ß-Catenin Is a Novel Marker of the Aggresome and Suggests Disruption of the Proteasome-Aggresome-Autophagasome Pathway in Multiple Myeloma In Vivo.

The ubiquitin-proteasome system (UPS) is responsible for protein catabolism by recognizing misfolded and ubiquitin-tagged polypeptides for degradation through the proteasome. In the event of translational errors or insufficient chaperone proteins, newly synthesized peptides fold improperly from their native conformation. In most cellular contexts, the UPS is sufficient to handle normal protein synthesis, misfolding, and turnover. However, exceeding the capacity of the UPS to degrade proteins causes misfolded and ubiquitinated polypeptides to accumulate in cytosolic aggregates, which can amass to form a structure termed the aggresome. While initially providing a cytoprotective effect, the prolonged presence of aggresomes impairs the UPS and eventually leads to cell death. Dense intracellular protein deposits in aggresomes have been linked to the etiology of a number of neurodegenerative disorders. Plasma cells offer a unique system to study a stressed UPS due to their extremely high rate of immunoglobulin synthesis and degree of hyper somatic mutation of variable light chain regions, increasing the probability of protein misfolding. Furthermore, emerging therapies targeting the proteasome, including bortezomib, have shown clinical effectiveness for patients with relapsed multiple myeloma (MM). In order to gain a more complete understanding of the UPS in myeloma, we investigated the presence of aggresomes both in vitro and in vivo. In addition, we document ß-catenin as a novel marker of aggresomes in tumor plasma cells. Double immunoflorescence studies in MM cell lines using a ß-catenin antibody revealed colocalization with established aggresomal markers ubiquitin and HDAC6. Aggresome frequency increased upon treatment of proteasome inhibitors. To further validate the biological significance of aggresome formation in vivo, we performed immunohistochemical analysis on tissue microarrays of MGUS and MM bone marrow biopsies. Using a CD138 antibody as a marker of plasma cells, we documented ß-catenin staining in aggresomes of malignant, but not normal plasma cells. Scanning electron microscopy confirmed the presence of aggresomes in tumor plasma cells. The frequency and intensity of aggresome staining was correlated with clinical evolution, as few were detected in the pre-malignant condition of MGUS, but clearly detectable in nearly all MM cases. We have found that the number of aggresomes in cell lines increases when cells are transplanted into the SCID-human xenograft mouse model of MM. This finding suggests that a signal emanating from the stroma may influence the UPS and aggresome formation in vivo. We extended our study to other plasma cell dyscrasia including plasmacytoma and lymphoplasmacytic lymphoma cases to demonstrate the presence of aggresomes, but to a lesser extent than MM. These data indicate possible defects in any of the junctures in the protein degradation pathway including the proteasome, aggresome, and autophagasome/lysosome. On-going experiments are investigating the correlation of the lysosomal defect in Gaucher’s disease associated with increased risk of MM. This study represents the first in vivo documentation of aggresome formation in lymphoid malignancies, providing new insights into disease pathogenesis.