Curcumin reverses cisplatin resistance in cisplatin-resistant lung caner cells by inhibiting FA/BRCA pathway.

Cisplatin (DDP) is the most widely used chemotherapy agent for treatment of malignancies including lung cancer. However, the effectiveness of DDP is often weakened by acquired resistance of tumor cells. DDP kills cancer cells primarily by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The Fanconi anemia (FA)/BRCA pathway is a DNA cross-link damage repair pathway, which regulates cellular resistance to DNA cross-link agents, such as DDP. Some study has shown that natural compound curcumin sensitize human ovarian and breast cancer cells to DDP. However, whether curcumin may reverse resistance to DDP in DDP-resistant lung cancer cells has not been understood. In this study, we showed that curcumin enhanced the proliferation inhibitory effect of DDP and promote DDP-induced apoptosis in A549/DDP cells (DDP-resistant lung adenocarcinoma cells). Moreover, we observed that FA/BRCA pathway DNA damage repair processes, such as DDP-induced FANCD2 monoubiquitination and nuclear foci formation were downregulated in the presence of curcumin, suggesting that curcumin enhanced sensitivity to DDP in A549/DDP cells through the inhibition of FA/BRCA pathway. Furthermore, the calculation of q value and apoptosis analyses revealed that curcumin in combination with DDP could exert a synergistic cytotoxic effect in A549/DDP cells, further demonstrating that curcumin can reverse cisplatin resistance of A549/DDP cells. In conclusion, by suppressing the FA/BRCA pathway DNA repair, curcumin potentiates DDP-induced proliferation inhibitory effect and apoptosis in A549/DDP cell, indicating that curcumin may serve as a chemosensitizer to cross-link-inducing anticancer drugs DDP.

Expression and purification of human FANCI and FANCD2 using Escherichia coli cells.

The DNA interstrand crosslink (ICL) is an extremely deleterious DNA lesion that covalently crosslinks complementary strands and prevents the strand-separation reaction. In higher eukaryotes, the Fanconi anemia proteins, FANCI and FANCD2, form a heterodimer and play essential roles in ICL repair. Human FANCI and FANCD2 are large proteins with molecular masses of 149kDa and 164kDa, respectively, and were reportedly purified using a baculovirus expression system with insect cells. We have established a novel expression and purification procedure for human FANCD2 and FANCI, using Escherichia coli cells. The human FANCD2 and FANCI proteins purified by this bacterial expression method formed a stable heterodimer, and exhibited DNA binding and histone chaperone activities, as previously reported for the proteins purified by the baculovirus system. Therefore, these purification methods for human FANCI and FANCD2 provide novel procedures to facilitate structural and biochemical studies of human FANCI and FANCD2.

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

Brain tumours kill more children and adults under 40 than any other cancer. Around half of primary brain tumours are glioblastoma multiforme (GBMs) where treatment remains a significant challenge, where survival rates have improved little over the last 40 years, thus highlighting an unmet need for the identification/development of novel therapeutic targets and agents to improve GBM treatment. Using archived and fresh glioma tissue, we show that in contrast to normal brain or benign schwannomas GBMs exhibit re-expression of FANCD2, a key protein of the Fanconi Anaemia (FA) DNA repair pathway, and possess an active FA pathway. Importantly, FANCD2 expression levels are strongly associated with tumour grade, revealing a potential exploitable therapeutic window to allow inhibition of the FA pathway in tumour cells, whilst sparing normal brain tissue. Using several small molecule inhibitors of the FA pathway in combination with isogenic FA-proficient/deficient glioma cell lines as well as primary GBM cultures, we demonstrate that inhibition of the FA pathway sensitises gliomas to the chemotherapeutic agents Temozolomide and Carmustine. Our findings therefore provide a strong rationale for the development of novel and potent inhibitors of the FA pathway to improve the treatment of GBMs, which may ultimately impact on patient outcome.

Gene expression profiling reveals activation of the FA/BRCA pathway in advanced squamous cervical cancer with intrinsic resistance and therapy failure.

BACKGROUND: Advanced squamous cervical cancer, one of the most commonly diagnosed cancers in women, still remains a major problem in oncology due to treatment failure and distant metastasis. Antitumor therapy failure is due to both intrinsic and acquired resistance; intrinsic resistance is often decisive for treatment response. In this study, we investigated the specific pathways and molecules responsible for baseline therapy failure in locally advanced squamous cervical cancer. METHODS: Twenty-one patients with locally advanced squamous cell carcinoma were enrolled in this study. Primary biopsies harvested prior to therapy were analyzed for whole human gene expression (Agilent) based on the patient's 6 months clinical response. Ingenuity Pathway Analysis was used to investigate the altered molecular function and canonical pathways between the responding and non-responding patients. The microarray results were validated by qRT-PCR and immunohistochemistry. An additional set of 24 formalin-fixed paraffin-embedded cervical cancer samples was used for independent validation of the proteins of interest. RESULTS: A 2859-gene signature was identified to distinguish between responder and non-responder patients. 'DNA Replication, Recombination and Repair' represented one of the most important mechanisms activated in non-responsive cervical tumors, and the 'Role of BRCA1 in DNA Damage Response' was predicted to be the most significantly altered canonical pathway involved in intrinsic resistance (p = 1.86E-04, ratio = 0.262). Immunohistological staining confirmed increased expression of BRCA1, BRIP1, FANCD2 and RAD51 in non-responsive compared with responsive advanced squamous cervical cancer, both in the initial set of 21 cervical cancer samples and the second set of 24 samples. CONCLUSIONS: Our findings suggest that FA/BRCA pathway plays an important role in treatment failure in advanced cervical cancer. The assessment of FANCD2, RAD51, BRCA1 and BRIP1 nuclear proteins could provide important information about the patients at risk for treatment failure.

Expression of DNA repair proteins in endometrial cancer predicts disease outcome.

OBJECTIVE: The consequences of defective homologous recombination and other DNA repair pathways are important in disease outcomes of numerous tumor types. The objective of this study was to explore BRCA1, PARP, FANCD2, PTEN, H2AX, and ATM protein expression in endometrial cancer (EC). METHODS: PARP1, γH2AX, ATM, FANCD2, PTEN, BRCA1, and p53 proteins were evaluated in EC tissue microarray (TMA) and their expressions were correlated with clinical and pathological parameters in 357 patients. RESULTS: In type I EC, PARP1(+), ATM(+), and FANCD2(+) were associated with high tumor grade (p 0.031, p 0.0045, p 0.0062 respectively); γH2AX(+) and FANCD2(+) with advanced tumor stage (p 0.0004, p 0.0085 respectively); γH2AX(+), FANCD2(+) and p53(+) with the presence of lympho-vascular invasion (p 0.0004, p 0.0042, p 0.0098 respectively); and γH2AX(+) and ATM(+) with tumor recurrence (p 0.0203, p 0.0465) respectively. In type II EC, only PARP1(+) was associated with tumor stage (p 0.0499). EC patients with p53(+) or FANCD2(+) were more likely to recur with 5year recurrence free survival (RFS) probability of 71.4% in comparison to 85.5% for the other patients and they were more likely to have shorter 5year overall survival (OS) of 66.46% in comparison to 78.5% of those other patients Finally, patients with ATM(+) and p53(+) or FANCD2(+) were more likely to recur with 5year RFS probability of 68% versus 80.3% for the other patients. CONCLUSION: DNA repair proteins seemed to play an important role in EC, and their expressions can forecast for poor outcomes.

Overabundant FANCD2, alone and combined with NQO1, is a sensitive marker of adverse prognosis in breast cancer.

BACKGROUND: Defective DNA repair is central to the progression and treatment of breast cancer. Immunohistochemically detected DNA repair markers may be good candidates for novel prognostic and predictive factors that could guide the selection of individualized treatment strategies. PATIENTS AND METHODS: We have analyzed nuclear immunohistochemical staining of BRCA1, FANCD2, RAD51, XPF, and PAR in relation to clinicopathological and survival data among 1240 paraffin-embedded breast tumors, and additional gene expression microarray data from 76 tumors. The antioxidant enzyme NQO1 was analyzed as a potential modifier of prognostic DNA repair markers. RESULTS: RAD51 [hazard ratio (HR) 0.81, 95% confidence interval (CI) 0.70-0.94, P = 0.0050] and FANCD2 expression (HR 1.50, 95% CI 1.28-1.76, P = 1.50 × 10(-7)) were associated with breast cancer survival. High FANCD2 expression correlated with markers of adverse prognosis but remained independently prognostic in multivariate analysis (HR 1.27, 95% CI 1.08-1.49, P = 0.0043). The FANCD2-associated survival effect was most pronounced in hormone receptor positive, HER2-negative tumors, and in tumors with above-median NQO1 expression. In the NQO1-high subset, patients belonging to the highest quartile of FANCD2 immunohistochemical scores had a threefold increased risk of metastasis or death (HR 3.10, 95% CI 1.96-4.92). Global gene expression analysis indicated that FANCD protein overabundance is associated with the upregulation of proliferation-related genes and a downregulated nucleotide excision repair pathway. CONCLUSION: FANCD2 immunohistochemistry is a sensitive, independent prognostic factor in breast cancer, particularly when standard markers indicate relatively favorable prognosis. Taken together, our results suggest that the prognostic effect is linked to proliferation, DNA damage, and oxidative stress; simultaneous detection of FANCD2 and NQO1 provides additional prognostic value.

Different DNA damage and cell cycle checkpoint control in low- and high-risk human papillomavirus infections of the vulva.

Human papillomavirus (HPV) infections may result in benign hyperplasia, caused by low-risk HPV types, or (pre)malignant lesions caused by high-risk HPV types. The molecular basis of this difference in malignant potential is not completely understood. Here, we performed gene profiling of different HPV infected vulvar tissues (condylomata acuminata (n = 5), usual type vulvar intraepithelial neoplasia (uVIN) (n = 9)) and control samples (n = 14) using Affymetrix Human U133A plus 2 GeneChips. Data were analyzed using OmniViz®, Partek® and Ingenuity® Software. Results were validated by real-time RT-PCR and immunostaining. Although similarities were observed between gene expression profiles of low- and high-risk HPV infected tissues (e.g., absence of estrogen receptor in condylomata and uVIN), high-risk HPV infected tissues showed more proliferation and displayed more DNA damage than tissues infected with low-risk HPV. These observations were confirmed by differential regulation of cell cycle checkpoints and by increased expression of DNA damage-biomarkers p53 and γH2AX. Furthermore, FANCA, FANCD2, BRCA1 and RAD51, key players in the DNA damage response, were significantly upregulated (p < 0.05). In addition, we compared our results with publicly available gene expression profiles of various other HPV-induced cancers (vulva, cervix and head-and-neck). This showed p16(INK4a) was the most significant marker to detect a high-risk HPV infection, but no other markers could be found. In conclusion, this study provides insight into the molecular basis of low- and high-risk HPV infections and indicates two main pathways (cell cycle and DNA damage response) that are much stronger affected by high-risk HPV as compared to low-risk HPV.

Targeting the Fanconi anemia/BRCA pathway circumvents drug resistance in multiple myeloma.

The Fanconi anemia/BRCA (FA/BRCA) DNA damage repair pathway plays a pivotal role in the cellular response to replicative stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA genes are overexpressed and causative for drug resistance in human melphalan-resistant multiple myeloma cell lines. However, the transcriptional regulation of the FA/BRCA pathway is not understood. In this report, we describe for the first time a novel function of the NF-kappaB subunits, RelB/p50, as transcriptional activators of the FA/BRCA pathway. Specifically, our findings point to constitutive phosphorylation of IkappaB kinase alpha and subsequent alterations in FANCD2 expression and function as underlying events leading to melphalan resistance in repeatedly exposed multiple myeloma cells. Inhibiting NF-kappaB by small interfering RNA, blocking the IkappaB kinase complex with BMS-345541, or using the proteasome inhibitor bortezomib drastically reduced FA/BRCA gene expression and FANCD2 protein expression in myeloma cells, resulting in diminished DNA damage repair and enhanced melphalan sensitivity. Importantly, we also found that bortezomib decreases FA/BRCA gene expression in multiple myeloma patients. These results show for the first time that NF-kappaB transcriptionally regulates the FA/BRCA pathway and provide evidence for targeting Fanconi anemia-mediated DNA repair to enhance chemotherapeutic response and circumvent drug resistance in myeloma patients.

HPV-16 E7 reveals a link between DNA replication stress, fanconi anemia D2 protein, and alternative lengthening of telomere-associated promyelocytic leukemia bodies.

Expression of the high-risk human papillomavirus (HPV-16) E7 oncoprotein extends the life span of primary human keratinocytes and partially restores telomere length in the absence of telomerase. The molecular basis of this activity is incompletely understood. Here, we show that HPV-16 E7 induces an increased formation of alternative lengthening of telomeres (ALT)-associated promyelocytic leukemia bodies (APBs) in early passage primary human keratinocytes as well as HPV-negative tumor cells. This activity was found to require sequences of HPV-16 E7 involved in degradation of the retinoblastoma tumor suppressor protein as well as regions in the COOH terminus. HPV-16 E7-induced APBs contained ssDNA and several proteins that are involved in the response to DNA replication stress, most notably the Fanconi anemia D2 protein (FANCD2) as well as BRCA2 and MUS81. In line with these results, we found that FANCD2-containing APBs form in an ATR-dependent manner in HPV-16 E7-expressing cells. To directly show a role of FANCD2 in ALT, we provide evidence that knockdown of FANCD2 rapidly causes telomere dysfunction in cells that rely on ALT to maintain telomeres. Taken together, our results suggest a novel link between replication stress and recombination-based telomere maintenance that may play a role in HPV-16 E7-mediated extension of host cell life span and immortalization.

Defects of FA/BRCA pathway in lymphoma cell lines.

The aim was to find the possible relationship between defects in the FA/BRCA pathway of genomic maintenance and potential pathogenesis of T and B cell lymphoma. We screened 29 cell lines derived from diverse subtypes of lymphoma for possible FA pathway defects. The results indicated: no defect in FANCD2 ubiquitination, BRCA2 and FANCJ expression; absence of FANCN protein in three cell lines: HT, Sudhl4 and JEKO-1. This absence was correlated with enhanced MMC-induced G2 arrest, growth inhibition and high chromosomal breakage rate in the three cell lines. We only found one substitution in HT and JEKO-1 exon-5a fragment: c.1769C > T, p. A590V. But in another lymphoma cell line Sudhl4 with FANCN absence, we have not found any mutation. In conclusion, this mutation maybe the reason which caused FANCN protein expression absent or made the protein very unstable and lose its function in HT and JEKO-1 cell lines.

First synthesis of N-[(aziridin-2-yl)methyl]benzimidazolequinone and analysis of toxicity towards normal and Fanconi anemia cells.

A diazole is N-substituted with 1-trityl-2-methylaziridine and demethylated and oxidised with NBS under acidic conditions to give a benzimidazolequinone; this novel anti-tumour agent is marginally more cytotoxic than mitomycin C (MMC) towards the normal human fibroblast cell line GM00637, while the MMC-hypersensitive human Fanconi anaemia (FA) cell line, PD20i, lacking the FANCD2 protein, is also hypersensitive to the benzimidazolequinone, with expression of FANCD2 protein decreasing sensitivity to both MMC and the benzimidazolequinone.

Loss of expression of FANCD2 protein in sporadic and hereditary breast cancer.

Fanconi anemia (FA) is a recessive disorder associated with progressive pancytopenia, multiple developmental defects, and marked predisposition to malignancies. FA is genetically heterogeneous, comprising at least 12 complementation groups (A-M). Activation of one of the FA proteins (FANCD2) by mono-ubiquitination is an essential step in DNA damage response. As FANCD2 interacts with BRCA1, is expressed in proliferating normal breast cells, and FANCD2 knockout mice develop breast tumors, we investigated the expression of FANCD2 in sporadic and hereditary invasive breast cancer patients to evaluate its possible role in breast carcinogenesis. Two tissue microarrays of 129 and 220 sporadic breast cancers and a tissue microarray containing 25 BRCA1 germline mutation-related invasive breast cancers were stained for FANCD2. Expression results were compared with several clinicopathological variables and tested for prognostic value. Eighteen of 96 (19%) sporadic breast cancers and two of 21 (10%) BRCA1-related breast cancers were completely FANCD2-negative, which, however, still showed proliferation. In the remaining cases, the percentage of FANCD2-expressing cells correlated strongly with mitotic index and percentage of cells positive for the proliferation markers Ki-67 and Cyclin A. In immunofluorescence double staining, coexpression of FANCD2 and Ki-67 was apparent. In survival analysis, high FANCD2 expression appeared to be prognostically unfavorable for overall survival (p = 0.03), independent from other major prognosticators (p = 0.026). In conclusion, FANCD2 expression is absent in 10-20% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.

Cytogenetic instability in ovarian epithelial cells from women at risk of ovarian cancer.

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.