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1.  PROGgeneV2: enhancements on the existing database 
BMC Cancer  2014;14(1):970.
Background
We recently published PROGgene, a tool that can be used to study prognostic implications of genes in various cancers. The first version of the tool had several areas for improvement. In this paper we present some major enhancements we have made on the existing tool in the new version, PROGgeneV2.
Results
In PROGgeneV2, we have made several modifications to enhance survival analysis capability of the tool. First, we have increased the repository of public studies catalogued in our tool by almost two folds. We have also added additional functionalities to perform survival analysis in a variety of new ways. Survival analysis can now be performed on a) single genes b) multiple genes as a signature, c) ratio of expression of two genes, and d) curated/published gene signatures in new version. Users can now also adjust the survival analysis models for available covariates. Users can study prognostic implications of entire gene signatures in different cancer types, which are searchable by keywords. Also, unique to our tool, in the new version, users will be able to upload and use their own datasets to perform survival analysis on genes of interest.
Conclusions
We believe, like its predecessor, PROGGeneV2 will continue to be useful for the scientific community for formulating research hypotheses and designing mechanistic studies. With added datasets PROGgeneV2 is the most comprehensive survival analysis tool available. PROGgeneV2 is available at http://www.compbio.iupui.edu/proggene.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-970) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-970
PMCID: PMC4300843  PMID: 25518851
Biomarker; Multiple cancer; Survival; Pan cancer; Prognostic; mRNA; Database; Kaplan; Meier; KM
2.  ANTXR1, a stem cell enriched functional biomarker, connects collagen signaling to cancer stem-like cells and metastasis in breast cancer 
Cancer research  2013;73(18):5821-5833.
Cancer stem-like cells are thought to contribute to tumor recurrence. The anthrax toxin receptor ANTXR1 has been identified as a functional biomarker of normal stem cells and breast cancer stem-like cells. Primary stem cell-enriched basal cells (CD49f+/EpCAM−/Lin−) expressed higher levels of ANTXR1 compared to mature luminal cells. CD49f+/EpCAM−, CD44+/EpCAM−, CD44+/CD24− or ALDEFLUOR-positive subpopulations of breast cancer cells were enriched for ANTXR1 expression. CD44+/CD24−/ANTXR1+ cells displayed enhanced self-renewal as measured by mammosphere assay compared to CD44+/CD24−/ANTXR1− cells. Activation of ANTXR1 by its natural ligand C5A, a fragment of collagen VI α3, increased stem cell self-renewal in mammosphere assays and Wnt signaling including the expression of the Wnt receptor LRP6, phosphorylation of GSK3α/β and elevated expression of Wnt target genes. RNAi-mediated silencing of ANTXR1 enhanced the expression of luminal-enriched genes but diminished Wnt signaling including reduced LRP6 and ZEB1 expression, self-renewal, invasion, tumorigenicity and metastasis. ANTXR1 silencing also reduced the expression of HSPA1A, which is overexpressed in metastatic breast cancer stem cells. Analysis of public databases revealed ANTXR1 amplification in medullary breast carcinoma and overexpression in estrogen receptor-negative breast cancers with the worst outcome. Further, ANTXR1 is among the 10% most overexpressed genes in breast cancer and is co-expressed with collagen VI. Thus, ANTXR1:C5A interactions bridge a network of collagen cleavage and remodeling in the tumor microenvironment, linking it to a stemness signaling network drives metastatic progression.
doi:10.1158/0008-5472.CAN-13-1080
PMCID: PMC3778138  PMID: 23832666
ANTXR1; breast cancer; cancer stem cells; collagen VI; metastasis
3.  HOXB13 Mediates Tamoxifen Resistance and Invasiveness in Human Breast Cancer by Suppressing ERα and Inducing IL-6 Expression 
Cancer research  2013;73(17):5449-5458.
Most breast cancers expressing the estrogen receptor α (ERα) are treated successfully with the receptor antagonist tamoxifen (TAM), but many of these tumors recur. Elevated expression of the homeodomain transcription factor HOXB13 correlates with TAM-resistance in ERα-positive (ER+) breast cancer, but little is known regarding the underlying mechanism. Our comprehensive evaluation of HOX gene expression using tiling microarrays, with validation, showed that distant metastases from TAM-resistant patients also displayed high HOXB13 expression, suggesting a role for HOXB13 in tumor dissemination and survival. Here we show that HOXB13 confers TAM resistance by directly downregulating ERα transcription and protein expression. HOXB13 elevation promoted cell proliferation in vitro and growth of tumor xenografts in vivo. Mechanistic investigations showed that HOXB13 transcriptionally upregulated interleukin (IL)-6, activating the mTOR pathway via STAT3 phosphorylation to promote cell proliferation and fibroblast recruitment. Accordingly, mTOR inhibition suppressed fibroblast recruitment and proliferation of HOXB13-expressing ER+ breast cancer cells and tumor xenografts, alone or in combination with TAM. Taken together, our results establish a function for HOXB13 in TAM resistance through direct suppression of ERα and they identify the IL-6 pathways as mediator of disease progression and recurrence.
doi:10.1158/0008-5472.CAN-13-1178
PMCID: PMC3967590  PMID: 23832664
4.  PROGgene: gene expression based survival analysis web application for multiple cancers 
Background
Identification of prognostic mRNA biomarkers has been done for various cancer types. The data that are published from such studies are archived in public repositories. There are hundreds of such datasets available for multiple cancer types in public repositories. Wealth of such data can be utilized to study prognostic implications of mRNA in different cancers as well as in different populations or subtypes of same cancer.
Description
We have created a web application that can be used for studying prognostic implications of mRNA biomarkers in a variety of cancers. We have compiled data from public repositories such as GEO, EBI Array Express and The Cancer Genome Atlas for creating this tool. With 64 patient series from 18 cancer types in our database, this tool provides the most comprehensive resource available for survival analysis to date. The tool is called PROGgene and it is available at http://www.compbio.iupui.edu/proggene.
Conclusions
We present this tool as a hypothesis generation tool for researchers to identify potential prognostic mRNA biomarkers to follow up with further research. For this reason, we have kept the web application very simple and straightforward. We believe this tool will be useful in accelerating biomarker discovery in cancer and quickly providing results that may indicate disease-specific prognostic value of specific biomarkers.
doi:10.1186/2043-9113-3-22
PMCID: PMC3875898  PMID: 24165311
Biomarker; Multiple cancer; Survival; Pan cancer; Prognostic; mRNA; Database; Kaplan; Meier; KM
5.  Identification of FDA-approved Drugs Targeting Breast Cancer Stem Cells Along With Biomarkers of Sensitivity 
Scientific Reports  2013;3:2530.
Recently developed genomics-based tools are allowing repositioning of Food and Drug Administration (FDA)-approved drugs as cancer treatments, which were employed to identify drugs that target cancer stem cells (CSCs) of breast cancer. Gene expression datasets of CSCs from six studies were subjected to connectivity map to identify drugs that may ameliorate gene expression patterns unique to CSCs. All-trans retinoic acid (ATRA) was negatively connected with gene expression in CSCs. ATRA reduced mammosphere-forming ability of a subset of breast cancer cells, which correlated with induction of apoptosis, reduced expression of SOX2 but elevated expression of its antagonist CDX2. SOX2/CDX2 ratio had prognostic relevance in CSC-enriched breast cancers. K-ras mutant breast cancer cell line enriched for CSCs was resistant to ATRA, which was reversed by MAP kinase inhibitors. Thus, ATRA alone or in combination can be tested for efficacy using SOX2, CDX2, and K-ras mutation/MAPK activation status as biomarkers of response.
doi:10.1038/srep02530
PMCID: PMC3965360  PMID: 23982413
6.  Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing 
BMC Medical Genomics  2013;6:21.
Background
Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown.
Methods
MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively.
Results
We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF.
Conclusion
E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.
doi:10.1186/1755-8794-6-21
PMCID: PMC3687557  PMID: 23758675
7.  PROGmiR: a tool for identifying prognostic miRNA biomarkers in multiple cancers using publicly available data 
Background
Identification of prognostic biomarkers is hallmark of cancer genomics. Since miRNAs regulate expression of multiple genes, they act as potent biomarkers in several cancers. Identification of miRNAs that are prognostically important has been done sporadically, but no resource is available till date that allows users to study prognostics of miRNAs of interest, utilizing the wealth of available data, in major cancer types.
Description
In this paper, we present a web based tool that allows users to study prognostic properties of miRNAs in several cancer types, using publicly available data. We have compiled data from Gene Expression Omnibus (GEO), and recently developed “The Cancer Genome Atlas (TCGA)”, to create this tool. The tool is called “PROGmiR” and it is available at http://www.compbio.iupui.edu/progmir. Currently, our tool can be used to study overall survival implications for approximately 1050 human miRNAs in 16 major cancer types.
Conclusions
We believe this resource, as a hypothesis generation tool, will be helpful for researchers to link miRNA expression with cancer outcome and to design mechanistic studies. We studied performance of our tool using identified miRNA biomarkers from published studies. The prognostic plots created using our tool for specific miRNAs in specific cancer types corroborated with the findings in the studies.
doi:10.1186/2043-9113-2-23
PMCID: PMC3564827  PMID: 23272654
miRNA; Prognostics; Cancer; Pan-cancer; Database; Signature; Biomaker
8.  Virtual Screening Targeting the Urokinase Receptor, Biochemical and Cell-Based Studies, Synthesis, Pharmacokinetic Characterization, and Effect on Breast Tumor Metastasis 
Journal of Medicinal Chemistry  2011;54(20):7193-7205.
Virtual screening targeting the urokinase receptor (uPAR) led to (3R)-4-cyclohexyl-3-(hexahydrobenzo[d][1,3]dioxol-5-yl)-N-((hexahydrobenzo[d][1,3]dioxol-5-yl)methyl)butan-1-aminium 1 (IPR-1) and 4-(4-((3,5-dimethylcyclohexyl)carbamoyl)-2-(4-isopropylcyclohexyl)pyrazolidin-3-yl)piperidin-1-ium 3 (IPR-69). Synthesis of an analog of 1, namely 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC50 near 30 μM. Both compounds blocked angiogenesis with IC50 of 3 μM. Compounds 2 and 3 inhibited cell growth with IC50 of 6 and 18 μM and induced apoptosis. Biochemical assays revealed lead-like properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 hours. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.
doi:10.1021/jm200782y
PMCID: PMC3280887  PMID: 21851064
9.  Delivery of nanoparticles to brain metastases of breast cancer using a cellular Trojan horse 
Cancer Nanotechnology  2012;3(1-6):47-54.
As systemic cancer therapies improve and are able to control metastatic disease outside the central nervous system, the brain is increasingly the first site of relapse. The blood–brain barrier (BBB) represents a major challenge to the delivery of therapeutics to the brain. Macrophages originating from circulating monocytes are able to infiltrate brain metastases while the BBB is intact. Here, we show that this ability can be exploited to deliver both diagnostic and therapeutic nanoparticles specifically to experimental brain metastases of breast cancer.
doi:10.1007/s12645-012-0029-9
PMCID: PMC3505533  PMID: 23205151
Breast cancer; Brain metastasis; Gold-silica nanoshell; Nanoparticle; Blood–brain barrier
10.  A water soluble parthenolide analogue suppresses in vivo tumor growth of two tobacco associated cancers, lung and bladder cancer, by targeting NF-κB and generating reactive oxygen species 
Dimethylaminoparthenolide (DMAPT) is a water soluble parthenolide analogue with preclinical activity in hematologic malignancies. Using NSCLC cell lines (A549, H522) and an immortalized human bronchial epithelial cell line (BEAS2B) and TCC cell lines (UMUC-3, HT-1197, HT-1376) and a bladder papilloma (RT-4), we aimed to characterize DMAPT's anti-cancer activity in tobacco associated neoplasms. Flow cytometric, electrophorectic mobility gel shift assays (EMSA), and western blot studies measured generation of reactive oxygen species (ROS), inhibition of NFκB DNA binding, and changes in cell cycle distribution and apoptotic proteins. DMAPT generated ROS with subsequent JNK activation and also decreased NFκB DNA binding and anti-apoptotic proteins, TRAF-2 and XIAP. DMAPT induced apoptotic cell death and altered cell cycle distribution with upregulation of p21 and p73 levels in a cell type dependent manner. DMAPT suppressed cyclin D1 in BEAS2B. DMAPT retained NFκB and cell cycle inhibitory activity in the presence of the tobacco carcinogen nitrosamine ketone, 4(methylnitrosamino)-1-(3–pyridyl)-1-butanone (NNK). Using a BrdU accumulation assay, 5 to 20μM of DMAPT was shown to inhibit cellular proliferation of all cell lines by more than 95%. Oral dosing of DMAPT suppressed in vivo A549 and UMUC-3 subcutaneous xenograft growth by 54% (p=0.015) and 63% (p<0.01) respectively and A549 lung metastatic volume by 28% (p=0.043). In total this data demonstrates DMAPT's novel anti-cancer properties in both early and late stage tobacco associated neoplasms as well as its significant in vivo activity. The data provides support for the conduct of clinical trials in TCC and NSCLC.
doi:10.1002/ijc.25587
PMCID: PMC2982935  PMID: 20669221
Dimethylaminoparthenolide; cell cycle; apoptosis; cancer
11.  Loss of ERα and FOXA1 expression in a progression model of luminal type breast cancer: Insights from PyMT transgenic mouse model 
Oncology reports  2010;24(5):1233-1239.
The classification of breast cancers into multiple molecular subtypes has necessitated the need for biomarkers that can assess tumor progression and the effects of chemo-preventive agents on specific breast cancer subtypes. The goal of this study was to identify biomarkers whose expression are altered along with estrogen receptor α (ERα) in the polyoma middle-T antigen (PyMT) transgenic model of breast cancer and to investigate the chemopreventive activity of phenethyl isothiocyanate (PEITC). The diet of PyMT female mice was fortified with PEITC (8 mmol/kg) and the mammary streak and/or gross tumors and metastases in lungs were subjected to immunohistochemical analyses for ERα, FOXA1, and GATA-3. FOXA1 is associated with luminal type A cancers, while GATA-3 is a marker of luminal progenitor cell differentiation. In both control and PEITC-treated groups, there was a progressive loss of ERα and FOXA1 but persistence of GATA-3 expression indicating that the tumors retain luminal phenotype. Overall, the PyMT induced tumors exhibited the entire gamut of phenotypes from ERα+/FOXA1+/GATA-3+ tumors in the early stage to ERα±/FOXA1−/GATA-3+ in the late stage. Thus, PyMT model serve as an excellent model for studying progression of luminal subtype tumors. PEITC treated animals had multiple small tumors, indicating delay in tumor progression. Although these tumors were histologically similar to those in controls, there was a lower expression of these biomarkers in normal luminal cells indicating delay in tumor initiation. In in vitro studies, PEITC depleted AldeFluor-positive putative stem/progenitor cells, which may partly be responsible for the delay in tumor initiation.
PMCID: PMC2948410  PMID: 20878115
estrogen receptor; GATA-3; FOXA1; breast cancer
12.  ITF2 is a target of CXCR4 in MDA-MB-231 breast cancer cells and is associated with reduced survival in estrogen receptor-negative breast cancer 
Cancer Biology & Therapy  2010;10(6):600-614.
CXCR4, a chemokine receptor, plays an important role in breast cancer growth, invasion, and metastasis. The transcriptional targets of CXCR4 signaling are not known. Microarray analysis of CXCR4-enriched and CXCR4-low subpopulations of the MDA-MB-231 breast cancer cell line, which has a constitutively active CXCR4 signaling network, revealed differential expression of ∼ 200 genes in the CXCR4-enriched subpopulation. ITF2, upregulated in CXCR4-enriched cells, was investigated further. Expression array datasets of primary breast tumors revealed higher ITF2 expression in estrogen receptor negative tumors, which correlated with reduced progression free and overall survival and suggested its relevance in breast cancer progression. CXCL12, a CXCR4 ligand, increased ITF2 expression in MDA-MB-231 cells. ITF2 is a basic helixloop-helix transcription factor that controls the epithelial-to-mesenchymal transition and the function of the ID family (inhibitor-of-differentiation) of transcription factors, such as ID2. ID2 promotes differentiation of breast epithelial cells and its reduced expression in breast cancer is associated with an unfavorable prognosis. Both CXCR4 and ITF2 repressed ID2 expression. In xenograft studies, CXCR4-enriched cells formed large tumors and exhibited significantly elevated lung metastasis. Short interfering RNA against ITF2 reduced invasion of the CXCR4-enriched MDA-MB-231 subpopulation, whereas ITF2 overexpression restored the invasive capacity of MDA-MB-231 cells expressing CXCR4shRNA. Furthermore, overexpression of ITF2 in these cells enhanced tumor growth. We propose that ITF2 is one of the CXCR4 targets, which is involved in CXCR4-dependent tumor growth and invasion of breast cancer cells.
doi:10.4161/cbt.10.6.12586
PMCID: PMC3040950  PMID: 20603605
CXCR4; breast cancer; ID2; ITF2B; invasion
13.  Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients 
Introduction
Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease.
Methods
Sera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease.
Results
U6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization.
Conclusions
Elevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose.
doi:10.1186/bcr2943
PMCID: PMC3262198  PMID: 21914171
14.  A large, consistent plasma proteomics data set from prospectively collected breast cancer patient and healthy volunteer samples 
Background
Variability of plasma sample collection and of proteomics technology platforms has been detrimental to generation of large proteomic profile datasets from human biospecimens.
Methods
We carried out a clinical trial-like protocol to standardize collection of plasma from 204 healthy and 216 breast cancer patient volunteers. The breast cancer patients provided follow up samples at 3 month intervals. We generated proteomics profiles from these samples with a stable and reproducible platform for differential proteomics that employs a highly consistent nanofabricated ChipCube™ chromatography system for peptide detection and quantification with fast, single dimension mass spectrometry (LC-MS). Protein identification is achieved with subsequent LC-MS/MS analysis employing the same ChipCube™ chromatography system.
Results
With this consistent platform, over 800 LC-MS plasma proteomic profiles from prospectively collected samples of 420 individuals were obtained. Using a web-based data analysis pipeline for LC-MS profiling data, analyses of all peptide peaks from these plasma LC-MS profiles reveals an average coefficient of variability of less than 15%. Protein identification of peptide peaks of interest has been achieved with subsequent LC-MS/MS analyses and by referring to a spectral library created from about 150 discrete LC-MS/MS runs. Verification of peptide quantity and identity is demonstrated with several Multiple Reaction Monitoring analyses. These plasma proteomic profiles are publicly available through ProteomeCommons.
Conclusion
From a large prospective cohort of healthy and breast cancer patient volunteers and using a nano-fabricated chromatography system, a consistent LC-MS proteomics dataset has been generated that includes more than 800 discrete human plasma profiles. This large proteomics dataset provides an important resource in support of breast cancer biomarker discovery and validation efforts.
doi:10.1186/1479-5876-9-80
PMCID: PMC3120690  PMID: 21619653
15.  Prognosis of hormone-dependent breast cancers: implications of the presence of dysfunctional transcriptional networks activated by insulin via the immune transcription factor T-bet 
Cancer research  2010;70(2):685-696.
Estrogen receptor alpha (ERα)-positive breast cancers that co-express trans cription factors GATA-3 and FOXA1 have a favorable prognosis. These transcription factors form an autoregulatory hormonal network that influences estrogen responsiveness and sensitivity to hormonal therapy. Disruption of this network may be a mechanism whereby ERα positive breast cancers become resistant to therapy. The transcription factor T-bet is a negative regulator of GATA-3 in the immune system. In this study, we report that insulin increases the expression of T-bet in breast cancer cells, which correlates with reduced expression of GATA-3, FOXA1 and the ERα:FOXA1:GATA-3 target gene GREB-1. The effects of insulin on GATA-3 and FOXA1 could be recapitulated through overexpression of T-bet in MCF-7 cells (MCF-7-T-bet). Chromatin immunoprecipitation assays revealed reduced ERα binding to GREB-1 enhancer regions in MCF-7-T-bet cells and in insulin treated MCF-7 cells. MCF-7-T-bet cells were resistant to tamoxifen in the presence of insulin and displayed prolonged ERK and AKT activation in response to epidermal growth factor treatment. ERα-positive cells with intrinsic tamoxifen resistance as well as MCF-7 cells with acquired tamoxifen and fulvestrant resistance expressed elevated levels of T-bet and/or reduced levels of FOXA1 and GATA-3. Analysis of publicly available databases revealed ERα-positive/T-bet-positive breast cancers expressing lower levels of FOXA1 (p=0.0137) and GATA-3 (p=0.0063) compared to ERα-positive/T-bet-negative breast cancers. Thus, T-bet expression in primary tumors and circulating insulin levels may serve as surrogate biomarkers to identify ERα-positive breast cancers with a dysfunctional hormonal network, enhanced growth factor signaling, and resistance to hormonal therapy.
doi:10.1158/0008-5472.CAN-09-1530
PMCID: PMC2807987  PMID: 20068169
estrogen receptor; GATA-3; FOXA1; T-bet; breast cancer
16.  Radiation resistance in breast cancer: are CD44+/CD24-/proteosomelow/PKH26+ cells to blame? 
Identification and characterization of cancer-initiating cells (CICs) enriched for stem cell-like functions and the establishment of a link between CICs and tumor recurrence, chemotherapy resistance and radiation resistance, and metastasis have been the focus of cancer research for the last eight years. Although this field has its share of controversies, it is becoming apparent that cells isolated from recurrent or residual tumors or both are enriched for cancer cells that have a specific phenotype compared with heterogeneous cells in the primary tumor. Enrichment of CICs in tumors subjected to radiation therapy could be due in part to the delivery of sublethal doses of treatment and the efficient radical scavenging system within CICs. Sublethal doses of radiation are sufficient to induce senescence of non-CICs while forcing CICs to gain several new properties related to cell cycle progression in addition to maintaining or enhancing stem cell characteristics of pre-treatment CICs. Characterizing pathways responsible for the increase in CICs after therapy and exploiting the unique characteristics of therapy-resistant CICs for developing targeted therapies are becoming a central focus of research in the rapidly evolving field of CICs.
doi:10.1186/bcr2559
PMCID: PMC2879566  PMID: 20377923
17.  SLUG/SNAI2 and Tumor Necrosis Factor Generate Breast Cells With CD44+/CD24- Phenotype 
BMC Cancer  2010;10:411.
Background
Breast cancer cells with CD44+/CD24- cell surface marker expression profile are proposed as cancer stem cells (CSCs). Normal breast epithelial cells that are CD44+/CD24- express higher levels of stem/progenitor cell associated genes. We, amongst others, have shown that cancer cells that have undergone epithelial to mesenchymal transition (EMT) display the CD44+/CD24- phenotype. However, whether all genes that induce EMT confer the CD44+/CD24- phenotype is unknown. We hypothesized that only a subset of genes associated with EMT generates CD44+/CD24- cells.
Methods
MCF-10A breast epithelial cells, a subpopulation of which spontaneously acquire the CD44+/CD24- phenotype, were used to identify genes that are differentially expressed in CD44+/CD24- and CD44-/CD24+ cells. Ingenuity pathway analysis was performed to identify signaling networks that linked differentially expressed genes. Two EMT-associated genes elevated in CD44+/CD24- cells, SLUG and Gli-2, were overexpressed in the CD44-/CD24+ subpopulation of MCF-10A cells and MCF-7 cells, which are CD44-/CD24+. Flow cytometry and mammosphere assays were used to assess cell surface markers and stem cell-like properties, respectively.
Results
Two thousand thirty five genes were differentially expressed (p < 0.001, fold change ≥ 2) between the CD44+/CD24- and CD44-/CD24+ subpopulations of MCF-10A. Thirty-two EMT-associated genes including SLUG, Gli-2, ZEB-1, and ZEB-2 were expressed at higher levels in CD44+/CD24- cells. These EMT-associated genes participate in signaling networks comprising TGFβ, NF-κB, and human chorionic gonadotropin. Treatment with tumor necrosis factor (TNF), which induces NF-κB and represses E-cadherin, or overexpression of SLUG in CD44-/CD24+ MCF-10A cells, gave rise to a subpopulation of CD44+/CD24- cells. Overexpression of constitutively active p65 subunit of NF-κB in MCF-10A resulted in a dramatic shift to the CD44+/CD24+ phenotype. SLUG overexpression in MCF-7 cells generated CD44+/CD24+ cells with enhanced mammosphere forming ability. In contrast, Gli-2 failed to alter CD44 and CD24 expression.
Conclusions
EMT-mediated generation of CD44+/CD24- or CD44+/CD24+ cells depends on the genes that induce or are associated with EMT. Our studies reveal a role for TNF in altering the phenotype of breast CSC. Additionally, the CD44+/CD24+ phenotype, in the context of SLUG overexpression, can be associated with breast CSC "stemness" behavior based on mammosphere forming ability.
doi:10.1186/1471-2407-10-411
PMCID: PMC3087321  PMID: 20691079
18.  Tissue transglutaminase protects epithelial ovarian cancer cells from cisplatin-induced apoptosis by promoting cell survival signaling 
Carcinogenesis  2008;29(10):1893-1900.
Tissue transglutaminase (TG2), an enzyme involved in protein cross-linking and overexpressed in ovarian tumors, has antiapoptotic effects in cancer cells and may play a role in response to chemotherapy. In this study, we investigated the role of TG2 in the sensitivity of ovarian cancer cells to cisplatin. By using stable knockdown and overexpression strategies, we demonstrate that the level of expression of TG2 regulates apoptosis induced by cisplatin in SKOV3 and OV-90 ovarian cancer cells. Interestingly, not only TG2 knockdown but also a TG2 enzymatic inhibitor (KCC009) sensitized SKOV3 cells to cisplatin. To understand the mechanism by which TG2 exerts its antiapoptotic role, we examined the effects of protein kinase B (Akt) and nuclear factor-kappa B (NF-κB), two survival pathways commonly involved in development of drug resistance. Overexpression of the constitutively active p65 subunit of NF-κB, but not constitutively active Akt, rescued cells with diminished TG2 expression from cisplatin-induced apoptosis. This implicates activation of NF-κB as the main cisplatin resistance mechanism downstream of TG2. Indeed, NF-κB activity is decreased and the level of the inhibitory subunit IκBα is increased in ovarian cancer cells engineered to express diminished levels of TG2 or treated with the enzymatic inhibitor, KCC009. Our data show that TG2 prevents apoptosis induced by cisplatin by activating the NF-κB survival pathway in ovarian cancer cells.
doi:10.1093/carcin/bgn158
PMCID: PMC2556973  PMID: 18667446
19.  Effects of HIV Protease Inhibitor Ritonavir on Akt-Regulated Cell Proliferation in Breast Cancer 
Purpose
These studies were designed to determine whether ritonavir inhibits breast cancer in vitro and in vitro and, if so, how.
Experimental Design
Ritonavir effects on breast cancer cell growth were studied in the estrogen receptor (ER)-positive lines MCF7 and T47D and in the ER-negative lines MDA-MB-436 and MDA-MB-231. Effects of ritonavir on Rb-regulated and Akt-mediated cell proliferation were studied. Ritonavir was tested for inhibition of a mammary carcinoma xenograft.
Results
ER-positive estradiol-dependent lines (IC50, 12–24 µmol/L) and ER-negative (IC50, 45 µmol/L) lines exhibit ritonavir sensitivity. Ritonavir depletes ER-α levels notably in ER-positive lines. Ritonavir causes G1 arrest, depletes cyclin-dependent kinases 2, 4, and 6 and cyclin D1 but not cyclin E, and depletes phosphorylated Rb and Ser473 Akt. Ritonavir induces apoptosis independent of G1 arrest, inhibiting growth of cells that have passed the G1 checkpoint. Myristoyl-Akt, but not activated K-Ras, rescues ritonavir inhibition. Ritonavir inhibited a MDA-MB-231 xenograft and intratumoral Akt activity at a clinically attainable serum Cmax of 22 ± 8 µmol/L. Because heat shock protein 90 (Hsp90) substrates are depleted by ritonavir, ritonavir effects on Hsp90 were tested. Ritonavir binds Hsp90 (KD, 7.8 µmol/L) and partially inhibits its chaperone function. Ritonavir blocks association of Hsp90 with Akt and, with sustained exposure, notably depletes Hsp90. Stably expressed Hsp90α short hairpin RNA also depletes Hsp90, inhibiting proliferation and sensitizing breast cancer cells to low ritonavir concentrations.
Conclusions
Ritonavir inhibits breast cancer growth in part by inhibiting Hsp90 substrates, including Akt. Ritonavir may be of interest for breast cancer therapeutics and its efficacy may be increased by sustained exposure or Hsp90 RNA interference.
doi:10.1158/1078-0432.CCR-05-1167
PMCID: PMC2727652  PMID: 16551874
20.  Estradiol-regulated microRNAs control estradiol response in breast cancer cells 
Nucleic Acids Research  2009;37(14):4850-4861.
Estradiol (E2) regulates gene expression at the transcriptional level by functioning as a ligand for estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). E2-inducible proteins c-Myc and E2Fs are required for optimal ERα activity and secondary estrogen responses, respectively. We show that E2 induces 21 microRNAs and represses seven microRNAs in MCF-7 breast cancer cells; these microRNAs have the potential to control 420 E2-regulated and 757 non-E2-regulated mRNAs at the post-transcriptional level. The serine/threonine kinase, AKT, alters E2-regulated expression of microRNAs. E2 induced the expression of eight Let-7 family members, miR-98 and miR-21 microRNAs; these microRNAs reduced the levels of c-Myc and E2F2 proteins. Dicer, a ribonuclease III enzyme required for microRNA processing, is also an E2-inducible gene. Several E2-regulated microRNA genes are associated with ERα-binding sites or located in the intragenic region of estrogen-regulated genes. We propose that the clinical course of ERα-positive breast cancers is dependent on the balance between E2-regulated tumor-suppressor microRNAs and oncogenic microRNAs. Additionally, our studies reveal a negative-regulatory loop controlling E2 response through microRNAs as well as differences in E2-induced transcriptome and proteome.
doi:10.1093/nar/gkp500
PMCID: PMC2724297  PMID: 19528081
21.  AKT Alters Genome-Wide Estrogen Receptor α Binding and Impacts Estrogen Signaling in Breast Cancer▿ † 
Molecular and Cellular Biology  2008;28(24):7487-7503.
Estrogen regulates several biological processes through estrogen receptor α (ERα) and ERβ. ERα-estrogen signaling is additionally controlled by extracellular signal activated kinases such as AKT. In this study, we analyzed the effect of AKT on genome-wide ERα binding in MCF-7 breast cancer cells. Parental and AKT-overexpressing cells displayed 4,349 and 4,359 ERα binding sites, respectively, with ∼60% overlap. In both cell types, ∼40% of estrogen-regulated genes associate with ERα binding sites; a similar percentage of estrogen-regulated genes are differentially expressed in two cell types. Based on pathway analysis, these differentially estrogen-regulated genes are linked to transforming growth factor β (TGF-β), NF-κB, and E2F pathways. Consistent with this, the two cell types responded differently to TGF-β treatment: parental cells, but not AKT-overexpressing cells, required estrogen to overcome growth inhibition. Combining the ERα DNA-binding pattern with gene expression data from primary tumors revealed specific effects of AKT on ERα binding and estrogen-regulated expression of genes that define prognostic subgroups and tamoxifen sensitivity of ERα-positive breast cancer. These results suggest a unique role of AKT in modulating estrogen signaling in ERα-positive breast cancers and highlights how extracellular signal activated kinases can change the landscape of transcription factor binding to the genome.
doi:10.1128/MCB.00799-08
PMCID: PMC2593438  PMID: 18838536
22.  CD44+/CD24- breast cancer cells exhibit enhanced invasive properties: an early step necessary for metastasis 
Breast Cancer Research  2006;8(5):R59.
Introduction
A subpopulation (CD44+/CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells has the unique ability to invade, home, and proliferate at sites of metastasis.
Methods
CD44 and CD24 expression was determined by flow cytometry. Northern blotting was used to determine the expression of proinvasive and 'bone and lung metastasis signature' genes. A matrigel invasion assay and intracardiac inoculation into nude mice were used to evaluate invasion, and homing and proliferation at sites of metastasis, respectively.
Results
Five among 13 breast cancer cell lines examined (MDA-MB-231, MDA-MB-436, Hs578T, SUM1315, and HBL-100) contained a higher percentage (>30%) of CD44+/CD24- cells. Cell lines with high CD44+/CD24- cell numbers express basal/mesenchymal or myoepithelial but not luminal markers. Expression levels of proinvasive genes (IL-1α, IL-6, IL-8, and urokinase plasminogen activator [UPA]) were higher in cell lines with a significant CD44+/CD24- population than in other cell lines. Among the CD44+/CD24--positive cell lines, MDA-MB-231 has the unique property of expressing a broad range of genes that favor bone and lung metastasis. Consistent with previous studies in nude mice, cell lines with CD44+/CD24- subpopulation were more invasive than other cell lines. However, only a subset of CD44+/CD24--positive cell lines was able to home and proliferate in lungs.
Conclusion
Breast cancer cells with CD44+/CD24- subpopulation express higher levels of proinvasive genes and have highly invasive properties. However, this phenotype is not sufficient to predict capacity for pulmonary metastasis.
doi:10.1186/bcr1610
PMCID: PMC1779499  PMID: 17062128

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