Towards an integrated proteomic and glycomic approach to finding cancer biomarkers

Advances in mass spectrometry have had a great impact on the field of proteomics. A major challenge of proteomic analysis has been the elucidation of glycan modifications of proteins in complex proteomes. Glycosylation is the most structurally elaborate and diverse type of protein post-translational modification and, because of this, proteomics and glycomics have largely developed independently. However, given that such a large proportion of proteins contain glycan modifications, and that these may be important for their function or may produce biologically relevant protein variation, a convergence of the fields of glycomics and proteomics would be highly desirable. Here we review the current status of glycoproteomic efforts, focusing on the identification of glycoproteins as cancer biomarkers

Detection of human somatic cell structural gene mutations by two-dimensional electrophoresis

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38517/1/340020103_ftp.pd

Biomarkers for Diagnosis and Prognosis of Sinusoidal Obstruction Syndrome after Hematopoietic Cell Transplantation.

Reliable, non-invasive methods for diagnosing and prognosing sinusoidal obstruction syndrome (SOS) early after hematopoietic cell transplantation (HCT) are needed. We used a quantitative mass spectrometry-based proteomics approach to identify candidate biomarkers of SOS by comparing plasma pooled from 20 patients with and 20 patients without SOS. Of 494 proteins quantified, we selected six proteins [L-Ficolin, vascular-cell-adhesion-molecule-1 (VCAM1), tissue-inhibitor of metalloproteinase-1, von Willebrand factor, intercellular-adhesion-molecule-1, and CD97] based on a differential heavy/light isotope ratio of at least 2 fold, information from the literature, and immunoassay availability. Next, we evaluated the diagnostic potential of these six proteins and five selected from the literature [suppression of tumorigenicity-2 (ST2), angiopoietin-2 (ANG2), hyaluronic acid (HA), thrombomodulin, and plasminogen activator inhibitor-1] in samples from 80 patients. The results demonstrate that together ST2, ANG2

High-throughput genomic technology in research and clinical management of breast cancer. Plasma-based proteomics in early detection and therapy

Protein-based breast cancer biomarkers are a promising resource for breast cancer detection at the earliest and most treatable stages of the disease. Plasma is well suited to proteomic-based methods of biomarker discovery because it is easily obtained, is routinely used in the diagnosis of many diseases, and has a rich proteome. However, due to the vast dynamic range in protein concentration and the often uncertain tissue and cellular origin of plasma proteins, proteomic analysis of plasma requires special consideration compared with tissue and cultured cells. This review briefly touches on the search for plasma-based protein biomarkers for the early detection and treatment of breast cancer

Proteomic Analysis of Ovarian Cancer Cells Reveals Dynamic Processes of Protein Secretion and Shedding of Extra-Cellular Domains

Background: Elucidation of the repertoire of secreted and cell surface proteins of tumor cells is relevant to molecular diagnostics, tumor imaging and targeted therapies. We have characterized the cell surface proteome and the proteins released into the extra-cellular milieu of three ovarian cancer cell lines, CaOV3, OVCAR3 and ES2 and of ovarian tumor cells enriched from ascites fluid. Methodology and Findings: To differentiate proteins released into the media from protein constituents of media utilized for culture, cells were grown in the presence of [ 13 C]-labeled lysine. A biotinylation-based approach was used to capture cell surface associated proteins. Our general experimental strategy consisted of fractionation of proteins from individual compartments followed by proteolytic digestion and LC-MS/MS analysis. In total, some 6,400 proteins were identified with high confidence across all specimens and fractions. Conclusions and Significance: Protein profiles of the cell lines had substantial similarity to the profiles of human ovarian cancer cells from ascites fluid and included protein markers known to be associated with ovarian cancer. Proteomic analysis indicated extensive shedding from extra-cellular domains of proteins expressed on the cell surface, and remarkably high secretion rates for some proteins (nanograms per million cells per hour). Cell surface and secreted proteins identified by indept

A Mouse to Human Search for Plasma Proteome Changes Associated with Pancreatic Tumor Development

Background: The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer. Methods and Findings: Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer. Conclusions: Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection

Int6 reduction activates stromal fibroblasts to enhance transforming activity in breast epithelial cells

Background: The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6's role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts. Results: We searched microarray databases of human tumors to assess Int6's role in breast cancer. While INT6 expression levels, as expected, were lower in breast tumors than in adjacent normal breast tissue samples, INT6 expression levels were also substantially lower in tumor stroma. By immunohistochemistry, we determined that the low levels of INT6 mRNA observed in the microarray databases most likely occurs in stromal fibroblasts, because far fewer fibroblasts in the tumor tissue showed detectable levels of the Int6 protein. To directly investigate the effects of Int6 repression on fibroblasts, we silenced INT6 expression in immortalized human mammary fibroblasts (HMFs). When these INT6-repressed HMFs were co-cultured with breast cancer cells, the abilities of the latter to form colonies in soft agar and to invade were enhanced. We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin. Furthermore, like CAFs, these INT6-repressed HMFs secreted more stromal cell-derived factor 1 (SDF-1), and the addition of an SDF-1 antagonist attenuated the INT6-repressed HMFs' ability to enhance soft agar colony formation when co-cultured with cancer cells. These INT6-repressed HMFs also expressed high levels of mesenchymal markers such as vimentin and N-cadherin. Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced. Conclusion: These data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis

Fibulin-2 Is a Driver of Malignant Progression in Lung Adenocarcinoma

The extracellular matrix of epithelial tumors undergoes structural remodeling during periods of uncontrolled growth, creating regional heterogeneity and torsional stress. How matrix integrity is maintained in the face of dynamic biophysical forces is largely undefined. Here we investigated the role of fibulin-2, a matrix glycoprotein that functions biomechanically as an inter-molecular clasp and thereby facilitates supra-molecular assembly. Fibulin-2 was abundant in the extracellular matrix of human lung adenocarcinomas and was highly expressed in tumor cell lines derived from mice that develop metastatic lung adenocarcinoma from co-expression of mutant K-ras and p53. Loss-offunction experiments in tumor cells revealed that fibulin-2 was required for tumor cells to grow and metastasize in syngeneic mice, a surprising finding given that other intra-tumoral cell types are known to secrete fibulin-2. However, tumor cells grew and metastasized equally well in Fbln2-null and -wildtype littermates, implying that malignant progression was dependent specifically upon tumor cellderived fibulin-2, which could not be offset by other cellular sources of fibulin-2. Fibulin-2 deficiency impaired the ability of tumor cells to migrate and invade in Boyden chambers, to create a stiff extracellular matrix in mice, to cross-link secreted collagen, and to adhere to collagen. We conclude that fibulin-2 is a driver of malignant progression in lung adenocarcinoma and plays an unexpected role in collagen cross-linking and tumor cell adherence to collagen

A Compendium of Potential Biomarkers of Pancreatic Cancer

Akhilesh Pandey and colleagues describe a compendium of potential biomarkers that can be systematically validated by the pancreatic cancer community

The Long Noncoding RNA CCAT2 Induces Chromosomal Instability Through BOP1-AURKB Signaling

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors