Phenotypic and Molecular Characterization of MCF10DCIS and SUM Breast Cancer Cell Lines.

We reviewed the phenotypic and molecular characteristics of MCF10DCIS.com and the SUM cell lines based on numerous studies performed over the years. The major signaling pathways that give rise to the phenotype of these cells may serve as a good resource of information when researchers in drug discovery and development use these cells to identify novel targets and biomarkers. Major signaling pathways and mutations affecting the coding sequence are also described providing important information when using these cells as a model in a variety of studies.

Prospective gene signature study using microRNA to identify the tissue of origin in patients with carcinoma of unknown primary.

PURPOSE: Accurate identification of tissue of origin (ToO) for patients with carcinoma of unknown primary (CUP) may help customize therapy to the putative primary and thereby improve the clinical outcome. We prospectively studied the performance of a microRNA-based assay to identify the ToO in CUP patients. EXPERIMENTAL DESIGN: Formalin-fixed paraffin-embedded (FFPE) metastatic tissue from 104 patients was reviewed and 87 of these contained sufficient tumor for testing. The assay quantitates 48 microRNAs and assigns one of 25 tumor diagnoses by using a biologically motivated binary decision tree and a K-nearest neighbors (KNN). The assay predictions were compared with clinicopathologic features and, where suitable, to therapeutic response. RESULTS: Seventy-four of the 87 cases were processed successfully. The assay result was consistent or compatible with the clinicopathologic features in 84% of cases processed successfully (71% of all samples attempted). In 65 patients, pathology and immunohistochemistry (IHC) suggested a diagnosis or (more often) a differential diagnosis. Out of those, the assay was consistent or compatible with the clinicopathologic presentation in 55 (85%) cases. Of the 9 patients with noncontributory IHC, the assay provided a ToO prediction that was compatible with the clinical presentation in 7 cases. CONCLUSIONS: In this prospective study, the microRNA diagnosis was compatible with the clinicopathologic picture in the majority of cases. Comparative effectiveness research trials evaluating the added benefit of molecular profiling in appropriate CUP subsets are warranted. MicroRNA profiling may be particularly helpful in patients in whom the IHC profile of the metastasis is nondiagnostic or leaves a large differential diagnosis.

hsa-miR-191 is a candidate oncogene target for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.

A diagnostic assay based on microRNA expression accurately identifies malignant pleural mesothelioma.

The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.

Differential diagnosis of hepatocellular carcinoma from metastatic tumors in the liver using microRNA expression.

Distinguishing hepatocellular carcinoma from metastatic tumors in the liver is of great practical importance, with significant therapeutic and prognostic implications. This differential diagnosis can be difficult because metastatic cancers in the liver, especially adenocarcinomas, may mimic the morphology and immunoexpression of hepatocellular carcinoma. Biomarkers that are specifically expressed in either hepatocellular carcinoma or metastatic adenocarcinoma can therefore be useful diagnostic tools. To find such biomarkers, we studied microRNA expression in 144 tumor samples using custom microarrays. Hsa-miR-141 and hsa-miR-200c, microRNAs that promote epithelial phenotypes, had significantly higher levels in non-hepatic epithelial tumors. In contrast, endothelial-associated hsa-miR-126 showed higher expression levels in hepatocellular carcinomas. Combinations of these microRNAs accurately identified primary hepatocellular carcinoma from metastatic adenocarcinoma in the liver. These findings were validated using quantitative real-time PCR to measure microRNA expression in additional samples. Thus, the tissue-specific expression patterns of microRNAs make them useful biomarkers for the diagnosis of liver malignancies.

Pro-tumorigenic effects of miR-31 loss in mesothelioma.

The human genome encodes several hundred microRNA (miRNA) genes that produce small (21-23n) single strand regulatory RNA molecules. Although abnormal expression of miRNAs has been linked to cancer progression, the mechanisms of this dysregulation are poorly understood. Malignant mesothelioma (MM) of pleura is an aggressive and highly lethal cancer resistant to conventional therapies. We and others previously linked loss of the 9p21.3 chromosome in MM with short time to tumor recurrence. In this study, we report that MM cell lines derived from patients with more aggressive disease fail to express miR-31, a microRNA recently linked with suppression of breast cancer metastases. We further demonstrate that this loss is due to homozygous deletion of the miR-31-encoding gene that resides in 9p21.3. Functional assessment of miR-31 activity revealed its ability to inhibit proliferation, migration, invasion, and clonogenicity of MM cells. Re-introduction of miR-31 suppressed the cell cycle and inhibited expression of multiple factors involved in cooperative maintenance of DNA replication and cell cycle progression, including pro-survival phosphatase PPP6C, which was previously associated with chemotherapy and radiation therapy resistance, and maintenance of chromosomal stability. PPP6C, whose mRNA is distinguished with three miR-31-binding sites in its 3'-untranslated region, was consistently down-regulated by miR-31 introduction and up-regulated in clinical MM specimens as compared with matched normal tissues. Taken together, our data suggest that tumor-suppressive propensity of miR-31 can be used for development of new therapies against mesothelioma and other cancers that show loss of the 9p21.3 chromosome.

hsa-miR-29c* is linked to the prognosis of malignant pleural mesothelioma.

The inability to forecast outcomes for malignant mesothelioma prevents clinicians from providing aggressive multimodality therapy to the most appropriate individuals who may benefit from such an approach. We investigated whether specific microRNAs (miR) could segregate a largely surgically treated group of mesotheliomas into good or bad prognosis categories. A training set of 44 and a test set of 98 mesothelioma tumors were analyzed by a custom miR platform, along with 9 mesothelioma cell lines and 3 normal mesothelial lines. Functional implications as well as downstream targets of potential prognostic miRs were investigated. In both the training and test sets, hsa-miR-29c* was an independent prognostic factor for time to progression as well as survival after surgical cytoreduction. The miR was expressed at higher levels in epithelial mesothelioma, and the level of this miR could segregate patients with this histology into groups with differing prognosis. Increased expression of hsa-miR-29c* predicted a more favorable prognosis, and overexpression of the miR in mesothelioma cell lines resulted in significantly decreased proliferation, migration, invasion, and colony formation. Moreover, major epigenetic regulation of mesothelioma is mediated by hsa-miR-29c* and was shown through downregulation of DNA methyltransferases as well as upregulation of demethylating genes. A single miR has the potential to be a prognostic biomarker in mesothelioma, and validation of these findings as well as investigation of its downstream targets may give insight for potential therapies in the future.

Diagnostic assay based on hsa-miR-205 expression distinguishes squamous from nonsquamous non-small-cell lung carcinoma.

PURPOSE: Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay. PATIENTS AND METHODS: High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples. RESULTS: We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set. CONCLUSION: Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.

MicroRNAs accurately identify cancer tissue origin.

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.

Histone deacetylase activities are required for innate immune cell control of Th1 but not Th2 effector cell function.

Histone deacetylases (HDACs) play a critical role in regulating gene expression and key biological processes. However, how HDACs are involved in innate immunity is little understood. Here, in this first systematic investigation of the role of HDACs in immunity, we show that HDAC inhibition by a small-molecule HDAC inhibitor (HDACi), LAQ824, alters Toll-like receptor 4 (TLR4)-dependent activation and function of macrophages and dendritic cells (DCs). Surprisingly, pan-HDAC inhibition modulates only a limited set of genes involved in distinct arms of immune responses. Specifically, it inhibited DC-controlled T helper 1 (Th1) effector but not Th2 effector cell activation and migration. It also inhibited macrophage- and DC-mediated monocyte but not neutrophil chemotaxis. These unexpected findings demonstrate the high specificity of HDAC inhibition in modulating innate and adaptive immune responses, and highlight the potential for HDACi to alter the Th1 and Th2 balance in therapeutic settings.

Activation of mitochondrial pathway is crucial for tumor selective induction of apoptosis by LAQ824.

HDAC inhibitors are promising antitumor drugs with several HDAC inhibitors already in clinical trials. LAQ824, a potent pan-HDAC inhibitor, has been shown to induce cell cycle arrest and cell death. However, the mechanism of its antitumor effects and specially its tumor selectivity are still poorly understood. The focus of this study is to elucidate LAQ824 mediated anti-proliferative effects in lung carcinoma cells and the mechanism underlying the different sensitivity of LAQ824 to cancer and normal cells. In this study, LAQ824 mediated apoptosis was found to occur mainly via activation of the mitochondrial death pathway by inducing Apaf1 and caspase 9 and promoting mitochondrial release of key proapoptotic factors in lung cancer cells, but not in normal fibroblast cells. Using chromatin immunoprecipitation assay, we found that RNA Pol II binding and histone H3 acetylation levels at Apaf1 promoter were increased following LAQ824 treatment, explaining LAQ824 induced expression of Apaf1 in lung cancer cells. Furthermore, we showed that LAQ824 only triggered the release of mitochondrial proapoptotic factors such as cytochrome C (Cyto C) and apoptosis inducing factor (AIF) in lung cancer cells but not in normal blast cells. In addition, LAQ824 was found to induce Bax translocation in lung cancer cell, which may play important role in the induction of the release of mitochondrial proapoptotic factors. These data provide insight into the mechanism underlying the selective induction of apoptosis by LAQ824 in cancer cells.

Genomic approaches to drug discovery.

Considerable progress has been made in exploiting the enormous amount of genomic and genetic information for the identification of potential targets for drug discovery and development. New tools that incorporate pathway information have been developed for gene expression data mining to reflect differences in pathways in normal and disease states. In addition, forward and reverse genetics used in a high-throughput mode with full-length cDNA and RNAi libraries enable the direct identification of components of signaling pathways. The discovery of the regulatory function of microRNAs highlights the importance of continuing the investigation of the genome with sophisticated tools. Furthermore, epigenetic information including DNA methylation and histone modifications that mediate important biological processes add to the possibilities to identify novel drug targets and patient populations that will benefit from new therapies.

Design of a genome-wide siRNA library using an artificial neural network.

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

Functional genomics to new drug targets.

The completion of the sequencing of the human genome, and those of other organisms, is expected to lead to many potential new drug targets in various diseases, and it is predicted that novel therapeutic agents will be developed against such targets. The role of functional genomics in modern drug discovery is to prioritize these targets and to translate that knowledge into rational and reliable drug discovery. Here, we describe the field of functional genomics and review approaches that have been applied to drug discovery, including RNA profiling, proteomics, antisense and RNA interference, model organisms and high-throughput, genome-wide overexpression or knockdowns, and outline the future directions that are likely to yield new drug targets from genomics.

Transcription regulation by histone deacetylases.

Dynamic changes in the post-translational modification pattern of histories such as acetylation, deacetylation, phosphorylation, methylation and ubiquitination are thought to provide a code for correct regulation of gene expression by affecting chromatin structure and interaction with regulatory factors. Our studies focus on the role of histone deacetylases (HDACs) in transcriptional regulation and addressing functional differences of class I and class II HDACs. To identify genes that were transcriptionally regulated by specific HDACs, genome scale expression profiles were performed in cancer cells following the inhibition of three HDAC family members by specific oligonucleotides. The modulated genes identified in this study represented a wide range of modifications in different cellular pathways. In addition, treatment of cancer cells with a HDAC inhibitor was found to induce the expression of the small GTPase RhoB through an inverted CCAAT box in the RhoB promoter. These studies identified a specific transcription element involved in HDAC-mediated gene transcription and genes that are transcriptionally regulated by specific HDACs, providing important insight into the potential therapeutic benefit of HDAC inhibition.

Selective growth inhibition of tumor cells by a novel histone deacetylase inhibitor, NVP-LAQ824.

We have synthesized a histone deacetylase inhibitor, NVP-LAQ824, a cinnamic hydroxamic acid, that inhibited in vitro enzymatic activities and transcriptionally activated the p21 promoter in reporter gene assays. NVP-LAQ824 selectively inhibited growth of cancer cell lines at submicromolar levels after 48-72 h of exposure, whereas higher concentrations and longer exposure times were required to retard the growth of normal dermal human fibroblasts. Flow cytometry studies revealed that both tumor and normal cells arrested in the G(2)-M phase of the cell cycle after compound treatment. However, an increased sub-G(1) population at 48 h (reminiscent of apoptotic cells) was observed only in the cancer cell line. Annexin V staining data supported our hypothesis that NVP-LAQ824 induced apoptosis in tumor and transformed cells but not in normal cells. Western blotting experiments showed an increased histone H3 and H4 acetylation level in NVP-LAQ824-treated cancer cells, suggesting that the likely in vivo target of NVP-LAQ824 was histone deacetylase(s). Finally, NVP-LAQ824 exhibited antitumor effects in a xenograft animal model. Together, our data indicated that the activity of NVP-LAQ824 was consistent with its intended mechanism of action. This novel histone deacetylase inhibitor is currently in clinical trials as an anticancer agent.

Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells.

This report describes an unbiased method for systematically determining gene function in mammalian cells. A total of 20,704 predicted human full-length cDNAs were tested for induction of the IL-8 promoter. A number of genes, including those for cytokines, receptors, adapters, kinases, and transcription factors, were identified that induced the IL-8 promoter through known regulatory sites. Proteins that acted through a cooperative interaction between an AP-1 and an unrecognized cAMP response element (CRE)-like site were also identified. A protein, termed transducer of regulated cAMP response element-binding protein (CREB) (TORC1), was identified that activated expression through the variant CRE and consensus CRE sites. TORC1 potently induced known CREB1 target genes, bound CREB1, and activated expression through a potent transcription activation domain. A functional Drosophila TORC gene was also identified. Thus, TORCs represent a family of highly conserved CREB coactivators that may control the potency and specificity of CRE-mediated responses.

Histone deacetylase 1 represses the small GTPase RhoB expression in human nonsmall lung carcinoma cell line.

The dynamic balance between histone acetylation and deacetylation plays a significant role in the regulation of gene transcription. Much of our current understanding of this transcriptional control comes from the use of HDAC inhibitors such as trapoxin A (TPX), which leads to hyperacetylated histone, alters local chromatin architecture and transcription and results in tumor cell death. In this study, we treated tumor cells with TPX and HDAC1 antisense oligonucleotides, and analysed the transcriptional consequences of HDAC inhibition. Among other genes, the small GTPase RhoB was found to be significantly upregulated by TPX and repressed by HDAC1. The induction of RhoB by HDAC inhibition was mediated by an inverted CCAAT box in the RhoB promoter. Interestingly, measurement of RhoB transcription in approximately 130 tumor-derived cell lines revealed low expression in almost all of these samples, in contrast to RhoA and RhoC. Accumulating evidence indicates that the small GTPase Rho proteins are involved in a variety of important processes in cancer, including cell transformation, survival, invasion, metastasis and angiogenesis. This study for the first time demonstrates a link between HDAC inhibition and RhoB expression and provides an important insight into the mechanisms of HDAC-mediated transcriptional control and the potential therapeutic benefit of HDAC inhibition.

Genome-scale functional profiling of the mammalian AP-1 signaling pathway.

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.

Histone deacetylase dHDAC4 is involved in segmentation of the Drosophila embryo and is regulated by gap and pair-rule genes.

Histone deacetylases (HDACs) are catalytic subunits of multiprotein complexes that are targeted to specific promoters through their interaction with different transcriptional repressors causing silencing of the corresponding genes. This study describes the isolation of dHDAC4, a novel, catalytically active class II Drosophila histone deacetylase, and the analysis of its role in embryonic development. In early embryos, dHDAC4 is expressed in several phases. Initial ubiquitous expression becomes localized to an anterior domain, then evolves into a pair-rule-like and finally into a segment-polarity-like pattern. Suppression of dHDAC4 during early embryogenesis by double-stranded RNA interference led to segmentation defects. Analysis of dHDAC4 expression in gap and pair-rule gene mutants demonstrated that hunchback, knirps, and giant activate, while even-skipped suppresses dHDAC4 expression. These data revealed dHDAC4 involvement in the segmentation regulatory pathway and suggested complex transcriptional regulation as a potential mechanism that controls its expression.