Julio E. Celis

Up-regulated Proteins in the Fluid Bathing the Tumour Cell Microenvironment as Potential Serological Markers for Early Detection of Cancer of the Breast

Breast cancer is by far the most common diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel‐based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective breast cancer patients. The goal of this study was to identify abundant cancer up‐regulated proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra‐sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well‐matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110 proteins that were up‐regulated by a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D gels of the remaining 68 TIF samples in order to identify TIF 46 up‐regulated proteins that were deregulated in 90% or more of all the available TIFs, thus representing common breast cancer markers. This second phase singled out a set of 26 breast cancer markers, most of which were also identified by a complementary analysis using LC‐MS/MS. The expression of calreticulin, cellular retinoic acid‐binding protein II, chloride intracellular channel protein 1, EF‐1‐beta, galectin 1, peroxiredoxin‐2, platelet‐derived endothelial cell growth factor, protein disulfide isomerase and ubiquitin carboxyl‐terminal hydrolase 5 were further validated using a tissue microarray containing 70 malignant breast carcinomas of various grades of atypia. A significant number of these proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include biomarker prioritization based on the hierarchal evaluation of these markers, antibody and antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and breast cancer patients, are discussed.

A fibroblast-associated antigen: characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells.

Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10 immunoreactivity was specific to fibroblasts and smooth muscle differentiated fibroblasts within the context of vascular smooth muscle cells.

Protein Detection in Gels by Silver Staining: A Procedure Compatible with Mass Spectrometry

Publisher Summary This chapter describes a procedure that facilitates protein detection in gels by silver staining. Silver staining is one of the procedures, in addition to Coomassie blue, R and G types, and fluorescent dyes. The basic mechanisms underlying silver staining of proteins in gels are relatively well understood. Basically, protein detection depends on the binding of silver ions to the amino acid side chains, primary the sulfhydril and carboxiyl groups of proteins. The protein bands are visualized as spots where the reduction occurs and, as a result, the image of protein distribution within the gel is based on the difference in oxidation reduction potential between the gel area occupied by the proteins and the free adjacent sites. The silver amine or alkaline methods usually have lower background and, as a result, are most sensitive but require extended procedures. Acidic protocols, however, are faster but slightly less sensitive. The gel fixation and washing procedure can be carried in a staining try, but make sure that these are only used for silver staining. The size of the container has to be big enough to perform free movement of the gel during the shaking.

Molecular pathology of breast apocrine carcinomas: A protein expression signature specific for benign apocrine metaplasia

Breast cancer is a heterogeneous disease that encompasses a wide range of histopathological types including: invasive ductal carcinoma, lobular carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, and apocrine carcinoma among others. Pure apocrine carcinomas represent about 0.5% of all invasive breast cancers according to the Danish Breast Cancer Cooperative Group Registry, and despite the fact that they are morphologically distinct from other breast lesions, there are at present no standard molecular criteria available for their diagnosis. In addition, the relationship between benign apocrine changes and breast carcinoma is unclear and has been a matter of discussion for many years. Recent proteome expression profiling studies of breast apocrine macrocysts, normal breast tissue, and breast tumours have identified specific apocrine biomarkers [15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) and hydroxymethylglutaryl coenzyme A reductase (HMG‐CoA reductase)] present in early and advanced apocrine lesions. These biomarkers in combination with proteins found to be characteristically upregulated in pure apocrine carcinomas (psoriasin, S100A9, and p53) provide a protein expression signature distinctive for benign apocrine metaplasias and apocrine cystic lesions. These studies have also presented compelling evidence for a direct link, through the expression of the prostaglandin degrading enzyme 15‐PGDH, between early apocrine lesions and pure apocrine carcinomas. Moreover, specific antibodies against the components of the expression signature have identified precursor lesions in the linear histological progression to apocrine carcinoma. Finally, the identification of proteins that characterize the early stages of mammary apocrine differentiation such as 15‐PGDH, HMG‐CoA reductase, and cyclooxygenase 2 (COX‐2) has opened a window of opportunity for pharmacological intervention, not only in a therapeutic manner but also in a chemopreventive setting. Here we review published and recent results in the context of the current state of research on breast apocrine cancer.

Towards discovery‐driven translational research in breast cancer

Discovery‐driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number of novel and powerful technologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diagnosed, treated and monitored in the future. Here we present a brief report on long‐term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and targets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform ‘omic’ data sets collected from tissue samples obtained from individual patients. The ultimate goal of this initiative is to coalesce knowledge‐based complementary procedures into a systems biology approach to fight breast cancer.

High-Resolution Two-Dimensional Gel Electrophoresis of Proteins: Isoelectric Focusing and Nonequilibrium pH Gradient Electrophoresis (NEPHGE)

Publisher Summary This chapter discusses high-resolution two-dimensional (2D) gel electrophoresis method. High-resolution two-dimensional (2D) gel electrophoresis is at present considered the method with the highest resolution for the separation of complex protein mixtures such as those present in eukaryotic cells. The technique, which separates proteins in terms of their isoelectric points and molecular weights, is commonly used to identify new cellular components for example cytoskeletal proteins, organelle components, etc., and to detect alterations in their expression using qualitative and quantitative comparisons. The method allows resolving proteins having apparent molecular weights between 8.5 and 230 kDa and pI values from 4 to 12.

Molecular characterization of apocrine carcinoma of the breast: validation of an apocrine protein signature in a well-defined cohort.

Invasive apocrine carcinomas (IACs), as defined by morphological features, correspond to 0.3–4% of all invasive ductal carcinomas (IDC), and despite the fact that they are histologically distinct from other breast lesions there are currently no standard molecular criteria available for their diagnosis and no unequivocal information as to their prognosis. In an effort to address these concerns we have been using protein expression profiling technologies in combination with mass spectrometry and immunohistochemistry (IHC) to discover specific biomarkers that could allow us to molecularly characterize these lesions as well as to dissect some of the steps in the processes underlying breast apocrine metaplasia and development of precancerous apocrine lesions. Establishing these apocrine‐specific markers as best practice for the routine pathology evaluation of breast cancer, however, will require their validation in large cohorts of patients. Towards this goal we have composed a panel of antibodies against components of an apocrine protein signature that includes probes against the apocrine‐specific markers 15‐prostaglandin dehydrogenase (15‐PGDH), and acyl‐CoA synthetase medium‐chain family member 1 (ACSM1), in addition to a set of categorizing markers that are consistently expressed (AR, CD24) or not expressed (ERα, PgR, Bcl‐2, and GATA‐3) by apocrine metaplasia in benign breast lesions and apocrine sweat glands. This panel was used to analyze a well‐defined cohort consisting of 14 apocrine ductal carcinoma in situ (ADCIS), and 33 IACs diagnosed at the Cancer Institute Hospital, Tokyo between 1997 and 2001. Samples were originally classified on the basis of cellular morphology with all cases having more than 90% of the tumour cells exhibiting cytological features typical of apocrine cells. Using the expression of 15‐PGDH and/or ACSM1 as the main criterion, but taking into account the expression of other markers, we were able to identify unambiguously 13 out of 14 ADCIS (92.9%) and 20 out of 33 (60.6%) IAC samples, respectively, as being of apocrine origin. Our results demonstrate that IACs correspond to a distinct, even if heterogeneous, molecular subgroup of breast carcinomas that can be readily identified in an unbiased way using a combination of markers that recapitulate the phenotype of apocrine sweat glands (15‐PGDH+, ACSM1+, AR+, CD24+, ERα−, PgR−, Bcl‐2−, and GATA‐3−). These results pave the way for addressing issues such as prognosis of IACs, patient stratification for targeted therapeutics, as well as research strategies for identifying novel therapeutic targets for developing new cancer therapies.

Omics-based profiling of carcinoma of the breast and matched regional lymph node metastasis

Axillary lymph node (ALN) status is currently used as an important clinical indicator of breast cancer prognosis. However, the molecular mechanisms underlying lymph node metastasis are poorly understood and the relationship between ALN metastasis and the primary tumor remains unclear. In an effort to reveal structural changes in the genome and related protein responses that may drive regional metastatic progression we have analyzed matched pairs of primary breast tumors and ALN metastases both at the genomic and proteomic levels using comparative genomic hybridization (CGH) array, quantitative high‐resolution 2‐D PAGE in combination with MS, and immunohistochemistry (IHC). Array CGH revealed a remarkable similarity in genomic aberration profiles between the matched primary tumors and the ALN metastases. Quantitative profiling of 135 known proteins also revealed striking similarities in their overall expression patterns, although we observed distinct changes in the levels of individual proteins in some sample pairs. The remarkable similarities of the overall genomic and proteomic profiles between primary tumors and matched ALN metastases are taken to suggest that, in general, key biological characteristics of the primary breast tumor are maintained in the corresponding lymph node metastases. Given that the omics‐based technologies are oblivious to changes that only occur in minor cellular subsets we validated the proteomic data using IHC, which provides protein expression information with a valuable topological component. Besides confirming the omics‐derived data, the IHC analysis revealed that in two cases the ALN metastases may have been derived from a distinct minor cell subpopulation present in the primary tumor rather than from the bulk of it.

Characterization of breast precancerous lesions and myoepithelial hyperplasia in sclerosing adenosis with apocrine metaplasia

The identification as well as the molecular characterization of breast precancerous lesions in terms of increased risk of progression and/or recurrence is becoming a critical issue today as improved non‐surgical procedures are detecting cancer at an earlier stage. The strategy we have been pursuing to identify early apocrine breast lesions is based on the postulate that invasive apocrine carcinomas evolve from epithelial cells in terminal duct lobular units (TDLUs) in a stepwise manner that involves apocrine metaplasia of normal breast epithelia, hyperplasia, atypia, and apocrine carcinoma in situ. First, we identify specific protein biomarkers for benign apocrine metaplasia and thereafter we search for biomarkers that are highly overexpressed by pure invasive apocrine carcinomas. Here we present studies in which we have used antibodies against components of a benign apocrine signature that includes 15‐prostaglandin dehydrogenase (15‐PGDH), a protein that is expressed by all benign apocrine lesions, and markers that are highly overexpressed by pure invasive apocrine carcinomas such as MRP14 (S100A9), psoriasin (S100A7), and p53 to identify precancerous lesions in sclerosing adenosis (SA) with apocrine metaplasia. The latter is a benign proliferative lesion of the breast that exhibits an increase in the size of the TDLUs and characterized by retained two‐cell lining, and myoepithelial (ME) and stromal hyperplasia. SA with apocrine metaplasia, i.e. apocrine adenosis (AA), presents with a higher degree of atypical apocrine hyperplasia, and these lesions are believed to be precursors of apocrine carcinoma, in situ and invasive. Analysis of 24 selected SA samples with apocrine metaplasia revealed non‐obligate putative apocrine precancerous lesions that displayed some, or in same cases all the three markers associated with pure invasive apocrine carcinomas. These studies also revealed p53 positive, non‐apocrine putative precancerous lesions as well as novel phenotypes for ME and some luminal cells characterized by the expression of cytokeratin 15.

A single lysis solution for the analysis of tissue samples by different proteomic technologies

Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a “one‐size fits all” solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array‐based proteomics (reverse‐phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel‐based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and tongue. This solution when combined with a specific sample preparation technique – cryostat sectioning of frozen specimens – simplifies tissue sample preparation and solves most of the difficulties associated with the integration of data generated by different proteomic technologies.