Stimulates Breast Cancer Cell Growth Research Articles

Abstract LB224: The potential effect of chronically increased endotoxin levels on breast carcinoma progression in obese patients.

Abstract Obesity serves as a risk factor for estrogen-dependent postmenopausal breast cancer (BC). While the exact association occurring between these two disease states remains unknown, obesity-associated inflammation is thought to be the most likely contributing factor. In addition, obesity has been correlated to changes in gut microbiota composition and a subsequent chronic increase in bacterial endotoxin (lipopolysaccharide [LPS] - canonic ligand of toll-like receptor 4 [TLR4]) blood levels. It was recently shown that BC cells intrinsically express TLR4 and such expression has been associated with decreased patient survival as well as increased tumor growth. We hypothesized that obesity-associated endotoxemia may contribute to BC progression by utilizing TLR-dependent mechanisms and exerting cancer-promoting effects directly (on carcinoma cells) and indirectly (triggering abnormal activation of macrophages). Utilizing a chronic metabolic endotoxemia and breast cancer murine model as well as in vitro experimental systems, we found that continuous exposure to low concentrations of LPS not only promotes BC progression in vivo but stimulates BC cell growth in culture through the activation of key breast cancer-promoting signaling pathways (Stat3, Akt, ERK1/2). Obesity has reached epidemic proportions globally, where elucidation of the molecular mechanisms underlying breast tumor-promoting action of obesity has become of vital importance. Improving the understanding of such mechanisms has the potential to reveal improved efficacious therapy regimens and prevention strategies in a rapidly growing population of obese, breast cancer patients. Citation Format: Amichay Meirovitz, Daniela Nahmias, Esther Hermano, Ofra Maimon, Tamar Peretz, Michael Elkin. The potential effect of chronically increased endotoxin levels on breast carcinoma progression in obese patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB224.

Role of betaine in inhibiting the induction of RNA Pol III gene transcription and cell growth caused by alcohol

Alcohol has been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). Studies have demonstrated that alcohol intake increases the risk of breast cancer, and alcohol also stimulates breast cancer cell growth. Deregulation of Pol III genes is tightly associated with tumour development. Transcription factor II-B (TFIIB)-related factor 1 (Brf1) is a transcription factor that specifically regulates Pol III gene transcription. Our in vivo and in vitro studies have indicated that alcohol enhances the transcription of Pol III genes to cause an alteration of cellular phenotypes, which is closely related with human breast cancer. Betaine is a vegetable alkaloid and has antitumor functions. Most reports about betaine show that the consumption level of betaine is inversely associated with a risk of breast cancer. Although different mechanisms of betaine against tumour have been investigated, nothing has been reported on the effect of betaine on the deregulation of Brf1 and Pol III genes. In this study, we determine the role of betaine in breast cancer cell growth and colony formation and explore its mechanism. Our results indicate that alcohol increases the rates of growth and colony formation of breast cancer cells, whereas betaine is able to significantly inhibit the effects of alcohol on these cell phenotypes. Betaine decreases the induction of Brf1 expression and Pol III gene transcription caused by ethanol to reduce the rates of cell growth and colony formation. Together, these studies provide novel insights into the role of betaine in alcohol-caused breast cancer cell growth and deregulation of Brf1 and Pol III genes. These results suggest that betaine consumption is able to prevent alcohol-associated human cancer development.

Repeated Exposure to 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), an Active Metabolite of Bisphenol A, Aggressively Stimulates Breast Cancer Cell Growth in an Estrogen Receptor β (ERβ)-Dependent Manner.

Bisphenol A (BPA), recognized as an endocrine disruptor, is thought to exert its activity through a mechanism involving the activation of estrogen receptors (ERs) α/β However, a major problem is that very high concentrations of BPA are required (i.e., those in excess of environmental levels) for effective activation of ERα/β-mediated transcriptional activities in vitro, despite the BPA-induced estrogenic effects observed in vivo. To elucidate the causal reasons, we successfully identified a BPA metabolite, 4-methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP), which exhibits highly potent estrogenic activity both in vivo and in vitro. We have focused on the biologic relationship between breast tumor promotion and MBP/BPA, because BPA is considered to be a human carcinogen owing to its breast tumor-promoting properties. In general, humans are exposed to many endocrine disruptors, including BPA. In the present study, we used the ERα/β-positive human breast cancer cell line MCF-7 as an experimental model to investigate the effects of repeated exposure to BPA/MBP at concentrations found in the environment on the expression of ERα/β and to determine the particular ER subtype involved. We demonstrated that repeated exposure to MBP, but not to BPA, significantly downregulated ERα protein expression and stimulated the proliferation of MCF-7 cells through the activation of ERβ-mediated signaling.

AKR1B10 activates diacylglycerol (DAG) second messenger in breast cancer cells.

Aldo-keto reductase 1B10 (AKR1B10) is upregulated in breast cancer and promotes tumor growth and metastasis. However, little is known of the molecular mechanisms of action. Herein we report that AKR1B10 activates lipid second messengers to stimulate cell proliferation. Our data showed that ectopic expression of AKR1B10 in breast cancer cells MCF-7 promoted lipogenesis and enhanced levels of lipid second messengers, including phosphatidylinositol bisphosphate (PIP2), diacylglycerol (DAG), and inositol triphosphate (IP3). In contrast, silencing of AKR1B10 in breast cancer cells BT-20 and colon cancer cells HCT-8 led to decrease of these lipid messengers. Qualitative analyses by liquid chromatography-mass spectrum (LC-MS) revealed that AKR1B10 regulated the cellular levels of total DAG and majority of subspecies. This in turn modulated the phosphorylation of protein kinase C (PKC) isoforms PKCδ (Thr505), PKCµ (Ser744/748), and PKCα/βII (Thr638/641) and activity of the PKC-mediated c-Raf/MEK/ERK signaling cascade. A pan inhibitor of PKC (Go6983) blocked ERK1/2 activation by AKR1B10. In these cells, phospho-p90RSK, phospho-MSK, and Cyclin D1 expression was increased by AKR1B10, and pharmacological inhibition of the ERK signaling cascade with MEK1/2 inhibitors U0126 and PD98059 eradicated induction of phospho-p90RSK, phospho-MSK, and Cyclin D1. In breast cancer cells, AKR1B10 promoted the clonogenic growth and proliferation of breast cancer cells in two-dimension (2D) and three-dimension (3D) cultures and tumor growth in immunodeficient female nude mice through activation of the PKC/ERK pathway. These data suggest that AKR1B10 stimulates breast cancer cell growth and proliferation through activation of DAG-mediated PKC/ERK signaling pathway.

Glyphosate induces growth of estrogen receptor alpha positive cholangiocarcinoma cells via non-genomic estrogen receptor/ERK1/2 signaling pathway

Previous studies showed that glyphosate stimulates breast cancer cell growth via estrogen receptors. The present study investigated the effect of glyphosate on the estrogen signaling pathway involved in the induction of cholangiocarcinoma (CCA) cell growth. HuCCA-1, RMCCA-1 and MMNK-1 were chosen for comparison. The effects of glyphosate on cell growth, cell cycle and molecular signaling pathways were measured. The results showed that HuCCA-1 cells expressed estrogen receptor alpha (ERα), while ERα was not detected in RMCCA-1 and MMNK-1 cells. ERα was mostly expressed in cytoplasmic compartment of HuCCA-1 cells. Estradiol (E2) (10−11-10−5 M) induced cell proliferation in HuCCA-1 but not in RMCCA-1 and MMNK-1 cells. Glyphosate at the same concentration range also induced HuCCA-1 cell proliferation. The S phase of the cell cycle, and protein levels of the cyclin family were significantly increased after treatment of glyphosate or E2. Both compounds also induced the expression of proliferative signaling–related proteins including ERα, VEGFR2, pERK, PI3K(p85), and PCNA. These effects of glyphosate and E2 were abolished by the ER antagonist, 4-hydroxytamoxifen and U0126, a MEK inhibitor. The data from this study indicate that glyphosate can induce cell growth in ERα positive CCA cells through non-genomic estrogen receptor/ERK1/2 signaling pathway.

Therapeutic Potentials of BDNF/TrkB in Breast Cancer; Current Status and Perspectives.

Brain-derived neurotrophic factor (BDNF) is a potent neurotrophic factor that has been shown to stimulate breast cancer cell growth and metastasis via tyrosine kinase receptors TrkA, TrkB, and the p75NTR death receptor. The aberrant activation of BDNF/TrkB pathways can modulate several signaling pathways, including Akt/PI3K, Jak/STAT, NF-kB, UPAR/UPA, Wnt/β-catenin, and VEGF pathways as well as the ER receptor. Several microRNAs have been identified that are involved in the modulation of BDNF/TrkB pathways. These include miR-206, miR-204, MiR-200a/c, MiR-210, MiR-134, and MiR-191; and these may be of value as prognostic and predictive biomarkers for detecting patients at high risk of developing breast cancer. It has been also been demonstrated that a high expression of genes involved in the BDNF pathway in breast cancer is associated with poor clinical outcome and reduced survival of patients. Several approaches have been developed for targeting this pathway, for example TKr inhibitors (AZD6918, CEP-701) and RNA interference. The aim of the current review was to provide an overview of the role of BDNF/TrkB pathways in the pathogenesis of breast cancer and its value as a potential therapeutic target. J. Cell. Biochem. 118: 2502-2515, 2017. © 2017 Wiley Periodicals, Inc.

Deubiquitinating enzyme USP22 positively regulates c-Myc stability and tumorigenic activity in mammalian and breast cancer cells.

The proto-oncogene c-Myc has a pivotal function in growth control, differentiation, and apoptosis and is frequently affected in human cancer, including breast cancer. Ubiquitin-specific protease 22 (USP22), a member of the USP family of deubiquitinating enzymes (DUBs), mediates deubiquitination of target proteins, including histone H2B and H2A, telomeric repeat binding factor 1, and cyclin B1. USP22 is also a component of the mammalian SAGA transcriptional co-activating complex. In this study, we explored the functional role of USP22 in modulating c-Myc stability and its physiological relevance in breast cancer progression. We found that USP22 promotes deubiquitination of c-Myc in several breast cancer cell lines, resulting in increased levels of c-Myc. Consistent with this, USP22 knockdown reduces c-Myc levels. Furthermore, overexpression of USP22 stimulates breast cancer cell growth and colony formation, and increases c-Myc tumorigenic activity. In conclusion, the present study reveals that USP22 in breast cancer cell lines increases c-Myc stability through c-Myc deubiquitination, which is closely correlated with breast cancer progression.

Abstract P3-05-03: A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts

Abstract Background: Progesterone (P) is an important hormone for development and normal function of the breast; however, its role in established breast cancers is less clear. P has been implicated in regulating tumor cell growth, signaling, differentiation state, and stem/progenitor properties in breast cancer cells. Progesterone receptors (PRs) are considered positive prognostic indicators yet potential targets for treatment, although the dilemma of positive or negative targeting persists. P can either inhibit or stimulate breast cancer cell growth in the absence of estrogens (E), and usually blocks E mediated growth. Recent studies using breast cancer cell lines have deciphered a mechanism by which P suppresses E dependent growth through modulation of the estrogen receptor (ER) cistrome through a physical ER/PR association. However, the ER/PR association is weak to undetectable in our ER+PR+ breast cancer patient-derived xenografts (PDX). We therefore hypothesize that PR can regulate E-dependent breast cancer growth independent of direct interference of ER transcription. The purpose of this study was to use unbiased genomics and proteomics of breast cancer PDX to uncover additional mechanisms of P repression of breast cancer growth. Methods: These studies used two luminal ER+PR+ PDX (UCD4 and UCD65) that contain high levels of ER and PR (>90%), and where P inhibits E-dependent growth. Tumors were grown in vivo in female NSG mice under continuous placebo, E (17b-estradiol), E plus P, or E plus the synthetic progestin medroxyprogesterone acetate (MPA) for 8-10 weeks. RNAseq, chromatin immunoprecipitation sequencing (ChIPseq), and rapid immunoprecipitation followed by mass spectrometry of endogenous proteins (RIME) were performed to analyze differential transcriptional, cistromic, and protein-protein interactions in E compared to E plus P or MPA treated tumors. Co-immunoprecipitation (co-IP) and ChIP were used to verify results. Results: Both P and MPA potently inhibited E-dependent growth of both tumor lines. Gene expression studies found that both hormones reversed transcription of over one third of the estrogen/ER transcriptome in both tumors. RIME for PR uncovered significant interactions between PR and multiple RNA polymerase III subunits (POLR3). Co-IP using POLR3A and POLR3B confirmed PR associates with the POLR3 holoenzyme. Furthermore, ChIPseq revealed that PR binds to one third of POLR3 regulated tRNA genes. PR also associated with Maf1 in one tumor, a known POLR3 suppressor. Conclusions: Here we describe a novel association of PR with POLR3 in two luminal breast cancer PDX, an interaction not described in breast cancer cell lines. Our data suggest this is a negative regulatory interaction that may occur through recruiting a POLR3 repressor. These data implicate multifaceted P control of E dependent tumor growth; in addition to antagonizing E regulated genes at the transcription level, P can regulate translation though depletion of amino acid carrying tRNAs, thus slowing protein synthesis. Identifying which tumors utilize this growth-suppressive mechanism may pinpoint appropriate candidates for progestin therapy and/or provide a prognostic tool for predicting tumor progression. Citation Format: Finlay-Schultz J, Gillen AE, Brechbuhl HM, Ivie J, Bentley DL, Kabos P, Sartorius CA. A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-03.

Estradiol-independent modulation of breast cancer transcript profile by 17beta-hydroxysteroid dehydrogenase type 1

17beta-hydroxysteroid dehydrogenase type 1 (17β-HSD1) is a steroidal enzyme which, in breast cancer cells, mainly synthesizes 17-beta-estradiol (E2), an estrogenic hormone that stimulates breast cancer cell growth. We previously showed that the enzyme increased breast cancer cell proliferation via a dual effect on E2 and 5α-dihydrotestosterone (DHT) levels and impacted gene expression and protein profile of breast cancer cells cultured in E2-contained medium. Here, we used RNA interference technique combined with microarray analyses to investigate the effect of 17β-HSD1 expression on breast cancer cell transcript profile in steroid-deprived condition. Our data revealed that knockdown of 17β-HSD1 gene, HSD17B1, modulates the transcript profile of the hormone-dependent breast cancer cell line T47D, with 105 genes regulated 1.5 fold or higher (p < 0.05) in estradiol-independent manner. Using Ingenuity Pathway Analysis (IPA), we additionally assessed functional enrichment analyses, including biological functions and canonical pathways, and found that, in concordance with the role of 17β-HSD1 in cancer cell growth, most regulated genes are cancer-related genes. Genes that primarily involved in the cell cycle progression, such as the cyclin A2 gene, CCNA2, are generally down-regulated whereas genes involved in apoptosis and cell death, including the pro-apoptotic gene XAF1, IFIH1 and FGF12, are on the contrary up-regulated by 17β-HSD1 knockdown, and 21% of the modulated genes belong to this latter functional category. This indicates that 17β-HSD1 may be involved in oncogenesis by favoring anti-apoptosis pathway in breast cancer cells and correborates with its previously shown role in increasing breast cancer cell proliferation. The gene regulation occurring in steroid-deprived conditions showed that 17β-HSD1 can modulate endogenous gene expression in steroid-independent manners. Besides, we tested the ability of estrogen to induce or repress endogenous genes of T47D by microarray analysis. Expression of a total of 130 genes were found to increase or decrease 1.5-fold or higher (p < 0.05) in response to E2 treatment (1 nM for 48 h), revealing a list of potential new estrogen-responsive genes and providing useful information for further studies of estrogen-dependent breast cancer mechanisms. In conclusion, in breast cancer cells, in addition to its implication in the E2-dependent gene transcription, the present study demonstrates that 17β-HSD1 also modulates gene expression via mechanisms independent of steroid actions. Those mechanisms that may include the ligand-independent gene transcription of estrogen receptor alpha (ERα), whose expression is positively correlated with that of the enzyme, and that may implicate 17β-HSD1 in anti-apoptosis pathways, have been discussed.

The Interactions of Obesity, Inflammation and Insulin Resistance in Breast Cancer

Obese postmenopausal women have an increased breast cancer risk, the principal mechanism for which is elevated estrogen production by adipose tissue; also, regardless of menstrual status and tumor estrogen dependence, obesity is associated with biologically aggressive breast cancers. Type 2 diabetes has a complex relationship with breast cancer risk and outcome; coexisting obesity may be a major factor, but insulin itself induces adipose aromatase activity and estrogen production and also directly stimulates breast cancer cell growth and invasion. Adipose tissue inflammation occurs frequently in obesity and type 2 diabetes, and proinflammatory cytokines and prostaglandin E2 produced by cyclooxygenase-2 in the associated infiltrating macrophages also induce elevated aromatase expression. In animal models, the same proinflammatory mediators, and the chemokine monocyte chemoattractant protein-1, also stimulate tumor cell proliferation and invasion directly and promote tumor-related angiogenesis. We postulate that chronic adipose tissue inflammation, rather than body mass index-defined obesity per se, is associated with an increased risk of type 2 diabetes and postmenopausal estrogen-dependent breast cancer. Also, notably before the menopause, obesity and type 2 diabetes, or perhaps the associated inflammation, promote estrogen-independent, notably triple-negative, breast cancer development, invasion and metastasis by mechanisms that may involve macrophage-secreted cytokines, adipokines and insulin.

P037 Ki67 assessment using a 5-grade scale revealed high reproducibility for luminal type breast cancer

Goals: Local application of estradiol (E2) to treat vulvovaginal atrophy in postmenopausal breast cancer patients receiving aromatase inhibitors is known to elevate serum estradiol levels and thereby might counteract breast cancer therapy. Thus, vaginal application of estriol (E3) has been recommended for these patients. However, it is unclear to what extent E3 stimulates breast cancer cell growth. In this study, we examined the effect of E3 on growth and gene expression of two human breast cancer cell lines. Methods: We used an established in vitro cell culture assay and compared the effect of E2 and E3 on growth of the estrogen receptor alpha-positive breast cancer cell lines MCF-7 and T-47D testing a wide range of hormone concentrations of 10–10M. E3 effects on gene expression were examined by means of reporter gene assays, RT-qPCR and Western blot analysis. Results: E3 acted as a potent estrogen and exerted a mitogenic effect on T-47D and MCF-7 cells at concentrations of 10M (288 pg/ml) and higher. With regard to activation of an estrogen response element (ERE) in breast cancer cells, effects of E3 were visible at 10M. The same concentrations of E3 activated expression of the estrogen-responsive gene PR and of the proliferation genes cyclin A2, cyclin B1, Ki-67, c-myc and b-myb, providing molecular mechanisms underlying the observed growth increase. Conclusion: Like E2, low levels of E3 were able to trigger a robust estrogenic response in breast cancer cells. Thus, our data suggest caution regarding use of E3 by breast cancer survivors. Disclosure of Interest: No significant relationships.

Nerve growth factor and proNGF simultaneously promote symmetric self-renewal, quiescence, and epithelial to mesenchymal transition to enlarge the breast cancer stem cell compartment.

The discovery of cancer stem cells (CSCs) fundamentally advanced our understanding of the mechanisms governing breast cancer development. However, the stimuli that control breast CSC self-renewal and differentiation have still not been fully detailed. We previously showed that nerve growth factor (NGF) and its precursor proNGF can stimulate breast cancer cell growth and invasion in an autocrine manner. In this study, we investigated the effects of NGF and proNGF on the breast CSC compartment and found that NGF or proNGF enrich for CSCs in several breast cancer cell lines. This enrichment appeared to be achieved by increasing the number of symmetric divisions of quiescent/slow-proliferating CSCs. Interestingly, in vitro NGF pretreatment of MCF-7 luminal breast cancer cells promoted epithelial to mesenchymal transition in tumors of severe combined immunodeficient mice. Furthermore, p75(NTR), the common receptor for both neurotrophins and proneurotrophins, mediated breast CSC self-renewal by regulating the expression of pluripotency transcription factors. Our data indicate, for the first time, that the NGF/proNGF/p75(NTR) axis plays a critical role in regulating breast CSC self-renewal and plasticity.

ChIP-seq predicted estrogen receptor biding sites in human breast cancer cell line MCF7

The aim of this study was to find estrogen receptor (ER) binding sites of estradiol (E2)-treated and control groups and discuss the roles of ER activation in the tumorigenesis and progression of various human cancers. The ER ChIP-seq data GSE19013 was downloaded from Gene Expression Omnibus database, including E2-treated data GSM470419 and control data GSM470418. MACS software was utilized to identify ER binding sites in two groups. R's ChIPpeakAnno was used to detect ER-regulated target genes. Motif finding was employed to analyze ER concordant transcription factors (TFs) in MCF7 cell. The Gene Ontology (GO) was used to conduct functional enrichment analysis. We identified 9,134 ER binding sites in E2 stimulation group and 1,969 in control group. GO enrichment analysis of target genes showed that ER-regulated target genes mainly participated in mRNA catabolic process, protein complex disassembly, and protein localization to organelle-related biology process; while in E2 stimulation group, the function of ER-regulated target genes sharply changed. The effect of E2 in MCF7 cell suggested that activated ER probably reacted with several TFs and then co-regulated related genes expression. Furthermore, several TFs, such as PAX6, SMAD3, and ESR2, had multiply cellular regulation function. Our results showed that E2 stimulates breast cancer cell growth through ER. This may infer the function of ER in occurrence and development of breast cancer. Together, our study would pave ways for discussing ER concordant TFs and studying other ER-recruited TFs.

Effects of estriol on growth, gene expression and estrogen response element activation in human breast cancer cell lines

ObjectiveLocal application of estradiol (E2) to treat vulvovaginal atrophy in postmenopausal breast cancer patients receiving aromatase inhibitors is known to elevate serum estradiol levels and thereby might counteract breast cancer therapy. Thus, vaginal application of estriol (E3) has been recommended for these patients. However, it is unclear to what extent E3 stimulates breast cancer cell growth. In this study, we examined the effect of E3 on growth and gene expression of two human breast cancer cell lines. MethodsWe used an established in vitro cell culture assay and compared the effect of E2 and E3 on growth of the estrogen receptor alpha-positive breast cancer cell lines MCF-7 and T-47D testing a wide range of hormone concentrations of 10−12–10−7M. E3 effects on gene expression were examined by means of reporter gene assays, RT-qPCR and Western blot analysis. ResultsE3 acted as a potent estrogen and exerted a mitogenic effect on T-47D and MCF-7 cells at concentrations of 10−9M (288pg/ml) and higher. With regard to activation of an estrogen response element (ERE) in breast cancer cells, effects of E3 were visible at 10−10M. The same concentrations of E3 activated expression of the estrogen-responsive gene PR and of the proliferation genes cyclin A2, cyclin B1, Ki-67, c-myc and b-myb, providing molecular mechanisms underlying the observed growth increase. ConclusionsLike E2, low levels of E3 were able to trigger a robust estrogenic response in breast cancer cells. Thus, our data suggest caution regarding use of E3 by breast cancer survivors.

Abstract P5-10-17: Radiotherapy-induced miR expression influences the formation of local recurrence in breast cancer

Abstract In early breast cancer (EBC) patients, local disease relapse occurs at the site of the original primary tumor in 90% of cases and the addition of external beam radiotherapy (EBRT) after surgery is necessary to reduce recurrence rate. This observation suggested that surgery itself could represent a perturbing factor, possibly by altering the local microenvironment by inducing a wound healing response. Recent evidences indicate that, in selected EBC patients, 5 weeks of EBRT can be successfully replaced by a single dose of Targeted Intraoperative Radiotherapy (TARGIT). Interestingly, TARGIT acts not only affecting tumor cell survival itself, but is also able to alter the tumor microenvironment, by modifying cytokines secretion and activation of several intracellular pathways in breast cancer cells. In order to understand the significance of these modifications in breast tumor microenvironment after exposure to TARGIT we set up a mouse model of TARGIT treatment. Our results highlighted that the act of surgery alone strongly stimulates breast cancer cell growth and the addition of local irradiation completely reverses the surgery-induced breast cancer cell growth. Moreover, we evaluated the impact of TARGIT by analyzing microRNA (miR) expression both in our mouse model and in human specimens. To this aim, we collected paired specimens of peri-tumoral mammary tissues from 30 early breast cancer (EBC) patients, before and after treatment with TARGIT. Microarray approach followed by qRT-PCR validation revealed the presence of specific TARGIT-regulated miRs. Among the others, miR-223 was the most highly and significantly modified. In silico analyses indicated that epidermal growth factor (EGF) is a potential candidate of mR-223 regulation. In vitro experiments, using both normal and tumor derived cell lines validated the bioinformatic prediction, demonstrating that the 3′ UTR of EGF is a target of miR-223 and this binding results in the control of EGF production by the cells. Importantly, miR-223 overexpression was able to impair, at least in part, the wound-induced growth of normal and tumor derived breast-cancer cells, eventually influencing breast cancer cell proliferation and survival. Our results suggest that the effects of radiotherapy in the wounded tissue go far beyond the mere killing of residual tumor cells and is able to balance the negative effects of the wound healing process, both in human and mouse breast tissue. TARGIT-induced microenvironment modifications, in particular miR-223 up-regulation, significantly impair breast cancer response to surgery-induced inflammation eventually resulting in recurrence control. Our experimental findings strongly support the notion that the timely application of radiotherapy to the tumor bed, immediately after tumor removal, is critical to counteract the effects of surgery and wound healing process on residual breast cancer cells and normal peri-tumoral breast tissue. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-17.

PRSS23 Is an Estrogen Induced, AP-1-Dependent Gene in MCF-7 Breast Cancer Cells.

Abstract Background: Breast cancer is the second leading cause of cancer related deaths among women in the United States. While estrogens have been documented to play a role in the etiology and progression of breast cancer through a “classical” pathway by binding to the estrogen receptor which in turn binds at estrogen response elements (EREs) within gene promoters, the mechanisms by which estrogens stimulate breast cancer growth are poorly understood. Estrogens have also been shown to stimulate transcription by interacting with other transcription factors such as AP-1, SP-1 or NFκB through a “non-classical” pathway. We have shown previously that estrogen can stimulate breast cancer cell growth by activating gene expression independently of ERα binding directly to the DNA. We also previously used microarray analysis to identify a set of genes that are induced by estrogen and that are dependent on AP-1 for their induction. One of these ER and AP-1-dependent genes is PRSS23. We hypothesized that many growth regulatory estrogen-induced genes are induced through these non-classical pathways via crosstalk with the AP-1 transcription factor. To test this hypothesis, we investigated the role of AP-1 in the up-regulation of one estrogen responsive gene, PRSS23.Methods: qPCR was used to establish the estrogen and AP-1 responsiveness of PRSS23 following treatment of MCF7 cells with 1nM estradiol (E2) and 100nM TPA respectively. Antibodies specific to ERα (Santa Cruz), cJun/AP-1 (Oncogene), cFos (Santa Cruz), and pan-Fos (Santa Cruz), with rabbit IgG (Santa Cruz) as a control, were used in chromatin immunoprecipitation (ChIP) assays to identify the transcription factors recruited to the PRSS23 promoter after estrogen stimulation. siRNA knockdown studies were used to assess the requirement of specific transcription factors for estrogen-induction of PRSS23.Results: qPCR analysis confirmed that PRSS23 mRNA levels are induced following both E2 and TPA stimulation. These studies also demonstrated that the induction of PRSS23 by estrogen mRNA is reduced by expression of a dominant-negative form of cJun (TAM67), showing that estrogen induction of PRSS23 is AP-1-dependent. siRNA experiments identified that members of both ERα and AP-1 (cFos and Jun) are required for estrogen induction of PRSS23 mRNA expression. ChIP assays demonstrated that estrogen induces ERα recruitment to an upstream enhancer region. We are now investigating the location of recruitment of the AP-1 transcription factors to the PRSS23 promoter after estrogen stimulation.Conclusion: Our results demonstrate that PRSS23 is a “non-classical”, estrogen-induced gene that requires ERα and AP-1 family members for its induction by estrogen. The mechanism by which ER and these AP-1 proteins cooperate to induce the expression of this gene is now being investigated. Data generated from this study may yield a new therapeutic target that could have a broad application to the diagnosis and treatment of breast cancer. Support: NCI R01 CA123246 (PHB), NIH/NCI T32 CA909221 (JAM) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1131.

Fibroblast growth factor 8 increases breast cancer cell growth by promoting cell cycle progression and by protecting against cell death

Fibroblast growth factor 8 (FGF-8) is expressed in a large proportion of breast cancers, whereas its level in normal mammary gland epithelium is low. Previous studies have shown that FGF-8b stimulates breast cancer cell growth in vitro and in vivo. To explore the mechanisms by which FGF-8b promotes growth, we studied its effects on cell cycle regulatory proteins and signalling pathways in mouse S115 and human MCF-7 breast cancer cells. We also studied the effect of FGF-8b on cell survival. FGF-8b induced cell cycle progression and up-regulated particularly cyclin D1 mRNA and protein in S115 cells. Silencing cyclin D1 with siRNA inhibited most but not all FGF-8b-induced proliferation. Inhibition of the FGF-8b-activated ERK/MAPK pathway decreased FGF-8b-stimulated proliferation. Blocking the constitutively active PI3K/Akt and p38 MAPK pathways also lowered FGF-8b-induced cyclin D1 expression and proliferation. Corresponding results were obtained in MCF-7 cells. In S115 and MCF-7 mouse tumours, FGF-8b increased cyclin D1 and Ki67 levels. Moreover, FGF-8b opposed staurosporine-induced S115 cell death which effect was blocked by inhibiting the PI3K/Akt pathway but not the ERK/MAPK pathway. In conclusion, our results suggest that FGF-8b increases breast cancer cell growth both by stimulating cell cycle progression and by protecting against cell death.

CCAAT/Enhancer Binding Protein δ Up-regulates Aromatase Promoters I.3/II in Breast Cancer Epithelial Cells

Aromatase is the enzyme responsible for the last step of estrogen synthesis. The female hormone, estrogen, is known to stimulate breast cancer cell growth. Because the expression of aromatase in breast cancer tissues is driven by unique promoters I.3 and II, a more complete understanding of the regulatory mechanism of aromatase expression through promoters I.3/II in breast tumors should be valuable in developing targeted therapies, which selectively suppress estrogen production in breast tumor tissue. Results from in vivo footprinting analyses revealed several protein binding sites, numbered 1 to 5. When site 2 (-124/-112 bp, exon I.3 start site as +1) was mutated, promoters I.3/II activity was dramatically reduced, suggesting that site 2 is a positive regulatory element. Yeast one-hybrid screening revealed that a potential protein binding to site 2 was CCAAT/enhancer binding protein delta (C/EBP delta). C/EBP delta was shown to bind to site 2 of aromatase promoters I.3/II in vitro and in vivo. C/EBP delta up-regulated promoters I.3/II activity through this site and, as a result, it also up-regulated aromatase transcription and enzymatic activity. p65, a subunit of nuclear factor-kappaB (NF-kappaB) transcription factor, inhibited C/EBP delta-up-regulated aromatase promoters I.3/II and enzymatic activity. This inhibitory effect of p65 was mediated, in part, through prevention of the C/EBP delta binding to site 2. This C/EBP delta binding site in aromatase promoters I.3/II seems to act as a positive regulatory element in non-p65-overexpressing breast cancer epithelial cells, whereas it is possibly inactive in p65 overexpressing cancer epithelial cells, such as estrogen receptor-negative breast cancer cells.

Doctors Seek To Prevent Breast Cancer Recurrence by Lowering Insulin Levels

E ven in this era of targeted molecular therapies, an old drug can sometimes provide a surprising new benefit. A case in point, the diabetes drug metformin is emerging as a potential treatment for breast cancer on the basis of evidence that cancer patients with lower insulin levels have better outcomes. But using a diabetes drug to lower insulin levels in patients who are not diabetic has caused concern among some researchers, who say that doctors first need to know more about how metformin works in the oncology setting. First isolated as an active ingredient in the medieval herbal remedy goat’s rue, metformin has been used to treat type II diabetes for more than 50 years. The U.S. Food and Drug Administration ap proved its use in the United States in 1995, and it is now the most prescribed drug for type II diabetes, the adult-onset form of the disease that accounts for 90% – 95% of all diabetes cases. After several epidemiological studies linked metformin use to lower cancer rates and reduced risk of cancer death, some physicians proposed adding metformin to cancer treatment regimens. Among the fi rst to advocate its use was Pamela Goodwin , M.D., a medical oncologist at Mount Sinai Hospital in Toronto. In a recent editorial in the Journal of Clinical Oncology , Goodwin laid out a rationale for adding metformin to adjuvant breast cancer treatment, citing evidence that elevated insulin levels, often associated with obesity, may be a biological link connecting obesity with lower survival. “If you look at the effect of insulin, the women in the highest quartile of insulin levels in the studies that have been done all yield the same result: They have a triple risk of death,” Goodwin said. “If we could lower those insulin levels by 25%, we might see a 5% – 6% absolute improvement in outcome, and that’s huge.” Goodwin bases her optimism on several published studies, including a prospective cohort study of 512 nondiabetic women with early-stage breast cancer that she published in 2002. The study showed that women with the highest fasting insulin levels had three times the risk of recurrence and death compared with women with the lowest insulin levels. Other large studies have replicated this fi nding. The NCIsponsored Health, Eating, Activity, and Lifestyle (HEAL) study confi rmed that higher body mass index and lower physical activity levels are associated with higher insulin levels in breast cancer survivors and that women with invasive disease and the highest insulin levels have three times the risk of death of women with the lowest insulin levels. Melinda Irwin, Ph.D., an epidemiologist at the Yale School of Public Health in New Haven, Conn., presented those data at the American Association for Cancer Research’s prevention meeting last year. “The high-insulin effect is large,” said Michael Pollak , M.D., professor of oncology at McGill University in Montreal. “The adverse effect of high insulin on outcome of postmenopausal breast cancer is in the same order of magnitude as the benefi cial effect of adjuvant chemotherapy. This is nothing subtle.” Pollak, who studies the association of high insulin levels with breast and colorectal cancer incidence, agrees with Goodwin that therapies to reduce high insulin levels in cancer patients could dramatically reduce cancer-related deaths. “In postmenopausal women, where obesity is associated with a bad [breast cancer] outcome, it’s a very reasonable hypothesis that the bad outcome is because obesity is associated with high insulin,” he said. Two other large studies, using data from Canadian and British diabetes registries, have shown that people taking metformin, and thereby lowering their insulin levels, had unexpectedly lower overall cancer rates than patients taking diabetes medications such as sulfonylureas. These drugs work by raising insulin levels to force blood glucose levels down. “Previously the endocrinologists who used metformin to treat diabetes called it an insulinsensitizing agent because when type II diabetic patients were given metformin, their insulin levels fell. So the endocrinologists thought the drug was making the insulin work better,” said Pollak. But if metformin actually worked by making cells more sensitive to insulin, he said, it would probably increase tumor growth because insulin stimulates breast cancer cell growth. Pollak found that the opposite was true. When he studied the effect of metformin in both estrogen receptor – positive and estrogen receptor – negative breast cancer cells in culture, the drug reduced breast cancer cell growth.

Phytoestrogen Supplement Use by Women

Phytoestrogens are weak estrogens found concentrated in soybeans. Americans consume phytoestrogens primarily in traditional soy foods, soymilk and isolated soy protein added during food processing or consumed as a beverage. Extracted phytoestrogens are also marketed in numerous forms as dietary supplements regulated under the Dietary Supplement Health and Education Act. Consumers of phytoestrogen supplements tend to be peri- and postmenopausal women looking for an alternative to hormone therapy. Although there are no approved health claims for phytoestrogens at this time, numerous claims are being made regarding benefits to heart, bone, breast and general menopausal health. The data supporting these claims are generally not strong. The strongest data show that phytoestrogens reduce the number and intensity of hot flashes, although the reduction is a modest 10-20%. The studies showing cholesterol lowering have used soy protein rather than phytoestrogen extracts. The soy protein appears to be required for this effect, although phytoestrogen extracts may have other beneficial effects on the cardiovascular system. The data on bone metabolism are suggestive of possible benefits whereas the effects on the breast are the most poorly understood. Although most animal studies have shown cancer-preventive effects, a few recent studies suggest that soy phytoestrogens may stimulate breast cancer cell growth under certain circumstances. Before recommendations regarding phytoestrogen supplements can be safely made, we must have more information on the effects of the extracts on bone, heart and breast health. Until safety with respect to breast cancer is established, phytoestrogen supplements should not be recommended, particularly for women at high risk of breast cancer.