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Inherent cancer phenotypes that are independent of fluctuating cross-talk with the surrounding tissue matrix are highly desirable candidates for targeting tumor cells. Our novel study design uses epithelial cell lines derived from low versus high histologic grade primary breast cancer to effectively diminish the breadth of transient variability generated within the tumor microenvironment of the host, revealing a “paracrine-independent expression of grade-associated” (PEGA) gene signature. PEGA members extended beyond “proliferation-driven” signatures commonly associated with aggressive, high-grade breast cancer. The calcium-binding protein S100P was prominent among PEGA genes overexpressed in high-grade tumors. A three-member fingerprint of S100P-correlated genes, consisting of GPRC5A, FXYD3, and PYCARD, conferred poor outcome in multiple breast cancer data sets, irrespective of estrogen receptor status but dependent on tumor size (P < 0.01). S100P silencing markedly diminished coregulated gene transcripts and reversed aggressive tumor behavior. Exposure to pathway-implicated agents, including the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, phenothiazine, and chlorpromazine, resulted in rapid apoptotic cell death in high-grade tumor cells resistant to the chemotherapeutic drug cisplatin. This is the first comprehensive study describing molecular phenotypes intimately associated with histologic grade whose expression remains relatively fixed despite an unavoidably changing environment to which tumor cells are invariably exposed. [Cancer Res 2009;69(19):7826–34]

Supplementary Methods, Figure Legend from Estrogen Receptor α Inhibits p53-Mediated Transcriptional Repression: Implications for the Regulation of Apoptosis

Supplementary Table 3 from Immutable Functional Attributes of Histologic Grade Revealed by Context-Independent Gene Expression in Primary Breast Cancer Cells

Supplementary Figure 1 from Estrogen Receptor α Inhibits p53-Mediated Transcriptional Repression: Implications for the Regulation of Apoptosis

Estrogen receptor α (ERα) and tumor suppressor protein p53 exert opposing effects on cellular proliferation. As a transcriptional regulator, p53 is capable of activating or repressing various target genes. We have previously reported that ERα binds directly to p53, leading to down-regulation of transcriptional activation by p53. In addition to transcriptional activation, transcriptional repression of a subset of target genes by p53 plays important roles in diverse biological processes, such as apoptosis. Here, we report that ERα inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERα interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERα binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERα is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERα to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERα to repress p53-mediated transcriptional activation. Further, RNA interference–mediated knockdown of ERα resulted in reduced Survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment, an effect that was blocked by exogenous expression of Survivin. These results unravel a novel mechanism by which ERα opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer. [Cancer Res 2007;67(16):7746–55]

Supplementary Table 2 from Immutable Functional Attributes of Histologic Grade Revealed by Context-Independent Gene Expression in Primary Breast Cancer Cells

Cells are known to release different types of vesicles such as small extracellular vesicles (sEVs) and large extracellular vesicles (LEVs). sEVs and LEVs play important roles in intercellular communication, pre-metastatic niche formation, and disease progression; both can be detected cell culture media and biological fluids. sEVs and LEVs contain a variety of protein and RNA cargo, and they are believed to impact many biological functions of the recipient cells upon their internalization or binding to cell surface proteins. It has recently been established that standard isolation techniques, such as differential ultracentrifugation, yield a mixed population of EVs. However, density gradient ultracentrifugation has been reported to allow the isolation of sEVs without cellular debris. Here, we describe the most common methods used to isolate sEVs from cell culture medium, mouse and human plasma, and a new technique for isolating sEVs from tissues as well. This article also provides detailed procedures to isolate LEVs.

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.

The human circulation contains cell-free DNA and non-coding microRNA (miRNA). Less is known about the presence of messenger RNA (mRNA). This report profiles the human circulating mRNA transcriptome in people with liver cirrhosis (LC) and hepatocellular carcinoma (HCC) to determine whether mRNA analytes can be used as biomarkers of liver disease. Using RNAseq and RT-qPCR, we investigate circulating mRNA in plasma from HCC and LC patients and demonstrate detection of transcripts representing more than 19,000 different protein coding genes. Remarkably, the circulating mRNA expression levels were similar from person to person over the 21 individuals whose samples were analyzed by RNAseq. Liver derived circulating transcripts such as albumin (ALB), apolipoprotein (APO) A1, A2 & H, serpin A1 & E1, ferritin light chain (FTL) and fibrinogen like 1 (FGL1) were significantly upregulated in HCC patient samples. Higher levels of some of these liver-specific transcripts in the plasma of HCC patients were confirmed by RT-qPCR in another cohort of 20 individuals. Several less abundant circulating transcripts associated with cancer were detected in most HCC samples, but not in healthy subjects. Liver specificity of circulating transcripts was confirmed by investigating their expression in HCC tumor and liver cancer cell lines. Liver specific mRNA sequences in the plasma were predominantly present outside circulating extracellular vesicles. Conclusions: The circulating "mRNA" transcriptome is remarkably consistent in diversity and expression from person to person. Detection of transcripts corresponding to disease selective polypeptides suggests the possibility that circulating mRNA can work as a biomarker analyte for cancer detection.

Treatment of chronic hepatitis B infection with nucleos(t)ide analogues induces a significant inhibition of HBV DNA titer. However, reduction of viral DNA load is not an ideal marker to predict sustainable antiviral effect. Circulating HBV RNA has been, recently, explored as a novel biomarker for monitoring viral persistence and the progression of liver disease. This review aims to discuss the characteristics of circulating HBV RNA and to evaluate its applications in hepatitis B infection management. HBV RNA is readily detectable in the blood of infected patients. Circulating viral RNA is originated from polyadenylated pgRNA that is packaged in, and secreted with, either naked core particles or virion-like particles. Serum pgRNA could be presented in full length or multiple-spliced forms. The amount and composition of pgRNA in blood can be used to gauge intrahepatic cccDNA transcriptional activity, predict drug response, and reflect liver histologic changes. Circulating HBV pgRNA is exclusively transcribed from cccDNA in the liver. As an alternative to liver biopsy, non-invasive measurement of serum HBV RNA in patients receiving antiviral treatment might provide clinicians a safe and effective tool to oversee therapeutic efficacy.

// Aejaz Sayeed 1,2 , Huimin Lu 1,2 , Qin Liu 1,3 , David Deming II 1,2 , Alexander Duffy 1,2 , Peter McCue 4 , Adam P. Dicker 5 , Roger J. Davis 6,7 , Dmitry Gabrilovich 1,8 , Ulrich Rodeck 5,9 , Dario C. Altieri 1,10 and Lucia R. Languino 1,2,5 1 Prostate Cancer Discovery and Development Program, Philadelphia, PA, USA 2 Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA 3 Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA 4 Department of Pathology, Thomas Jefferson University, Philadelphia, PA, USA 5 Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, USA 6 Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA 7 Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, MA, USA 8 Translational Tumor Immunology Program, The Wistar Institute, Philadelphia, PA, USA 9 Department of Dermatology and Cutaneous Biology, Thomas Jefferson University, Philadelphia, PA, USA 10 Tumor Microenvironment and Metastasis Program, The Wistar Institute, Philadelphia, PA, USA Correspondence to: Lucia R. Languino, email: // Keywords : TRAMP mice, prostate cancer, β1 integrins, FAK, insulin-like growth factor receptor Received : November 25, 2015 Accepted : June 15, 2016 Published : July 11, 2016 Abstract Radiation therapy is an effective cancer treatment modality although tumors invariably become resistant. Using the transgenic adenocarcinoma of mouse prostate (TRAMP) model system, we report that a hypofractionated radiation schedule (10 Gy/day for 5 consecutive days) effectively blocks prostate tumor growth in wild type (β1 wt /TRAMP) mice as well as in mice carrying a conditional ablation of β1 integrins in the prostatic epithelium (β1 pc-/- /TRAMP). Since JNK is known to be suppressed by β1 integrins and mediates radiation-induced apoptosis, we tested the effect of SP600125, an inhibitor of c-Jun amino-terminal kinase (JNK) in the TRAMP model system. Our results show that SP600125 negates the effect of radiation on tumor growth in β1 pc-/- /TRAMP mice and leads to invasive adenocarcinoma. These effects are associated with increased focal adhesion kinase (FAK) expression and phosphorylation in prostate tumors in β1 pc-/- /TRAMP mice. In marked contrast, radiation-induced tumor growth suppression, FAK expression and phosphorylation are not altered by SP600125 treatment of β1 wt /TRAMP mice. Furthermore, we have reported earlier that abrogation of insulin-like growth factor receptor (IGF-IR) in prostate cancer cells enhances the sensitivity to radiation. Here we further explore the β1/IGF-IR crosstalk and report that β1 integrins promote cell proliferation partly by enhancing the expression of IGF-IR. In conclusion, we demonstrate that β1 integrin-mediated inhibition of JNK signaling modulates tumor growth rate upon hypofractionated radiation.

Transforming growth factor (TGF) β1 activity depends on a complex signalling cascade that controls expression of several genes. Among others, TGFβ1 regulates expression of matrix metalloproteinases (MMPs) through activation of Smads. In the present study, we demonstrate for the first time that the αvβ6 integrin interacts with TGFβ receptor II (TβRII) through the β6 cytoplasmic domain and promotes Smad3 activation in prostate cancer (PrCa) cells. Another related αv integrin, αvβ5, as well as the αvβ6/3 integrin, which contains a chimeric form of β6 with a β3 cytoplasmic domain, do not associate with TβRII and fail to show similar responses. We provide evidence that αvβ6 is required for up-regulation of MMP2 by TGFβ1 through a Smad3-mediated transcriptional programme in PrCa cells. The functional relevance of these results is underscored by the finding that αvβ6 modulates cell migration in an MMP2-dependent manner on an αvβ6-specific ligand, latency-associated peptide (LAP)-TGFβ. Overall, these mechanistic studies establish that expression of a single integrin, αvβ6, is sufficient to promote activation of Smad3, regulation of MMP2 levels and consequent catalytic activity, as well as cell migration. Our study describes a new TGFβ1-αvβ6-MMP2 signalling pathway that, given TGFβ1 pro-metastatic activity, may have profound implications for PrCa therapy.

Breast cancer is an estrogen-driven disease. Consequently, hormone replacement therapy correlates with disease incidence. However, increasing male breast cancer rates over the past three decades implicate additional sources of estrogenic exposure including wide spread estrogen-mimicking chemicals or xenoestrogens (XEs), such as bisphenol-A (BPA). By exposing renewable, human, high-risk donor breast epithelial cells (HRBECs) to BPA at concentrations that are detectable in human blood, placenta and milk, we previously identified gene expression profile changes associated with activation of mammalian target of rapamycin (mTOR) pathway genesets likely to trigger prosurvival changes in human breast cells. We now provide functional validation of mTOR activation using pairwise comparisons of 16 independent HRBEC samples with and without BPA exposure. We demonstrate induction of key genes and proteins in the PI3K-mTOR pathway--AKT1, RPS6 and 4EBP1 and a concurrent reduction in the tumor suppressor, phosphatase and tensin homolog gene protein. Altered regulation of mTOR pathway proteins in BPA-treated HRBECs led to marked resistance to rapamycin, the defining mTOR inhibitor. Moreover, HRBECs pretreated with BPA, or the XE, methylparaben (MP), surmounted antiestrogenic effects of tamoxifen showing dose-dependent apoptosis evasion and induction of cell cycling. Overall, XEs, when tested in benign breast cells from multiple human subjects, consistently initiated specific functional changes of the kind that are attributed to malignant onset in breast tissue. Our observations demonstrate the feasibility of studying renewable human samples as surrogates and reinforce the concern that BPA and MP, at low concentrations detected in humans, can have adverse health consequences.

An improved understanding of cell immortalization and its manifestation in clinical tumors could facilitate novel therapeutic approaches. However, only rare tumor cells, which maintain telomerase expression in vitro, immortalize spontaneously. By expression-profiling analyses of limited-life primary breast tumor cultures pre- and post-hTERT transduction, and spontaneously immortalized breast cancer cell lines, we identified a common signature characteristic of tumor cell immortalization. A predominant feature of this immortalization signature (ImmSig) was the significant overexpression of oxidoreductase genes. In contrast to epithelial cells derived from low histologic grade primary tumors, which required hTERT transduction for the acquisition of ImmSig, spontaneously immortalizing high-grade tumor cultures displayed similar molecular changes independent of exogenous hTERT. Silencing the hTERT gene reversed ImmSig expression, increased cellular reactive oxygen species levels, altered mitochondrial membrane potential and induced apoptotic and proliferation changes in immortalized cells. In clinical breast cancer samples, cell-proliferation-pathway genes were significantly associated with ImmSig. In these cases, ImmSig expression itself was inversely correlated with patient survival (P=0), and was particularly relevant to the outcome of estrogen receptor-positive tumors. Our data support the notion that ImmSig assists in surmounting normal barriers related to oxidative and replicative stress response. Targeting a subset of aggressive breast cancers by reversing ImmSig components could be a practical therapeutic strategy.

This study was carried out to dissect the mechanism by which β1 integrins promote resistance to radiation. For this purpose, we conditionally ablated β1 integrins in the prostatic epithelium of transgenic adenocarcinoma of mouse prostate (TRAMP) mice. The ability of β1 to promote resistance to radiation was also analyzed by using an inhibitory antibody to β1, AIIB2, in a xenograft model. The role of β1 integrins and of a β1 downstream target, c-Jun amino-terminal kinase 1 (JNK1), in regulating radiation-induced apoptosis in vivo and in vitro was studied. We show that β1 integrins promote prostate cancer (PrCa) progression and resistance to radiation in vivo. Mechanistically, β1 integrins are shown here to suppress activation of JNK1 and, consequently apoptosis, in response to irradiation. Downregulation of JNK1 is necessary to preserve the effect of β1 on resistance to radiation in vitro and in vivo. Finally, given the established crosstalk between β1 integrins and type1 insulin-like growth factor receptor (IGF-IR), we analyzed the ability of IGF-IR to modulate β1 integrin levels. We report that IGF-IR regulates the expression of β1 integrins, which in turn confer resistance to radiation in PrCa cells. In conclusion, this study demonstrates that β1 integrins mediate resistance to ionizing radiation through inhibition of JNK1 activation. J. Cell. Physiol. 228: 1601–1609, 2013. © 2013 Wiley Periodicals, Inc.

Dynamic crosstalk between growth factor receptors, cell adhesion molecules and extracellular matrix is essential for cancer cell migration and invasion. Integrins are transmembrane receptors that bind extracellular matrix proteins and enable cell adhesion and cytoskeletal organization. They also mediate signal transduction to regulate cell proliferation and survival. The type 1 insulin-like growth factor receptor (IGF-IR) mediates tumor cell growth, adhesion and inhibition of apoptosis in several types of cancer. We have previously demonstrated that β1 integrins regulate anchorage-independent growth of prostate cancer (PrCa) cells by regulating IGF-IR expression and androgen receptor-mediated transcriptional functions. Furthermore, we have recently reported that IGF-IR regulates the expression of β1 integrins in PrCa cells. We have dissected the mechanism through which IGF-IR regulates β1 integrin expression in PrCa. Here we report that IGF-IR is crucial for PrCa cell growth and that β1 integrins contribute to the regulation of proliferation by IGF-IR. We demonstrate that β1 integrin regulation by IGF-IR does not occur at the mRNA level. Exogenous expression of a CD4 - β1 integrin cytoplasmic domain chimera does not interfere with such regulation and fails to stabilize β1 integrin expression in the absence of IGF-IR. This appears to be due to the lack of interaction between the β1 cytoplasmic domain and IGF-IR. We demonstrate that IGF-IR stabilizes the β1 subunit by protecting it from proteasomal degradation. The α5 subunit, one of the binding partners of β1, is also downregulated along with β1 upon IGF-IR knockdown while no change is observed in the expression of the α2, α3, α4, α6 and α7 subunits. Our results reveal a crucial mechanistic role for the α5β1 integrin, downstream of IGF-IR, in regulating cancer growth.

Whether stromal components facilitate growth, invasion, and dissemination of cancer cells or suppress neoplastic lesions from further malignant progression is a continuing conundrum in tumor biology. Conceptualizing a dynamic picture of tumorigenesis is complicated by inter-individual heterogeneity. In the post genomic era, unraveling such complexity remains a challenge for the cancer biologist. Towards establishing a functional association between cellular crosstalk and differential cancer aggressiveness, we identified a signature of malignant breast epithelial response to stromal signaling. Proximity to fibroblasts resulted in gene transcript alterations of >2-fold for 107 probes, collectively designated as Fibroblast Triggered Gene Expression in Tumor (FTExT). The hazard ratio predicted by the FTExT classifier for distant relapse in patients with intermediate and high grade breast tumors was significant compared to routine clinical variables (dataset 1, n = 258, HR--2.11, 95% CI 1.17-3.80, p-value 0.01; dataset 2, n = 171, HR--3.07, 95% CI 1.21-7.83, p-value 0.01). Biofunctions represented by FTExT included inflammatory signaling, free radical scavenging, cell death, and cell proliferation. Unlike genes of the 'proliferation cluster', which are overexpressed in aggressive primary tumors, FTExT genes were uniquely repressed in such cases. As proof of concept for our correlative findings, which link stromal-epithelial crosstalk and tumor behavior, we show a distinctive differential in stromal impact on prognosis-defining functional endpoints of cell cycle progression, and resistance to therapy-induced growth arrest and apoptosis in low vs. high grade cancer cells. Our experimental data thus reveal aspects of 'paracrine cooperativity' that are exclusively contingent upon the histopathologically defined grade of interacting tumor epithelium, and demonstrate that epithelial responsiveness to the tumor microenvironment is a deterministic factor underlying clinical outcome. In this light, early attenuation of epithelial-stromal crosstalk could improve the management of cases prone to be clinically challenging.

Despite the findings that β1 integrins play a vital role in the regulation of cell proliferation and survival, the mechanisms through which they operate and lead to cancer progression remain elusive. Previously, our laboratory has shown that β(1A) integrins support insulin-like growth factor 1 (IGFI)-mediated mitogenic and transforming activities. Here, we report that β(1A) integrins regulate basal levels of IGF-IR, although they are not critical for maintaining cancer cell morphology. Upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, we show inhibition of anchorage-independent growth of prostate cancer cells, a function which is dependent on IGF-IR expression. In addition, we demonstrate that IGFI-mediated activation of androgen receptor (AR), known to occur in prostate cancer cells, requires expression of β(1A) integrins as evaluated by luciferase reporter assays and immunoblotting analysis. Since β(1A) integrin levels are increased by R1881 or dihydrotestosterone (DHT), our results imply that β(1A) integrins support an androgen-enhanced feedback loop that regulates the expression of IGF-IR. β(1A) integrins also regulate inducible levels of IGF-IR in cells stimulated by androgen or by a combination of androgen and IGFI, as evaluated by flow cytometric analysis and immunoblotting. Furthermore, upon transfection of β(1A) siRNA and consequent downregulation of IGF-IR, neither activation of AKT, an effector of IGF-IR, nor AR levels are affected. We conclude that β(1A) integrin expression is critical for maintaining the regulatory crosstalk between IGF-IR and AR.

Inherent cancer phenotypes that are independent of fluctuating cross-talk with the surrounding tissue matrix are highly desirable candidates for targeting tumor cells. Our novel study design uses epithelial cell lines derived from low versus high histologic grade primary breast cancer to effectively diminish the breadth of transient variability generated within the tumor microenvironment of the host, revealing a “paracrine-independent expression of grade-associated” (PEGA) gene signature. PEGA members extended beyond “proliferation-driven” signatures commonly associated with aggressive, high-grade breast cancer. The calcium-binding protein S100P was prominent among PEGA genes overexpressed in high-grade tumors. A three-member fingerprint of S100P-correlated genes, consisting of GPRC5A, FXYD3, and PYCARD, conferred poor outcome in multiple breast cancer data sets, irrespective of estrogen receptor status but dependent on tumor size (P < 0.01). S100P silencing markedly diminished coregulated gene transcripts and reversed aggressive tumor behavior. Exposure to pathway-implicated agents, including the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, phenothiazine, and chlorpromazine, resulted in rapid apoptotic cell death in high-grade tumor cells resistant to the chemotherapeutic drug cisplatin. This is the first comprehensive study describing molecular phenotypes intimately associated with histologic grade whose expression remains relatively fixed despite an unavoidably changing environment to which tumor cells are invariably exposed. [Cancer Res 2009;69(19):7826–34]

Breast cancer outcome is highly variable. Whether inadvertent exposure to environmental xenobiotics evokes a biological response promoting cancer aggressiveness and a higher probability of tumor recurrence remains unknown. To determine specific molecular alterations which arise in high-risk breast tissue in the presence of the ubiquitous xenoestrogen, bisphenol A (BPA), we used nonmalignant random periareolar fine-needle aspirates in a novel functional assay. Early events induced by BPA in epithelial-stromal cocultures derived from the contralateral tissue of patients with breast cancer included gene expression patterns which facilitate apoptosis evasion, endurance of microenvironmental stress, and cell cycle deregulation without a detectable increase in cell numbers. This BPA response profile was significantly associated with breast tumors characterized by high histologic grade (P < 0.001) and large tumor size (P = 0.002), resulting in decreased recurrence-free patient survival (P < 0.001). Our assays show a biological “fingerprint” of probable prior exposure to endocrine-disrupting agents, and suggest a scenario in which their presence in the microenvironmental milieu of high-risk breast tissue could play a deterministic role in establishing and maintaining tumor aggressiveness and poor patient outcome. [Cancer Res 2008;68(7):2076–80]

Estrogen receptor α (ERα) and tumor suppressor protein p53 exert opposing effects on cellular proliferation. As a transcriptional regulator, p53 is capable of activating or repressing various target genes. We have previously reported that ERα binds directly to p53, leading to down-regulation of transcriptional activation by p53. In addition to transcriptional activation, transcriptional repression of a subset of target genes by p53 plays important roles in diverse biological processes, such as apoptosis. Here, we report that ERα inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERα interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERα binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERα is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERα to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERα to repress p53-mediated transcriptional activation. Further, RNA interference–mediated knockdown of ERα resulted in reduced Survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment, an effect that was blocked by exogenous expression of Survivin. These results unravel a novel mechanism by which ERα opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer. [Cancer Res 2007;67(16):7746–55]

Toward the goal of identifying early genetic losses, which mediate the release of human breast epithelium from replicative suppression leading to cellular immortalization, we have used a newly developed in vitro model system. This system consists of epithelial cultures derived from noncancerous breast tissue, treated with the chemical carcinogen N-ethyl-N-nitrosourea, and continuously passaged to yield cell populations culminating in the immortal phenotype. Genome-wide allelotyping of early passage N-ethyl-N-nitrosourea-exposed cell populations revealed aberrations at10% (18 of 169) loci examined. Allelic losses encompassing chromosomes 6q24-6q27, implicating immortalization-associated candidate genes, hZAC and SEN6, occurred in two independently derived cell lines before the Hayflick limit. Additional LOH sites were present in one cell line at 3p11-3p26, 11p15, and 20p12-13. Allelic losses reported in this cell line preceded detectable levels of telomerase activity and the occurrence of p53-related aberrations. Information gained from the search for early immortalization-associated genetic deletions in cultured cells was applied in a novel approach toward the analysis of morphologically normal terminal ductal lobular units microdissected from 20 cases of ductal carcinoma in situ. Notably, clonal allelic losses at chromosome 3p24 and 6q24 were an early occurrence in adjoining terminal ductal lobular units of a proportion of primary tumors, which displayed loss of heterozygosity (3 of 11 and 3 of 6, respectively). The biological insights provided by the new model system reported here strongly suggest that early allelic losses delineated in immortalized cultures and validated in vivo could serve as surrogate endpoints to assist in the identification and intervention of high-risk benign breast tissue, which sustains the potential for continuous proliferation.

BACKGROUND: Antiestrogens are important for treatment and prevention of breast cancer. Such drugs influence multiple pathways involved in regulation of cell survival — including apoptosis. In previous work, exposure of human, benign, high-risk donor breast epithelial cells (HRBECs) to the xenoestrogen (XE) bisphenol-A (BPA) induced gene expression changes predicting disruption of apoptotic signaling (Dairkee et al, Cancer Research 2008). Therefore, we hypothesized that antiestrogenic, pro-apoptotic effects of tamoxifen are likely modulated under the influence of XEs. Because the environmental XE, BPA is routinely detected in a large percentage of random human blood samples, we employed functional tests to determine whether BPA interferes with tamoxifen-induced apoptosis in XE-exposed HRBECs. METHODS: With IRB approval and written informed consent, HRBECs were obtained by random periareolar fine needle aspiration (RPFNA) from women who were high risk on the basis of personal or family history of breast cancer, breast density, or high-risk histology. Primary HRBEC cultures were treated for one week with a low concentrations of BPA corresponding to levels reported in human body fluids. Estrogen Receptor (ER)-α and -β were measured by Western blot (n=6). Altered response to tamoxifen toxicity was measured by analyzing quantitative changes in the Annexin V-positive apoptotic cell population (n=8). RESULTS: After one-week exposure to BPA, ER-α increased in six of six cases, ER-β decreased in 5 of 6 cases, and we observed a 25 percent reduction in tamoxifen-induced apoptosis. Prior exposure to BPA at the high concentration range detected in the human population reduced tamoxifen efficacy by 50 percent. There was a dose response of evasion of apoptosis to increasing concentrations of BPA (P CONCLUSION: BPA and MP, in a range of concentrations measured in humans, prevent tamoxifen-induced apoptotic cell death. For BPA this involves upregulation of ER-α and downregulation of ER-β. Because these XEs are widespread in the American population, they may play a role in limiting the prophylactic and therapeutic efficacy of tamoxifen and other hormone-based therapies. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-05-04.

The histological manifestation of growth-regulating and differentiation-inducing signals in cancer cells is considered as a key component for clinical outcome prediction and commonly defined as tumor differentiation grade. However, the molecular and functional framework underlying this clinical parameter remains poorly understood. Our correlative data display a significant association (P>0.001) between mitochondrial uncoupling protein 2 (UCP2) and tumor grade in primary breast cancer (n=234). Through mechanistic analyses, we show a synergistic link between UCP2 and established cellular pathways in conferring grade-associated functional phenotypes. Here, the application of well to moderately differentiated primary tumor cell lines has enabled direct observation of SMAD recruitment to the UCP2 promoter underlying repression of gene transcription. In contrast, poorly differentiated tumor cells, known to be TGFβ resistant, displayed aberrant UCP2 regulation, and consequently, gene overexpression, which reduced mitochondrial calcium and facilitated the maintenance of mitochondrial membrane potential, thereby significantly decreasing oxidative stress and inhibiting cell death. Conversely, UCP2 silencing in such cells rapidly led to the induction of apoptosis and cell differentiation, concurrent with reduced cell survival and proliferation, confirming gene-specific effects. Demonstration of a biologically driven role for UCP2 dysregulation in promoting multiple characteristics of tumor aggressiveness strongly endorses assessment of gene expression at clinical presentation to augment therapeutic decision-making and improve patient outcome through personalized targeting approaches.