Quantitative Proteomic Profiling of Primary Cutaneous CD30+ T-Cell Lymphoma Progression.

Primary cutaneous CD30+ T-cell lymphoproliferative disorders (LPD) represent a complex spectrum of related conditions that are not very well understood. Many may progress to more aggressive or systemic lymphomas. The factors involved in the progression of these LPDs are largely unknown. Advances in robust high throughput proteomics techniques permit elucidation of the molecular mechanisms underlying disease development and progression. In this study we applied a quantitative proteomics methodology involving isotope coded affinity tag reagents (ICAT™), 3-dimensional liquid chromatography (3-D LC), and tandem mass spectrometry (MS/MS) to evaluate the differential protein expression patterns of two unique T-cell lymphoma derived cell lines. The Mac-1 and Mac-2A are clonally related cell lines derived from the same cutaneous T-cell lymphoma (CTCL) at relatively indolent versus aggressive stages of tumor progression, respectively. Equivalent quantities of total cell lysates obtained from the two cell lines were labeled with cleavable ICAT™ and dialyzed prior to digestion with proteinase K. The labeled peptides were then subjected to strong cation-exchange (SCX) chromatography followed by further purification of each offline fraction using avidin affinity chromatography. These cysteine enriched ICAT™ labeled peptides were analyzed by reverse phase nanospray LC-MS/MS. Differential expression of 490 unique proteins was determined. Of these, a total of 59 proteins showed a 1.5 fold or greater increase in abundance while 86 proteins exhibited a similar decrease in the clinically aggressive lymphoma cells as compared to the more indolent form. Functional classification of the differentially expressed proteins revealed increased expression of those involved in cell signaling (neurotrophic tyrosine kinase receptor, ATP binding cassette transporter 1, SH3 protein interacting with NCK), transcription (HFK1, general transcription factor IIIC), and cell adhesion (ICAM, protocadherin) in the advanced lymphoma. There was also decreased expression in the advanced lymphoma of proteins which were involved in apoptosis (caspase 8), cell signaling (STAT4, LIM kinase), cell cycle regulation (CDC10 homolog, ankyrin 3), cell adhesion (laminin, periostin), and ion transport (voltage-gated potassium channel). Overexpression of MALT1/paracaspase that leads to activation of the NF-kappaB pathway was observed in the advanced lymphoma. Proteins such as Smad4 which is involved in regulation of the TGF-beta signaling pathway and ubiquitin specific protease of the ubiquitin-proteasomal degradation pathway that in turn regulates Smads were differentially expressed. Some of the proteins identified by MS were selected for confirmation by Western blot analysis. This study implicates deregulation of multiple signal transduction pathways during progression of CTCL and provides novel insights into the underlying molecular pathogenetic mechanisms involved.