Deciphering the ovarian cancer ascites fluid peptidome

BACKGROUND: Conventional proteomic approaches have thus far been unable to identify novel serum biomarkers for ovarian cancer that are more sensitive and specific than the current clinically used marker, CA-125. Because endogenous peptides are smaller and may enter the circulation more easily than proteins, a focus on the low-molecular-weight region may reveal novel biomarkers with enhanced sensitivity and specificity. In this study, we deciphered the peptidome of ascites fluid from 3 ovarian cancer patients and 3 benign individuals (ascites fluid from patients with liver cirrhosis). RESULTS: Following ultrafiltration of the ascites fluids to remove larger proteins, each filtrate was subjected to solid phase extraction and fractionated using strong cation exchange chromatography. The resultant fractions were analyzed using an Orbitrap mass spectrometer. We identified over 2000 unique endogenous peptides derived from 259 proteins. We then catalogued over 777 peptides that were found only in ovarian cancer ascites. Our list of peptides found in ovarian cancer specimens includes fragments derived from the proteins vitronectin, transketolase and haptoglobin. CONCLUSIONS: Peptidomics may uncover previously undiscovered disease-specific endogenous peptides that warrant further investigation as biomarkers for ovarian cancer

Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM\u27s absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes

Major milestones in translational oncology.

Translational oncology represents a bridge between basic research and clinical practice in cancer medicine. Today, translational research in oncology benefits from an abundance of knowledge resulting from genome-scale studies regarding the molecular pathways involved in tumorigenesis. In this Forum article, we highlight the state of the art of translational oncology in five major cancer types. We illustrate the use of molecular profiling to subtype colorectal cancer for both diagnosis and treatment, and summarize the results of a nationwide screening program for ovarian cancer based on detection of a tumor biomarker in serum. Additionally, we discuss how circulating tumor DNA can be assayed to safely monitor breast cancer over the course of treatment, and report on how therapy with immune checkpoint inhibitors is proving effective in advanced lung cancer. Finally, we summarize efforts to use molecular profiling of prostate cancer biopsy specimens to support treatment decisions. Despite encouraging early successes, we cannot disregard the complex genetics of individual susceptibility to cancer nor the enormous complexity of the somatic changes observed in tumors, which urge particular attention to the development of personalized therapies

Recycling of the Membrane-anchored Chemokine, CX 3 CL1

CX(3)CL1 (fractalkine) plays an important role in inflammation by acting as both chemoattractant and as an adhesion molecule. As for other chemokines, expression of CX(3)CL1 is known to be regulated at the level of transcription and translation. The unique transmembrane structure of CX(3)CL1 raises the possibility of additional functional regulation by altering its abundance at the cell surface. This could be accomplished in principle by changes in traffic between subcellular compartments. To analyze this possibility we examined the subcellular distribution of CX(3)CL1 in human ECV-304 cells stably expressing untagged or green fluorescent protein-tagged forms of the chemokine. CX(3)CL1 was present in two distinct compartments, diffusely on the plasma membrane and in a punctate juxtanuclear compartment. The latter shared some features with, yet was distinct from the conventional endocytic pathway and may represent a specialized recycling subcompartment. Accordingly, surface CX(3)CL1 was found to be in dynamic equilibrium with the juxtanuclear vesicular compartment. Intracellular CX(3)CL1 co-localized with the SNARE (soluble N-ethylmaleimide factor attachment protein receptor) proteins syntaxin-13 and VAMP-3. Cleavage of VAMP-3 by tetanus toxin or impairment of syntaxin-13 function by expression of a dominant-negative allele inhibited the ability of internalized CX(3)CL1 to traffic back to the plasma membrane. These data demonstrate the existence of a dynamic, SNARE-mediated recycling of CX(3)CL1 from the cell surface to and from an endomembrane storage compartment. The intracellular storage depot may serve as a source of the chemokine that could be rapidly mobilized by stimuli

Identification and Validation of Candidate Breast Cancer Biomarkers: A Mass Spectrometric Approach

One of the best ways to diagnose breast cancer early or to predict therapeutic response is to use serum biomarkers. Unfortunately, for breast cancer, we do not have effective serological biomarkers. We hypothesized that novel candidate tumor markers for breast cancer may be secreted or shed proteins that can be detected in tissue culture supernatants of human breast cancer cell lines. A two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) strategy was utilized to identify and compare levels of extracellular and membrane-bound proteins in the conditioned media. Proteomic analysis of the media identified in excess of 600, 500 and 700 proteins in MCF-10A, BT474 and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6 and 10 (ranging in concentration from 2-50 µg/L), as validated by ELISA and confidently identified HER-2/neu in BT474 cells. Sub-cellular localization was determined based on Genome Ontology (GO) for the 1,139 proteins, of which 34% were classified as extracellular and membrane-bound. Tissue specificity, functional classifications and label-free quantification were performed. The levels of eleven promising molecules were measured in biological samples to determine its discriminatory ability for control versus cases. This screen yielded activated leukocyte cell adhesion molecule (ALCAM) as a promising candidate. The levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were examined in 300 serum samples by quantitative ELISA. All three biomarkers effectively separated cancer from non-cancer groups. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p <0.05). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.Ph

Fascin-1 is a novel biomarker of aggressiveness in some carcinomas

Abstract Tremendous progress has been made in recent years towards the understanding, prevention and management of malignant disease, yet cancer remains a leading cause of global mortality and morbidity. Current approaches towards combating this disease include prevention, early detection and various treatment modalities. However, even with implementation of novel therapeutic options and preventative measures, most cancers are currently diagnosed at late stages, when treatment therapies are least effective. In a recent study published in BMC Medicine, Tan et al. performed a systematic review and meta-analysis to show that fascin-1, an actin-bundling protein, is associated with increased risk of mortality and metastasis in various cancer types. Although the study examined the association of fascin-1 with mortality, time-to-disease progression, lymph node metastasis and distant metastasis in five major cancer types, the clinical implications of these findings are still unclear and many unanswered questions remain. Please see related research article here http://www.biomedcentral.com/1741-7015/11/5

SPME-LC/MS-based serum metabolomic phenotyping for distinguishing ovarian cancer histologic subtypes : a pilot study

Epithelial ovarian cancer (EOC) is the most common cause of death from gynecological cancer. The outcomes of EOC are complicated, as it is often diagnosed late and comprises several heterogenous subtypes. As such, upfront treatment can be highly challenging. Although many significant advances in EOC management have been made over the past several decades, further work must be done to develop early detection tools capable of distinguishing between the various EOC subtypes. In this paper, we present a sophisticated analytical pipeline based on solid-phase microextraction (SPME) and three orthogonal LC/MS acquisition modes that facilitates the comprehensive mapping of a wide range of analytes in serum samples from patients with EOC. PLS-DA multivariate analysis of the metabolomic data was able to provide clear discrimination between all four main EOC subtypes: serous, endometrioid, clear cell, and mucinous carcinomas. The prognostic performance of discriminative metabolites and lipids was confirmed via multivariate receiver operating characteristic (ROC) analysis (AUC value > 88% with 20 features). Further pathway analysis using the top 57 dysregulated metabolic features showed distinct differences in amino acid, lipid, and steroids metabolism among the four EOC subtypes. Thus, metabolomic profiling can serve as a powerful tool for complementing histology in classifying EOC subtypes

Genomic profiling for copy number changes in plasma of ovarian cancer patients – a new era for cancer diagnostics?

Abstract A blood test that can detect human malignancy with high clinical sensitivity and specificity is highly desirable. To achieve this, a tumor marker is needed that correlates with tumor burden and that can be measured with high analytical sensitivity and specificity. Over the past decades, a number of different types of tumor markers have emerged, including proteins such as enzymes, glycoproteins, and oncofetal antigens. Besides proteins, genetic abnormalities such as mutations, amplifications, and circulating tumor DNA have served as tumor markers. Despite the diversity of such biomarkers, their acceptance and implementation into routine clinical practice requires that their use results in improvements in patient outcome. Current tumor markers used in the clinic have limited utility. As such, innovative approaches to identifying tumor markers are highly desirable and one such approach may be to look for sub-chromosomal changes in the blood of patients with ovarian cancer, as is routinely performed in prenatal screening. Please see related article: http://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-016-0667-

A Solid-Phase Microextraction—Liquid Chromatography-Mass Spectrometry Method for Analyzing Serum Lipids in Psoriatic Disease

Approximately 25% of psoriasis patients have an inflammatory arthritis termed psoriatic arthritis (PsA). There is strong interest in identifying and validating biomarkers that can accurately and reliably predict conversion from psoriasis to PsA using novel technologies such as metabolomics. Lipids, in particular, are of key interest in psoriatic disease. We sought to develop a liquid chromatography-mass spectrometry (LC-MS) method to be used in conjunction with solid-phase microextraction (SPME) for analyzing fatty acids and similar molecules. A total of 25 chromatographic methods based on published lipid studies were tested on two LC columns. As a proof of concept, serum samples from psoriatic disease patients (n = 27 psoriasis and n = 26 PsA) were processed using SPME and run on the selected LC-MS method. The method that was best for analyzing fatty acids and fatty acid-like molecules was optimized and applied to serum samples. A total of 18 tentatively annotated features classified as fatty acids and other lipid compounds were statistically significant between psoriasis and PsA groups using both multivariate and univariate approaches. The SPME-LC-MS method developed and optimized was capable of detecting fatty acids and similar lipids that may aid in differentiating psoriasis and PsA patients