Signaling In MCF-7 Cells Research Articles

Cellular prion protein regulates invasion and migration of breast cancer cells through MMP-9 activity

Function of cellular prion protein (PrPc) in cancer progression has not been elucidated yet. Ectopic expression of PrPc increases the invasion and migration of breast cancer cell line, MCF-7 cells. Overexpressed PrPc increases matrix metalloprotease-9 (MMP-9) expression by enhancing association of NF-κB in promoter of MMP-9 gene and ERK signaling in MCF-7 cells. Whereas, silencing of PrPc by siRNA suppresses ERK activation and MMP-9 expression resulting the down-regulation of MD-MB231 cell migration and invasion. Overall, these results suggest that PrPc contribute the breast cancer invasion and migration via MMP-9.

Human Umbilical Cord Mesenchymal Stem Cells Promote Breast Cancer Metastasis by Interleukin-8- and Interleukin-6-Dependent Induction of CD44(+)/CD24(-) Cells.

Although emerging evidence links mesenchymal stem cells (MSCs) with cancer metastasis, the underlying mechanisms are poorly understood. In the present study, we found that human umbilical cord-derived MSCs (UC-MSCs) promoted MCF-7 cell migration in vitro and metastasis in vivo. To explore the mechanisms, the characteristics of MCF-7 cells cocultured with UC-MSCs were assessed. The expression and secretion of interleukin-8 (IL-8) and IL-6 were induced in MCF-7 cells cocultured with UC-MSCs. However, neutralization of IL-8 or IL-6 secreted by UC-MSCs could attenuate the enhanced expression of IL-8 and IL-6 in MCF-7 cells cocultured with UC-MSCs, which subsequently alleviated the enhanced migration. Similar to UC-MSCs, exogenous human recombinant IL-8 or IL-6 also promoted IL-8 and IL-6 expression and MCF-7 cell migration. In addition to enhanced IL-8 and IL-6 expression, MCF-7 cells cocultured with UC-MSCs displayed enhanced mammosphere-forming ability and increased percentage of CD44(+)/CD24(-) cells. However, epithelial-to-mesenchymal transition (EMT) was not observed in MCF-7 cells cocultured with UC-MSCs. Taken together, these results suggested that IL-8 and IL-6 secreted by UC-MSCs activated the autocrine IL-8 and IL-6 signaling in MCF-7 cells and induced CD44(+)/CD24(-) cells, which subsequently promoted MCF-7 cell migration in vitro and metastasis in vivo.

Modulation of 17β‐Estradiol Signaling on Cellular Proliferation by Caveolin‐2

The sex hormone 17β-estradiol (E2) exerts pleiotropic effects by binding to the ligand-activated transcription factor estrogen receptor α (ERα). The E2:ERα complex regulates several physiological processes, including cell survival and proliferation, through transcriptional effects (i.e., estrogen responsive element [ERE]-based gene transcription) and non-transcriptional membrane-initiated effects (i.e., the activation of extra-nuclear signaling cascades), which derive from the activation of the pool of ERα that is localized to plasma membrane caveolae. Caveolae are ω-shaped membrane sub-domains that are composed of scaffold proteins named caveolins (i.e., caveolin-1, caveolin-2, and caveolin-3). Although caveolin-3 is exclusively expressed in muscles, caveolin-1 and caveolin-2 are co-expressed in all human tissues. From a functional point of view, caveolin-2 can operate both dependently on and independently of caveolin-1, which is the main coat component of caveolae. Interestingly, while a functional interplay between caveolin-1 and ERα has been reported in the control of E2-induced physiological effects, the role of caveolin-2 in E2:ERα signaling within the cell remains poorly understood. This study shows that siRNA-mediated caveolin-2 depletion in breast ductal carcinoma cells (MCF-7) reduces E2-induced ERα phosphorylation at serine residue 118 (S118), controls intracellular receptor levels, precludes ERα-mediated extra-nuclear activation of signaling pathways, reduces ERα transcriptional activity, and prevents cellular proliferation. Meanwhile, the impact of caveolin-1 depletion on ERα signaling in MCF-7 cells is shown to be similar to that elicited by siRNA-mediated caveolin-2 depletion. Altogether, these data demonstrate that caveolin-2 expression is necessary for the control of E2-dependent cellular proliferation.

Nuclear receptor 4A1 as a drug target for breast cancer chemotherapy.

The orphan nuclear receptor 4A1 (NR4A1) is overexpressed in mammary tumors and breast cancer cell lines. The functional activity of this receptor was investigated by RNA interference with oligonucleotides targeted to NR4A1 (siNR4A1) and by treatment with NR4A1 antagonists. Breast cancer cells were treated with NR4A1 antagonists or transfected with siNR4A. Effects on cell proliferation and apoptosis as well as specific genes associated with these responses were investigated in MCF-7, SKBR3, and MDA-MB-231 cells, and in athymic nude mice bearing MDA-MB-231 cells as xenografts. Transfection of MCF-7, MDA-MB-231, and SKBR3 breast cancer cells with siNR4A1 decreased cell proliferation and induced apoptosis in these cell lines. Transfection of breast cancer cells with siNR4A1 also decreased expression of Sp-regulated genes including survivin, bcl-2, and epidermal growth factor receptor, inhibited mTOR signaling in MCF-7 cells that express WT p53, and activated oxidative and endoplasmic reticulum stress through downregulation of thioredoxin domain-containing 5 and isocitrate dehydrogenase 1. 1,1-Bis(3'-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) are NR4A1 ligands that act as NR4A1 antagonists. Treatment with selected analogs also inhibited breast cancer cell and tumor growth and induced apoptosis. The effects of C-DIM/NR4A1 antagonists were comparable to those observed after NR4A1 knockdown. Results with siNR4A1 or C-DIMs/NR4A1 antagonists in breast cancer cells and tumors were similar to those previously reported in pancreatic, lung, and colon cancer cells. They demonstrate the potential clinical applications of NR4A1 antagonists in patients with tumors that overexpress this receptor.

Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells.

PurposeIsocryptotanshinone (ICTS) is a natural bioactive product that is isolated from the roots of the widely used medical herb Salvia miltiorrhiza. However, few reports exist on the mechanisms underlying the therapeutic effects of ICTS. Here, we report that ICTS has anticancer activity and describe the mechanism underlying this effect.MethodsThe antiproliferative effect of ICTS was determined using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and clonogenic assays. The effect of ICTS on the cell cycle was measured using flow cytometry. Apoptosis was determined by Hoechst 33342 staining, DNA fragmentation assays, and Western blotting for apoptotic proteins. Finally, the effect of ICTS on mitogen-activated protein kinases (MAPKs) was determined by Western blotting.ResultsICTS significantly inhibited proliferation of MCF-7 and MDA-MB-231 human breast cancer cells, HepG2 human liver cancer cells, and A549 human lung cancer cells in vitro. Among the tested cell lines, MCF-7 cells showed the highest sensitivity to ICTS. ICTS significantly inhibited colony formation by MCF-7 cells. Furthermore, exposure of MCF-7 cells to ICTS induced cell cycle arrest at the G1 phase and decreased mitochondrial membrane potential. Hoechst 33342 staining and Western blot analysis for apoptotic proteins suggested that ICTS induced apoptosis in MCF-7 cells. In addition, ICTS activated MAPK signaling in MCF-7 cells by inducing time- and concentration-dependent phosphorylation of JNK, ERK, and p38 MAPK.ConclusionOur results suggest that ICTS inhibited MCF-7 cell proliferation by inducing apoptosis and activating MAPK signaling pathways.

The chemokine receptor CXCR7 interacts with EGFR to promote breast cancer cell proliferation.

BackgroundRecent advances have revealed a significant contribution of chemokines and their receptors in tumor growth, survival after chemotherapy, and organ-specific metastasis. The CXC chemokine receptor-7 (CXCR7) is the latest chemokine receptor implicated in cancer. Although over expressed in breast cancer cell lines and tumor tissues, its mechanism of action in breast cancer (BrCa) growth and metastasis is unclear. Studies in other cancers have implicated CXCR7 in cell proliferation, anti-apoptotic activity and cell-cell adhesion. The present study was initiated to examine the pattern of CXCR7 expression and its role in regulation of growth signaling in breast cancer.MethodsThe contribution of CXCR7 in BrCa cell proliferation was investigated in representative cell lines using real time quantitative PCR (q-PCR), proliferation assays, immunohistochemistry and immunoblotting. Phenotypic changes were examined after CXCR7 specific cDNA and siRNA transfection and expression levels were monitored by q-PCR. Further, the association of CXCR7 with epidermal growth factor receptor (EGFR) and modulation of its activity were investigated by western blotting, immunofluorescence, and in-situ proximity ligation assays in human BrCa cells and tissues.ResultsCXCR7 was expressed in both, estrogen receptor (ER) positive and negative BrCa cell lines. CXCR7 was also expressed unevenly in normal breast tissues and to a much higher extent in ER + cancer tissues. Depletion of CXCR7 in MCF7 BrCa cells by RNA interference decreased proliferation and caused cell cycle arrest. Further, proximity ligation assay (PLA) revealed colocalization of CXCR7 with EGFR in cancer tissues and cancer cell lines. CXCR7 depletion reduced levels of phospho-EGFR at Tyrosine1110 after EGF-stimulation and also reduced phosphorylation of ERK1/2, indicating a potentially direct impact on mitogenic signaling in MCF7 cells. Using siRNA to knockdown β-arrestin2 in cells with EGFR over expression we were able to nearly deplete the CXCR7-EGFR colocalization events, suggesting that β-arrestin2 acts as a scaffold to enhance CXCR7 dependent activation of EGFR after EGF stimulation.ConclusionsThese results demonstrate coupling of CXCR7 with EGFR to regulate proliferation of BrCa cells and suggest an important ligand-independent role of CXCR7 in BrCa growth. Thus, the CXCR7-EGFR axis is a promising target for breast cancer therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-198) contains supplementary material, which is available to authorized users.

Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells

4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent.

Abstract C210: Atypical chemokine receptor 3/CXCR7 and EGFR interact to control breast cancer growth.

Abstract Introduction: Breast cancer (BrCa) is a major burden for women in the US, ranking second in both incidence and cancer related deaths. Many studies comparing cancer vs. normal gene expression have identified CXCR7 as being preferentially expressed in several different cancer types, including breast cancer. CXCR7 is the latest CXC-chemokine receptor implicated in breast cancer growth. The chemokine receptor CXCR7 is an atypical G-protein coupled receptor, in that it does not bind G-proteins as GPCRs do. It is so atypical, that convention by HUGO has recently changed the name of CXCR7 to ACKR3, atypical chemokine receptor 3. Methods: We used the database Oncomine to confirm the correlation of CXCR7 with breast cancer at both mRNA and tissue level. We also show functional expression of CXCR7 on relevant human breast cancer cell lines and tissue. To identify the mechanism of function of CXCR7 in BrCa we studied its interaction with EGFR and beta-arrestin2 (beta-ARR2), which are relevant partners important in the mitogenic growth cascade. We used a proximity ligation assay (PLA) to visualize CXCR7-EGFR co-localization both in cell culture and in human normal and breast cancer tissue. To visualize interaction with beta-ARR2, we used live cell confocal microscopy. Results: CXCR7 was over-expressed in ER-α positive BrCa cells, compared to normal breast epithelial cells. ER-negative cells also expressed CXCR7, however, at lower levels. Depletion of CXCR7 in MCF-7 BrCa cells by RNAi decreased cell proliferation and caused cell cycle arrest. Since CXCR7 is an atypical G-protein coupled receptor where ligand activation does not elicit conventional intracellular signaling, we hypothesized that CXCR7 regulates BrCa proliferation through interaction with EGFR or activation of other potential targets of the MAP-kinase pathway. CXCR7 depletion reduced site-specific phosphorylation of EGFR at Tyrosine 1110 after EGF-stimulation. CXCR7 depletion also reduced phosphorylation of ERK-1/2, indicating a potentially direct interaction with mitogenic signaling in MCF-7 cells. We found EGFR and CXCR7 co-localize as demonstrated by confocal microscopy, with increased co-localization after EGF stimulation. To clarify whether CXCR7-EGFR interaction is direct or indirect, we analyzed the role beta-ARR2 plays in this transactivation. SDF1 addition to MCF7 cells over expressing beta-ARR2-EGFP induced minimal movement of beta-ARR2-EGFP over a 30 min window. EGF addition induced an obvious shift of beta-ARR2-EGFP localization. When CXCR7 was down-regulated by siRNA, there was decreased rearrangement of beta-ARR2-EGFP after EGF stimulation, suggesting this CXCR7-EGFR interaction is beta-ARR2 mediated. Conclusion: These data demonstrate significant molecular interactions between two cell surface receptors that have high clinical significance. The impact of this work may potentially lead to better treatment strategies based on cell surface targets. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C210. Citation Format: Nicole Salazar, Daniel Muñoz, Bal L. Lokeshwar. Atypical chemokine receptor 3/CXCR7 and EGFR interact to control breast cancer growth. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C210.

G Protein–Coupled Estrogen Receptor Is Apoptotic and Correlates with Increased Distant Disease-Free Survival of Estrogen Receptor–Positive Breast Cancer Patients

G protein-coupled estrogen receptor 1 (GPER1), previously named GPR30, is a membrane receptor reported to mediate nongenomic estrogen responses. We investigated if GPER1 expression correlates with any clinicopathologic variables and distant disease-free survival (DDFS) in patients with breast cancer, if any prognostic impact of the receptor is dependent on estrogen receptor-α (ER-α) status, and if the receptor impacts apoptotic signaling in ER-positive breast cancer cells. GPER1 expression was analyzed by immunohistochemistry in breast tumors from 273 pre- and postmenopausal stage II patients, all treated with adjuvant tamoxifen for 2 years (cohort I) and from 208 premenopausal lymph node-negative patients, of which 87% were not subjected to any adjuvant systemic treatment (cohort II). GPER1-dependent proapoptotic signaling was analyzed in MCF7 cells with and without GPER1 knockdown, T47D cells, HEK293 cells (HEK), and HEK stably expressing GPER1 (HEK-R). GPER1 positively correlates with ER and progesterone receptor expression. Multivariate analysis showed that GPER1 is an independent prognostic marker of increased 10-year DDFS in the ER-positive subgroup. HEK-R has higher basal proapoptotic signaling compared with HEK including increased cytochrome C release, caspase-3 cleavage, PARP cleavage, and decreased cell viability. Treating HEK-R with the proteasome inhibitor epoxomicin, to decrease GPER1 degradation, further increases receptor-dependent proapoptotic signaling. Also, GPER1 knockdown decreases basal and agonist-stimulated proapoptotic receptor signaling in MCF7 cells. GPER1 is a prognostic indicator for increased DDFS in ER-positive breast cancer, which may be associated with constitutive GPER1-dependent proapoptotic signaling in ER-positive breast cancer cells.

The effect of prolactin and estrogen cross-talk on prolidase– dependent signaling in MCF-7 cells

Estrogen and prolactin play important role in mammary carcinogenesis. The present study was undertaken to evaluate the effect of prolactin and estrogen cross-talk on HIF-1 α level and expression of some HIF-1 α- dependent signaling proteins. Since up-regulation of prolidase activity inhibits HIF-1 α degradation, the enzyme was considered as an interface of estrogen/prolactin signaling. The experiments were performed on MCF-7 cells cultured with prolactin in the presence or absence of estradiol. It was found that in the presence of estradiol, prolactin inhibits prolidase activity and its down-stream signaling proteins: HIF-1α, mTOR, AKT and MAPK p-38, while in the absence of estradiol, an opposite effect was observed. These results suggest that prolactin/estrogen cross-talk exert beneficial effect on prolidase-dependent down regulation of HIF-1α. It suggests that dual action of prolactin and estrogen may be considered as a strategy in therapy of breast cancer.

Abstract P5-07-01: Defining the mechanism of breast cancer growth by chemokine receptor CXCR7 and Epidermal Growth Factor Receptor coupling interaction.

Abstract Introduction: Breast cancer (BrCa) ranks second in both incidence and cancer deaths for women in the USA. Recent advances have revealed a significant contribution of chemokines and their receptors in tumor growth, survival after chemotherapy, and organ-specific metastasis. CXCR7 is the latest CXC-chemokine receptor implicated in BrCa growth. It is endogenously over expressed in BrCa, but its mechanism of tumor growth enhancement is still not clearly defined. CXCR7 heterodimerizes with CXCR4 and binds to SDF-1 (CXCL12) and CXCL11. No direct ligand mediated physiological action has been implicated for CXCR7 in BrCa other than facilitating CXCR4-mediated cell motility, however, studies in other cancers have implicated CXCR7 in cell proliferation, anti-apoptotic activity and cell-cell adhesion. The expression pattern of CXCR7 in BrCa tissues and its potential contribution in BrCa cell proliferation and survival were investigated in representative BrCa cell lines using knock down and knock-in experiments. Results: CXCR7 was only expressed in ER-α positive BrCa cells (e.g., MCF-7) and was not found in BrCa cells with known metastatic potential (MDA-MB231 and SKBr3). Depletion of CXCR7 in MCF-7 BrCa cells by both siRNA and shRNA decreased cell proliferation and caused cell cycle arrest. Since CXCR7 is an atypical G-protein coupled receptor where ligand activation does not elicit conventional intracellular signaling, we hypothesized that CXCR7 regulates BrCa proliferation through interaction with EGFR or activating other potential targets of MAP-kinase activation. CXCR7 depletion reduced site-specific phosphorylation of EGFR at Tyrosine 1110 after EGF-stimulation. CXCR7 depletion also reduced phosphorylation of ERK-1/2, indicating a potentially direct interaction with mitogenic signaling in MCF-7 cells. We tested for direct interaction of CXCR7 with EGFR by co-immunoprecipitation and immunofluorescence studies. We found EGFR and CXCR7 co-immunoprecipitate and co-localize as demonstrated by confocal microscopy. Interestingly, our results also demonstrate CXCR7 and EGFR are co-localized before EGF stimulation. Further corroboration of CXCR7-EGFR interaction was established using the proximity ligation assay (PLA) technique that allowed us to visualize CXCR7-EGFR co-localization both in cell culture and in human normal and breast cancer tissue. Conclusion: This data demonstrates an important role for CXCR7 in BrCa growth and explores the mechanism of action of direct coupling of EGFR with CXCR7. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-01.

The TCF4/β-catenin pathway and chromatin structure cooperate to regulate D-glucuronyl C5-epimerase expression in breast cancer

D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MCF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-deoxyazacytidine (5-aza-dC) treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. Chromatin activation by Trichostatin A (TSA) or 5-aza-dC/TSA treatment increased GLCE expression by two to 3-fold due to an increased interaction between the GLCE promoter and the TCF4/β-catenin transactivation complex, or H3K9ac and H3K4Me3 histone modifications. However, ectopic expression of TCF4/β-catenin was not sufficient to activate GLCE expression in MCF7 cells, suggesting that chromatin structure plays a key role in GLCE regulation. Although TSA treatment significantly repressed canonical WNT signaling in MCF7 cells, it did not influence endogenous TCF4/β-catenin mRNA levels and activated TCF4/β-catenin-driven transcription from the GLCE promoter, indicating GLCE as a novel target for TCF4/β-catenin complex in breast cancer cells. A correlation was observed between GLCE, TCF4 and β-catenin expression in breast cancer cells and primary tumors, suggesting an important role for TCF4/β-catenin in regulating GLCE expression both in vitro and in vivo. Taken together, the results indicate that GLCE expression in breast cancer is regulated by a combination of chromatin structure and TCF4/β-catenin complex activity.

Human pituitary tumor-transforming gene 1 overexpression reinforces oncogene-induced senescence through CXCR2/p21 signaling in breast cancer cells

IntroductionhPTTG1 (human pituitary tumor-transforming gene 1) is an oncogene overexpressed in breast cancer and several other types of cancer. Increased hPTTG1 expression has been shown to be associated with poor patient outcomes in breast cancer. Although hPTTG1 overexpression plays important roles in promoting the proliferation, invasion, and metastasis of cancer cells, it also has been suggested to induce cellular senescence. Deciphering the mechanism by which hPTTG1 overexpression induces these contradictory actions in breast cancer cells is critical to our understanding of the role of hPTTG1 in breast cancer development.MethodsMCF-10A and MCF-7 cells were used to identify the mechanism of hPTTG1-induced senescence. The interplay between hPTTG1 overexpression and chemokine C-X-C motif receptor 2 (CXCR2)/p21-dependent senescence in tumor growth and metastasis of MCF-7 cells was investigated by orthotopic transplantation of severe combined immunodeficiency (SCID) mice. Additionally, human invasive ductal carcinoma (IDC) tissue arrays were used to confirm the hPTTG1/CXCR2/p21 axis established in vitro.ResultsIn this study, we investigated the mechanism of hPTTG1-induced senescence as well as its role in breast cancer progression and metastasis. Herein, we showed that hPTTG1 overexpression reinforced senescence through the CXCR2/p21 signaling. Furthermore, hPTTG1 overexpression activated NF-κB signaling to transactivate the expression of interleukin (IL)-8 and growth-regulated oncogene alpha (GROα) to execute CXCR2 signaling in MCF-7 cells. When CXCR2 expression was knocked down in hPTTG1-overexpressing MCF-7 cells, hPTTG1-induced senescence was abrogated by alleviating CXCR2-induced p21 expression. In a mouse model, CXCR2-mediated senescence limited hPTTG1-induced tumor growth and metastasis. Moreover, CXCR2 knockdown in hPTTG1-overexpressing MCF-7 tumors dramatically accelerated tumor growth and metastasis. Our in vitro and in vivo results demonstrated that hPTTG1 overexpression reinforces senescence through CXCR2 signaling, and the evasion of CXCR2/p21-dependent senescence was critical to hPTTG1 exerting its oncogenic potential. Interestingly, although CXCR2-dependent senescence restrained hPTTG1-induced tumor progression, when MCF-7 cells and hPTTG1-overexpressing MCF-7 cells were co-transplanted into the mammary fat pads of SCID mice, hPTTG1-overexpressing senescent cells created a metastasis-promoting microenvironment that promoted lung metastasis of the MCF-7 cells. Immunohistochemical analysis of human breast tumor samples also confirmed the importance of the hPTTG1/CXCR2 axis in promoting breast cancer metastasis.ConclusionsOur findings provide novel molecular insights into hPTTG1-induced senescence and identify a novel mechanism by which hPTTG1 promotes metastasis by regulating the senescence-associated microenvironment.

S2-5: An Anti-HER3 Antibody That Stabilizes the Inactive Conformation Inhibits Both HER2 and Ligand Driven Tumor Growth.

Abstract Background: HER3 (ErbB3) is a member of the ErbB family of receptor tyrosine kinases (RTK). In normal physiology, ligands (e.g. neuregulin) activate HER3 by promoting dimerization with other RTK's such as HER2 (ErbB2). Inappropriate HER2/HER3 dimerization as a result of HER2 over-expression in cancer results in HER3 mediated activation of the oncogenic PI3K pathway. The HER2− targeted antibody trastuzumab inefficiently inhibits HER2 mediated HER3 activation allowing persistent HER3 signaling that is speculated to limit clinical responses. Consequently, combination of a HER3- targeted agent with trastuzumab may be of clinical benefit. Furthermore, ectopic HER3 activation has recently been implicated in the relief of a feedback loop induced by PI3K inhibitors that may perhaps limit their efficacy in HER2 driven tumors. Since HER3 activation in HER2 driven cancers occurs in a ligand- independent manner, antibodies that primarily inhibit ligand- induced HER3 activation are largely inactive in HER2 driven xenograft models. Results: H3F15, a high affinity (26pM) HER3- targeted fully human IgG1 antibody was selected from the Human Combinatorial Antibody Library (HuCAL) using phage display technology. In a broad range of HER2 amplified breast and gastric cell lines, H3F15 displayed potent inhibition (IC50 <1nM) of HER3/AKT phosphorylation and proliferation. Interestingly, H3F15 also effectively inhibited neuregulin stimulated HER3 phosphorylation and downstream signaling in MCF7 cells indicating that H3F15 inhibits multiple modes of HER3 activation. Determining the H3F15/HER3 crystal structure revealed that H3F15 binds a novel conformational epitope that traps HER3 in the inactive conformation thus preventing its activation by either HER2 or neuregulin. In vivo testing of H3F15 using xenograft tumor models driven by either HER2 (BT474) or HER3 ligands (BxPC3) confirmed that single agent H3F15 (20mg/kg) significantly inhibited tumor growth (83 and 77% inhibition respectively). Furthermore, combinations of H3F15 with either trastuzumab or PI3K- targeted agents were synergistic in a panel of HER2 driven cell lines whilst the in vivo combination of H3F15 (20mg/kg) with trastuzumab (1mg/kg) was sufficient to induce tumor regression. Discussion: H3F15 is a HER3- targeted antagonist IgG1 that stabilizes the inactive form of HER3. This novel mechanism of action enables H3F15 to uniquely inhibit both ligand-induced and HER2−mediated activation of HER3. Thus H3F15 is the first HER3 antibody to display single-agent efficacy in both HER2 and ligand driven xenograft models whilst also inducing tumor regressions in combination with trastuzumab. Based on preclinical data, combining H3F15 with either trastuzumab or PI3K- targeted agents fully inhibits the HER2/HER3 signaling pathway, which may lead to greater and more sustained clinical efficacy in HER2 driven cancers. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S2-5.

TRIM29 Functions as a Tumor Suppressor in Nontumorigenic Breast Cells and Invasive ER+ Breast Cancer

Tripartite motif-containing 29 (TRIM29) is a member of the TRIM protein family that has been implicated in hematologic and solid tumor cancers. We found that TRIM29 functions as a tumor suppressor in both the nontumorigenic MCF10A [estrogen receptor (ER)-/TRIM29+] breast cell line and the invasive MCF7 (ER+/TRIM29-) breast cell line. Silencing TRIM29 in MCF10A cells resulted in preneoplastic changes that included loss of polarity in three-dimensional culture, increased proliferation, anchorage-independent growth, and increased migration and invasion. Conversely, the introduction of TRIM29 into MCF7 cells caused reversion to a less aggressive phenotype by antagonizing the growth effect of 17β-estradiol. The interaction between TRIM29 and ER signaling in MCF7 cells was supported by a reduction in ERE binding in the presence of TRIM29 and suppression of ER-dependent gene expression of TFF1, FOS, and GREB1. By microarray analyses, we showed that younger women (<55 years of age) with early-stage, ER+ breast cancer who were given no adjuvant systemic therapy had a significantly lower risk of relapse when their tumor had high TRIM29 expression (P = 0.02). This effect was not observed in older women (>55 years of age) and thus may be due to menopause and loss of circulating estrogens. Our results suggest that loss of TRIM29 expression in normal breast luminal cells can contribute to malignant transformation and lead to progression of ER+ breast cancer in premenopausal women.

Specific targeting of insulin-like growth factor 1 receptor signaling in human estrogen dependent breast cancer cell by a novel tyrosine-based benzoxazepine derivative

The present study sought to investigate the in vitro and in vivo effects of a tyrosine-based benzoxazepine, 4-[4-(toluene-4-sulfonyl)-2,3,4,5-tetrahydro-benzo[f][1,4]oxazepin-3-ylmethyl]-phenol) [THBP] in human breast cancer cells, with a focus on determining its molecular target. THBP had growth inhibitory effect on MCF-7 and MDA-MD-231 cells. At IC 50 value (∼20 μM), THBP resulted in G1 arrest, decrease in cyclin D1 levels and induction of apoptosis of MCF-7 cells. Mechanistically, activation of caspase 8 contributes critically to the induction of apoptotic cell death as copresence of selective inhibition of caspase 8 effectively abrogates the cytotoxic effect of THBP in MCF-7 cells. Further, THBP increased pro-apoptotic protein, Bax; decreased anti-apoptotic protein, Bcl-2; and decreased mitochondrial membrane potential in MCF-7 cells, indicating involvement of an intrinsic pathway of apoptosis following caspase 8 activation. Out of the various growth factors/hormones, THBP selectively abrogated increased viability of MCF-7 cells by insulin-like growth factor 1 (IGF-1). Molecular docking studies revealed that THBP occupied the ATP binding pocket of IGF-1 receptor (IGF-1R). Accordingly THBP was found to inhibit IGF-1-induced phosphorylation of IGF-1R and insulin receptor substrate-1 (IRS-1) without inhibiting insulin signaling in MCF-7 cells. In athymic nude mice, compared with vehicle, THBP treatment significantly reduced the growth of MCF-7 xenograft tumors through inhibition of cancer cell proliferation as well as promotion of cell death that correlated with reduced phospho-IGF-1R levels. We suggest that interfering with the IGF-1R signaling by the benzoxazepine THBP offers a novel and selective therapeutic strategy for estrogen receptor-positive, postmenopausal breast cancer patients.

Abstract 3819: Activation of toll-like receptor 5 on breast cancer cells by flagellin suppresses tumor development and growth

Abstract Toll-like receptors (TLRs) play key roles in both the innate and adaptive immune systems, particularly in inflammatory responses against pathogen infection. Recent evidence showed that functional TLRs are also expressed on a wide variety of tumor cells; however, their activation exhibits either antitumor or pro-tumor effects. In order to investigate the function of TLR signaling pathways in breast cancers, we analyzed the expression of TLRs and their functional status in breast cancer cells by RT-PCR and NF-κB reporter gene assays in response to synthetic ligands for these receptors. We found that only TLR5 signaling is highly responsive in breast cancer cells. The phosphorylation levels of IκB, ERK, and JNK after flagellin (an agonist of TLR5) stimulation were increased in tumor cells. Activation of TLR5 signaling in tumor cells by flagellin induces the secretion of various cytokines and chemokines including interleukin-6, interleukin-8, and TNF-α. Moreover, flagellin/TLR5 signaling in MCF-7 cells inhibits an anchorage-independent growth, a hallmark of tumorigenic transformation. In addition, the secretion of soluble factors induced by flagellin contributed to the growth-inhibitive activity in an autocrine fashion. The inhibitive activity was further confirmed in mouse xenografts of human breast cancer model. Administration of flagellin inhibits tumor growth and therefore prolongs the survival of tumor-bearing mice. These findings indicate that TLR5 activation by flagellin mediates innate immunity to elicit potent antitumor activity in breast cancers, which may serve as a novel therapeutic target for human breast cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3819.

Hexachlorobenzene induces cell proliferation and IGF-I signaling pathway in an estrogen receptor α-dependent manner in MCF-7 breast cancer cell line

Hexachlorobenzene (HCB) is an organochlorine pesticide widely distributed in the biosphere. ERα regulates the expression of genes involved in growth and development, and plays an important role in breast cancer. The present study focuses attention on the effect of HCB (0.005, 0.05, 0.5, 5 μM) on cell proliferation in estrogen receptor α (ERα)(+) MCF-7, and ERα(−) MDA-MB-231 breast cancer cell lines. We also studied the insulin growth factor-I (IGF-I) signaling pathway in MCF-7 cells. HCB (0.005 and 0.05 μM) stimulated cell proliferation in MCF-7, but not in MDA-MB-231 cells. The pesticide increased apoptosis in MCF-7, at HCB (0.5 and 5 μM). At these doses, HCB induced the expression of the aryl hydrocarbon receptor (AhR)-regulated gene cytochrome P4501A1. MCF-7 cells exposed to HCB (0.005 and 0.05 μM) overexpressed IGF-IR and insulin receptor (IR). IRS-1-phosphotyrosine content was increased in a dose dependent manner. ICI 182,780 prevented HCB-induced cell proliferation and IGF-I signaling in MCF-7 cells incubated in phenol-red free media. In addition, HCB (0.005 μM) increased c-Src activation, Tyr537-ERα phosphorylation and ERα down-regulation. Taken together, our data indicate that HCB stimulation of cell proliferation and IGF-I signaling is ERα dependent in MCF-7 cells.

TCDD mediates inhibition of p53 and activation of ERα signaling in MCF-7 cells at moderate hypoxic conditions

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is known to promote cancer initiation and progression and accumulates in mammary fat tissue. Effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). Physiological conditions of moderate hypoxia in breast cancer also activate another transcription factor, hypoxia-inducible factor-1 alpha (HIF-1alpha). In addition, the transcription factors p53 and the estrogen receptor alpha (ERalpha) are important key players in breast cancer progression. Here, human breast cancer cells cultured under mild hypoxic conditions were exposed to TCDD and analyzed for regulation of p53 signaling and ERalpha transactivation. Simultaneous exposure to TCDD and hypoxia resulted in a moderate but reproducible inhibition of p53 expression. Both the direct activation of the ERalpha and the transcriptional regulation of Hdm2 mediated this inhibition. As consequence the p53-mediated target gene expression (Dusp5) was reduced. Silencing of Dusp5 by simultaneous exposure of TCDD and hypoxia or by RNAi led to increased phosphorylation of ERK1/2. This increase resulted in transactivation of ERalpha and induction of ERalpha-mediated transcription of Hdm2 and SOCS3. Specificity of ERalpha-transactivation by ERK1/2 was confirmed by treatment with MAPKK-inhibitor PD98059. The combination of inhibition of functional p53 protein and induction of ERalpha signaling could serve as a model for the operational sequence of TCDD effects to prevent cell death and promote breast tumor progression.

C-FLIP knockdown induces ligand-independent DR5-, FADD-, caspase-8-, and caspase-9-dependent apoptosis in breast cancer cells

Cellular-FLICE inhibitory protein (c-FLIP) is an inhibitor of apoptosis downstream of the death receptors Fas, DR4, and DR5, and is expressed as long (c-FLIP L) and short (c-FLIP S) splice forms. We found that the knockdown of c-FLIP using small interfering RNA (siRNA) triggered ligand-independent caspase-8- and -9-dependent spontaneous apoptosis and decreased the proliferation of MCF-7 breast cancer cells. Further analysis revealed that an apoptotic inhibitory complex (AIC) comprised of DR5, FADD, caspase-8, and c-FLIP L exists in MCF-7 cells, and the absence of c-FLIP L from this complex induces DR5- and FADD-mediated caspase-8 activation in the death inducing signaling complex (DISC). c-FLIP S was not detected in the AIC, and using splice form-specific siRNAs we showed that c-FLIP L but not c-FLIP S is required to prevent spontaneous death signaling in MCF-7 cells. These results clearly show that c-FLIP L prevents ligand-independent death signaling and provides direct support for studying c-FLIP as a relevant therapeutic target for breast cancers.