Differentiation Of Breast Epithelial Cells Research Articles

Notch signaling pathway: a comprehensive prognostic and gene expression profile analysis in breast cancer

Breast cancer is a complex disease exhibiting a great degree of heterogeneity due to different molecular subtypes. Notch signaling regulates the differentiation of breast epithelial cells during normal development and plays a crucial role in breast cancer progression through the abnormal expression of the Notch up-and down-stream effectors. To date, there are only a few patient-centered clinical studies using datasets characterizing the role of Notch signaling pathway regulators in breast cancer; thus, we investigate the role and functionality of these factors in different subtypes using publicly available databases containing records from large studies. High-throughput genomic data and clinical information extracted from TCGA were analyzed. We performed Kaplan–Meier survival and differential gene expression analyses using the HALLMARK_NOTCH_SIGNALING gene set. To determine if epigenetic regulation of the Notch regulators contributes to their expression, we analyzed methylation levels of these factors using the TCGA HumanMethylation450 Array data. Notch receptors and ligands expression is generally associated with the tumor subtype, grade, and stage. Furthermore, we showed gene expression levels of most Notch factors were associated with DNA methylation rate. Modulating the expression levels of Notch receptors and effectors can be a potential therapeutic approach for breast cancer. As we outline herein, elucidating the novel prognostic and regulatory roles of Notch implicate this pathway as an essential mediator controlling breast cancer progression.

Abstract P5-08-16: Molecular landscape of the breasts of women at high risk for breast cancer

Abstract Background: Despite advances in breast cancer diagnosis and treatment, there is a critical need to identify the determinants of breast cancer susceptibility in order to define new strategies to target cancer initiation and progression. The Susan G. Komen Tissue Bank at Indiana University (KTB), the only repository worldwide of non-malignant breast samples, can address key scientific questions by examining molecular changes, including epigenetic (DNA methylation) and transcriptomic alterations that occur in breasts of cancer-free women who are considered at high risk for developing breast cancer. Methods: Disease-free breast tissue cores donated by 80 high-risk (Tyrer-Cuzick lifetime risk ≥20%) and 80 average-risk women were processed for whole methylome (Diagenode MeCap Seq Library and NextSeq 75_sequencing) and whole transcriptome (Illumina TruSeq Stranded mRNA HT Library followed by NextSeq 75_sequencing) profiling. The two groups were matched according to age, racial background, and menopausal status. The cohort included 131 pre-menopausal and 29 post-menopausal women (mean age 38.6, SD 12.1). Frozen breast tissue cores with either abundant or moderate epithelial cellularity were selected. For the gene expression analysis, the reads were mapped to the human genome reference, GRCh38.p12 using the alignment software STAR ver. 2.5.2b and the read counts per gene were obtained using featureCounts ver. 1.6.3. For the evaluation of the epigenetically regulated genes, we focused our investigation on two types of functionally relevant epigenetic panels: the first panel includes 666 cancer-related markers (or pan-cancer) described by Shegafinia et al 2018, and the second panel includes 16 epigenetic markers identified in normal breast tissues-derived cells as connected with breast epithelial cell differentiation*. Results: In a preliminary analysis, we searched for transcriptomic differences in the breast tissues of high-risk women (N=43) as compared with that of matched average-risk subjects (N=48).The differential expression analysis identified 126 differentially expressed genes (DEGs, FDR<0.05, fold change >2), including 117 upregulated and 9 downregulated in the high-risk group. Interestingly, pathway analysis (Ingenuity Pathway Analysis, IPA v06_01) showed that most of these genes are involved with cancer. The sub-category “tumorigenesis of tissues” as defined by IPA included 71 genes. Through TCGA database analysis, using the UALCAN portal, we searched among our candidate genes for those targets that showed both overexpression and hypomethylation in breast tumors as compared with the normal breast, or vice versa, downregulation and hypermethylation in breast cancer. This pipeline allowed us to identify 17 genes potentially epigenetically regulated and involved in breast cancer susceptibility and the early phases of cancer development. Among those, two genes, FAM83A and PLA2G3, belong to a family of genes previously described as epigenetically dysregulated in multiple cancers (TCGA dataset, and Shegafinia et al 2018) and transform breast epithelial cell in vitro (FAM83A), thus reaffirming robustness of our approach in identifying genes associated with breast cancer risk Conclusion: The discovery of epigenetically regulated genes associated with breast cancer risk will open the doors to a deeper understanding of the process of cancer initiation and progression. The present study identified 17 gene alterations, two of which are epigenetically regulated in cancer. Our findings highlight an opportunity to address molecular alterations potentially linked with breast cancer susceptibility and risk, using a unique resource of normal breast samples. *Unpublished data by Dr. H. Nakshatri Citation Format: Natascia Marino, Rana German, Harikrishna Nakshatri, Ashley Vode, Jun Liu, Jie Huang, Douglas B Rush, Sha Cao, Anna Maria V Storniolo. Molecular landscape of the breasts of women at high risk for breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-08-16.

The Notch Pathway in Breast Cancer Progression.

Objective Notch signaling pathway is a vital parameter of the mammalian vascular system. In this review, the authors summarize the current knowledge about the impact of the Notch signaling pathway in breast cancer progression and the therapeutic role of Notch's inhibition. Methods The available literature in MEDLINE, PubMed, and Scopus, regarding the role of the Notch pathway in breast cancer progression was searched for related articles from about 1973 to 2017 including terms such as “Notch,” “Breast Cancer,” and “Angiogenesis.” Results. Notch signaling controls the differentiation of breast epithelial cells during normal development. Studies confirm that the Notch pathway has a major participation in breast cancer progression through overexpression and/or abnormal genetic type expression of the notch receptors and ligands that determine angiogenesis. The cross-talk of Notch and estrogens, the effect of Notch in breast cancer stem cells formation, and the dependable Notch overexpression during breast tumorigenesis have been studied enough and undoubtedly linked to breast cancer development. The already applied therapeutic inhibition of Notch for breast cancer can drastically change the course of the disease. Conclusion Current data prove that Notch pathway has a major participation and multiple roles during breast tumor progression. Inhibition of Notch receptors and ligands provides innovative therapeutic results and could become the therapy of choice in the next few years, even though further research is needed to reach safe conclusions.

Abstract 4493: p190B RhoGAP modifies growth factor signaling in breast cancer in a non-cell autonomous manner

Abstract Estrogen mediates the growth and differentiation of breast epithelial cells and plays an important role in breast cancer progression. The upregulation of estrogen receptors has been linked to p190B RhoGAP in vivo. p190B regulates the activity of several Rho GTPases in vitro. To better understand the molecular mechanism of p190B's role in tumor progression, we sought to develop a cell culture model that would recapitulate the effects of p190B on various growth factor signaling pathways. Protein expression of p190B was knocked down using siRNA in MCF-7 breast cancer cells cultured in the absence of steroid hormones. Upon stimulation with estrogen, cell proliferation did not significantly differ between MCF-7 cells expressing reduced p190B and those expressing normal levels of p190B, suggesting that the effects of p190B on estrogen receptor expression are not cell autonomous and that additional components of the tumor microenvironment are required to recapitulate the in vivo findings. Citation Format: Eduardo Castro, Aaron Preston, Mykal Quinlan, Peter McHenry. p190B RhoGAP modifies growth factor signaling in breast cancer in a non-cell autonomous manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4493.

Regulation of HC11 mouse breast epithelial cell differentiation by the E-cadherin/Rac axis

It was previously demonstrated that differentiation of some established breast epithelial cell lines requires confluence and stimulation with hydrocortisone, insulin and prolactin inducers. We and others previously demonstrated that E-cadherin engagement, which is favored under conditions of confluence, increases the levels and activity of the Rac small GTPase. To investigate the functional relationship between the transforming ability of Rac and its role as an integral component of the differentiative E-cadherin signaling pathway, we introduced a mutationally activated form of Rac, RacV12, into the mouse breast epithelium-derived cell line, HC11. Our results demonstrate that the strength of the Rac signal is key for the outcome of the differentiation process; cRac1 is critically required for differentiation, and at low levels, mutationally activated RacV12 is able to increase differentiation, presumably reinforcing the E-cadherin/Rac differentiative signal. However, high RacV12 expression blocked differentiation concomitant with E-cadherin downregulation, while inducing neoplastic transformation. Therefore, the intensity of the Rac signal is a central determinant in the balance between cell proliferation vs differentiation, two fundamentally opposed processes, a finding which could also have important therapeutic implications.

Intermittent energy restriction induces changes in breast gene expression and systemic metabolism.

BackgroundObservational studies suggest weight loss and energy restriction reduce breast cancer risk. Intermittent energy restriction (IER) reduces weight to the same extent as, or more than equivalent continuous energy restriction (CER) but the effects of IER on normal breast tissue and systemic metabolism as indicators of breast cancer risk are unknown.MethodsWe assessed the effect of IER (two days of 65 % energy restriction per week) for one menstrual cycle on breast tissue gene expression using Affymetrix GeneChips, adipocyte size by morphometry, and systemic metabolism (insulin resistance, lipids, serum and urine metabolites, lymphocyte gene expression) in 23 overweight premenopausal women at high risk of breast cancer. Unsupervised and supervised analyses of matched pre and post IER biopsies in 20 subjects were performed, whilst liquid and gas chromatography mass spectrometry assessed corresponding changes in serum and urine metabolites in all subjects after the two restricted and five unrestricted days of the IER.ResultsWomen lost 4.8 % (±2.0 %) of body weight and 8.0 % (±5.0 %) of total body fat. Insulin resistance (homeostatic model assessment (HOMA)) reduced by 29.8 % (±17.8 %) on the restricted days and by 11 % (±34 %) on the unrestricted days of the IER. Five hundred and twenty-seven metabolites significantly increased or decreased during the two restricted days of IER. Ninety-one percent of these returned to baseline after 5 days of normal eating. Eleven subjects (55 %) displayed reductions in energy restriction-associated metabolic gene pathways including lipid synthesis, gluconeogenesis and glycogen synthesis. Some of these women also had increases in genes associated with breast epithelial cell differentiation (secretoglobulins, milk proteins and mucins) and decreased collagen synthesis (TNMD, PCOLCE2, TIMP4). There was no appreciable effect of IER on breast gene expression in the other nine subjects. These groups did not differ in the degree of changes in weight, total body fat, fat cell size or serum or urine metabolomic markers. Corresponding gene changes were not seen in peripheral blood lymphocytes.ConclusionThe transcriptional response to IER is variable in breast tissue, which was not reflected in the systemic response, which occurred in all subjects. The mechanisms of breast responsiveness/non-responsiveness require further investigation.Trial registrationISRCTN77916487 31/07/2012.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0714-4) contains supplementary material, which is available to authorized users.

Abstract 1018: Role of Stat3 vs Stat5 in the differentiation of HC11, mouse breast epithelial cells

Abstract The Signal Transducer and Activator of Transcription (Stat)3 and Stat5 are transcription factors involved in normal breast development, and are hyperactivated in 30-50% of breast cancers. Stat5 is required for breast epithelial cell differentiation and is over-expressed in breast cancers with a more differentiated phenotype. On the other hand, constitutive over-activation of Stat3 can drive expression of genes involved in survival, migration and angiogenesis, while mutationally activated Stat3 (Stat3C) can transform cultured cells, indicating that Stat3 may play an important role in cancer etiology. Following activation of a number of growth factor or cytokine receptors such as interleukin-6 or expression of oncogenes such as Src, Stat3 is phosphorylated at tyr-705, dimerizes and migrates to the nucleus where it activates transcription from a number of genes involved in cell division and survival, such as survivin, Bcl-xL, Mcl1 and myc, while it downregulates expresssion of the tumor suppressor p53. We recently discovered a novel pathway of activation of Stat3: Engagement of cadherins (E-, N-cadherin or cadherin-11), cell to cell adhesion proteins induces a dramatic increase in the levels of the Rac1 and Cdc42 small GTPases, and this leads to interleukin-6 transcription, hence Stat3 activation. The differentiation of HC11, nonneoplastic mouse breast epithelial cells requires prolactin, hydrocortisone and insulin, added at confluence. Since confluence activates Stat3, we examined the role of Stat3 upon breast epithelial cell differentiation, measured by cellular morphology and expression of the milk proteins, β-casein or whey acidic protein. Through pharmacological inhibition with CPA7 or S3I-201, our results demonstrate that Stat3 is, in fact, required for HC11 cell differentiation. In contrast, constitutive expression of mutationally activated Rac1 was found to block differentiation while inducing transformation. In sharp contrast, our results indicate that Stat5 is upregulated by the differentiation cocktail but is unaffected by cell density, while expression of activated Stat5 promotes a more differentiated phenotype. Taken together, our results demonstrate that Stat3 and Stat5 may constitute independent prognostic markers as well as treatment targets for breast cancer. Citation Format: Jamaica Cass, Maximilian Niit, Rozanne Arulanandam, Bruce Elliott, Leda Raptis. Role of Stat3 vs Stat5 in the differentiation of HC11, mouse breast epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1018. doi:10.1158/1538-7445.AM2015-1018

The Status of STAT3 and STAT5 in Human Breast Atypical Ductal Hyperplasia

Signal Transducer and Activation of Transcription factors (STAT3 and STAT5) play important roles in breast epithelial cell differentiation, proliferation, and apoptosis. They have been investigated extensively in established breast cancer, but their activation status in precancerous lesions has not been reported. Formalin-fixed, paraffin-embedded archival tissues from 59 cases of atypical ductal hyperplasia (ADH) and 31 cases of normal human breast tissue as well as 21 cases of usual ductal hyperplasias (UDH) were obtained from the First Hospital of Jilin University, China, and stained for pSTAT3 and pSTAT5 by immunohistochemistry. The median percentage of pSTAT5+ cells in ADH was 12%, not significantly deviant from that in normal breast. The median percentage of pSTAT3+ cells in ADH was 30%, significantly higher than that of normal breast. pSTAT3 and pSTAT5 were exclusive of each other—they were detected in different ADHs or in different cells within the same ADHs. In addition, both pSTAT3 and pSTAT5 were produced in similar percentages of cells in ADHs from cancer-free patients vs. ADHs that were adjacent to an invasive cancer. Our finding of a complementary expression pattern of pSTAT3 and pSTAT5 in ADH suggests that these two transcription factors may have feedback inhibitory effects on each other during early stages of breast cancer evolution, and that disruption of this inverse relationship may be important in the progression from early lesions to cancer, which exhibits positive association between pSTAT3 and pSTAT5.

Abstract 2040: Adenomatous polyposis coli mediated signaling, self renewal and differentiation of breast epithelial cells

Abstract Breast cancer is the leading cause of cancer-related death in non-smoking women in the United States. Despite tremendous amounts of information about the etiology of breast cancer, many questions remain unanswered. It has been demonstrated that specific subtypes of breast cancer differ in their sensitivity to chemotherapeutic and targeted therapies. The oncogenic events and signaling pathways driving these tumor subtypes are distinct. This indicates that increased knowledge of the molecular events defining the unique subtypes will provide opportunities for novel therapeutic approaches tailored to fit the specific subtypes and will therefore lead to improvement in patient outcomes. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated or hypermethylated in 18-70% of sporadic breast cancers depending on subtype. Oncomine database analysis revealed reduced expression of APC in aggressive estrogen receptor (ER) negative breast cancers compared to ER-positive breast cancer or normal breast. Using the ApcMin/+ mouse model, we identified pre-neoplastic lesions in the breast and enhanced breast tumorigenesis in the presence of the Polyoma middle T antigen (PyMT) oncogene. Apc mutation changed the tumor histopathology from solid to squamous adenocarcinomas, resembling the highly aggressive human metaplastic breast cancer. In this model of mouse mammary tumorigenesis, these changes occurred independent of Wnt/b-catenin signaling and required activation of focal adhesion kinase (FAK)/Src signaling, with a subsequent increase in cyclooxygenase-2 (COX-2). The applicability of these signaling pathways to human breast cancer remains unclear. Using the epithelial cell reprogramming assay for growing normal breast epithelial cells, we have begun to characterize expression of cell surface markers to delineate subtypes of epithelial cells. We have also assessed APC expression and correlated APC expression with risk and well-established clinical markers, such as ErbB2. Future studies will modulate APC expression in specific compartments of breast epithelial hierarchy to accomplish our objectives of dissecting the molecular alterations specifically downstream of APC loss and its role in the self-renewal and differentiation of breast epithelial cells. Citation Format: Alyssa Lesko, Harikrishna Nakshatri, Manjushree Anjanappa, Poornima Bhat-Nakshatri, Jenifer Prosperi. Adenomatous polyposis coli mediated signaling, self renewal and differentiation of breast epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2014-2040

The Aryl-hydrocarbon Receptor (AhR) as a Therapeutic Target in Human Breast Cancer

Breast cancer is currently the most prevalent malignancy among women in industrialized countries, and 1 in 8 women living in the United States will develop the disease at some point in her lifetime. Although the incidence of breast cancer has increased 0.3% per year since 1990, the mortality rate has decreased by 2% per year since 1990 due to improvements in treatment and early detection. New treatment strategies and a better molecular understanding of the disease will be important to continue the progress science has made against this disease. The aryl-hydrocarbon receptor (AhR) has been traditionally associated with activation by environmental contaminants, acute toxicity, and cancer risks associated with exposures. However, the AhR has been highly conserved throughout evolution suggesting an important biological role for the receptor independent of its response to environmental contaminants. There is a significant body of evidence indicating the AhR plays a role in breast epithelial cell differentiation and that receptor agonists can inhibit breast cancer growth. A 50% reduction in estrous-induced terminal end buds was observed in the mammary glands of AhR knockout animals when compared to wild type suggesting a role for the AhR in mammary development. In 2 independent rodent cancer bioassays, treatment with the AhR agonist 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) significantly reduced the incidence of spontaneous rat mammary tumors. These and other studies support the premise that the AhR plays a fundamental role in breast epithelial cell differentiation. This review provides a brief summary of the current evidence that the AhR may be an important pharmacological target for treating human breast cancer.

Abstract 693: Development of a protein-based functional assay for the determination of BRCA1 carrier status

Abstract While most breast cancers are sporadic, 5-10% are hereditary and attributable to the presence of a mutation in the breast cancer associated gene 1 (BRCA1). The BRCA1 gene encodes a tumour suppressor with several well described roles in maintaining genomic integrity, such as cell cycle checkpoint control and DNA repair. BRCA1 is also known to play a role in the differentiation of breast epithelial cells. Given the high risk of cancer development associated with BRCA1 mutation carriers, it is important that they be identified early and accurately. Previous work in our lab developed a novel functional assay to predict BRCA1 status based on gene expression profiles. That study analyzed EBV-transformed lymphocyte cell lines (LCLs) of BRCA1 mutation carriers (BRCA1+/−) and controls (BRCA1+/+) and found that lymphocytes from BRCA1 carriers could be distinguished with high fidelity (∼90% accuracy) from individuals with two wild type copies of the gene. Interestingly, it was found that many of the genes used to distinguish BRCA1 mutation carriers were also markers of differentiation in blood cells. We are now using this information to develop a flow cytometric protein-based biomarker assay. To do this, our transcriptome microarray data was analyzed to identify genes with strong edictability of BRCA1 status, including genes with known roles in lymphocyte differentiation such as TBX21 and CXCR3. These genes were then used to model the assay in-silico using a training set of data comprised of 53 LCLs. The model was designed by selecting genes both up- and down-regulated in BRCA1 carriers, placing them in pairs and looking at the ratio of their expression values. This model was subsequently validated using a test data set comprising 16 LCLs. Overall accuracy of the model was 94%, correctly predicting 54/59 LCLs. Based on in-silico modelling, we chose six antibodies to develop the assay. To identify other candidate proteins, LCL lysates were analyzed by mass spectrometry (MS). The results identified several other candidate proteins for assay development including IgG1, IgD, BCR and MX1. Interestingly, as was the case in our transcriptome analysis, these MS identified proteins also suggested that BRCA1 mutation carriers displayed defects in cellular differentiation. This was indicated by the higher expression of surface markers in responding to less differentiated B-lymphocytes in comparison with controls. Ultimately, the assay will be applied to fresh patient blood samples with the expectation that it will become an assay that can be implemented in a clinical setting to provide accurate identification of BRCA1 mutation carriers and aid in disease management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 693. doi:1538-7445.AM2012-693

Abstract 22: Differential IGF-II response of AA and CA normal breast cell lines to prolactin treatment

Abstract African-American (AA) breast cancer patients have lower survival compared with Caucasian-American (CA) patients. This survival disparity is significant in view of the lower incidence of breast cancer among AA. Lower incidence of breast cancer is associated with parity and lactation in young women. Lactation's protective effect is mediated by fatty-acid binding protein 3 (FABP3), a protein that promotes differentiation of breast epithelial cells. We have demonstrated that AA normal women below 50 years have lower FABP3 levels as compared to CA women (p = 0.030). FABP3 is a protective tumor suppressor that increases during pregnancy and lactation, in response to the lactation hormone prolactin (PRL). PRL effects on the breast are mediated by IGF-II. Thus, we propose that PRL treatment of normal epithelial breast cell lines would increase IGF-II to promote differentiation, increase FABP3, and decrease FABP5. FABP5 is associated with metastasis and may compensate for FABP3 decrease. We treated MCF10A (CA) and AG11132 (AA) normal breast epithelial cell lines with recombinant human PRL (0-200 ng/ml) for 24 hours. Cell lysates were examined for protein and mRNA levels of IGF-II, FABP3, and FABP5. In MCF10A cells, PRL stimulated an increase in protein levels of IGF-II (p=0.044), and a decrease in protein levels of FABP5 (p=0.044). Protein levels of FABP3 significantly decreased with increasing PRL concentration (p=0.030). IGF-II mRNA levels also increased at 50 and 200 ng/ml PRL (2.59 and 2.58 fold, respectively). In AG11132 cells, protein levels of FABP3 significantly increased with increasing PRL concentration (p=0.016). IGF-II and FABP5 mRNA levels increased significantly at 10 ng/ml PRL (49.91 and 2.13 fold, respectively); however, no corresponding changes were seen in protein levels. Though both cell lines respond to PRL treatment, only the MCF10A line displays increased IGF-II and decreased FABP5, which are protective changes in normal mammary cells. FABP3 levels decreased in MCF10A cells at higher PRL concentrations; this may be due to untreated MCF10A cells expressing significantly more FABP3 and prolactin, relative to AG11132 cells. The higher levels of PRL and FABP3 in untreated MCF10A cells indicate that they are more differentiated and thus require less PRL stimulation, relative to the AG11132 cells, to achieve a differentiated state. The dissimilar responsiveness of these cell lines to PRL may suggest decreased protection against breast carcinogenesis in AA patients, and may help us to further understand this health disparity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 22. doi:1538-7445.AM2012-22

ITF2 is a target of CXCR4 in MDA-MB-231 breast cancer cells and is associated with reduced survival in estrogen receptor-negative breast cancer

CXCR4, a chemokine receptor, plays an important role in breast cancer growth, invasion, and metastasis. The transcriptional targets of CXCR4 signaling are not known. Microarray analysis of CXCR4-enriched and CXCR4-low subpopulations of the MDA-MB-231 breast cancer cell line, which has a constitutively active CXCR4 signaling network, revealed differential expression of ~ 200 genes in the CXCR4-enriched subpopulation. ITF2, upregulated in CXCR4-enriched cells, was investigated further. Expression array datasets of primary breast tumors revealed higher ITF2 expression in estrogen receptor negative tumors, which correlated with reduced progression free and overall survival and suggested its relevance in breast cancer progression. CXCL12, a CXCR4 ligand, increased ITF2 expression in MDA-MB-231 cells. ITF2 is a basic helix-loop-helix transcription factor that controls the epithelial-to-mesenchymal transition and the function of the ID family (inhibitor-of-differentiation) of transcription factors, such as ID2. ID2 promotes differentiation of breast epithelial cells and its reduced expression in breast cancer is associated with an unfavorable prognosis. Both CXCR4 and ITF2 repressed ID2 expression. In xenograft studies, CXCR4-enriched cells formed large tumors and exhibited significantly elevated lung metastasis. Short interfering RNA against ITF2 reduced invasion of the CXCR4-enriched MDA-MB-231 subpopulation, whereas ITF2 overexpression restored the invasive capacity of MDA-MB-231 cells expressing CXCR4shRNA. Furthermore, overexpression of ITF2 in these cells enhanced tumor growth. We propose that ITF2 is one of the CXCR4 targets, which is involved in CXCR4-dependent tumor growth and invasion of breast cancer cells.

Predictive value of a gene signature evaluated in multiple chemotherapy regimens for breast cancer.

545 Background: Approximately 12,800 U.S. women present with locally advanced breast cancer each year. Primary (or neoadjuvant) chemotherapy is a treatment to shrink a large tumor to a size that allows it to be removed with breast conserving surgery. A woman who would otherwise need a mastectomy (surgical removal of the breast) may instead elect to receive primary chemotherapy and breast- conserving lumpectomy. Unfortunately, primary chemotherapy has a low response rate (30%-50%) and a significant negative impact on a patient's quality of life. If a tumor does not respond to primary chemotherapy, a mastectomy may be necessary after all. Currently, there is no widely used clinical test to determine before treatment if a given tumor will respond. We have developed a signature of 22 genes whose expression pattern accurately predicts response to primary chemotherapy in breast cancer. This 22-gene signature was discovered by a unique approach using an in vitro model of normal breast epithelial cell differentiation. Here we present data showing that the 22-gene signature predicts response to multiple chemotherapies. Methods: We performed analysis of two published microarray datasets to test whether the 22- gene signature would be predictive of response to primary chemotherapy by hierarchical clustering, Fischer's exact tests, and ROC analysis. The dataset of Chang et al. includes 24 breast cancer core biopsies (Lancet. 2003;362:362-369) and Hess et al. includes 133 fine-needle aspirates from breast cancer patients (JCO. 2006;24:4236-4244) taken before primary treatment with docetaxel or combination treatment respectively and response was assessed after chemotherapy. Results: The 22-gene signature accurately predicted chemotherapy response in both independent datasets (ROC p = 0.0001; Fisher's Exact p < 0.0001). ROC analysis values for sensitivity and specificity showed that the 22-gene signature was superior to clinical parameters and other published gene signatures. Conclusions: This gene signature has the potential to fill the existing need for an in vitro diagnostic to provide accurate personalized information to guide primary chemotherapy treatment. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bioarray Therapeutics Inc. Bioarray Therapeutics Inc. Bioarray Therapeutics Inc. Bioarray Therapeutics Inc.

Adipocyte differentiation influences the proliferation and migration of normal and tumoral breast epithelial cells

Stromal tissue regulates the development and differentiation of breast epithelial cells, with adipocytes being the main stromal cell type. The aim of the present study was to evaluate the effect of adipocyte differentiation on proliferation and migration, as well as to assess the activity of heparanase and metalloproteinase-9 (MMP-9), in normal (NMuMG) and tumoral (LM3) murine breast epithelial cells. NMuMG and LM3 cells were grown on irradiated 3T3-L1 cells (stromal support, SS) at various degrees of differentiation [preadipocytes (preA), poorly differentiated adipocytes (pDA) and mature adipocytes (MA)] and/or were incubated in the presence of conditioned medium (CM) derived from each of these three types of differentiated cells. Cells grown on a plastic support or in fresh medium served as the controls. Cell proliferation was measured with a commercial colorimetric kit, and the motility of the epithelial cells was evaluated by means of a wound-healing assay. Heparanase activity was assessed by quantifying heparin degradation, and the expression of MMP-9 was determined using Western blotting. The results indicate that cell proliferation was increased after 24 and 48 h in the NMuMG and LM3 cells grown on preA, pDA and MA SS. In the NMuMG cells cultured on SS in the presence of all three types of CM, proliferation was enhanced. LM3 cell migration was increased in the presence of all three types of CM and in cells grown on preA SS. Heparanase activity was increased in the NMuMG cells incubated with all three types of CM, and in the LM3 cells incubated with the CM from pDA and MA. Both the NMuMG and LM3 cell lines presented basal expression of MMP-9; however, a significant increase in MMP-9 expression was observed in the LM3 cells incubated with each of the three types of CM. In conclusion, adipocyte differentiation influences normal and tumoral breast epithelial cell proliferation and migration. Heparanase and MMP-9 appear to be involved in this regulation. The experimental model presented in this study is in keeping with the characteristics of the physiological environment of breast epithelial cells, in terms of both the soluble and insoluble factors present and the stromal structure per se.

Breast epithelial differentiation is altered in BRCA1mut/+ carriers prior to the onset of cancer and contributes to the basal tumor phenotype.

Abstract Abstract #3083 Human breast tumors are broadly divided into either luminal-like or basal-like cancers. This distinction is significant since basal-like tumors are more aggressive and afford a poor patient prognosis relative to luminal-like tumors. For reasons that are unclear, germline mutations in BRCA1 strongly predispose for poor prognosis basal-like tumors. The predisposition for basal-like tumors in BRCA1mut/+ patients could be due to (1) differences in underlying target cell populations between BRCA1mut/+ and BRCA1+/+ women or (2) differences in the genetic mutations arising within a single shared target cell type. This basic question has remained unresolved due to a lack of experimental models in which it can be addressed. We describe here a novel in vivo breast cancer system that enables the generation of tumors by introducing oncogenes into normal breast epithelium derived directly from human breast tissue. This system is unique in that it enables human-derived epithelial cells to be sorted for cell surface markers and transformed without requiring in vitro culture prior to implantation in vivo. Using this experimental system, we show that epithelial cells from BRCA1mut/+ patients give rise to tumors that exhibit multiple features of basal differentiation, in contrast to epithelial cells transformed with identical oncogenes from BRCA1+/+ patients. We show further that non-cancerous epithelial cells from BRCA1mut/+ patients already exhibit atypical differentiation even prior to the onset of cancer, in contrast to cells from BRCA1+/+ women. Remarkably, some of these differences are observable in the context of unperturbed breast tissue obtained from disease-free BRCA1mut/+ and BRCA1+/+ patients. Collectively, these findings show that the increased incidence of basal-like tumors in BRCA1mut/+ patients is a reflection of the altered differentiation of breast epithelial cells in BRCA1mut/+ patients. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3083.

Cyclin‐dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis

The cyclin-dependent kinase inhibitors (CDKIs), p21(CIP1) and p27(KIP1) regulate growth and differentiation in diverse tissue types. We aimed to determine whether p21(CIP1) or p27(KIP1) could induce a terminally differentiated phenotype in breast cells, and to examine if CDKI expression is regulated by basement membrane interactions. Effects of increased CDKI expression on the phenotype of MCF-10A breast epithelial cells were examined by retroviral transduction of p21(CIP1) or p27(KIP1) cDNA. Overexpression of p21(CIP1) or p27(KIP1) reduced MCF-10A growth rates in monolayer cultures, altered cellular morphology and stimulated accumulation of neutral lipid droplets, suggesting partial lactational differentiation. However, markers of luminal differentiation (oestrogen and progesterone receptors, alpha-lactalbumin, beta-casein and adipophilin) were absent when examined by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Cell-basement membrane contacts are known to be essential for full mammary epithelial cell differentiation and therefore parental MCF-10A cells were cultured on a basement membrane preparation (Matrigel) in which they form acini. Immunocytochemistry showed that Ki67, the cell proliferation marker, was initially expressed at high levels and as growth decreased p27(KIP1) expression steadily increased. Surprisingly, p21(CIP1) was highest at the early stages of acinus growth and was detected in proliferating cells, as demonstrated by colocalization in dual Ki67/p21(CIP1) immunofluorescence. Overexpression of p21(CIP1) or p27(KIP1) impaired formation of acini, whereas their knockdown, using siRNA, increased acinus formation. We conclude that both p21(CIP1) and p27(KIP1) induce partial secretory differentiation of mammary cells in monolayer, but during acinus morphogenesis in 3D culture they have a highly regulated temporal expression pattern.

GATA3 protein as a MUC1 transcriptional regulator in breast cancer cells

IntroductionRecent studies have demonstrated that members of the GATA-binding protein (GATA) family (GATA4 and GATA5) might have pivotal roles in the transcriptional upregulation of mucin genes (MUC2, MUC3 and MUC4) in gastrointestinal epithelium. The zinc-finger GATA3 transcription factor has been reported to be involved in the growth control and differentiation of breast epithelial cells. In SAGE (serial analysis of gene expression) studies we observed an intriguing significant correlation between GATA3 and MUC1 mRNA expression in breast carcinomas. We therefore designed the present study to elucidate whether MUC1 expression is regulated by GATA3 in breast cancer cells.MethodsPromoter sequence analysis of the MUC1 gene identified six GATA cis consensus elements in the 5' flanking region (GATA1, GATA3 and four GATA-like sequences). Chromatin immunoprecipitation and electrophoretic mobility-shift assays were employed to study the presence of a functional GATA3-binding site. GATA3 and MUC1 expression was analyzed in vitro with a GATA3 knockdown assay. Furthermore, expression of GATA3 and MUC1 genes was analyzed by real-time RT-PCR and immunohistochemistry on breast cancer-specific tissue microarrays.ResultsWe confirmed the presence of a functional GATA3-binding site on the MUC1 promoter region in the MCF7 cell line. We determined that GATA3 knockdown assays led to a decrease in MUC1 protein expression in MCF7 and T47D cells. In addition, we detected a statistically significant correlation in expression between GATA3 and MUC1 genes at the mRNA and protein levels both in normal breast epithelium and in breast carcinomas (p = 0.01). GATA3 expression was also highly associated with estrogen receptor and progesterone receptor status (p = 0.0001) and tumor grade (p = 0.004) in breast carcinomas.ConclusionOur study provides evidence indicating that GATA3 is probably a mediator for the transcriptional upregulation of MUC1 expression in some breast cancers.

Retinoic Acid Exerts Dual Regulatory Actions on the Expression and Nuclear Localization of Interferon Regulatory Factor-1

Interferon regulatory factor-1 (IRF-1), a transcription factor and tumor suppressor involved in cell growth regulation and immune responses, has been shown to be induced by all-trans retinoic acid (ATRA). However, the factors controlling the cellular location and activity of IRF-1 are not well understood. In this study, we examined the expression of IRF-1 and its nuclear localization, DNA-binding activity, and target gene expression in human mammary epithelial MCF10A cells, a model of breast epithelial cell differentiation and carcinogenesis. Following initial treatment with ATRA, IRF-1 mRNA and protein were induced within 2 hrs, reached a peak (>30-fold induction) at 8 hrs, and declined afterwards. IRF-1 protein was predominantly cytoplasmic during this treatment. Although a second dose of ATRA or Am580 (a related retinoid selective for retinoic acid receptor-alpha [RARalpha]), given 16 hrs after the first dose, restimulated IRF-1 mRNA and protein levels to a similar level to that obtained by the first dose, IRF-1 was predominantly concentrated in the nucleus after restimulation. ATRA and Am580 also increased nuclear RARalpha, whereas retinoid X receptor-alpha (RXRalpha)--a dimerization partner for RARalpha, was localized to the nucleus upon second exposure to ATRA. However, ATRA and Am580 did not regulate the expression or activation of signal transducer and activator of transcription-1 (STAT-1), a transcription factor capable of inducing the expression of IRF-1, indicating an STAT-1-independent mechanism of regulation by ATRA and Am580. The increase in nuclear IRF-1 after retinoid restimulation was accompanied by enhanced binding to an IRF-E DNA response element, and elevated expression of an IRF-1 target gene, 2',5'-oligoadenylate synthetase-2. The dual effect of retinoids in increasing IRF-1 mRNA and protein and in augmenting the nuclear localization of IRF-1 protein may be essential for maximizing the tumor suppressor activity and the immunosurveillance functions of IRF-1 in breast epithelial cells.

Bone Marrow Stromal Cell-derived Growth Inhibitor Inhibits Growth and Migration of Breast Cancer Cells via Induction of Cell Cycle Arrest and Apoptosis

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.