The application of state-of-the-art proteomics and functional genomics technologies to the study of cancer is rapidly shifting toward the analysis of clinically relevant samples derived from ...patients, as the ultimate aim of translational research is to bring basic discoveries closer to the bedside. Here we describe the essence of a long-term initiative undertaken by The Danish Centre for Translational Breast Cancer Research and currently underway for cancer biomarker discovery using fresh tissue biopsies and bio-fluids. The Centre is a virtual hub that brings together scientists working in various areas of basic cancer research such as cell cycle control, invasion and micro-environmental alterations, apoptosis, cell signaling, and immunology, with clinicians (oncologists, surgeons), pathologists, and epidemiologists, with the aim of understanding the molecular mechanisms underlying breast cancer progression and ultimately of improving patient survival and quality of life. The unifying concept behind our approach is the use of various experimental paradigms for the prospective analysis of clinically relevant samples obtained from the same patient, along with the systematic integration of the biological and clinical data.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The galectins are a family of β-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this ...family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a λgt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone comigrated with IEF 17 as determined by two-dimensional (two-dimensional gel electrophoresis) analysis of proteins expressed by transiently transfected COS-1 cells, and bound lactose. Alignment of the amino acid sequences with other members of the family showed that the amino acids central to the β-galactoside interaction are conserved. Galectin-7 was partially externalized to the medium by keratinocytes although it has no typical secretion signal peptide. Immunoblotting as well as immunofluorescence analysis of human tissues with a specific galectin-7 antibody revealed a narrow distribution of the protein which was found mainly in stratified squamous epithelium. The antigen localized to basal keratinocytes, although it was also found, albeit at lower levels, in the suprabasal layers where it concentrated to areas of cell to cell contact. Both, its cellular localization as well as its striking down-regulation in K14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
We have applied protein expression profiling technologies in combination with immunohistochemistry, using fresh tissue and urine samples, to assess bladder cancer heterogeneity and prognosis as well ...as to generate protein markers for tumor progression and early diagnosis of the disease. Here, we review some selected lines of investigation and approaches undertaken by our laboratory, drawing on more than 15 years of experience in bladder cancer proteomics, to highlight a number of issues that may be useful for researchers entering the field. In particular, we address the identification of markers for bladder cancer progression and exemplify the potential of gel‐based proteomic profiling of urine samples for the early detection of urothelial carcinomas. In addition, we provide a brief description of a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the tumor microenvironment.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Apocrine morphology in breast is observed in a wide variety of lesions ranging from simple cysts and atypical hyperplasia to invasive metastatic stages of disease. The accurate diagnosis of breast ...apocrine carcinoma remains controversial, mainly due to the subjectivity of histopathological criteria and the lack of sensitive and specific biomarkers for reliable categorization of this subtype of breast carcinoma. Thus, many efforts are currently being made to identify novel molecular marker signature(s) that can define apocrine carcinoma with high levels of accuracy and reliability, and determine with certainty the true clinical significance of these lesions. The purpose of this article is to review the data on apocrine lesions, with an emphasis on borderline apocrine differentiation. In particular, we address relevant issues in the context of the current state of research on benign and malignant apocrine lesions of the breast, with a focus on parameters for diagnosis and potential-targeted therapeutic options.
Clinical cancer proteomics aims at the identification of markers for early detection and predictive purposes, as well as to provide novel targets for drug discovery and therapeutic intervention. ...Proteomics-based analysis of traditional sources of biomarkers, such as serum, plasma, or tissue lyzates, has resulted in a wealth of information and the finding of several potential tumor biomarkers. However, many of these markers have shown limited usefulness in a clinical setting, underscoring the need for new clinically relevant sources. Here we present a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the breast tumor microenvironment. We collected TIFs from small pieces of freshly dissected invasive breast carcinomas and analyzed them by two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Western immunoblotting, as well as by cytokine-specific antibody arrays. This approach provided for the first time a snapshot of the protein components of the TIF, which we show consists of more than one thousand proteins--either secreted, shed by membrane vesicles, or externalized due to cell death--produced by the complex network of cell types that make up the tumor microenvironment. So far, we have identified 267 primary translation products including, but not limited to, proteins involved in cell proliferation, invasion, angiogenesis, metastasis, inflammation, protein synthesis, energy metabolism, oxidative stress, the actin cytoskeleton assembly, protein folding, and transport. As expected, the TIF contained several classical serum proteins. Considering that the protein composition of the TIF reflects the physiological and pathological state of the tissue, it should provide a new and potentially rich resource for diagnostic biomarker discovery and for identifying more selective targets for therapeutic intervention.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also on factors present in the tumor microenvironment. Of the cell types that ...constitute the mammary stroma, the adipocytes are perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells. Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response; transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary heart disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Tumor interstitial fluid (TIF) is a proximal fluid that, in addition to the set of blood soluble phase-borne proteins, holds a subset of aberrantly externalized components, mainly proteins, released ...by tumor cells and tumor microenvironment through various mechanisms, which include classical secretion, non-classical secretion, secretion via exosomes and membrane protein shedding. Consequently, the interstitial aqueous phase of solid tumors is a highly promising resource for the discovery of molecules associated with pathological changes in tissues. Firstly, it allows one to delve deeper into the regulatory mechanisms and functions of secretion-related processes in tumor development. Secondly, the anomalous secretion of molecules that is innate to tumors and the tumor microenvironment, being associated with cancer progression, offers a valuable source for biomarker discovery and possible targets for therapeutic intervention. Here we provide an overview of the features of tumor-associated interstitial fluids, based on recent and updated information obtained mainly from our studies of breast cancer. Data from the study of interstitial fluids recovered from several other types of cancer are also discussed. This article is a part of a Special Issue entitled: The Updated Secretome.
► Tumor interstitial fluid (TIF) is a novel source for cancer biomarker discovery. ► Methods for recovering TIF from fresh tissue specimens are critically discussed. ► Multiple proteomic technologies that profiled TIF are discussed. ► The main key studies of TIF proteomes for several types of cancers are overviewed.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In our laboratories we are exploring the possibility of using proteome expression profiles of fresh bladder tumors (transitional cell carcinomas, TCCs; squamous cell carcinomas, SCCs) and random ...biopsies as fingerprints to subclassify histopathological types and as a starting point to search for protein markers that may form the basis for diagnosis, prognosis, and treatment. Ultimately, the goal of these studies is to identify signaling pathways and components that are affected at various stages of bladder cancer progression and that may provide novel leads in drug discovery. Here we present our ongoing efforts to establish comprehensive two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) databases of TCCs and SCCs which are being constructed based on the proteomic and immunohistochemical analysis of hundreds of fresh tumors, random biopsies and cystectomies received shortly after operation (http://biobase.dk/cgi‐bin/celis).
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Abstract
BACKGROUND
Glioblastoma (GBM) remains one of the most lethal tumors and is associated with a median survival of only approx. 15 months despite aggressive radio-chemo-therapy. One distinct ...challenge in GBM management includes diffuse location of the tumor within the brain and migration of cancerous cells into the healthy surrounding tissue preventing complete surgical removal and promoting recurrence. A subpopulation within the tumor mass, the so-called stem cells (or glioma initiating cells, GICs) are further complicating treatment strategies as this pool of cells is more invasive and is known to possess resistance to traditional therapy. It is commonly known that cancer cells utilize glucose as their main energy source, a phenomenon known as the Warburg effect. Phosphofructokinase-1 (PFK-1) is one of two rate-limiting enzymes in glycolysis and presents a potential critical check point for these glucose “addictive” cells. Additional to its glycolytic role we here propose a regulatory role for PFK-1 in motility in GICs.
METHODS
To elucidate the role of PFK-1 in GBM, we abrogated its function using shRNA-mediated knockdown or chemical inhibition. Next, we evaluated the cellular responses utilizing a broad range of in vitro assays including cell viability, apoptosis, cytoskeleton assembly, migration and invasion assay.
RESULTS
Depletion of PFK-1 in GICs led to decreased viability and increased rates of apoptosis. Furthermore, impaired function of PFK-1 affected cytoskeleton assembly and decreased migration and invasion capacity, a phenotype we were able to mimic with a chemical PFK-1 inhibitor. PFK-1-regulated motility was found to be through translational regulation of KIF11. CONCLUSION: PFK-1 regulates GBM stem cell motility and invasion through the regulation of KIF11, a motor protein belonging to the kinesin-like protein family. The novel role of PFK-1 identified in our studies, together with successful therapeutic targeting in vitro and in vivo, expands our understanding of GBM maintenance and cell motility.
It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also
on factors present in the tumor microenvironment. Of the cell types that ...constitute the mammary stroma, the adipocytes are
perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast
tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some
studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells.
Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may
come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional
gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh
fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent
mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous
signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal
transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response;
transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive
overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes
in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results
open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary
heart disease.
Full text
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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