A short history of SFRR-Asia,its current status and significance

Antineoplastic agents are known to induce the production of free radicals leading to cell damage. These adverse effects may fuel the acquisition of new mutations and the development of treatment resistances. We selected 30 metastatic breast cancer patients receiving palliative chemotherapy, and paired blood samples, before and after chemotherapy, were extracted. We analysed DNA, lipid and protein oxidative damage markers and determined the extent of antioxidant and repair defences activation at the systemic level. We found that the DNA repair activity of the KU86 enzyme was significantly lower after chemotherapy and the antioxidant capacity of the plasma was significantly higher after treatment. Cox regression analysis revealed a significant effect of KU86 activity on the survival rates of those patients who received anthracyclines as part of their treatment. The high clinical heterogeneity of metastatic breast cancer patients warrants further studies to clarify the role of DNA repair and systemic antioxidant capacities during chemotherapy.     

Basal, oxidative and alkylative DNA damage, DNA repair efficacy and mutagen sensitivity in breast cancer

Impaired DNA repair may fuel up malignant transformation of breast cells due to the accumulation of spontaneous mutations in target genes and increasing susceptibility to exogenous carcinogens. Moreover, the effectiveness of DNA repair may contribute to failure of chemotherapy and resistance of breast cancer cells to drugs and radiation. The breast cancer susceptibility genes BRCA1 and BRCA2 are involved in DNA repair. To evaluate further the role of DNA repair in breast cancer we determined: (1) the kinetics of removal of DNA damage induced by hydrogen peroxide and the anticancer drug doxorubicin, and (2) the level of basal, oxidative and alkylative DNA damage before and during/after chemotherapy in the peripheral blood lymphocytes of breast cancer patients and healthy individuals. The level of DNA damage and the kinetics of DNA repair were evaluated by alkaline single cell gel electrophoresis (comet assay). Oxidative and alkylative DNA damage were assayed with the use of DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg), recognizing oxidized DNA bases and 3-methyladenine-DNA glycosylase II (AlkA) recognizing alkylated bases. We observed slower kinetics of DNA repair after treatment with hydrogen peroxide and doxorubicin in lymphocytes of breast cancer patients compared to control individuals. The level of basal, oxidative and alkylative DNA damage was higher in breast cancer patients than in the control and the difference was more pronounced when patients after chemotherapy were engaged, but usually the level of DNA damage in these patients was too high to be measured with our system. Our results indicate that peripheral blood lymphocytes of breast cancer patients have more damaged DNA and display decreased DNA repair efficacy. Therefore, these features can be considered as risk markers for breast cancer, but the question whether they are the cause or a consequence of the illness remains open. Nevertheless, our results suggest that research on the mutagen sensitivity and efficacy of DNA repair could impact the development of new diagnostic and screening strategies as well as indicate new targets to prevent and cure cancer. Moreover, the comet assay may be applied to evaluate the suitability of a particular mode of chemotherapy to a particular cancer patient.     

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).

Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.

Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.

DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.     

Potentiated DNA Damage Response in Circulating Breast Tumor Cells Confers Resistance to Chemotherapy

Circulating tumor cells (CTCs) are seeds for cancer metastasis and are predictive of poor prognosis in breast cancer patients. Whether CTCs and primary tumor cells (PTCs) respond to chemotherapy differently is not known. Here, we show that CTCs of breast cancer are more resistant to chemotherapy than PTCs because of potentiated DNA repair. Surprisingly, the chemoresistance of CTCs was recapitulated in PTCs when they were detached from the extracellular matrix. Detachment of PTCs increased the levels of reactive oxygen species and partially activated the DNA damage checkpoint, converting PTCs to a CTC-like state. Inhibition of checkpoint kinases Chk1 and Chk2 in CTCs reduces the basal checkpoint response and sensitizes CTCs to DNA damage in vitro and in mouse xenografts. Our results suggest that DNA damage checkpoint inhibitors may benefit the chemotherapy of breast cancer patients by suppressing the chemoresistance of CTCs and reducing the risk of cancer metastasis.     

Nuclear excision repair-based personalized therapy for non-small cell lung cancer: from hypothesis to reality

The crucial 'flaw' in the existing treatment paradigm for non-small cell lung cancer (NSCLC) is the 'one size fits all approach'. Consequently, adjuvant chemotherapy is given to all patients to benefit a minority and, in the metastatic setting doublet chemotherapy only provides modest improvements in response rates and survival. A personalized approach of treatment selection is therefore desperately needed. Genetic information is stored in the chemical structure of DNA. To maintain the structural integrity of DNA, an intricate network of DNA repair systems have evolved. One of these is the nucleotide excision repair (NER), a highly versatile and sophisticated DNA damage removal pathway. We show here that this DNA repair mechanism is instrumental in defining prognosis and response to treatment. ERCC1, one of the proteins in this pathway, is measured to assess its functional status of the NER pathway. In patients with early stage NSCLC, low ERCC1 predicts for relapse and selects for patients who will benefit from adjuvant cisplatin-based chemotherapy. Conversely, ERCC1-positive resected patients have a better intrinsic prognosis and are not likely to benefit from platinum based chemotherapy. In a phase II trial in metastatic disease, we show that by tailoring chemotherapy using ERCC1 and RRM1 we can obtain 1-year survival of 60% (versus approximately 36% in historical controls) and response rates of 42% (versus 25% in historical controls). This approach is currently being validated in a prospective phase III trial. In the future, assessment of NER function may play a central role in NSCLC treatment decision making.     

Dermatomyositis as paraneoplastic syndrome of peritoneal and ovarian relapse after long-term complete remission in patient with metastatic bilateral breast cancer

Dermatomyositis is a rare disease characterised by inflammatory muscle affection and characteristic cutaneous changes. When occuring in a patient with cancer, dermatomyositis may indicate recurrence or progression and poor outcome. Herein, the treatment of metastatic breast cancer, metastatic pattern, characteristics of long-term survivors, and link between dermatomyositis and breast cancer are discussed and the literature reviewed. We report a 57-year old female patient with metastatic bilateral breast cancer whose ovarian and peritoneal relapse after long-term remission was disclosed by occurence of paraneoplastic dermatomyositis. The patient previously had a 15-year long disease free-period after primary treatment for breast cancer before onset of pulmonary dissemination. Following antracycline-based chemotherapy, the complete remission lasting another 15 years was accomplished. Dermatomyositis had been resolved upon induction of second-line taxane-based chemotherapy. After completion of six cycles of gemcitabine and paclitaxel chemotherapy, check-up revealed further progression. The patient subsequently underwent six cycles of third-line CAP chemotherapy (cyclofosfamide, doxorubicine, cisplatin) but disease progressed and oral capecitabine chemotherapy was initiated. The patient received four cycles of capecitabine followed by further vast progression and finally expired following massive pulmonary embolism. Our case stresses the need of thorough staging and check-up when dermatomyositis arises in patients with breast cancer, regardless of previous stable long-term complete remission. Furthermore, we believe that treatment with curative intent in young patients with metastatic breast cancer, who have good performance statuses and no comorbidities is required, because it is more likely to produce long-term complete remission. However, following disease relapse a poor outcome can be expected.     

Association of ionizing radiation-induced foci of NBS1 with chromosomal instability and breast cancer susceptibility

NBS1, a protein essential for DNA double-strand break repair, relocalizes into subnuclear structures upon induction of DNA damage by ionizing radiation, forming ionizing radiation-induced foci. We compared radiation-induced NBS1 foci in peripheral blood lymphocytes (PBLs) from 46 sporadic breast cancer patients and 30 healthy cancer-free volunteers. The number of persistent radiation-induced NBS1 foci per nucleus at 24 h after irradiation for patients with invasive cancer was significantly higher than for normal healthy volunteers. The frequency of spontaneous chromosome aberration increased as the number of persistent radiation-induced NBS1 foci increased, indicating that the number of persistent radiation-induced NBS1 foci might be associated with chromosome instability. There was also an inverse correlation between the number of radiation-induced NBS1 foci and the activity of DNA-dependent protein kinase (DNA-PK), which plays an important role in the nonhomologous end-joining (NHEJ) pathway, another mechanism of DNA DSB repair, indicating a close interrelationship between homologous recombination (HR) and NHEJ in DNA DSB repair. In conclusion, the number of persistent radiation-induced NBS1 foci is associated with chromosomal instability and risk of sporadic breast cancer and hence might be used to select individuals for whom a detailed examination is necessary because of their increased susceptibility to breast cancer, although refinement of the techniques for technical simplicity and accuracy will be required for clinical use.     

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.     

Lack of Genomic Heterogeneity at High-Resolution aCGH between Primary Breast Cancers and Their Paired Lymph Node Metastases

Lymph-node metastasis (LNM) predict high recurrence rates in breast cancer patients. Systemic treatment aims to eliminate (micro)metastatic cells. However decisions regarding systemic treatment depend largely on clinical and molecular characteristics of primary tumours. It remains, however, unclear to what extent metastases resemble the cognate primary breast tumours, especially on a genomic level, and as such will be eradicated by the systemic therapy chosen. In this study we used high-resolution aCGH to investigate DNA copy number differences between primary breast cancers and their paired LNMs. To date, no recurrent LNM-specific genomic aberrations have been identified using array comparative genomic hybridization (aCGH) analysis. In our study we employ a high-resolution platform and we stratify on different breast cancer subtypes, both aspects that might have underpowered previously performed studies.To test the possibility that genomic instability in triple-negative breast cancers (TNBCs) might cause increased random and potentially also recurrent copy number aberrations (CNAs) in their LNMs, we studied 10 primary TNBC–LNM pairs and 10 ER-positive (ER+) pairs and verified our findings adding additionally 5 TNBC-LNM and 22 ER+-LNM pairs. We found that all LNMs clustered nearest to their matched tumour except for two cases, of which one was due to the presence of two distinct histological components in one tumour. We found no significantly altered CNAs between tumour and their LNMs in the entire group or in the subgroups. Within the TNBC subgroup, no absolute increase in CNAs was found in the LNMs compared to their primary tumours, suggesting that increased genomic instability does not lead to more CNAs in LNMs. Our findings suggest a high clonal relationship between primary breast tumours and its LNMs, at least prior to treatment, and support the use of primary tumour characteristics to guide adjuvant systemic chemotherapy in breast cancer patients.     

RDM1 promotes critical processes in breast cancer tumorigenesis

Breast cancer is currently among the most common cancers in women, with almost 200,000 new cases diagnosed annually. Dysregulation of DNA repair pathways allows cells to accumulate damage and eventually mutations, with a subsequent reduction in DNA repair capacity in breast tissue, leading to tumorigenesis. One component of the DNA damage repair pathway is RAD52 motif‐containing 1 (RDM1), but the specific role of RDM1 in breast cancer and the underlying mechanism remain unclear. Here, we examined the role played by RDM1 in breast cancer cell culture using the HBL100 and MCF‐7 breast cancer cell lines. Disruption of RDM1 reduced in vitro cell proliferation and promoted apoptosis. Knockdown of RDM1 also induced up‐regulation of p53 levels, whereas RAD51 and RAD52, both involved in DNA repair, were down‐regulated. In addition, the in vivo growth of RDM1‐deficient cells was significantly repressed, suggesting that RDM1 is a novel oncogenic protein in human breast cancer cells. This study reveals a link between the DNA damage response pathway and oncogenic functionality in breast cancer. Accordingly, therapeutic targeting of RDM1 is a potential treatment strategy for breast cancer and overcoming drug resistance.     

Selenium and GPx-1 overexpression protect mammalian cells against UV-induced DNA damage

Supplementation of the culture media of human MCF-7 breast carcinoma cells or mouse fibroblasts with low levels of selenium (30 nM) provided as sodium selenite was shown to protect these cells from ultraviolet (UV)-induced chromosome damage, as quantified by micronucleus assay. Selenium supplementation was also effective in reducing UV-induced gene mutations as measured in the lacI shuttle vector model. Protection was dependent on functional BRCA1 activity, a protein implicated in breast cancer risk and DNA damage repair. In addition, overexpression of GPx-1, a selenoprotein with antioxidant activity, also attenuated UV induced micronuclei formation in the absence of selenium supplementation. Combining selenium supplementation with GPx-1 overexpression further reduced UV-induced micronucleus frequency. These data provide evidence that the benefits of selenium supplementation might be either through the prevention or repair of DNA damage, and they implicate at least one selenoprotein (GPx-1) in the process.     

ABT-888 and quinacrine induced apoptosis in metastatic breast cancer stem cells by inhibiting base excision repair via adenomatous polyposis coli

PARP inhibitors in combination with other agents are in clinical trial against cancer, but its effect on cancer stem cells (CSCs) is limited. CSCs are responsible for drug resistance, metastasis and cancer relapse due to high DNA repair capacity. Here, we present preclinical effects of Quinacrine (QC) with ABT-888, a PARP inhibitor, on highly metastatic breast cancer stem cells (mBCSCs). An increased level of Adenomatous polyposis coli (APC) was noted after treatment with ABT-888 in QC pre-treated mBCSCs cells. Increased APC physically interacts with PARP-1 and inhibits PARylation causing the non assembly of base excision repair (BER) multiprotein complex, resulting in an irreparable DNA damage and subsequent apoptosis. Knockdown of APC in mBCSCs inhibited DNA damage, increased BER and PARylation, reduces apoptosis while the over-expression of APC in BT20 (APC low expressing) cells reversed the effect. Thus, combination of QC and ABT-888 decreased mBCSCs growth by activating APC and inhibiting BER within the cells.     

Cancer pharmacogenomics: role of DNA repair genetic polymorphisms in individualizing cancer therapy

The evolving field of cancer pharmacogenomics uses genetic profiling to predict the response of tumor and normal tissue to therapy. The narrow therapeutic index and heterogeneity of patient responses to chemotherapy and radiotherapy implies that the efficacy of these treatments could, potentially, be significantly enhanced by improving our understanding of the genetic bases for interindividual differences in their effects. The cytotoxicity of both chemotherapy and radiotherapy is to a large extent directly related to their ability to induce DNA damage. The ability of cancer cells to recognize and repair this damage contributes to therapeutic resistance. On the other hand, suboptimal DNA repair in normal tissue may negatively impact on normal tissue tolerance.More than 130 genes have been identified that are associated with human DNA repair, and single nucleotide polymorphisms of several of the DNA repair genes have been described recently. In this article, we present the current evidence implicating variations within DNA repair genes as important predictive and prognostic markers in cancer. We review evidence suggesting DNA repair genetic polymorphisms may significantly influence the clinical response to chemotherapy and radiotherapy, and may influence normal tissue tolerance to cancer treatments.     

DNA repair: relationship to drug and radiation resistance, metastasis and growth factors

DNA repair mechanisms are important for the recovery of both normal and malignant tissues from radiation and chemotherapy. Drug 'resistance' may merely reflect the similarity of cancer to normal tissues. Investigating the normal repair mechanisms by cloning human DNA repair genes will permit a much better comparison. Therapeutic inhibition of DNA repair may be possible with poly-ADP-ribose polymerase inhibitors. A differential effect may be obtained since less-differentiated cells have a higher poly-ADP-ribose polymerase activity. Clinical application of repair inhibitors can be achieved by using antimetabolites such as high-dose hydroxyurea which produces levels of 1-3 mmol litre -1/24 hours. The whole cell and tissue response to DNA damage is more complex than removal of adducts and joining strand breaks. DNA damage can result in an increase in growth-factor receptors, the release of soluble mediators that affect undamaged cells and stimulation of plasminogen activator. These changes may enhance growth and recovery as well as bypass or repair the damage. The generation of heterogeneity in a tumour population may be mediated by DNA rearrangements. Genetic instability is much higher in metastatic clones and a comparison of DNA strand-break repair in a metastatic and a non-metastatic line showed more rapid repair in the former. Aberrant use of DNA repair stimulated by growth factors may mediate tumour progression and heterogeneity as well as drug resistance.     

A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses

DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We here report a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine-tyrosine-arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with gammaH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes.     

Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and nonmalignant MCF-10A cells

Oxidatively induced stress and DNA damage have been associated with various human pathophysiological conditions, including cancer and aging. Complex DNA damage such as double-strand breaks (DSBs) and non-DSB bistranded oxidatively induced clustered DNA lesions (OCDL) (two or more DNA lesions within a short DNA fragment of 1-10 bp on opposing DNA strands) are hypothesized to be repair-resistant lesions challenging the repair mechanisms of the cell. To evaluate the induction and processing of complex DNA damage in breast cancer cells exposed to radiotherapy-relevant gamma-ray doses, we measured single-strand breaks (SSBs), DSBs, and OCDL in MCF-7 and HCC1937 malignant cells as well as MCF-10A nonmalignant human breast cells. For the detection and measurement of SSBs, DSBs, and OCDL, we used the alkaline single-cell gel electrophoresis, gamma-H2AX assay, and an adaptation of pulsed-field gel electrophoresis with E. coli repair enzymes as DNA damage probes. Increased levels for most types of DNA damage were detected in MCF-7 cells while the processing of DSBs and OCDL was deficient in these cells compared to MCF-10A cells. Furthermore, the total antioxidant capacity of MCF-7 cells was lower compared to their nonmalignant counterparts. These findings point to the important role of complex DNA damage in breast cancer and its potential association with breast cancer development especially in the case of deficient BRCA1 expression.     

Concurrent or sequential use of cytotoxic chemotherapy and hormone treatment in advanced breast cancer: report of the Swiss Group for Clinical Cancer Research.

In a trial of combined hormone treatment and cytotoxic chemotherapy 464 patients with advanced breast cancer were randomly allocated to either concurrent or sequential treatment. Cytotoxic drugs were given only if the antitumour activity of the hormone treatment was inadequate. Hormone treatment consisted of oophorectomy for premenopausal and tamoxifen administration for postmenopausal patients. Length of survival was better, though not significantly, in premenopausal patients (p = 0.29) treated concurrently and in postmenopausal women (p = 0.17) treated sequentially; the difference was highly significant (p = 0.003) only for postmenopausal women in the low-risk category. The findings suggest that postmenopausal women with metastatic breast cancer should probably be treated primarily by carefully monitored hormone treatment.