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BCRL Home » Research Projects » Chromatin remodeling as a preventive strategy in breast cancer

Chromatin remodeling in breast cancer

Epigenetic changes in breast cancer prevention

Cancer initiation was profoundly inhibited in rats that had completed one pregnancy, or had received a 21-day treatment with the placental hormone chorionic gonadotropin (hCG) prior to DMBA administration. In addition, hCG also inhibits tumor progression when administered to tumor-bearing animals. The inhibitory effect of hCG on both cancer initiation and progression involved the expression of differentiation markers in breast epithelial cells, including the synthesis of inhibin, a secreted protein with tumor suppressor activity that is hypermethylated in hormone-dependent carcinomas. In human breast epithelial cells invitro,hCG induces the synthesis of inhibin and significantly increases the levels of acetylation of histones H3 and H4. Our observation that hCG activates the expression of tumor suppressors, such as p53 and inhibin, that it induces histones H3 and H4 acetylation, and the fact that DMBA-induced mammary tumors overexpress DNA methyltransferase, lend support to the postulate that hCG might inhibit mammary carcinogenesis by inducing re-expression of genes silenced by DNA methylation, particularly at CpG islands. Our laboratory was the first one to report that the induction of mammary gland differentiation by hCG in vivo and in vitro treatment of human breast epithelial cells both stimulated the synthesis of inhibins in the mammary epithelium. Traditionally, inhibins have been considered to be gonadal hormones whose production by granulose cells is considered to be a marker of follicular responsiveness to hCG treatment in infertility treatments. Inhibins are heterodimeric glycoproteins belonging to the inhibin/activin family that are composed of a common 18 Kd α-subunit and one of two 14 Kd β-subunits (βA or βB), thus forming the heterodimers inhibin A (α-βA), and inhibin B (α-βB) respectively. Although inhibins are known to selectively suppress the synthesis and release of the pituitary folliclestimulating hormone (FSH), they have been recognized more recently to be part of a large group of morphogenesis and differentiation - related proteins, the transforming growth factor β (TGF-β) superfamily which also includes the β-homodimer activin .

We had previously postulated that hCG induces differentiation and activation of apoptosis related genes via activation of inhibins, which in turn might act through autocrine or paracrine mechanisms for furthering cell differentiation and inhibiting the progression of neoplastic cells. For elucidating these mechanisms we tested the in vitro effect of recombinant hCG (r-hCG) and inhibin β-subunit added to the culture medium of the immortalized human breast epithelial cells (HBEC) MCF-10 F. Cells were treated with 1, 10, 50, or 100 IU r-hCG or 1, 10, 100, or 1000 ng inhibin-b-subunit per ml and cells were harvested at 1, 4, 12, or 24 hours of treatment with each one of the hormones. Control cells were treated with the same volume of buffer in which the hormones were dissolved. For Western blot analysis total cell extracts were prepared using standard procedures in our laboratory. After quantification of protein concentration 20 mg of each extract was separated by electrophoresis in an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with a 1:200 dilution of anti-Acetylated histones H3 or H4 IgGs. Horseradish peroxidase-conjugated goat anti-rabbit IgG at 1:5000 dilution was used as a secondary antibody for ECL detection. Western blot analysis revealed that hCG acetylated both histones H3 and H4 at all the doses tested and the levels significantly increased at 12 and 24 hours of treatment. Inhibin b-subunit induced the accumulation of acetylated histone H3 after 4 hr treatment with 1 ng and at all time points with the higher concentrations. A slight acetylated histone H4 increase was detected in cells treated with 100 ng/ml for 12 and 24 hs. Our data indicate that both hCG and inhibin increase acetylation levels in histone H4 in the HBEC MCF-10F. It is possible to postulate that the induction of histone acetylation by hCG and inhibin plays a role in the activation and transcription of early genes, such as c-myc and c-jun, as well as the tumor suppressors, such as p53 and inhibin, previously described. The fact that DMBA-induced mammary tumors over express DNA methyltransferase lend support to the postulate that hCG might inhibit mammary carcinogenesis by inducing re-expression of genes silenced by DNA methylation, particularly at CpG islands. These findings lead us to consider a new paradigm in the mechanism of action of hCG in breast cancer prevention and therapy, since this hormone might act not only as an activator of differentiation associated genes, but might also reactive the expression of genes transcriptionally silenced by promoter hypermethylation during the process of carcinogenesis.

Main Publications Generated from this Project
  1. Jiang, X., Russo, IH., Russo J. Human chorionic gonadotropin and inhibin induce histone acetylation in human breast cancer cells. Int. J. Oncol. 20(1):77-79, 2002.
  2. Alvarado, M.V., Ho, T-Y., Russo, J. and Russo, I.H. Human chorionic gonadotropin regulates the synthesis of inhibin in the ovary and the mammary gland of rats. Endocrine 2:1-10, 1994.
  3. Srivastava, P., Russo, J. and Russo, I.H. Inhibition of rat mammary tumorigenesis by human chorionic gonadotropin is associated with increased expression of inhibin. Mol. Carcinog. 26: 10-19, 1999.
  4. Alvarado, M.E., Alvarado, N.E., Russo, J. and Russo, I.H. Human chorionic gonadotropin inhibits proliferation and induces expression of inhibin in human breast epithelial cells in vitro. In Vitro 30A:4-8, 1994.
  5. Russo, I.H. and Russo, J. Role of hCG and inhibin in breast cancer (Review). Int. J. Oncology 4:297-306, 1994.
Chromatin remodeling during Human breast epithelial cell transformation

Treatment of the human breast epithelial cells MCF-10F with 17-β-estradiol (E2) induces transformation and tumorigenesis. E2-transformed MCF-10F cells are known to exhibit progressive loss of ductulogenesis, and invasive and tumorigenic phenotypes. DNA amounts and chromatin supraorganization change in E2-transformed MCF cells. We have shown that Feulgen-DNA content and chromatin supraorganization are involved during E2-induced transformation and tumorigenesis of the MCF-10F cells (1). Image analysis was performed for nontransformed and E2-transformed MCF cells, highly invasive cells (C5), and for cell lines (C5-A6-T6 and C5-A8-T8) derived from tumors generated by injection of C5 cells in SCID mice (2). A decrease in Feulgen-DNA amounts and nuclear sizes induced by E2 treatment was accented with selection of the highly invasive tumorigenesis potential. However, in the tumor-derived cells a high variability in cellular phenotypes resulted inclusive in near-polyploidy. Significant changes in textural parameters, including nuclear entropy, indicated chromatin structural remodeling with advancing tumorigenesis. An increased variability in the degree of chromatin packing states in the E2-transformed MCF cells is followed by reduction in chromatin condensation and in contrast between condensed and noncondensed chromatin in the highly invasive C5 cells and tumor-derived cell lines (2).

Main Publications Generated from this Project
  1. Mello, M.L. Russo, P.A., Russo, J and Vidal B.C.17-ß-estradiol affects nuclear image properties in MCF-10Fhuman breast epithelial cells with tumorigenesis. Oncology Reports 18: 1475-1481, 2007
  2. Russo, J., Fernandez, S.V., Russo, P.A., Fernbaugh, R., Sheriff, F.S., Lareef, H.M., Garber, J., and Russo,I.H. 17 beta estradiol induces transformation and tumorigenesis in human breast epithelial cells. FASEB J 20:1622-1634, 2006.
Epigenomic control of Epithelial Mesenchymal Transition (EMT) leading to Tumor invasion

While the regulation of EMT is not fully understood, a network of several signaling pathways affecting the expression and/or function of a complex hierarchical network of transcription factors (TFs) has been partially elaborated. Known signaling pathways include multiple tyrosine kinase receptors leading to Ras-mediated activation of MAPK and PI3K pathways, TGF-β, Notch and Wnt. Evidence for enhanced TGF-β and Wnt signaling pathways was found in the EMT expressing bcMCF and caMCF cells (1). TGF-β acting though Smad transcriptional complexes can repress expression of the Id TFs (Id1, Id2, Id3) and activate HMGA2, a DNA binding protein important for chromatin architecture. Expression of HMGA2 is known to regulate several EMT controlling TFs including TWIST1, SNAI1, and SNAI2 (Slug). Figure 28:

Figure28

TGF-β and Wnt signaling also affect the expression of several additional EMT-regulating TFs including ZEB1 (TCF8), TCF3 (E2A encoding E12 and E47), and LEF1. Analysis of the EMT expressing bcMCF cell line revealed the absence of expression of the secreted frizzled-related protein 1 (SFRP1), a repressor of Wnt signaling (1). One allele of SFRP1 was deleted in these cells, with the remaining apparently silenced by methylation, accounting for the 28-fold reduction of this transcript. Loss and epigenetic inactivation of SFRP1 occurs often in invasive breast cancer and is associated with poor prognosis. Inspection of the SFRP1 expression levels in Basal B cell lines showed absent calls for 4 of the 8 invasive cell lines; and 8-fold decreases in another 3 invasive cell lines relative to the non-invasive MCF-10A cells (data not shown). Inspection of the expression files for bcMCF cells and the 8 invasive Basal B cell lines (15) revealed that LEF1 was always absent, while TCF 3 and TCF 8 were expressed (data not shown).

Main Publications Generated from this Project
Breast cancer metastasis and SATB1 as a reprogrammer of gene expression to promote breast tumour growth and metastasis

Breast cancer is the number one cause of cancer death in women in Europe and North America and mortality is related to metastatic spread of the primary tumor, with approximately 50% of all cancer patients showing evidence of metastasis at first presentation. Metastasis is a multi-step process involving both invasion and migration. It begins with migration from the primary tumor site followed by intravasation of the invading tumor cells to vessels of the circulatory systems, from where the surviving cells extravasate into a distant organ establishing a secondary tumor. It is suggested that the formation of distant metastases may be due to the presence of cells within the primary tumor which have the ability to form metastases very early in the disease process and recently has been demonstrated that SATB1 gene may play and important role ion this process.

SATB1 binds to specialized DNA sequences in the genome and functions as a 'genome organizer' by tethering these DNA sites to a functional nuclear architecture that has a 'cage-like' protein distribution surrounding heterochromatin. SATB1 regulate a large number of genes during T-cell development and activation. Once SATB1 is expressed in breast cancer cells, it coordinates expression of a large number of genes to induce metastasis. Removal of SATB1 from aggressive breast cancer cells not only reverses metastatic phenotypes but also inhibits tumor growth, indicating its key role in breast cancer progression. The prognostic significance of SATB1 was determined by assessing its nuclear staining using tissue microarrays containing 1,318 breast cancer specimens with known clinical follow-up records. Using Kaplan–Meier survival analysis have identified a correlation between higher SATB1 expression levels and shorter overall survival times (P<0.001). This correlation was also observed with all breast cancer types. SATB1 expression in primary tumors serves as a prognostic marker independent of other established prognostic factors for breast cancer, including tumor stage, the histological grade and nodal stage. SATB1's role in breast cancer as a global gene regulator was clearly demonstrated by using a breast cancer cell line (MDA-MB-231) in which silencing of SATB1 induces up- or down regulation of genes that in breast tumors are associated with poor prognoses. Among the upregulated genes are those known to have important functions in promoting metastasis including metastasin (S100A4) and VEGFB, which have roles in metastasis and angiogenesis; matrix metalloproteases (MMPs), which degrade ECM and promote tumor invasion; transforming growth factor-b1 (TGFB1), which stimulates invasion; and connective tissue growth factor (CTGF), which mediates angiogenesis and bone metastasis. Notably, SATB1 upregulated genes involved in epidermal growth factor (EGF) signaling, such as the EGF receptor subfamily members ERBB1, ERBB2 (HER-2 orNEU), ERBB3, ERBB4 and the ligands NRG and AREG. ERBB2, the most oncogenic member of the ERBB family, is an important regulator of breast cancer progression, and drugs that intercept ERBB2 signaling are in routine clinical application.

We have postulated that epithelial mesenchymal transition or EMT occurs during carcinoma progression, particularly at specific stages such as invasion and intravasation, where tumor cells disassemble and migrate to tissue/organ sites distant from the primary tumors. As the epithelial cells lose their polarity and cell-cell junctions, regulated in part by the expression of E-cadherin, they acquire characteristics of mesenchymal cells, which lack stable intercellular junctions. This EMT leads to enhanced motility and invasiveness in many cell types, and is often considered a prerequisite for tumor infiltration and metastasis. Of particular importance to this pathological process is that SATB1 could be an important player not previously sought. The first indication is that SATB1 expression induces a marked change in the gene expression pattern in cancer cells and promotes their acquisition of aggressive phenotypes SATB1 depletion blocks the upregulation of cell-structure genes typical in invasive breast cancers, consistent with our observations that SATB1 depletion from MDA-MB-231 cells restores normal cell morphology. Such cell structure genes include the extracellular matrix (ECM) protein fibronectin (FN), the intermediate filament protein vimentin (VIM) and the cell–ECM interacting protein 4 integrin (ITGB4). Dysregulated expression of cadherin and catenins, which mediate cell-to-cell adhesion, has been associated with breast cancer. OB-cadherin (CDH11), VE-cadherin (CDH5) and N-cadherin (CDH2), often upregulated in invasive breast cancer, were all upregulated by SATB1. In contrast, genes downregulated by SATB1 included claudin 1 (CLDN1), a tight junction protein that is commonly lost or mislocalized in invasive tumors; b-catenin (CTNNB1), a critical member of the canonical Wnt pathway; and E-cadherin (CDH1), an adherens junction protein and tumour suppressor. Loss of E-cadherin is a hallmark for epithelial to mesenchymal transition, a process whereby epithelial cells lose polarity, cell-to-cell contacts, and cytoskeletal integrity contributing to the dissemination of carcinoma cells from epithelial tumors. On SATB1 depletion, the observed upregulation of E-cadherin and down regulation of fibronectin and repressors of E-cadherin such as SNAIL and SIP1 indicate that the epithelial to mesenchymal transition process is reversed, resulting in the restoration of acinar-like morphology. Altogether this novel information clearly indicates that SATB1 is an important step in the cascade that initiates EMT and therefore a justified target for therapeutic intervention.

Because SATB1 expression in cancer cells necessarily alters the gene expression profile to promote tumor growth and metastasis, SATB1 can serve as a sentinel indicating that cells have acquired the aggressive phenotype. The mechanism by which SATB1 globally reprogrammes gene expression during metastasis could be by tethering hundreds of gene loci onto its regulatory network, assembling them with chromatin modifying and transcription factors. SATB1 expression is not restricted to late clinical stages of disease, but is also observed in a subset of primary breast tumors at early clinical stages before lymph node metastasis. The SATB1 protein levels in the nuclei of cancer cells has high prognostic significance, independent of the lymph node status (P<0.0001), indicating its utility in predicting the likelihood of disease progression in patients with early-stage breast cancer. Silencing SATB1 in those tumors that are highly expressive of this protein may open a new target for cancer therapy.

Main Publications Generated from this Project