Clinicopathological significance of KU70/KU80, a key DNA damage repair protein in breast cancer

Although the role of BRCA1 and the homologous recombination (HR) pathway in breast cancer (BC) has been extensively studied, the alternative repair pathway for DNA double-strand breaks (DSBs), non-homologous end-joining (NHEJ) remains to be defined. Ku proteins bind to DNA DSB ends and play a key role in NHEJ. In this study we aimed to assess the expression and biological significance of the KU70/KU80 heterodimer in the different molecular classes of BC. The expression of KU70/KU80 was assessed immunohistochemically in a well-characterised and annotated series of 1302 unselected invasive BC cases with a long-term follow-up together with 25 cases with known BRCA1 mutations. The results were correlated with clinicopathological parameters, other DNA repair proteins and patient outcome. The expression of KU70/KU80 protein was further evaluated in various BC cell lines using western blotting and reverse-phase protein microarray (RPPA). Nuclear KU70/KU80 expression was correlated with features of poor prognosis including higher histological grade, lymphovascular invasion, negative oestrogen receptor expression, basal-like phenotype, P53 and CHK1 positivity. KU70/KU80 was expressed in all BRCA1-associated tumours and showed an inverse correlation with nuclear BRCA1 protein and aberrant cytoplasmic RAD51 expression. RPPA confirmed these results and showed higher expression of KU70/KU80 in BRCA1-deficient cell line compared to BRCA1-proficient cell line. KU70/KU80 expression showed an association with disease-free interval; however, it was not an independent predictor of outcome. As a conclusion, KU70/KU80 may play a role in DNA DSBs repair in HR-deficient tumours. Further study of other NHEJ markers in sporadic BC is warranted.     

Total prolactin binding sites in human breast cancer biopsies

Summary Total and free prolactin receptors were assayed in 72 human breast tumor biopsies. Forty-nine percent of the tumors are positive (specific binding greater than 0.8% of total radioactivity) when assaying free receptors, while 71% are positive when total receptors levels are determined. No clear relationship exists between the prolactin receptor positivity and the presence of either progesterone or estradiol receptors. Measurement of total prolactin receptors could be an important and independent criterion of the hormonal sensitivity of the tumors. Address for reprints: Dr. J.P. Peyrat, Laboratoire d'Endocrinologie Expérimentale, Centre de Anticancéreux de Lille (Centre Oscar Lambret), BP 307, 59020 Lille Cédex, France.     

Chromodomain helicase DNA binding protein 5 plays a tumor suppressor role in human breast cancer

Introduction

The chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as a tumor suppressor in a mouse model. The CHD5 locus at 1p36 is deleted, and its mutation has been detected in breast cancer. We, therefore, evaluated whether CHD5 plays a role in human breast cancer.

Methods

We screened mutations in 55 tumors, determined promoter methylation in 39 tumors, measured RNA expression in 90 tumors, analyzed protein expression in 289 tumors, and correlated expression changes with clinicopathological characteristics of breast cancer. Functional effects of CHD5 on cell proliferation, invasion and tumorigenesis were also tested.

Results

Although only one mutation was detected, CHD5 mRNA expression was significantly reduced, accompanied by frequent genomic deletion and promoter methylation, in breast cancer. The extent of methylation was significantly associated with reduced mRNA expression, and demethylating treatment restored CHD5 expression. Lower CHD5 mRNA levels correlated with lymph node metastasis (P = 0.026). CHD5 protein expression was also reduced in breast cancer, and lack of CHD5 expression significantly correlated with higher tumor stage, ER/PR-negativity, HER2 positivity, distant metastasis and worse patient survival (P ≤ 0.01). Functionally, ectopic expression of CHD5 in breast cancer cells inhibited cell proliferation and invasion in vitro and tumorigenesis in nude mice. Consistent with the inhibition of invasion, CHD5 down-regulated mesenchymal markers vimentin, N-cadherin and ZEB1 in breast cancer cells.

Conclusion

Down-regulation of CHD5, mediated at least in part by promoter methylation, contributes to the development and progression of human breast cancer.     

Elevated protein kinase C expression in human breast tumor biopsies relative to normal breast tissue

The Ca2+- and phospholipid-dependent protein kinase, protein kinase C (PKC), is a critical enzyme in the regulation of cell growth. In this report, we demonstrate elevated expression of PKC activity in surgical specimens of eight of nine spontaneous human breast tumors, as compared with the expression of PKC activity in normal breast tissue obtained from the same patients. The mean PKC specific activity in histologically normal breast tissue was 166 +/- 63 pmol 32P/min/mg, whereas the mean PKC specific activity in the breast tumors was 460 +/- 182 pmol 32P/min/mg (P = 0.0003; Student's t test). The low interpatient variability among the PKC levels observed in the histologically normal breast tissue specimens and the significant elevation of PKC levels observed in the tumors indicate that elevated expression of PKC activity in breast tissue is a potential marker for malignant disease in the breast.     

Anticancer drug activity in human bladder tumor cell lines

A panel of ten human bladder tumor cell lines were tested for drug sensitivity to ten standard or investigational anticancer drugs using a tumor colony assay. The activity of these anticancer agents in vitro was then compared with the clinical activity of these agents in bladder cancer. Drug activity was found in only five of the ten cell lines. In only 9 of 100 drug assays was the inhibition of colony growth lower than 30% of the controls. The activity of the more active anticancer drugs in bladder cancer (i.e., methotrexate and cisplatin) was not predicted using the tumor colony assay. Overall, the low level of activity of most anticancer drugs tested paralleled the clinical experience of drug resistance found in human bladder cancer.     

DNA mismatch binding in human lung tumor cell lines

Defects in mismatch repair (MMR) genes have been involved in several types of sporadic and hereditary cancers. In order to elucidate the role of MMR in human lung carcinogenesis we examined DNA mismatch binding in cell-free extracts of seven lung tumor cell lines and five corresponding lymphoblastoid cell lines from lung cancer patients. Using the technique of bandshift assay we have demonstrated that 2/7 of the tumor cell lines are aberrant in binding to specific DNA mismatches while all lymphoblastoid cell lines were proficient in binding to all tested mismatches. Both extracts were aberrant in binding to G/T mismatch whereas one of the cell lines showed deficiency in binding to the C:A mismatches as well. Immunoblotting analysis showed that all known DNA mismatch repair (MMR) proteins were present in these extracts. The cell line deficient in binding to both G:T and C:A mismatches showed microsatellite instability (MSI) in tumor DNA and higher resistance to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). This report indicates that DNA mismatch binding deficiencies may be implicated in at least a subgroup of human lung cancer.     

Expression and DNA binding activity of the Ku heterodimer in bladder carcinoma.

BACKGROUND: The Ku protein is a tightly associated heterodimer, comprised of 70-kilodalton (kD) and 86-kD subunits, that forms the DNA-dependent protein kinase (DNA-PK) complex together with the 470-kD DNA-PKcs catalytic subunit, and is involved mainly in DNA double-strand breaks (DSBs) repair. The objective of the current study was to investigate the expression and DNA-binding activity of the Ku protein in fresh tissues from patients with bladder carcinoma and to compare it with that in nontumor tissues obtained from the same organ. Moreover, the DNA-binding activity of Ku was assessed after exposure of the tumor cells to 1 or 2 grays (Gy) of X-rays. Furthermore, the level of phosphorylated Ku was analyzed in both the nuclear and cytoplasmic compartment of normal tissue after exposure to 2 Gy of X-rays. METHODS: The expression and DNA-binding activity of Ku protein were assessed in tumor samples from patients who all were diagnosed with transitional cell carcinoma (TCC) of the bladder using Western blot analysis and the electrophoretic mobility shift assay, respectively. RESULTS: Enhanced Ku activity and expression were found in tumor tissue compared with normal tissue for each patient. Moreover, variations in Ku activity were found in a dose-dependent manner after the tumor cells were exposed to 1 or 2 Gy of X-rays. A decrease in phosphorylated Ku in the cytoplasm and a parallel increase in the nucleus of normal tissue cells were observed after exposure to X-rays. CONCLUSIONS: The results of the current study suggest a possible role of Ku in regulating the DNA-PK activity of DSBs repair in bladder tumors.     

Tumor cell specific expression of MMP-2 correlates with tumor vascularisation in breast cancer

The metastatic potential of tumors is dependent on the ability of tumor cells to degrade extracellular matrix components by the expression of matrix metalloproteinases (MMPs) and to induce vascularisation of the tumor tissue. Thus, expression of MMPs and the number of blood vessel in tumor tissue may serve as prognostic markers of aggressive and metastasizing tumor growth. We have determined the vascularisation and the expression of MMP-2 by immuno-histochemical staining of 19 benign and 75 malignant breast tissue specimens with CD31- and MMP-2 specific antisera. The degree of vascularisation was expressed by intratumoral microvascular density (IMD), which takes into account all vessels present in a hot spot irrespective of their size. In addition, we have introduced a novel parameter, vascular grading (VG), which describes the percentage of small microvessels of <20 microm in diameter in the total number of blood vessels. IMD tended to indicate an elevated risk for metastasis formation and disease recurrence, while VG did not correlate with metastasis formation. Similarly, MMP-2 expression neither correlated with the clinical outcome of the disease nor with the classical histo-pathological parameters such as stage, grade, lymph node involvement and estrogen receptor status. Tumor cell-specific MMP-2 expression, however, showed a highly significant correlation with VG but not with IMD. These results indicate that MMP-2 expression is rather involved in the formation of small capillaries than in vessel maturation and tumor cell invasion. Thus, MMP-2 expression by tumor cells may serve as indicator of strong angiogenic induction potential of breast tumor cells.     

Characterization of binding sites for a GnRH-agonist (buserelin) in human breast cancer biopsies and their distribution in relation to tumor parameters

Summary Gonadotropin-releasing hormone analogs (GnRH-A) have been added to the armentarium in the therapy of hormone-dependent breast cancer in premenopausal women. The effect of chronic GnRH-A-treatment in premenopausal women is based on the suppression of the hypothalamus-pituitary-ovarian axis and the reduction of sex-steroid serum levels. In addition, a number of experimental and clinical data have been accumulated indicating a direct action of GnRH-A on breast cancer cells and tissue. In this study we analyzed 235 human breast cancer biopsies for specific GnRH-A-binding. We demonstrate high affinity GnRH-A binding sites in human breast cancer tissues. The evaluation of clinical data showed no correlation of the level of GnRH-A-binding with classical tumor parameters.     

Epidermal growth factor binding by breast tumor biopsies and relationship to estrogen receptor and progestin receptor levels

Epidermal growth factor (EGF) may be important in regulating the growth of some breast cancer cells in vivo because of its mitogenic action on some breast cancer cell lines in vitro. Epidermal growth factor receptors (EGF-R) were measured in a series of breast tumors to determine what percentage of breast tumors express EGF-R and whether EGF-R was independent of expression of estrogen receptor and progestin receptor. Specific binding of 125I-EGF to membranes from pooled homogenates of breast tumors reached equilibrium after 45 min at 25 degrees and remained constant. Scatchard analysis of 125I-EGF binding indicated a single class of receptors with an apparent Kd of 2 nM and a binding capacity of 28 fmol/mg of membrane protein, and the binding of 125I-EGF was not effectively competed for by insulin, fibroblast growth factor, growth hormone, or prolactin. Specific binding of 125I-EGF of 1 fmol or greater/mg of membrane protein and 15% or greater specific binding was detected in 48% of 137 unselected primary and metastatic breast tumors. The frequency distribution of EGF binding values was unimodal, with a progressive decrease in the proportion of patients with high EGF binding values. The values of EGF binding ranged from 1 to 121 fmol/mg of protein, with an arithmetic mean of 8.4 fmol/mg of protein and a geometric mean of 3.2 fmol/mg of protein. Forty-two % of 24 metastatic breast tumors were positive for EGF binding, with an arithmetic mean of 6.3 fmol/mg of protein and a geometric mean of 4.1 fmol/mg of protein. The magnitude of EGF binding in individual tumors was independent of either estrogen receptor or progestin receptor levels, although the highest quantities of EGF binding were expressed by tumors lacking steroid receptors. Approximately 20% of the tumors in the study were EGF-R-positive and ER-negative, suggesting that the growth of these tumors may be regulated predominantly by a peptide hormone (EGF) rather than a steroid hormone (estrogen). EGF binding did not correlate significantly with age of the patients. Correlation analysis between EGF binding and the percentage of malignant and nonmalignant cell types present in sections of tumor adjacent to the area assayed for EGF binding indicated that the percentage of malignant cells is an important factor in determining the amount of EGF binding in tumor homogenates. The recent discovery of transforming growth factors which interact with the EGF-receptor system suggests additional roles for EGF receptors in breast cancer.     

人脑星形细胞瘤组织中NF-κB的DNA结合活性

背景与目的:人类肿瘤细胞的转录因子可在转录水平调控许多恶性相关基因的表达,与肿瘤细胞的低分化和高转移有关,并抑制肿瘤细胞凋亡。本研究旨在观察人脑星形细胞瘤组织中核因子-κB(nuclearfactor-kappaB,NF-κB)的DNA结合活性,探讨其在人脑星形细胞瘤发生和发展中的意义。方法:采用电泳迁移率(electrophoreticmobilityshiftassay,EMSA)同位素放射自显影方法分别检测12例正常脑组织及37例人脑星形细胞瘤组织中NF-κB的DNA结合活性。结果:NF-κB的DNA结合活性在人脑星形细胞瘤组织光密度值为134.2±24.1,高于正常脑组织的97.5±1.9(P<0.05),其中多形胶质母细胞瘤(Ⅳ级)NF-κB的DNA结合活性最高,为106.8±7.4,依次为间变性星形细胞瘤(Ⅲ级)123.2±10.1,星形母细胞瘤(Ⅱ级)139.3±16.8,星形细胞瘤(Ⅰ级)160.2±18.6(P<0.05)。结论:NF-κB的DNA结合活性与人脑星形细胞瘤分级有关,并随恶性程度增加而增强。     

Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer

BackgroundMolecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies.Patients and methodsThe Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations.ResultsEvaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0–2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44–90%) patients had ≥1 mutation (median 1 mutation per patient, range 0–2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information.ConclusionPlasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs.     

Estrogen receptor and proteolytic activity in human breast tumor nuclei.

We have previously found that human breast cancer cells in tissue culture contain estrogen receptor in their nuclei despite the absence of estrogen. We have now investigated solid human breast cancer biopsies and find that proteolytic activities in extracts from the nuclear pellets of these biopsies interfere with or prevent the measurement of nuclear estrogen receptor when the protamine assay is used. However, the problems of receptor degradation can be avoided by the use of a hydroxylapatite assay.     

Aldehyde dehydrogenase activity in xenografted human brain tumor in nude mice. Preliminary results in human glioma biopsies

ALDH activity measured fluorimetrically using a high concentration of aliphatic aldehyde as substrate was studied in human glioblastomas grafted in nude mice. Compared with normal brain, ALDH activity is significantly increased in malignant glioma tissue, especially in the cytosolic subcellular fraction. Correlatively, in comparison with normal brain tissue, MDA levels were significantly reduced in whole homogenates and in cytosolic fractions of xenografted glioblastoma tissue. Preliminary results concerning human malignant glioma biopsies are in good agreement with our experimental data. In view of previous works, these results suggest a relationship between alterations in ALDH iso-enzymes activities and cytosolic aldehyde concentrations with respect to normal or tumoral cell growth.     

Detection of DNA fragmentation in human breast cancer tissue by an antibody specific to single-stranded DNA

While there have been many reports concerning the clinical significance of bcl-2 expression in human breast cancer, little is known about apoptosis in primary breast cancers. We immunohistochemically examined DNA fragmentation in 107 primary human breast cancers from Japanese women using an antibody specific to single-stranded DNA. The apoptosis index, calculated as the product of the positive cell number and the cellularity coefficient, ranged from 0 to 48. The average incidence of apoptosis was calculated as 0.1% of tumor cells. No relationships were observed among the apoptosis index, expression of bcl-2, and the histological grade of the tumors. Almost all apoptotic cells were phagocytosed by surrounding tumor cells immediately after DNA fragmentation. Apoptotic body formation was rare. The apoptotic cells seemed to be degraded within phagocytes, leaving no trace of apoptosis except the tiny shells of nuclei. The intensive phagocytic reaction might be one of the main reasons for the low incidence of apoptosis in human breast cancers.     

Metabolism and DNA binding of benzo[a]pyrene in cultured human bladder and bronchus

The metabolism of benzo[a]pyrene (BP) was examined in explantcultures of human bladder and bronchus. Three-day cultures wereexposed to radiolabeled BP for 24 h, and the metabolism wasdetermined by analysis of the level of binding of reactive metabolitesto DNA, and by the release of metabolites into the medium. Fora given individual, the DNA binding level and extent of metabolismwas usually higher in the bladder than in the bronchus. In specimensobtained from 16 individuals, the average DNA-binding levelsfor BP-DNA adducts following a 24 h exposure to 1 µM BPwere 6.4 ± 5.0 µmol BP/mol deoxyribonucleotidefor the bladder and 3.1 ± 1.9 µmol BP/mol deoxyribonucleotidefor the bronchus. The major BP-DNA adduct in both tissues co-chromatographedwith one of the adducts formed by reaction of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrenewith deoxyguanosine using high-pressure liquid chromatography.In tissues obtained from the same individual, the binding levelsof BP metabolites to bladder cell DNA was not strongly correlatedto that of bronchial cell DNA (r=0.54). The medium of both tissuescontained small amounts of free, unconjugated metabolites ofBP (<3% of the total) and large amounts (30–85% ofthe total) of unidentified, highly polar material. Human bladderappears to be the most active human explant tissue yet studiedwith respect to its ability to activate BP to DNA binding forms.The relevance of this observation to human bladder cancer is,as yet, unknown.     

Detection of human papillomavirus DNA in biopsies of human oral tissue

We have employed molecular probes produced from DNA fragments of human papillomavirus, cloned into prokaryotic vectors, to detect virus nucleic acid sequences in extracts of human oral tissues. The study was conducted with duplicate coded snap-frozen tissue biopsies from which frozen sections had been taken to accurately assess the pathology of each particular sample. The results show that a large proportion of the oral biopsies contained DNA which hybridized to the viral DNA probes, even under conditions of high stringency. The presence of virus did not correlate with neoplasia in the tissues examined, but HPV like sequences were found in a high proportion (80%) of biopsies taken from areas of keratosis and lichen planus and also in 41 to 46% of normal and tumour tissues.