Elucidating the molecular mechanisms underlying pSS and pSS/MALT lymphoma remains an important challenge. As a result of the dearth of molecular markers for pSS, the diagnosis of this devastating disease often lags behind disease onset by almost a decade. The objective diagnostic criterion of lymphocytic infiltration requires an invasive biopsy procedure, though it is currently a routine procedure in the diagnostic work-up for SS (3
). The gain in knowledge regarding the cellular mechanisms of T and B lymphocyte activity in pathogenesis of pSS has resulted in novel opportunities (e.g., anti-TNF-alpha, IFN- alpha, anti-CD20, anti-CD22 and anti-B cell-activating factor) for therapeutic intervention, however none of these agents have been approved yet for treatment of pSS and pSS/MALT (23
). Understanding the molecular basis of pSS and pSS/MALT is critical for improving target-based therapies and developing diagnostic and prognostic criteria (e.g., lymphoma development). We show that a systems-level analysis of high-throughput gene expression and proteomics data can implicate disease-related pathways and their key constituents.
In this study, we first identified gene co-expression modules related to pSS or pSS/MALT based on analysis of gene expression data with WGCNA. Using this method, we then determined the intramodular connectivity for each gene, which measures how connected, or coexpressed, a given gene is with respect to the genes of a particular module (6
). This provided highly connected intramodular hub genes that are most significant for maintaining the structure and function of the disease modules. This was followed by conducting GO and functional pathway analysis to understand the most significant biological processes and pathways associated with the disease modules. Finally, a panel of candidate key target genes for pSS and pSS/MALT pathogenesis, as implicated by both WGCNA and proteomic analysis are presented for future biological validation.
GO analysis indicated that the pSS-associated modules (Turquoise, Brown and Red) are significantly enriched with genes known to be involved in immune/defense response, antigen presentation/processing, apoptosis and cell death pathways. This observation is consistent with our previous study, which revealed the salivary mRNAs associated with apoptosis and immune responses in pSS patients (27
). The most significant GO terms (biological processes) were well preserved between pSS/MALT-associated modules (Turquoise, Red, Yellow, Brown and Green) and pSS-associated modules (Turquoise, Red and Bronw), although they showed dissimilar significances. The fact that unsupervised clustering based on a co-expression measure resulted in modules enriched for biologically important processes suggests that these modules are a robust feature of the molecular architecture of pSS and pSS/MALT.
Proteasome degradation appears to be the most significant pathway associated with the pSS modules. Since the proteasome system has a pivotal role in the control of the immune response, it might be involved in the pathogenesis of autoimmune disorders such as pSS. In fact, recent studies have shown that the expression of proteasome subunits LMP2 and LMP7 were significantly altered in pSS (28
). Functional pathway analysis also suggested that apoptosis of epithelial cells in parotid glands significantly contributes to the pathogenesis of pSS. Apoptotic cell death may be induced by cytotoxic T cells through the release of perforin and granzymes. Cleavage of certain autoantigens during apoptosis such as caspase-mediated proteolysis of alpha-fodrin and poly(ADP-ribose) polymerase (PARP) may reveal immunocryptic epitopes that could potentially induce autoimmune response and lead to tissue destruction on the development of pSS (30
). Apoptosis can also be induced by the interaction of Fas ligand (FasL/CD95L), expressed by T lymphocytes, with Fas (Apo-1/CD95) on epithelial cells. Fas ligand (FasL), and its receptor Fas are essential in the homeostasis of the peripheral immune system (32
). All of these previously reported genes, including CD40, Fas, FasL, perforin, caspases and PARP, were found significantly altered in pSS in our study, which further confirmed the role of apoptotic pathways for impaired function of salivary glandular cells in pSS. In pSS/MALT-associated modules, two most significant pathways are translation and ribosome pathways. However, most of the other pathways (e.g., proteasome degradation pathway) were highly preserved between pSS and pSS/MALT modules. Considering the limited sample size and heterogeneous cell population in the disease patients’ parotid gland biopsies, further studies are required to confirm these findings regarding key target genes and activated signal pathways. Since MALT lymphoma only constitutes about half the lymphomas, it may also be interesting to look into those discovered genes between lymphoma with pSS and without pSS.
The proteins elevated in pSS patients are functionally related to immune/defense response, apoptosis, cell-cell adhesion and anti-oxidative stress whereas many of the proteins up-regulated in pSS/MALT lymphoma are related to signal transduction, gene regulation, apoptosis, immune response, and oxidative stress. Some of these targets have been linked to lymphoma previously, and two cancer-related proteins, Rho-GDP dissociation inhibitor (Rho-GDI) and cyclophilin A (CypA), are particularly of biological significance. The over-expression of Rho-GDI has been observed in most of the human tumors and may associate with the development of drug resistance in breast and lymphoma cancer cells (33
). In fact, Rho-GDI is an anti-apoptotic molecule that mediates cellular resistance to chemotherapy agents. The mechanism for the anti-apoptotic activity of Rho-GDI may derive from its ability to inhibit caspase-mediated cleavage of Rac1 GTPase. CypA has been reported to be over-expressed in cancer cells, especially in solid tumors. Over-expression of CypA prevented hypoxia- and chemo-induced apoptosis, and this was associated with the suppression of reactive oxygen species generation and depolarization of mitochondrial membrane potential (34
). Both Rho-GDI and CypA were highly connected genes based on WGCNA, and they showed concordant over-expression in pSS/MALT lymphoma than in pSS and non-pSS controls based on proteomic and transcriptomic analyses.
The systems approach led to the discovery and identification of a number of candidate genes that are bears the potential to be key molecular targets for pSS and pSS/MALT pathogenesis in parotid glands. The fact that integrated WGCNA with proteome analysis identified the known disease-related genes provides a proof-of-principle of the systems biologic strategy in studying pSS and its progression. These candidate genes, previously linked or unlinked to pSS or pSS/MALT lymphoma, can now be tested for their biological involvement by developing models for in vitro and in vivo testing. This process will be iterative (model building and testing) and will eventually lead to the identification of key molecular targets of pSS and pSS/MALT pathogenesis.
In summary, this paper provides proof-of-concept data that a systems analysis of pSS and pSS/MALT lymphoma using tools of transcriptomics, proteomics and WGCNA have led to the identification of distinct biological pathways and key target candidate genes related to pSS pathogenesis and lymphoma progression. We found consistent disease-related features with respect to molecular networks involved in various aspects of proteasome degradation, immune/defense response, apoptosis, cell signaling, gene regulation, and oxidative stress. It has become clear in the past decade that the initiation of autoimmunity is a multifactorial and complex process that requires genetic components to synergize with multi-etiological events (35
). With the large scale gene expression and proteomic profiling and the use of systems biologic methods such as WGCNA to integrate multi-dimensional data, we have narrowed down the co-expression modules and associated gene determinants based on their interaction, concordance, and connectivity. The synergetic role of the identified candidate key target genes in modules/pathways can now be further validated with the use of animal or cell models, and will help elucidate the molecular mechanisms. The discovered candidate genes, when validated, can be translated into early diagnosis/prognosis biomarkers and molecular targets for therapeutic intervention.