MDA-MB231 Breast Cancer Cell Lines Research Articles

Abstract 5266: TGF-β1 modulates the homeostasis between MMPs and MMPs inhibitors through p38 MAPK and ERK½ in highly invasive human breast cancer cells

Abstract Matrix metalloproteinases (MMPs) and their specific inhibitors, known as tissue inhibitors of MMPs (TIMPs) and the membrane-associated MMP inhibitor (RECK), are involved in several steps of the metastatic process. Previous results from our laboratory showed a positive correlation between high mRNA expression levels of MMPs and their inhibitors upon breast cancer progression, both in cellular models and in tumor tissue samples. However, the molecular mechanisms underlying this coordinate regulation of MMPs, TIMPs and RECK expression remain unknown. Since transforming growth factor beta 1 (TGF-β1) has been related to mechanisms which regulate the expression of some MMPs and MMPs inhibitors in different models, we investigated whether this multifunctional cytokine could have a role as a common regulator of MMPs, TIMPs and RECK in the human breast cancer model. The mRNA expression levels of TGF-β isoforms and their receptors were examined, by qRT-PCR, in a panel of five human breast cancer cell lines displaying different degrees of invasiveness and metastatic potential. We demonstrated that TGF-β1, TGF-β2 and TβRII are significantly overexpressed in more aggressive mammary cell lines. Furthermore, using the Spearman correlation test, we showed a significantly positive correlation between the mRNA expression levels of TGF-β1 and MMP-2, MMP-14, TIMP-1, TIMP-3 and RECK. In addition, upon treatment with TGF-β1, the MDA-MB-231 breast cancer cell line presented significantly increased mRNA expression levels of MMP-2, MMP-9, MMP-14, TIMP-2 and RECK. TGF-β1 was also able to induce the protein expression levels of MMP-2, MMP-9, MMP-14 and TIMP-2. On the other hand, RECK protein expression was significantly downregulated in TGF-β1-treated-MDA-MB-231 cells. In addition to the classical TGF-β-induced signal transduction by Smads, it is well known that this cytokine also signals in a Smad-independent manner, by induction of other pathways. Here, we analyzed the involvement of p38 MAPK and ERK½, two well established Smad-independent pathways, in the proposed mechanism of coordinate regulation of MMPs, TIMPs and RECK by TGF-β1 in the breast cancer model. Firstly, we confirmed that TGF-β1 is able to induce phosphorylation of p38MAPK and ERK½ proteins in the MDA-MB-231 cell line. Inhibition of ERK½ pathway led to increased levels of the MMPs inhibitors (TIMP-2 and RECK) mRNA in MDA-MB-231 cells treated (or not) with TGF-β1. In contrast, cells treated with a pharmacological p38MAPK inhibitor showed blockage of TGF-β1-induced mRNA expression of MMPs (MMP-2, MMP-9 and MMP-14) and MMPs inhibitors (TIMP-2 and RECK). Altogether, our results suggest that the balance between p38MAPK and ERK½ activities mediates the role of TGF-β1 as an important homeostasis regulator between MMPs and MMPs inhibitors in the MDA-MB231 breast cancer cell line. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5266. doi:10.1158/1538-7445.AM2011-5266

Abstract 2469: ATM inhibitor 55933 abolishes the radiation resistance of CD44+/CD24−or low subsets of breast cancer cells

Abstract CD44+/CD24− subsets of breast cancer cells are enriched in cancer stem-like cells (CSC) and are radiation resistant. We isolated ESA+/CD44+/CD24− or low subsets by flow cytometry from MCF7, MDA-MB231, and HCC1937 breast cancer cell lines and primary cultures of breast cancer cells from two patients. The isolated CD44+/CD24−orlow subsets showed a 50 to 70% increase of sphere formation by in suspension culture in serum-free medium compared to CD44−/CD24high subsets. CD44+/CD24−or low subsets of the cell lines and primary cultures showed a significantly increased radiation resistance compared to the CD44−/CD24high cell line subsets or the unsorted primary cells with > 40% increased survival at 2Gy. Post-radiation survival showed that the radiation resistance was dependent on the CD44+/CD24 −or low cell phenotype. The correlation of CD44/CD24 status with radiation resistance was demonstrated by culture of the subsets. After 2 weeks of culture the following transitions occurred: 1) CD44+/CD24−or low cells changed to CD44−/CD24high cells (MCF-7 and HCC1937) or to CD44+/CD24+ (MDA-MB-231); and 2) CD44−/CD24 high (MCF-7 and HCC1937) and CD44+/CD24+ (MDA-MB-231) cells became CD44+/CD24−or low cells. Cells that lost the CD44+/CD24 −or low phenotype were no longer radiation resistant. The mechanisms of increased radiation resistance in CD44+/CD24−orlow subsets was not a function of non-homologous end joining (NHEJ) as there was no in vivo and in vitro change in NHEJ activity in the resistant cells nor altered expression of the NHEJ related proteins Ku 80, Ku70, PARP-1, and DNA-PK. However, there were differences ATM activity between CD44+/CD24−or low and the CD44−/CD24 high (MCF-7 and primary breast cancer) or CD44+/CD24+ (MDA-MB-231) subsets. Depending on the cell type and radiation dose, ATM phsophorylation increased by 2-5 fold in radiated CD44+/CD24−or low subsets of breast cancer cell lines and primary cultured breast cancer cells compared to the control subsets. The ATM inhibitor 55933 in a dose-dependent manner increased radiation sensitivity of all subsets including significantly abolishing the radiation resistance of CD44+/CD24−or low subsets of cells. After 2Gy of radiation, the survival fractions of cells treated with10μM of 55933 were 8-10 fold lower than that of untreated cells and were now similar between CD44+/CD24−or low subsets and control subsets for the cell lines and primary cultures. In conclusion, ATM is a candidate target for eliminating the radiation resistance of CSC's of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2469. doi:10.1158/1538-7445.AM2011-2469

Phosphoproteomics and mass-action modeling to predict optimal drug combination in signaling networks

Abstract Targeted therapeutics hold tremendous promise in inhibiting cancer cell proliferation. However, targeting proteins individually can be compensated for by bypass mechanisms and activation of regulatory loops. Designing optimal therapeutic combination must therefore take into consideration the complex dynamic networks in the cell. In this study, we analyzed the insulinlike growth factor (IGF-1) signaling network in the MDA-MB231 breast cancer cell line using reverse phase protein arrays to quantify the dynamic crosstalk between the phosphoinositide 3-kinase (PI3K), the mitogen-activated protein kinase (MAPK) and the mammalian target of rapamycin (mTOR) pathways. We developed a computational procedure that integrated mass-action modeling with particle swarm optimization to train the model against the experimental data and infer the unknown model parameters. The trained model was used to predict how targeting individual signaling proteins altered the rest of the network and identify drug combination that minimally increased phosphorylation of other proteins elsewhere in the network. Experimental testing of the modeling predictions showed that optimal drug combination inhibited cell signaling and proliferation, while non-optimal combination of inhibitors increased phosphorylation of non-targeted proteins and rescued cells from cell death. The integrative approach described here is useful for generating experimental intervention strategies that could optimize drug combination and discover novel pharmacologic targets for cancer therapy.

Targeting Prosurvival Bcl-2 Family Members Increases Radiation Sensitivity in Breast Cancer Cells

Targeting Prosurvival Bcl-2 Family Members Increases Radiation Sensitivity in Breast Cancer Cells

A-kinase-interacting Protein 1 (AKIP1) Acts as a Molecular Determinant of PKA in NF-κB Signaling

A-kinase-interacting Protein 1 (AKIP1) Acts as a Molecular Determinant of PKA in NF-κB Signaling

Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation

Rho GTPases impact a number of activities important for oncogenesis. We describe a small molecule inhibitor that blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NF-κB activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

Identification of optimal drug combinations targeting cellular networks: integrating phospho-proteomics and computational network analysis.

Targeted therapeutics hold tremendous promise in inhibiting cancer cell proliferation. However, targeting proteins individually can be compensated for by bypass mechanisms and activation of regulatory loops. Designing optimal therapeutic combinations must therefore take into consideration the complex dynamic networks in the cell. In this study, we analyzed the insulin-like growth factor (IGF-1) signaling network in the MDA-MB231 breast cancer cell line. We used reverse-phase protein array to measure the transient changes in the phosphorylation of proteins after IGF-1 stimulation. We developed a computational procedure that integrated mass action modeling with particle swarm optimization to train the model against the experimental data and infer the unknown model parameters. The trained model was used to predict how targeting individual signaling proteins altered the rest of the network and identify drug combinations that minimally increased phosphorylation of other proteins elsewhere in the network. Experimental testing of the modeling predictions showed that optimal drug combinations inhibited cell signaling and proliferation, whereas nonoptimal combination of inhibitors increased phosphorylation of nontargeted proteins and rescued cells from cell death. The integrative approach described here is useful for generating experimental intervention strategies that could optimize drug combinations and discover novel pharmacologic targets for cancer therapy.

Interleukin-1β regulates the migratory potential of MDAMB231 breast cancer cells through the hypoxia-inducible factor-1α

The casual relationship between inflammation and tumour progression has been widely accepted and the etiology of breast cancer has been associated with inflammatory processes. Interleukin (IL)-1β, besides its central role in inflammation, has also been recognised as a powerful player in tumour progression, angiogenesis and invasiveness. Recently, there has been considerable interest in understanding the non-hypoxic upregulation of the hypoxia-inducible factor (HIF)-1α by IL-1 in neoplastic cells since aberrant expression of HIF-1α correlates with tumour progression. Here, using the highly invasive human breast cancer cell line MDAMB231, we studied the effect of IL-1β on tumour cell migration along with HIF-1α accumulation. We observed that non-hypoxic induction of HIF-1α by IL-1β in MDAMB231 was associated with increased cell migration, paralleled by upregulation of p38 MAPK phosphorylation and CXCL8/CXCR1 expression. Inhibition of HIF-1α by siRNA resulted in a significant reduction of CXCR1 expression and IL-1β-induced cell migration in MDAMB231 cells, thus confirming a role of HIF-1α in the non-hypoxic-IL-1β-dependent induction of migratory potentials. Our observation that IL-1 induces HIF-1α accumulation in MDAMB231 cells was confirmed in tumour cells growing in vivo using an experimental approach, mimicking the endogenous release of IL-1 in mice bearing MDAMB231 xenografts. Our in vivo data, along with the fact that inhibition of HIF-1α resulted in the decrease of IL-1β-promoted cell migration, further support the link between inflammation and cancer. The overall results may have important implications in those therapeutic approaches aimed to inhibit IL-1-mediated activities in tumour cells, specifically in breast cancer.

Tumour suppressor function of MDA-7/IL-24 in human breast cancer

IntroductionMelanoma differentiation associated gene-7 (MDA-7), also known as interleukin (IL)-24, is a tumour suppressor gene associated with differentiation, growth and apoptosis. However, the mechanisms underlying its anti-neoplastic activity, tumour-specificity and efficacy across a spectrum of human cancers have yet to be fully elucidated. In this study, the biological impact of MDA-7 on the behavior of breast cancer (BC) cells is evaluated. Furthermore, mRNA expression of MDA-7 is assessed in a cohort of women with BC and correlated with established pathological parameters and clinical outcome.MethodsThe human BC cell line MDA MB-231 was used to evaluate the in-vitro impact of recombinant human (rh)-MDA-7 on cell growth and motility, using a growth assay, wounding assay and electric cell impedance sensing (ECIS). Localisation of MDA-7 in mammary tissues was assessed with standard immuno-histochemical methodology. BC tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription, MDA-7 transcript levels were determined using real-time quantitative PCR. Transcript levels were analyzed against tumour size, grade, oestrogen receptor (ER) status, nodal involvement, TNM stage, Nottingham Prognostic Index (NPI) and clinical outcome over a 10 year follow-up period.ResultsExposure to rh-MDA-7 significantly reduced wound closure rates for human BC cells in-vitro. The ECIS model demonstrated a significantly reduced motility and migration following rh-MDA-7 treatment (p = 0.024). Exposure to rh-MDA-7 was only found to exert a marginal effect on growth. Immuno-histochemical staining of human breast tissues revealed substantially greater MDA-7 positivity in normal compared to cancer cells. Significantly lower MDA-7 transcript levels were identified in those predicted to have a poorer prognosis by the NPI (p = 0.049) and those with node positive tumours. Significantly lower expression was also noted in tumours from patients who died of BC compared to those who remained disease free (p = 0.035). Low levels of MDA-7 were significantly correlated with a shorter disease free survival (mean = 121.7 vs. 140.4 months, p = 0.0287) on Kaplan-Meier survival analysis.ConclusionMDA-7 significantly inhibits the motility and migration of human BC cells in-vitro. MDA-7 expression is substantially reduced in malignant breast tissue and low transcript levels are significantly associated with unfavourable pathological parameters, including nodal positivity; and adverse clinical outcomes including poor prognosis and shorter disease free survival. MDA-7 offers utility as a prognostic marker and potential for future therapeutic strategies.

RNA interference for inhibiting expression of epidermal growth factor receptor in breast cancer cells

Objective To investigate whether RNA interference (RNAi) produced by small inter- ference RNA (siRNA) could induce gene silencing in breast cancer cells and to assess the degree of epidermal growth factor (EGF) receptor gene silencing and its effect on functional outcome. Methods Overexpression of EGF receptor gene was determined in breast cancer cells and a variety of other types of cancer cells. Then breast cancer cell lines MDA-MB231, MDA-MB-453S, ZR-75-30, and ZR-75 were transfected with target se- quence-specific dsRNA as well as various controls. Immune fluorescent labeling, flowcytometry and Western blot were used to monitor the reduction in production of the EGF receptor protein. Quantitative reverse- transcriptase PCR was used to detect the silencing of EGF receptor gene levels. Cell count, colony assay, scratch assay, MTT assay, cell cycle analysis, and ELISA were used to assess the functional effects of RNAi. Results EGF receptor gene was overexpressed in breast cancer cells. In cell line MDA-MB231 and MDA-MB-453S, we displayed sequence specific silencing of the EGF receptor with 71.31% of downregulation of EGF receptor protein production and 37.04% of silencing of EGF receptor gene. The reduction in EGF receptor caused growth inhibition of the cells, reducing the total cell numbers by 85.0% and colony number by 63.3%. This also retarded the migration of MDA-MB231 and MDA-MB-453S by more than 80% at 24h and 48h, enhanced the chemosensitivity to cisplatin by four-fold. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in Go-Gi phase by 12.67% with a decrease in the percentage of cells in S-phase by 6.56% relative to control. The results from ELISA showed EGF level was reduced by 27.74% and 11.07% in medium and cell extraction respectively. In cell line ZR-75-30 and ZR-75, we displayed sequence specific silencing of the EGF receptor with 71.78% of downregulation of EGF receptor protein production and 50.00% of silencing of EGF receptor gene. The reduction in EGF receptor caused growth inhibition of the cells, reducing the total cell numbers by 78.3% and colony number by 66.8%. This also retarded the migration of ZR-75-30 and ZR-75 by more than 80% at 24h and 48h, enhanced the chemosensitivity to cisplatin by seven-fold. Cell cycle analysis showed that dsRNA-EGFR induced accumulation of cells in G0-G1 phase by 17.48% with a significant decrease in the percentage of cells in S-phase by 19.20% relative to control. ELISA showed EGF level was reduced by 45.65% and 22.45% in medium and cell extraction, respectively. Conclusions These sequence specific siRNAs showed a blockbuster effect in downregulation of EGF receptor gene expression, inhibition of the cellular proliferation and motility, arrest of the cell cycle, and enhancement of the cellular chemosensitization. The successful application of dsRN A-EGFR to reverse the neoplastic phenotype of EGF receptor overexpressing cells extends the list of available therapeutic modalities in the treatment of human cancer. Key words: Epidermal growth factor receptor; RNA interference; Small interference RNA; Double stranded RNA; Breast cancer

Synthesis of 2- and 7- Substituted C19 Steroids Having a 1,4,6-Triene or 1,4-Diene Structure and Their Cytotoxic Effects on T47D and MDA-MB231 Breast Cancer Cells

2-Chloro-, 2-bromo- and 2-azido-1,4,6-androstatriene-3,17-diones were synthesized from 1α,2α-epoxy-4,6-androstadiene-3,17-dione (2) using HCl, HBr and NaN3, respectively. Compound 2 was also reacted with NaCN to give 2-cyano-1,4,6-androstatriene-3,17-dione (5) and 2β-cyano-1α-hydroxy-4,6-androstadiene-3,17-dione (6). 6α,7α-Epoxy-1,4-androstadiene-3,17-dione (8) was reacted with HCl, HBr and NaN3 to form the corresponding 7β-chloro-, 7β-bromo- and 7β-azido-6α-hydroxy-1,4-androstadiene-3,17-diones. The cytotoxic activity of these compounds towards T47D (estrogen-dependent) and MDA-MB231 (estrogen-independent) breast cancer cell lines was evaluated. The 6α-hydroxy-7β-substituted analogs were more active than the 2-substituted analogs on both cell lines. Compound 2 showed the highest selective activity against the T47D (IC507.1 μM) cell line and 5 showed good cytotoxic activity on MDA-MB231 (IC5018.5 μM) cell line, respectively. The 6α,7α-epoxy analog 8 also showed high cytotoxic activity on both cell lines (IC50 17.3 μM on T47D and IC50 26.9 μM on MDA-MB231).

Effect of a combination of tyrosine kinase inhibitor imatinib with doxorubicin shows in breast cancer cell lines.

e13657 Background: In the need to optimize breast cancer therapies, constantly new targeted agents are examined for their potential clinical use. In a broad range of solid and haematological cancers tyrosine kinase inhibitors, which target growth factor receptors like PDGFR α and β as well as c-kit, have already shown to be of clinical benefit with tolerable side effects. Here effects of the tyrosine kinase inhibitor imatinib as a single agent and in combination with standard chemotherapy (doxorubicin) have been investigated in vitro. Methods: Biological effects of imatinib in breast cancer cell lines MDAMB 231 and MCF 7 were tested using proliferation assays, TUNEL assays and migration assays. Cells were incubated with different concentrations of doxorubicin (0,25 nmol-500μmol) and imatinib (1-15 μmol) each as single agent and in combination. Results: Incubation with imatinib as a single agent reduces cell proliferation in a dose-dependent manner with an IC 50 of 6uM for both breast cancer cell lines. Moreover, imatinib induces apoptosis and reduces cell migration. Combining imatinib with doxorubicin leads to an additive effect on cell growth inhibition in MDA MB 231 and MCF 7 cells. A further reduction in cell migration can be detected when imatinib is combined with doxorubicin. In the hormone receptor positive cell line MCF 7, a significant increase in apoptotic cells can be detected when combining both drugs compared to each single treatment. The concentrations of doxorubicin as well as the imatinib concentrations can be applied in clinical use. Conclusions: Combining imatinib with doxorubicin shows a significant improvement in inhibiting breast cancer cell proliferation and migration, as well as apoptosis induction. These data suggest that the combination of standard chemotherapy with imatinib could be beneficial for breast cancer patients. No significant financial relationships to disclose.

Abstract 4838: Alpha-methyltryptophan, a blocker of the amino acid transporter SLC6A14, induces autophagy and apoptosis in SLC6A14-positive breast cancer and colon cancer cell lines

Abstract SLC6A14 (also known as ATB0,+) is a Na+/Cl− -coupled amino acid transporter that transports 18 of the 20 proteinogenic amino acids, including all essential amino acids. Normal tissues express this transporter at low levels, whereas tumor tissues (colon, breast, and cervix) upregulate this transporter several-fold. The unique functional features of SLC6A14 (broad substrate specificity and multiple driving forces) make it an ideal transporter for tumor cells to maintain amino acid nutrition necessary for enhanced cell proliferation. We speculate that SLC6A14 could not only be an ideal tumor-specific delivery system for amino acid-based anti-cancer drugs but also be a direct drug target for cancer chemotherapy. Recently we identified alpha-methyltryptophan (ALT) as a blocker of SLC6A14. Here we examine the potential of this blocker as an anti-cancer agent in SLC6A14-positive breast and colon cancer cells. Treatment of MCF7 (a breast cancer cell line), HCT116 and LS174T (colon cancer cell lines) with ALT causes amino acid starvation as evidenced by the upregulation of asparagine synthetase and CHOP. These three cell lines are SLC6A14-positive. In contrast, ALT treatment of breast cancer cell line MDA-MB231, which is SLC6A14-negative, does not show any evidence of amino acid starvation. Similarly, ALT treatment has no effect on normal breast and colon epithelial cell lines (MCF10A and CCD841, respectively). These cell lines also do not express SLC6A14. These data show that ALT-induced amino acid starvation is mediated specifically through the blockade of SLC6A14. Since nutrient signaling is also associated with changes in mTOR pathway, we examined the influence of ALT treatment on phosphorylation of S6 and S6kinase. Blockade of SLC6A14 with ALT in SLC6A14-positive cancer cell lines leads to decreased mTOR signaling as evidenced by decreased phosphorylation of S6 and S6kinase. Again, such changes are not seen in SLC6A14-negative cancer or normal cell lines. The initial response of the SLC6A14-positive cancer cell lines to ALT treatment is induction of autophagy. ALT-induced autophagy is documented in these cells by light and electron microscopic examination as well as by biochemical means. Inhibition of autophagy with 3-methyladenine under these conditions leads to apoptosis. We conclude that SLC6A14 is a potential drug target for cancer chemotherapy and that ALT could serve as a lead compound for the future design of high-affinity blockers of SLC6A14. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4838.

Abstract 469: Functional imaging of the hypoxic tumor microenvironment in metastatic progression

Abstract In the last years, the need for visualization of tumoral processes has become a high-top priority in molecular imaging. This is especially true for those methods dedicated to functional imaging that focus on revealing phenomena associated with biological processes such as hypoxia to cancer. Among them, optical imaging methods such as fluorescence are provided with a broad range of proteins and dyes used to visualize many types of these biological processes widely used in cell biology studies. Although the best-known example of these fluorochromes is the green fluorescent protein (GFP), tissue autofluorescence and signal dispersion raise doubts about its suitability as an in vivo tracer. However, a number of groups have recently developed several NIR fluorescent proteins that enable real-time imaging free of autofluorescence interference thus making possible to take a deep view into the tissues. With all of this in mind, we devised a novel fluorescence-bioluminescence genetically encoded biosensor activated by the neoangiogenesis-related transcription factor HIF-1α, which appears upregulated in growing tumors. At the same time, by fusing a fluorescent to a bioluminescent protein we obtained a bioluminescence resonance energy transfer (BRET) phenomenon turning this fusion protein into a new class of hypoxia-sensing genetically encoded biosensor. We also tested the inducibility and performance of this hypoxia sensor by imaging artificially activated cells both in culture (in vitro) and inside an animal model (in vivo). Finally, the breast cancer cell line MDA-MB 231, which is known by its invasiveness and ability to metastasize, was modified to carry the hypoxia sensor and subsequently injected intravenously in live mice. Metastases were detected in lung and brain by fluorescence and/or bioluminescence. In conclusion, we have described the designing and development of a functional dual hypoxia-sensing system able to display BRET activity between the firefly luciferase and the NIR fluorochrome. In addition a metastatic tumoral cell line was genetically modified to demonstrate that the biosensor is able to detect hypoxic conditions in metastasis in vivo, yielding valuable functional information about the tumor besides size and spatial localization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 469.

Abstract 5277: S100A7 modulates EGFR signaling in estrogen receptor negative breast cancer cell lines

Abstract Purpose of Study: S100A7 (psoriasin) is highly expressed in estrogen receptor alpha negative (ER-α−) and EGFR+ basal like phenotype, especially triple negative, that have been associated with worse prognosis and outcome. However, not much is known about the role of S100A7 in promoting tumorigenesis of ER-α− and EGFR+ breast cancers. We hypothesize that S100A7 secreted by tumor cells may modulate tumor growth by binding to receptor for advanced glycation end-products (RAGE) and activating EGFR in ER-α−. In the present study, we analyzed recombinant S100A7-mediated EGFR signaling that enhances breast tumorigenesis and metastasis in ER-α− cells. Methods: We examined the effect of recombinant S100A7 on cell migration and motility of ER-α− MDA-MB231 and MDA-MB453 breast cancer cell lines. In addition, we analyzed S100A7-mediated signaling mechanisms by monitoring changes in phosphorylation and expression of various signaling molecules by Western blot, immunoprecipitation and immunofluorescence. We also co-transfected HEK-293 with EGFR and RAGE plasmids to analyze crosstalk between S100A7/RAGE and EGF/EGFR in these cells. Results: We observed high expression of RAGE in ER-α− MB-231 and MB-453 breast cancer cell lines by FACS analysis. We have also shown that S100A7 is secreted in the conditioned media of ER-α− MB-231 overexpressing S100A7 cells. Recombinant soluble S100A7 was shown to induce cell migration and wound healing in MB-231 and MB-453 cells. These effects were mediated through RAGE, as RAGE neutralizing antibody blocked S100A7-mediated functional effects. To further analyze the molecular mechanism of S100A7-mediated signaling, we observed that recombinant S100A7 transactivates EGFR. Further analysis of molecular mechanism revealed that S100A7 enhances AKT and Erk phosphorylation as well as increases association between Jab-1 and EGFR. The crosstalk between S100A7/RAGE and EGF/EGFR was further confirmed by co-transfecting HEK-293 cells with EGFR and RAGE. Recombinant S100A7 was shown to induce EGFR phosphorylation in these transfected cells, confirming S100A7 may transactivate EGFR by binding to RAGE. Conclusion: These studies reveal that S100A7 may enhance metastatic potential of ER-α− cells by binding to RAGE and activating EGFR signaling. Furthermore, these studies may help in the development of S100A7/RAGE as novel therapies target for ER-α− breast cancer, especially triple-negative and EGFR+ basal-like cancers, which are highly aggressive and difficult to treat. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5277.

Effect of Physiological Levels of Sodium Selenite on the Expression and Regulation of Hypermethylated Tumor Supressor Gene in Human Breast Cancer Cell Line

Breast cancer (BC) was the 4th‐leading cause of cancer death in Taiwan with the rising incidence as malignant type in young premenopausal women during the recent decades. The aim of our study was to investigate the effects of physiological levels of selenite on regulation of BC tumor suppression gene through hypermethylation. Exposure of human breast cancer cell line MDA‐MB 231 to 1.5 and 2.5 μM of sodium selenite for 7 days significantly decreased cell invasion and migration ability compared to control. Selenite treatment (2.5 μM) reactivated the anti‐invasion gene CST6 (cystatin E/M), of which the promoter region hypomethylation was confirmed by methylation‐specific PCR. The protein expression of DNA methyltransferase £L (DMNT1) and histone deacetyl transferase I were also significantly decreased by selenite treatment. In conclusion, the suppressed invasion ability of MDA‐MB 231 cells by physiological levels of selenite may be due to the suppressed protein expression of DNMT1 as the result of reactivating the anti‐invasion genes such as CST6, of which the promoter region were hypomethylated. The results of this study are expected to explain the role of selenium in regulating the re‐expression of specific tumor suppressor genes and to be applied in improving the prognosis of breast cancer.(NSC98‐2320‐B‐030‐004)Grant Funding Source: NSC98‐2320‐B‐030‐004

Estrogen receptor beta participate in the regulation of metabolizm of extracellular matrix in estrogen alpha negative breast cancer.

The biology of breast cancer is closely releted to sex steroid hormones. Estrogen receptor beta is overexpressed in around 70% breast cancer cases, referrd to as "ER positive". Estrogens bind to estrogen receptor and stimulate the transcription of genes involved in control of cell proliferation. Moreover, estrogens may induce growth factors and components of extracellular matrix and interact with them in a complex manner. Extracellular matrix and integrins play an important role in cell functions and their aberrant expressions are implicated in breast cancer development, invasion and metastasis. ER beta is certainly associated with more differentiated tumors, while evidence of role of ER beta is controversial. The highly invasive breast cancer ER beta negative cell line MDA-MB 231 can be the model of exam the role of ER beta in breast cancer. The aim of this study was to examine the role of activation of ER beta on the metabolism of the extracellular matrix and the expression of beta-1 integrin in the breast cancer cell line MDA-MB 231. The cells were exposed on the estradiol, tamoxifen, raloxifen and genisteina in dose dependent concentrations. To determine the relative rate of collagen syntesis we measured the time-dependent reduction of collagen-bound radioactivity after pulse-chase labeling with [3 H] prolina by Peterkofsky methods. The expression of beta-1 integrin was determine by Western blot analysis. The activity of MMP2 and 9 were measured using gelatin zymography with an image analysis system. Our data suggest on the role of estrogen receptor beta on the metabolism of extracellular matrix in the breast cancer line MDA - MB 231. Estradiol and SERMs regulate the expression of ECM proteins: collagen, integrins and enhance activity of metaloproteinases 2 and 9.

Treatment of Breast Cancer Cells with Proteasome Inhibitor Lactacystin Increases the Level of Sensitivity to Cell Death Induced by Human Immunodeficiency Virus Type 1

Upon binding to CD4, the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 undergoes conformational changes that facilitate subsequent interactions with the chemokine coreceptor CXCR4 on the T cells. Our previous study showed that HIV-1 induces breast cancer cell death through gp120-CXCR4 interaction without CD4-induced conformational change of gp120. To characterize the structural properties of CXCR4 on breast cancer cells, the structural differences in CXCR4 between breast cancer cell lines and T cells were investigated. Immunoblots of whole cell lysates from breast cancer cell and T cell lines demonstrated that the predominant forms of CXCR4 on the breast cancer cell lines and T cell lines were three species (45, 61, 100 kDa) and one species (45 kDa), respectively. Cell surface biotin labeling revealed that the 100-kDa polyubiquitinated form of CXCR4 is specifically expressed on the surface of breast cancer cell line DU4475 but not T cell line Molt4#8. The treatment of breast cancer cell lines MDA-MB231 and DU4475 with proteasome inhibitor lactacystin leads to increased surface expression of the 100-kDa polyubiquitinated form of CXCR4 and increases the level of sensitivity to cell death induced by HIV-1. These data suggest that the 100-kDa polyubiquitinated form of CXCR4 plays an important role as a trigger for gp120-induced breast cancer cell death.

Palladium(II) complexes with thiosemicarbazones: syntheses, characterization and cytotoxicity against breast cancer cells and Anti-Mycobacterium tuberculosis activity

Three PdII complexes were prepared from N(4)-substituted thiosemicarbazones: [Pd(aptsc)(PPh3)](NO3)•H2O, 1, [Pd(apmtsc)(PPh3)](NO3), 2, and [Pd(apptsc)(PPh3)](NO3)•H2O, 3, where PPh3 = triphenylphosphine; Haptsc = 2-acetylpyridine-thiosemicarbazone; Hapmtsc = 2-acetylpyridine-N(4)-methyl-thiosemicarbazone and Happtsc = 2-acetylpyridine-N(4)-phenyl-thiosemicarbazone. All complexes were characterized by elemental analysis, IR, UV-Vis, ¹H and 31P{¹H} NMR spectroscopies, and had their crystalline structures determined by X-ray diffractometry from single crystals. The monoanionic thiosemicarbazonate ligands act in a tridentate mode, binding to the metal through the pyridine nitrogen, the azomethine nitrogen and the sulfur atoms. The cytotoxic activity against the breast cancer cell line MDA-MB231 and the anti-Mycobacterium tuberculosis H37Rv ATCC 27294 activity were evaluated for the compounds. All PdII complexes were highly active against the studied cell line, presenting similar values of IC50, around 5 µmol L-1, while the clinically applied antitumor agent cisplatin was inactive. The compounds show remarkable anti-M. tuberculosis activities, presenting MIC values comparable or better than some commercial anti-M tuberculosis drugs.

Cytotoxicity of Atriplex confertifolia.

The search for cancer treatment continues to be a global effort. As part of this global effort, many natural products have been tested against cancer cell lines, mostly from tropically located plants. This study reports that extracts of Atriplex confertifolia (Torr. and Frem.) S. Watson (Chenopodiaceae), a native North American plant (also known as shadscale or saltbush), has significant bioactivity against human breast cancer cell lines MCF-7, MDA-MB 435, MDA-MB 231, and HeLa cells (cervical cancer cells). The bioactivity of A. confertifolia extracts on these cells lines was compared to an FDA-approved cancer drug (Onxol®) and an industry-standard leukocyte control cell line. Active portions of the extracts were found primarily in the polar fractions of the plant. A dose-response curve of the extracts displayed significant cell death similar to Onxol®. The plant extracts did not significantly inhibit the viability of the leukocyte cell line. In a timed study, over 90% of cell lines MDA-MB 435 and HeLa died after 24 hours. Cell death appears to result from apoptosis.