Differentiation Of Breast Cancer Cells Research Articles

Cisplatin induces differentiation of breast cancer cells

Breast tumours are heterogeneous, including cells with cancer stem cell properties and more differentiated tumour cells. As breast cancer stem cells are often resistant to chemotherapy, recent efforts have been focusing on treatments that may shift them towards a more differentiated phenotype, making them more susceptible to treatment. Here, we examined whether t cisplatin induces cell differentiation in four breast cancer cell lines that represent different types of breast tumours (BT549, MDA‐MB‐468, MDA‐MB‐231, MCF7). Cisplatin treatment of 20 μM reduced cell viability from 100% to 50–80%, depending on the cell line. Flow cytometric quantification showed that treatment with cisplatin induced a 18–65% decrease of stem cell marker expression (CD49f, SSEA4) and a 4–97% increase in expression of differentiation markers (CK18, Tubulin). It is concluded that in addition to its effects on cell survival, cisplatin shifts breast cancer cell lines towards a more differentiated phenotype.Research Support: Malaysian Ministry of Higher Education Postgraduate Scholarship

793 MICRO RNA DIFFERENTIALLY EXPRESSED IN BIOCHEMICAL RECURRENCE IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012793 MICRO RNA DIFFERENTIALLY EXPRESSED IN BIOCHEMICAL RECURRENCE IN PROSTATE CANCER Katia Leite, Sabrina Reis, Caio Moura, Nayara Viana, Alberto Antunes, Jose Pontes, Alexandre Sant'Anna, Adriano Nesrallah, Marcos Dall'Oglio, Luiz Heraldo Camara-Lopes, and Miguel Srougi Katia LeiteKatia Leite Sao Paulo, Brazil More articles by this author , Sabrina ReisSabrina Reis Sao Paulo, Brazil More articles by this author , Caio MouraCaio Moura Sao Paulo, Brazil More articles by this author , Nayara VianaNayara Viana Sao Paulo, Brazil More articles by this author , Alberto AntunesAlberto Antunes Sao Paulo, Brazil More articles by this author , Jose PontesJose Pontes Sao Paulo, Brazil More articles by this author , Alexandre Sant'AnnaAlexandre Sant'Anna Sao Paulo, Brazil More articles by this author , Adriano NesrallahAdriano Nesrallah Sao Paulo, Brazil More articles by this author , Marcos Dall'OglioMarcos Dall'Oglio Sao Paulo, Brazil More articles by this author , Luiz Heraldo Camara-LopesLuiz Heraldo Camara-Lopes Sao Paulo, Brazil More articles by this author , and Miguel SrougiMiguel Srougi Sao Paulo, Brazil More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.881AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Micro RNA (miRNA) are small RNAs involved in the control of gene expression by degrading mRNA repressing protein translation. We previously have shown change in miRNA expression levels related to the development of prostate cancer (PC) and to biochemical recurrence after radical prostatectomy (RP). Now, using miRNA microarray we report a miRNA profile related to biochemical recurrence (BR). METHODS 56 men underwent RP to treat PC from August 2000 to June 2002. A fragment of surgical specimen representative of tumor was storage in liquid nitrogen for molecular studies. BR was considered when PSA>0.2 ng/mL. The GeneChip® miRNA microarray (Affymetrix, CA, USA) containing 46,228 probes derived from Sanger miRBase miRNA database v11 was used to identify differentially expressed miRNAs between patients who have recurred from those who have not. RESULTS The differentially expressed miRNA related to BR are exposed in Table 1. For underexpressed miRNA in patients who have suffered BR we considered those with less than -1.40 in fold change and for overexpressed, those with more than +1.70 fold change. The level of significance was p<0.05. There were 14 miRNA underexpressed, and 17 overexpressed. CONCLUSIONS β Among the 14 underexpressed miRNAs are miR-129 and 145 both having SOX2 as target, involved in cell differentiation, invasiveness and metastatization in gastric and breast cancer. miR-340 is a controller of c-met and its underexpression is related to increase in cell migration and invasiveness. miR-16 is a pro-apoptotic miRNA controlling Bcl2. 17 miRNAs were overexpressed, among them miR-373 that has been related to invasivess, development of metastases and stem cell maintenance in cancers inducing MMP9 expression and activating the Ras/Raf/Mek/Erk pathway. miR-1238 has been involved in colon carcinogenesis,miR-370 that has been described in advanced gastric cancer and miR-181a and c induced by Wnt/βcatenin related to maintenance of stem cell state in hepatocellular cancer. The knowledge of miRNAs involved in PC progression could provide us a profile that could identify patients for adjuvant therapy or even propose new target drugs to avoid disease progression or treat metastatic PC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e324-e325 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Katia Leite Sao Paulo, Brazil More articles by this author Sabrina Reis Sao Paulo, Brazil More articles by this author Caio Moura Sao Paulo, Brazil More articles by this author Nayara Viana Sao Paulo, Brazil More articles by this author Alberto Antunes Sao Paulo, Brazil More articles by this author Jose Pontes Sao Paulo, Brazil More articles by this author Alexandre Sant'Anna Sao Paulo, Brazil More articles by this author Adriano Nesrallah Sao Paulo, Brazil More articles by this author Marcos Dall'Oglio Sao Paulo, Brazil More articles by this author Luiz Heraldo Camara-Lopes Sao Paulo, Brazil More articles by this author Miguel Srougi Sao Paulo, Brazil More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Fresh Garlic Extract Induces Growth Arrest and Morphological Differentiation of MCF7 Breast Cancer Cells

Consumption of diets rich in fruits and vegetables is often associated with a reduced risk of developing cancer, particularly breast cancer. Considering that 1 in 8 women in the United States will develop breast cancer in the course of her lifetime, dietary manipulation could have a major impact on the incidence of breast cancer. We report here that fresh extracts of garlic (not boiled) arrested the growth and altered the morphology of MCF7 breast cancer cells. Deregulated levels of E-cadherin, cytokeratin8/18, and β-catenin correlated with the altered phenotype. We propose that early down-regulation of cyclin D1, reduced phosphorylation of ERK1, and increased phosphorylation of eIF2-α triggered the phenotypical changes. Reduced expression of hsp27 and sam68 and elevated levels of Rb and p21 further contributed to the sustained growth reduction. These findings provide a better understanding of the cellular responses to dietary supplements and provide potential options to treat breast cancer.

BMP2 promotes migration and invasion of breast cancer cells via cytoskeletal reorganization and adhesion decrease: an AFM investigation

Bone morphogenetic protein 2 (BMP2) has been shown to modulate the proliferation and differentiation of breast cancer cells. However, the biochemical effects and mechanisms remain unknown. In this paper, the effects of recombinant human BMP2 on the migration of MCF-7 cells-one breast cancer cell line, using transwell and wound healing experiments, as well as on the cellular morphology, cytoskeleton, cell surface adhesion, and stiffness detected at subcellular level by an atomic force microscope, were investigated. After BMP2 treatment, the untreated round-shaped MCF-7 cells transformed to a spindle-like shape with lots of specialized structures, such as lamellipodia, filopodia, membrane protrusions, and others, which are essential for cellular migration or spreading. Moreover, flow cytometry quantitatively detected the BMP2-induced changes in the expression of adhesion molecules, a significant rise of CD44, and a remarkable drop of E-cadherin. The data indicated that BMP2 promoted the migration and invasion of MCF-7 cells by regulating the reorganization of cytoskeleton and the expression of adhesion molecules in/on the cells. Thus, it is very imperative to evaluate the oncogenicity of BMP2 when used in tissue engineering.

Fluorescent polymer-based post-translational differentiation and subtyping of breast cancer cells

Herein, we report the application of synthesized fluorescent, water soluble polymers for post-translational subtyping and differentiation of breast cancer cells in vitro. The fluorescence emission spectra from these polymers were modulated differently in the presence of conditioned cell culture media from various breast cancer cells. These polymers differentiate at a post-translation level possibly due to their ability to interact with extracellular enzymes that are over-expressed in cancerous conditions.

Regulation of Sodium Iodide Symporter Gene Expression by Rac1/p38β Mitogen-activated Protein Kinase Signaling Pathway in MCF-7 Breast Cancer Cells

Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide (131)I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of (131)I concentration and treatment of breast cancer.

Identification of Potential Tumor Differentiation Factor (TDF) Receptor from Steroid-responsive and Steroid-resistant Breast Cancer Cells

Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.

Abstract B42: Jak2/Stat5a overexpression synergizes with mistletoe (Viscum album) extracts, and/or diallyl trisulfide (DATS) to inhibit breast cancer stem cell characteristics, epithelial-mesenchymal transition, and invasion potential.

Abstract Epithelial-mesenchymal transition (EMT) is a key event in the tumor invasion process and poor prognosis of breast cancer, the main leading cause of cancer-related deaths among women, is associated with a high potential of cancer cells invasion and metastasis. Recently, a growing body of evidence is supporting the idea that human breast cancer can be considered as a stem cell disease and the metastatic cell state is strongly correlated to (EMT) and the CD44+/CD24- stem cell phenotype. We have recently uncovered novel evidence that signal transducers and activators of transcription 5a (Stat5a) and its Janus tyrosine kinase, (Jak2), suppress early invasion and promote human breast cancer cells differentiation through inhibition of (EMT) dedifferentiation. Understanding and identifying the role of Prolactin/Stat5a/Jack2 pathway in the process of early metastasis and differences between invasive and non-invasive breast cancer related to (EMT) or CD44+/CD24- breast cancer stem cell (BCSC) characteristics could lead to more effective treatment. In the present study, a comparative analysis of epithelial and mesenchymal marker expression was performed, using a panel of human breast cancer cell lines that display different phenotypic and differentiation patterns ranging from poor to high differentiation and expressing different levels of CD44+/CD24- (BCSC) characteristics, such as MDA-231, MDA-468, MDA-157, T-47D, MCF-7, BT-20, SKBr3, and ZR-75–1 cell lines. Gene expression profiles of the tested cell lines were examined to identify a set of genes that could play a role in (EMT) and evaluated by RT-PCR and flow Cytometry. Our data indicated that human breast cancer cell lines contain a small population of CD44+/CD24- (BCSC) and their self-renewal capacity is inhibited by overexpression of Jak2/Stat5a signaling combined with Mistletoe (Viscum album) plant extracts and/or diallyl trisulfide (DATS) pre-treatment. Our data showed that the increased invasive potential was proportional to increased CD44 expression and downregulated CD24 expression. Using specific CD44 antibody treatment significantly decreased (EMT) and cell invasion potential in MDA-MB-231, MDA-468 and MDA-157 cells. Moreover, Jak2/Stat5a overexpressing cells synergizes with diallyl trisulfide (DATS) in apoptosis induction by activating capase-3 and inhibiting the expression of Bcl-2 in CD44+/CD24- (BCSC) population. Furthermore, the cell lines that overexpressing Jak2/Stat5a and pretreated with (DATS) inhibits (EMT) by blocking the expression of nuclear -catenin, vimentin, slug, and snail, and also decreased population of CD44+/CD24- (BCSC) invasion capacity by induction of E-cadherin expression, suggesting the blockade of signaling involved in early process of metastasis through elimination of cancer stem cell-characteristics. All-in-all, our data support a novel role of a combination between Jak2/Stat5a overexpression and nontoxic agents that can delay onset and/or progression of breast cancer as a new strategy to inhibit (EMT) and in turn breast cancer invasion through inhibition of the CD44+/CD24- (BCSC) characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B42.

Lack of Angiogenesis in Experimental Brain Metastases

Angiogenesis is believed to be essential for the growth of metastatic tumors in the brain. We analyzed the vascularization of tumors formed by 4 epithelial cell lines (C38, ZR75, HT25, and H1650) and a fibrosarcoma (HT1080) cell line injected into the brains of mice. No peritumoral angiogenesis was observed. Tumors apparently acquired their vasculature by incorporation of native vessels. Vessel density was lower, but vessel diameter and vascular cell proliferation were higher within all tumors versus those in the peritumoral tissue. There was an inverse correlation between the number of incorporated vessels and vascular cell proliferation. Epithelial tumors with pushing growth patterns had lower vessel density and elevated vascular cell proliferation compared with invasive tumors. The incorporated vessels retained their normal structure, with the exception of astrocyte foot processes that were replaced by tumor cells. Attachment to the vascular basement membrane led to the differentiation of the ZR75 breast cancer cells. In the HT1080 metastases, there was intussusceptive angiogenesis, that is, the fibrosarcoma cells that were attached to the vessel caused lumen splitting and filled the developing pillars. Branching angiogenesis was not observed either in the tumors or in control cerebral wounds. These data suggest that sprouting angiogenesis is not needed for the incipient growth of cerebral metastases and that tumor growth in this model is a result of incorporation of host vessels.

Quantification of Epithelial Cell Differentiation in Mammary Glands and Carcinomas from DMBA- and MNU-Exposed Rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer.

Contribution of Xanthine Oxidoreductase to Mammary Epithelial and Breast Cancer Cell Differentiation In Part Modulates Inhibitor of Differentiation-1

Loss of xanthine oxidoreductase (XOR) has been linked to aggressive breast cancer in vivo and to breast cancer cell aggressiveness in vitro. In the present study, we hypothesized that the contribution of XOR to the development of the normal mammary gland may underlie its capacity to modulate breast cancer. We contrasted in vitro and in vivo developmental systems by differentiation marker and microarray analyses. Human breast cancer microarray was used for clinical outcome studies. The role of XOR in differentiation and proliferation was examined in human breast cancer cells and in a mouse xenograft model. Our data show that XOR was required for functional differentiation of mammary epithelial cells both in vitro and in vivo. Poor XOR expression was observed in a mouse ErbB2 breast cancer model, and pharmacologic inhibition of XOR increased breast cancer tumor burden in mouse xenograft. mRNA microarray analysis of human breast cancer revealed that low XOR expression was significantly associated with time to tumor relapse. The opposing expression of XOR and inhibitor of differentiation-1 (Id1) during HC11 differentiation and mammary gland development suggested a potential functional relationship. While overexpression of Id1 inhibited HC11 differentiation and XOR expression, XOR itself modulated expression of Id1 in differentiating HC11 cells. Overexpression of XOR both inhibited Id1-induced proliferation and -stimulated differentiation of Heregulin-β1-treated human breast cancer cells. These results show that XOR is an important functional component of differentiation whose diminished expression contributes to breast cancer aggressiveness, and they support XOR as both a breast cancer biomarker and a target for pharmacologic activation in therapeutic management of aggressive breast cancer.

Abstract 1961: PKCΔ mediates glycodelin-induced differentiation of breast cancer cells

Abstract Glycodelin is a secreted lipocalin family glycoprotein, expressed mainly in differentiated epithelia in reproductive tissues. The expression of glycodelin in breast and endometrial cancer cells induces cell differentiation. We have found that transfection of glycodelin cDNA into MCF-7 breast cancer cells changes cellular morphology and gene expression towards less malignant direction. In pre-clinical xenograft mouse model glycodelin-producing cells formed significantly smaller tumors with lower proliferation rate than control cells. This is in keeping with the association of glycodelin with well-differentiated and less proliferative tumors in sporadic breast cancer patients. The aim of the present study is to elucidate the mechanism(s) mediating glycodelin-induced differentiation of breast cancer cells. The gene expression changes between glycodelin-producing and control cells were compared using a cDNA micro array. The cells were grown on Matrigel with and without protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), PKCΔ inhibitor Rottlerin and various other kinase inhibitors. PKCΔ was also down-regulated with RNAi. The levels of PKCΔ were measured using Western Blotting. The gene expression changes induced by glycodelin were enriched in several pathways, including mitogen-activated protein kinase (MAPK) -pathway. Among various studied proteins of MAPK-pathway, we found that phosphorylated PKCΔ was down-regulated in glycodelin-producing cells. Further studies with activators and inhibitors indicated that addition of PKC activator PMA to cell culture medium induced dramatic morphological changes and increased motility of the control cells, while glycodelin-producing cells were unresponsive. The effect of PMA was abolished by Rottlerin, an inhibitor of PKCΔ, as well as by using siRNA for PKCΔ. These results suggest that the glycodelin-induced differentiation of breast cancer cells is mediated by PKCΔ, which in other studies has been proposed to be a potential target for cancer treatment. 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 1961. doi:10.1158/1538-7445.AM2011-1961

Abstract 3097: Global transcript profiling for prolactin-stat5 dependent target genes in breast cancer

Abstract Transcription factor Stat5 is a key mediator of prolactin receptor signaling and plays important roles in both normal mammary development and breast cancer. Although numerous studies support tumor-promoting roles for Stat5, accumulating evidence also points to a role of Stat5 in promoting cell differentiation in breast cancer cells. In fact, high levels of tyrosine phosphorylated Stat5 (pY-Stat5) in human breast cancer specimens are correlated with favorable prognosis. Identifying Stat5 target genes in human breast cancer may shed new light on the roles of Stat5 as well as provide new candidate prognostic biomarkers. This study was designed to identify candidate Stat5 target genes using tumor extracts of the breast cancer cell line T47D xenotransplanted into nude mice that were subsequently treated with or without human prolactin. Global transcript profiling on the Affymetrix U133 chip platform was employed. The analysis revealed 97 genes that were significantly modulated by prolactin, based on fold difference greater than 1.7 and a p-value less than 0.01. Among these, 61 genes were upregulated and 36 genes were downregulated by prolactin. A total of 18 transcripts were selected based on their relevance to breast cancer and these genes were further investigated using qRT-PCR analysis. Of the 18 candidate targets, 15 were regulated by prolactin in vivo. Of these, 13 were also regulated by prolactin in T47D cells in vitro. Given that prolactin signals through multiple pathways other than Stat5, we examined the ability of Stat5 to regulate the newly identified prolactin-dependent genes by over-expressing Stat5a or Stat5b in the T47D cells. Twelve of the 13 genes tested responded by increased degree of prolactin-modulation following ectopic over-expression of Stat5a or Stat5b, supporting the notion that these genes are downstream targets of Stat5. The identified candidate Stat5 target genes are known to have diverse functions that may critically impact breast cancer growth and progression. For instance, NTN4 is a transmembrane protein whose expression levels positively correlates with better prognosis in breast cancer. AREG and EREG are known to activate the ErbB receptors, whose functions are essential for breast cancer progression. FN1 is a known factor involved in breast cancer invasion. Lastly, PTHrP is well established as being functionally involved in breast cancer metastasis. Fluorescence-based quantitative immunohistochemistry revealed a highly positive correlation between levels of pY-Stat5 and PTHrP proteins in human clinical breast cancer specimens, providing proof-of-principle that the combination of global transcript profiling and quantitative immunohistochemistry analysis is valuable for identifying potential breast cancer biomarkers. Efforts are underway to examine the function of these and additional Stat5 target genes in order to further elucidate the roles of Stat5 in breast cancer. 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 3097. doi:10.1158/1538-7445.AM2011-3097

Silencing of the icb-1 gene inhibits the induction of differentiation-associated genes by vitamin D3 and all-trans retinoic acid in gynecological cancer cells

Icb-1 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. To further elucidate the function of the icb-1 gene in differentiation of breast and endometrial cancer cells, we knocked down its expression by means of shRNA transfection. Knockdown of icb-1 inhibited the vitamin D3-induced up-regulation of E-cadherin expression in both MCF-7 and HEC-1B cells. Induction of E-cadherin expression by all-trans retinoic acid (ATRA) was also blocked in both cell lines expressing icb-1 siRNA. Examination of icb-1 and E-cadherin expression in 66 breast cancer tissue samples revealed a significant positive correlation between the two genes. In MCF-7 cells, silencing of the icb-1 gene inhibited the ATRA- and the vitamin D3-induced up-regulation of lactoferrin and estrogen receptor β expression. The data of our knockdown study suggest that icb-1 may act as a mediator of differentiation signals in breast cancer cells induced by ATRA or vitamin D3. These findings together with the observed co-expression of icb-1 with E-cadherin in breast cancer samples support an important role of the icb-1 gene in cancer cell differentiation.

CARM1 Is an Important Determinant of ERα-Dependent Breast Cancer Cell Differentiation and Proliferation in Breast Cancer Cells

Breast cancers with estrogen receptor α (ERα) expression are often more differentiated histologically than ERα-negative tumors, but the reasons for this difference are poorly understood. One possible explanation is that transcriptional cofactors associated with ERα determine the expression of genes which promote a more differentiated phenotype. In this study, we identify one such cofactor as coactivator-associated arginine methyltransferase 1 (CARM1), a unique coactivator of ERα that can simultaneously block cell proliferation and induce differentiation through global regulation of ERα-regulated genes. CARM1 was evidenced as an ERα coactivator in cell-based assays, gene expression microarrays, and mouse xenograft models. In human breast tumors, CARM1 expression positively correlated with ERα levels in ER-positive tumors but was inversely correlated with tumor grade. Our findings suggest that coexpression of CARM1 and ERα may provide a better biomarker of well-differentiated breast cancer. Furthermore, our findings define an important functional role of this histone arginine methyltransferase in reprogramming ERα-regulated cellular processes, implicating CARM1 as a putative epigenetic target in ER-positive breast cancers.

Conjugated Polymer Loaded Nanospheres with Surface Functionalization for Simultaneous Discrimination of Different Live Cancer Cells under Single Wavelength Excitation

Two conjugated polymers, poly[9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)ethyl) fluorenyldivinylene] (PFV) and the PFV derivative containing 10 mol % 2,1,3-benzothiadiazole (BT) units (PFVBT), have been synthesized and employed to fabricate conjugated polymer loaded nanospheres for simultaneous discrimination of mixed live cancer cells in one solution. The incorporation of BT units into the PFV backbone leads to PFVBT with a similar absorption maximum but significantly red-shifted emission in film state as compared to those of PFV, due to aggregation enhanced energy transfer from the fluorenevinylene segments to electron-deficient BT units. Both conjugated polymer loaded nanospheres have shown optical features that are similar to their film states, which allow simultaneous multichannel signal collection with negligible interference upon excitation at a single wavelength. After further surface functionalization with antihuman epidermal growth factor receptor 2 (HER2) affibody or arginine-glycine-aspartic acid (RGD) peptide, the distinct fluorescence from PFV or PFVBT loaded nanospheres allows differentiation of SKBR-3 breast cancer cells (HER2 overexpression) from HT-29 colon cancer cells (integrin receptor overexpression) in live cell mixtures. The conjugated polymer loaded nanospheres with high quantum yield, low cytotoxicity, and multiple color emission upon single laser excitation are ideal for simultaneous multiple-target imaging and detection.

Mycophenolic acid induces adipocyte-like differentiation and reversal of malignancy of breast cancer cells partly through PPARγ

Mycophenolic acid (MPA) has been known for decades to be an anticancer and immunosuppressive agent and has significant anticancer properties, but its underlying molecular mechanisms are poorly characterized. Peroxisome proliferator-activated receptor gamma (PPARγ) has a central role in adipocyte differentiation, and MPA has been shown to be a potent PPARγ agonist. Whether PPARγ activation has a putative role in the anticancer efficacy of MPA via induction of adipocyte-like differentiation has not been elucidated. In the present study, MPA was demonstrated to dose-dependently activate PPARγ transcription in the GAL4-hPPARγ (LBD) chimeric receptor assay and PPRE-luc reporter gene assay with an EC 50 of 5.2–9.3 μM. Treatment of the breast cancer cell lines MDA-MB-231 and MCF-7 with MPA resulted in differentiation of adipose tissue that was characterized by accumulation of intracellular lipids, enlargement of cell volume, and permanent withdrawal from the cell cycle at the G1/G0 stage. At a molecular level, the expression of three adipocyte differentiation markers (PPARγ, adipsin D, and aP2) was remarkably induced in differentiated breast cancer cells. However, RNA interference experiments showed that PPARγ-knockdown cannot completely reverse the differentiated state of MDA-MB-231 cells after MPA treatment. These data suggest that the effects of MPA on adipocyte-like terminal differentiation of breast cancer cells are (at least in part) due to PPARγ activation, which is a novel anticancer mechanism of MPA.

Functional Significance of Vitamin D Receptor FokI Polymorphism in Human Breast Cancer Cells

BackgroundThe FokI vitamin D receptor (VDR) polymorphism results in different translation initiation sites on VDR. In the VDRff variant, initiation of translation occurs at the first ATG site, giving rise to a full length VDR protein of 427 amino acids. Conversely, in the VDRFF variant, translation begins at the second ATG site, resulting in a truncated protein with three less amino acids. Epidemiological studies have paradoxically implicated this polymorphism with increased breast cancer risk. 1α,25 (OH)2D3, the active metabolite of vitamin D, is known to inhibit cell proliferation, induce apoptosis and potentiate differentiation in human breast cancer cells. It is well documented that 1α,25 (OH)2D3 downregulates estrogen receptor α expression and inhibits estrogen mediated signaling in these cells. The functional significance of the VDR FokI polymorphism in vitamin D action is undefined.Methods/FindingsTo elucidate the functional role of FokI polymorphism in breast cancer, MCF-7-Vector, MCF-7-VDRff and MCF-7-VDRFF stable cell lines were established from parental MCF-7 cells as single-cell clones. In response to 1α,25 (OH)2D3 treatments, cell growth was inhibited by 60% in VDRFF cells compared to 28% in VDRff cells. The induction of the vitamin D target gene CYP24A1 mRNA was 1.8 fold higher in VDRFF cells than in VDRff cells. Estrogen receptor-α protein expression was downregulated by 62% in VDRFF cells compared to 25% in VDRff cells. VDR protein stability was greater in MCF-7-VDRFF cells in the presence of cycloheximide. PCR array analyses of VDRff and VDRFF cells revealed increased basal expression levels of pro-inflammatory genes Cyclooxygenase-2, Interleukin-8 and Chemokine (C-C Motif) Ligand 2 in MCF-7-VDRff cells by 14, 52.7 and 5 fold, respectively.Conclusions/SignificanceThese results suggest that a VDRff genotype may play a role in amplifying aggressive breast cancer, paving the way for understanding why some breast cancer cells respond inefficiently to vitamin D treatment.

N-myc Downstream-regulated Gene 1 (NDRG1) a Differentiation Marker of Human Breast Cancer

N-myc downstream-regulated gene 1 (NDRG1), also called differentiation-related gene-1 (Drg1) and Cap43, is expressed in various normal tissues and suppressed in several malignancies. In this study, whether NDRG1 expression was correlated with differentiation of human breast cancer cells has been investigated. Endogenous expression level of NDRG1 was closely correlated with differentiation status of breast cancer cell lines. Furthermore, sodium butyrate (NaB), an inducer of cellular differentiation, increased the expression of β-casein, a milk-related differentiation marker, together with up-regulation of NDRG1 in breast cancer cells. In contrast, inhibition of NDRG1 by its siRNA resulted in reduced accumulation of β-casein. Immunohistochemical analysis showed co-expression of NDRG1 and β-casein or milk fat protein (MFP), another differentiation marker of breast tissue, in the mouse xenograft model of breast cancer. Furthermore, overexpression of NDRG1 expanded the differentiated area in the xenograft model of breast cancer. In human breast cancer, using samples from 45 patients, we also showed close relationship between NDRG1 and β-casein or MFP expression. Altogether, in vitro and in vivo data demonstrated a possible role of NDRG1 in differentiation of breast cancer. We concluded that NDRG1 could be used as a biomarker for differentiation of breast cancer for both diagnostic and therapeutic purposes.

Comparative analysis of nuclear estrogen receptor alpha and beta interactomes in breast cancer cells

Estrogen Receptor alpha and beta (ER-α and -β) are members of the nuclear receptor family of transcriptional regulators with distinct roles in mediating estrogen dependent breast cancer cell growth and differentiation. Following activation by the hormone, these proteins undergo conformation changes and accumulate in the nucleus, where they bind to chromatin at regulatory sites as homo- and/or heterodimers and assemble in large multiprotein complexes. Although the two ERs share a conserved structure, they exert specific and distinct functional roles in normal and transformed mammary epithelial cells and other cell types. To investigate the molecular bases of such differences, we performed a comparative computational analysis of the nuclear interactomes of the two ER subtypes, exploiting two datasets of receptor interacting proteins identified in breast cancer cell nuclei by Tandem Affinity Purification for their ability to associate in vivo with ligand-activated ER-α and/or ER-β. These datasets comprise 498 proteins, of which only 70 are common to both ERs, suggesting that differences in the nature of the two ER interactomes are likely to sustain the distinct roles of the two receptor subtypes. Functional characterization of the two interactomes and their topological analysis, considering node degree and closeness of the networks, confirmed this possibility. Indeed, clustering and network dissection highlighted the presence of distinct and ER subtype-specific subnetworks endowed with defined functions. Altogether, these data provide new insights on the protein-protein interaction networks controlled by ER-α and -β that mediate their ability to transduce estrogen signaling in breast cancer cells.