Calpain-Associated Proteolytic Regulation of the Stromal Microenvironment in Cancer.

BACKGROUND: Normalization of the stromal microenvironment is a promising strategy for cancer control. Cancer-associated fibroblasts, tumor-associated macrophages, and mesenchymal stromal cells have a central role in stromal functions. Accordingly, understanding these stromal cells is indispensable for the development of next-generation cancer therapies. Growing evidence suggests that calpain-induced intracellular proteolysis is responsible for cancer growth and stromal regulation. Calpain is a family of stress-responsive intracellular proteases and is inducible in cancer and stromal cells during carcinogenesis. OBJECTIVE: Here, we shed light on the recent advances that have been made in understanding how calpain contributes to stromal regulation in cancer. CONCLUSION: Calpains are activated in stromal cells, including pancreatic stellate cells and mesenchymal cells. They induce fibrogenic responses in cancer stroma. Moreover, these molecules contribute to epithelial-mesenchymal transition and endothelial-mesenchymal transition to provide mesenchymal stromal cells in the microenvironment and concomitantly participate in cancer angiogenesis. In addition to the conventional calpains, the unconventional calpain-9 is associated with epithelial-mesenchymal transition. Animal experiments showed that targeting calpain systems antagonizes cancer development; thus, this approach is promising for cancer control.

Hypothalamic Monoaminergic Pathology in a Neurodevelopmental Rat Model Showing Prenatal 5-Bromo-2'-Deoxyuridine Treatment-Induced Hyperactivity and Hyporeproductivity.

OBJECTIVE: Prenatal treatment of rats with 5-bromo-2'-deoxyuridine (BrdU) is a neurodevelopmental model showing hyperactivity and impaired sexual activity. Human neurodevelopmental disorders, such as autism, exhibit sex-related pathology, but sex-related neurodevelopment has not been fully investigated in this model. We conducted this study to facilitate the understanding of the pathophysiology of neurodevelopmental disorders. METHODS: Pregnant rats received 50 mg/kg BrdU on gestational days 9-15. The tissue content of dopamine (DA), serotonin (5-HT), and their metabolites dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid were measured in male and female offspring at 3 weeks (juveniles) and 10 weeks (adults) of age. RESULTS: Prenatally BrdU-treated rats had reduced DA metabolism or DA content in the hypothalamus from the juvenile through the adult period without sex differences, but sex-specific striatal DA abnormalities emerged after maturation. A reduction in 5-HT metabolism was measured in the hypothalamus without sex differences throughout development. Developmental alterations in the striatal 5-HT states were sex-dependent. Temporal changes in DA or 5-HT metabolism were found in the frontal cortex and midbrain. CONCLUSION: The sex-specific influence of a genotoxic factor on the development of the DA and 5-HT systems was clarified in the hypothalamus and striatum. The results suggest that the observed sex dependence and region specificity are related to the pathology of social dysfunction in neurodevelopmental disorders.

Impact of Dysfunctional Protein Catabolism on Macrophage Cholesterol Handling.

Protein catabolism in macrophages, which is accomplished mainly through autophagy- lysosomal degradation, ubiquitin-proteasome system, and calpains, is disturbed in atheroprone vessels. Moreover, growing evidence suggests that defects in protein catabolism interfere with cholesterol handling in macrophages. Indeed, decreases in autophagy facilitate the deposition of cholesterol in atheroprone macrophages and the subsequent development of vulnerable atherosclerotic plaques due to impaired catabolism of lipid droplets and limited efferocytic clearance of dead cells. The proteasome is responsible for the degradation of ATP-binding cassette transporters, which leads to impaired cholesterol efflux from macrophages. Overactivation of conventional calpains contributes to excessive processing of functional proteins, thereby accelerating receptor-mediated uptake of oxidized low-density lipoproteins (LDLs) and slowing cholesterol efflux. Furthermore, calpain-6, an unconventional nonproteolytic calpain in macrophages, potentiates pinocytotic uptake of native LDL and attenuates the efferocytic clearance of dead cells. Herein, we focus on recent progress in understanding how defective protein catabolism is associated with macrophage cholesterol handling and subsequent atherogenesis.

Dysregulation of Calpain Proteolytic Systems Underlies Degenerative Vascular Disorders.

Chronic vascular diseases such as atherosclerosis, aneurysms, diabetic angiopathy/retinopathy as well as fibrotic and proliferative vascular diseases are generally complicated by the progression of degenerative insults, which are characterized by endothelial dysfunction, apoptotic/necrotic cell death in vascular/immune cells, remodeling of extracellular matrix or breakdown of elastic lamella. Increasing evidence suggests that dysfunctional calpain proteolytic systems and defective calpain protein metabolism in blood vessels contribute to degenerative disorders. In vascular endothelial cells, the overactivation of conventional calpains consisting of calpain-1 and -2 isozymes can lead to the disorganization of cell-cell junctions, dysfunction of nitric oxide synthase, sensitization of Janus kinase/signal transducer and activator of transcription cascades and depletion of prostaglandin I2, which contributes to degenerative disorders. In addition to endothelial cell dysfunctions, calpain overactivation results in inflammatory insults in macrophages and excessive fibrogenic/proliferative signaling in vascular smooth muscle cells. Moreover, calpain-6, a non-proteolytic unconventional calpain, is involved in the conversion of macrophages to a pro-atherogenic phenotype, leading to the pinocytotic deposition of low-density lipoprotein cholesterol in the cells. Here, we discuss the recent progress that has been made in our understanding of how calpain contributes to degenerative vascular disorders.

Emerging roles of calpain proteolytic systems in macrophage cholesterol handling.

Calpains are Ca2+-dependent intracellular proteases that play central roles in the post-translational processing of functional proteins. In mammals, calpain proteolytic systems comprise the endogenous inhibitor calpastatin as well as 15 homologues of the catalytic subunits and two homologues of the regulatory subunits. Recent pharmacological and gene targeting studies in experimental animal models have revealed the contribution of conventional calpains, which consist of the calpain-1 and -2 isozymes, to atherosclerotic diseases. During atherogenesis, conventional calpains facilitate the CD36-dependent uptake of oxidized low-density lipoprotein (LDL), and block cholesterol efflux through ATP-binding cassette transporters in lesional macrophages, allowing the expansion of lipid-enriched atherosclerotic plaques. In addition, calpain-6, an unconventional non-proteolytic calpain, in macrophages reportedly potentiates pinocytotic uptake of native LDL, and attenuates the efferocytic clearance of apoptotic and necrotic cell corpses from the lesions. Herein, we discuss the recent progress that has been made in our understanding of how calpain contributes to atherosclerosis, in particular focusing on macrophage cholesterol handling.

Calpain-6 confers atherogenicity to macrophages by dysregulating pre-mRNA splicing.

Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL-derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex-driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor-deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems.

Early Tumor Shrinkage Under Treatment with First-line Tyrosine Kinase Inhibitors as a Predictor of Overall Survival in Patients with Metastatic Renal Cell Carcinoma: a Retrospective Multi-Institutional Study in Japan.

BACKGROUND: Prognostic significance of early tumor shrinkage following treatment with tyrosine kinase inhibitors (TKIs) in patients with metastatic renal cell carcinoma (mRCC) has not been fully elucidated. OBJECTIVE: The aim of this study was to assess the impact of early tumor shrinkage induced by first-line TKIs on overall survival (OS) in mRCC patients. PATIENTS AND METHODS: This study retrospectively included 185 consecutive Japanese patients with mRCC treated with either sunitinib or sorafenib for at least 3 months as first-line molecular-targeted therapy between April 2011 and December 2014 at Kobe University Hospital and its affiliated institutions. RESULTS: Median OS in the 185 patients was 33.6 months. At 12 weeks after the introduction of TKIs, 9 patients had achieved tumor shrinkage from -100 to -50 %, 43 from -49 to -25 %, 61 from -24 to 0 %, and the remaining 72 patients showed an increase in tumor size. The median OS stratified according to tumor shrinkage as shown above was 59.2, 39.1, 31.4, and 16.1 months, respectively. Univariate analysis identified prior nephrectomy, Memorial Sloan Kettering Cancer Center (MSKCC) risk classification, C-reactive protein (CRP) level, liver metastasis, number of metastatic organs, histological subtype, sarcomatoid feature, and early tumor shrinkage as significant predictors of OS. Of these significant factors, only the MSKCC classification, CRP level, liver metastasis, and early tumor shrinkage were shown to be independently associated with OS on multivariate analysis. CONCLUSIONS: Early tumor shrinkage could be a useful predictor of OS in mRCC patients receiving TKIs as a first-line molecular-targeted agent.

Improved health-related quality of life of patients with metastatic renal cell carcinoma treated with a 2 weeks on and 1 week off schedule of sunitinib.

The objective of this study was to investigate the significance of changes from the standard dosing schedule of sunitinib, which is 4 weeks of treatment and 2 weeks off (schedule 4/2), to an alternative schedule with 2 weeks of treatment and 1 week off (schedule 2/1), after encountering dose-limiting toxicity in 45 consecutive Japanese patients with metastatic renal cell carcinoma (mRCC). Despite a definitively improved relative dose intensity of sunitinib by changing from schedule 4/2 to 2/1, this difference was not significant. Adverse events (AEs) occurred in all patients on both schedules 4/2 and 2/1; however, the proportion of patients experiencing AEs ≥ grade 3 on schedule 2/1 was significantly lower than that on schedule 4/2. Quality of life (QOL) analysis using SF-36 revealed that all eight scores during schedule 2/1 were more favorable than those during schedule 4/2, and there were significant differences in 2 of the 8 scores between these two schedules. Furthermore, multivariate analyses, which were performed to evaluate the contribution of several AEs on schedule 2/1 to the improvement of each score in SF-36, revealed that fatigue had independent impacts on two scores, despite the lack of an independent association between any scores and the remaining AEs examined. These findings suggest that schedule 2/1 is the optimal dosing schedule of sunitinib against mRCC that balances efficacy and toxicity, since treatment on schedule 2/1 resulted in a markedly improved QOL compared with that on schedule 4/2 by relieving the profile of sunitinib-related AEs.

Bacillus Calmette-Guérin-pulsed dendritic cells stimulate natural killer T cells and gammadeltaT cells.

BACKGROUND: Immunotherapy with bacillus Calmette-Guérin (BCG) for bladder cancer is successful, although the precise mechanism is unclear. Natural killer (NK) cells are a candidate for BCG-activated killer cells, but the roles of other T lymphocytes, such as NKT cells and gammadeltaT cells, are not fully understood. Mycobacterium tuberculosis is a potent activator of both NKT cells and gammadeltaT cells. However, it is known that the patient's prognosis is good if there are increased numbers of dendritic cells (DCs) in the urine after BCG therapy. Therefore, we investigated whether DCs are matured by BCG and whether BCG-pulsed DCs stimulate NKT cells and gammadeltaT cells. METHODS: Naïve Pan T cells were isolated form peripheral blood mononuclear cells (PBMCs) and DCs were obtained by culturing CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor and interleukin-4. The DCs were pulsed with BCG and their maturation was measured by fluorescence-activated cell sorter analysis using the CD86 antibody. Naïve T lymphocytes were stimulated by coculture with BCG-pulsed DCs in vitro, followed by FACS analysis to estimate the ratio and activation of NKT cells and the ratio of gammadeltaT cells. The (51)Cr (chromium) release assay was used to estimate the cytotoxic activity of the stimulated T cells. Cytolytic proteins in the patient's PBMCs were measured during BCG therapy using semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: The DCs were matured by BCG stimulation and the number of NKT cells and gammadeltaT cells increased after culturing with BCG-pulsed DCs. The BCG-pulsed DCs also activated the NKT cells and gammadeltaT cells. Also, the lymphocytes that were cocultured with the BCG-pulsed DCs showed unspecific cytotoxic activity against a bladder cancer cell line. CONCLUSION: Sensitization of NKT cells and gammadeltaT cells by BCG-pulsed DCs might be one of the mechanisms of BCG immunotherapy.