MCF-10A Human Mammary Epithelial Cells Research Articles

Abstract 3422: Requirement of the histone demethylase LSD1 in Snail-mediated transcriptional repression during epithelial-mesenchymal transition

Abstract Cellular transitions between epithelial and mesenchymal states have crucial roles in embryonic development and tumor metastasis. Members of the Snai1 family of zinc finger transcription factors have emerged as key regulators of epithelial-mesenchymal transition (EMT) and induce EMT in part by directly repressing epithelial markers such as E-cadherin, a gate keeper of the epithelial phenotype. However, the mechanisms underlying Snai1-mediated transcriptional repression remains incompletely understood. Our study shows Snai1 physically interacts with histone demethylase LSD1 (KDM1A) via the repressive SNAG domain located at the N-terminus. Stable over-expression of Snai1 in human mammary epithelial cell MCF10A recruits LSD1 to the E-cadherin promoter and reduce the level of dimethylation of lysine 4 in histone 3 (H3K4m2), a covalent histone modification commonly associated with active promoters. LSD1 is essential for Snai1-mediated transcriptional repression and for maintenance of the silenced state of Snai1 target genes in invasive cancer cells. In the absence of LSD1, Snai1 fails to repress E-cadherin. In cancer cells in which E-cadherin is silenced, depletion of LSD1 results in partial de-repression of epithelial genes and elevated H3K4m2 levels at the E-cadherin promoter, These results underline the critical role of LSD1 in Snai1-dependent transcriptional repression of epithelial markers and suggest that the LSD1 complex could be a potential therapeutic target for prevention of Snai1-dependent EMT and tumor invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3422. doi:10.1158/1538-7445.AM2011-3422

NBA1/MERIT40 and BRE Interaction Is Required for the Integrity of Two Distinct Deubiquitinating Enzyme BRCC36-containing Complexes

BRCC36-deubiquitinating enzyme (DUB) forms two different complexes through interactions with two different adaptor proteins Abraxas and ABRO1 in cells. Abraxas mainly localizes in the nucleus, mediating the interaction of BRCC36 with BRCA1. ABRO1 is mainly localized in the cytoplasm. Because it lacks the BRCA1-interacting motif, the ABRO1 complex does not interact with BRCA1. Both BRCC36-containing complexes contain common components including BRE and NBA1/MERIT40. Here, we found that the two complexes are assembled in a similar manner and NBA1 and BRE interaction is critical for maintaining the integrity of both of the complexes. Knockdown of NBA1 or BRE leads to decreased levels of components of the two BRCC36-containing complexes. We provided evidence that NBA1 interacts with BRE through a C-terminal conserved motif of the NBA1 protein and a C-terminal UEV domain of the BRE protein. Furthermore, the NBA1-BRE interaction is required for cellular resistance to ionizing irradiation and NBA1's role in recruiting BRCA1 to DNA damage sites. Together, these studies reveal critical interactions required for the formation and function of BRCC36-containing DUB complexes.

Trastuzumab has preferential activity against breast cancers driven by HER2 homodimers.

In breast cancer cells with HER2 gene amplification, HER2 receptors exist on the cell surface as monomers, homodimers, and heterodimers with EGFR/HER3. The therapeutic antibody trastuzumab, an approved therapy for HER2(+) breast cancer, cannot block ligand-induced HER2 heterodimers, suggesting it cannot effectively inhibit HER2 signaling. Hence, HER2 oligomeric states may predict the odds of a clinical response to trastuzumab in HER2-driven tumors. To test this hypothesis, we generated nontransformed human MCF10A mammary epithelial cells stably expressing a chimeric HER2-FKBP molecule that could be conditionally induced to homodimerize by adding the FKBP ligand AP1510, or instead induced to heterodimerize with EGFR or HER3 by adding the heterodimer ligands EGF/TGFα or heregulin. AP1510, EGF, and heregulin each induced growth of MCF10A cells expressing HER2-FKBP. Trastuzumab inhibited homodimer-mediated but not heterodimer-mediated cell growth. In contrast, the HER2 antibody pertuzumab, which blocks HER2 heterodimerization, inhibited growth induced by heregulin but not AP1510. Lastly, the HER2/EGFR tyrosine kinase inhibitor lapatinib blocked both homodimer- and heterodimer-induced growth. AP1510 triggered phosphorylation of Erk1/2 but not AKT, whereas trastuzumab inhibited AP1510-induced Erk1/2 phosphorylation and Shc-HER2 homodimer binding, but not TGFα-induced AKT phosphorylation. Consistent with these observations, high levels of HER2 homodimers correlated with longer time to progression following trastuzumab therapy in a cohort of patients with HER2-overexpressing breast cancer. Together, our findings confirm the notion that HER2 oligomeric states regulate HER2 signaling, also arguing that trastuzumab sensitivity of homodimers may reflect their inability to activate the PI3K (phosphoinositide 3-kinase)/AKT pathway. A clinical implication of our results is that high levels of HER2 homodimers may predict a positive response to trastuzumab.

Drug-loaded biodegradable microspheres for image-guided combinatory epigenetic therapy in cells

We synthesize drug-loaded poly (lactic-co-glycolic acid) (PLGA) microspheres for image-guided combinatory epigenetic therapy in MCF-10A human mammary epithelial cells. LY294002 and Nile Red are encapsulated in microspheres for sustained drug release and fluorescence microscopic imaging. Drug-loaded microspheres target MCF-10A cells through a three-step binding process involving biotinylated antibody, streptavidin, and biotinylated microspheres. LY294002 loaded microspheres and 5-Aza-2-deoxycytidine are applied to MCF-10A cells for combinatory PI3K∕AKT inhibition and deoxyribonucleic acid (DNA) demethylation. Our study implies the technical potential of disease targeting and image-guided combinatory epigenetic therapy using drug-loaded multifunctional biodegradable PLGA microspheres.

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

Abstract A47: Establishing candidate biomarkers for the pharmacodynamic action of sulforaphane in the breast

Abstract The goal of this study is the discovery of biomarkers reflecting the pharmacodynamic action of sulforaphane (SFN), initially using normal human mammary epithelial MCF10A cells and then in tissue obtained from healthy patients undergoing reduction mammoplasty. The isothiocyanate SFN is formed by the hydrolysis of glucoraphanin, a water soluble glucosinolate found in cruciferous vegetables with especially high levels measured in 3 day old broccoli sprouts. Chemoprevention by SFN is achieved in part through the upregulation of cytoprotective enzymes via the Keap1/Nrf2 pathway. Preliminary dose response and time course studies in MCF10A cells established that SFN upregulated cytoprotective enzymes as indicated by increased levels of NQO1 transcripts, protein and activity. NQO1 induction was dependent on the Keap1/Nrf2 pathway since NQO1 transcripts, protein and activity were enhanced by KEAP1 siRNA knockdown in MCF10As. For the biomarker discovery phase SFN treated and KEAP1 knockdown MCF10A cells were analyzed by microarray to determine transcriptomic changes in response to pharmacological and genetic stimulation of the Keap1/Nrf2 pathway. The two experimental approaches were analyzed independently and then compared. The predominant class of upregulated genes common to both SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidant genes including NQO1, heme oxygenase (decycling) 1, sequestosome 1, thioredoxin reductase 1 and peroxiredoxin 1. The most highly upregulated genes in this class were the aldo-keto reductase family members including AKR1B1, AKR1B10, AKR1C1 and AKR1C3. Other classes of genes with overlapping regulation were glutathione metabolism genes such as GCLC and GCLM, molecular transport, cell growth and cell death regulation genes. Some members of the xenobiotic metabolism, molecular transport, cell growth and cell death regulation classes of genes were also regulated exclusively by SFN. Additionally histone cluster and chromatin remodeling genes were downregulated by SFN. Potential biomarker genes from the microarray study include NQO1, GCLC, SQSTM1 and AKR1C1. An accompanying proteomic study for biomarker discovery using Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC) is underway. The transcriptomic and proteomic changes in response to SFN treatment and KEAP1 knockdown will be compared to determine if the changes seen on the transcriptomic level are translated to the proteomic level. Candidate biomarkers identified from microarray and SILAC studies reflecting the pharmacodynamic action of SFN in human cells will be analyzed by Western blot, qRT-PCR and enzyme assays in tissue from reduction mammoplasty patients. These women have been randomized in a clinical trial to receive either a broccoli sprout preparation containing 100 micromole SFN or a placebo beverage daily for 10 days prior to surgery. Supported by DOD grant BC073262 and NIH grants P50 CA088843 and U54 RR020839. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A47.

Metastasis promoted by caveolin-1 gene silence in human mammary epithelial cell MCF10A

Metastasis promoted by caveolin-1 gene silence in human mammary epithelial cell MCF10A

Heat-shock transcription factor HSF1 has a critical role in human epidermal growth factor receptor-2-induced cellular transformation and tumorigenesis

The heat shock transcription factor HSF1 was recently demonstrated to play a key role in the development of tumors associated with activation of Ras or inactivation of p53. Here we show that HSF1 is required for cell transformation and tumorigenesis induced by HER2 oncogene responsible for aggressive breast tumors. Upon expression of HER2, untransformed human mammary epithelial cells MCF-10A underwent neoplastic transformation, formed foci in culture and tumors in nude mouse xenografts. However, expression of HER2 in MCF-10A cells with knockdown of HSF1 did not cause either foci formation or tumor growth in xenografts. The anti-tumorigenic effect of downregulation of HSF1 was associated with HER2-induced accumulation of the CDK inhibitor p21 and decrease of the mitotic regulator survivin, which resulted in growth inhibition and cell senescence. In fact, either knockout of p21 or overexpression of survivin alleviated these effects of HSF1 knockdown. Proliferation of certain human HER2-postitive breast cancer lines also requires HSF1, since its knockdown led to upregulation of p21 and/or drop of survivin, precipitating growth arrest. Similar effects were observed with a small molecular weight inhibitor of the heat shock response NZ28. Effects of HSF1 knockdown on growth arrest and senescence of HER2-expressing cells were associated with downregulation of Hsp72 and Hsp27. Therefore, HSF1 is critical for proliferation of HER2-expressing cells, most likely since it maintains levels of HSPs, which in turn control regulators of senescence p21 and survivin.

H1047R phosphatidylinositol 3-kinase mutant enhances HER2-mediated transformation by heregulin production and activation of HER3

Hyperactivation of phosphatidylinositol-3 kinase (PI3K) can occur as a result of somatic mutations in PIK3CA, the gene encoding the p110α subunit of PI3K. The HER2 oncogene is amplified in 25% of all breast cancers and some of these tumors also harbor PIK3CA mutations. We examined mechanisms by which mutant PI3K can enhance transformation and confer resistance to HER2-directed therapies. We introduced the PI3K mutations E545K and H1047R in MCF10A human mammary epithelial cells that also overexpress HER2. Both mutants conferred a gain of function to MCF10A/HER2 cells. Expression of H1047R PI3K but not E545K PI3K markedly upregulated the HER3/HER4 ligand heregulin (HRG). HRG siRNA inhibited growth of H1047R but not E545K-expressing cells and synergized with the HER2 inhibitors trastuzumab and lapatinib. The PI3K inhibitor BEZ235 markedly inhibited HRG and pAKT levels and, in combination with lapatinib, completely inhibited growth of cells expressing H1047R PI3K. These observations suggest that PI3K mutants enhance HER2-mediated transformation by amplifying the ligand-induced signaling output of the ErbB network. This also counteracts the full effect of therapeutic inhibitors of HER2. These data also suggest that mammary tumors that contain both HER2 gene amplification and PIK3CA mutations should be treated with a combination of HER2 and PI3K inhibitors.

The Ras oncogene signals centrosome amplification in mammary epithelial cells through cyclin D1/Cdk4 and Nek2

Centrosome amplification (CA) contributes to carcinogenesis by generating aneuploidy. Elevated frequencies of CA in most benign breast lesions and primary tumors suggest a causative role for CA in breast cancers. Clearly, identifying which and how altered signal transduction pathways contribute to CA is crucial to breast cancer control. Although a causative and cooperative role for c-Myc and Ras in mammary tumorigenesis is well documented, their ability to generate CA during mammary tumor initiation remains unexplored. To answer that question, K-Ras(G12D) and c-Myc were induced in mouse mammary glands. Although CA was observed in mammary tumors initiated by c-Myc or K-Ras(G12D), it was detected only in premalignant mammary lesions expressing K-Ras(G12D). CA, both in vivo and in vitro, was associated with increased expression of the centrosome-regulatory proteins, cyclin D1 and Nek2. Abolishing the expression of cyclin D1, Cdk4 or Nek2 in MCF10A human mammary epithelial cells expressing H-Ras(G12V) abrogated Ras-induced CA, whereas silencing cyclin E1 or B2 had no effect. Thus, we conclude that CA precedes mammary tumorigenesis, and interfering with centrosome-regulatory targets suppresses CA.

PTEN and p53 cross-regulation induced by soy isoflavone genistein promotes mammary epithelial cell cycle arrest and lobuloalveolar differentiation

The tumor suppressors phosphatase and tensin homologue deleted on chromosome ten (PTEN) and p53 are closely related to the pathogenesis of breast cancer, yet pathway-specific mechanisms underlying their participation in mediating the protective actions of dietary bioactive components on breast cancer risk are poorly understood. We recently showed that dietary exposure to the soy isoflavone genistein (GEN) induced PTEN expression in mammary epithelial cells in vivo and in vitro, consistent with the breast cancer preventive effects of soy food consumption. Here, we evaluated PTEN and p53 functional interactions in the nuclear compartment of mammary epithelial cells as a mechanism for mammary tumor protection by GEN. Using the non-tumorigenic human mammary epithelial cells MCF10-A, we demonstrate that GEN increased PTEN expression and nuclear localization. We show that increased nuclear PTEN levels initiated an autoregulatory loop involving PTEN-dependent increases in p53 nuclear localization, PTEN-p53 physical association, PTEN-p53 co-recruitment to the PTEN promoter region and p53 transactivation of PTEN promoter activity. The PTEN-p53 cross talk induced by GEN resulted in increased cell cycle arrest; decreased pro-proliferative cyclin D1 and pleiotrophin gene expression and the early formation of mammary acini, indicative of GEN promotion of lobuloalveolar differentiation. Our findings provide support to GEN-induced PTEN as both a target and regulator of p53 action and offer a mechanistic basis for PTEN pathway activation to underlie the antitumor properties of dietary factors, with important implications for reducing breast cancer risk.

Krüppel-like Factor 4 Inhibits Epithelial-to-Mesenchymal Transition through Regulation of E-cadherin Gene Expression

The Krüppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.

RNA inference-mediated caveolin-1 down-regulation decrease estrogen receptor alpha (ERα) signaling in human mammary epithelial cells

Several evidences support that caveolin-1 is associated with mammary cell transformation and oncogenesis. We have previously reported that a cell line, named MCF10A-ST1 (ST1), which only expressed 30% Cav-1 compared with parental cells MCF10A (human mammary epithelial cell line) was isolated by gene trapping. The decreased expression of Cav-1 is sufficient for phenotypic transformation of MCF10A cells, which involved in the loss of anchorage-dependent growth and migration in nude mice with the existence of E2. The previous study in our lab on microarray assay showed that the expression of cyclin D1 (cell cycle protein) was up-regulated in ST1 cell line. Here, we not only confirmed the results by Western Blot but also demonstrated that Cav-1 down-regulation accelerate the progression of mammary cells from G1 phase into S phase by Flow Cytometry (FCM). This proposed that the Cav-1 down-regulation could change the progress of cell cycle in the mammary cells. Otherwise, microarray assay also showed that the transcription factor (c-Jun) was up-regulated. But, the original carcinoma gene (c-Fos) and transcription factor (AP-1) have not obviously changed compared with MCF10A. ST1 cells obtained the morigenicity in nude mice with the existence of E2, and the immunoprecipitation showed the interactions of Cav-1 with ERα in both MCF10A and ST1. ERα expression was increased as further down-regulation of Cav-1. So, we hypothesize that Cav-1 down-regulation could induce the activation of ERα-associated signaling pathway, in order to adjust the development and proliferation. By siRNA technology, the down regulation of Cav-1 could activate MAP kinase and Akt signaling pathway, including the phosphorylation of ERK1/2 and Akt. However, the mechanism of Cav-1 down-regulation in the early transformation and signaling transduction of mammary epithelial cells is unclear. Here, we report that down-regulation of caveolin-1 protein expression leads to deregulate estrogen receptor alpha (ERα) signaling and consequently early transformation in mammary epithelia.

Abstract 1: Role of the splicing factor SF2/ASF in mammary epithelial cell transformation

Abstract Cancer cells often display aberrant profiles of alternative splicing, leading to the production of isoforms that can stimulate cell proliferation and migration or improve resistance to apoptosis. Some of these alterations are caused by mutations at splice sites or in splicing-regulatory elements, but changes can also be caused by alterations in the expression of proteins involved in splice-site selection. Changes in the expression of splicing factors have been reported in different types of cancers, such as colon, ovarian, and breast. Our lab recently demonstrated that the splicing factor SF2/ASF can be oncogenic and is often upregulated in human cancers. Several studies have suggested a role for splicing factors in the development of breast tumors in both human and mice. Thus, our goal is to examine the role of splicing factors in transformation and tumor maintenance of mammary epithelial cells. We have analyzed the ability of the splicing factor SF2/ASF to promote transformation of MCF-10A human mammary epithelial cells, which form organized acinar structures in three-dimensional (3D) culture. Cells slightly overexpressing SF2/ASF form significantly larger acini than the control, but retain a proper acinar organization with a hollow lumen. They show increased proliferation and delayed apoptosis in 3D culture, and can also form colonies in soft agar, a measure of anchorage-independent growth. Interestingly, these features require the first RNA-recognition motif (RRM1) of SF2/ASF, as a deletion mutant lacking this motif has the same phenotype as the control. Furthermore, we find that SF2/ASF can cooperate with the myc oncogene but not with ErbB2 to transform mammary epithelial cells grown in 3-D, and also to promote anchorage-independent growth in soft agar. In contrast, when SF2/ASF is overexpressed together with the HPV16 E7 oncogene, MCF-10A acini are smaller than the controls, suggesting competitive pathways. We are currently investigating if epithelial cells overexpressing SF2/ASF alone or with the above oncogenes can form tumors in a xenograft model, after transplantation into epithelium-free fat pads of three-week-old mice. We are also investigating the signaling pathways involved in SF2/ASF-mediated transformation, focusing on mTOR signaling, as well as on the isoforms of the downstream kinase S6K1, which has been linked to SF2/ASF's oncogenic properties in murine fibroblasts. In addition, we are in the process of identifying the splicing targets of SF2/ASF in MCF-10A in 3D culture. This study will allow us to assess the role of SF2/ASF in breast cancer. By identifying oncogenic splicing factors and their specific targets, we hope in the future to be able to contribute to the development of alternative cancer therapies based on modulating the expression or activity of these factors or their targets. 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 1.

Abstract 4928: 14-3-3ζ-mediated hypomethylation of c-myc oncogene by downregulation of Dnmts contributes to initiation and progression of breast cancer

Abstract The development of breast cancer is a multistep process and the molecular mechanisms underlying this process are still elusive. Previously, we discovered that 14-3-3ζ overexpression not only contribute to breast cancer progression but also play an important role in breast cancer initiation, including an early stage of breast disease-atypical ductal hyperplasia (ADH). However, the underlying mechanisms remain unclear. Recently, 14-3-3ζ has been shown to bind to Histone H3, Methylation Binding Proteins (MBPs) and HDAC family members, suggesting 14-3-3ζ may play a role in epigenetic regulation. As an initial effort to investigate whether 14-3-3ζ has a role in epigenetic regulation and whether epigenetic regulation by 14-3-3ζ plays a role in breast cancer initiation, we established 14-3-3ζ stable overexpressing lines (10A.14-3-3ζ) in non-tumorigenic MCF10A human mammary epithelial cells (HMEC) and analyzed molecular alterations by cDNA microarray in the 10A.14-3-3ζ cells versus the 10A.vec control cells in a 3D culture system. Interestingly, we found that c-myc, a well known oncogene which is overexpressed in many types of cancer, was upregulated at the mRNA level in the 10A.14-3-3ζ cells versus the 10A.vec cells. We further confirmed c-Myc upregulation at both mRNA level by RT-PCR and protein level by Western Blotting. Additionally, we performed Reverse Phase Protein Array (RPPA) analysis with a series of validated antibodies, and we found that Dnmt1, the key DNA methyltransferase in mammals, was downregulated in 10A.14-3-3ζ cells compared to 10A.vec cells. We have confirmed Dnmt1 downregulation by western blotting. Since c-myc oncogene are well known to be upregulated by hypomethylation as a consequence of Dnmt1 downregulation, these data suggest that 14-3-3ζ may contribute to breast cancer initiation and progression by upregulation of oncogene c-myc via promoter hypomethylation. We will continue this study to provide a better understanding on how 14-3-3ζ contributes to breast cancer initiation and progression via epigenetic deregulation (demethylation) of c-myc. This study will bring insight into how 14-3-3ζ overexpression elicits the phenotypic changes during the development of breast carcinomas through global deregulation of transcriptome as well as specific gene expressions. Our studies could provide a better understanding of the role of 14-3-3ζ in breast cancer initiation and progression. 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 4928.

Abstract 4006: Soluble ALK4 extracellular domain antagonizes oncogenic Cripto signaling and inhibits tumor growth

Abstract Cripto is a small GPI-anchored glycoprotein that plays key roles during normal development and in tumorigenesis. Cripto is expressed at aberrantly high levels in human tumors and blocking its oncogenic effects on activin/Nodal/TGF-beta, Erk/MAPK and PI3K/Akt pathways represents a promising therapeutic strategy. Cripto functions as a Nodal co-receptor and binds directly to the extracellular domain (ECD) of the activin/Nodal type I receptor ALK4. We have shown that a kinase-deleted ALK4 mutant deficient in activin binding (ALK4-L75A) retains Cripto binding and can block Cripto effects on activin and Nodal signaling. We hypothesized that soluble ALK4-L75A ECD proteins would similarly bind Cripto and disrupt oncogenic Cripto function. To test this, we generated two soluble constructs incorporating the ALK4-L75A ECD, one with a C-terminal His tag (sALK4-L75A-His) and the other consisting of an ALK4-L75A ECD-Fc fusion with an N-terminal FLAG tag (sALK4-L75A-Fc). We expressed the sALK4-L75A-His and sALK4-L75A-Fc proteins in 293T cells and then purified them from conditioned media using Ni-NTA agarose and Protein-A agarose, respectively. The sALK4-L75A-Fc protein dose-dependently inhibited 125I-Cripto binding to 293T cells transfected with full length ALK4. In addition, sALK4-L75A-Fc blocked 125I-Cripto binding to untransfected human mammary epithelial MCF10A cells. In accordance with these binding data, sALK4-L75A-His inhibited Cripto-dependent Nodal signaling in 293T cells and sALK4-L75A-Fc inhibited Nodal-induced Smad2 phosphorylation in human NCCIT cells. sALK4-L75A-His also blocked Cripto-induced PI3K signaling while sALK4-L75A-Fc inhibited Cripto-induced MCF10A cell proliferation. Importantly, we have shown that sALK4-L75A-Fc can also inhibit the growth of tumor xenografts derived from NCCIT cells at doses where no toxicity was observed. Thus, we provide the first evidence that soluble forms of ALK4 inhibit oncogenic Cripto signaling. The efficacy of a soluble ALK4 receptor as an antitumor agent points to the utility of Cripto antagonists in the treatment of human cancer. 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 4006.

Abstract 5056: Signaling pathways critical for insulin receptor substrate (IRS)-mediated disruption of MCF-10A acini formation

Abstract Insulin receptor substrate (IRS)-1 and IRS-2 are important adaptor proteins in insulin-like growth factor-I receptor (IGF-IR) signaling. IRS-1 and IRS-2 are expressed during mammary gland development and also in human breast tumors. In order to study the role of IRS-1 and IRS-2 in breast tumorigenesis, we overexpressed both IRS proteins in immortalized, non-transformed human mammary epithelial MCF-10A cells. It has been shown that MCF-10A cells form uniform, empty-lumen acini in three dimensional (3D) culture. We previously showed that overexpression of either IRS-1 or IRS-2 in MCF-10A cells disrupted the acini in 3D culture and resulted in extensive proliferation and disrupted apical-basal polarity. In both IRS-1 and IRS-2 overexpressing MCF-10A cells, an increase in tyrosine phosphorylation and association with p85 of both IRS proteins indicated that IRS-mediated signaling was activated. siRNA knockdown of the overexpressed IRS proteins reversed the disruption of MCF-10A acini, confirming that this is indeed an IRS-related event. In order to identify the critical effectors in IRS-mediated disruption of MCF-10A polarity and growth, we used small molecule inhibitors to screen multiple kinase signaling pathways upstream and downstream of the IRS proteins. Inhibition of Rac or atypical PKC (aPKC) didn't affect MCF-10A cell acini formation, however it abolished the IRS-mediated disruption of MCF-10A acini formation. Consistent with this, knockdown of Rac and aPKC by siRNA suppressed IRS-mediated disruption in acini formation. Rac belongs to the family of GTPase that regulates cell motility. aPKC is a key component of the PAR polarity complex, and has previously been shown to be involved in IRS-signaling in adipocytes. While the signaling mechanism of Rac and aPKC in the IRSs-induced disruption of acini is still unknown, we found that another component in the PAR polarity complex Par3 may associate with IRS proteins and leads to dissociation of the polarity complex. Taken together, overexpression of IRS-1 and IRS-2 disrupts the MCF-10A acini in 3D culture in part through Rac and aPKC pathways. 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 5056.

PCB-153 exposure coordinates cell cycle progression and cellular metabolism in human mammary epithelial cells

2,2',4,4',5,5'-Hexachlorobiphenyl (PCB-153) is a non-metabolizable environmental chemical contaminant commonly found in breast milk of PCB exposed individuals, suggesting that chronic exposure to PCB-153 could have adverse health effects. We have shown previously that PCB-153 increased reactive oxygen species levels in non-tumorigenic MCF-10A human mammary epithelial cells, which were associated with DNA damage, growth inhibition, and cytotoxicity. This study investigates the hypothesis that PCB-153 exposure coordinates cell cycle progression and cellular metabolism by inhibiting cyclin D1 accumulation. PCB-153 treated MCF-10A cells exhibited a dose and time dependent decrease in cyclin D1 protein levels. The decrease in cyclin D1 protein levels was associated with an inhibition in AKT and GSK-3beta phosphorylation, which correlated with an increase in cyclin D1-T286 phosphorylation. Fibroblasts carrying a mutant form of cyclin D1 (T286A) were resistant to PCB-153 induced degradation of cyclin D1. Pre-treatment of cells with a proteasome inhibitor (MG132) suppressed PCB-153 induced decrease in cyclin D1 protein levels. Interestingly, suppression in cyclin D1 accumulation was associated with an increase in cellular glucose consumption, and hexokinase II and pyruvate kinase protein levels. These results suggest that cyclin D1 coordinates cell cycle progression and cellular metabolism in PCB-153 treated non-tumorigenic human mammary epithelial cells.

Arachidonic acid promotes epithelial-to-mesenchymal-like transition in mammary epithelial cells MCF10A

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. Cancer progression requires the development of metastasis, which is characterized by an increase in cell motility and invasion. Epithelial-to-mesenchymal transition (EMT) is a process, by which epithelial cells are transdifferentiated to a more mesenchymal state. A similar process takes place during tumor progression, when carcinoma cells stably or transiently lose epithelial polarities and acquire a mesenchymal phenotype. Arachidonic acid (AA) is a fatty acid that mediates cellular processes, such as cell survival, angiogenesis, chemotaxis, mitogenesis, migration and apoptosis. However, the role of AA on the EMT process in human mammary epithelial cells remains to be studied. We demonstrate here that AA promotes an increase in vimentin and N-cadherin expression, MMP-9 secretion, a decrease in E-cadherin junctional levels, and the activation of FAK, Src and NF-κB in MCF10A cells. Furthermore, AA also promotes cell migration in an Src kinase activity-dependent fashion. In conclusion, our results demonstrate, for the first time, that AA promotes an epithelial-to-mesenchymal-like transition in MCF10A human mammary non-tumorigenic epithelial cells.

Squalene protects against oxidative DNA damage in MCF10A human mammary epithelial cells but not in MCF7 and MDA-MB-231 human breast cancer cells

Until now, very little has been known about the antioxidant capacity of squalene and its effect on human breast tumourigenesis. In the present work, we investigated squalene’s scavenging properties and its effect on cell proliferation, cell cycle profile, apoptosis, reactive oxygen species (ROS) level and oxidative DNA damage, using human breast cell lines. Our results showed that squalene neither possesses scavenging activity nor significantly alters cell proliferation rates, the cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A), minimally invasive (MDA-MB-231) breast cancer cells, and highly invasive (MCF7) breast cancer cells. However, we found that squalene did exert the following effects on MCF10A epithelial cells in a dose-dependent manner: (a) it decreased intracellular ROS level, (b) it prevented H 2O 2-induced oxidative injury, and (c) it protected against oxidative DNA damage. Interestingly, squalene did not exert these effects on MCF7 and MDA-MB-231 cancer cells. Therefore, our data suggest that squalene, found in high amounts in virgin olive oils, could be partially responsible for the lower incidence of breast cancer in populations that consume the Mediterranean diet due to its protective activity against oxidative DNA damage in normal mammary cells.