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1.  The Fibroblast Growth Factor–Inducible 14 Receptor Is Highly Expressed in HER2-Positive Breast Tumors and Regulates Breast Cancer Cell Invasive Capacity 
Molecular cancer research : MCR  2008;6(5):725-734.
Genomic characterization is beginning to define a molecular taxonomy for breast cancer; however, the molecular basis of invasion and metastasis remains poorly understood. We report a pivotal role for the fibroblast growth factor–inducible 14 (Fn14) receptor in this process. We examined whether Fn14 and its ligand tumor necrosis factor–like weak inducer of apoptosis (TWEAK) were expressed in breast tumors and whether deregulation of Fn14 levels affected malignant behavior of breast cancer cell lines. Analysis of TWEAK and Fn14 in publicly available gene expression data indicated that high Fn14 expression levels significantly correlated with several poor prognostic indicators (P < 0.05). Fn14 expression was highest in the HER2-positive/estrogen receptor–negative (HER2+/ER−) intrinsic subtype (P = 0.0008). An association between Fn14 and HER2 expression in breast tumors was confirmed by immunohistochemistry. Fn14 levels were elevated in invasive, ER− breast cancer cell lines. Overexpression of Fn14 in weakly invasive MCF7 and T47D cells resulted in a marked induction of invasion and activation of nuclear factor-κB (NF-κB) signaling. Ectopic expression of Fn14tCT, a Fn14 deletion mutant that cannot activate NF-κB signaling, was not able to induce invasion. Moreover, ectopic expression of Fn14tCT in highly invasive MDA-MB-231 cells reduced their invasive capability. RNA interference–mediated inhibition of Fn14 expression in both MDA-MB-231 and MDA-MB-436 cells reduced invasion. Expression profiling of the Fn14-depleted cells revealed deregulation of NF-κB activity. Our findings support a role for Fn14-mediated NF-κB pathway activation in breast tumor invasion and metastasis.
PMCID: PMC3519279  PMID: 18505918
2.  PAR6B is required for tight junction formation and activated PKCζ localization in breast cancer 
Dysregulation of mechanisms that govern the control of epithelial cell polarity, morphology and plasticity are emerging as key processes in tumor progression. In this study we report amplification and overexpression of PAR6B, an essential component in epithelial cell tight junction (TJ) formation and maintenance of apico-basal polarity, in breast cancer cell lines. Analysis of chromosome 20q13.13 in 11 breast cancer cell lines by fluorescence in situ hybridization (FISH) identified a novel small amplicon centered at PARD6B in 5 cell lines, with copy number ranging from 7 to 27. The presence of the PARD6B amplicon correlated with PARD6B transcript and PAR6B protein abundance. Expression of related isoforms PARD6A and PARD6G were detectable at significantly lower levels. PARD6B overexpression correlated with TJ network formation in cultured cell monolayers. SiRNA-mediated inhibition of PAR6B in MCF7 resulted in loss of TJ assembly and membrane localization of atypical PKCζ (aPKC), but did not affect adherens junction formation. SiRNA-mediated inhibition of CDC42 in MCF7 also resulted in loss of TJ networks, confirming the requirement of a complete PAR6-aPKC-CDC42-PAR3 complex to activate and stabilize TJs. Immunohistochemical analysis of PAR6B expression on breast tumor microarrays indicated exquisite epithelial cell-specificity. Few quantitative differences in staining were observed between normal epithelium and adjacent tumor margins. However staining appeared reduced and cytoplasmic in more poorly differentiated tumors. We propose that quantitative imbalances in the components of pathways governing normal epithelial cell polarity arising from gain or loss of function may radically alter epithelial cell architecture and contribute to tumor progression.
PMCID: PMC3433109  PMID: 22957302
Breast Cancer; DNA amplification; tight junction; siRNA; polarity; adhesion; PARD6B; PAR6B; CDC42; PKCζ
3.  Small cell carcinoma of the ovary, hypercalcemic type, displays frequent inactivating germline and somatic mutations in SMARCA4 
Nature genetics  2014;46(5):427-429.
Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is an extremely rare, aggressive cancer affecting children and young women. We identified germline and somatic inactivating mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 69% (9/13) of SCCOHT cases in addition to SMARCA4 protein loss in 82% (14/17) of SCCOHT tumors but in only 0.4% (2/485) of other primary ovarian tumors. These data implicate SMARCA4 in SCCOHT oncogenesis.
PMCID: PMC4332808  PMID: 24658001
4.  ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer 
BMC Cancer  2014;14(1):749.
The role and clinical value of ERβ1 expression is controversial and recent data demonstrates that many ERβ antibodies are insensitive and/or non-specific. Therefore, we sought to comprehensively characterize ERβ1 expression across all sub-types of breast cancer using a validated antibody and determine the roles of this receptor in mediating response to multiple forms of endocrine therapy both in the presence and absence of ERα expression.
Nuclear and cytoplasmic expression patterns of ERβ1 were analyzed in three patient cohorts, including a retrospective analysis of a prospective adjuvant tamoxifen study and a triple negative breast cancer cohort. To investigate the utility of therapeutically targeting ERβ1, we generated multiple ERβ1 expressing cell model systems and determined their proliferative responses following anti-estrogenic or ERβ-specific agonist exposure.
Nuclear ERβ1 was shown to be expressed across all major sub-types of breast cancer, including 25% of triple negative breast cancers and 33% of ER-positive tumors, and was associated with significantly improved outcomes in ERα-positive tamoxifen-treated patients. In agreement with these observations, ERβ1 expression sensitized ERα-positive breast cancer cells to the anti-cancer effects of selective estrogen receptor modulators (SERMs). However, in the absence of ERα expression, ERβ-specific agonists potently inhibited cell proliferation rates while anti-estrogenic therapies were ineffective.
Using a validated antibody, we have confirmed that nuclear ERβ1 expression is commonly present in breast cancer and is prognostic in tamoxifen-treated patients. Using multiple breast cancer cell lines, ERβ appears to be a novel therapeutic target. However, the efficacy of SERMs and ERβ-specific agonists differ as a function of ERα expression.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-749) contains supplementary material, which is available to authorized users.
PMCID: PMC4196114  PMID: 25288324
Estrogen receptor beta; Breast cancer; Estrogen receptor alpha; Triple negative breast cancer; Therapy
5.  Expression of quiescin sulfhydryl oxidase 1 is associated with a highly invasive phenotype and correlates with a poor prognosis in Luminal B breast cancer 
Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. Tumor specific expression of QSOX1 has been reported for numerous tumor types. In this study, we investigate QSOX1 as a marker of breast tumor progression and evaluate the role of QSOX1 as it relates to breast tumor growth and metastasis.
Correlation of QSOX1 expression with breast tumor grade, subtype and estrogen receptor (ER) status was gathered through informatic analysis using the "Gene expression based Outcome for Breast cancer Online" (GOBO) web-based tool. Expression of QSOX1 protein in breast tumors tissue microarray (TMA) and in a panel of breast cancer cell lines was used to confirm our informatics analysis. To investigate malignant cell mechanisms for which QSOX1 might play a key role, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in ER+ Luminal A-like MCF7, ER+ Luminal B-like BT474 and ER- Basal-like BT549 breast cancer cell lines.
GOBO analysis revealed high levels of QSOX1 RNA expression in ER+ subtypes of breast cancer. In addition, Kaplan Meyer analyses revealed QSOX1 RNA as a highly significant predictive marker for both relapse and poor overall survival in Luminal B tumors. We confirmed this finding by evaluation of QSOX1 protein expression in breast tumors and in a panel of breast cancer cell lines. Expression of QSOX1 in breast tumors correlates with increasing tumor grade and high Ki-67 expression. Suppression of QSOX1 protein slowed cell proliferation as well as dramatic inhibition of MCF7, BT474 and BT549 breast tumor cells from invading through Matrigel™ in a modified Boyden chamber assay. Inhibition of invasion could be rescued by the exogenous addition of recombinant QSOX1. Gelatin zymography indicated that QSOX1 plays an important role in the function of MMP-9, a key mediator of breast cancer invasive behavior.
Taken together, our results suggest that QSOX1 is a novel biomarker for risk of relapse and poor survival in Luminal B breast cancer, and has a pro-proliferative and pro-invasive role in malignant progression partly mediated through a decrease in MMP-9 functional activity.
PMCID: PMC3738157  PMID: 23536962
6.  In Vitro Assessment of the Inflammatory Breast Cancer Cell Line SUM 149: Discovery of 2 Single Nucleotide Polymorphisms in the RNase L Gene 
Journal of Cancer  2013;4(2):104-116.
Background: Inflammatory breast cancer (IBC) is a rare, highly aggressive form of breast cancer. The mechanism of IBC carcinogenesis remains unknown. We sought to evaluate potential genetic risk factors for IBC and whether or not the IBC cell lines SUM149 and SUM190 demonstrated evidence of viral infection.
Methods: We performed single nucleotide polymorphism (SNP) genotyping for 2 variants of the ribonuclease (RNase) L gene that have been correlated with the risk of prostate cancer due to a possible viral etiology. We evaluated dose-response to treatment with interferon-alpha (IFN-α); and assayed for evidence of the putative human mammary tumor virus (HMTV, which has been implicated in IBC) in SUM149 cells. A bioinformatic analysis was performed to evaluate expression of RNase L in IBC and non-IBC.
Results: 2 of 2 IBC cell lines were homozygous for RNase L common missense variants 462 and 541; whereas 2 of 10 non-IBC cell lines were homozygous positive for the 462 variant (p= 0.09) and 0 of 10 non-IBC cell lines were homozygous positive for the 541 variant (p = 0.015). Our real-time polymerase chain reaction (RT-PCR) and Southern blot analysis for sequences of HMTV revealed no evidence of the putative viral genome.
Conclusion: We discovered 2 SNPs in the RNase L gene that were homozygously present in IBC cell lines. The 462 variant was absent in non-IBC lines. Our discovery of these SNPs present in IBC cell lines suggests a possible biomarker for risk of IBC. We found no evidence of HMTV in SUM149 cells. A query of a panel of human IBC and non-IBC samples showed no difference in RNase L expression. Further studies of the RNase L 462 and 541 variants in IBC tissues are warranted to validate our in vitro findings.
PMCID: PMC3563072  PMID: 23386909
inflammatory breast cancer; SUM149; HMTV; interferon-alpha; MMTV; RNase L.
7.  Deep Clonal Profiling of Formalin Fixed Paraffin Embedded Clinical Samples 
PLoS ONE  2012;7(11):e50586.
Formalin fixed paraffin embedded (FFPE) tissues are a vast resource of annotated clinical samples. As such, they represent highly desirable and informative materials for the application of high definition genomics for improved patient management and to advance the development of personalized therapeutics. However, a limitation of FFPE tissues is the variable quality of DNA extracted for analyses. Furthermore, admixtures of non-tumor and polyclonal neoplastic cell populations limit the number of biopsies that can be studied and make it difficult to define cancer genomes in patient samples. To exploit these valuable tissues we applied flow cytometry-based methods to isolate pure populations of tumor cell nuclei from FFPE tissues and developed a methodology compatible with oligonucleotide array CGH and whole exome sequencing analyses. These were used to profile a variety of tumors (breast, brain, bladder, ovarian and pancreas) including the genomes and exomes of matching fresh frozen and FFPE pancreatic adenocarcinoma samples.
PMCID: PMC3511535  PMID: 23226320
8.  Context-specific gene regulatory networks subdivide intrinsic subtypes of breast cancer 
BMC Bioinformatics  2011;12(Suppl 2):S3.
Breast cancer is a highly heterogeneous disease with respect to molecular alterations and cellular composition making therapeutic and clinical outcome unpredictable. This diversity creates a significant challenge in developing tumor classifications that are clinically reliable with respect to prognosis prediction.
This paper describes an unsupervised context analysis to infer context-specific gene regulatory networks from 1,614 samples obtained from publicly available gene expression data, an extension of a previously published methodology. We use the context-specific gene regulatory networks to classify the tumors into clinically relevant subgroups, and provide candidates for a finer sub-grouping of the previously known intrinsic tumors with a focus on Basal-like tumors. Our analysis of pathway enrichment in the key contexts provides an insight into the biological mechanism underlying the identified subtypes of breast cancer.
The use of context-specific gene regulatory networks to identify biological contexts from heterogenous breast cancer data set was able to identify genomic drivers for subgroups within the previously reported intrinsic subtypes. These subgroups (contexts) uphold the clinical relevant features for the intrinsic subtypes and were associated with increased survival differences compared to the intrinsic subtypes. We believe our computational approach led to the generation of novel rationalized hypotheses to explain mechanisms of disease progression within sub-contexts of breast cancer that could be therapeutically exploited once validated.
PMCID: PMC3073183  PMID: 21489222
9.  The G Protein-Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor-Positive Breast Cancer Cells 
Cancer research  2010;70(3):1184-1194.
The G protein-coupled receptor GPR30 (GPER) binds 17β-estradiol (E2), yet differs from classical estrogen receptors (ERα and ERβ). GPR30 can mediate E2-induced non-genomic signaling, but its role in ERα-positive breast cancer remains unclear. Gene expression microarray data from 5 cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol, ligands which selectively activate GPR30 and ER, respectively, and small interfering RNAs (siRNAs). In expression studies, E2 and diethylstilbestrol but not G-1 down-regulated both ER and GPR30, indicating this was ER mediated. In Ca2+ mobilization studies, GPR30 but not ERα mediated E2-induced Ca2+ responses, since E2, 4-hydroxytamoxifen (also activates GPR30) and G-1, but not DES, elicited cytosolic Ca2+ increases not only in MCF-7 cells, but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E2- and G-1-induced Ca2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca2+ responses were sustained and inositol triphosphate receptor-mediated in ER-positive MCF-7 cells, but transitory and ryanodine receptor-mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion indicated that GPR30's role was to promote SKBr3 cell growth, but reduce MCF-7 cell growth. Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further, flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G(1)-phase. Thus, GPR30 antagonizes growth of ERα-positive breast cancer, and may represent a new target to combat this disease.
PMCID: PMC2879282  PMID: 20086172
GPR30; estrogen receptor α; G-1; calcium mobilization; MCF-7 cells; SKBr3 cells
10.  Overexpression of CEACAM6 promotes migration and invasion of oestrogen deprived breast cancer cells 
Carcinocinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an intercellular adhesion molecule that is overexpressed in a wide variety of human cancers, including colon, breast, and lung and is associated with tumourigenesis, tumour cell adhesion, invasion, and metastasis. In the present study, we showed that CEACAM6 was overexpressed in a panel of oestrogen receptor (ERα)-positive human breast cancer cell lines (MCF-7:5C and MCF-7:2A) that have acquired resistance to oestrogen deprivation and this overexpression was associated with a more aggressive invasive phenotype in vitro. Expression array analysis revealed that MCF-7:5C and MCF-7:2A cells overexpressed CEACAM6 mRNA by 27-fold and 12-fold, respectively, and were 6 to 15-times more invasive compared to non-invasive wild-type MCF-7 cells which expressed low levels of CEACAM6. Suppression of CEACAM6 expression using small interfering RNA (siRNA) completely reversed migration and invasion of MCF-7:5C and MCF-7:2A cells and it significantly reduced E-cadherin, Akt, and expression in these cells. In conclusion, our findings establish CEACAM6 as a unique mediator of migration and invasion of drug resistant oestrogen deprived breast cancer cells and suggest that this protein could be an important biomarker of metastasis.
PMCID: PMC2778047  PMID: 18614350
Breast cancer; CEACAM6; Invasion and migration; Oestrogen deprivation; Endocrine-resistance
12.  Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa 
Infection and Immunity  2001;69(9):5385-5394.
The expression of many virulence factors in Pseudomonas aeruginosa is dependent upon environmental conditions, including iron levels, oxygen, temperature, and osmolarity. The virulence of P. aeruginosa PAO1 is influenced by the iron- and oxygen-regulated gene encoding the alternative sigma factor PvdS, which is regulated through the ferric uptake regulator (Fur). We observed that overexpression of PvdS in strain PAO1 and a ΔpvdS::Gm mutant resulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the ΔpvdS::Gm mutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from ΔpvdS::Gm was investigated. Amino acid sequence analysis and examination of the genomic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV of P. aeruginosa strain PA103-29 and is homologous to an endoprotease produced by Lysobacter enzymogenes. In this study, the gene encoding an endoprotease was cloned from PAO1 and designated prpL (PvdS-regulated endoprotease, lysyl class). All (n = 41) but one of the strains of P. aeruginosa, including clinical and environmental isolates, examined carry prpL. Moreover, PrpL production among these strains was highly variable. Analysis of RNase protection assays identified the transcription initiation site of prpL and confirmed that its transcription is iron dependent. In the ΔpvdS::Gm mutant, the level of prpL transcription was iron independent and decreased relative to the level in PAO1. Furthermore, transcription of prpL was independent of PtxR, a PvdS-regulated protein. Finally, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin and contributes to PAO1's ability to persist in a rat chronic pulmonary infection model.
PMCID: PMC98648  PMID: 11500408

Results 1-12 (12)