<div>Abstract<p><b>Purpose:</b> High-frequency microsatellite-instable (MSI-H) tumors account for ∼15% of colorectal cancers. Therapeutic decisions for colorectal cancer are empirically based and currently do not emphasize molecular subclassification despite an increasing collection of gene expression information. Our objective was to identify low molecular weight compounds with preferential activity against MSI colorectal cancers using combined gene expression data sets.</p><p><b>Experimental Design:</b> Three expression/query signatures (discovery data set) characterizing MSI-H colorectal cancer were matched with information derived from changes induced in cell lines by 164 compounds using the systems biology tool “Connectivity Map.” A series of sequential filtering and ranking algorithms were used to select the candidate compounds. Compounds were validated using two additional expression/query signatures (validation data set). Cytotoxic, cell cycle, and apoptosis effects of validated compounds were evaluated in a panel of cell lines.</p><p><b>Results:</b> Fourteen of the 164 compounds were validated as targeting MSI-H cell lines using the bioinformatics approach; rapamycin, LY-294002, 17-(allylamino)-17-demethoxygeldanamycin, and trichostatin A were the most robust candidate compounds. <i>In vitro</i> results showed that MSI-H cell lines due to hypermethylation of <i>MLH1</i> are preferentially targeted by rapamycin (18.3 versus 4.4 μmol/L; <i>P</i> = 0.0824) and LY-294002 (15.02 versus 10.37 μmol/L; <i>P</i> = 0.0385) when compared with microsatellite-stable cells. Preferential activity was also observed in <i>MSH2</i> and <i>MSH6</i> mutant cells.</p><p><b>Conclusion:</b> Our study shows that the phosphatidylinositol 3-kinase-AKT-mammalian target of rapamycin pathway is of special relevance in mismatch repair-deficient colorectal cancer. In addition, we show that amalgamation of gene expression information across studies provides a robust approach for selection of potential therapies corresponding to specific groups of patients.</p></div>
To better understand the contribution of the chemokine system in immune senescence, we determined the aging effect on CD4+ and CD8+ T-cell chemokine expression by microarray screening and ribonuclease protection assays. Compared with young C57BL/6 mice, freshly isolated CD4+ cells from aged mice express increased level of interferon-gamma-inducible protein 10 (IP-10), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated upon activation, normal T-cell expressed and secreted (RANTES), and lymphotactin (Ltn). T-cell receptor (TCR)/coreceptor stimulation up-regulates MIP-1alpha, MIP-1beta, and Ltn, and down-regulates IP-10 and RANTES expression in CD4+ T cells. A similar increase in chemokine expression was demonstrated in the CD8+ T cell. Enzyme-linked immunosorbent assays confirmed increased T-cell chemokine protein production in old CD4+ and CD8+ T cells. Finally, supernatant of cultured T cells from old animals caused an enhanced leukocyte chemotaxis response compared with that from young animals, suggesting that the age-related difference in T-cell chemokine expression has an important functional consequence.
Sequencing of the human genome has greatly impacted the proteomics-based analysis of disease by providing a framework for understanding the proteome of diseased cells, tissues, and biological fluids. Consequently, there is a growing interest in applying proteomics technologies to define protein pathways involved in various diseases, to identify new biomarkers that correlate with diseases, ideally in their early stages, and to accelerate the development of new therapeutic targets. However, disease-related proteomics applications require that we improve our ability to separate and characterize the components of complex protein mixtures in such a way as to boost both throughput and sensitivity. In response to these demands, the proteomics technologies have been improved markedly over recent years. Today, proteomics, in all its various forms, is proving to be invaluable to our understanding of the biochemistry of health and disease and will likely play a central role in the evolution of personalized medicine. In this special issue, we include reports of novel research findings together with several reviews that highlight advances in key areas.
The first two papers of this special issue focus on lung cancer. The first paper, by H. C. Gong et al., addresses the profiling of receptor tyrosine kinase pathway activation and the role of key genetic mutations in human lung tumor cell lines and human lung tumors. The authors defined molecular pathways which may assist in development of targeted lung tumor therapies. Within the second paper, Q. Zhang et al. used proteomic profiling to delineate expression and subcellular localization of multiple forms of aldehyde dehydrogenase in lung adenocarcinoma cell lines. The next two papers focus on pancreatic cancer. The third paper, by R. S. Kwon and D. M. Simeone, reviews the use of protein-based biomarkers for the diagnosis of cystic tumors of the pancreas. The fourth paper, by M. Abulaizi et al., utilizes a three-step proteomic protocol (immunodepletion of abundant serum proteins, followed by fractionation by RP-HPLC and further separation by 2D-PAGE) to discover candidate early detection biomarkers of pancreatic cancer.
The next two papers focus on breast cancer, with the fifth paper, by D. E. Misek and E. H. Kim, reviewing the development of protein biomarkers for the early detection of breast cancer. The sixth paper, by J. He et al., addresses LC-MS/MS identification of protein biosignatures in breast tumors, as protein-based markers that correctly classify tumor subtypes and predict therapeutic response would be of great clinical utility in guiding patient treatment. The next two papers are both by M. S. Sabel et al., and focus on melanoma. The seventh paper reviews the use of proteomics for the discovery of new prognostic and predictive biomarkers. The eighth paper explores the clinical utility of serum autoantibodies that were detected in melanoma patients. The investigators profiled serum antibodies against melanoma-associated antigens to identify those that may predict nodal positivity, a widely accepted index of metastatic disease.
The ninth paper, by E. H. Kim and D. E. Misek, reviews the use of glycoproteomics to identify cancer biomarkers. The tenth paper, by A. Vivekanandan-Giri et al., utilized glycoproteomics to identify novel urinary glycoprotein biomarkers of chronic kidney disease. The issue concludes with two papers that report on novel approaches and related considerations. The eleventh paper, by I. Kiyokawa et al., describes the development of a new surface coating for urinary collection tubes that minimizes the amount of urine protein adsorption onto the walls of the collection tube. Within the final paper of this special issue, T. Hagiwara et al. examine the utility of a solid-phase hexapeptide ligand library in combination with conventional plasma proteomics modalities for comprehensive profiling of intact plasma proteins.
David E. Misek
Tadashi Kondo
Mark W. Duncan
The goal of this study was to identify potential protein markers in lung adenocarcinomas.A series of 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung samples were examined for quantitative differences in protein expression using two-dimensional PAGE. Candidate proteins were identified using matrix-assisted laser desorption/ionization mass spectrometry or peptide sequencing. The levels of the individual isoforms of nine proteins found to be overexpressed in the lung tumors were examined. Potential mechanisms for overexpression were examined by comparing mRNA expression levels, assessed using oligonucleotide arrays, to the protein values in the same samples.Antioxidant enzyme AOE372, ATP synthase subunit d (ATP5D), beta1,4-galactosyltransferase, cytosolic inorganic pyrophosphatase, glucose-regulated M(r) 58,000 protein, glutathione-S-transferase M4, prolyl 4-hydroxylase beta subunit, triosephosphate isomerase, and ubiquitin thiolesterase (UCHL1) were identified as being significantly overexpressed in lung adenocarcinomas. The expression of these proteins was increased from 1.4- to 10.6-fold as compared with uninvolved lung tissue. The expression of the individual protein isoforms was correlated with 10 clinicopathological variables as well as with each gene's mRNA level in the same sample. Both isoforms of glucose-regulated M(r) 58,000 protein were found to be significantly correlated with their mRNA expression profiles (P < 0.05), indicating that increased transcription likely underlies the increased expression of these proteins.Two-dimensional PAGE and mass spectrometry can identify proteins showing increased expression in lung adenocarcinoma. The association of specific isoforms of these proteins with clinical variables and understanding the regulation of their expression will aid in determination of their potential use as biomarkers in this cancer.
The molecular basis of pancreatic cancer is not understood. Previous attempts to determine the specific genes expressed in pancreatic cancer have been hampered by similarities between adenocarcinoma and chronic pancreatitis. In the current study, microarrays (Affymetrix) were used to profile gene expression in pancreatic adenocarcinoma (10), pancreatic cancer cell lines (7), chronic pancreatitis (5), and normal pancreas (5). Molecular profiling indicated a large number of genes differentially expressed between pancreatic cancer and normal pancreas but many fewer differences between pancreatic cancer and chronic pancreatitis, likely because of the shared stromal influences in the two diseases. To specifically identify genes expressed in neoplastic epithelium, we selected genes more highly expressed (>2-fold, p < 0.01) in adenocarcinoma compared with both normal pancreas and chronic pancreatitis and which were also highly expressed in pancreatic cancer cell lines. This strategy yielded 158 genes, of which 124 were not previously associated with pancreatic cancer. Quantitative-reverse transcription-PCR for two molecules, S100P and 14-3-3sigma, validated the microarray data. Support for the success of the neoplastic cell gene expression identification strategy was obtained by immunocytochemical localization of four representative genes, 14-3-3sigma, S100P, S100A6, and beta4 integrin, to neoplastic cells in pancreatic tumors. Thus, comparisons between pancreatic adenocarcinoma, pancreatic cancer cell lines, normal pancreas, and chronic pancreatitis have identified genes that are selectively expressed in the neoplastic epithelium of pancreatic adenocarcinoma. These data provide new insights into the molecular pathology of pancreatic cancer that may be useful for detection, diagnosis, and treatment.
The L1 larvae of the parasitic nematode Trichinella spiralis invade skeletal muscle and initiate a process that has been interpreted to represent skeletal muscle dedifferentiation.In this process, the infected region of the muscle cell is converted into a unique structure, called the Nurse cell.The nematode T. spiralis can survive for tens of years within the cytoplasm of the Nurse cell and secretes proteins into the cytoplasm that are believed to play a role in mediating the Nurse cell formation or maintenance.We have cloned a cDNA encoding the T. spiralis-derived, 43-kDa secreted protein.Structural analysis of the predicted 344-amino acid sequence revealed an N terminally located signal peptide and a potential helix-loop-helix motif in the main body of the protein.Antibodies raised against the 43-kDa recombinant protein were used in immunocytolocalizations of T. spiralis-infected skeletal muscle sections.These antibodies strongly stained the Nurse cell nuclei and the nematode itself.Specific, though slightly weaker staining also occurred in the Nurse cell cytoplasm.In Western blots, the antibodies react with the 43-kDa protein but also detected at least two other T. spiralis-secreted proteins.DNA hybridizations reveal at least one additional 43-kDa-related sequence encoded in the T. spiralis genome.We conclude that either the 43-kDa protein and/or a closely related 43-kDa family member is secreted into the muscle and translocates to the muscle-derived nuclei.This model may provide insights into the mechanisms involved in Nurse cell formation.The parasitic nematode Trichinella spiralis is infective to a wide range of hosts including mice, rats, swine, and humans.Host infection begins upon ingestion of raw or undercooked
We hypothesized that genes expressed in pancreatic acinar cells during the initiation of acute pancreatitis determine the severity of the disease. Therefore, we utilized microarrays to identify those genes commonly induced in rat pancreatic acinar cells within 1–4 h in two in vivo models, caerulein and taurocholate administration. This strategy yielded 51 known genes representing a complex array of molecules, including those that are likely to either reduce or increase the severity of the disease. Novel genes identified in the current study included ATF3, BRF1, C/EBPβ, CGRP, EGR-1, ephrinA1, villin2, ferredoxin, latexin, lipocalin, MKP-1, NGFI-B, RhoA, tissue factor (TF), and syndecan. To validate these microarray results, the role of EGR-1 was further investigated using quantitative RT-PCR, Western blotting, and immunocytochemistry. EGR-1 expression occurred within acinar cells and correlated with the development of caerulein-induced acute pancreatitis in rats. Furthermore, the levels of the inflammation-related genes MCP-1, PAI, TF, IL-6, and ICAM-1 and the extent of lung inflammation were reduced during the initiation of caerulein-induced acute pancreatitis in EGR-1-deficient mice. Thus this study identified EGR-1 and several other novel genes likely to be important in the development and severity of acute pancreatitis.
