Autophagy is an evolutionally conserved "self-eating" process. Although the genes essential for autophagy (termed Atg) have been identified in yeast, the molecular mechanism of how these Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through UVRAG and acts as a positive mediator of the class III PI3-kinase (PI3KC3). In response to nutrition deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as LC3. Furthermore, loss of Bif-1 suppresses autophagosome formation. While the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI3KC3 and induce autophagosome formation. We also found that Bif-1 ablation prolongs cell survival under nutrient starvation. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumors in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumor suppressor.Autophagy is a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles that appears to be essential for many physiological processes such as cellular homeostasis, development, differentiation, tissue remodeling, cell survival and death, innate immunity, and pathogenesis in various organisms 1-4 . The process of autophagic degradation is initiated when a portion of the cytosolic components are sequestered in cupshaped membrane structures called isolation membranes 1, 2, 5, 6 . The isolation membranes are elongated and eventually sealed to become double-membrane vesicles called autophagosomes, which are then fused with lysosomes resulting in degradation of the enclosed components. Eighteen autophagy-related (Atg) genes have been characterized in S. cerevisiae and can be categorized into four functional groups: (1) the Atg1 protein kinase complex regulating the induction of autophagy, (2) the class III PI3-kinase (PI3KC3) lipid kinase complex controlling vesicle nucleation, (3) the Atg12-Atg5 and Atg8-phosphatidylethanolamine conjugation pathways for vesicle expansion and completion, and (4) the Atg protein retrieval system 2, 7 . Beclin 1, the mammalian homologue of yeast Atg6, is a key component of the PI3KC3 complex, which plays an essential role in autophagosome formation 8-11 . Although the phosphatidylinositol 3-phosphate (PtdIns-3-P) generated by PI3KC3 has been proposed to control membrane dynamics during autophagosome formation 3 , the molecular mechanism underlying this process remains unknown. Results Loss of Bif-1 suppresses caspase-independent cell deathWe have previously reported that Bif-1 localizes to mitochondria and regulates the activation of Bax and Bak during apoptosis induced by intrinsic death stimuli 21 . To examine Bif-1 localization in mouse embryonic fibroblast (MEF) cells during serum deprivation, we added a pancaspase inhibitor, z-VAD-fmk, to ...

There has been recent interest in the depletion of regulatory T cells (Tregs) as part of a multi-faceted approach to the immunotherapy of melanoma patients. This is in part due recent findings that convincingly show that Tregs are an integral part of regulating and even suppressing an immune response to growing tumor cells. We therefore compared three methods of Treg depletion and/or elimination, utilizing low dose cyclophosphamide (CY), a specific antibody directed against the IL-2 receptor found on Tregs (PC61) and the use of denileukin diftitox (DD), which is a fusion protein designed to have a direct cytocidal action on cells which express the IL-2 receptor. We show that CY administration resulted in the highest reduction in Tregs among the three reagents. However, the reduction in Tregs with CY was also associated with the concomitant reduction of CD8(+) T cells and a lack of tumor antigen priming. Utilization of DD resulted in a >50% Treg cell reduction without parallel cytocidal effects upon other T cell subsets but did not enhance anti-tumor immunity against B16 melanoma. Lastly, the PC61 showed a moderate reduction of Tregs that lasted longer than the other reagents, without a reduction in the total number of CD8(+) T cells. Furthermore, PC61 treatment did not abrogate tumor antigen-specific immunity elicited by dendritic cells (DC). We therefore conclude that PC61 administration was the most effective method of reducing Tregs in a murine melanoma model in addition to providing evidence of a synergistic effect when combined with DC-based immunotherapy.

BackgroundThe prognosis of patients with advanced biliary tract cancer (BTC) is extremely poor and only a few standard treatments are available for this condition. We performed a phase I trial to investigate the safety, immune response and anti-tumor effect of vaccination with three peptides derived from cancer-testis antigens.MethodsThis study was conducted as a phase I trial. Nine patients with advanced BTC who had unresectable tumors and were refractory to standard chemotherapy were enrolled. Three HLA-A*2402 restricted epitope peptides-cell division cycle associated 1 (CDCA1), cadherin 3 (CDH3) and kinesin family member 20A (KIF20A)-were administered subcutaneously, and the adverse events and immune response were assessed. The clinical effects observed were the tumor response, progression-free survival (PFS) and overall survival (OS).ResultsThe three-peptide vaccination was well-tolerated up to a dose of 3 mg per peptide (9 mg total). No grade 3 or 4 adverse events were observed after vaccination. Peptide-specific T cell immune responses were observed in all patients and stable disease was observed in 5 of 9 patients. The median PFS and OS were 3.4 and 9.7 months. The Grade 2 injection site reaction and continuous vaccination after PD judgment appeared to be prognostic of OS.ConclusionsMultiple-peptide vaccination was well tolerated and induced peptide-specific T-cell responses.Trial registrationThis study was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR000003229).

Purpose: The prognosis of patients with advanced biliary tract cancer (BTC) is extremely poor and there are only a few standard treatments. We conducted a phase I trial to investigate the safety, immune response, and antitumor effect of vaccination with four peptides derived from cancer-testis antigens, with a focus on their fluctuations during long-term vaccination until the disease had progressed.Experimental Design: Nine patients with advanced BTC who had unresectable tumors and were refractory to standard chemotherapy were enrolled. HLA-A Ã 2402-restricted epitope peptides, lymphocyte antigen 6 complex locus K, TTK protein kinase, insulin-like growth factor-II mRNA-binding protein 3, and DEP domain containing 1 were vaccinated subcutaneously once a week at doses of 0.5, 1, or 2 mg and continued until disease progression. The adverse events were assessed by Common Terminology Criteria for Adverse Events and the immune response was monitored by an enzyme-linked immunospot assay or by flow cytometry. The clinical effects observed were tumor response, progression-free survival (PFS), and overall survival (OS).Results: Four-peptide vaccination was well tolerated. No grade 3 or 4 adverse events were observed. Peptide-specific T-cell immune responses were observed in seven of nine patients and clinical responses were observed in six of nine patients. The median PFS and OS were 156 and 380 days. The injection site reaction and CTL induction seemed to be prognostic factors of both PFS and OS.Conclusions: Four-peptide vaccination was well tolerated and seemed to provide some clinical benefit to some patients. These immunologic and clinical responses were maintained over the long term through continuous vaccinations.

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We reported that murine tumor lysate-pulsed dendritic cells (TP-DC) could elicit tumor-specific CD4 + and CD8 + T cells in vitro and in vivo. In some limited cases, TP-DC treatments in vivo could also result in regression of established subcutaneous tumors and lung metastases. By gene array analysis, we reported a high level of expression of a novel member of the cell surface class A scavenger receptor family, MARCO, by murine TP-DC compared to unpulsed DC. MARCO is thought to play an important role in the immune response by mediating binding and phagocytosis, but also in the formation of lamellipodia-like structures and dendritic processes. We have now examined the biologic and therapeutic implications of MARCO expressed by TP-DC. In vitro exposure of TP-DC to a monoclonal anti-MARCO antibody resulted in a morphologic change of rounding with disappearance of dendritic-like processes. TP-DC remained viable after anti-MARCO antibody treatment; had little, if any, change in production of IL-10, IL-12p70 and TNF-alpha; but demonstrated enhanced migratory capacity in a microchemotaxis assay. The use of a selective inhibitor showed MARCO expression to be linked to the p38 mitogen-activated protein kinase (MAPK) pathway. In vivo, anti-MARCO antibody treated TP-DC showed better trafficking from the skin injection site to lymph node, enhanced generation of tumor-reactive IFN-gamma producing T cells, and improved therapeutic efficacy against B16 melanoma. These results, coupled with our finding that human monocyte-derived DC also express MARCO, could have important implications to human clinical DC vaccine trials.

Recently, analysis of tumor antigens using micro-arrays has revealed upregulation of cancer-testis antigens RNF43 and TOMM34 and vascular endothelial growth factor receptors VEGFR1 and VEGFR2 in colorectal cancer. A phase I clinical trial of peptide vaccine therapy together with oral anticancer drugs was conducted to treat advanced colorectal cancer using synthesized peptides of these tumor antigens in order to confirm the safety, immunogenicity and activity of this treatment. The subjects were patients with a human leukocyte antigen (HLA) type of A2402 who had inoperable colorectal cancer but had failed to respond to or were unable to undergo standard chemotherapy. Four peptides (RNF43, TOMM34, VEGFR1 and VEGFR2) were emulsified with incomplete Freund's adjuvant (Montanide), and the resulting solution was administered subcutaneously once a week. Patients received the oral anticancer drug tegafur-uracil plus leucovorin for four weeks continuously as part of one course followed by one week of rest. The primary endpoint of the trial was observation of adverse events as determined by the NCI-CTCAE criteria, and the secondary endpoints were the size of the tumor and the number of cytotoxic T lymphocytes (CTLs) in the peripheral blood after treatment. Vaccine therapy was administered 148 times to 10 patients from July 2008 to December 2009. The adverse events were grade 1 redness and induration, a grade 2 skin ulcer at the vaccination site and grade 1 pyrexia. All patients tolerated treatment. Tumor imaging revealed that after 1 course of treatment 1 patient had partial response (PR), 7 had stable disease (SD) and 2 had progressive disease. A CTL assay of 10 patients revealed an increase in peptide-specific CTLs in patients with PR and SD, and the clinical responses of those patients were observed. Kaplan‑Meier analysis indicated that patients who had a strong CTL reaction had a tendency to have longer progression‑free survival and overall survival.

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