Host response to human breast Invasive Ductal Carcinoma (IDC) as observed by changes in the stromal proteome.

Following initial transformation, tumorigenesis, growth, invasion, and metastasis involves a complex interaction between the transformed tissue and the host, particularly in the microenvironment adjacent to the developing tumor. The tumor microenvironment itself is a unique outcome of the host reacting to the tumor and perhaps the tumor reacting to the host and in turn the tumor altering the host's response to give rise to an environment that ultimately promotes tumor progression. The tumor-adjacent stromal, sometimes referred to as "reactive stromal" or the desmoplastic stroma, has received some investigative studies, but it is incomplete, and likely different tumors promote a varied response and hence different reactive stroma. In this study, we have investigated the proteomics of the host response, both in vitro and in vivo, to breast epithelial cancer, in the former using tissue culture and in the latter laser microdissection of stromal tissue both adjacent and distal to breast invasive ductal cancer (IDC). From proteomic analysis of in vitro tissue culture studies, we observed that the stroma produced is related to the invasiveness of the stimulating breast cancer cell lines but different from that observed from the stromal proteome of archival tissue. In vivo we have identified several potential markers of a reactive stroma. Furthermore, we observed that the proteome of tumor-adjacent stroma differs from that of tumor-distal stroma. The proteomic description of human breast IDC stroma may serve to enhance our understanding of the role of stroma in the progression of cancer and may suggest potential mechanisms of therapeutic interdiction.

Proteomic anatomy of human skin.

The extracellular matrix is composed of a variety of proteins which are essential for growth, wound healing, and fibrosis. It provides both structural support as well as contributing to the regulation of the local microenvironment. To further characterize the molecular composition of human skin we have undertaken a proteomic approach to identify proteins in three skin regions from two locations. Using laser microdissection, extracellular matrix was obtained from three distinct regions (basement membrane, papillary dermis, and reticular dermis) of formalin-fixed, paraffin embedded tissue from normal human leg and breast skin. The proteome of these regions was determined by mass spectrometric analysis. This study provides a relative quantitative assessment, including protein composition and relative abundance, of the proteins found in different skin regions giving rise to a "proteomic anatomy" of skin. Our findings indicate that there was little difference detected in the subproteomes of the dermal and papillary regions and little difference between the proteomes of leg skin compared to breast skin. One finding of interest is the identification of tenascin-X only in the breast dermis and serum amyloid P-component in the leg dermis. The results of this proteomic analysis corroborate much of the information on the protein composition identified by other methodologies found in the literature but provide additional insight as to localization of skin proteins in the various regions of skin. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments. BIOLOGICAL SIGNIFICANCE: This investigation underscores the power of proteomics to bring semiquantitative, non-presumptive molecular characterization to the field of histological anatomy. Traditionally, anatomy relied on visual or histochemical structural characterization which generally involved some level of understanding of the area of interest. With the advent of laser microdissection or laser capture microscopy localization of anatomical structures of interest can be correlated to molecular composition by virtue of mass spectrometric determination of the proteome of that structure. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments.

Evaluation of molecular markers of mesenchymal phenotype in melanoma.

The epithelial to mesenchymal transition is a developmental process allowing epithelial cells to dedifferentiate into cells displaying mesenchymal phenotypes. The pathological role of epithelial to mesenchymal transition has been implicated in invasion and metastasis for numerous carcinomas, yet limited data exist addressing whether mesenchymal transition (MT) occurs in malignant melanoma cells. Our group developed an in-vitro three-dimensional culture system to address MT in melanoma cells upon transforming growth factor-ß/ tumor necrosis factor-α treatment. Loss of E-cadherin is one of the best indicators of MT in epithelial cells. Not surprisingly, E-cadherin was expressed in only three of 12 (25%) melanoma cell lines and all three mesenchymal proteins, N-cadherin, vimentin, and fibronectin, were expressed by seven (58%) melanoma cell lines. However, after cytokine treatment, two or more mesenchymal proteins were elevated in nine (75%) melanoma cell lines. Data support the transforming growth factor-ß production by melanoma cells which may induce/support MT. Evaluation of E-cadherin, N-cadherin, and Snail expression in melanoma tissue samples are consistent with an inverse coupling of E-cadherin and N-cadherin expression, however, there are also examples suggesting a more complex control of their expression. These results indicate that malignant melanoma cell lines are susceptible to MT after cytokine treatment and highlight the importance of understanding the effects of cytokines on melanoma to undergo MT.

Predominant occupation of the class I MHC molecule H-2Kwm7 with a single self-peptide suggests a mechanism for its diabetes-protective effect.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing pancreatic beta cells. In both humans and the non-obese diabetic (NOD) mouse model of T1D, class II MHC alleles are the primary determinant of disease susceptibility. However, class I MHC genes also influence risk. These findings are consistent with the requirement for both CD4(+) and CD8(+) T cells in the pathogenesis of T1D. Although a large body of work has permitted the identification of multiple mechanisms to explain the diabetes-protective effect of particular class II MHC alleles, studies examining the protective influence of class I alleles are lacking. Here, we explored this question by performing biochemical and structural analyses of the murine class I MHC molecule H-2K(wm7), which exerts a diabetes-protective effect in NOD mice. We have found that H-2K(wm7) molecules are predominantly occupied by the single self-peptide VNDIFERI, derived from the ubiquitous protein histone H2B. This unexpected finding suggests that the inability of H-2K(wm7) to support T1D development could be due, at least in part, to the failure of peptides from critical beta-cell antigens to adequately compete for binding and be presented to T cells. Predominant presentation of a single peptide would also be expected to influence T-cell selection, potentially leading to a reduced ability to select a diabetogenic CD8(+) T-cell repertoire. The report that one of the predominant peptides bound by T1D-protective HLA-A*31 is histone derived suggests the potential translation of our findings to human diabetes-protective class I MHC molecules.

The utility of ETD mass spectrometry in proteomic analysis.

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.

Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy.

Alterations in phosphorylation of cellular proteins are a hallmark of malignant transformation. Degradation of these phosphoproteins could generate cancer-specific class I MHC-associated phosphopeptides recognizable by CD8+ T lymphocytes. In a comparative analysis of phosphopeptides presented on the surface of melanoma, ovarian carcinoma, and B lymphoblastoid cells, we find 5 of 36 that are restricted to the solid tumors and common to both cancers. Differential presentation of these peptides can result from differential phosphorylation of the source proteins. Recognition of the peptides on cancer cells by phosphopeptide-specific CD8+ T lymphocytes validates the potential of these phosphopeptides as immunotherapeutic targets.