Breast Cancer Cell Lines BT474 Research Articles

ATRA-hydrazonate derivatives and their copper complexes against hormone-dependent (MCF-7), hormone-independent (MDA-MB-231and BT-20) breast cancer and androgen-independent (PC3) prostate cancer cell lines

Abstract Retinoids, the derivatives of vitamin A, are known to exhibit anticancer properties through binding to nuclear retinoic acid receptors (RARs). However, several factors such as high lipophilicity, short half life, associated toxicity and appearance of resistant cells during differentiation therapy has limited their therapeutic potential. Here we report synthesis of aryl/heterocyclic analogs of retinoid trans-2-octenal with and without copper complexation. The biological activity of these novel synthetic compounds was tested against multiple human cancer cell lines: breast cancer cell lines MCF-7, BT-20, MDA-MB-231 and prostate cancer cell line PC-3, all of which differ in their expression of RAR subunits. The retinoid-derivatives were found to be effective in inhibiting the growth of all the cell lines, particularly those that express RAR α. Synthesis of hydrazonate analogs of retinoic acid and their copper conjugation is therefore an effective strategy to design novel anticancer compounds.

Susceptibility of HER2+ breast cancer cells to poly (ADP-ribose) polymerase (PARP) inhibition independent of an inherent DNA repair defect.

621 Background: HER2 overexpression in breast cancer confers increased tumor aggressiveness. Targeted therapy against HER2 has improved outcomes, but resistance and disease progression ultimately occurs, thus necessitating novel therapeutic strategies. PARP inhibitors target homologous recombination (HR) deficient tumors, such as the BRCA-associated breast and ovarian cancers. In this study, we report unexpected susceptibility of HER2+ breast cancer cells to PARP inhibition alone independent of an inherent HR deficiency. Methods: Cell viability was measured using colony formation and ATPLite assays. Tumor growth delay was assessed in vivo in mice bearing breast cancer xenografts. Proteins were detected by immunoblotting. HR was assayed using radiation (IR)-induced Rad51 foci or a GFP-based HR assay. NFκB activity was measured using a NFκB-driven luciferase assay. Results: Surprisingly, PARP inhibition with ABT-888 alone reduced the colony forming ability and cell viability of the HER2+ breast cancer cell lines BT474, SKBR3, MDA-MB361, and HCC1954 (~70 – 99% reduction at 10μM). This susceptibility did not correlate with an inherent HR deficit. Interestingly, HER2 overexpression itself may be one mechanism of susceptibility to ABT-888 as evidenced by increased cellular cytotoxicity and in vivo tumor growth delay of MCF7 cells stably expressing HER2. Further dissection of the mechanism revealed that NFκB transcriptional activity was significantly inhibited by ABT-888 (>95%) which corresponded with reduced levels of phosphorylated p65 and total IKKα, and a concomitant increase in IkBα. Furthermore, overexpression of p65 abrogated cellular sensitivity to ABT-888. Conclusions: HER2+ breast cancer cells are highly susceptible to PARP inhibition despite being HR proficient. This may be, in part, due to inhibition of NFκB. Further investigation of whether the addition of PARP inhibition to HER2 targeted therapy will delay the onset of resistance to therapy is warranted to potentially improve outcomes in HER2+ breast cancer patients.

Evaluation of the clinical utility of kallikrein-related peptidase 6 gene (KLK6) downregulation in breast cancer.

10606 Background: Breast cancer (BC), the most common malignancy among the female population, remains a crucial public health problem. The identification and exploitation of novel molecular biomarkers can contribute in the multidimensional approach which is required for optimal management of BC patients. The abnormal expression of several KLK members has been documented for breast cancer. Interestingly, many of these genes are found to be potential prognostic markers for this malignancy. KLK6 expression is positively associated with tumor progression in various human malignancies; however in breast cancer, it can restrain tumor progression. We therefore sought to investigate the possible clinical value of KLK6 as a breast cancer biomarker. Methods: Total RNA was isolated from 107 breast tumors and their matched normal compartments. After testing the quality of the extracted RNA, cDNA was prepared by reverse transcription. Quantitative Real-time PCR was performed, for KLK6 mRNA quantification, using the SYBR Green chemistry. Relative quantification analysis was made using the comparative Ct (2-ΔΔCT) method. HPRT1 was used as an endogenous control gene and BT-474 breast cancer cell line served as a calibrator. Results: KLK6 mRNA levels were significantly downregulated in the cancerous breast tissue specimens compared to their normal counterparts (p<0.001). Additionally, a negative association was observed between KLK6 expression status and tumor stage, given the fact that 54.2% of less advanced breast tumors (TNM stage≤ T2a) were KLK6-positive compared to the 34.6% of more advanced-stage tumors (TNM >T2a) (p=0.034). Moreover, a statistically significant negative correlation was documented between KLK6 mRNA levels and estrogen (p<0.001) and progesterone receptor (p=0.003) statuses. Conclusions: The downregulation of KLK6 in cancerous compared to normal sections of breast tissue samples reflects the reported tumor suppressor properties of KLK6 gene in breast malignancies. Furthermore, the negative association of KLK6 mRNA expression with tumor stage, ER and PR statuses reveals the putative role of KLK6 as a promising prognostic breast cancer biomarker.

Dysadherin expression promotes the motility and survival of human breast cancer cells by AKT activation

High dysadherin expression has been recognized as a biological predictor of metastasis and poor prognosis for many different cancer types; however, the molecular mechanisms of how dysadherin affects cancer progression are still poorly understood. In this study, we examined whether AKT signaling could link dysadherin expression with downstream events that promote the metastatic potential of human breast cancer cells. Immunohistochemical analysis of breast cancer tissues showed that dysadherin expression was highly associated with elevated expression of phospho-AKT. The introduction of dysadherin cDNA into BT-474, MCF-7 and T-47D breast cancer cell lines enhanced their levels of AKT phosphorylation, while knockdown of dysadherin in MDA-MB-231 and Hs578T breast cancer cell lines suppressed AKT phosphorylation. Treatment with the AKT inhibitor triciribine suppressed dysadherin-mediated pro-metastatic effects, including epithelial-mesenchymal transition, cell motility and drug resistance. These findings suggest that dysadherin might contribute to breast cancer progression through AKT activation.

Regulation of HRG-β1-induced proliferation, migration and invasion of MCF-7 cells by upregulation of GPR30 expression

The cooperation and communication between different cell signaling transduction pathways are considered critical in the development of various types of cancer as well as drug resistance. There is evidence of crosstalk between the G protein-coupled receptor 30 (GPR30), the newly discovered estrogen receptor (ER), and the ErbB family. Heregulin (HRG)-β1, the ligand for ErbB3 and ErbB4, upregulates GPR30 expression in MCF-7, T-47D and BT-474 breast cancer cell lines that express ERα. In the present study, recombinant human HRG-β1 was used to investigate the upregulation of GPR30 expression by HRGs in MCF-7 breast cancer cells which were ERα-positive. In MCF-7 cells, the ErbB2 inhibitor, AG825, the MAPK inhibitor, PD98059, and the MEK1/2 inhibitor, U0126, blocked the HRG-β1-induced GPR30 expression. 17-β-estradiol (E2) boosted the HRG-β1-induced proliferation, migration and invasion of MCF-7 cells. Similar to E2, the specific GPR30 agonist, G-1, promoted HRG-β1-induced migration and invasion, but inhibited growth. Using the specific GPR30 antagonist, G-15, or the small interfering RNA for GPR30, the functions of GPR30 after treatment with HRG-β1 were further investegated. The results from our study indicate that the interruption between GPR30 signaling and the ErbB family system may serve as a promising therapeutic strategy for breast cancer.

Abstract 2163: RSK signaling in HER2-positive breast cancer cells

Abstract Heregulin (HRG) is a soluble growth factor involved in the control of proliferation, survival, invasion, and differentiation of normal and malignant mammary epithelial cells. HRG binds its receptor, HER3, leading to activation of the HER2-HER3 heterodimer, which acts as an oncogenic factor in breast cancers. Pharmacological inhibitors to HER2 are currently being used in the clinic to treat HER2-positive breast cancer, however, they are associated with resistance after one year of therapy. A more comprehensive understanding of the signaling network downstream of HRG, in HER2-positive breast cancer cells, will be crucial for further development of inhibitors that do not elicit resistance. This study assessed HRG signaling in the HER2-positive breast cancer cell lines BT474 and SKBR3. The RSK (p90 ribosomal S6 Kinase) pathway was activated in an ERK-dependent manner. HRG stimulation increased phosphorylation of RSK at S380, T359, and S363. HRG stimulated the phosphorylation of CREB (cAMP Response-Element Binding Protein), an established substrate of RSK, at S133 and this was blocked by pharmacological inhibition of RSK. HRG induced the expression of c-FOS in an RSK-dependent manner. These data demonstrate that RSK has an important role in transducing HRG signaling to the gene expression program and suggest that inhibitors of RSK may be useful in the treatment of HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2163. doi:1538-7445.AM2012-2163

Abstract 296: The expression and potential role of MUC7 in pancreatic adenocarcinoma

Abstract Human mucin 7 (MUC7) is the smallest of the mucin core proteins at 39 kD and its fully glycosylated form ranges from 125-250 kD. This secreted, non-gel forming mucin is normally expressed by the mucousal and serosal salivary glands and sub-mucosal glands in the lower respiratory tract. Recently MUC7 has been observed to be expressed in such diseases as allergic-asthma, COPD, cystic fibrosis and bladder cancer. The tandem repeat domain consists of 5-6 repeats of a 23 amino acid sequence, which contains 3 SEA domains which may serve as sites of auto-cleavage. Our immunohistochemical studies of pancreatic adenocarcionoma revealed that MUC7 was expressed in over 50% of primary tumors, liver metastases, and lymph node metastases. MUC7 was found to be primarily expressed by the immune cells in the lymph node metastases rather than tumor cells. In contrast, MUC7 was not expressed by infiltrating immune cells in primary tumors and liver metastases, but it was present in the tumors themselves. Further characterization of the immune cells expressing MUC7 in the lymph nodes (by means of immunofluorescence and flow cytometry) revealed that the majority were macrophages and myeloid-derived suppressor cells together with a small population of dendritic cells. MUC7 has been posited to play a role in inflammation. This function is likely due to a unique histatin-like domain allowing it to bind numerous microbes to promote their clearance.MUC7 has been shown to be upregulated by IL-1β, IL-4, IL-13, TNF-α, LPS, and EGF in inflammed airways, and its minimal proximal promoter contains binding sites for NF-κB, AP1 and STAT transcription factors; all of which are involved in promoting inflammation. We found that MUC7 expression was up-regulated in the pancreatic cancer cell line Hs766T and the breast cancer cell line BT474 upon incubation with Th1 cytokines IFN-γ, IL-1β and TNF-α and the Th2 cytokines IL-4, IL-10 and IL-13. MUC7 over-expression was achieved in a time-dependent manner when incubated with the Th1 cytokines; however immediate and sustained expression was observed when cells were treated with the Th2 cytokines. Global gene expression by microarray was evaluated on samples of primary and metastatic pancreatic adenocarcinoma that expressed or did not express MUC7 to identify candidate genes affected by MUC7 expression. The microarray data revealed that a number of genes involved in tissue remodeling and inflammation were differentially regulated by MUC7 expressing tumors. Together, the data from the analyses presented here led us to propose that MUC7 has a role in establishing a pro-tumorigenic microenvironment through enhancing the Th2 inflammatory response and aiding in tissue remodeling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 296. doi:1538-7445.AM2012-296

Abstract 2539: The PARP inhibitor AZD2281 induces autophagy and complete cell death in BRCA mutant breast cancer cell lines

Abstract BRCA1 and BRCA2 are well-known DNA repair proteins that are usually mutated or deleted in breast tumors. According to the literature, 5 (12%) of 41 breast cancer cell lines have BRCA mutations and 28 (68%) of the 41 cell lines have BRCA1 allelic loss. PARP inhibitors are known to inhibit tumor growth in BRCA mutant tumors. The purpose of this study was to investigate the effects of PARP inhibitors in BRCA mutant breast cancer cell lines. We found that AZD2281 as a single agent induced complete cell death in BRCA1 mutant (HCC-1937, MDA-MB-436, and SUM-149PT) and BRCA2 mutant (HCC-1428) cell lines. AZD2281 induced an average growth inhibition of 33% in BRCA wild-type cell lines with BRCA1 allelic loss. Downregulation of BRCA1 or BRCA2 by shRNA sensitized the triple-negative breast cancer cell lines BT-20 and MDA-MB-231 to AZD2281 treatment. We also investigated whether autophagy is induced in response to PARP inhibition. AZD2281 treatment induced LC3-II expression, an indication of autophagy. Transmission electron micrographs demonstrated autophagosome formation after AZD2281 treatment. On transmission electron micrographs, we observed a decrease in mitochondria related to autophagosome formation after AZD2281 treatment. To show AZD2281 induced autophagy is targeting mitochondria, we labeled mitochondria with yellow fluorescein protein using lentiviral stable transfection. Flow cytometry measurement demonstrated a decrease in fluorescein-labeled mitochondria after AZD2281 treatment. Inhibition of autophagy in a AZD2281-treated BRCA mutant breast cancer cell line rescued mitochondrial degradation and partially inhibited apoptosis. In conclusion, we showed that autophagy plays an important role in AZD2281-induced cell death in BRCA mutant breast cancer cell lines by degradation of mitochondria besides DNA degradation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2539. doi:1538-7445.AM2012-2539

Abstract LB-345: Differences in metformin transporter expression between breast tumor and breast cancer cell lines: selecting a relevant breast cancer cell model for metformin therapy

Abstract Purpose: Recent studies show that the anti-diabetic drug metformin induces significant anticancer effects in several cancers including breast cancer via activation of intracellular AMPK. Because of its hydrophilic nature and net positive charge at physiologic pHs, metformin requires cation-selective transporters such as OCT1-3, PMAT and MATE1-2 to enter cells and activate AMPK. Expression of these transporters in cancer cells would determine the intracellular concentrations of metformin that can be achieved for anticancer effects. Therefore, elucidating metformin transport in breast cancer is central to understanding optimum dosing and transporter-associated inter-subject variability in its anticancer efficacy. However, there is limited information on metformin transport in breast cancer. Studies in our laboratory suggest that cation-selective transporters are expressed at negligible levels in MCF-7 cells, a widely used in vitro breast cancer model, which is not consistent with the robust anticancer effect of metformin in xenograft mouse models and clinical studies. Therefore, this study aims at determining a) metformin transporter expression in human breast tumor, b) if metformin transporter expression in breast cancer cell lines is relevant to that in breast tumor tissue and c) there is differential metformin transporter expression in tumor and non-malignant breast tissue. Methods: Total RNA isolated from breast cancer cell lines MCF-7, SK-BR-3, MDA-MB-231, ZR-75-1, BT-474, MDA-MB-435S, MDA-MB-468, BT-20 and BT-549 and from 14 pairs of human breast tumor and adjacent non-malignant breast tissues was subjected to.real-time PCR to determine relative expression levels of OCT1-3, PMAT, MATE1 and 2. Results: Most cell lines expressed low levels of candidate cation-selective transporters. MDA-MB-468 and BT-549 showed high expression of MATE1 and MDA-MB-231 expressed high levels of OCT3 and MATE1. In tumor and non-malignant breast tissue, the predominant transporters were OCT3 and PMAT. Nine of the total 14 tumor samples showed downregulation of OCT3 by 85% and PMAT by 66% compared to non-malignant tissue. Conclusions: The expression profile of cation-selective transporters is different between tumor and normal breast tissue and between breast tumor tissue and cell lines. Therefore, one needs to use caution in choosing a relevant in vitro cell model for investigating anticancer efficacy of metformin whose uptake is transporter-mediated. Expression data suggest that OCT3 and PMAT may play a major role in metformin intracellular uptake in breast cancer. MDA-MB-231 may be a useful model to assess the metformin anticancer effects in breast cancer. Clearly, genetic variability in metformin transporters may lead to differential responses to metformin anticancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-345. doi:1538-7445.AM2012-LB-345

Abstract 1063: The ErbB3/4 receptor ligand Heregulin negatively regulates ErbB2 and ErbB3 receptor levels

Abstract ErbB2 is overexpressed in 30% of breast cancers and its overexpression is associated with poor prognosis. In addition, the ErbB2/3 heterodimer forms the most potent mitogenic receptor in vitro and is key to the proliferation of breast cancer cells. Despite the importance of this heterodimer in breast cancer progression, the negative regulation of these ErbB tyrosine kinases is still poorly understood. We demonstrate here for the first time that the ErbB3/4 ligand Heregulin (HRG) reduces mRNA and protein levels of both ErbB2 and ErbB3 in human breast cancer cell lines. We have found that after 24h of HRG treatment, ErbB2 expression decreased at both the RNA and protein levels in ErbB2 overexpressing AU565, BT474 and LTLT-Ca breast cancer cell lines. In contrast, EGFR levels were unaffected by HRG treatment. An initial decline in steady state mRNA levels was first noted at 1 hour after HRG treatment. ErbB3 levels also decreased in LTLT-Ca cells after HRG treatment. Lapatinib, which inhibits ErbB2 and EGFR kinase activity and HRG induced-ErbB3 phosphorylation, diminished the HRG- induced decrease in ErbB2 and ErbB3 in AU565 and LTLT-Ca cells. This finding suggests that the kinase activity of EGFR/ErbB2 may be involved in the HRG-induced down-regulation. HRG did not change the activity of ErbB2 promoter reporter constructs spanning 215 kb to 6007 kb upstream of the ErbB2 transcription start site as indicated by luciferase assays. An ongoing study suggests that HRG could be involved in decreasing ErbB2 mRNA stability. Our findings suggest that resistance to Lapatinib may be due in part to the fact that inhibition of ErbB2/ EGFR kinase activity dampens the negative effect of HRG on ErbB2 expression, resulting in elevated levels of ErbB2 in breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1063. doi:1538-7445.AM2012-1063

Antitumor effect of the mTOR inhibitor everolimus in combination with trastuzumab on human breast cancer stem cells in vitro and in vivo

This study evaluated the effects of a mammalian target of mTOR inhibitor everolimus alone or in combination with trastuzumab on stem cells from HER2-overexpressing primary breast cancer cells and the BT474 breast cancer cell line in vitro and in vivo. For the in vitro studies, we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells from primary breast cancer cells and BT474 cells using flow cytometry. The MTT assay was used to quantify the inhibitory effect of the drugs on total cells and stem cells specifically. Stem cell apoptosis, cell cycle distributions, and their tumorigenicity after treatment were investigated by flow cytometry or soft agar colony formation assays. For the in vivo studies, BALB/c mice were injected with BT474 stem cells, and the different treatments were administered. After necropsy, the expression of Ki67, CD31, AKT1, and phospho-AKT (Thr308) was analyzed by immunohistochemistry. For the in vitro studies, Treatment with everolimus resulted in stem cell growth inhibition in a dose-dependent manner. The combination of everolimus with trastuzumab was more effective at inhibiting cell growth (P < 0.001) and tumorigenicity (P < 0.001) compared with single-agent therapy. In addition, an increase in G1 cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either of the single-agent groups (P < 0.01). For the in vivo studies, everolimus plus trastuzumab therapy was much more effective at reducing tumor volume in mice compared with either single agent alone (P < 0.05). Compared with everolimus alone, the combination of everolimus and trastuzumab reduced the expression of Ki67, AKT1, and phospho-AKT (Thr308) (P < 0.05). We conclude that everolimus has effective inhibitory effects on HER2-overexpressing stem cells in vitro and vivo. Everolimus plus trastuzumab is a rational combination treatment that may be promising in human clinical trials.

NRF2 inhibition represses ErbB2 signaling in ovarian carcinoma cells: Implications for tumor growth retardation and docetaxel sensitivity

NF-E2-related factor 2 (NRF2) is a transcription factor that regulates the expression of various antioxidant and detoxifying enzymes. Although the benefit of NRF2 in cancer prevention is well established, its role in cancer pathobiology was recently discovered. In this study, the role of NRF2 in tumor growth and docetaxel sensitivity was investigated in ErbB2-overexpressing ovarian carcinoma SKOV3 cells. Interfering RNA-mediated stable inhibition of NRF2 in SKOV3 cells repressed NRF2 signaling, resulting in cell growth arrest at G0/G1 phase and tumor growth retardation in mouse xenografts. Microarray analysis revealed that ErbB2 expression is substantially reduced in NRF2-inhibited SKOV3 and this was further confirmed by RT-PCR and immunoblot analysis. Repression of ErbB2 led to a decrease in phospho-AKT and enhanced p27 protein, reinforcing the effect of NRF2 knockdown on SKOV3 growth. Furthermore, NRF2 inhibition-mediated ErbB2 repression increases the sensitivity of these cells to docetaxel cytotoxicity and apoptosis. The linkage between NRF2 and ErbB2 was confirmed in the ErbB2-positive breast cancer cell line BT-474: NRF2 knockdown suppressed ErbB2 expression and enhanced docetaxel sensitivity. Our results provide insight into the coordinated regulation of signaling molecules responding to environmental stress and suggest that NRF2 modulation might be a therapeutic strategy to limit tumor growth and enhance sensitivity to taxane-based chemotherapy.

P1-12-08: Induction of Adhesion Molecules on Target Cells Maximizes Antibody-Dependent Cell-Mediated Cytotoxicity of Anti-Her2 Therapeutic Antibody with High Fucose Glycan.

Abstract Background: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one pivotal effector function of certain therapeutic antibodies. ADCC is the lysis of antibody-coated target cells by effector cells expressing Fc receptors (FcR) that recognize the fragment crystallizable (Fc) region of the bound antibody. Higher fucosylated terminal glycan on Fc results in significantly lower ADCC activity because core fucose sterically hinders the binding of Fc region to FcR on effector cells. The role of adhesion molecules in ADCC is unclear but it has been reported that adhesion molecules help better orientation of effector cells (natural killer cells) toward adhesion molecule-coated beads. We hypothesize that a preferable association of effector cells and target cells mediated by adhesion molecules may compensate ADCC loss due to the steric hindrance of high core fucose on Fc. Recombinant humanized monoclonal anti-Her2 antibodies (anti-Her2 rhuMAb) at various fucosylation levels have been used in this study to evaluate the role of adhesion molecules in ADCC of antibodies with high fucose glycan. Materials and Methods: Natural killer (NK) cell line transfected with FcR were used as effector cells. Her2-expressing breast cancer cell lines BT-474 and SK-BR3 were used as target cells. Adhesion molecules were induced in target cells by incubation with 50ng/ml of recombinant human tumor necrosis factor (TNFa) for 1–2 days. Anti-Her2 rhuMAbs with different Fc fucosylation (G0-F from &amp;lt;1% to &amp;gt;2%) were added in the co-culture of effector cells and target cells. ADCC activities were quantitated by target cells lysis assessment using Europium-TDA release assay. Results: Untreated BT-474 or SK-BR3 target cells had very little adhesion molecule expression. A high fucosylated anti-Her2 rhuMAb induced 52% of target cell lysis, which represented two-thirds of cell lysis by a low fucosylated Ab. Increment of E:T ratio could not maximize ADCC activities. With TNFa treatment, target cell lysis was increased from 52% to 100% by high fucose rhuMAb. This enhancement of ADCC activities is probably due to the expression of adhesion molecules such as ICAM-1 and VCAM-1, which was shown to be up-regulated by 100-fold on target cells in the presence of TNFa. Our data suggested that ADCC activities in Her2-expressing breast cancer cell lines were HER2−specific, anti-HER2 rhuMAb-dependent and adhesion molecules were indispensible for the enhanced ADCC activities. Discussion: rhuMAbs produced in Chinese hamster ovary (CHO) cell lines may contain various Fc fucosylation, hence various ADCC activities. Although up-regulation of adhesion molecules alone cannot lyse target cells directly, our data suggested maximized ADCC activities by rhuMAbs with high Fc fucosylation level could be achieved by induction of adhesion molecule expression on target cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-08.

P4-01-11: Autocrine Human Growth Hormone (hGH) Reduces the Sensitivity of Breast Cancer Cells to Treatment with Tamoxifen and Fulvestrant.

Abstract Despite recent advances in breast cancer treatment regimes, intrinsic or development of resistance to conventional breast cancer therapies is still a significant clinical challenge. Expression of growth factors by tumour cells has been proposed as one mechanism for developing resistance to radiotherapy and chemotherapeutic agents. The growth hormone/insulin-like growth factor-1 axis is emerging as an important mediator of tumour development in human breast cancer. Recent studies have demonstrated that humans with growth hormone receptor deficiency are protected from developing cancer. Studies investigating human growth hormone (hGH) expression in human breast cancer have demonstrated that hGH expression is associated with specific histopathological features and survival outcomes for patients. Increased hGH expression is significantly associated with lymph node metastasis, tumour stage, proliferative index, worse relapse free survival and reduced overall survival in mammary carcinoma. In recent studies we have demonstrated that autocrine hGH promotes cell proliferation, cell survival and oncogenicity of human mammary carcinoma cells, enhancing anchorage independent cell growth, and supporting tumour formation in immunodeficient mice. Autocrine hGH also promotes mammary carcinoma cell migration/invasion and epithelial to mesenchymal transition and tumour vascularisation in xenograft studies. In this study we investigated whether autocrine hGH promotes resistance to anti-estrogen therapeutic drugs, sensitivity to tamoxifen and fulvestrent using the human breast cancer cell lines MCF-7, T47D and BT474. Stable transfection with an expression vector containing the hGH gene enhanced MCF-7, T47D and BT474 cell viability, total cell number and anchorage independent growth following treatment with tamoxifen or fulvestrant when compared to control transfected cells. Conversely, treatment of BT474 cells with an hGH receptor antagonist (B2036), increased BT474 cell sensitivity to treatment with tamoxifen or fulvestrant. Using an estrogen response element (ERE) luciferase assay, we observed that autocrine hGH enhanced basal estrogen receptor (ER)-mediated transcriptional activity. Our results demonstrate that forced expression of hGH in mammary carcinoma cells enhances ER-mediated signal tranduction and promotes resistance to tamoxifen and fulvestrant, while functional antagnonism of hGH increased cell sensitivity. Consequently, antagonism of the hGH receptor may help maintain sensitivity to anti-estrogen therapy and improve the prognosis of patients with hormone sensitive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-11.

Preclinical antitumor activity of the novel heat shock protein 90 inhibitor CH5164840 against human epidermal growth factor receptor 2 (HER2)‐overexpressing cancers

Heat shock protein 90 (Hsp90), a molecular chaperone that plays a significant role in the stability and maturation of client proteins, including oncogenic targets for cell transformation, proliferation, and survival, is an attractive target for cancer therapy. We identified the novel Hsp90 inhibitor, CH5164840, and investigated its induction of oncogenic client protein degradation, antiproliferative activity, and apoptosis against an NCI-N87 gastric cancer cell line and a BT-474 breast cancer cell line. Interestingly, CH5164840 demonstrated tumor selectivity both in vitro and in vivo, binding to tumor Hsp90 (which forms active multiple chaperone complexes) in vitro, and being distributed effectively to tumors in a mouse model, which, taken together, supports the decreased levels of phosphorylated Akt by CH5164840 that we observed in tumor tissues, but not in normal tissues. As well as being well tolerated, the oral administration of CH5164840 exhibited potent antitumor efficacy with regression in NCI-N87 and BT-474 tumor xenograft models. In addition, CH5164840 significantly enhanced antitumor efficacy against gastric and breast cancer models when combined with the human epidermal growth factor receptor 2 (HER2)-targeted agents, trastuzumab and lapatinib. These data demonstrate the potent antitumor efficacy of CH5164840 when administered alone, and its significant combination efficacy when combined with trastuzumab or lapatinib, supporting the clinical development of CH5164840 as an Hsp90 inhibitor for combination therapy with HER2-targeted agents against HER2-overexpressing tumors.

C-Src-mediated RANKL-induced breast cancer cell migration by activation of the ERK and Akt pathway

The receptor activator for nuclear factor κB ligand/receptor activator for nuclear factor κB (RANKL/RANK) pathway is critical for RANK-expressing cancer cells to home to bones, and c-Src is critical for cancer progression. The objective of this study was to explore the effect of c-Src in the RANKL/RANK pathway and migration activity in human breast cancer cells. Breast cancer cell lines MCF-7, MDA-MB-231 and BT-474 were obtained and cultured. Flow cytometry was used to examine RANK expression. The results showed that RANK was expressed in breast cancer cell lines MCF-7, MDA-MB-231 and BT-474, and soluble RANKL (sRANKL)-triggered migration of breast cancer cells by activating ERK1/2, Akt and c-Src. The sRANKL-induced migration was blocked with RANKL inhibitor osteoprotegerin (OPG), MEK inhibitor PD98059, PI3K inhibitor LY294002 and Src inhibitor PP2. Inhibition of c-Src function with PP2 blocked the activation of Akt and ERK1/2, resulting in the inhibition of RANKL-induced migration. In conclusion, RANKL was found to increase the migration of breast cancer cells by activating the c-Src-Akt and c-Src-ERK signaling pathways.

Comparing the inhibitory effect of Silybin-phosphatidylcholine with Silybin in BT474 breast cancer cell line

Comparing the inhibitory effect of Silybin-phosphatidylcholine with Silybin in BT474 breast cancer cell line

FOXO3a represses VEGF expression through FOXM1-dependent and -independent mechanisms in breast cancer

Vascular endothelial growth factor (VEGF) plays a central role in breast cancer development and progression, but the mechanisms that control its expression are poorly understood. Breast cancer tissue microarrays revealed an inverse correlation between the Forkhead transcription factor FOXO3a and VEGF expression. Using the lapatinib-sensitive breast cancer cell lines BT474 and SKBR3 as model systems, we tested the possibility that VEGF expression is negatively regulated by FOXO3a. Lapatinib treatment of BT474 or SKBR3 cells resulted in nuclear translocation and activation of FOXO3a, followed by a reduction in VEGF expression. Transient transfection and inducible expression experiments showed that FOXO3a represses the proximal VEGF promoter whereas another forkhead member, FOXM1, induces VEGF expression. Chromatin immunoprecipitation and oligonucleotide pull-down assays demonstrated that both FOXO3a and FOXM1 bind a consensus Forkhead response element (FHRE) in the VEGF promoter. Upon lapatinib stimulation, activated FOXO3a displaces FOXM1 bound to the FHRE before recruiting histone deacetylase 2 (HDAC2) to the promoter, leading to decreased histones H3 and H4 acetylation, and concomitant transcriptional inhibition of VEGF. These results show that FOXO3a-dependent repression of target genes in breast cancer cells, such as VEGF, involves competitive displacement of DNA-bound FOXM1 and active recruitment of transcriptional repressor complexes.

Reduction-Sensitive Liposomes from a Multifunctional Lipid Conjugate and Natural Phospholipids: Reduction and Release Kinetics and Cellular Uptake

The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Förster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.

Modular anti‐EGFR and anti‐Her2 targeting of SK‐BR‐3 and BT474 breast cancer cell lines in the presence of ErbB receptor‐specific growth factors

Over the last decade, a number of monoclonal antibodies and small molecule inhibitors emerged as potent therapeutic agents in the treatment of Her2/neu overexpressing breast cancer. Numerous patients, however, do not adequately respond to anti-epidermal growth factor receptor (EGFR)/Her2 receptor targeting. Receptor- and, in turn, growth-stimulating effects, which potentially hamper antiproliferative cell treatment, have barely been investigated. BT474 and SK-BR-3 breast cancer cell lines were treated with Trastuzumab, Pertuzumab, and Lapatinib alone using different combinations and concentrations. Moreover, epidermal growth factor (EGF) or heregulin (HRG) was added to reveal potential growth factor-mediated compensatory effects. Receptor and intracellular signaling were analyzed as a function of cell treatment. Read-out parameters were cell proliferation and apoptosis. BT474 cells were efficiently driven into quiescence by Trastuzumab, but not by Pertuzumab treatment. Simultaneous EGF or HRG administration, however, restored the BT474 cell proliferation capacity. In contrast, neither therapeutic antibody treatment caused a profound inhibition of SK-BR-3 cell-cycle progress. Lapatinib turned out to be the most potent cell-cycle inhibitor in both cell lines even though its impact was significantly abrogated in the presence of EGF and HRG. The compensatory effect of EGF on Lapatinib-induced cell-cycle inhibition was reversed by Trastuzumab as well as by Pertuzumab treatment. Most importantly, HRG-caused compensation of Lapatinib-induced cell-cycle exit was reversed by Pertuzumab but not by Trastuzumab. Apparently, multiple anti-EGFR/Her2 targeting by using Trastuzumab, Pertuzumab, and Lapatinib more efficiently affects receptor function (interaction and activation) and consequently enhances their antiproliferative capacity. Growth inhibition by anticancer drugs targeted to Her/ErbB receptors, however, can be significantly undermined in the presence of EGF and in particular by HRG treatment, which suggests that specific therapeutic growth factor sequestration might further enhance anti-EGFR/Her2 targeting.