Ezra Vadai

DAP kinase links the control of apoptosis to metastasis

DAP kinase is a new type of calcium/calmodulin-dependent enzyme that phosphorylates serine/threonine residues on proteins. Its structure contains ankyrin repeats and the ‘death’ domain, and it is associated with the cell cytoskeleton. The gene encoding DAP kinase was initially isolated as a positive mediator of apoptosis induced by interferon-γ, by using a strategy of functional cloning. We have now tested whether this gene has tumour-suppressive activity. We found that lung carcinoma clones, characterized by their highly aggressive metastatic behaviour and originating from two independent murine lung tumours, did not express DAP kinase, in contrast to their low-metastatic counterparts. Restoration of DAP kinase to physiological levels in high-metastatic Lewis carcinoma cells suppressed their ability to form lung metastases after intravenous injection into syngeneic mice, and delayed local tumour growth in a foreign ‘microenvironment’. Conversely, in vivo selection of rare lung lesions following injection into syngeneic mice of low-metastatic Lewis carcinoma cells or of DAP kinase transfectants, was associated with loss of DAP kinase expression. In situ TUNEL staining of tumour sections revealed that DAP kinase expression from the transgene raised the incidence of apoptosis in vivo. DAP-kinase transfectants also showed increased sensitivity in vitro to apoptotic stimuli, of the sort encountered by metastasizing cells at different stages of malignancy. We propose that loss of DAP kinase expression provides a unique mechanism that links suppression of apoptosis to metastasis.

Regression of established murine carcinoma metastases following vaccination with tumour-associated antigen peptides

The cure of micrometastases following surgery is the major goal of cancer immunotherapy. We have recently isolated tumour-associated antigen (TAA) peptides, MUT 1 and MUT 2, derived from a mutated connexin 37 gap-junction protein, from the malignant 3LL-D122 murine lung carcinoma. We now report that synthetic MUT 1 or MUT 2 induces effective antitumour cytoxic T lymphocytes. Peptide vaccines protect mice from spontaneous metastases of 3LL-D122 tumours. Moreover, peptide vaccines reduce metastatic loads in mice carrying pre-established micrometastases. Tumour-specific immunity was primarily mediated by CD8+ T cells. This is the first evidence that peptide therapy may be effective in treatment of residual tumours and provides a rationale for the development of peptide vaccines as a modality for cancer therapy.

Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL

Senescent cells, formed in response to physiological and oncogenic stresses, facilitate protection from tumourigenesis and aid in tissue repair. However, accumulation of such cells in tissues contributes to age-related pathologies. Resistance of senescent cells to apoptotic stimuli may contribute to their accumulation, yet the molecular mechanisms allowing their prolonged viability are poorly characterized. Here we show that senescent cells upregulate the anti-apoptotic proteins BCL-W and BCL-XL. Joint inhibition of BCL-W and BCL-XL by siRNAs or the small-molecule ABT-737 specifically induces apoptosis in senescent cells. Notably, treatment of mice with ABT-737 efficiently eliminates senescent cells induced by DNA damage in the lungs as well as senescent cells formed in the epidermis by activation of p53 through transgenic p14ARF. Elimination of senescent cells from the epidermis leads to an increase in hair-follicle stem cell proliferation. The finding that senescent cells can be eliminated pharmacologically paves the way to new strategies for the treatment of age-related pathologies.

Cu/Zn superoxide dismutase plays a role in angiogenesis.

Endothelial cells produce oxygen radicals spontaneously and this process is augmented by hypoxia/reoxygenation. Cu/Zn superoxide dismutase (SOD‐1) is an important enzyme in cellular oxygen metabolism. To determine whether alterations in SOD‐1 activity affect angiogenesis we used transgenic SOD‐1 (Tg‐SOD) mice with elevated level of SOD‐1. Angiogenesis induced subcutaneously by bFGF in Tg‐SOD mice was 3‐fold higher than in control non‐transgenic (ntg) mice. Oral administration of disulfiram (DSF), an inhibitor of SOD‐1, inhibited angiogenesis in Tg‐SOD mice as well as in CD1 nude mice. Effects of DSF on cultured cells were also tested. Application of DSF to cultured bovine capillary endothelial (BCE) cells caused inhibition of DNA synthesis and induction of apoptosis. These effects were prevented by addition of antioxidants, further indicating involvement of reactive oxygen species. DSF also reduced the level of glutathione and the production of H2O2 in BCE cells. Moreover, PC12‐SOD cells with elevated SOD‐1 were less sensitive to DSF treatment then control cells. These data indicate that the effects of DSF are mediated by inhibition of SOD‐1 activity. Tumor development is known to largely depend on angiogenesis. We found that oral administration of DSF to mice caused significant inhibition of C6 glioma tumor development and marked reduction (by 10–19‐fold) in metastatic growth of Lewis lung carcinoma. The data suggest a role for SOD‐1 in angiogenesis, establish DSF as a potential inhibitor of angiogenesis and raise the possibility that attenuating SOD‐1 activity may be important in treatment of angiogenesis‐dependent pathologies.

MHC class I-restricted epitope spreading in the context of tumor rejection following vaccination with a single immunodominant CTL epitope

Epitope spreading is a process whereby epitopes distinct from and non‐cross‐reactive with an inducing epitope become targets of an evolving immune response. This phenomenon has been associated most notably with the progression of naturally occurring or experimentally induced chronic autoimmune diseases. We have investigated the potential occurrence of epitope spreading in the context of antitumor cytotoxic T cell (CTL) responses using chicken ovalbumin (OVA) as a model antigen. Our results indicate that following rejection of OVA‐expressing EG.7 tumor cells effectuated by a CTL response which is induced against the MHC class I‐restricted immunodominant epitope OVA257 – 264, there occurs intramolecular diversification of the CTL response to two additional OVA‐derived epitopes, OVA176 – 183 and OVA55 – 62, as well as intermolecular spreading to other endogenous tumor‐derived determinants. It seems that CTL‐mediated tumor cell destruction in vivo favors cross‐presentation of additional epitopes with the consequent activation of additional tumor‐reactive lymphocytes. The process of epitope spreading in that context has obvious important implications for the design of antigen‐specific antitumor immunotherapies.

Combined Dendritic Cell Cryotherapy of Tumor Induces Systemic Antimetastatic Immunity

Purpose: Cryotherapy of localized prostate, renal, and hepatic primary tumors and metastases is considered a minimally invasive treatment demonstrating a low complication rate in comparison with conventional surgery. The main drawback of cryotherapy is that it has no systemic effect on distant metastases. We investigated whether intratumoral injections of dendritic cells following cryotherapy of local tumors (cryoimmunotherapy) provides an improved approach to cancer treatment, combining local tumor destruction and systemic anticancer immunity. Experimental Designs: The 3LL murine Lewis lung carcinoma clone D122 and the ovalbumin-transfected B16 melanoma clone MO5 served as models for spontaneous metastasis. The antimetastatic effect of cryoimmunotherapy was assessed in the lung carcinoma model by monitoring mouse survival, lung weight, and induction of tumor-specific CTLs. The mechanism of cryoimmunotherapy was elucidated in the melanoma model using adoptive transfer of T cell receptor transgenic OT-I CTLs into the tumor-bearing mice, and analysis of Th1/Th2 responses by intracellular cytokine staining in CD4 and CD8 cells. Results: Cryoimmunotherapy caused robust and tumor-specific CTL responses, increased Th1 responses, significantly prolonged survival and dramatically reduced lung metastasis. Although intratumor administration of dendritic cells alone increased the proliferation rate of CD8 cells, only cryoimmunotherapy resulted in the generation of effector memory cells. Furthermore, cryoimmunotherapyprotected mice that had survived primary MO5 tumors from rechallenge with parental tumors. Conclusions: These results present cryoimmunotherapy as a novel approach for systemic treatment of cancer. We envisage that cryotherapy of tumors combined with subsequent in situ immunotherapy by autologous unmodified immature dendritic cells can be applied in practice.

Human CTL Epitopes Prostatic Acid Phosphatase-3 and Six-Transmembrane Epithelial Antigen of Prostate-3 as Candidates for Prostate Cancer Immunotherapy

Specific immunotherapy of prostate cancer may be an alternative or be complementary to other approaches for treatment of recurrent or metastasized disease. This study aims at identifying and characterizing prostate cancer-associated peptides capable of eliciting specific CTL responses in vivo. Evaluation of peptide-induced CTL activity in vitro was done following immunization of HLA-A2 transgenic (HHD) mice. An in vivo tumor rejection was tested by adoptive transfer of HHD immune lymphocytes to nude mice bearing human tumors. To confirm the existence of peptide-specific CTL precursors in human, lymphocytes from healthy and prostate cancer individuals were stimulated in vitro in the presence of these peptides and CTL activities were assayed. Two novel immunogenic peptides derived from overexpressed prostate antigens, prostatic acid phosphatase (PAP) and six-transmembrane epithelial antigen of prostate (STEAP), were identified; these peptides were designated PAP-3 and STEAP-3. Peptide-specific CTLs lysed HLA-A2.1+ LNCaP cells and inhibited tumor growth on adoptive immunotherapy. Furthermore, peptide-primed human lymphocytes derived from healthy and prostate cancer individuals lysed peptide-pulsed T2 cells and HLA-A2.1+ LNCaP cells. Based on the results presented herein, PAP-3 and STEAP-3 are naturally processed CTL epitopes possessing anti-prostate cancer reactivity in vivo and therefore may constitute vaccine candidates to be investigated in clinical trials.

Senescent cells communicate via intercellular protein transfer

Mammalian cells mostly rely on extracellular molecules to transfer signals to other cells. However, in stress conditions, more robust mechanisms might be necessary to facilitate cell-cell communications. Cellular senescence, a stress response associated with permanent exit from the cell cycle and the development of an immunogenic phenotype, limits both tumorigenesis and tissue damage. Paradoxically, the long-term presence of senescent cells can promote tissue damage and aging within their microenvironment. Soluble factors secreted from senescent cells mediate some of these cell-nonautonomous effects. However, it is unknown whether senescent cells impact neighboring cells by other mechanisms. Here we show that senescent cells directly transfer proteins to neighboring cells and that this process facilitates immune surveillance of senescent cells by natural killer (NK) cells. We found that transfer of proteins to NK and T cells is increased in the murine preneoplastic pancreas, a site where senescent cells are present in vivo. Proteomic analysis and functional studies of the transferred proteins revealed that the transfer is strictly dependent on cell-cell contact and CDC42-regulated actin polymerization and is mediated at least partially by cytoplasmic bridges. These findings reveal a novel mode of intercellular communication by which senescent cells regulate their immune surveillance and might impact tumorigenesis and tissue aging.

O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma

Due to the fact that many cellular proteins are extensively glycosylated, processing and presentation mechanisms are expected to produce a pool of major histocompatibility complex (MHC) class I‐bound protein‐derived peptides, part of which retain sugar moieties. The immunogenic properties of the presented glycosylated peptides in comparison to their non‐glycosylated counterparts have not been determined clearly. We assessed the cellular immunogenicity of MUC1 (mucin)‐derived peptides O‐glycosylated with a Tn epitope (GalNAc) using HLA‐A*0201 single chain (HHD)‐transfected cell lines and transgenic mice. For part of the compounds Tn moiety did not interfere with the HLA‐A*0201 binding. Moreover, part of the glycopeptides elicited effective cytotoxic responses, indicating recognition of the glycopeptide‐HLA‐A*0201 complex by the T cell receptor (TCR) and subsequent cytotoxic T lymphocyte (CTL) activation. The CTLs exhibited a substantial degree of cross‐reactivity against target cells loaded with glycosylated and non‐glycosylated forms of the same peptide. The studied (glyco)peptides showed cellular immunogenicity in both MUC1‐HHD and HHD mice and induced effective lysis of (glyco)peptide‐loaded target cells in CTL assays. However, the elicited CTLs did not induce selective lysis of human MUC1‐expressing murine cell lines. Moreover, immunization with (glyco)peptide‐loaded dendritic cells (DCs) did not induce significant immunotherapeutic effects. We conclude that Tn glycosylated MUC1‐derived peptides can be presented by MHC class I molecules, and may be recognized by specific TCR molecules resulting in cytotoxic immune responses. However, the studied glycopeptides did not offer significant benefit as targets for cytotoxic immune response due apparently to (a) cross‐reactivity of the elicited CTLs against the glycosylated and non‐glycosylated forms of the same peptide and (b) low abundance of glycopeptides on tumour target cells.

A Systems Immunology Approach to the Host-Tumor Interaction: Large-Scale Patterns of Natural Autoantibodies Distinguish Healthy and Tumor-Bearing Mice

Traditionally, immunology has considered a meaningful antibody response to be marked by large amounts of high-affinity antibodies reactive with the specific inciting antigen; the detection of small amounts of low-affinity antibodies binding to seemingly unrelated antigens has been considered to be beneath the threshold of immunological meaning. A systems-biology approach to immunology, however, suggests that large-scale patterns in the antibody repertoire might also reflect the functional state of the immune system. To investigate such global patterns of antibodies, we have used an antigen-microarray device combined with informatic analysis. Here we asked whether antibody-repertoire patterns might reflect the state of an implanted tumor. We studied the serum antibodies of inbred C57BL/6 mice before and after implantation of syngeneic 3LL tumor cells of either metastatic or non-metastatic clones. We analyzed patterns of IgG and IgM autoantibodies binding to over 300 self-antigens arrayed on slides using support vector machines and genetic algorithm techniques. We now report that antibody patterns, but not single antibodies, were informative: 1) mice, even before tumor implantation, manifest both individual and common patterns of low-titer natural autoantibodies; 2) the patterns of these autoantibodies respond to the growth of the tumor cells, and can distinguish between metastatic and non-metastatic tumor clones; and 3) curative tumor resection induces dynamic changes in these low-titer autoantibody patterns. The informative patterns included autoantibodies binding to self-molecules not known to be tumor-associated antigens (including insulin, DNA, myosin, fibrinogen) as well as to known tumor-associated antigens (including p53, cytokeratin, carbonic anhydrases, tyrosinase). Thus, low-titer autoantibodies that are not the direct products of tumor-specific immunization can still generate an immune biomarker of the body-tumor interaction. System-wide profiling of autoantibody repertoires can be informative.