Project title: New analytical and diagnostic approaches towards identification of apocrine and holocrine mechanisms

Grant designation: EEA-NFM SK-0086/3655/2009/ORINFM

Outline

The proposed research is expected to bring essential new information on apocrine and holocrine secretion in health and disease. In contrast to classical and well known merocrine secretion (exocytosis) which is an example of very intensely studied secretory mechanism with many dozens of identified factors to be involved, until today no protein components, factors or genes affecting apocrine and holocrine secretion were identified. Whole group of various infectious, eczema and tumour diseases of dermatogenic origin is accompanied with substantial change in their apocrine/holocrine secretory activity, however, without any knowledge of their etiology and causality. In some cases presence and extend of the apocrine secretory activity, so far identified routinely only by histological observation, can serve as important differential diagnostic marker. However, to use apocrine/holocrine activity as potentially main diagnostic tool or even to find the way how to treat its unwanted and adverse effects, it is necessary to characterize molecular and genetic clues of this process. Initially, studying programmed cell death in Drosophila, widely used and well-characterized genetic model, we have found that final executive phase of their salivary glands that is controlled by tumour suppressor genes, is preceded by very intense apocrine secretory cycle. This opened a new vistas for researching this so far uncharacterized process. This project proposal is aimed to characterize proteins and their coding genes that are involved in the apocrine and holocrine secretion by using very modern and update genomic and proteomic tools. We hope to identify determinants that are common to apocrine and holocrine activity in both, the humans and the model metazoan organism, like Drosophila, which we expect that share high degree of orthology similarly to exocytosis. Since this is based on our own findings, the first data of which were published, and it has not been investigated elsewhere until now, the project proposal is unique and innovative, and bears high potential to bring novel and signifcant data that are directly related to specific pathologies. We hope that project can importantly contribute to solving problems associated with apocrine and holocrine diseases, and that crucial new data that will help to address problems associated with these secretory disorders.

Brief project description

In the course of our research on programmed cell death we have unexpectedly discovered that several years ago for the first time described extrusion of cellular components from doomed Drosophila salivary glands is a process of apocrine secretion when whole pieces of cellular contents are released into extracellular space. The apocrine secretion as a type of cell contents release has been described morphologically and defined as such some 178 years ago, however, until today no protein components, factors or genes affecting apocrine secretion were identified. In contrast, widespread and well known exocytosis is an example of very intensely studied secretory mechanism with many dozens of identified factors to be involved (Jahn, 2004; Rutter and Tsuboi, 2004; Sudhof, 2004; Chieregatti a Meldolesi, 2005; Barclay et al., 2005; Snyder a Kelly, 2006; Westerink, 2006). In textbooks, the apocrine secretion is frequently described in association with lactation activity of mammary glands, Harderian gland, and of some exocrine glands (Satoh et al., 1996; Gesase a Satoh, 2003) or notably as a differential diagnostic marker for some benign metaplasias and in many dermatogenic cancers (Griffith, 2005; Khalbuss, 2005; Shah, 2005; Crowson et al., 2006). It should be noted that, for example, epithelial tissue of the mammary gland in humans where milk is released by apocrine mechanism is known to undergo widespread involution after lactation is completed, and this resembles strongly many features of apoptosis. However, to use apocrine/holocrine activity as potentially main diagnostic tool or even to find the way how to treat its unwanted and adverse effects, it is necessary to characterize molecular and genetic clues of this process.
Proposed project is based on ah experience of the research team in this field including methodology that have been build during basic research activity. The main goal of the project is to significanlty contribute to selfsustaining development and wide-spread application of new knowledge and novel methods in biomedical research and practical medicine itself. The proposed research is expected to bring essential new information on apocrine and holocrine secretion in health and disease. Project is based on broad application of proteomic and subsequently genomic methods to identify main and crucial components of apocrine and holocrine secretion that as we expect will share common determinants between different cell types and even evolutionary different species.
Similarly as incidence of skin diseases, notably skin tumours, also incidence of breast cancer is steadily increasing not only in Slovakia but world-wide. Several skin diseases are listed among those which are cited to be apocrine-coupled: apocrine hydrocystoma, cylindromatosis, various apocrine gland cysts with hemosiderotic dermatofibromas, lipomatous mixed tumor of the skin, bilateral axillary metachronous apocrine carcinomas, poromatosis, focal axillary hyperhidrosis, hypohidrotic ectodermal dysplasia, and various forms of almost two centuries known hidradenitis suppurativa. There are also several apocrine-associated types of breast cancer: deciduoid-like myofibroblastoma, fibroadenomatous cancer, and mammary-type fibroepithelial neoplasms. Some salivary gland tumours like intraductal carcinoma, micropapillary mucinous carcinoma, pleomorphic adenoma or sclerosing polycystic adenosis are also known to show positive apocrine histology. This short list provide strong evidence how important basic as well as applied research to this field is to facilitate understanding of apocrine and/or holocrine diseases. The role of research, preventive and clinical medicine from this point of view is to gain insights into molecular mechanism of the apocrine and holocrine processes which finally can contribute to highly individualized and specific treatment of every diagnosed patient that is in good accordance with world-leading trends in this field.
One of the main overal goals is to identify molecular determinant (markers) of listed diseases with special emphasis on factors common to apocrine and holocrine secretion. There is no doubt that in spite of different etiology and histogenetic origin of mentioned diagnoses, the unifying or common feature is apo/holocrine secretory activity, and it is assumed that this mechanism of cellular contents release is controlled by identical or highly related components of the proteome. This assumption is based on our findings from initial phases of the study we have been undertaking several months ago in model organisms as a part of basic research. For the proposed project, in collaboration with clinicians, material collection (exogenous secretions, mucus, sweat, cerumens, milk etc.) from healthy volunteers and from patients is planned. Because all these samples are exosecretory materials, their collecting is straightforward, very easy and noninvasive. After collecting and storage of the material (for the shortest necessary time at -80°C), samples are being extracted for their protein and lipid components, followed by separation of the fractions (using appropriate analytical methods including SDS-PAGE, 2D PAGE and nano-HPLC, GC), and subsequently identified, where especially in the case of proteins mass spectrometry (MALDI/TOF-TOF) will be employed. 
The regular part of this project should be also research to identify orthologous molecules in model organisms that are more amenable to genetic analysis and to characterize previously unknown but during this study uncovered proteins at their functional genetic level. It will greatly help to understand molecular mechanisms underlying diseases associated with apocrine and holocrine glands, and potentially further facilitate diagnosis and improve treatments.

References

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