Plasma Membrane Proteomics of Human Breast Cancer Cell Lines Identifies Potential Targets for Breast Cancer Diagnosis and Treatment

The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.     

Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

Background

Prostate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.

Methodology/Principal Findings

In this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.

Conclusion/Significance

Our data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.     

Expression of Polycomb Targets Predicts Breast Cancer Prognosis

Global changes in the epigenome are increasingly being appreciated as key events in cancer progression. The pathogenic role of enhancer of zeste homolog 2 (EZH2) has been connected to its histone 3 lysine 27 (H3K27) methyltransferase activity and gene repression; however, little is known about relationship of changes in expression of EZH2 target genes to cancer characteristics and patient prognosis. Here we show that through expression analysis of genomic regions with H3K27 trimethylation (H3K27me3) and EZH2 binding, breast cancer patients can be stratified into good and poor prognostic groups independent of known cancer gene signatures. The EZH2-bound regions were downregulated in tumors characterized by aggressive behavior, high expression of cell cycle genes, and low expression of developmental and cell adhesion genes. Depletion of EZH2 in breast cancer cells significantly increased expression of the top altered genes, decreased proliferation, and improved cell adhesion, indicating a critical role played by EZH2 in determining the cancer phenotype.     

JISTIC: Identification of Significant Targets in Cancer

Background  

Cancer is caused through a multistep process, in which a succession of genetic changes, each conferring a competitive advantage for growth and proliferation, leads to the progressive conversion of normal human cells into malignant cancer cells. Interrogation of cancer genomes holds the promise of understanding this process, thus revolutionizing cancer research and treatment. As datasets measuring copy number aberrations in tumors accumulate, a major challenge has become to distinguish between those mutations that drive the cancer versus those passenger mutations that have no effect.     

The Antihelmintic Drug Pyrvinium Pamoate Targets Aggressive Breast Cancer

WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.     

Metabolic-Pathway-Based Subtyping of Triple-Negative Breast Cancer Reveals Potential Therapeutic Targets

1. Download : Download high-res image (199KB)
2. Download : Download full-size image

     

Systematic Identification of Combinatorial Drivers and Targets in Cancer Cell Lines

There is an urgent need to elicit and validate highly efficacious targets for combinatorial intervention from large scale ongoing molecular characterization efforts of tumors. We established an in silico bioinformatic platform in concert with a high throughput screening platform evaluating 37 novel targeted agents in 669 extensively characterized cancer cell lines reflecting the genomic and tissue-type diversity of human cancers, to systematically identify combinatorial biomarkers of response and co-actionable targets in cancer. Genomic biomarkers discovered in a 141 cell line training set were validated in an independent 359 cell line test set. We identified co-occurring and mutually exclusive genomic events that represent potential drivers and combinatorial targets in cancer. We demonstrate multiple cooperating genomic events that predict sensitivity to drug intervention independent of tumor lineage. The coupling of scalable in silico and biologic high throughput cancer cell line platforms for the identification of co-events in cancer delivers rational combinatorial targets for synthetic lethal approaches with a high potential to pre-empt the emergence of resistance.     

基于合成致死策略寻找ARID1A突变肝细胞癌的治疗靶点

ARID1A编码的BAF250a蛋白是SWI/SNF(SWItch/Sucrose Non-Fermentable)染色质重组复合物BAF(BRG1-associated factors)的亚基之一,参与改变染色体的结构和可接近性。ARID1A在肝细胞癌(hepatocellular carcinoma,HCC)中的突变率高达13%,但目前尚无有效的治疗药物。本研究旨在利用合成致死策略寻找携带ARID1A突变HCC的治疗新靶标。首先,本研究通过分析ARID1A突变与肿瘤恶性程度的相关性发现ARID1A突变的肿瘤恶性度增加;进而分析Achilles和NCI-60癌症细胞系中ARID1A突变和野生型细胞系的基因表型值(gene phenotype value,GPV)和高表达基因,获得ARID1A突变细胞低GPV和高表达的重叠基因,再扩大样本使用CCLE(Cancer Cell Line Encyclopedia)细胞系的高表达基因进行重叠基因分析;最后并在TCGA(the Cancer Genome Atlas)肝癌数据库中进行筛选,获得116个潜在的ARID1A合成致死基因。本研究运用生物信息学方法计算获得多个ARID1A的潜在合成性致死基因,为ARID1A突变HCC患者提供新的治疗靶点,也为靶向药物研发提供了新靶标和新策略。     

Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (P_trend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2^nd, 3^rd, 4^th, and 5^th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1^st quintile (P_trend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (P_trend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk.     

Identification of Natural CRISPR Systems and Targets in the Human Microbiome

1. Download : Download high-res image (223KB)
2. Download : Download full-size image

     

Comprehensive Analysis of MicroRNA (miRNA) Targets in Breast Cancer Cells

MicroRNAs (miRNAs) regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of ∼70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3′-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the 50 most abundantly expressed miRNAs. Together, these results suggested that the majority of the AGO2-associated mRNAs were bona fide miRNA targets. Functional enrichment analysis uncovered that the AGO2-IP mRNAs were involved in regulation of cell cycle, apoptosis, adhesion/migration/invasion, stress responses (e.g. DNA damage and endoplasmic reticulum stress and hypoxia), and cell-cell communication (e.g. Notch and Ephrin signaling pathways). A role of miRNAs in regulating cell migration/invasion and stress response was further defined by examining the impact of DROSHA knockdown on cell behaviors. We demonstrated that DROSHA knockdown enhanced cell migration and invasion, whereas it sensitized cells to cell death induced by suspension culture, glucose depletion, and unfolding protein stress. Data from an orthotopic xenograft model showed that DROSHA knockdown resulted in reduced growth of primary tumors but enhanced lung metastasis. Taken together, these results suggest that miRNAs collectively function to promote survival of tumor cells under stress but suppress cell migration/invasion in breast cancer cells.     

TARGETgene: A Tool for Identification of Potential Therapeutic Targets in Cancer

The vast array of in silico resources and data of high throughput profiling currently available in life sciences research offer the possibility of aiding cancer gene and drug discovery process. Here we propose to take advantage of these resources to develop a tool, TARGETgene, for efficiently identifying mutation drivers, possible therapeutic targets, and drug candidates in cancer. The simple graphical user interface enables rapid, intuitive mapping and analysis at the systems level. Users can find, select, and explore identified target genes and compounds of interest (e.g., novel cancer genes and their enriched biological processes), and validate predictions using user-defined benchmark genes (e.g., target genes detected in RNAi screens) and curated cancer genes via TARGETgene. The high-level capabilities of TARGETgene are also demonstrated through two applications in this paper. The predictions in these two applications were then satisfactorily validated by several ways, including known cancer genes, results of RNAi screens, gene function annotations, and target genes of drugs that have been used or in clinical trial in cancer treatments. TARGETgene is freely available from the Biomedical Simulations Resource web site (http://bmsr.usc.edu/Software/TARGET/TARGET.html).     

乳腺癌增强子-miRNA调控网络识别与分析

乳腺癌发病率位列女性恶性肿瘤之首.先前研究表明增强子可通过调控miRNA影响肿瘤的发生发展.然而,增强子与miRNA在乳腺癌中形成何种调控网络目前尚不清楚.为了探究乳腺癌中增强子与miRNA形成的调控网络,通过分析 112个乳腺浸润癌样本和104个正常组织样本,共识别了 220对增强子-miRNA调控关系,其中包括22...