Breast Cancer Cell Lines BT474 Research Articles

Abstract 4079: Reversal of immune evasion mediated by HER2 requires both humoral and cellular HER-2 targeted immune interventions

Abstract Objectives: HER2 over-expression in breast cancer is associated with a poor prognosis and HER2 is an intense target of immunotherapy with both humoral and T cell -base approaches. HER2 expression is also known to facilitate the escape of tumor cells from immune surveillance by down-regulating MHC class I, resulting in a reduced sensitivity to CTL lysis. Restoring tumor cell MHC Class I expression and reversing their immune evasion are therefore important strategies in the design of immunotherapy against HER2 expressing tumors. In this study we investigated the effects of monoclonal antibody trastuzumab and T help-1 associated cytokines IFN-γ and TNF-A on HLA Class I expression and the susceptibilities to CTL lysis of human HER2 expressing tumor cell lines. Methods: Breast cancer cell lines MCF7, SKBR3, BT474 and ovarian cancer cell line SKOV3 were treated with trastuzumab only, combination of IFN-γ and TNF-A or trastuzumab with IFN-γ and TNF-A. Cell surface expressions of HLA ABC were examined by flow cytometric analysis. The susceptibility of tumor cells to HER2 specific CD8+ T cell recognition and CTL lysis were assessed by measurement of CD8+ T cell IFN-γ secretion and flow cytometric analysis respectively. Results: Th1 cytokines IFN-γ and TNF-A dramatically increased HLA ABC expressions on all tumor lines compared to untreated cells, [ MCF7 (p=0.04); SKBR3 (p=0.02); SCOV3 (p=0.02) and BT474 (p=0.04)], while trastuzumab alone showed little impact on HLA ABC up-regulation. When treated with a combination of trastuzumab, IFN-γ and TNF-A tumor cells displayed synergistically enhanced HLA ABC expression [MCF7 (p=0.02); SKBR3 (p=0.004); SKOV3 (0.006) and BT474 (p=0.03)]. We observed that IFN-γ and TNF-A treatment was able to remarkably increase CD8+ T cell recognition and CTL lysis of low and intermediate HER2 expressing tumors [ MCF7(p=0.03) and SKOV3(0.02)], but not High HER2 tumor[ SKBR3(p=0.07)]. Only a combined treatment of trastuzumab, IFN-γ and TNF-A rendered SKBR3 susceptible to a significant killing by CTL (p=0.02). To investigate the impact of other HER family members EGFR and HER3 on MHC class I expression , HER2 over-expressing breast cancer cell lines BT474 and SKBR3 were pre-treated with EGF or Heregulin, then subjected to IFN-γ and TNF-A treatments. Activation of HER1 and HER3 signaling made HER2 over-expressing cells resistant to the effect of MHC class I up-regulation by IFN-γ and TNF-A. Conclusion: Both humoral and cellular immune HER-2 targeted interventions are required to reverse immune escape mediated by HER2; in addition to targeting HER2, targeting HER1 and HER3 signaling elements should also be included in order to abrogate the cross talk between HER family members and more effectively inhibit the HER2 signaling pathway. Citation Format: Shuwen Xu, Jessica Cintolo, Jashodeep Datta, Cinthia Rosemblit, Erik Berk, Julia Terhune, Elizabeth Fitzpatrick, Brian Czerniecki. Reversal of immune evasion mediated by HER2 requires both humoral and cellular HER-2 targeted immune interventions. [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 4079. doi:10.1158/1538-7445.AM2014-4079

Abstract 5469: Trastuzumab resistant HER2+ breast cancer cells retain sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition

Abstract Background: Overexpression of HER2 in breast cancer is associated with aggressive tumor features and poor patient survival. Current therapeutic agents targeting this receptor have increased the survival rate of patients with HER2+ breast cancer; however some patients eventually develop resistance to these therapies. We have previously reported that HER2+ breast cancers are susceptible to poly (ADP-Ribose) polymerase inhibitors (PARPi) alone independent of a basal or induced DNA repair deficiency but instead via suppression of NF-κB signaling. In this study, we investigated PARPi sensitivity in HER2+ breast cancer cells that are resistant to the HER2 targeted agent trastuzumab. Materials and Methods: HER2+ breast cancer cell lines BT-474, UACC812, SKBRR3 and their trastuzumab resistant counterparts were used in this study. Cells were treated with vehicle or 10µM ABT-888 or transfected with scrambled or PARP1 siRNA. NF-κB transcriptional activity was measured using a NF-κB-driven luciferase reporter assay. Protein expression was measured by Western blot analysis. Cellular cytotoxicity and viability following PARPi was assessed with colony formation and ATPlite assays, respectively. Results: HER2+ parent and trastuzumab resistant breast cancer cells showed similar susceptibility to the PARPi ABT-888. There was a dose response reduction in survival fraction (greater than 70% at 10μM) and a greater than 40% decrease in cell viability with 10μM ABT-888. This cytotoxicity was associated with more than 50% attenuation of NF-κB transcriptional activity and reduced expression of the NF-κB activator IKKα. Suppression of NF-κB signaling was also observed utilizing PARP1 siRNA. Conclusions: HER2+ breast cancer cells resistant to trastuzumab continue to be sensitive to PARP inhibition through attenuation of the NF-κB signaling pathway. These results support the use of PARPi as part of a therapeutic strategy for patients with HER2+ breast cancer. Citation Format: Monica E. Wielgos, Tiffiny Cooper, Andres Forero, James A. Bonner, Francisco J. Esteva, C K. Osborne, Rachel Schiff, Albert F. LoBuglio, Eddy S. Yang. Trastuzumab resistant HER2+ breast cancer cells retain sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition. [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 5469. doi:10.1158/1538-7445.AM2014-5469

Abstract 1971: Inhibition of CXCR4 pathway augments trastuzumab sensitivity in HER2 positive breast cancer cells with intrinsic and acquired trastuzumab resistance

Abstract The clinical benefit from trastuzumab (T) in HER2-positive breast cancers is limited by de novo or acquired resistance. By serial exposure of HER2+ breast cancer cell lines BT474 and SKBr3 to T at 200 µg/ml for 1 year and clonogenic selection, we have developed T-resistant cell lines and identified differentially expressing genes including consistent overexpression of CXCR4 at the mRNA and protein levels. CXCR4 is involved in breast cancer metastasis and proliferation and its overexpression in human breast cancer specimens is linked to poor prognosis. We sought to functionally validate CXCR4 as a resistance mechanism to T. CXCR4 mRNA was quantified using qRT-PCR, and CXCR4 protein expression was quantified by Western blotting. Stable clones overexpressing CXCR4 were selected by transfecting CXCR4 full length sequence-containing lentivirus into parental trastuzumab sensitive BT474 and SKBR3 cells. Stable CXCR4 knockdown clones were selected by transfecting three CXCR4 sequence specific shRNA containing lentivirus along with scrambled sequence as a control, into acquired T-resistant cell lines termed BT474R1 and SKBR3R1 and intrinsically T-resistant HCC-1419 cell line. Stable clones of BT474R1 with tetracycline controlled (tet-on) CXCR4 expression, were created using the pSLIK-hygro lentiviral system. CXCR4 expression was assessed by Western blot assay and by flow cytometry. MTT cell viability analysis was performed on these transfected cell lines to characterize T dose responsiveness with parental sensitive and T-resistant BT474R1 cells as control. BT474R cells demonstrated a 100-fold increase in IC50 to T treatment when compared to parental BT474 cells and also exhibited a 4-fold increase in CXCR4 mRNA and a corresponding increase in CXCR4 protein. Transfection of CXCR4 into sensitive BT474 and SkBR3 cell lines showed a greater than 100 fold increase in the IC50 value by MTT assay after treatment with T as compared to the parental cell lines. Semi-quantitative analysis by Western-blotting confirmed a 7 to 8 fold increase in CXCR4 expression in CXCR4-transfected cells as compared to parental cells. shRNA-mediated knockdown of CXCR4 in BT474R1 as well as primarily resistant HCC-1419 cell line showed a significantly improved responsiveness to trastuzumab with a 100 fold decrease in the IC50 value whereas a moderate response was seen in the SKBR3R1 cells. CXCR4 overexpression and activation of the CXCR4/CXCL-12 pathway contributes to trastuzumab resistance in human breast cancer cell lines. shRNA-mediated inhibition of the CXCR4 pathway in resistant cells significantly augments trastuzumab sensitivity. CXCR4 targeting may represent a therapeutic strategy to reverse de novo and acquired resistance and to improve the efficacy of trastuzumab in HER2-positive breast cancer. Citation Format: Arjun Mehta, Gloria Yang-Kolodji, Debu Tripathy. Inhibition of CXCR4 pathway augments trastuzumab sensitivity in HER2 positive breast cancer cells with intrinsic and acquired trastuzumab resistance. [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 1971. doi:10.1158/1538-7445.AM2014-1971

Taspase1 cleaves MLL1 to activate cyclin E for HER2/neu breast tumorigenesis.

Taspase1, a highly conserved threonine protease, cleaves nuclear transcriptional regulators mixed-lineage leukemia (MLL, MLL1), MLL2, TFIIA, and ALF to orchestrate a wide variety of biological processes. In vitro studies thus far demonstrated that Taspase1 plays important roles in the proliferation of various cancer cell lines, including HER2-positive breast cancer cells. To investigate the role of Taspase1 in breast tumorigenesis in vivo, we deleted Taspase1 from mouse mammary glands by generating MMTV-neu;MMTV-cre;Tasp1F/− mice. We demonstrate that initiation of MMTV-neu- but not MMTV-wnt-driven breast cancer is blocked in the absence of Taspase1. Importantly, Taspase1 loss alone neither impacts normal development nor pregnancy physiology of the mammary gland. In mammary glands Taspase1 deficiency abrogates MMTV-neu-induced cyclins E and A expression, thereby preventing tumorigenesis. The mechanisms were explored in HER2-positive breast cancer cell line BT474 and HER2-transformed MCF10A cells and validated using knockdown-resistant Taspase1. As Taspase1 was shown to cleave MLL which forms complexes with E2F transcription factors to regulate Cyclins E, A, and B expression in mouse embryonic fibroblasts (MEFs), we investigated whether the cleavage of MLL by Taspase1 constitutes an essential in vivo axis for HER2/neu-induced mammary tumorigenesis. To this end, we generated MMTV-neu;MLLnc/nc transgenic mice that carry homozygous non-cleavable MLL alleles. Remarkably, these mice are also protected from HER2/neu-driven breast tumorigenesis. Hence, MLL is the primary Taspase1 substrate whose cleavage is required for MMTV-neu-induced tumor formation. As Taspase1 plays critical roles in breast cancer pathology, it may serve as a therapeutic target for HER2-positive human breast cancer.

Abstract 3142: Derivation and analysis of preclinical models of human her-2 positive breast cancer

Abstract Although a number of Her-2 targeting drugs are now available for the treatment of Her-2 positive breast cancers, resistance to these therapies rapidly emerges. There is therefore a need to study how Her-2 targeting strategies work in vivo, and the mechanisms by which tumors eventually become resistant to anti-Her-2 therapies. We report the in vivo selection of variants of the human Her-2 positive breast cancer cell lines BT474 and MDA-MB361. Both cell lines were initially implanted orthotopically into Severely Compromised Immunodeficient mice, and the resulting tumors were serially passaged into new hosts over a 4 year period. This selection process produced the BT474V3 and MDA361V3 variants, which grow rapidly in vivo and which retain their Her-2 over-expression, and which respond (p<0.05, relative to controls) to trastuzumab (20mg/kg i.p. every 3 days). Tumor variants were also derived from selected tumors that relapsed after 3 months of continuous trastuzumab monotherapy. MDA361V3 tumors showed a significant (p<0.05) response to the combination therapy of trastuzumab plus metronomic cyclophosphamide (20mg/kg/day, p.o.). Tumors eventually progressed under this combination therapy, and cell lines were derived from the drug resistant tumors. Non-invasive monitoring of the Her-2 positive models in vivo was achieved by luciferase transfection of the tumor variants, or by transfection of chorionic gonadotropin (hCG) cDNA which allows for relative tumor growth to be evaluated via the resulting hCG levels in the mouse urine. Luciferase transfected BT474V3 were implanted intracranially (coordinates for implantation from Bregma were AP, +1.1; LM, -2.0; and DV, -3), and bioluminescence was detectable 20 days later. The implanted tumors in the brain eventually showed a doubling of bioluminescence every 2 weeks, which provides ample time for therapeutic intervention in this model of Her-2 positive breast cancer brain metastasis. To further model the response of Her-2 positive metastatic breast cancer, we employed our met2.hCG model, derived from the human MDA-MB-231 human breast cancer cell line. Thus, MDA-MB-231 were transduced to overexpress Her-2, then tagged with hCG, and then selected for high spontaneous metastatic capacity. Orthotopically implanted met2.hCG tumors responded to trastuzumab as a monotherapy, and when given in combination with metronomic cyclophosphamide. Following surgical removal of the orthotopically implanted met2.hCG tumors, urine hCG levels indicated that the metastases also responded (p<0.05, compared to saline treated controls) to the combination of metronomic cyclophosphamide plus trastuzumab, but they did not respond to the trastuzumab monotherapy. Collectively, our data shows that we have derived models of Her-2 positive breast cancer that can be used to evaluate anti-Her-2 therapeutic strategies, and to study the emergence of resistance to anti-Her-2 based therapies. Citation Format: Paloma A. Valenzuela, Sarah N. Jallad, Karla Parra, Natzidielly Lerma, Irving Miramontes, Alejandra Gallegos, Ping Xu, William Cruz-Munoz, Shan Man, Robert S. Kerbel, Giulio Francia. Derivation and analysis of preclinical models of human her-2 positive breast cancer. [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 3142. doi:10.1158/1538-7445.AM2014-3142

Abstract 4338: The lipogenic phenotype of HER2/neu-positive breast cancer cells

Abstract 15 - 30 % of all breast cancer cases are classified as HER2/neu-positive, a very aggressive form of breast cancer that correlates with a poor prognosis, reduced survival time and increased incidence of metastasis. We have shown that the HER2-positive breast cancer cell line BT474 relies on a unique Warburg-like metabolism for survival and aggressive behavior. These cells are dependent on fatty acid synthesis, presumably as a means to regenerate NAD+ for aerobic glycolysis. The cells show markedly increased levels of stored fats compared to other breast cancer cell lines and disruption of the synthetic process results in apoptosis. This metabolic phenotype is operating at its limits in HER2-positive cells, as addition of exogenous palmitate results in cell death, whereas HER2-normal cells are not affected by this treatment. Microarray analysis of palmitate-treated HER2/neu-positive SKBR3 cells and HER2-normal MCF7 cells suggests that palmitate induces an ER-stress response in the HER2-positive cells. This is congruent with protein markers for stress-related translational stalling, which ultimately result in CHOP-dependent cell death. Palmitate distinctly reduces the expression of the HER3 protein and combination treatments with palmitate and HER2-targeted therapeutic compounds show synergistic effects and markedly increase the cytotoxic effects of either compound alone. In addition to these translational changes, palmitate treatment of HER2/neu-positive SKBR3 cells results in AMPK activation and subsequent inhibition of fatty acid synthesis. Transcriptome and global metabolite profiling analyses of the effect of increased palmitate on these cells indicate that palmitate alters the metabolic phenotype by re-routing the glucose carbon flux in the cells. Supported by CDMRP Award W81XWH-11-1-0094 to Jan Baumann and NCI Award 5R01CA136658-03 to Douglas Conklin. Citation Format: Jan M. Baumann, Douglas S. Conklin. The lipogenic phenotype of HER2/neu-positive breast cancer cells. [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 4338. doi:10.1158/1538-7445.AM2014-4338

Abstract 4870: Proteomics of phyllodes tumor revealed that decorin increase in the extracellular matrix by periostin deficiency decreased cancer cell motility and invasion

Abstract The ability of cancer cells to metastasize is dependent on the interactions of their cell surface molecules with microenvironment. However the tumor microenvironment, especially cancer-associated stroma, is poorly understood. To search for proteins which are present in the stroma, we investigated the phyllodes tumor, which contains breast stromal tissue, specific expression proteins compared with normal tissue by using iTRAQ-based quantitative proteomic approach. Periostin and versican core protein were up-regulated in phyllodes tumor. Decorin, mimecan, hemoglobin subunit alpha, hemoglobin subunit beta and ketatin, type1 cytoskeletal 19 were up-regulated in normal tissue. Periostin and decorin are one of component of the extracellular matrix. Periostin upregulation has been reported in many cancer types and is consequently defined as a tumor-enhancing factor. On the other hand, decorin upregulation inhibits tumor growth by antagonizing tumor angiogenesis. Periostin upregulation in phyllodes tumor and decorin upregulation in normal tissue was validated by immunohistochemical analysis from phyllodes tumor thirty five patients. We determined the interaction between decorin and periostin in phyllodes tumor tissues and breast cancer cell line BT-20 cells, by using immunoprecipitation and mass spectrometry analysis. Furthermore, we uncovered the plasma membrane translocation of decorin from cytoplasm in BT-20 or T-47D cells by siRNA-mediated knockdown of periostin, and detected the secreted decorin to the extracellular medium using Multiple Reaction Monitoring (MRM) method. The periostin siRNA-treated BT-20 cells motility and invasion were prevented by the secreted decorin into the extracellular medium. The overexpression of decorin suppressed the metastatic breast cancer cell line, MDA-MB-231 cells motality and invasion. These results demonstrated the secreted decorin by periostin deficiency decreased cancer cells motality and invasion. This molecular mechanism may become the new target for anti-cancer therapy. Citation Format: Toshiyuki Ishiba, Akira Nakanishi, Takanobu Sato, Tsuyoshi Nakagawa, Makoto Nagahara, Ryo Oono, Hiroyuki Uetake, Kenichi Sugihara, Yoshio Miki. Proteomics of phyllodes tumor revealed that decorin increase in the extracellular matrix by periostin deficiency decreased cancer cell motility and invasion. [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 4870. doi:10.1158/1538-7445.AM2014-4870

Abstract 532: Evaluation of the role of miRNA-4728-3p, a miRNA located in an intron of HER2 gene, in breast cancer stem cells

Abstract Background: Cancer stem cells (CSCs) contribute to tumor metastasis, chemotherapy resistance, and are highly tumorigenic when injected into immunodeficient mice. High levels of aldehyde dehydrogenase (ALDH1) are characteristic of stem cells and allow the isolation of subpopulations of CSCs, including those derived from breast cancer. HER2+ tumors, which are more aggressive than tumors with no amplification of this oncogene, are enriched in CSCs, a finding that suggests the participation of this oncogene in the generation and maintenance of CSCs. miR-4728-3p is a poorly investigated miRNA, which is located inside an intron of HER2. Its expression is increased in HER2+ tumors and cell lines, but its role in CSC formation and tumor behavior has not been investigated. Aim: to contribute to the better understanding of the role of miRNA-4728-3p in the biology of CSCs in HER2+ breast cancer cell lines. Methods: miR-4728-3p levels were evaluated in breast cancer cell lines MCF-7, BT-474 and SKBR3 grown in adherent conditions and in conditions that favor the formation of mammospheres for 7, 14, 21 and 28 days. Overexpression of miR-4728-3p was performed using lentiviral transduction with plasmids for the overexpression of this miRNA. Flow cytometry was performed to analyze ALDH1 expression in adherent cells and cells derived from mammospheres and also in cells overexpressing miRNA-4728-3p. Results: Mammospheres in culture for 7 days showed high levels of ALDH1 for all cells lines evaluated. The overexpression of miR-4728-3p induced a significant increment (>10-fold) of ALDH1+ subpopulations in MCF-7 and SKBR3, reinforcing its possible role in the biology of CSCs. Conclusion: HER2-amplification is an event that intrinsically leads to the amplification of miR-4728-3p. The effects of the amplification of this oncogene and this miRNA have to be evaluated together as both may have important consequences for tumor behavior and CSC formation. This increased expression of ALDH1 after miR-4728-3p overexpression suggest the importance of this miRNA in the biology of CSC formation. Note: This abstract was not presented at the meeting. Citation Format: Juliana Laino do Val Carneiro, Diana Noronha Nunes, Emmanuel Dias-Neto. Evaluation of the role of miRNA-4728-3p, a miRNA located in an intron of HER2 gene, in breast cancer stem cells. [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 532. doi:10.1158/1538-7445.AM2014-532

Radioprotective effects of Antrodia cinnamomea are enhanced on immune cells and inhibited on cancer cells

Purpose: The radioprotective effects of Antrodia cinnamomea (AC) were investigated for understanding the potential usefulness of AC as an adjunct treatment for reducing radiation side-effects.Materials and methods: In this study, we determined the ability of AC extracts (AC539) to reduce radiation side-effects by analyzing cellular viability in normal mouse spleen immune cells and human cancer cells with different radiosensitivity. We further detected the effect of AC on radiation-induced changes in cytokine- and inflammatory-related gene expressions. Furthermore, apoptosis assay was performed to determine whether AC could inhibit radiation-induced cytotoxicity.Results: We found that an AC dose of 100–150 μg/ml in a time-dependent manner was the most effective in blocking radiation-induced cytotoxicity, in vitro. Radiation-induced cytotoxicity was inhibited in spleen immune cells by 37–56%; however, pretreatment of human colorectal cancer cell line HT-29 with AC did not have any effect on radiation-induced cytotoxicity, while pretreatment of radiosensitive human breast cancer cell lines BT-474 with AC caused a moderate enhancement of radiation-induced damage. Furthermore, AC pretreatment differentially regulated the mRNA expression of several important immunomodulatory genes in response to irradiation in normal and cancer cells.Conclusions: Our data indicate that AC may inhibit important immunoregulatory signaling which could be vital in the avoidance of an over-activated cytotoxic and inflammatory response of the immune system caused by radiation-induced tissue damage. Additionally, AC does not provide a radioprotective effect to tumor cells but instead enhances radiation-induced inflammation and cytotoxicity in cancer.

Evaluation of functional genetic variants at 6q25.1 and risk of breast cancer in a Chinese population.

IntroductionSingle-nucleotide polymorphisms (SNPs) at 6q25.1 that are associated with breast cancer susceptibility have been identified in several genome-wide association studies (GWASs). However, the exact causal variants in this region have not been clarified.MethodsIn the present study, we genotyped six potentially functional single-nucleotide polymorphisms (SNPs) within the CCDC170 and ESR1 gene regions at 6q25.1 and accessed their associations with risk of breast cancer in a study of 1,064 cases and 1,073 cancer-free controls in Chinese women. The biological function of the risk variant was further evaluated by performing laboratory experiments.ResultsBreast cancer risk was significantly associated with three SNPs located at 6q25.1—rs9383935 in CCDC170 and rs2228480 and rs3798758 in ESR1—with variant allele attributed odds ratios (ORs) of 1.38 (95% confidence interval (CI): 1.20 to 1.57, P = 2.21 × 10-6), 0.84 (95% CI: 0.72 to 0.98, P = 0.025) and 1.19 (95% CI: 1.04 to 1.37, P = 0.013), respectively. The functional variant rs9383935 is in high linkage disequilibrium (LD) with GWAS-reported top-hit SNP (rs2046210), but only rs9383935 showed a strong independent effect in conditional regression analysis. The rs9383935 risk allele A showed decreased activity of reporter gene in both the MCF-7 and BT-474 breast cancer cell lines, which might be due to an altered binding capacity of miR-27a to the 3' untranslated region (3' UTR) sequence of CCDC170. Real-time quantitative reverse transcription PCR confirmed the correlation between rs9383935 genotypes and CCDC170 expression levels.ConclusionsThe results of this study suggest that the functional variant rs9383935, located at the 3' UTR of CCDC170, may be one candidate of the causal variants at 6q25.1 that modulate the risk of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0422-x) contains supplementary material, which is available to authorized users.

Lidocaine and ropivacaine, but not bupivacaine, demethylate deoxyribonucleic acid in breast cancer cells in vitro

Lidocaine demethylates deoxyribonucleic acid (DNA) in breast cancer cells. This modification of epigenetic information may be of therapeutic relevance in the perioperative period, because a decrease in methylation can reactivate tumour suppressor genes and inhibit tumour growth. The objectives of this study were to determine the effect of two amide local anaesthetics, ropivacaine and bupivacaine, on methylation in two breast cancer cell lines and to detect whether the combination of lidocaine with the chemotherapy agent 5-aza-2'-deoxycytidine (DAC) would result in additive demethylating effects. Breast cancer cell lines BT-20 [oestrogen receptor (ER)-negative] and MCF-7 (ER-positive) were incubated with lidocaine, bupivacaine, and ropivacaine to assess demethylating properties. Then, we tested varying concentrations of lidocaine and DAC to assess whether their demethylating effects were additive. Cell numbers and global methylation status were analysed. Lidocaine decreased methylation in BT-20 and MCF-7 cells, ropivacaine decreased methylation in BT-20 cells, and bupivacaine had no demethylating effect. When combined, lidocaine and DAC had additive demethylating effects. At clinically relevant doses, lidocaine and ropivacaine exert demethylating effects on specific breast cancer cell lines, but bupivacaine does not. The demethylating effects of lidocaine and DAC are indeed additive.

Boolean ErbB network reconstructions and perturbation simulations reveal individual drug response in different breast cancer cell lines.

BackgroundDespite promising progress in targeted breast cancer therapy, drug resistance remains challenging. The monoclonal antibody drugs trastuzumab and pertuzumab as well as the small molecule inhibitor erlotinib were designed to prevent ErbB-2 and ErbB-1 receptor induced deregulated protein signalling, contributing to tumour progression. The oncogenic potential of ErbB receptors unfolds in case of overexpression or mutations. Dimerisation with other receptors allows to bypass pathway blockades. Our intention is to reconstruct the ErbB network to reveal resistance mechanisms. We used longitudinal proteomic data of ErbB receptors and downstream targets in the ErbB-2 amplified breast cancer cell lines BT474, SKBR3 and HCC1954 treated with erlotinib, trastuzumab or pertuzumab, alone or combined, up to 60 minutes and 30 hours, respectively. In a Boolean modelling approach, signalling networks were reconstructed based on these data in a cell line and time course specific manner, including prior literature knowledge. Finally, we simulated network response to inhibitor combinations to detect signalling nodes reflecting growth inhibition.ResultsThe networks pointed to cell line specific activation patterns of the MAPK and PI3K pathway. In BT474, the PI3K signal route was favoured, while in SKBR3, novel edges highlighted MAPK signalling. In HCC1954, the inferred edges stimulated both pathways. For example, we uncovered feedback loops amplifying PI3K signalling, in line with the known trastuzumab resistance of this cell line. In the perturbation simulations on the short-term networks, we analysed ERK1/2, AKT and p70S6K. The results indicated a pathway specific drug response, driven by the type of growth factor stimulus. HCC1954 revealed an edgetic type of PIK3CA-mutation, contributing to trastuzumab inefficacy. Drug impact on the AKT and ERK1/2 signalling axes is mirrored by effects on RB and RPS6, relating to phenotypic events like cell growth or proliferation. Therefore, we additionally analysed RB and RPS6 in the long-term networks.ConclusionsWe derived protein interaction models for three breast cancer cell lines. Changes compared to the common reference network hint towards individual characteristics and potential drug resistance mechanisms. Simulation of perturbations were consistent with the experimental data, confirming our combined reverse and forward engineering approach as valuable for drug discovery and personalised medicine.

Role of CD24 in HER2-positive breast cancer.

e11572 Background: Human epidermal growth factor receptor 2 (HER2) is a transmembrane kinase receptor, which promotes the RAS/RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. The standard treatment for HER2-positive breast cancer is a combination of chemotherapy plus an anti-HER2 agent, such as trastuzumab and lapatinib. HER2-positive breast cancers are predominantly positive for CD24, which is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein. Overexpression of CD24 in breast cancer is associated with a poor prognosis. We hypothesize that the co-expression of HER2 with CD24 is associated with the therapeutic resistance of HER2-positive breast cancer cells. Methods: We established HER2-expressing cell lines by introducing a wild type HER2 gene into MDA-MB-231 triple-negative (ER-/PgR-/HER2-) breast cancer cells. We used the HER2-positive breast cancer cell lines BT-474, HCC202, SKBR3, and HCC1569. To investigate the relevance of CD24 expression in HER2-positive breast cancer, we analyzed HER2 and...

IL-7 splicing variant IL-7δ5 induces EMT and metastasis of human breast cancer cell lines MCF-7 and BT-20 through activation of PI3K/Akt pathway.

Our previous study has confirmed that IL-7δ5 (an IL-7 variant lacking exon 5) promotes breast cancer growth. However, whether IL-7δ5 is involved in tumor cell EMT and metastasis remains unclear. In this study, we investigated the preclinical effects and molecular mechanisms of IL-7δ5 on EMT and metastasis in human MCF-7 and BT-20 breast cancer cells in vitro and in vivo. The results showed that IL-7δ5 induced EMT and invasion in tumor cells, associated with up-regulation of N-cadherin and the down-regulation of E-cadherin. Furthermore, we found that IL-7δ5 induced the activation of Akt. Inhibition of PI3K/Akt pathway by LY294002 reversed the EMT transition in breast cancer cell lines MCF-7 and BT-20 induced by IL-7δ5. In addition, IL-7δ5 enhanced cancer metastasis and shortened survival time, with increased level changes of activated Akt in nude mice with breast cancer. In conclusion, our findings demonstrate that IL-7δ5 induces human breast cancer cell lines EMT and metastasis via activation of PI3K/Akt pathway. Thus, IL-7δ5 may be a potential target against human breast cancer.

English

The seeds of flax (binomial name: Linum usitatissimum L.) are well known for their high content of phytoestrogens. In the present study, extracts from roots, leaves and stems of flax were analysed for their content of compounds, which might have phytoestrogen-like properties, by pyrolysis field ionisation mass spectrometry. All extracts were tested on the human breast cancer cell lines MCF7 (estrogen receptor positive) and BT20 (estrogen receptor negative). Specific tests were applied for cell vitality ((3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay (MTT) test), proliferation (BrdU test) and cytotoxicity (lactate dehydrogenase (LDH) test). In the flax root extract, the amounts of monolignols and polyphenols were three times higher than in the stem and leaf extracts. Even at higher concentrations, the root extract also was the least cytotoxic one of all extracts in MCF7 cells, while it showed a dose-dependent and much higher cytotoxicity in BT20 cells. Furthermore, at higher concentrations (> 100 µg/ml), the root extract reduced cell vitality in MCF7 significantly less than in BT20 cells and inhibited proliferation in MCF7 by up to 85%. Since flax root extracts induce significant inhibition of cell vitality and proliferation without performing strong cytotoxicity in the human mamma carcinoma cell lines MCF7, the potential phytoestrogens in flax root extracts could have beneficial effects in hormone-dependent tumours. Key words: Flax, Linum usitatissimum, phytoestrogens, MCF7, cell proliferation, breast cancer.

Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis

BackgroundThe MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism.ResultsA range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines.ConclusionsOur study provides a comprehensive and specific characterization of complex chromosomal aberrations of MCF7, T47D, BT474 and SKBR3 cell lines.The chromosomal pattern of ER+/HER2- cells is less complex than that of ER+/HER2+ and ER-/HER2+ cells. These chromosomal abnormalities could influence the biologic and pharmacologic response of cells. Finally, although gene expression profiling and aCGH studies have classified these four cell lines as luminal, our results suggest that they are heterogeneous at the cytogenetic level.

S100A14, a Member of the EF-hand Calcium-binding Proteins, Is Overexpressed in Breast Cancer and Acts as a Modulator of HER2 Signaling

HER2 is overexpressed in 20–25% of breast cancers. Overexpression of HER2 is an adverse prognostic factor and correlates with decreased patient survival. HER2 stimulates breast tumorigenesis via a number of intracellular signaling molecules, including PI3K/AKT and MAPK/ERK.S100A14,one member of the S100 protein family, is significantly associated with outcome of breast cancer patients. Here, for the first time, we show that S100A14 and HER2 are coexpressed in invasive breast cancer specimens,andthere is a significant correlation between the expression levels of the two proteins by immunohistochemistry. S100A14 and HER2 are colocalized in plasma membrane of breast cancer tissue cells and breast cancer cell lines BT474 and SK-BR3. We demonstrate that S100A14 binds directly to HER2 by co-immunoprecipitation and pull-down assays. Further study shows that residues 956–1154 of the HER2 intracellular domain and residue 83 of S100A14 are essential for the two proteins binding.Moreover,we observe a decrease of HER2 phosphorylation, downstream signaling, and HER2-stimulated cell proliferation in S100A14-silenced MCF-7, BT474, and SK-BR3 cells. Our findings suggest that S100A14 functions as a modulator of HER2 signaling and provide mechanistic evidence for its role in breast cancer progression.

Synthesis of 5-arylidine amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamides endowed with potent antioxidants and anticancer activity induces growth inhibition in HEK293, BT474 and NCI-H226 cells

A series of imines 5-amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamide derivatives were synthesized from various aromatic aldehydes and substituted with benzoyl acetazolamides under different reaction conditions and were evaluated for their antioxidant and free radical scavenging, antimitotic activity by Allium cepa meristem root model and cytotoxicity activity against HEK 293 (human epidermal kidney cell line), BT474 (breast cancer cell line) and NCI-H226 (lung cancer cell line) by MTT assay. Some of the synthesized compounds showed moderately potent cytotoxicity compared to indisulam. Graphical abstractA series of imines 5-amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamide derivatives (9a–j); 5-amino-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (4a–g); 5-(4-acetamido phenyl sulphonamido)-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (6a–g); and 5-(4-amino phenyl sulphonamido)-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (7a–g) were synthesized from acetazolamide and were investigated for the in vitro anticancer by MTT assay, free radical scavenging and antimitotic activity by Allium cepa root meristem model. Experimental observations indicate that synthesized compounds were moderately potent anticancer agents.

In Vitro Assessment of the Effects of Anti-HER2 Monoclonal Antibodies on Proliferation of HER2-Overexpressing Breast Cancer Cells

HER2 proto-oncogene is critical in the biology of breast cancer and an important therapeutic target of monoclonal antibodies (mAbs). We have recently established a panel of anti-HER2 mAbs recognizing different epitopes within the extracellular domain of HER2. In the present study the antiproliferative effect of these mAbs was investigated on HER2-overexpressing human breast cancer cell line BT474, using radioactive thymidine incorporation assay. Our results demonstrated that while two of the mAbs (1T0 and 2A8) inhibited cell proliferation dose dependently, similar to trastuzumab, six mAbs (1F2, 1B5, 1H9, 4C7, 1H6 and 2A9) augmented cell proliferation. Treatment of BT474 cells with different combinations of two mAbs induced either synergistic inhibitory or stimulatory effects. Our findings indicate that combination of some stimulatory mAbs could completely abolish the inhibitory effect of other mAbs against HER2. Employment of some combinations of mAbs with significant synergistic inhibitory function may improve the therapeutic efficacy of HER2-specific mAbs.

Abstract P5-08-03: In vitro resistance to ERBB2-targeted therapies does not bestow cross-resistance to antibody dependent cellular cytotoxicity

Abstract Background: The advent of ERBB2-directed therapies, including trastuzumab and lapatinib, has changed the clinical management of ERBB2+ breast cancer and improved patient outcomes. However, both de novo and acquired resistance to ERBB2-targeting agents remain significant clinical problems; thus, it is important to understand the mechanisms that tumor cells use to resist and/ or escape ERBB2-directed therapeutic agents. A growing body of evidence supports the role of immune effector cells in trastuzumab-induced tumor cell lysis in vivo, which is hypothesized to occur through antibody dependent cellular cytotoxicity (ADCC) mediated by natural killer cells bearing FC gamma receptors. Existing in vitro models of trastuzumab and lapatinib resistance are limited in that they are most often generated by incubating tumor cells with drug alone, outside the context of the host immune system. Consequently, the impact of in vitro generated drug resistance on ADCC susceptibility has not been defined. In this study, we tested existing trastuzumab and lapatinib resistant breast cancer cell lines for ADCC sensitivity. Materials and Methods: The ERBB2 amplified breast cancer cell lines BT474 and SKBR3, which are sensitive to trastuzumab-mediated ADCC, were selected for trastuzumab and lapatinib resistance. Drug resistant cells were generated by serial culture in the presence of escalating doses of trastuzumab and/or lapatinib until complete tolerance was acquired at clinically achievable concentrations (100μg/ml trastuzumab and 2.6μM lapatinib). Drug sensitivity was assessed by Alamar Blue assay. Cell resistance to trastuzumab-mediated ADCC was measured by Calcein-AM release assay, using 1μg/ml antibody concentration and four different effector target ratios (20:1, 10:1, 5:1, and 1:1), with a minimum of five replicates. Human peripheral blood lymphocytes freshly isolated from buffy coat were used as effector cells. Results and Discussion: Neither BT474 nor SKBR3 cells with acquired resistance to single agent trastuzumab (HerR), lapatinib (LapR), or both in combination (DualR) demonstrated cross-resistance to effector cell-mediated ADCC at any of the E:T ratios tested. At the highest E:T ratio tested (20:1), trastuzumab-induced ADCC of control BT474 cells resulted in 61.92% cell lysis (95% CI = 3.97), compared to 64.97% lysis for BT-HerR cells (95% CI = 2.87, Student's t-test p > 0.1). Similarly, BT-LapR and DualR cell lysis was 58.13% and 63.66%, respectively (95% CI = 2.62 and 6.57, Student's t-test p > 0.1). Additionally, trastuzumab-induced ADCC of control SKBR3 cells resulted in 89.98% cell lysis (95% CI = 4.29), compared to 93.53% lysis for SK-HerR cells (95% CI = 0.81, Student's t-test p > 0.1). SK-LapR and DualR cell lysis was 94.81% and 92.09%, respectively (95% CI = 0.6 and 2.66, Student's t-test p > 0.1). Our results indicate the importance of incorporating immune effector cells to in vitro models of trastuzumab resistance. Accordingly, we have begun modeling ADCC resistance in vitro to further investigate the relationship between targeted antibody therapies, effector cell-mediated cytolysis, and mechanisms of tumor immune escape. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-03.