Inflammasome activation by altered proteostasis.

The association between altered proteostasis and inflammatory disorders has been increasingly recognized, but the underlying mechanisms are not well understood. In this study, we show that deficiency of either autophagy or sequestosome 1 (p62 or SQSTM) led to inflammasome hyperactivation in response to LPS and ATP in primary macrophages and in mice in vivo. Importantly, induction of protein misfolding by puromycin, thapsigargin, or geldanamycin resulted in inflammasome activation that was more pronounced in autophagy- or p62-deficient macrophages. Accumulation of misfolded proteins caused inflammasome activation by inducing generation of nonmitochondrial reactive oxygen species and lysosomal damage, leading to release of cathepsin B. Our results suggest that altered proteostasis results in inflammasome activation and thus provide mechanisms for the association of altered proteostasis with inflammatory disorders.

Transient aggregation of ubiquitinated proteins is a cytosolic unfolded protein response to inflammation and endoplasmic reticulum stress.

Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins in the cytosol. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that occurs under these conditions. This response was aided by rapid transcriptional up-regulation of polyubiqutin-binding protein p62. NF-κB and mTOR activation were also required for ALIS formation. Importantly, we show a cross talk between UPR in the ER and cytosolic ALIS. Down-regulation of ER UPR in XBP1 deficient cells increases cyotosolic ALIS formation. Furthermore, lysosomal activity but not macroautophagy is responsible for ALIS clearance. This study reveals the underlying regulatory mechanisms of ALIS formation and clearance, and provides a previously unrecognized common adaptive mechanism for cellular responses against inflammation and ER stress.

Defining the Inflammatory Plasma Proteome in Pediatric Hodgkin Lymphoma.

Hodgkin lymphoma (HL) histopathology is characterized by rare malignant Reed-Sternberg cells among an inflammatory infiltrate. We hypothesized that characteristics of inflammation in pediatric HL lesions would be reflected by the levels of inflammatory cytokines or chemokines in pre-therapy plasma of children with HL. The study objectives were to better define the inflammatory pre-therapy plasma proteome and identify plasma biomarkers associated with extent of disease and clinical outcomes in pediatric HL. Pre-therapy plasma samples were obtained from pediatric subjects with newly diagnosed HL and healthy pediatric controls. Plasma concentrations of 135 cytokines/chemokines were measured with the Luminex platform. Associations between protein concentration and disease characteristics were determined using multivariate permutation tests with false discovery control. Fifty-six subjects with HL (mean age: 13 years, range 3-18) and 47 controls were analyzed. The cytokine/chemokine profiles of subjects with HL were distinct from controls, and unique cytokines/chemokines were associated with high-risk disease (IL-10, TNF-α, IFN-γ, IL-8) and slow early response (CCL13, IFN-λ1, IL-8). TNFSF10 was significantly elevated among those who ultimately relapsed and was significantly associated with worse event-free survival. These biomarkers could be incorporated into biologically based risk stratification to optimize outcomes and minimize toxicities in pediatric HL.

Sustained long-term immune responses after in situ gene therapy combined with radiotherapy and hormonal therapy in prostate cancer patients.

PURPOSE: To explore long-term immune responses after combined radio-gene-hormonal therapy. METHODS AND MATERIALS: Thirty-three patients with prostate specific antigen 10 or higher or Gleason score of 7 or higher or clinical stage T2b to T3 were treated with gene therapy that consisted of 3 separate intraprostatic injections of AdHSV-tk on Days 0, 56, and 70. Each injection was followed by 2 weeks of valacyclovir. Intensity-modulated radiation therapy was delivered 2 days after the second AdHSV-tk injection for 7 weeks. Hormonal therapy was initiated on Day 0 and continued for 4 months or 2.3 years. Blood samples were taken before, during, and after treatment. Lymphocytes were analyzed by fluorescent antibody cell sorting (FACS). RESULTS: Median follow-up was 26 months (range, 4-48 months). The mean percentages of DR+CD8+ T cells were increased at all timepoints up to 8 months. The mean percentages of DR+CD4+ T cells were increased later and sustained longer until 12 months. Long-term (2.3 years) use of hormonal therapy did not affect the percentage of any lymphocyte population. CONCLUSIONS: Sustained long-term (up to 8 to 12 months) systemic T-cell responses were noted after combined radio-gene-hormonal therapy for prostate cancer. Prolonged use of hormonal therapy does not suppress this response. These results suggest the potential for sustained activation of cell-mediated immune responses against cancer.

Harnessing of TLR-mediated autophagy to combat mycobacteria in macrophages.

Autophagy, an evolutionary highly conserved process in virtually all eukaryotic cells, involves the sequestration of cytosol regions within double-membrane bound compartments and delivery of the contents to the lysosomes for degradation. Rapidly accumulating evidence has shown that autophagy is a component of innate immunity and is involved in host defense elimination of pathogens. Our previous studies show that Toll-like receptor 4 (TLR4) is a sensor for autophagy associated with innate immunity. We, now, further demonstrate that LPS or poly(I:C)-treatment significantly reduced mycobacterial viability in mouse macrophages. In addition, LPS reduction of mycobacterial viability was abrogated with the use of autophagy inhibitor 3-MA and in autophagy deficient macrophages. These findings demonstrate that TLR3 or TLR4 stimulation induces autophagy-mediated elimination of mycobacteria in macrophages. These results provide groundwork for therapeutic strategies directed at elimination of mycobacterial infections in macrophages.

GLIPR1 suppresses prostate cancer development through targeted oncoprotein destruction.

Downregulation of the proapoptotic p53 target gene glioma pathogenesis-related protein 1 (GLIPR1) occurs frequently in prostate cancer, but the functional meaning of this event is obscure. Here, we report the discovery of functional relationship between GLIPR1 and c-Myc in prostate cancer where c-Myc is often upregulated. We found that the expression of GLIPR1 and c-Myc were inversely correlated in human prostate cancer. Restoration of GLIPR1 expression in prostate cancer cells downregulated c-myc levels, inhibiting cell-cycle progression. Downregulation was linked to a reduction in ß-catenin/TCF4-mediated transcription of the c-myc gene, which was caused by GLIPR1-mediated redistribution of casein kinase 1α (CK1α) from the Golgi apparatus to the cytoplasm where CK1α could phosphorylate ß-catenin and mediate its destruction. In parallel, GLIPR1 also promoted c-Myc protein ubiquitination and degradation by glycogen synthase kinase-3α- and/or CK1α-mediated c-Myc phosphorylation. Notably, genetic ablation of the mouse homolog of Glipr1 cooperated with c-myc overexpression to induce prostatic intraepithelial neoplasia and prostate cancer. Together, our findings provide evidence for CK1α-mediated destruction of c-Myc and identify c-Myc S252 as a crucial CK1α phosphorylation site for c-Myc degradation. Furthermore, they reveal parallel mechanisms of c-myc downregulation by GLIPR1 that when ablated in the prostate are sufficient to drive c-Myc expression and malignant development.

Functional analysis of secreted caveolin-1 in mouse models of prostate cancer progression.

Previously, we reported that caveolin-1 (cav-1) is overexpressed in metastatic prostate cancer and that virulent prostate cancer cells secrete biologically active cav-1. We also showed that cav-1 expression leads to prosurvival activities through maintenance of activated Akt and that cav-1 is taken up by other cav-1-negative tumor cells and/or endothelial cells, leading to stimulation of angiogenic activities through PI-3-K-Akt-eNOS signaling. To analyze the functional consequences of cav-1 overexpression on the development and progression of prostate cancer in vivo, we generated PBcav-1 transgenic mice. Adult male PBcav-1 mice showed significantly increased prostatic wet weight and higher incidence of epithelial hyperplasia compared with nontransgenic littermates. Increased immunostaining for cav-1, proliferative cell nuclear antigen, P-Akt, and reduced nuclear p27(Kip1) staining occurred in PBcav-1 hyperplastic prostatic lesions. PBcav-1 mice showed increased resistance to castration-induced prostatic regression and elevated serum cav-1 levels compared with nontransgenic littermates. Intraprostatic injection of androgen-sensitive, cav-1-secreting RM-9 mouse prostate cancer cells resulted in tumors that were larger in PBcav-1 mice than in nontransgenic littermates (P = 0.04). Tail vein inoculation of RM-9 cells produced significantly more experimental lung metastases in PBcav-1 males than in nontransgenic male littermates (P = 0.001), and in cav-1(+/+) mice than in cav-1(-/-) mice (P = 0.041). Combination treatment with surgical castration and systemic cav-1 antibody dramatically reduced the number of experimental metastases. These experimental data suggest a causal association of secreted cav-1 and prostate cancer growth and progression.