Factors In Breast Cancer Cells Research Articles

Glucose impairs tamoxifen responsiveness modulating connective tissue growth factor in breast cancer cells.

Type 2 diabetes and obesity are negative prognostic factors in patients with breast cancer (BC). We found that sensitivity to tamoxifen was reduced by 2-fold by 25 mM glucose (High Glucose; HG) compared to 5.5 mM glucose (Low Glucose; LG) in MCF7 BC cells. Shifting from HG to LG ameliorated MCF7 cell responsiveness to tamoxifen. RNA-Sequencing of MCF7 BC cells revealed that cell cycle-related genes were mainly affected by glucose. Connective Tissue Growth Factor (CTGF) was identified as a glucose-induced modulator of cell sensitivity to tamoxifen. Co-culturing MCF7 cells with human adipocytes exposed to HG, enhanced CTGF mRNA levels and reduced tamoxifen responsiveness of BC cells. Inhibition of adipocyte-released IL8 reverted these effects. Interestingly, CTGF immuno-detection in bioptic specimens from women with estrogen receptor positive (ER+) BC correlated with hormone therapy resistance, distant metastases, reduced overall and disease-free survival. Thus, glucose affects tamoxifen responsiveness directly modulating CTGF in BC cells, and indirectly promoting IL8 release by adipocytes.

The Role of Interleukin-8 and Its Mechanism in Patients with Breast Cancer: Its Relation with Oxidative Stress and Estrogen Receptor

Context: Breast cancer is one of the leading causes of death among women all over the world. Prognostic markers can be used for patient survival. Likewise, these biomarkers are able to generate main information related to clinical manner of breast gland tumors. Interleukin-8 (IL-8) is a chemotactic cytokines caused initiation of an inflammatory response. Evidence Acquisition: We performed a computerized search in Medline/PubMed databases with key words: IL-8, breast cancer, and oxidative stress. Results: IL-8 secretion motivates the expression of adhesion molecule, including fibronectin in human inflammatory breast cancer cells. IL-8 activates PI3K/Akt pathway, which in turn activates NF-κB resulted up-regulation of integrin β3 expression and increases the invasion of breast cancer cells. Therefore, it seems that IL-8/PI3K/Akt/NF-κB/integrin β3 axis may be used for therapeutic intervention of breast cancer with metastasis status. NF-κB plays an important role in IL-8 gene transcription via several signals, such as LPS, TNF-alpha, and oxidative stress. Oxidative stress caused angiogenic factor production, such as vascular endothelial growth factor and IL-8 via cancer cells. Moreover, there is inverse relation between ER level and IL-8, but the mechanism of IL-8 action in estrogen receptor-negative breast cancer is largely unknown. Estrogen receptor and growth factor in breast cancer cells is influenced by mitogen activated protein kinase (MAPK) cascade, as a part of IL-8 signaling pathway. Conclusions: According to these studies, interleukin-8 may be as a main biomarker for breast cancer. It seems that signaling pathway of IL-8 may be used for therapeutic intervention of breast cancer. Also oxidative stress and breast cancer cells with lacking estrogen receptor generate more interleukin-8.

Exposure to 60% oxygen promotes migration and upregulates angiogenesis factor secretion in breast cancer cells.

Peri-operative factors, including anaesthetic drugs and techniques, may affect cancer cell biology and clinical recurrence. In breast cancer cells, we demonstrated that sevoflurane promotes migration and angiogenesis in high fractional oxygen but not in air. Follow-up analysis of the peri-operative oxygen fraction trial found an association between high inspired oxygen during cancer surgery and reduced tumor-free survival. Here we evaluated effects of acute, high oxygen exposure on breast cancer cell viability, migration and secretion of angiogenesis factors in vitro. MDA-MB-231 and MCF-7 breast cancer cells were exposed to 21%, 30%, 60%, or 80% v/v O2 for 3 hours. Cell viability at 24 hours was determined by MTT and migration at 24 hours with the Oris™ Cell Migration Assay. Secretion of angiogenesis factors at 24 hours was measured via membrane-based immunoarray. Exposure to 30%, 60% or 80% oxygen did not affect cell viability. Migration of MDA-MB-231 and MCF-7 cells was increased by 60% oxygen (P = 0.012 and P = 0.007, respectively) while 30% oxygen increased migration in MCF-7 cells (P = 0.011). These effects were reversed by dimethyloxaloylglycine. In MDA-MB-231 cells high fractional oxygen increased secretion of angiogenesis factors monocyte chemotactic protein 1, regulated on activation normal T-cell expressed and vascular endothelial growth factor. In MCF-7 cells, interleukin-8, angiogenin and vascular endothelial growth factor secretion was significantly increased by high fractional oxygen. High oxygen exposure stimulates migration and secretion of angiogenesis factors in breast cancer cells in vitro.

VLDL and LDL, but not HDL, promote breast cancer cell proliferation, metastasis and angiogenesis

Abnormal lipoprotein profiles are associated with breast cancer progression. However, the mechanisms linking abnormal lipoprotein levels to breast cancer progression, especially metastasis, remain unclear. Herein, we found that L1 and L5 subfractions of LDL and VLDL, but not HDL, enhanced breast cancer cell viability. L1, L5, and VLDL also increased the in vitro tumorigenesis of breast cancer cells in anchorage-independent soft agar assay. In addition, L1, L5, and VLDL, but not HDL, increased the levels of mesenchymal markers Slug, Vimentin, and β-Catenin, and promoted breast cancer cell migration and invasion. L1, L5, and VLDL increased Akt Ser473 phosphorylation and promoted cell migration, which were reversed by the PI3K/Akt inhibitor wortmannin. Further in vitro angiogenesis assay and cytokine array analysis demonstrated that L1, L5, and VLDL enhanced secretion of angiogenic factors in breast cancer cells and promoted angiogenic activity. However, only VLDL reduced anchorage-dependent cell death and promoted lung metastasis in nude mice. In summary, our data suggest that L1, L5, and especially VLDL promote breast cancer progression and metastasis through Akt-induced EMT and angiogenesis, and provide a novel mechanism of how dyslipoproteinemia promotes breast cancer progression.

Interleukin-30 Promotes Breast Cancer Growth and Progression

The inflammatory tissue microenvironment that promotes the development of breast cancer is not fully understood. Here we report a role for elevated IL30 in supporting the breast cancer cell viability and invasive migration. IL30 was absent in normal mammary ducts, ductules, and acini of histologically normal breast and scanty in the few stromal infiltrating leukocytes. In contrast, IL30 was expressed frequently in breast cancer specimens where it was associated with triple-negative and HER2+ molecular subtypes. In stromal leukocytes found in primary tumors or tumor-draining lymph nodes, which included mainly CD14+ monocytes, CD68+ macrophages, and CD33+/CD11b+ myeloid cells, IL30 levels increased with disease stage and correlated with recurrence. A negative correlation was determined between IL30 expression by nodal stromal leukocytes and overall survival. In vitro studies showed that human recombinant IL30 upregulated expression of a pro-oncogenic program, including especially IL6 in both triple-negative and HER2+ breast cancer cells. In triple-negative breast cancer cells, IL30 boosted a broader program of proliferation, invasive migration, and an inflammatory milieu associated with KISS1-dependent metastasis. Silencing of STAT1/STAT3 signaling hindered the regulation of the primary growth and progression factors in breast cancer cells. IL30 administration in vivo fostered the growth of triple-negative breast cancer by promoting proliferation and vascular dissemination of cancer cells and the accumulation of intratumoral CD11b+/Gr1+ myeloid cell infiltrates. Overall, our results show how IL30 regulates breast cancer cell viability, migration, and gene expression to promote breast cancer growth and progression and its impact on patient outcome. Cancer Res; 76(21); 6218-29. ©2016 AACR.

Abstract P3-06-12: Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?

Abstract Proteasome, the essential giant protease, is the hub of the ubiquitin-proteasome pathway critical for maintaining intracellular proteostasis. Inhibitors of the proteasome cause overload of cells with non-degraded protein debris and activate proapoptotic signaling. The effects are especially dramatic in fast metabolizing cancer cells; hence the proteasome inhibitors are used as anti-cancer drugs. There are two FDA-approved drugs directly blocking the proteasome activity, bortezomib and carfilzomib, and several others are in trials. So far, the high effectiveness of these drugs is limited to multiple myeloma and other hematological malignancies. Unfortunately, breast and other solid cancers are much more resistant to apoptosis triggered by attenuation of the ubiquitin-proteasome pathway than majority of blood cancers. The relatively low effectiveness of proteasome inhibitors has been noted even for cancers that have been identified in genome-wide screens as addicted to the proteasome activity, such as triple negative breast cancers. The source of the resistance, noted in cell culture, animal studies and clinical trials, remains unknown. Here we propose that, at least in part, the resistance can be tracked down to protein-protein interactions between the drug target, the proteasome, and a cytosolic heat shock protein, Hsp72 (inducible Hsp70). Indeed, high levels of Hsp72 have been observed in many cancers, including breast, and generally correlate with poor prognosis. The intracellular roles of the ubiquitous chaperone are undoubtedly very diverse, however we postulate a novel molecular action: direct protection of the proteasome from competitive inhibitors of its enzymatic activity. The hypothesis has been inspired by our recent work exploring the very effective ubiquitin-proteasome pathway in the naked mole rats. These small African rodents do not develop cancers and maintain unusually long lifespan and health-span. Interestingly, the proteasome in naked mole rats is exceptionally resistant to inhibition. We discovered that the resistance is bestowed by a cytosolic protein factor containing Hsp72 and Hsp40 chaperones (Biochim. Biophys. Acta-MBD 1842; 2060-2072; 2014). We partially purified the factor and tested its in vitro effectiveness with human proteasomes. The molecular mechanism beneficial for naked mole rats may be also protective for human cancer cells. We used pifithrin μ, a specific small molecule inhibitor blocking the interactions of Hsp72 with other proteins, to test the hypothesis. We prepared lysates from cultured triple negative breast cancer cells MDA-MB-231. Such lysates are rich in proteasome activity and preserve interactions of the proteasomes with natural protein ligands. Interestingly, upon the treatment with pifithrin μ proteasomes in lysates maintained their high activity while becoming significantly more sensitive to inhibition with bortezomib. The studies are in progress to assess the identity of a putative chaperone-based proteasome resistance factor in breast cancer cells. Citation Format: Gaczynska M, Osmulski PA. Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-12.

Bone Morphogenetic Protein-7 Inhibits EMT-Associated Genes in Breast Cancer

Background/Aims: Bone morphogenetic protein-7 (BMP7) has been shown to reduce the severity of injury-induced fibrosis through counteracting the fibrotic effects of transforming growth factor β 1 (TGFβ1). However, this model in the carcinogenesis of breast cancer is unknown. Methods: We analyzed the effects of BMP7 and TGFβ1 on gene transcripts and protein levels of EMT-related factors in breast cancer cells by RT-qPCR and Western blot, respectively. The effects of BMP7 and TGFβ1 on cell invasiveness and migration were evaluated by scratch wound healing assay and transwell cell migration assay. The cell growth was measured by MTT assay. Results: BMP7 did not alter the TGFβ1-stimulated phosphorylation of TGFβ receptor, but significantly inhibited the TGFβ1-activated epithelial-mesenchymal transition (EMT)-related genes in breast cancer cells, resulting in a significant reduction in TGFβ1-triggered cell growth and cell metastasis. Conclusion: Our data suggest that besides being a well-known antagonist for TGFβ1 in fibrosis, BMP7 may also antagonize TGFβ1 in tumorigenesis-associated EMT in breast cancer. Thus, BMP7 may be a promising therapeutic target for treating breast cancer.

Overexpression of granulocyte macrophage colony stimulating factor in breast cancer cells leads towards drug sensitization.

This report describes the effect of overexpressing granulocyte macrophage colony stimulating factor (GMCSF) in breast cancer cells, which otherwise is involved in proliferation and differentiation of granulocyte and macrophage lineages. The purified recombinant GMCSF cytokine is known to exert dose-dependent proliferative response on various cancer cells, but its effect during overexpression is yet to be evaluated. In our present study, we have generated MCF-7 (breast cancer) cells overexpressing GMCSF. Interestingly, cell viability studies showed pronounced sensitivity of GMCSF overexpressing MCF-7 cells towards anticancer drugs, such as, doxorubicin, 5FU and cisplatin. These findings were substantiated by cell cycle analysis of the drug-treated GMCSF overexpressing MCF-7 cells. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) results revealed differential expressions of cyclins, and the carboxyfluorescein succinimidyl ester (CFSE)-based assay established decrease in doubling time of GMCSF overexpressed cells with respect to the control populations. Thus, overexpressing of proliferative GMCSF cytokine in breast cancer cells may increase susceptibility to anticancer drugs.

Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells.

Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

Calcitriol reduces thrombospondin-1 and increases vascular endothelial growth factor in breast cancer cells: Implications for tumor angiogenesis

Calcitriol, a potent antineoplastic vitamin D metabolite, inhibits proliferation, induces apoptosis and slows the growth of tumors. Calcitriol also may exert either antiangiogenic or proangiogenic effects depending on the tissue. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (Tsp-1) are key factors involved in promoting and inhibiting angiogenesis, respectively. The effects of calcitriol on Tsp-1 have not been studied in the mammary gland, while VEGF regulation is not clear, since opposite outcomes have been demonstrated. Therefore, the present study was undertaken to investigate the effects of calcitriol on VEGF and Tsp-1 expression in primary breast tumor-derived cells and a panel of established breast cancer cell lines. In vivo studies in athymic mice were also performed in order to gain further insight into the biological effects of calcitriol on angiogenesis.Real time-PCR and ELISA analyses showed that calcitriol stimulated VEGF mRNA expression and protein secretion while elicited the opposite effect on Tsp-1 in 7 out of 8 cell lines studied, independently of the cell phenotype (P<0.05 in n=5). In vivo, calcitriol significantly inhibited the relative tumoral volume after 4 weeks of treatment; however, serum VEGF was higher in calcitriol-treated animals compared to controls (P<0.05). The integrated fluorescence intensity analysis of CD31, a vessel marker, showed that xenografted breast cancer cells developed tumors with similar vascular density regardless of the treatment. Nevertheless, larger necrotic areas were observed in the tumors of calcitriol-treated mice compared to controls. Since the antineoplastic activity of calcitriol has been consistently demonstrated in several studies including this one, our results suggest that the antitumoral effect of calcitriol in vivo involve different mechanisms not necessarily related to the inhibition of tumor vascularization. Overall, our findings indicate that calcitriol can impact the angiogenic process in breast cancer by regulating VEGF and Tsp-1 expression.This article is part of a Special Issue entitled ‘16th Vitamin D Workshop’.

Epigenetic modifications of prostate-derived Ets transcription factor in breast cancer cells

The importance of epigenetic alterations such as DNA methylation, histone modification and nucleosome remodeling in breast cancer is well established. Epigenetic alterations are reversible, and much research has been focused on understanding these alterations with the aim of developing effective therapies. Prostate-derived Ets factor (PDEF) is a member of the Ets family of transcription factors and has long been under investigation for its key role in tumor development and progression. To date, no studies have been conducted to elucidate the epigenetic modifications of PDEF in cancer progression. Using breast and prostate cancer cells, we investigated the effect of the methylation inhibitor 5' azacytidine (AZA) on the expression of PDEF in these cells. The inhibition of methylation observed was specific to breast cancer cells as experiments with prostate cells did not exhibit any significant change. Notably, the expression of p21, a cyclin-dependent kinase (CDK) inhibitor 1 and also a target gene of PDEF, was found to be positively correlated with PDEF expression following 5'AZA treatment. Inhibition of methylation led to a decrease in the proliferation rate of MDA-MB-468 cells as revealed by MTT proliferation assay. Other epigenetic alterations such as histone modifications were not observed in these breast cancer cells following treatment with specific HDAC inhibitors. Our data suggest the possibility of epigenetic modification of PDEF due to DNA methylation and involvement of the cell cycle inhibitor p21. Future studies on the epigenetic alterations of PDEF in correlation with p21 or other targets may facilitate the development of effective therapies for the treatment of breast cancer.

Effect of estrogen and tamoxifen on the expression pattern of AP-1 factors in MCF-7 cells: role of c-Jun, c-Fos, and Fra-1 in cell cycle regulation

The activated transcription factor ERα plays an important role in the breast development and progression of cancer. In a non-classical pathway ER interacts with other transcription factors AP-1, NFkB, SP1, etc. AP-1 transcription factors control rapid responses of mammalian cells to stimuli that impact proliferation, differentiation, and transformation. AP-1 factors are leucine zipper proteins belonging to members of the Jun family (c-Jun, JunB, and JunD) and Fos family (c-Fos, FosB, Fra-1, and Fra-2) proteins. Although AP-1 factors are well characterized, not much is known about the expression pattern of the AP-1 factors in breast cancer cells. Hence to determine which AP-1 factors are expressed and regulated by estrogen, we used human breast cancer MCF-7 cells as in vitro model system. The MCF-7 cells were treated with or without estradiol-17β (E2) or antiestrogen tamoxifen (TMX) and the cell proliferation and viability was assessed by MTT assay. The expression of different AP-1 factors was analyzed by semi-quantitative RT-PCR. The cells treated with E2 found to increase the cell proliferation by more than 35 % and TMX an antiestrogen decreased by 29 % compared to control. The E2 found to induce the expression of c-Jun, Fra-1, and c-Fos, while TMX decreased the expression. In addition TMX also decreased the mRNA levels of Jun-D and Fra-2. These results suggest that the AP-1 factors c-Jun, c-Fos, and Fra-1 may be involved in the proliferation and transformation of MCF-7 cells. E2 also found to induce cyclin D1 and cyclin E1 mRNA transcripts of cell cycle regulators while TMX significantly decreased compared to control. Further E2 induced the anti-apoptotic Bcl-2 and TMX decreased mRNA transcripts. The data presented here support the E2-ERα-mediated MCF-7 cell proliferation and confirms the role of AP-1 factors in cell cycle regulation.

Analysis of survivin, XIAP and FoxM1 as potential chemoresistance factors in breast cancer cells

Materials and methods Human breast carcinoma cell lines MCF7 (wild-type p53) and MDA-MB-231 (mutant p53) were exposed to dox and citotoxicity was assessed through the MTT assay. Apoptosis was detected through analysis of flow cytometry DNA content and caspases-3, -7 and -9 levels. Survivin, XIAP, p53 and FoxM1 levels were assessed by Western blotting and Survivin and XIAP mRNA levels, by Real Time PCR. Transfections with the Survivin-encoding plasmid and siRNA against Survivin and XIAP were performed using Lipofectamine reagent.

Effects of PPARγ agonists on the expression of leptin and vascular endothelial growth factor in breast cancer cells

The obesity hormone leptin has been implicated in breast cancer development. Breast cancer cells express the leptin receptor and are able to synthesize leptin in response to obesity-related stimuli. Furthermore, leptin is a positive regulator of vascular endothelial growth factor (VEGF) and high levels of both proteins are associated with worse prognosis in breast cancer patients. Peroxisome proliferator-activated receptor γ (PPARγ) ligands are therapeutic agents used in patient with Type 2 diabetes and obesity which have recently been studied for their potential anti-tumor effect. Here, we studied if these compounds, ciglitazone and GW1929, can affect the expression of leptin and VEGF in breast cancer cells. In MDA-MB-231 and MCF-7 breast cancer cells, treatment with submolar concentrations of ciglitazone and GW1929 elevated the expression of leptin and VEGF mRNA and protein, and increased cell viability and migration. These effects coincided with increased recruitment of PPARγ to the proximal leptin promoter and decreased association of a transcriptional factor Sp1 with this DNA region.

Integrating leptin and cAMP signalling pathways in triple-negative breast cancer cells

Breast cancer is a major cause of cancer death in women in the world. Triple-negative breast cancers, which accounts for 10-20% of all mammary tumours, are characterised by an aggressive phenotype, are often found in younger women and have been associated with poor prognosis. Obesity increases the risk for triple-negative breast cancer occurrence. Because triple-negative breast cancer patients are unresponsive to current targeted therapies and other treatment options are only partially effective, new pharmacological approaches are warranted. The obesity-linked adipokine, leptin, is a well known mitogen/survival factor in breast cancer cells and several studies have addressed the role of leptin in breast cancer pathogenesis and progression. Surprisingly, recent in vitro studies have shown that leptin enhances the anti-proliferative effects of cAMP elevation in triple-negative breast cancer cells by apoptosis induction. In the current review, we discuss on the role of cAMP as a growth suppressor and of leptin as a growth promoting factor in breast cancer cells and we will focus on the molecular pathways involved in the antiproliferative interaction between leptin and cAMP elevation. The rationale for the possible development of a simple, cheap and innovative approach for therapeutic intervention in triple-negative breast cancer, based on the use of cAMP elevating drugs at lower and tolerable doses, will be also discussed.

Abstract PD03-05: Analysis of tumour cell signaling in response to neoadjuvant metformin in women with early stage breast cancer.

Abstract Background: The anti-diabetic drug metformin, commonly used to treat type 2 diabetes due to its ability to reduce circulating glucose and insulin, has emerged as a potential anti-cancer agent. Observational studies have reported decreased cancer incidence and mortality in diabetics receiving metformin. Metformin's ability to reduce insulin may be particularly important for breast cancer (BC) because hyperinsulinemia is an adverse prognostic factor and most cells express the insulin receptor (IR). The anti-cancer effects of metformin are associated with both direct (insulin-independent) and indirect (insulin-dependent) actions. Direct effects are linked to activation of AMPK and an inhibition of mTOR signalling, while indirect effects are mediated by reductions in circulating insulin levels, leading to reduced IR-activated PI3K signalling. We conducted a neoadjuvant, single arm, “window of opportunity” trial examining the clinical and biological effects of metformin on thirty-nine locoregional BC patients awaiting definitive surgery. Methods: Non-diabetic women with newly diagnosed, untreated BC were given metformin 500 mg tid for ≥2 weeks post diagnostic core biopsy until surgery. Fasting blood and tumour samples were collected at diagnosis and surgery. Blood glucose and insulin were assayed to assess the physiologic effects of metformin, while IHC analysis of tumours was used to characterize cellular markers before and after metformin. Specifically, IR levels and the phosphorylation status of proteins involved in AMPK and PI3K/AKT/mTOR signalling, including AMPK (T172) and AKT (S473), were examined. Results: 39 patients with a mean age of 51 years received metformin for a median of 18 days (range 13–40) with minor GI toxicities. The clinical effects (previously reported) included significant (p &amp;lt; 0.05) decreases in body mass index (−0.5 kg/m2), weight (−1.2 kg), glucose (−0.14 mM) and HOMA (an estimate of insulin resistance, −0.21), and a decrease in insulin (−4.7 pmol/L) that approached significance (p = 0.0686). Ki67 staining in tumour tissue decreased significantly and TUNEL increased significantly. Levels of IR expression decreased significantly (from 4.39 to 3.82, p = 0.0375) as did the phosphorylation status of AKT (S473) and AMPK (T172) (from 9.82 to 7.08, p = &amp;lt;0.0001; from 6.2 to 5.1, p = 0.0034, respectively). Conclusions: Metformin impact was consistent with beneficial anti-cancer effects. Reduced AKT phosphorylation, coupled with decreased insulin and IR levels, suggest insulin-dependent effects are important in the clinical setting. Assessment of additional factors in BC cells, including OCT1 expression (required for metformin uptake), and the phosphorylation of ACC (a marker of AMPK activation), is underway and will be reported. Integrated analysis of these factors combined with the physiological and molecular data described above will further enhance understanding of metformin action in the clinical setting. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-05.

P01-18 IGFBP-2 acts as a survival factor for breast cancer cells: Role of ER-α

P01-18 IGFBP-2 acts as a survival factor for breast cancer cells: Role of ER-α

Response of angiogenic factors to the treatment with melatonin in breast cancer cell lines.

120 Background: Tumor growth and metastasis are the leading cause of death in breast cancer patients. The tumor growth requires neoangiogenesis, stimulated by vascular endothelial growth factor (VEGF) expressed under control of hypoxia-inducible factor-1α (HIF-1α). Melatonin, a natural hormone has been suggested as a new therapeutic agent to inhibit angiogenesis factors in several types of cancers. We evaluated the HIF-1α and VEGF expression in breast cancer cell line after hypoxia and treatment with melatonin. Methods: Breast cancer cell lines ER-positive (MCF-7) and ER-negative (MDA-MB-231) were cultured in DMEM at 37°C in 5% CO2. The cells were treated with CoCl2 to mimic hypoxia, and then treated with melatonin. Cell viability was measured by MTT assay with five melatonin concentrations (0.5mM, 1mM, 2mM, 5mM, 10mM). For the pharmacological concentration of melatonin (1mM) the protein and gene expression of HIF-1α and VEGF were detected by immunocytochemistry and real time PCR, respectively. Results: There was a significantly decrease in MCF-7 cells viability after treatment with all concentrations of melatonin and in MDA-MB-231 cells just with 1mM (p&lt;0.05). The melatonin treatment did not alter the protein and gene expression of HIF-1α in MCF-7 cells (p&gt;0.05), however, it significantly decreased the VEGF protein expression (p&lt;0.05). In MDA-MB-231 cells there was significant decreased in the HIF-1α protein expression (p&lt;0.05). Conclusions: Our results showed that 1mM of melatonin decreased the cell viability and HIF-1α protein expression in ER-negative cells, and VEGF protein expression in ER-positive cells. This study is the first that evaluated the expression of these angiogenic factors in breast cancer cells after treatment with melatonin that can be a potential therapeutic agent. Thus, we are performing in vivo study to confirm the potential effectiveness of melatonin in the control of angiogenesis in breast cancer.

Abstract 2256: PDEF regulation and role as a survival factor in ER positive breast cancer cells

Abstract PDEF (Prostate Derived Ets Factor) is a member of the ‘Ets’ family. These transcription factors regulate a number of biological processes, including cell proliferation, differentiation, invasion and are thought to play an important role in oncogenesis. PDEF is expressed exclusively in tissues with a high epithelial content, especially hormone-regulated epithelium, such as prostate, mammary gland, endometrium and ovary. Furthermore, several studies showed PDEF to be one of the most highly over expressed mRNA in human and mouse mammary tumors. PDEF is interestingly significantly co-expressed with ER in ER positive breast carcinomas, together with known ER cofactors such as FOXA1, XBP1 and GATA3. PDEF was previously characterized as an Androgen Receptor cooperating factor in prostate, however its potential function as an ER cofactor is not explored yet. We investigated the mechanism of this ER/PDEF co expression and the role of PDEF in breast cancer cells. Using siRNA downregulations, mRNA quantification, and Chromatin IP, we discovered that the mechanism of ER/PDEF co expression implies a critical role for GATA3 as a negative regulator of PDEF expression. Moreover PDEF and GATA3 expressions are inversely correlated in ER positive Breast Cancer and cell lines. Kaplan-Meier survival analyses showed that these two factors could be useful prognosis markers for patients. We performed growth curves, cell cycle (FACs) and apoptosis (Annexin V) analyses of MCF7 cells transfected with a PDEF targeting siRNAs. The growth curves revealed an impaired proliferation rate of PDEF downregulated cells. Quantification of apoptosis in various conditions (basal, stress induced) demonstrated that PDEF is an essential survival factor for breast cancer cells. The loss of PDEF expression indeed triggers an increased proportion of apoptotic cells, especially in stress conditions such as hormone depletion or tamoxifen / fulvestrant treatments. The findings presented in this poster are evidences that designate PDEF as a potential new cofactor of ER and as an interesting therapeutic target, since its down regulation makes breast cancer cells more prone to undergo apoptosis in stress conditions. 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 2256. doi:1538-7445.AM2012-2256

Abstract 3067: Regulation of PAX2 expression/activity by hypoxia and EGF in breast cancer cells

Abstract Breast cancer is the most common cancer in women and is associated with the highest mortality rate after lung cancer. The leading cause of deaths associated with this cancer is metastasis, but cellular and molecular mechanisms controlling the metastatic ability of breast cancer cells are only partially elucidated. There is direct and indirect evidence suggesting that PAX2, a transcription factor preferentially activated in breast cancer cells of the luminal subtype, could reduce the invasive and metastatic ability of these cells, by downregulating the expression of ERBB2. It is thus primordial to understand the mechanisms regulating the expression and activity of PAX2 in breast cancer cells. Specifically, because hypoxic conditions prevailing in the center of solid primary tumours are known to regulate the expression of many genes controlling invasion and metastasis, we are investigating the impact of hypoxia-mimicking agent cobalt chloride (CoCl2) on PAX2 expression in breast cancer cells. Similarly, because EGF growth factor regulates the expression and/or activity of many genes controlling invasion and metastasis, we are determining the impact of EGF exposure on PAX2 activation and/or gene expression in ER+ MCF-7 cell line. The identification of factors and mechanisms regulating the expression and activity of PAX2 in breast cancer cells will help understand the biology of this emerging factor in breast cancer cells and may provide multiple strategies to control invasion and metastasis of these 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 3067. doi:1538-7445.AM2012-3067