Nora D. Mineva

Histone Deacetylases: Unique Players in Shaping the Epigenetic Histone Code

Abstract: The epigenome is defined by DNA methylation patterns and the associated posttranslational modifications of histones. This histone code determines the expression status of individual genes dependent upon their localization on the chromatin. The silencing of gene expression is associated with deacetylated histones, which are often found to be associated with regions of DNA methylation as well as methylation at the lysine 4 residue of histone 3. In contrast, the activation of gene expression is associated with acetylated histones and methylation at the lysine 9 residue of histone 3. The histone deactylases play a major role in keeping the balance between the acetylated and deacetylated states of chromatin. Histone deacetylases (HDACs) are divided into three classes: class I HDACs (HDACs 1, 2, 3, and 8) are similar to the yeast RPD3 protein and localize to the nucleus; class II HDACs (HDACs 4, 5, 6, 7, 9, and 10) are homologous to the yeast HDA1 protein and are found in both the nucleus and cytoplasm; and class III HDACs form a structurally distinct class of NAD‐dependent enzymes that are similar to the yeast SIR2 proteins. Since inappropriate silencing of critical genes can result in one or both hits of tumor suppressor gene (TSG) inactivation in cancer, theoretically the reactivation of affected TSGs could have an enormous therapeutic value in preventing and treating cancer. Indeed, several HDAC inhibitors are currently being developed and tested for their potency in cancer chemotherapy. Importantly, these agents are also potentially applicable to chemoprevention if their toxicity can be minimized. Despite the toxic side effects and lack of specificity of some of the inhibitors, progress is being made. With the elucidation of the structures, functions and modes of action of HDACs, finding agents that may be targeted to specific HDACs and potentially reactivate expression of only a defined set of affected genes in cancer will be more attainable.

Oestrogen signalling inhibits invasive phenotype by repressing RelB and its target BCL2

Aberrant constitutive expression of c-Rel, p65 and p50 NF-κB subunits has been reported in over 90% of breast cancers. Recently, we characterized a de novo RelB NF-κB subunit synthesis pathway, induced by the cytomegalovirus (CMV) IE1 protein, in which binding of p50–p65 NF-κB and c-Jun–Fra-2 AP-1 complexes to the RELB promoter work in synergy to potently activate transcription. Although RelB complexes were observed in mouse mammary tumours induced by either ectopic c-Rel expression or carcinogen exposure, little is known about RelB in human breast disease. Here, we demonstrate constitutive de novo RelB synthesis is selectively active in invasive oestrogen receptor alpha (ERα)-negative breast cancer cells. ERα signalling reduced levels of functional NF-κB and Fra-2 AP-1 and inhibited de novo RelB synthesis, leading to an inverse correlation between RELB and ERα gene expression in human breast cancer tissues and cell lines. Induction of Bcl-2 by RelB promoted the more invasive phenotype of ERα-negative cancer cells. Thus, inhibition of de novo RelB synthesis represents a new mechanism whereby ERα controls epithelial to mesenchymal transition (EMT).

RelB NF-κB Represses Estrogen Receptor α Expression via Induction of the Zinc Finger Protein Blimp1

ABSTRACT Aberrant constitutive expression of NF-κB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERα)-negative breast cancers versus ERα-positive ones, due in part to repression of RelB synthesis by ERα signaling. Notably, RelB promoted a more invasive phenotype in ERα-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERα synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERα (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERα-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-κB subunit mediates repression, specifically of ERα, thereby promoting a more migratory phenotype.

Epigallocatechin-3-gallate inhibits stem-like inflammatory breast cancer cells.

Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by high rates of proliferation, lymphangiogenesis and metastasis, and an overall poor survival. As regular green tea consumption has been associated with improved prognosis of breast cancer patients, including decreased risk of recurrence, here the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) were tested on two IBC lines: SUM-149 and SUM-190. EGCG decreased expression of genes that promote proliferation, migration, invasion, and survival. Consistently, growth, invasive properties, and survival of IBC cells were reduced by EGCG treatment. EGCG also reduced lymphangiogenesis-promoting genes, in particular VEGF-D. Conditioned media from EGCG-treated IBC cells displayed decreased VEGF-D secretion and reduced ability to promote lymphangiogenesis in vitro as measured by hTERT-HDLEC lymphatic endothelial cell migration and tube formation. Tumorsphere formation by SUM-149 cells was robustly inhibited by EGCG, suggesting effects on self-renewal ability. Stem-like SUM-149 cells with high aldehyde dehydrogenase (ALDH) activity, previously implicated in poor patient prognosis, were isolated. EGCG treatment reduced growth and induced apoptosis of the stem-like SUM-149 cells in culture. In an orthotopic mouse model, EGCG decreased growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells and their expression of VEGF-D, which correlated with a significant decrease in peritumoral lymphatic vessel density. Thus, EGCG inhibits the overall aggressive IBC phenotype. Reduction of the stem-like cell compartment by EGCG may explain the decreased risk of breast cancer recurrence among green tea drinkers. Recent clinical trials demonstrate the efficacy of green tea polyphenol extracts in treatment of prostate cancer and lymphocytic leukemia with low toxicity. Given the poor prognosis of IBC patients, our findings suggest further exploration of EGCG or green tea in combinatorial treatments against active IBC disease or in maintenance regimens to avoid recurrence is warranted.

c‐Myc represses FOXO3a‐mediated transcription of the gene encoding the p27Kip1 cyclin dependent kinase inhibitor

The p27Kip1 (p27) cyclin‐dependent kinase inhibitor and c‐Myc oncoprotein play essential roles in control of cell cycle progression and apoptosis. Induction of p27 (CDKN1B) gene transcription by Forkhead box O proteins such as FOXO3a leads to growth arrest and apoptosis. Previously, we observed that B cell receptor (surface IgM) engagement of WEHI 231 immature B lymphoma cells with an anti‐IgM antibody results in activation of FOXO3a, growth arrest and apoptosis. As ectopic c‐Myc expression in these cells prevented anti‐IgM induction of p27 and cell death, we hypothesized that c‐Myc represses FOXO3a‐mediated transcription. Here we show that c‐Myc inhibits FOXO3a‐mediated activation of the p27 promoter in multiple cell lines. The mechanism of this repression was explored using a combination of co‐immunoprecipitation, oligonucleotide precipitation, and chromatin immunoprecipitation experiments. The studies demonstrate a functional association of FOXO3a and c‐Myc on a proximal Forkhead binding element in the p27 promoter. This association involves the Myc box II domain of c‐Myc and the N‐terminal DNA‐binding portion of FOXO3a. Analysis of publicly available microarray datasets showed an inverse pattern of c‐MYC and p27 RNA expression in primary acute myeloid leukemia, prostate cancer and tongue squamous cell carcinoma samples. The inhibition of FOXO3a‐mediated activation of the p27 gene by the high aberrant expression of c‐Myc in many tumor cells likely contributes to their uncontrolled proliferation and invasive phenotype. J. Cell. Biochem. 104: 2091–2106, 2008.

CD40 Ligand-mediated Activation of the de Novo RelB NF-κB Synthesis Pathway in Transformed B Cells Promotes Rescue from Apoptosis

CD40, a tumor necrosis factor receptor family member, is expressed on B lymphocytes. Interaction between CD40 and its ligand (CD40L), expressed on activated T lymphocytes, is critical for B cell survival. Here, we demonstrate that CD40 signals B cell survival in part via transcriptional activation of the RelB NF-κB subunit. CD40L treatment of chronic lymphocytic leukemia cells induced levels of relB mRNA. Similarly, CD40L-mediated rescue of WEHI 231 B lymphoma cells from apoptosis induced upon B cell receptor (surface IgM) engagement led to increased relB mRNA levels. Recently, we characterized a new de novo synthesis pathway for the RelB NF-κB subunit, induced by the cytomegalovirus IE1 protein, in which binding of p50/p65 NF-κB and c-Jun/Fra-2 AP-1 complexes to the relB promoter works in synergy to potently activate transcription (Wang, X., and Sonenshein, G. E. (2005) J. Virol. 79, 95–105). CD40L treatment of WEHI 231 cells caused induction of AP-1 family members Fra-2, c-Jun, JunD, and JunB. Cotransfection of Fra-2 with the Jun AP-1 subunits and p50/c-Rel NF-κB led to synergistic activation of the relB promoter. Ectopic expression of relB or RelB knockdown using small interfering RNA demonstrated the important role of this subunit in control of WEHI 231 cell survival and implicated activation of the anti-apoptotic factors Survivin and manganese superoxide dismutase. Thus, CD40 engagement of transformed B cells activates relB gene transcription via a process we have termed the de novo RelB synthesis pathway, which protects these cells from apoptosis.

Inhibition of RelB by 1,25-Dihydroxyvitamin D3 Promotes Sensitivity of Breast Cancer Cells to Radiation

Aberrant constitutive expression of the NF‐κB c‐Rel and RelA subunits in breast cancer cells was shown to promote their survival. Recently, we demonstrated that aggressive breast cancers constitutively express high levels of the RelB subunit, which promotes their more invasive phenotype via induction of the BCL2 gene. As these cancers are frequently resistant to therapy, here we tested the hypothesis that RelB promotes their survival. High RelB expressing Hs578T and MDA‐MB‐231 breast cancer cells were more resistant to γ‐radiation than MCF7 and ZR‐75 cells, which express lower RelB levels. Knockdown of RelB in Hs578T led to decreased survival in response to γ‐irradiation, while conversely ectopic expression of RelB in MCF7 cells protected these cells from radiation. Similar data were obtained upon treatment of Hs578T or MCF7 cells with the chemotherapeutic agent doxorubicin. High serum levels of 25‐hydroxyvitamin D are associated with decreased breast cancer risk and mortality, although, the mechanisms of its protective actions have not been fully elucidated. Treatment of Hs578T and Her‐2/neu‐driven NF639 cells with 1,25‐dihydroxyvitamin D3 decreased RelB/RELB gene expression and levels of pro‐survival targets Survivin, MnSOD and Bcl‐2, while increasing their sensitivity to γ‐irradiation. Thus, RelB, which promotes survival and a more highly invasive phenotype of breast cancer cells, is a target of 1,25‐dihydroxyvitamin D3, providing one mechanism for the observed protective role of 25‐hydroxyvitamin D in patients with breast cancer. J. Cell. Physiol. 220: 593–599, 2009.

ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

The transmembrane metalloprotease‐disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple‐negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF‐A and transendothelial cell migration via β1‐integrin activation. In vivo, treatment with an anti‐ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non‐essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

Endogenous light scattering as an optical signature of circulating tumor cell clusters.

Circulating tumor cell clusters (CTCCs) are significantly more likely to form metastases than single tumor cells. We demonstrate the potential of backscatter-based flow cytometry (BSFC) to detect unique light scattering signatures of CTCCs in the blood of mice orthotopically implanted with breast cancer cells and treated with an anti-ADAM8 or a control antibody. Based on scattering detected at 405, 488, and 633 nm from blood samples flowing through microfluidic devices, we identified 14 CTCCs with large scattering peak widths and intensities, whose presence correlated strongly with metastasis. These initial studies demonstrate the potential to detect CTCCs via label-free BSFC.

Abstract B166: MicroRNAs downstream of ADAM8 as therapeutic targets and non-invasive biomarkers for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) represents ∼20% of breast cancers, is highly aggressive and lacks targeted therapies due to the absence of hormone and HER2 receptors. TNBCs respond only poorly to chemotherapy and recur more frequently than other breast cancers. Their recurrence often involves distant metastasis, especially to the brain and lungs with a poor prognosis, which lead to approximately 25% of the half million yearly deaths worldwide from breast cancer. Thus, there is a critical unmet need for early detection and new therapeutic targets for TNBC treatment, which was the focus of our current study. Recently, our lab has shown that the transmembrane protein ADAM8 (A Disintegrin And Metalloproteinase 8) plays a key role in invasion and metastasis of TNBCs and high ADAM8 levels correlate with a poor prognosis in breast cancer patients. MicroRNAs (miRNAs) play important functions in both normal and disease development. It is estimated that more than 60% of the protein-coding genes in the human genome are regulated by miRNAs. Furthermore, circulating tumor cell-associated miRNAs and free circulating miRNAs have been identified in whole blood, plasma and serum in various cancer subtypes. Here we tested two related hypotheses, that: (1) miRNAs regulated by the ADAM8 pathway are important mediators promoting invasion and migration of TNBC cells; and (2) these miRNAs represent potential therapeutic targets and/or noninvasive biomarkers for detection of TNBC recurrence. A microarray analysis was performed using TaqMan microRNA cards on RNA isolated from MDA-MB-231 cells after 72 h of transient siRNA-mediated knockdown of ADAM8. We identified 68 miRNAs that were differentially regulated upon ADAM8 knockdown. Thus far we have confirmed 13 miRNAs using real-time PCR. Of the miRNAs confirmed, miR-720 was selected as the initial candidate of study, since it has been previously identified as an oncomiR and also detected in blood from breast cancer patients. Transient ectopic ADAM8 overexpression in MDA-MB-231 and SUM149 TNBC cell lines induced levels of miR-720 whereas ADAM8 siRNA reduced them. Furthermore, knockdown of miR-720 using an antagomiR led to a significant decrease of the migratory and invasive phenotype of TNBC cells as judged by Boyden chamber and 3D Matrigel outgrowth assays, respectively. Consistently, we used a miRNA mimic to overexpress miR-720 activity and observed increased migration and invasion of TNBC cells. Importantly, a profound increase in levels of miR-720 was detected in the plasma of mice bearing orthotopic mammary tumors derived from control shRNA-MDA-MB-231 cells in contrast to shADAM8-MDA-MB-231 cells and non-tumor bearing mice, which suggests that miR-720 levels in blood correlate with ADAM8 expression in tumors. Taken together our data indicates that the miR-720 is induced by ADAM8 and miRNAs regulated downstream of ADAM8 have a strong potential as both biomarkers and therapeutic targets for the treatment of TNBC. Research was funded by grants from the DOD (W81XWH-11-1-0814) and the NIH (R01 CA129129) Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B166. Citation Format: Sonia G. Das, Mathilde Romagnoli, Nora D. Mineva, Gail E. Sonenshein. MicroRNAs downstream of ADAM8 as therapeutic targets and non-invasive biomarkers for triple-negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B166.