Selective autophagy receptor p62 promotes antibacterial and antiviral immunity in common carp (Cyprinus carpio).

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.

Preparation of polyclonal antibodies to chicken P62 protein and its application in nephropathogenic infectious bronchitis virus-infected chickens.

p62, also known as SQSTM1, has been shown to be closely related to the coronavirus. However, it remains unclear on the relationship between p62 and NIBV infection. Moreover, there are no available antibodies against the chicken p62 protein. Thus, this study aimed to prepare p62 polyclonal antibody and investigate the correlation between the p62 protein and NIBV infection. Here, PET-32a-p62 prokaryotic fusion expression vector was constructed for prokaryotic protein expression, and then p62 polyclonal antibody was prepared by immunizing rabbits. Lastly, these antibodies were then utilized in Western blotting (WB), immunohistochemistry (IHC), and immunofluorescence (IF) assays. The results showed that we successfully prepared chicken p62 polyclonal antibody. Meanwhile, WB and IF demonstrated that the expression of p62 showed a trend of first increase and then decrease after NIBV infection. IHC showed that the expression of p62 in the spleen, lung, kidney, bursa of Fabricius and trachea of chickens infected with NIBV in 11 dpi was significantly higher than that of normal chickens. Taken together, this study successfully prepared a polyclonal antibody for chicken p62 protein and confirmed its application and expression in chickens, as well as the expression of p62 in tissues after NIBV infection.

The identification of high-performing antibodies for Sequestosome-1 for use in Western blot, immunoprecipitation and immunofluorescence.

Sequestosome-1, encoded by the gene SQSTM1, functions as a bridge between ubiquitinated proteins and the proteasome or autophagosome, thereby regulating protein degradation pathways. Loss of Sequestosome-1 is hypothesized to enhance neurodegeneration progression in several diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal disorders (FTD). Sequestosome-1 reproducible research would be facilitated with the availability of well-characterized anti-Sequestosome-1 antibodies. In this study, we characterized seventeen Sequestosome-1 commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Single-domain antibody screening by isPLA-seq.

Single-domain antibody (sdAb) holds the promising strategies for diverse research and translational applications. Here, we describe a method for the adaptation of the in situ proximity ligation assay (isPLA) followed by sequencing (isPLA-seq) to facilitate screening of a high-sensitive, high-throughput sdAb library for a given protein at subcellular and single-cell resolution. Based on the sequence of complementarity-determining region 3 (CDR3), the recombinant sdAb can be produced for in vitro and in vivo utilities. This method provides a general means to identify the functional measure of sdAb and its complementary epitopes and its potential applications to investigate cellular processes.

Clostridium butyricum Alleviates Enterotoxigenic Escherichia coli K88-Induced Oxidative Damage Through Regulating the p62-Keap1-Nrf2 Signaling Pathway and Remodeling the Cecal Microbial Community.

Clostridium butyricum (CB) can enhance antioxidant capacity and alleviate oxidative damage, but the molecular mechanism by which this occurs remains unclear. This study used enterotoxigenic Escherichia coli (ETEC) K88 as a pathogenic model, and the p62-Keap1-Nrf2 signaling pathway and intestinal microbiota as the starting point to explore the mechanism through which CB alleviates oxidative damage. After pretreatment with CB for 15 d, mice were challenged with ETEC K88 for 24 h. The results suggest that CB pretreatment can dramatically reduce crypt depth (CD) and significantly increase villus height (VH) and VH/CD in the jejunum of ETEC K88-infected mice and relieve morphological lesions of the liver and jejunum. Additionally, compared with ETEC-infected group, pretreatment with 4.4×106 CFU/mL CB can significantly reduce malondialdehyde (MDA) level and dramatically increase superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in the serum. This pretreatment can also greatly increase the mRNA expression levels of tight junction proteins and genes related to the p62-Keap1-Nrf2 signaling pathway in the liver and jejunum in ETEC K88-infected mice. Meanwhile, 16S rDNA amplicon sequencing revealed that Clostridium disporicum was significantly enriched after ETEC K88 challenge relative to the control group, while Lactobacillus was significantly enriched after 4.4×106 CFU/mL CB treatment. Furthermore, 4.4×106 CFU/mL CB pretreatment increased the short-chain fatty acid (SCFA) contents in the cecum of ETEC K88-infected mice. Moreover, we found that Lachnoclostridium, Roseburia, Lactobacillus, Terrisporobacter, Akkermansia, and Bacteroides are closely related to SCFA contents and oxidative indicators. Taken together, 4.4×106 CFU/mL CB pretreatment can alleviate ETEC K88-induced oxidative damage through activating the p62-Keap1-Nrf2 signaling pathway and remodeling the cecal microbiota community in mice.

Autophagy receptors as viral targets.

Activation of autophagy is part of the innate immune response during viral infections. Autophagy involves the sequestration of endogenous or foreign components from the cytosol within double-membraned vesicles and the delivery of their content to the lysosomes for degradation. As part of innate immune responses, this autophagic elimination of foreign components is selective and requires specialized cargo receptors that function as links between a tagged foreign component and the autophagic machinery. Pathogens have evolved ways to evade their autophagic degradation to promote their replication, and recent research has shown autophagic receptors to be an important and perhaps previously overlooked target of viral autophagy inhibition. This is a brief summary of the recent progress in knowledge of virus-host interaction in the context of autophagy receptors.

Targeting XPO1 enhances innate immune response and inhibits KSHV lytic replication during primary infection by nuclear stabilization of the p62 autophagy adaptor protein.

Nucleocytoplasmic transport of signaling modulators is essential for regulating cellular responses to extracellular stimulation and stress, as well as pathogen infection. Exportin 1 (XPO1), also known as chromosomal maintenance 1 (CRM1), mediates nuclear export of proteins, rRNAs, snRNAs, and some mRNAs. In this study, we have identified an essential role of XPO1 in regulating Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication during primary infection of primary human umbilical vein endothelial cells. Treatment with an XPO1 inhibitor KPT-8602 and short hairpin RNA (shRNA)-mediated knockdown of XPO1 reduced KSHV lytic replication but had no effect on KSHV entry and trafficking. XPO1 inhibition induced retention of autophagy adaptor protein p62 (SQSTM1) in the nucleus, which enhanced activation of TBK1 and IRF3. As a result, nuclear accumulation of p62 increased expression of innate immune-related genes including IRF7, ISG15, IFIT1, IFIT2, and IFIT3, leading to a reduction of KSHV lytic replication. These results illustrate a novel mechanism by which XPO1 mediates innate immune response and KSHV replication, and identify XPO1 as a potential therapeutic target and KPT-8602 as a promising therapeutic agent for KSHV infection.

Legionella pneumophila Excludes Autophagy Adaptors from the Ubiquitin-Labeled Vacuole in Which It Resides.

Xenophagy targets intracellular pathogens for destruction by the host autophagy pathway. Ubiquitin chains are conjugated to xenophagic targets and recruit multiple autophagy adaptors. The intracellular pathogen Legionella pneumophila resides in a vacuole that is ubiquitinated; however, this pathogen avoids xenophagic detection. Here, the mechanisms by which L. pneumophila can prevent the host xenophagy pathway from targeting the vacuole in which it resides were examined. Ubiquitin-labeled vacuoles containing L. pneumophila failed to recruit autophagy adaptors by a process that was independent of RavZ function. Coinfection studies were conducted using a strain of Listeria monocytogenes that served as a robust xenophagic target. Legionella pneumophila infection blocked xenophagic targeting of L. monocytogenes by a RavZ-dependent mechanism. Importantly, when coinfection studies were conducted with a RavZ-deficient strain of L. pneumophila, L. monocytogenes was targeted by the host xenophagy system but vacuoles containing L. pneumophila avoided targeting. Enhanced adaptor recruitment to the vacuole was observed by using a strain of L. pneumophila in which all of the effector proteins in the SidE family were deleted; however, this strain was still not targeted by the host autophagy pathway. Thus, there are at least two pathways by which L. pneumophila can disrupt xenophagic targeting of the vacuole in which it resides. One mechanism involves global disruption of the host autophagy machinery by the effector protein RavZ. A second cis-acting mechanism prevents the binding of autophagy adaptors to the ubiquitin-decorated surface of the L. pneumophila-containing vacuole.

Generation and Characterization of Novel Monoclonal Antibodies Targeting p62/sequestosome-1 Across Human Neurodegenerative Diseases.

Human neurodegenerative diseases can be characterized as disorders of protein aggregation. As a key player in cellular autophagy and the ubiquitin proteasome system, p62 may represent an effective immunohistochemical target, as well as mechanistic operator, across neurodegenerative proteinopathies. In this study, 2 novel mouse-derived monoclonal antibodies 5G3 and 2A5 raised against residues 360-380 of human p62/sequestosome-1 were characterized via immunohistochemical application upon human tissues derived from cases of C9orf72-expansion spectrum diseases, Alzheimer disease, progressive supranuclear palsy, Lewy body disease, and multiple system atrophy. 5G3 and 2A5 reliably highlighted neuronal dipeptide repeat, tau, and α-synuclein inclusions in a distribution similar to a polyclonal antibody to p62, phospho-tau antibodies 7F2 and AT8, and phospho-α-synuclein antibody 81A. However, antibodies 5G3 and 2A5 consistently stained less neuropil structures, such as tau neuropil threads and Lewy neurites, while 2A5 marked fewer glial inclusions in progressive supranuclear palsy. Both 5G3 and 2A5 revealed incidental astrocytic tau immunoreactivity in cases of Alzheimer disease and Lewy body disease with resolution superior to 7F2. Through their unique ability to highlight specific types of pathological deposits in neurodegenerative brain tissue, these novel monoclonal p62 antibodies may provide utility in both research and diagnostic efforts.

Common Neurodegeneration-Associated Proteins Are Physiologically Expressed by Human B Lymphocytes and Are Interconnected via the Inflammation/Autophagy-Related Proteins TRAF6 and SQSTM1.

There is circumstantial evidence that, under neurodegenerative conditions, peptides deriving from aggregated or misfolded specific proteins elicit adaptive immune responses. On another hand, several genes involved in familial forms of neurodegenerative diseases exert key innate immune functions. However, whether or not such observations are causally linked remains unknown. To start addressing this issue, we followed a systems biology strategy based on the mining of large proteomics and immunopeptidomics databases. First, we retrieved the expression patterns of common neurodegeneration-associated proteins in two professional antigen-presenting cells, namely B lymphocytes and dendritic cells. Surprisingly, we found that under physiological conditions, numerous neurodegeneration-associated proteins are abundantly expressed by human B lymphocytes. A survey of the human proteome allowed us to map a unique protein-protein interaction network linking common neurodegeneration-associated proteins and their first shell interactors in human B lymphocytes. Interestingly, network connectivity analysis identified two major hubs that both relate with inflammation and autophagy, namely TRAF6 (TNF Receptor Associated Factor 6) and SQSTM1 (Sequestosome-1). Moreover, the mapped network in B lymphocytes comprised two additional hub proteins involved in both inflammation and autoimmunity: HSPA8 (Heat Shock Protein Family A Member 8 also known as HSC70) and HSP90AA1 (Heat Shock Protein 90 Alpha Family Class A Member 1). Based on these results, we then explored the Immune Epitope Database "IEDB-AR" and actually found that a large share of neurodegeneration-associated proteins were previously reported to provide endogenous MHC class II-binding peptides in human B lymphocytes. Of note, peptides deriving from amyloid beta A4 protein, sequestosome-1 or profilin-1 were reported to bind multiple allele-specific MHC class II molecules. In contrast, peptides deriving from microtubule-associated protein tau, presenilin 2 and serine/threonine-protein kinase TBK1 were exclusively reported to bind MHC molecules encoded by the HLA-DRB1 1501 allele, a recently-identified susceptibility gene for late onset Alzheimer's disease. Finally, we observed that the whole list of proteins reported to provide endogenous MHC class II-binding peptides in human B lymphocytes is specifically enriched in neurodegeneration-associated proteins. Overall, our work indicates that immunization against neurodegeneration-associated proteins might be a physiological process which is shaped, at least in part, by B lymphocytes.

Autophagy precedes apoptosis during degeneration of the Kölliker's organ in the development of rat cochlea.

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.

LncRNA Lethe protects sepsis-induced brain injury via regulating autophagy of cortical neurons.

OBJECTIVE: To investigate the role of long non-coding RNA (lncRNA) Lethe in mediating autophagy of cortical neurons in mice with sepsis-induced brain injury (SIBI). MATERIALS AND METHODS: A total of 60 wild-type C57BL/6 mice were divided into sham-operated wild-type (SWT) group and wild-type model (MWT) group. Sixty Lethe-/- mice were divided into sham-operated knockout (SKO) group and model knockout (MKO) group. Each group had 30 mice. Sepsis model in mice was established by cecal ligation and puncture (CLP). Neurobiological score was recorded at 6 h after CLP. Mice with lower than 6 scores of neurobehavioral tests were diagnosed with SIBI. Quantitative Real-time polymerase chain reaction (qRT-PCR) was performed to determine mRNA levels of Lethe and interferon-γ (INF-γ) in cortical neurons of SIBI mice. Western blot was conducted to detect protein levels of LC3-II, LC3-I and SQSTM1 in mice. Neuronal impairment in mouse brain was evaluated by hematoxylin and eosin (HE) staining. RESULTS: Expressions of LC3-I and LC3-II in cerebral cortex of MWT group began to increase at 6 h after CLP, and remained at high levels until 96 h. On the contrary, SQSTM1 expression in cerebral cortex of MWT group began to decrease at 6 h after CLP. Compared with SWT group, expressions of Lethe and IFN-γ were remarkably upregulated in cortex of MWT group at 12 h after CLP. Expression of LC3-II in MWT group was remarkably upregulated, while SQSTM1 was downregulated at 12 h after CLP, which were contrary to those in MKO group. At 12 h after CLP, the neurobiological scores of the MKO group (4.97±0.71) were markedly lower than those of the MWT group (5.43±0.86). HE staining showed worse damage in cerebral cortex and fewer neurons of MKO group relative to MWT group. CONCLUSIONS: Lethe has a protective effect on SIBI mice by regulating autophagy in mouse cortical neurons.

Type I interferon enhances necroptosis of Salmonella Typhimurium-infected macrophages by impairing antioxidative stress responses.

Salmonella enterica serovar Typhimurium exploits the host's type I interferon (IFN-I) response to induce receptor-interacting protein (RIP) kinase-mediated necroptosis in macrophages. However, the events that drive necroptosis execution downstream of IFN-I and RIP signaling remain elusive. In this study, we demonstrate that S Typhimurium infection causes IFN-I-mediated up-regulation of the mitochondrial phosphatase Pgam5 through RIP3. Pgam5 subsequently interacts with Nrf2, which sequesters Nrf2 in the cytosol, thereby repressing the transcription of Nrf2-dependent antioxidative genes. The impaired ability to respond to S Typhimurium-induced oxidative stress results in reactive oxygen species-mediated mitochondrial damage, energy depletion, transient induction of autophagy, and autophagic degradation of p62. Reduced p62 levels impair interaction of p62 with Keap1, which further decreases Nrf2 function and antioxidative responses to S Typhimurium infection, eventually leading to cell death. Collectively, we identify impaired Nrf2-dependent redox homeostasis as an important mechanism that promotes cell death downstream of IFN-I and RIP3 signaling in S Typhimurium-infected macrophages.

NF-κB Signaling Activation Induced by Chloroquine Requires Autophagosome, p62 Protein, and c-Jun N-terminal Kinase (JNK) Signaling and Promotes Tumor Cell Resistance.

Macroautophagy (hereafter autophagy) is a catabolic cellular self-eating process by which unwanted organelles or proteins are delivered to lysosomes for degradation through autophagosomes. Although the role of autophagy in cancer has been shown to be context-dependent, the role of autophagy in tumor cell survival has attracted great interest in targeting autophagy for cancer therapy. One family of potential autophagy blockers is the quinoline-derived antimalarial family, including chloroquine (CQ). However, the molecular basis for tumor cell response to CQ remains poorly understood. We show here that in both squamous cell carcinoma cells and melanoma tumor cells, CQ induced NF-κB activation and the expression of its target genes HIF-1α, IL-8, BCL-2, and BCL-XL through the accumulation of autophagosomes, p62, and JNK signaling. The activation of NF-κB further increased p62 gene expression. Either genetic knockdown of p62 or inhibition of NF-κB sensitized tumor cells to CQ, resulting in increased apoptotic cell death following treatment. Our findings provide new molecular insights into the CQ response in tumor cells and CQ resistance in cancer therapy. These findings may facilitate development of improved therapeutic strategies by targeting the p62/NF-κB pathway.

Combinational Immunotherapy with Allo-DRibble Vaccines and Anti-OX40 Co-Stimulation Leads to Generation of Cross-Reactive Effector T Cells and Tumor Regression.

It is well-known that vaccines comprising of irradiated whole tumor cells or tumor-derived heat shock proteins can generate tumor-specific immune responses. In contrast, we showed recently that vaccines composed of autophagosomes (DRibbles) derived from syngeneic sarcomas could induce cross-reactive T-cell responses and cross-protection against the tumor. This unusual property of DRibbles was related to the selective recruitment of defective ribosomal products (DRiPs) and other short-lived proteins (SLiPs) into autophagosomes via sequestosome (SQSTM1, p62) mediated association of ubiquitinated SLiPs to the autophagy gene product LC3. Here, we extend our observations to mammary carcinomas from mice of different genetic background. We demonstrated that combined of intranodal administration of autologous or allogeneic DRibbles together with anti-OX40 antibody led to robust proliferation, expansion, and differentiation of memory and effector T cells. We also showed that SLiPs is an excellent source of antigen for cross-priming of CD8+ T-cells that recognize shared tumor antigens in the context of host MHC class I molecules. Thus, our results provide a strong basis for novel clinical trials that combine allogeneic "off-the-shelf" DRibble vaccines together with antibodies against co-stimulatory molecules.

TRIM14 Inhibits cGAS Degradation Mediated by Selective Autophagy Receptor p62 to Promote Innate Immune Responses.

Cyclic GMP-AMP synthase (cGAS) is an essential DNA virus sensor that triggers type I interferon (IFN) signaling by producing cGAMP to initiate antiviral immunity. However, post-translational regulation of cGAS remains largely unknown. We report that K48-linked ubiquitination of cGAS is a recognition signal for p62-depdendent selective autophagic degradation. The induction of TRIM14 by type I IFN accelerates cGAS stabilization by recruiting USP14 to cleave the ubiquitin chains of cGAS at lysine (K) 414. Knockout of TRIM14 impairs herpes simplex virus type 1 (HSV-1)-triggered antiviral responses in a cGAS-dependent manner. Due to impaired type I IFN production, Trim14-/- mice are highly susceptible to lethal HSV-1 infection. Taken together, our findings reveal a positive feedback loop of cGAS signaling generated by TRIM14-USP14 and provide insights into the crosstalk between autophagy and type I IFN signaling in innate immunity.

TRIM11 Suppresses AIM2 Inflammasome by Degrading AIM2 via p62-Dependent Selective Autophagy.

The AIM2 inflammasome is a key cytosolic signaling complex that is activated by double-stranded DNA, leading to the maturation of proinflammatory cytokines such as interleukin-1ß (IL-1ß) and IL-18. Dysregulated AIM2 inflammasome activity is associated with human inflammatory diseases and cancers, suggesting that its activity must be tightly regulated. However, the precise molecular mechanisms that control AIM2 levels and activity are still poorly understood. Here, we report tripartite motif 11 (TRIM11) as a key negative regulator of the AIM2 inflammasome. Upon DNA virus infection, TRIM11 binds to AIM2 via its PS domain and undergoes auto-polyubiquitination at K458 to promote an association between TRIM11 and the autophagic cargo receptor p62 to mediate AIM2 degradation via selective autophagy. These findings identify a role for TRIMs in AIM2 inflammasome activation where TRIM11 acts as a secondary receptor to deliver AIM2 to the autophagosomes for degradation in a p62-dependent manner.

Mycotoxin Patulin Suppresses Innate Immune Responses by Mitochondrial Dysfunction and p62/Sequestosome-1-dependent Mitophagy.

Innate immune responses are important for pathogen elimination and adaptive immune response activation. However, excess inflammation may contribute to immunopathology and disease progression (e.g. inflammation-associated hepatocellular carcinoma). Immune modulation resulting from pattern recognition receptor-induced responses is a potential strategy for controlling immunopathology and related diseases. This study demonstrates that the mycotoxin patulin suppresses Toll-like receptor- and RIG-I/MAVS-dependent cytokine production through GSH depletion, mitochondrial dysfunction, the activation of p62-associated mitophagy, and p62-TRAF6 interaction. Blockade of autophagy restored the immunosuppressive activity of patulin, and pharmacological activation of p62-dependent mitophagy directly reduced RIG-I-like receptor-dependent inflammatory cytokine production. These results demonstrated that p62-dependent mitophagy has an immunosuppressive role to innate immune response and might serve as a potential immunomodulatory target for inflammation-associated diseases.

p62/Sequestosome-1: Mapping Sites of Protein-Handling Stress in Canine Cutaneous Mast Cell Tumors.

Canine cutaneous mast cell tumors (MCT) are common, frequently malignant neoplasms that are currently graded histologically for provision of prognostic information. Continuing evidence of subsets of MCT within certain grades (with differing survival times) indicate the need for biomarkers that will facilitate better patient stratification and also provide further information on the biological processes involved in progression. We decided to investigate the expression of p62/sequestosome-1 (p62/SQSTM1), a stress-inducible "hub protein" found in all cell types that shuttles rapidly between the nucleus and cytoplasm and is known to play important roles in protein handling and tumorigenesis. The identity of canine p62/SQSTM1 was confirmed in silico and by validation of a commercial antibody using both Western blotting and functional (pharmaceutical-based) analyses in cell culture. Using immunohistochemistry, 3 patterns of p62 expression were identified based on the predominant intracellular localization, that is, nuclear, mixed (nuclear and cytoplasmic), and cytoplasmic. There was a highly significant association with the 2-tier (Kiupel) grade (P < .0001), with all p62-nuclear immunoreactivity being associated with low grade and most p62-cytoplasmic immunoreactivity (93%) with high grade. Most but not all mixed nuclear-cytoplasmic labeling occurred in low-grade MCT; in other (human) tumor types, this pattern has been interpreted as borderline malignant. These data indicate that there is a shift in protein-handling stress from the nucleus to the cytoplasm in association with increasing malignancy in MCT. Studies to identify the processes and drug-able targets involved in this progression are ongoing.