Therapeutic Strategy For Breast Cancer Research Articles

Abstract P4-06-19: Astemizole and calcitriol: A novel targeted therapeutic strategy for breast cancer

Abstract Calcitriol antiproliferative effects include inhibition of the oncogenic ether-à-go-go-1 potassium channel (Eag1), which expression and activity are necessary for cell cycle progression and tumorigenesis. Astemizole, a new promising antineoplastic drug, targets Eag1 by blocking ion currents. In vitro, we showed that astemizole synergized calcitriol antiproliferative effects by downregulating CYP24A1, the enzyme that degrades calcitriol, and upregulating the vitamin D receptor (VDR), which mediates calcitriol actions. In this study we investigated the antineoplastic effect of astemizole and calcitriol in a murine model xenografted with T-47D and invasive ductal carcinoma-derived breast cancer cells (IDC). Tumor-bearing nude mice were treated with oral astemizole (50 mg/kg/day) and/or i.p. calcitriol (30 μg/kg/twice a week) during 3 weeks. Astemizole and calcitriol significantly inhibited, while the coadministration of both drugs further reduced, tumor growth compared to untreated controls. These results were supported by immunohistochemistry studies of Ki-67, a proliferation marker, which revealed less staining in tumors from mice with the co-treatment compared to controls and calcitriol or astemizole alone. In a similar manner as reported in vitro, we observed that the concomitant administration of astemizole and calcitriol upregulated VDR tumor expression (P < 0.05). Conclusion: Coadministration of calcitriol and astemizole increased the antineoplastic effects of both drugs. Our results indicate that astemizole may improve calcitriol bioactivity by upregulating tumoral VDR. In addition, the findings in this study suggest that VDR-negative tumors could be sensitized to calcitriol antineoplastic effects by concomitant administration with astemizole, which would also allow for less calcitriol dosing without sacrificing overall therapeutic efficacy. Herein we suggest a novel combined adjuvant therapy for the management of breast cancer tumors. These results provide scientific bases for further studies designed to test both compounds simultaneously in patients bearing solid or metastatic tumors. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-19.

Erratum to: Targeting the PELP1-KDM1 axis as a potential therapeutic strategy for breast cancer

Author details 1Department of Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, TX 78229, USA. 2Department of Cell and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229, USA. 3Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 13 Taishogun Nishitakatsukasa-Cho, Kita-ku, Kyoto 403-8334, Japan. 4PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan. 5Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8673, Japan. 6Department of Gynecologic Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 78030, USA. 7Center for RNA Interference and Noncoding RNA, University of Texas MD Anderson Cancer Center, Houston, TX 78030, USA. 8Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX 78030, USA. 9Cancer Therapy and Research Center, University of Texas Health Science Center, San Antonio, TX 78229, USA. Published: 21 December 2012

Heparins modulate the IFN-γ-induced production of chemokines in human breast cancer cells

Heparins seem to improve survival in patients with advanced malignancies independently of their anticoagulatory function. As the treatment options in advanced and metastatic breast cancer are still very limited, heparins might be an interesting addition to the existing systemic therapies. The interferon (IFN)-γ-inducible chemokines CXCL9 and CXCL10 play an essential role in the regulation of the immune milieu in malignant tumours, thereby being interesting targets for an immunological intervention. We therefore wanted to test whether heparins have an impact on the chemokines CXCL9 and CXCL10 as well as the IFN-γ signalling in human breast cancer cells in vitro. The well-established cell lines BT-474, MCF-7, SK-BR-3 and MDA-MB-231 were incubated with IFN-γ, unfractionated heparin (UFH), different low molecular weight heparins (LMWHs) and the heparin-related polyanions danaparoid and dextran sulphate. The production of CXCL9 and CXCL10 was measured by ELISA and real-time RT-PCR, the phosphorylation of signal transducer and activator of transcription (STAT) 1 was detected by an in-cell western assay and the amount of cellular bound IFN-γ was analysed by a high sensitivity ELISA. We observed that IFN-γ induced CXCL9 and CXCL10 production in MCF-7, SK-BR-3 and MDA-MB-231 cells but not in BT-474. UFH dose dependently inhibited the effect of IFN-γ on the secretion and expression of CXCL9 and CXCL10. LMWHs and heparin-related compounds differentially modulated IFN-γ-effects-the results depended on their molecular size and charge, but were independent of their anticoagulatory properties. As a reason for these heparin effects, we could show that the IFN-γ-induced phosphorylation of STAT1 was modulated by heparins, caused by an interaction with the cellular binding of IFN-γ. In conclusion, these results support the significance of the immunomodulatory properties of heparins independently of their classical anticoagulatory function. Heparin-derived sulphated polysaccharides with distinct molecular properties might thus be interesting candidates for new therapeutic strategies in breast cancer.

Macrophages Promote Fibroblast Growth Factor Receptor-Driven Tumor Cell Migration and Invasion in a Cxcr2-Dependent Manner

Infiltration of immune cells, specifically macrophages, into the tumor microenvironment has been linked to increased mammary tumor formation and progression. Activation of growth factor receptor signaling pathways within mammary epithelial cells, such as the fibroblast growth factor receptor 1 (FGFR1) pathway, induces recruitment of macrophages to the mammary epithelium. These macrophages promote increased epithelial cell proliferation and angiogenesis. However, the specific mechanisms by which these macrophages are regulated by the preneoplastic epithelial cells and the mechanisms of action of the macrophages within the developing FGFR1-driven tumor microenvironment remain unknown. In this study, we show that activation of inducible FGFR1 in mammary glands leads to decreased activity of the TGFβ/Smad3 pathway in macrophages associated with early stage lesions. Further studies show that macrophages have increased expression of inflammatory chemokines that bind Cxcr2 following exposure to conditioned media from mammary epithelial and tumor cells in which the FGF pathway had been activated. The increase in these ligands is inhibited following activation of the TGFβ pathway, suggesting that decreased TGFβ signaling contributes to the upregulation of these chemokines. Using coculture studies, we further show that macrophages are capable of promoting epithelial and tumor cell migration and invasion through activation of Cxcr2. These results indicate that macrophage-derived Cxcr2 ligands may be important for promoting mammary tumor formation regulated by FGFR signaling. Furthermore, these results suggest that targeting Cxcr2 may represent a novel therapeutic strategy for breast cancers that are associated with high levels of infiltrating macrophages.

Das Mammakarzinom - Therapiestandards und neue Therapiemöglichkeiten 2012, ein onkologisches Review

Breast cancer is the leading cancer in women with an annual incidence rate of 110/100.000 women in all age groups in Europe. The incidence rate increases in older women. Treatment includes surgery, radiotherapy, chemotherapy with or without antibody therapy and hormonal treatment. During the last 40 years the survival rate has significantly improved. From the 1970ies to 2010 the five year survival rate has risen from 85% to 98% early breast cancer. Similarly, in metastatic breast cancer 5-year survival has increased from below 10% in 1970 to 26% in 2010. This was possible because of better treatment options and better screening causing earlier diagnosis of breast cancer. This article reviews the standard treatment of breast cancer according to ESMO/ASCO guidelines and discusses new treatment options and new therapeutic strategies for breast cancer.

1660P - Epigenetic Silencing of Arginino-Succinate Synthase (ASS1) Defines Arginine Depletion Therapy as a Novel Treatment Strategy for Breast Cancer

ABSTRACT Background Loss of argininosuccinate synthetase (ASS1) or lyase (ASL) expression, critical for the biosynthesis of arginine in normal tissues, due to methylation-dependent silencing sensitises ovarian or glioblastoma cells to arginine depletion therapy (ADT). Cells not expressing sufficient levels of ASS1 or ASL become auxotrophic for arginine and require exogenous supply. Arginine deiminase (ADI), a novel arginine-degrading enzyme, can eliminate arginine from the circulation and has shown activity in cancers with low ASS1 and/or ASL activity. This study was performed to establish whether ASS1 or ASL are subject to methylation-dependent silencing in breast cancer to validate ADT as a novel therapeutic strategy for breast cancer. Methods Methylation of ASS1 and/or ASL was analysed by pyrosequencing and methylation-specific PCR (MSP) using primer sets validated by bisulphite sequencing. We used a panel of 19 breast cancer cell lines and two independent series of stage I-III primary breast cancers with linked mature clinical outcome that were randomly selected from the Tayside (n = 224) and Cuneo (n = 150) Tissue Banks. Tissue samples were subject to histopathological review to ensure adequate representation of cancer cells. Results Although ASS1 or ASL methylation could not be verified in breast cancer cell lines, ASS1 methylation was evident in a significant proportion of primary or metastatic breast cancer samples. In two independent series, aberrant methylation of ASS1 was detected in 4.5% and 16% of primary breast cancers. The incidence was even higher in metastatic breast cancer, with 47% of resected brain metastases (n = 19) showing methylation of ASS1 and 26% showing additional methylation of ASL, a combination that has shown to be highly sensitive to ADT. ASS1 methylation status as called by MSP agreed with that determined by pyrosequencing in all cases assessed by both methods. No significant association with a specific tumour type or outcome was found. Conclusions A significant proportion of primary and metastatic breast cancers show methylation-dependent transcriptional silencing of ASS1 or ASL and might be candidates for arginine depletion therapy. Disclosure All authors have declared no conflicts of interest.

Targeting the PELP1-KDM1 axis as a potential therapeutic strategy for breast cancer

IntroductionThe estrogen receptor (ER) co-regulator proline glutamic acid and leucine-rich protein 1 (PELP1) is a proto-oncogene that modulates epigenetic changes on ER target gene promoters via interactions with lysine-specific histone demethylase 1 (KDM1). In this study, we assessed the therapeutic potential of targeting the PELP1-KDM1 axis in vivo using liposomal (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DOPC) siRNA to downregulate PELP1 expression and KDM1 inhibitors, pargyline and N-((1S)-3-(3-(trans-2-aminocyclopropyl)phenoxy)-1-(benzylcarbamoyl)propyl)benzamide using preclinical models.MethodsPreclinical xenograft models were used to test the efficacy of drugs in vivo. Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end-labeling immunohistochemical analysis of epigenetic markers was performed on tumor tissues. The in vitro effect of PELP1-KDM axis blockers was tested using proliferation, reporter gene, chromatin immunoprecipitation and real-time RT-PCR assays. The efficacy of the KDM1 targeting drugs alone or in combination with letrozole and tamoxifen was tested using therapy-resistant model cells.ResultsTreatment of ER-positive xenograft-based breast tumors with PELP1-siRNA-DOPC or pargyline reduced tumor volume by 58.6% and 62%, respectively. In a postmenopausal model, in which tumor growth is stimulated solely by local estrogen synthesis, daily pargyline treatment reduced tumor volume by 78%. Immunohistochemical analysis of excised tumors revealed a combined decrease in cellular proliferation, induction of apoptosis and upregulation of inhibitory epigenetic modifications. Pharmacological inhibition of KDM1 in vitro increased inhibitory histone mark dimethylation of histone H3 at lysine 9 (H3K9me2) and decreased histone activation mark acetylation of H3K9 (H3K9Ac) on ER target gene promoters. Combining KDM1 targeting drugs with current endocrine therapies substantially impeded growth and restored sensitivity of therapy-resistant breast cancer cells to treatment.ConclusionOur results suggest inhibition of PELP1-KDM1-mediated histone modifications as a potential therapeutic strategy for blocking breast cancer progression and therapy resistance.

The role of AIB1 in breast cancer

Amplified in breast cancer 1 (AIB1) is a member of the p160 steroid receptor coactivator family that mediates the transcriptional activities of nuclear receptors including estrogen receptor (ER) and progesterone receptor (PR), as well as certain other transcription factors, including E2F1 and p53. AIB1 is widely implicated in nuclear receptor-mediated diseases, particularly malignant diseases, including breast, prostate, gastric and pancreatic cancers. AIB1 was initially implicated in hormone-dependent breast cancer, where increasing levels of AIB1 mRNA and protein were detected in some of these specimens and the overexpression of AIB1 in mice led to an increased incidence of tumors. More recent studies revealed that AIB1 also affects the growth of hormone-independent breast cancer via signaling pathways such as those of E2F1, IGF-I, EGF and PI3K/Akt/mTOR. The pleiotropic effect of AIB1 and the roles it plays in both normal development and cancer have presented a great challenge to formulating an effective therapeutic strategy for breast cancer. In this review, we highlight the significant progress made with the recent findings and present an overview of the current understanding of the influence of AIB1 on breast cancer via hormone-dependent and -independent signaling pathways.

992 Targeting ErbB2 (neu) Using Transmembrane Peptides is a Promising Novel Therapeutic Strategy for Breast Cancer

992 Targeting ErbB2 (neu) Using Transmembrane Peptides is a Promising Novel Therapeutic Strategy for Breast Cancer

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 LB-198: Ryl hydrocarbon receptor agonist Aminoflavone disrupts mammosphere formation via alpha 6 integrin inhibition

Abstract Introduction: Emerging evidence has suggested that cancer stem cells (CSCs) play a prominent role in the initiation and dissemination of a myriad of malignancies including breast cancer. CSCs have the capacity to self-renew within heterogeneous tumor cells. Currently available therapeutic options for breast cancer are incapable of suppressing CSC growth. Alpha 6 integrin contributes to CSC growth while aryl hydrocarbon receptor (AhR) activation impedes CSC proliferation. AhR agonist Aminoflavone, a synthetic flavonoid, is currently undergoing clinical evaluation for the treatment of breast cancer. We hypothesize that Aminoflavone alters the integrity and formation of cell suspension clusters initiated by breast cancer stem cells (mammospheres) by modulating alpha 6 integrin expression in an AhR-dependent manner Experimental Procedures: Mammospheres derived from MCF-7 breast cancer cells were exposed to Aminoflavone at varying times. We also treated mammospheres with Tamoxifen an anticancer agent frequently used in the treatment of breast cancer. Following Aminoflavone treatment, cells were analyzed microscopically to evaluate alterations in mammosphere structure. In addition, the expression of molecular targets associated with CSC growth in addition to alpha 6 integrin were evaluated by Western blotting and quantitative RT-PCR analysis. Results: We observed that Aminoflavone, but not Tamoxifen, disaggregates MCF-7 mammospheres. In contrast, Aminoflavone did not affect mammospheres produced from AhR defective MCF-7 (AHR100) cells. Aminoflavone abrogated the ability of secondary mammospheres to form. In addition, alpha-6-integrin expression was abolished following Aminoflavone treatment in MCF-7 but not AHR100 cells. The expression of several genes whose products regulate alpha-6 integrin expression were also modulated following Aminoflavone treatment in MCF-7 cells. Conclusion: These data suggest that Aminoflavone disrupts mammosphere formation via AhR signaling activation that results in alpha 6 integrin inhibition. These findings support continued efforts to develop novel AhR ligands with efficacy against breast CSCs as a viable therapeutic strategy for 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 LB-198. doi:1538-7445.AM2012-LB-198

Abstract 3411: A pilot feasibility study to evaluate the efficacy of lapatinib in the elimination of HER2-positive circulating tumor cells in peripheral blood of women with metastatic breast cancer

Abstract Background: The monitoring of circulating tumor cells (CTCs) detected in peripheral blood (PB) of patients (pts) with metastatic breast cancer (BC) has been proven a potent prognostic tool. CTCs detected after the completion of therapy are theoretically chemo- and/or hormone-resistant and could lead to disease progression. EGFR and HER2 expression has been demonstrated in CTCs from pts with BC. In the present trial we aimed to evaluate the efficacy of lapatinib, a dual EGFR and HER2 tyrosine kinase inhibitor, in eliminating HER2 positive (+) CTCs of women with metastatic BC. Pts and methods: Pts with metastatic BC and HER2+ CTCs detected despite disease stabilization or response after the completion of prior therapy were eligible. Pts received lapatinib, 1500 mg/day orally till disease or CTC progression, whichever occurred first. HER2 expression was evaluated in cytospins of peripheral blood mononuclear cells’ (PBMCs) prepared from PB obtained at baseline and monthly thereafter. Cytospins were stained with HER2 along with cytokeratin antibody; a total of 10^6 PBMCs/pt were analyzed by double immunofluorescence. Results: Nineteen pts were enrolled from September 2008 to October 2011. Median age was 62 years, 12 (63.2%) pts were post-menopausal and 2 had HER2+ tumors. Twelve (63.2%) pts had visceral disease and 7 (36.8%) had received β3 lines of treatment. One pt discontinued treatment prior to the completion of the first cycle due to toxicity and 2 pts are not evaluable yet. The median duration of treatment was 4.0 (range, 0.5 - 34.0) months (mo). CTC counts declined in 15 out of 16 pts during treatment with a maximum percentage of decrease of 100% in all of them. The median time to and the median duration of nadir CTC counts were 1 mo (range, 1 - 3 mo) and 1 mo (range, 0 - 7 mo), respectively. Prior to the second treatment cycle, a decrease in CTC counts was observed in 14 (88%) pts, in 1 (7%) the CTC count increased by 33% and in 1 (7%) remained stable. Median percentage of CTC count decrease per pt was 100% (range, 45-100%). At baseline, a total of 2642 HER2+ CTCs were detected (median: 79/pt, range 2 - 617), whereas after the completion of first cycle a total of 430 HER2+ CTCs were identified (median: 0, range 0 - 223). Disease evaluation revealed stable disease in 5 pts and progressive disease in 11. In pts with stable disease a sustained decrease in CTC counts over 2 to 7 mo was observed. On the contrary, in 9 out of 11 pts with disease progression, the decrease in CTC counts was short-lasting (median 1.0 mo, range 0 - 4). Conclusions: Lapatinib is effective in decreasing HER2+ CTCs in pts with metastatic BC, irrespective of the HER2 status of the primary tumor. Sustained CTC reduction was associated with disease stabilization. The above results suggest that tailoring therapy according to targets present on CTCs could be an effective therapeutic strategy in BC. 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 3411. doi:1538-7445.AM2012-3411

Progesterone receptor B through Sp1 induces the phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene promoter activity and cell death in breast cancer cell lines

Phosphatase and tensin homolog on chromosome 10 (PTEN) is a tumor suppressor attracting considerable attention as new target for effective therapeutic strategies in breast cancer. The antitumors effect of progesterone were envisaged, however the mechanism thorough which this occurs was not clearly established. In the present study we report that OHPg, through the PR‐B isoform, produces a significant inhibition of MCF‐7 breast cancer cell survival with a concomitant increase of PTEN protein, mRNA and gene promoter activity. By using different deleted constructs of the PTEN promoter, we found that the OHPg failed to enhance the transcriptional activity of the PTEN promoter lacking the region between −1398 bp to −1118 bp. This DNA sequence contains several DNA motifs known to bind the transcription factors Sp1that has been shown to interact with PRs. In our experimental conditions mithramycin, prevented the PTEN promoter transactivation on OHPg treatment. ChIP and EMSA analyses showed specific recruitment of PR, Sp1 and RNA polymerase II upon OHPg treatment at PTEN promoter. The persistence of such effects in T47 cells which express high PR levels, addresses how OHPg/PR signalling may be considered as useful pharmacological tool to be exploited for innovative adjuvant strategies in breast cancer treatment.

P2-01-26: TIMPv: a novel downstream transcriptional target gene that underpins CD146-suppressed breast tumor invasion

Abstract Background: CD146, a marker of endothelial cells, promotes tumor progression of many cancers including melanoma and prostate. Strikingly, several lines of evidence suggest that it is a suppressor of breast cancer (BC) progression. Not only the ligand(s) has not been identified, but CD146-downstream signaling mechanisms remain unknown. Material and Methods: Here, we report the functional importance of CD146 and that of a novel transcriptional target of CD146-signaling, variant of tissue inhibitor of metalloproteinases (TIMPv) identified by microarray analysis, in underpinning the suppression of BC invasion, using novel validated Enhanced Green Fluorescent Protein (EGFP)-inducible systems of CD146 expression both in vitro and in vivo. Results: In functional experiments, induction of CD146 inhibited BC cell migration and invasion. TIMPv was identified by expression profiling as a novel transcriptional target of CD146-signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast and melanoma cancer cells. However, siRNA inhibition of CD146 in the SKMel-28 melanoma cell line increased TIMPv expression, suggesting that while TIMPv is a positive transcriptional target of CD146 in BC cells, it is negatively regulated in melanoma cells. Furthermore, the functional relevance of TIMPv to CD146-suppressed metastasis was demonstrated by selective suppression of TIMPv in CD146-expressing BC inducible cells using RNAi. More interestingly, induction of CD146 expression in vivo using mouse EGFP-inducible system of CD146 expression resulted in suppression of breast tumor growth. Discussion: Our study is the first report to provide a functional molecular link of TIMPv to cancer via unique axis that underpin CD146-suppressed BC progression, thus identifying TIMPv as a potential target for guiding the development of novel therapeutic strategies for BC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-26.

Abstract B46: Effective therapeutic response of MDA-MB-231 cell line to rapamycin.

Abstract The assessment of alternative therapeutics agents can develop more effective treatments in breast cancer, resulting in molecular-targeted drugs, developed from the knowledge of molecules involved in specific pathways that provide growth and progression of cancer. A large number of studies have suggested that immunosuppressive agents can act as powerful anti-cancer drugs. Rapamycin, a potent immunosupressor used routinely in transplant patients, also has antiproliferative, antifungal and antimicrobial activity. Recent studies show that rapamycin can alter the function of blood vessels by promoting thrombosis in tumor vessels and thus prevents the formation of tumor vasculature. In addition, experiments have focused on the antiangiogenic effects, antiproliferative and antimetastatic target protein inhibitors of rapamycin (mTOR), affecting the expression of genes involved in metabolism and protein synthesis. Thus, believing in the inhibitory effect of rapamycin in tumor progression in diferent tumor types, this study evaluated its antitumor activity in mammary tumoral cell line. Samples of breast cancer cell line, MDA-MB-231 (estrogen receptor negative) were cultured in medium DMEM with high glucose concentration (Cultilab), and incubated at 37° C in 5% CO2. All culture medium used was enriched with 20% BSA, 1% penicillin/streptomycin and 1% L-glutamine. It was established five groups, four of them received different concentrations of rapamycin (group I - 1nM, group II - 10nM, group III - 50nM and group IV - 100nM) and one group was classified as control group (control - group V). After the interval of 72 hours of application of rapamycin, cells were stained with Trypan Blue (cell vitality). The growth curve was performed by counting cells in a Neubauer chamber, in order to evaluate the effects of drug. After 72 hours, the group V contained 200 cells. In the treated groups I and II, there was a small decrease in cell number, reaching 180 and 150 cells, respectively. In group III, there was a reduction in cell number reached 118 cells and in group IV was observed 50% reduction in the number of cells compared to group V. These results prove that rapamycin is effective in controlling cell proliferation and causes a decrease in the number of cells. In addition, the action of rapamycin may be dose dependent, as increasing the dose applied in the same cell concentration there was a decrease in the amount of cells. This fact suggests that rapamycin can be a possible alternative to the establishment of therapeutic strategies for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B46.

Abstract C239: Progesterone receptor B through Sp1 induces the phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene promoter activity and cell death in breast cancer cell lines.

Abstract Phosphatase and tensin homolog on chromosome 10 (PTEN) is a tumor suppressor attracting considerable attention as new target for effective therapeutic strategies in breast cancer. While recent studies suggest the down-regulation of PTEN by Estrogen Receptor alpha, the role of Progesterone Receptor (PR) is not clearly established. In the present study we report that PR produces a significant inhibition of MCF-7 breast cancer cell survival with a concomitant increase of PTEN protein, mRNA and gene promoter activity. On the contrary PRs failed to enhance the transcriptional activity of the PTEN promoter lacking the region between −1398 bp to −1118 bp. By analyzing this DNA sequence to search potential binding sites for transcription factors, we observed that this region contains several DNA motifs known to bind the transcription factors Sp1that has been shown to interact with PRs, activating specific gene transcription. In our experimental conditions mithramycin, prevented the PTEN promoter transactivation on OHPg treatment. ChIP analyses showed specific recruitment of PR, Sp1 and RNA polymerase II upon progesterone treatment at PTEN promoter. The persistence of such effects in T47 cells which express high PR levels, addresses how PR/PTEN signalling may be considered as useful pharmacological tool to be exploited for innovative adjuvant strategies in breast cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C239.

P53-Binding protein 1: A new player for tumorigenesis and a new target for breast cancer treatment

Breast cancer remains the most common and fatal cancer in women and has been recognized as a genetic disease. Recently, accumulating evidences have showed that p53-binding protein 1 (53BP1) plays an important role in DNA double-strand breaks (DSBs) repair induced by radiation. In vitro experiments have indicated its interaction with many other genes or proteins for tumor suppression or tumorigenesis via pathways associated with DNA repair, cell-cycle control, apoptosis and cell senescence. In vivo studies also showed suppressive effect of 53BP1 on tumor initiation and progression. Therefore, we hypothesize that 53BP1 has a profound effect on suppressing breast cancer as a tumor suppressor and will be an important new biomarker for breast cancer prognosis. Furthermore, 53BP1 gene therapy will be a potential therapeutic strategy for breast cancer.

LDH-A silencing suppresses breast cancer tumorigenicity through induction of oxidative stress mediated mitochondrial pathway apoptosis

LDH-A, as the critical enzyme accounting for the transformation from pyruvate into lactate, has been demonstrated to be highly expressed in various cancer cells and its silencing has also been approved relating to increased apoptosis in lymphoma cells. In this study, we intend to investigate the correlation between LDH-A and other clinicopathological factors of breast cancer and whether LDH-A silencing could suppress breast cancer growth, and if so the potential mechanisms. 46 breast cancer specimens were collected to study the relation between LDH-A expression and clinicopathological characteristics including menopause, tumor size, node involvement, differentiation, and pathological subtypes classified by ER, PR, and Her-2. shRNAs were designed and applied to silence LDH-A expression in breast cancer cell lines MCF-7 and MDA-MB-231. The effects of LDH-A reduction on cancer cells were studied by a series of in vitro and in vivo experiments, including cell growth assay, apoptosis evaluation, oxidative stress detection, transmission electron microscopy observation, and tumor formation assay on nude mice. LDH-A expression was found to correlate significantly with tumor size and to be independent for other clinicopathological factors. LDH-A reduction resulted in an inhibited cancer cell proliferation, elevated intracellular oxidative stress, and induction of mitochondrial pathway apoptosis. Meanwhile, the tumorigenic ability of LDH-A deficient cancer cells was significantly limited in both breast cancer xenografts. The Ki67 positive cancer cells were significantly reduced in LDH-A deficiency tumor samples, while the apoptosis ratio was enhanced. Our results suggested that LDH-A inhibition might offer a promising therapeutic strategy for breast cancer.

Fertility preservation in women—a practical guide to preservation techniques and therapeutic strategies in breast cancer, Hodgkin’s lymphoma and borderline ovarian tumours by the fertility preservation network FertiPROTEKT

PurposeFertility preservation methods are playing an increasing role in women up to the age of 40 years because of rising survival rates in those affected by cancer. However, balanced practical recommendations concerning all relevant fertility preservation, to support doctors in counselling and treating patients, are still rare.MethodsThese recommendations were prepared by the network FertiPROTEKT (http://www.fertiprotect.eu), a collaboration of around 70 centres in Germany, Switzerland and Austria. The recommendations were developed by specialists in reproductive medicine, reproductive biology and oncology, which gave a comprehensive overview of all named techniques as well as their benefits and risks. Furthermore, practice-orientated recommendations for the individual use of fertility preservation methods for various indications such as breast cancer, Hodgkin’s lymphoma and borderline ovarian tumours are given.ResultsVarious options such as ovarian stimulation and cryopreservation of unfertilised or fertilised oocytes, cryopreservation and transplantation of ovarian tissue, GnRH-agonist administration and transposition of the ovaries can be offered. All the techniques can be performed alone or in combination within a maximum of 2 weeks with low risk and different success rates.ConclusionsFertility preservation in women has become an option with realistic chances to become pregnant after cytotoxic therapies. The information provided allows a well balanced and realistic counselling and treatment.

Inhibition of the Rho GTPase, Rac1, decreases estrogen receptor levels and is a novel therapeutic strategy in breast cancer

Rac1, a Rho GTPase, modulates diverse cellular processes and is hyperactive in some cancers. Estrogen receptor-alpha (ERα) in concert with intracellular signaling pathways regulates genes associated with cell proliferation, tumor development, and breast cancer cell survival. Therefore, we examined the possibility of Rac1 and ERα crosstalk in breast cancer cells. We found that Rac1 enhanced ERα transcriptional activity in breast cancer cells. Vav3, a Rho guanine nucleotide exchange factor that activates Rac1, was an upstream mediator, and P21/Cdc42/Rac1 activating kinase-1 (Pak-1) was a downstream effector of Rac1 enhancement of ERα activity. These results suggest that Rac1 may prove to be a therapeutic target. To test this hypothesis, we used a small molecule Rac inhibitor, EHT 1864, and found that EHT 1864 inhibited ERα transcriptional activity. Furthermore, EHT 1864 inhibited estrogen-induced cell proliferation in breast cancer cells and decreased tamoxifen-resistant breast cancer cell growth. EHT 1864 decreased activity of the promoter of the ERα gene resulting in down-regulation of ERα mRNA and protein levels. Therefore, ERα down-regulation by EHT 1864 is the likely mechanism of EHT 1864-mediated inhibition of ERα activity and estrogen-stimulated breast cancer cell proliferation. Since ERα plays a critical role in the pathogenesis of breast cancer and the Rac inhibitor EHT 1864 down-regulates ERα expression and breast cancer cell proliferation, further investigation of the therapeutic potential of Rac1 targeting in the treatment of breast cancer is warranted.