1 and UAF1 cooperate to enhance homologous DNA pairing mediated by the recombinase RAD51 in DNA repair via the homologous recombination (HR) pathway. However, whereas the DNA-binding activity of RAD51AP1 has been shown to be important for RAD51-mediated homologous DNA pairing and HR-mediated DNA repair, the role of DNA binding by UAF1 in these processes is unclear. We have isolated mutant UAF1 variants that are impaired in DNA binding and tested them together with RAD51AP1 in RAD51-mediated HR. This biochemical analysis revealed that the DNA-binding activity of UAF1 is indispensable for enhanced RAD51 recombinase activity within the context of the UAF1-RAD51AP1 complex. In cells, DNA-binding deficiency of UAF1 increased DNA damage sensitivity and impaired HR efficiency, suggesting that UAF1 and RAD51AP1 have coordinated roles in DNA binding during HR and DNA damage repair. Our findings show that even though UAF1's DNA-binding activity is redundant with that of RAD51AP1 in FANCD2 deubiquitination, it is required for efficient HR-mediated chromosome damage repair.

Subcellular localization of FANCD2 is associated with survival in ovarian carcinoma.

Objective: Ovarian cancer is a leading cause of death from gynecological cancers. Late diagnosis and resistance to therapy results in mortality and effective screening is required for early diagnosis and better treatments. Expression of the Fanconi Anemia complementation group D2 protein (FANCD2) is reduced in ovarian surface epithelial cells (OSE) in patients with ovarian cancer. FANCD2 has been studied for its role in DNA repair; however multiple studies have suggested that FANCD2 has a role outside the nucleus. We sought to determine whether subcellular localization of FANCD2 correlates with patient outcome in ovarian cancer.Methods: We examined the subcellular localization of FANCD2 in primary OSE cells from consenting patients with ovarian cancer or a normal ovary. Ovarian tissue microarray was stained with anti-FANCD2 antibody by immunohistochemistry and the correlation of FANCD2 localization with patient outcomes was assessed. FANCD2 binding partners were identified by immunoprecipitation of cytoplasmic FANCD2.Results: Nuclear and cytoplasmic localization of FANCD2 was observed in OSEs from both normal and ovarian cancer patients. Patients with cytoplasmic localization of FANCD2 (cFANCD2) experienced significantly longer median survival time (50 months), versus patients without cytoplasmic localization of FANCD2 (38 months; p < 0.05). Cytoplasmic FANCD2 was found to bind proteins involved in the innate immune system, cellular response to heat stress, amyloid fiber formation and estrogen mediated signaling.Conclusions: Our results suggest that the presence of cytoplasmic FANCD2 modulates FANCD2 activity resulting in better survival outcome in ovarian cancer patients.

Structural basis of the fanconi anemia-associated mutations within the FANCA and FANCG complex

Monoubiquitination of the Fanconi anemia complementation group D2 (FANCD2) protein by the FA core ubiquitin ligase complex is the central event in the FA pathway. FANCA and FANCG play major roles in the nuclear localization of the FA core complex. Mutations of these two genes are the most frequently observed genetic alterations in FA patients, and most point mutations in FANCA are clustered in the C-terminal domain (CTD). To understand the basis of the FA-associated FANCA mutations, we determined the cryo-electron microscopy (EM) structures of Xenopus laevis FANCA alone at 3.35 Å and 3.46 Å resolution and two distinct FANCA–FANCG complexes at 4.59 and 4.84 Å resolution, respectively. The FANCA CTD adopts an arc-shaped solenoid structure that forms a pseudo-symmetric dimer through its outer surface. FA- and cancer-associated point mutations are widely distributed over the CTD. The two different complex structures capture independent interactions of FANCG with either FANCA C-terminal HEAT repeats, or the N-terminal region. We show that mutations that disturb either of these two interactions prevent the nuclear localization of FANCA, thereby leading to an FA pathway defect. The structure provides insights into the function of FANCA CTD, and provides a framework for understanding FA- and cancer-associated mutations.

Homocysteine aggravates DNA damage by impairing the FA/Brca1 Pathway in NE4C murine neural stem cells.

There is existing evidence that elevated homocysteine (Hcy) levels are risk factors for some neurodegenerative disorders. The pathogenesis of neurological diseases could be contributed to excessive cell dysfunction and death caused by defective DNA damage response (DDR) and accumulated DNA damage. Hcy is a neurotoxic amino acid and acts as a DNA damage inducer. However, it is not clear whether Hcy participates in the DDR. To investigate the effects of Hcy on DNA damage and the DDR, we employed mitomycin C (MMC) to cause DNA damage in NE4C murine neural stem cells (NSCs). Compared to treatment with MMC alone, we found that co-treatment with MMC and Hcy worsened DNA damage and increased death in NE4C cells. Intriguingly, in this DNA damage model mimicked by MMC, immunoblotting results showed that the monoubiquitination levels of Fanconi anemia complementation group I (Fanci) and Fanconi anemia complementation group D2 (Fancd2) were decreased to about 60.3% and 55.7% by supplementing cell culture medium with Hcy, indicating Hcy inactivates the function of Fanci and Fancd2 in DNA damage conditions. Given Breast Cancer 1 (BRCA1) is an important downstream of FANCD2, we next detected the interaction between Fancd2 and Brca1 in NE4C cells. Compared to treatment with MMC alone, the Fancd2-Brca1 interaction and the amount of Brca1 on chromatin were decreased when cells were co-exposed to MMC and Hcy, suggesting Hcy could impair the Fanconi anemia (FA)/Brca1 pathway. Taken together, our study demonstrates that Hcy may enhance cell death, which contributes to the accumulation of DNA damage and promotion of hypersensitivity to cytotoxicity by impairing the FA/Brca1 pathway in murine NSCs in the presence of DNA damage.