Breast Cancer Cell Lines HCC1937 Research Articles

Design, synthesis, and biological evaluation of novel chrysin derivatives as poly(ADP-ribose) polymerase 1 (PARP1) inhibitors for the treatment of breast cancer

In this study, we reported the discovery and structure-activity relationship analysis of chrysin derivatives as a new class of inhibitors targeting poly (ADP-ribose) polymerase 1 (PARP1). Among these derivatives, compound 5d emerged as the most effective chrysin-based inhibitor of PARP1, with an IC50 value of 108 nmol·L−1. This compound significantly inhibited the proliferation and migration of breast cancer cell lines HCC-1937 and MDA-MB-436 by inducing DNA damage. Furthermore, 5d induced apoptosis and caused an extended G1/S-phase in these cell lines. Molecular docking studies revealed that 5d possesses a strong binding affinity toward PARP1. In vivo, in a xenograft model, 5d effectively reduced tumor growth by downregulating PARP1 expression. Overall, compound 5d shows promise as a potential therapeutic agent for the treatment of BRCA wild-type breast cancer.

PLA2G7/PAF-AH as Potential Negative Regulator of the Wnt Signaling Pathway Mediates Protective Effects in BRCA1 Mutant Breast Cancer.

Past studies have confirmed that aberrant activation of the Wnt/β-catenin signaling is associated with tumorigenesis and metastasis in breast cancer, while the role of platelet-activating factor acetylhydrolase (PLA2G7/PAF-AH) in this signaling pathway remains unclear. In this study, we analyze the functional impact of PAF-AH on BRCA1 mutant breast cancer and explore its relationship to the Wnt signaling pathway. By performing immunohistochemistry, PAF-AH expression and β-catenin expression were examined in both BRCA1 WT and BRCA1 mutant breast cancer specimens. The BRCA1 mutant breast cancer cell line HCC1937 was used for in vitro experiments to assess the impact of PAF-AH on cellular functions. The intracellular distribution of β-catenin depending on PLA2G7/PAF-AH expression was investigated by immunocytochemistry. Significantly higher nuclear expression levels of PAF-AH were found in BRCA1 mutant tissue specimens than in BRCA1 WT samples. Cell viability, proliferation, and the motility rate of HCC1937 were significantly enhanced after PLA2G7 silencing, which indicated a protective role of PAF-AH in breast cancer. Nuclear PAF-AH expressed correlatedly with membranous β-catenin. PLA2G7 silencing provoked the β-catenin translocation from the membrane to the nucleus and activated Wnt signaling downstream genes. Our data showed a protective effect of high PAF-AH expression in BRCA1 mutant breast cancer. PAF-AH may achieve its protective effect by negatively regulating the Wnt pathway. In conclusion, our research sheds new light on the regulatory pathways in BRCA1 mutant breast cancer.

High miR-30 Expression Associates with Improved Breast Cancer Patient Survival and Treatment Outcome.

Simple SummaryPrevious research on the miR-30 family and breast cancer patient survival and on miR-30-related chemosensitivity prompted us to design a comprehensive study on the role of the miR-30 family in general and on miR-30d in particular in breast cancer. We present a study consisting of a tumor microarray analysis of 1238 breast cancer patients, a survival analysis, a drug-sensitivity screen with six breast cancer cell lines, and an in-silico pathway analysis. In our analysis, high miR-30d expression was associated with improved survival in breast cancer patients with aggressive tumor phenotypes. In the drug-sensitivity analysis, ectopic expression of miR-30 family members sensitized the cell lines to the treatment. The pathway analysis based on miRNA and mRNA expression in the METABRIC data suggested that the miR-30 family may have an inhibitory role in pathways contributing to EMT and metastasis. Our results suggest prognostic and predictive potential for the miR-30 family for further investigation.Deregulated miRNA expression has been suggested in several stages of breast cancer pathogenesis. We have studied the miR-30 family, in particular miR-30d, in relation to breast cancer patient survival and treatment outcomes. With tumor specimens from 1238 breast cancer patients, we analyzed the association of miR-30d expression with tumor characteristics with the 5-year occurrence of breast cancer-specific death or distant metastasis (BDDM), and with 10-year breast cancer survival (BCS). We conducted a two-stage drug-screen to investigate the impact of miR-30 family members (miR-30a-30e) on sensitivity to doxorubicin and lapatinib in six breast cancer cell lines HCC1937, HCC1954, MDA-MB-361, MCF7, MDA-MB-436 and CAL-120, using drug sensitivity scores (DSS) to compare the miR-30 family mimics to their specific inhibitors. The study was complemented with Ingenuity Pathway Analysis (IPA) with the METABRIC data. We found that while high miR-30d expression is typical for aggressive tumors, it predicts better metastasis-free (pBDDM = 0.035, HR = 0.63, 95% CI = 0.4–0.9) and breast cancer-specific survival (pBCS = 0.018, HR = 0.61, 95% CI = 0.4–0.9), especially in HER2-positive (pBDDM = 0.0009), ER-negative (pBDDM = 0.003), p53-positive (pBDDM = 0.011), and highly proliferating (pBDDM = 0.0004) subgroups, and after adjuvant chemotherapy (pBDDM = 0.035). MiR-30d predicted survival independently of standard prognostic markers (pBDDM = 0.0004). In the drug-screening test, the miR-30 family sensitized the HER2-positive HCC1954 cell line to lapatinib (p < 10−2) and HCC1937, MDA-MB-361, MDA-MB-436 and CAL120 to doxorubicin (p < 10−4) with an opposite impact on MCF7. According to the pathway analysis, the miR-30 family has a suppressive effect on cell motility and metastasis in breast cancer. Our results suggest prognostic and predictive potential for the miR-30 family, which warrants further investigation.

Abstract 2036: Alternative activation of TGFβ signaling pathways in breast cancer tumors with different subtype is related to a differential expression of transcription factors that induce EMT

Abstract Breast cancer is the leading cause of cancer death among women worldwide being distant metastases the main cause of disease. Patients with triple negative breast cancer tumors develop distant metastases earlier compared to luminal tumors. Epithelial-mesenchymal transition (EMT) induced by TGFβ signaling pathways, is the main mechanism to promote metastasis through the expression of the transcription factors TWIST, SNAIL, SLUG and ZEB1. Expression of EMT transcription factors has been studied in cancer cell lines however studies in breast cancer tumors with different subtype have not been reported. In addition, the molecular mechanism that induce EMT program is not known yet. In this study we evaluated the expression of the four transcription factors as well as the state of TGFβ/SMAD, ERK/MAPK and PI3K/AKT pathways in breast cancer cell lines HCC1937 (triple negative) and T47D (luminal) after TGFβ treatment. The same analysis was done by immunohistochemistry in 100 breast cancer tumors with different subtype. After TGFβ treatment T47D showed activation of ERK/MAPK and PI3K/AKT pathways and expression of only two transcription factors: SNAIL and SLUG. HCC1937 cells showed activation of TGFβ/SMAD and ERK/MAPK pathways and expression of TWIST, SLUG and ZEB1 but not SNAIL. Similar results were observed in breast cancer tumors, being TGFβ/SMAD pathway active in the majority of triple negative tumors, whereas an active state of PI3K/AKT was observed in luminal tumors. Active ERK/MAPK pathway was observed in both triple negative and luminal tumors. In relation to expression of transcription factors triple negative tumors with an active state of the TGFβ/SMAD pathway showed expression of transcription factors TWIST, SNAIL and SLUG, whereas those tumors with an active state of ERK/MAPK pathway showed only ZEB1 expression. Luminal tumors with an active PI3K/AKT pathway frequently showed ZEB1 expression. Our results showed that expression of a specific EMT transcription factor induced by TGFβ is related to the tumor subtype and depends on the activated signaling pathway. These results suggest different mechanisms of EMT induction by TGFβ in relation to breast cancer tumor subtype. Citation Format: Victoria Ortega-Hernández, Wanda Fernandez, Pilar Carvallo. Alternative activation of TGFβ signaling pathways in breast cancer tumors with different subtype is related to a differential expression of transcription factors that induce EMT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2036.

Abstract 4319: Epigenetic reprogramming associated with BRCA1 loss of function in ovarian cancer induces enhanced interferon gamma signaling

Abstract Inactivating mutations of the breast cancer type 1 susceptibility protein (BRCA1) have been implicated in breast and ovarian cancer (OC) initiation. Having observed differential response of OC cells to histone deacetylase inhibitors (HDACi) dependent on the presence of a functional BRCA1, we mapped promoters marked by acetylation of lysine 9 or lysine 27 of histone H3 (H3K9ac, H3K27ac) by ChIP-sequencing, and measured gene expression by RNA-sequencing in the isogenic OC cell lines UWB1.289 carrying an inactivating mutation of BRCA1 (BRCA1-null) and UWB1.289 cells with restored BRCA1 gene (BRCA1+). Significant differences in promoter levels of H3K9 and H3K27 acetylation were associated with marked alteration of gene expression levels between BRCA-null and BRCA1+ cells and in response to the HDACi, entinostat. Pathway analyses revealed cellular movement, cellular development, cellular growth and proliferation, TGFβ1, TNF, and INF-γ among the top cellular/molecular functions and upstream regulators enriched by the absence of BRCA1 among H3K9ac differentially marked and differentially expressed genes. Importantly, the same pathways and regulators were enriched in response to entinostat in BRCA1+ cells, indicating that changes in gene expression affecting these functions and regulators in BRCA1-null cells depend on promoter-associated histone acetylation. Integrated analyses revealed that TGFβ1 and IFN-γ were also activated in BRCA1-null/or low vs. BRCA1-normal ovarian tumors in the TCGA database. IFN-γ target genes were upregulated at baseline and responded less to IFN-γ stimulation in BRCA1-null vs. BRCA1+ cells. Binding of HDAC1 to promoters of IFN-γ target genes was increased in BRCA1-null cells. Similarly, the BRCA1-null breast cancer cell line HCC1937 responded less to IFN-γ stimulation compared with HCC1937 cells in which BRCA1 was restored. The knockdown of STAT1, the main IFN-γ signaling transduction molecule, inhibited BRCA1+ cell proliferation, but did not affect BRCA1-null cells. We conclude that the transcriptome of BRCA1-null cells and tumors is altered through changes in histone acetylation affecting cancer-related pathways, including IFN-γ cellular responses. These changes could alter tumor progression in BRCA-null OC and impact response to immunotherapy. Citation Format: Horacio Cardenas, Jessica Thomes Pepin, Guanglong Jiang, Salvatore Condello, Kenneth P. Nephew, Yunlong Liu, Daniela Matei. Epigenetic reprogramming associated with BRCA1 loss of function in ovarian cancer induces enhanced interferon gamma signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4319.

Induced Expression of Cancer Stem Cell Markers ALDH1A3 and Sox-2 in Hierarchical Reconstitution of Apoptosis-resistant Human Breast Cancer Cells.

We established an experimental system that can induce p53-dependent apoptosis by doxycycline treatment to analyze characteristics of the apoptosis-resistant cancer cell subpopulation in the human breast cancer cell line HCC1937. Expression patterns of the stem cell markers, ALDH1A3 and Sox-2, the luminal differentiation marker, GATA3 and the proliferation index marker, Ki-67 were analyzed using immunostaining and fluorescence-activated cell sorting (FACS). After doxycycline treatment, the number of viable cells was gradually decreased over seven days in a time-dependent manner due to p53-induced apoptosis; however, the number of smaller-sized ALDH1A3+ cells assessed by immunostaining increased sharply after 1 day of doxycycline treatment, suggesting their apoptosis-resistant nature. The expression of ALDH1A3 was also detected in 78% of small-sized Ki-67+ proliferating progenitor cells, followed by the transient expression of GATA3, which presumably indicated the ability to differentiate into luminal progenitor cells. Although 42.2–58.5% of residual cells were positive for both ALDH1A3 and GATA3, their expression patterns exhibited an inverse correlation. The expression pattern of another stem cell marker, Sox-2, was similar, but more drastically altered after p53 induction compared with ALDH1A3. These findings may aid in understanding the hierarchical responses of cancer stem cells to therapeutic stresses.

Effects of microRNA-194 on proliferation, migration, invasion and epithelial- mesenchymal transition of basal- like breast cancer cell line HCC1937

Objective To explore the role of microRNA- 194(miR- 194)in proliferation, migration,invasion and the epithelial- mesenchymal transition(EMT)of human basal- like breast cancer cell line HCC1937. Methods Three groups were set up:experimental group[HCC1937 with lentivirus- miR - 194- enhanced green fluorescent protein(EGFP), MOI= 50], negative control group(HCC1937 with lentivirus- CON157- EGFP), and blank control group(HCC1937 without lentivirus). The miR- 194 expression was detected by real- time quantitative reverse transcriptase- polymerase chain reaction(RT-qPCR). Cell growth ability of HCC1937 was determined by cell counting kit- 8(CCK- 8)assay. Cell migration and invasion ability of HCC1937 were mearsured by a Transwell assay, and the protein of epithelial - mesenchymal transition phenotype was measured by Western blotting. Results The expression of miR-194 in experimental group(432.93±0.01) was higher than in negative control group(0.90±0.02) and blank control group(1)(P< 0.01) The cells in experimental group grew more slowly[(44.81± 0.11)%]than negative control group[(104.29±0.13)%]and blank control group[(100±0)%, P< 0.01].The number of migration and invasion was both less in experimental group than in negative control group and blank control group(P< 0.05).Western blotting revealed that the expression of E- cadherin in experimental group(0.175±0.053) was significantly decreased as compared with negative control group (0.516±0.032)and blank control group(0.433±0.047, P< 0.05), and the expression of N- cadherin in experimental group(0.342±0.012)was significantly increased as compared with negative control group (0.137±0.007)and blank control group(0.142±0.021)(P<0.05) Conclusion miR- 194 can inhibit proliferation, migration and invasion of basal- like breast cancer cells(HCC1937), and also affect EMT process in HCC1937 cells. Key words: Basal- like breast cancer; MicroRNA- 194; Proliferation

Abstract 1686: Synergistic combination of Trabectedin and Olaparib in breast cancer tumor cell lines

Abstract Trabectedin (Yondelis, ET-743) is a marine-derived antitumor agent that is currently used for the treatment of sarcoma and, in combination with pegylated-liposomal doxorubicin, of platinum-sensitive ovarian cancer patients. After binding to the DNA minor groove, trabectedin induces a potent transcription inhibition on tumor cells and tumor-associated macrophages. The resolution of functional DNA adducts generated by the drug is known to occur through the coordinated action of multiple DNA repair pathways, including homologous recombination (HR), a process that gives rise to double-strand breaks (DSBs). Trabectedin treatment at nanomolar concentrations does indeed produce a high proportion of DSB-positive tumor cells with abundant γ-H2AX foci and an S-phase delay/arrest by activation of the DNA replication checkpoints. We had previously shown the synergism (CI &amp;lt; 1) of the combination of Trabectedin with Olaparib (AZD-2281) in the breast cancer cell lines HCC-1937, MCF-7, MDA-MB-231 and MDA-MB-436. In this work we have investigated the mechanism of this synergistic effect at the molecular level. While the treatment with either compound alone (0.05 nM Trabectedin or 5 μM Olaparib for 72 hours) induced only a mild antiproliferative effect, the combination of both compounds at the mentioned concentrations induced strong apoptosis in all the breast cancer cell lines. Exposure to 0.5 and 2.5 nM Trabectedin for 72 hours induced a clear accumulation of γ-H2AX foci. Exposure to Olaparib alone induced only a mild DNA damage at 72 hours with all the concentrations tested. Olaparib induced a clear inhibition of PARylation at concentrations from 1-2.5 μM of the compound. The combination of both drugs, keeping Olaparib at 5 μM and varying the concentrations of Trabectedin from 0.05 to 2.5 nM, demonstrated a synergistic effect on the accumulation of histone H2AX phosphorylation, as analyzed both by western blotting and immunofluorescence microscopy. When comparing all these results, it was clearly seen that, although Trabectedin or Olaparib generated a considerable amount of DSBs, the combination of both drugs induced a higher proportion of DNA damage that remained at very high levels even after 72 hours of treatment. Thus, the observed synergistic effect seems to be the result of higher accumulation of DSBs after the administration of the combination of Trabectedin with Olaparib. Citation Format: Sonia Avila, Marta Martínez, Victoria Moneo, Juan F. Martínez-Leal, Carmen Cuevas, Luis F. Garcia-Fernández, Carlos M. Galamarini. Synergistic combination of Trabectedin and Olaparib in breast cancer tumor cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1686. doi:10.1158/1538-7445.AM2014-1686

Immediate early response gene X-1 promotes the apoptosis of the breast cancer cell line HCC1937 induced by tumor necrosis factor-related apoptosis inducing ligand

Objective To observe the effect of immediate early response gene X-1 (IER3) on the apoptosis of the breast cancer cell line HCC1937 induced by tumor necrosis factor-related apoptosis inducing ligand (TRAIL).Methods The expression of gene IRAIL and IER3 was detected before and after treatment by real-time quantitative polymerase chain reaction (Real-time PCR) at 48 h,The proliferation of breast cancer cells HCC1937 was mearsured by using Methylthiazol tetrazolium (MTT) assay at 24,48,72,96,and 120 h,The clone formation ability was mearsuerd by clone formation assay after 10-14 d,breast cancer cells HCC1937 were examined by Hoechst 33258 stain to detect the apoptpsis after 24 h.Results Compared with the negative control group,the relative expression of TRAIL in the TRAIL and the IER3 + TRAIL groups were 42.69 ± 3.88 and 46.36 ± 3.55 times respectively.And the relative expression of the IER3 in the IER3 and the IER3 + TRAIL groups were 5.08 ± 1.40 and 9.66 ± 2.25 times respectively.The inhibition rate (％) of cells in TRAIL,IER3 and TRAIL + IER3 groups were (50.95 ± 7.45),(25.39 ± 4.49) and (72.01 ± 2.95) respectively.The cell clone formation ability in IER3 +TRAIL group was obviously reduced.Compared with the negative control group,the apoptosis rate of cells in IER3 + TRAIL group was also increased significantly.Conclusion IER3 could promote the apoptosis of the breast cancer cell line HCC1937 induced by TRAIL. Key words: Immediate early response gene X-1 ; Tumor necrosis factor-related apoptosis inducing ligand ; Breast carcinoma

A novel cytotoxic terpenoid from the flowers of Kaunia lasiophthalma Griseb

A phytochemical study of the flowers of Kaunia lasiophthalma G. (Asteraceae) yielded a novel triterpene (1) together with several known sesquiterpenoids. The structure of the new compound was elucidated by analysis of the spectroscopic data. The biosynthetic origin of 1 is proposed to be a dimerization of an oxidized derivative (3) of the germacrane sesquiterpene costunolide (2), also present in the flowers. The anticancer activity of 1 in the five breast cancer cell lines HCC1937, JIMT-1, L56Br-C1, MCF-7 and SK-BR-3 was compared with the cytotoxicity in the normal-like breast epithelial cell line MCF-10A. 1 exhibited high cytotoxicity in all investigated cancer cell lines with IC50 values ranging from 0.67 to 7.0μM, although it is lacking selectivity as the MCF-10A cells were almost as sensitive.

Cytotoxic Sesquiterpene Lactones from Kauna lasiophthalma Griseb

Two new eudesmane derivatives (3 and 8) were isolated from the ethanol extract of the aerial parts of Kaunia lasiophthalma Griseb, together with 14 known eudesmane, germacrane, and guaiane sesquiterpenes, and four flavones. The structures and relative configurations of all the compounds were established by NMR spectroscopy and high-resolution mass spectrometry. The anticancer activity of sesquiterpenes 1, 3, 6–9, 11, 12, 14, and 16 was evaluated in vitro with the breast cancer cell lines HCC1937, JIMT-1, L56Br-C1, MCF-7, and SK-BR-3, and compared with the cytotoxicity in the non-cancerous breast epithelial cell line MCF-10A. All compounds were found to possess anticancer activity, and compound 1 was the most potent in all of the investigated cancer cell lines with IC50 values ranging between 2.0 and 6.2 μM. In order to demonstrate the importance of the α-methylene-γ-lactone/ester moiety present in all compounds for the effects on the cells, the methyl cysteine adduct 21 was prepared from 9 and found to be inactive or considerably less potent.

Anti-apoptotic mechanism for p21(waf1) in human basal like breast cancer cell line HCC1937

To explore the anti-apoptotic mechanism of p21(waf1) in human basal like breast cancer cell line HCC1937. There were 3 groups, i.e. experimental group HCC1937 with lentivirus -p21(waf1)-shRNA-RFP, control group 1 HCC1937 without lentivirus and control group 2 HCC1937 with lentivirus -RFP. The p21(waf1) and bim mRNA and protein expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respectively. And apoptosis of HCC1937 in different groups was assayed by terminal deoxynucleotidyl transferase mediated C-dUTP nick end labeling (TUNEL). After interference with lentivirus-p21(waf1)-shRNA-RFP, p21(waf1) mRNA and protein expressions declined significantly in the experimental group versus the control groups (experiment group: 0.260 ± 0.004, 0.293 ± 0.006, control group 1: 0.879 ± 0.028, 0.483 ± 0.071, control group 2: 0.870 ± 0.025, 0.469 ± 0.047, all P < 0.01). The bim mRNA and protein expressions increased. And there was significant difference between the experiment and control groups (experiment group: 0.420 ± 0.013, 0.355 ± 0.007, control group 1: 0.258 ± 0.005, 0.142 ± 0.012, control group 2: 0.259 ± 0.002, 0.147 ± 0.013, all P < 0.001); apoptotic index increased (experiment group: 0.279 ± 0.012, control group 1: 0.145 ± 0.008, control group 2: 0.148 ± 0.012, both P < 0.001). In human basal-like breast cancer cell line HCC1937, p21(waf1) exerts anti-apoptotic activity by inhibiting the expression of bim, a mediator of mitochondrial apoptosis.

Abstract P2-07-01: BRCA1 regulates RHAMM function in breast cancer

Abstract Objectives: The receptor for hyaluronan-mediated motility (RHAMM, intracellular hyaluronan binding protein [IHABP], CD168), whose over-expression in tumors is associated with poor prognosis and early age at diagnosis, localizes to the centrosome, interacts with microtubules, functions in the maintenance of spindle pole stability and is required for centrosomal targeting. A previous study has shown that genetic variation at the HMMR locus (encoding for RHAMM) modifies breast cancer risk among BRCA1 mutation carriers and interplay between BRCA1, AURKA (aurora kinase A), RHAMM and phosphorylated RHAMM (pRHAMM) plays an important role in epithelial apicobasal polarization and breast carcinogenesis. Aim of this study was to investigate mechanisms by which BRCA1 regulates the function of RHAMM. Methods: Four pairs of shBRCA1 oligonucleotides containing target sequences against BRCA1 transcript were designed and synthesized. After annealing, these four double-strand shBRCA1 fragments were inserted into pLVTH vector and stably transfected into MCF-12A cells for knock-down of BRCA1 expression. The BRCA1 knock-out breast cancer cell line HCC1937 with homozygous BRCA1 c.5382insC mutation was transfected with the full length wt-BRCA1 to restore BRCA1 function. HCC1937 cells were also transfected with the BRCA1-delExon11 isoform that lacks BRCA1 exon 11 in its entirety. Western blotting analysis and immunohistochemical staining with anti-BRCA1, anti-RHAMM, anti-pRHAMM and anti-AURKA antibodies were performed. Real-time PCR was used to quantify RHAMM and AURKA expression in these different cell lines and transfectants. Results: Stable transfection of MCF-12A cells with the different shRNAs resulted in a significant decrease of BRCA1 transcript and protein levels. Real-time PCR showed increased RHAMM expression in MCF-12A cells transfected with shRNA BRCA1 knock-down constructs. Increased RHAMM gene expression was also determined in HCC1937 cells lacking BRCA1 function due to homozygous deleterious BRCA1 mutation c.5382insC. Restored wt-BRCA1 expression in HCC1937 cells transfected with the full length BRCA1 cDNA results in was significantly reduced RHAMM expression. Inhibition of RHAMM gene expression was even more pronounced in HCC1937 cells transfected with the BRCA1-delExon11variant lacking 1,143 amino acids of the BRCA1 protein. On the protein level loss of BRCA1 function affects nuclear localization of pT703-RHAMM the product of AURKA activity: pT703-RHAMM staining was revealed to be strong at the nuclear envelope in MCF12A BRCA1 knock-down transfectants compared to the homogenous and less intense but clearly nuclear pT703-RHAMM staining in MCF12A wt-BRCA1 cells. Conclusions: BRCA1 negatively regulates RHAMM gene expression and might be involved in the nuclear localization of pRHAMM. Interestingly on the transcription level the BRCA1delexon11 isoform of BRCA1 displayed a stronger RHAMM repressor function than BRCA1 wild type. Taken together, our data provide insight into new functions of BRCA1 regulating RHAMM expression and mediating pRHAMM function. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-07-01.

Detection of circulating tumor cells in blood of metastatic breast cancer patients using a combination of cytokeratin and EpCAM antibodies.

BackgroundCirculating tumor cells (CTCs) are detectable in peripheral blood of metastatic breast cancer patients (MBC). In this paper we evaluate a new CTC separation method based on a combination of anti-EpCAM- and anti-cytokeratin magnetic cell separation with the aim to improve CTC detection with low target antigen densities.MethodsBlood samples of healthy donors spiked with breast cancer cell line HCC1937 were used to determine accuracy and precision of the method. 10 healthy subjects were examined to evaluate specificity. CTC counts in 59 patients with MBC were measured to evaluate the prognostic value on overall survival.ResultsRegression analysis of numbers of recovered vs. spiked HCC1937 cells yielded a coefficient of determination of R2 = 0.957. The average percentage of cell recovery was 84%. The average within-run coefficient of variation for spiking of 185, 85 and 30 cells was 14%. For spiking of 10 cells the within-run CV was 30%. No CTCs were detected in blood of 10 healthy subjects examined.A standard threshold of 5 CTC/7.5 ml blood as a cut-off point between risk groups led to a highly significant prognostic marker (p < 0.001). To assess the prognostic value of medium CTC levels we additionally considered a low (CTC-L: 0 CTC), a medium (CTC-M: 1–4 CTC) and a high risk group (CTC-H: ≥5 CTC). The effect of this CTC-LMH marker on overall survival was significant as well (p < 0.001). A log-ratio test performed to compare the model with 3 vs. the model with 2 risk groups rejected the model with 2 risk groups (p = 0.026). For CTC as a count variable, we propose an offset reciprocal transformation 1/(1 + x) for overall survival prediction (p < 0.001).ConclusionsWe show that our CTC detection method is feasible and leads to accurate and reliable results. Our data suggest that a refined differentiation between patients with different CTC levels is reasonable.

Transduction of E2F-1 TAT fusion proteins represses expression of hTERT in primary ductal breast carcinoma cell lines

BackgroundTelomerase expression is detectable in 81–95% of breast carcinomas and may serve as a therapeutic target. The objective of this study was to investigate repression of telomerase activity in primary ductal breast cancer cells through transcriptional regulation of the catalytic subunit hTERT. We hypothesized that inhibition of telomerase expression could be achieved via Tat mediated protein transduction of the repressor protein E2F-1.MethodsProtein purification techniques were refined to yield biologically active Tat fusion proteins (TFPs) capable of transducing the breast cancer cell lines HCC1937 and HCC1599. Cell lines were treated with wildtype E2F-1 (E2F-1/TatHA), mutant E2F-1 (E132/TatHA) and a control Tat peptide (TatHA) for 24 hours. Total RNA was isolated from treated cells, reverse transcribed and fold changes in gene expression for hTERT determined via real-time RT-qPCR.ResultsSignificant repression of the catalytic subunit of telomerase (hTERT) was present in both HCC1937 and HCC1599 cells following treatment with E2F-1/TatHA. In HCC1937 cells, hTERT was repressed 3.5-fold by E2F-1/TatHA in comparison to E132/TatHA (p < 0.0012) and the TatHA peptide controls (p < 0.0024). In HCC1599 cells, hTERT was also repressed with E2F-1/TatHA treatment by 4.0-fold when compared to the E132/TatHA control (p < 0.0001). A slightly lower hTERT repression of 3.3-fold was observed with E2F-1/TatHA in the HCC1599 cells when compared to the TatHA control (p < 0.0001).ConclusionThese results suggest that transduction of E2F-1/TatHA fusion proteins in vitro is an effective repressor of hTERT expression in the primary ductal breast cancer cell lines HCC1937 and HCC1599.

Functional Characterization of a Novel BRCA1-Null Ovarian Cancer Cell Line in Response to Ionizing Radiation

The breast and ovarian cancer susceptibility gene BRCA1 plays a major role in the DNA damage response pathway. The lack of well-characterized human BRCA1-null cell lines has limited the investigation of BRCA1 function, particularly with regard to its role in ovarian cancer. We propagated a novel BRCA1-null human ovarian cancer cell line UWB1.289 from a tumor of papillary serous histology, the most common form of ovarian carcinoma. UWB1.289 carries a germline BRCA1 mutation within exon 11 and has a deletion of the wild-type allele. UWB1.289 is estrogen and progesterone receptor negative and has an acquired somatic mutation in p53, similar to the commonly used BRCA1-null breast cancer cell line HCC1937. We used ionizing radiation to induce DNA damage in both UWB1.289 and in a stable UWB1.289 line in which wild-type BRCA1 was restored. We examined several responses to DNA damage in these cell lines, including sensitivity to radiation, cell cycle checkpoint function, and changes in gene expression using microarray analysis. We observed that UWB1.289 is sensitive to ionizing radiation and lacks cell cycle checkpoint functions that are a normal part of the DNA damage response. Restoration of wild-type BRCA1 function in these cells partially restores DNA damage responses. Expression array analysis not only supports this partial functional correction but also reveals interesting new information regarding BRCA1-positive regulation of the expression of claudin 6 and other metastasis-associated genes and negative regulation of multiple IFN-inducible genes.

The sensitivity of human mesenchymal stem cells to ionizing radiation

Recent studies have shown that mesenchymal stem cells (MSCs) obtained from bone marrow transplantation patients originate from the host. This clinical observation suggests that MSCs in their niches could be resistant to irradiation. However, the biologic responses of bone marrow MSCs to irradiation have rarely been described in the literature. In this study, human bone marrow-derived, clonally expanded MSCs were used to investigate their sensitivity to irradiation in vitro, and the cellular mechanisms that may facilitate resistance to irradiation. The human lung cancer cell line A549 and the breast cancer cell line HCC1937 were used as controls for radiosensitivity; the former line has been shown to be radioresistant and the latter radiosensitive. We then examined their in vitro biologic changes and sensitivities to radiation therapy. Our results suggest that MSCs are characterized as resistant to irradiation. Several cellular mechanisms were demonstrated that may facilitate resistance to irradiation: ATM protein phosphorylation, activation of cell-cycle checkpoints, double-strand break repair by homologous recombination and nonhomologous end joining (NHEJ), and the antioxidant capacity for scavenging reactive oxygen species. As demonstrated, MSCs possess a better antioxidant reactive oxygen species-scavenging capacity and active double-strand break repair to facilitate their radioresistance. These findings provide a better understanding of radiation-induced biologic responses in MSCs and may lead to the development of better strategies for stem cell treatment and cancer therapy.

An hGCN5/TRRAP Histone Acetyltransferase Complex Co-activates BRCA1 Transactivation Function through Histone Modification

It is well established that genetic mutations that impair BRCA1 function predispose women to early onset of breast and ovarian cancer. However, the co-regulatory factors that support normal BRCA1 functions remain to be identified. Using a biochemical approach to search for such co-regulatory factors, we identified hGCN5, TRRAP, and hMSH2/6 as BRCA1-interacting proteins. Genetic mutations in the C-terminal transactivation domain of BRCA1, as found in breast cancer patients (Chapman, M. S., and Verma, I. M. (1996) Nature 382, 678-679), caused the loss of physical interaction between BRCA1 and TRRAP and significantly reduced the co-activation of BRCA1 transactivation function by hGCN5/TRRAP. The reported transcriptional squelching between BRCA1 and estrogen receptor alpha (Fan, S., Wang, J., Yuan, R., Ma, Y., Meng, Q., Erdos, M. R., Pestell, R. G., Yuan, F., Auborn, K. J., Goldberg, I. D., and Rosen, E. M. (1999) Science 284, 1354-1356) was rescued by the overexpression of TRRAP or hGCN5. Histone acetyltransferase hGCN5 activity appeared to be indispensable for coregulator complex function in both BRCA1-mediated gene regulation and DNA repair. Biochemical purification of the hGCN5/TRRAP-containing complex suggested that hGCN5/TRRAP formed a complex with hMSH2/hMSH6, presumably as a novel subclass of hGCN5/TRRAP-containing known TFTC (TBP-free TAF-containing)-type histone acetyltransferase complex (hTFTC, hPCAF, and hSTAGA) (Yanagisawa, J., Kitagawa, H., Yanagida, M., Wada, O., Ogawa, S., Nakagomi, M., Oishi, H., Yamamoto, Y., Nagasawa, H., McMahon, S. B., Cole, M. D., Tora, L., Takahashi, N., and Kato, S. (2002) Mol. Cell 9, 553-562). Unlike other subclasses, the isolated complex harbored a previously unknown combination of components including hMSH2 and hMSH6, major components of the BRCA1 genome surveillance repair complex (BASC). Thus, our results suggested that the multiple BRCA1 functions require a novel hGCN5/TRRAP histone acetyltransferase complex subclass.

Molecular profiles of BRCA1-mutated and matched sporadic breast tumours: relation with clinico-pathological features

About 5–10% of breast cancers are hereditary; a genetically and clinically heterogeneous disease in which several susceptibility genes, including BRCA1, have been identified. While distinct tumour features can be used to estimate the likelihood that a breast tumour is caused by a BRCA1 germline mutation it is not yet possible to categorize a BRCA1 mutated tumour. The aim of the present study is to molecularly classify BRCA1 mutated breast cancers by resolving gene expression patterns of BRCA1 and matched sporadic surgical breast tumour specimens. The expression profiles of 6 frozen breast tumour tissues with a proven BRCA1 gene mutation were weighed against those from 12 patients without a known family history but who had similar clinico-pathological characteristics. In addition two fibroblast cultures, the breast cancer cell-line HCC1937 and its corresponding B-lymphoblastoid cell line (heterozygous for mutation BRCA1 5382insC) and an epithelial ovarian cancer cell line (A2780) were studied. Using a high density membrane based array for screening of RNA isolated from these samples and standard algorithms and software, we were able to distinguish subgroups of sporadic cases and a group consisting mainly of BRCA1-mutated breast tumours. Furthermore this pilot analysis revealed a gene cluster that differentially expressed genes related to cell substrate formation, adhesion, migration and cell organization in BRCA1-mutated tumours compared to sporadic breast tumours. © 2001 Cancer Research Campaign http://www.bjcancer.com