Kazuaki Tanabe

Tumor-Driven Evolution of Immunosuppressive Networks during Malignant Progression

Tumors evolve mechanisms to escape immune control by a process called immune editing, which provides a selective pressure in the tumor microenvironment that could lead to malignant progression. A variety of tumor-derived factors contribute to the emergence of complex local and regional immunosuppressive networks, including vascular endothelial growth factor, interleukin-10, transforming growth factor-beta, prostaglandin E(2), and soluble phosphatidylserine, soluble Fas, soluble Fas ligand, and soluble MHC class I-related chain A proteins. Although deposited at the primary tumor site, these secreted factors could extend immunosuppressive effects into the local lymph nodes and the spleen, promoting invasion and metastasis. Vascular endothelial growth factors play a key role in recruiting immature myeloid cells from the bone marrow to enrich the microenvironment as tumor-associated immature dendritic cells and tumor-associated macrophages. The understanding of the immunosuppressive networks that evolve is incomplete, but several features are emerging. Accumulation of tumor-associated immature dendritic cells may cause roving dendritic cells and T cells to become suppressed by the activation of indoleamine 2,3-dioxygenase and arginase I by tumor-derived growth factors. Soluble phosphatidylserines support tumor-associated macrophages by stimulating the release of anti-inflammatory mediators that block antitumor immune responses. Soluble Fas, soluble FasL, and soluble MHC class I-related chain A proteins may help tumor cells escape cytolysis by cytotoxic T cells and natural killer cells, possibly by counterattacking immune cells and causing their death. In summary, tumor-derived factors drive the evolution of an immunosuppressive network which ultimately extends immune evasion from the primary tumor site to peripheral sites in patients with cancer.

Role of mitochondria as the gardens of cell death

Mitochondria play a crucial role in regulating cell death, which is mediated by outer membrane permeabilization in response to death triggers such as DNA damage and growth factor deprivation. Mitochondrial membrane permeabilization induces the release of cytochrome c, Smac/DIABLO, and AIF, which are regulated by proapoptotic and antiapoptotic proteins such as Bax/Bak and Bcl-2/xL in caspase-dependent and caspase-independent apoptosis pathways. Mitochondrial dysfunction is mediated in two ways. The first is by increased calcium in mitochondria derived from endoplasmic reticulum (ER); this calcium increase is regulated by Bcl-2 and Bax through the ER-mitochondria connection and the unfolded protein response in the ER. The second is by the lysosomal enzyme cathepsin, which activates Bid through lysosome–mitochondria cross-signaling. The genomic responses in intracellular organelles after DNA damage are controlled and amplified in the cross-signaling via mitochondria; such signals induce apoptosis, autophagy, and other cell death pathways. This review discusses the recent advancements in understanding the molecular mechanism of mitochondria-mediated cell death.

Current status of the molecular mechanisms of anticancer drug-induced apoptosis. The contribution of molecular-level analysis to cancer chemotherapy.

Abstract. Apoptosis is an important phenomenon in cytotoxicity induced by anticancer drugs. Here, we review the current status of the molecular mechanisms of anticancer drug-induced apoptosis in order to assess the contribution of molecular-level analysis to cancer chemotherapy. It is apparent that the molecular mechanisms by which anticancer drugs induce apoptosis are mediated by death receptor-dependent and -independent pathways, which are related to the release of cytochrome c through voltage-dependent anion channels in the mitochondrial inner membrane. The release of cytochrome c is the central gate in turning on/off apoptosis, and is regulated by the interaction of proapoptotic proteins, including Bid, Bax and Bak, and antiapoptotic proteins including Bcl-2 and Bcl-XL, and a specific class of inhibitors of apoptosis proteins (IAPs) including Akt, survivin, and heat-shock proteins. The caspase cascade is activated by the release of cytochrome c, which is initiated by the formation of apoptosomes consisting of procaspase-9, Apaf-1 and cytochrome c in the presence of dATP, and results in the activation of caspase-9 and caspase-3, thereby leading to apoptosis. Drug sensitivity can be enhanced by the introduction of proapoptotic genes and the inhibition of antiapoptotic proteins. The latter process is mediated by antisense oligonucleotides and is associated with apoptosis. The signal transduction pathways that are triggered by the central gate in mitochondria play a critical role in anticancer drug-induced apoptosis. The modulation of signal transduction pathways targeting the proteins involved in these signal transduction pathways using antisense IAPs, and growth factor antibodies may be a good strategy for enhancing therapeutic efficacy of anticancer drugs in cancer chemotherapy.

Cancer immunosuppression and autoimmune disease : beyond immunosuppressive networks for tumour immunity

Cancer immunosuppression evolves by constitution of an immunosuppressive network extending from a primary tumour site to secondary lymphoid organs and peripheral vessels and is mediated by several tumour‐derived soluble factors (TDSFs) such as interleukin‐10 (IL‐10), transforming growth factor‐β (TGF‐β) and vascular endothelial growth factor (VEGF). TDSFs induce immature myeloid cells and regulatory T cells in accordance with tumour progression, resulting in the inhibition of dendritic cell maturation and T‐cell activation in a tumour‐specific immune response. Tumour cells grow by exploiting a pro‐inflammatory situation in the tumour microenvironment, whereas immune cells are regulated by TDSFs during anti‐inflammatory situations—mediated by impaired clearance of apoptotic cells—that cause the release of IL‐10, TGF‐β, and prostaglandin E2 (PGE2) by macrophages. Accumulation of impaired apoptotic cells induces anti‐DNA antibodies directed against self antigens, which resembles a pseudo‐autoimmune status. Systemic lupus erythematosus is a prototype of autoimmune disease that is characterized by defective tolerance of self antigens, the presence of anti‐DNA antibodies and a pro‐inflammatory response. The anti‐DNA antibodies can be produced by impaired clearance of apoptotic cells, which is the result of a hereditary deficiency of complements C1q, C3 and C4, which are involved in the recognition of phagocytosis by macrophages. Thus, it is likely that impaired clearance of apoptotic cells is able to provoke different types of immune dysfunction in cancer and autoimmune disease in which some are similar and others are critically different. This review discusses a comparison of immunological dysfunctions in cancer and autoimmune disease with the aim of exploring new insights beyond cancer immunosuppression in tumour immunity.

Therapeutic potential of antisense Bcl‐2 as a chemosensitizer for cancer therapy

Bcl‐2 protein plays a critical role in inhibiting anticancer drug‐induced apoptosis, which is mediated by a mitochondria‐dependent pathway that controls the release of cytochrome c from mitochondria through anion channels. Constitutive overexpression of Bcl‐2 or unchanged expression after treatment with anticancer drugs confers drug resistance not only to hematologic malignancies but also to solid tumors. The down‐regulation of Bcl‐2 protein by the antisense (AS) Bcl‐2 (oblimesen sodium) may be a useful method for targeting the antiapoptotic protein and thereby increasing the chemotherapeutic effect of anticancer drugs. Several randomized, controlled, Phase III trials have compared standard chemotherapy with a combination of AS Bcl‐2 and standard chemotherapy for the treatment of patients with chronic lymphocytic leukemia, multiple myeloma, malignant melanoma, and nonsmall cell lung carcinoma. Nonrandomized clinical trials and preclinical evaluations of AS Bcl‐2 also are underway for patients with other malignancies. Here, the authors review the current clinical and preclinical evaluations of AS Bcl‐2 and discuss its potential to act as a chemosensitizer and to enhance the therapeutic effect of cancer chemotherapy. Cancer 2004.

The Role of Fas Ligand and Transforming Growth Factor in Tumor Progression Molecular Mechanisms of Immune Privilege via Fas-Mediated Apoptosis and Potential Targets for Cancer Therapy

Despite the fact that expression of Fas ligand (FasL) in cytotoxic T lymphocytes (CTLs) and in natural killer (NK) cells plays an important role in Fas‐mediated tumor killing, During tumor progression FasL‐expressing tumor cells are involved in counterattacking to kill tumor‐infiltrating lymphocytes (TILs). Soluble FasL levels also increase with tumor progression in solid tumors, and this increase inhibits Fas‐mediated tumor killing by CTLs and NK cells. The increased expression of FasL in tumor cells is associated with decreased expression of Fas; and the promoter region of the FASL gene is regulated by transcription factors, such as neuronal factor κB (NF‐κB) and AP‐1, in the tumor microenvironment. Although the ratio of FasL expression to Fas expression in tumor cells is not strongly related to the induction of apoptosis in TILs, increased expression of FasL is associated with decreased Fas levels in tumor cells that can escape immune surveillance and facilitate tumor progression and metastasis. Transforming growth factor β (TGF‐β) is a potent growth inhibitor and has tumor‐suppressing activity in the early phases of carcinogenesis. During subsequent tumor progression, the increased secretion of TGF‐β by both tumor cells and, in a paracrine fashion, stromal cells, is involved in the enhancement of tumor invasion and metastasis accompanied by immunosuppression. Herein, the authors review the clinical significance of FasL and TGF‐β expression patterns as features of immune privilege accompanying tumor progression in the tumor microenvironment. Potential strategies for identifying which molecules can serve as targets for effective antitumor therapy also are discussed. Cancer 2004.

Targeted therapy against Bcl-2-related proteins in breast cancer cells.

IntroductionBcl-2 and Bcl-xL confer resistance to apoptosis, thereby reducing the effectiveness of chemotherapy. We examined the relationship between the expression of Bcl-2 and Bcl-xL and chemosensitivity of breast cancer cells, with the aim of developing specific targeted therapy.MethodsFour human breast cancer cell lines were examined, and the effects of antisense (AS) Bcl-2 and AS Bcl-xL phosphorothioate oligodeoxynucleotides (ODNs) on chemosensitivity were tested in vitro and in vivo. Chemosensitivity was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, and the antitumor effect was assessed in vivo by the success of xenograft transplantation into athymic mice.ResultsTreatment with AS Bcl-2 and Bcl-xL ODNs resulted in a sequence-specific decrease in protein expression, compared with controls. Treatment of BT-474, ZR-75-1, and MDA-MB-231 cells with AS Bcl-2 increased chemosensitivity to doxorubicin (DOX), mitomycin C (MMC), paclitaxel (TXL), and docetaxel (TXT). Transfection of the Bcl-2 gene into MDA-MB-453 cells decreased sensitivity to DOX and MMC. Treatment of MDA-MB-231, BT-474, and ZR-75-1 cells with AS Bcl-xL increased chemosensitivity to DOX, MMC and taxanes to a smaller extent than AS Bcl-2. This occurred in the setting of increased Bax and cleaved poly(ADP-ribose) polymerase, as well as decreased Bcl-2 and pAkt. AS Bcl-2 ODNs induced splenomegaly in association with increased serum IL-12, which was attenuated by methylation of the CpG motifs of AS Bcl-2; however, methylated CpG failed to negate the increased antitumor effect of AS Bcl-2. Bcl-2 and Bcl-xL, to a smaller extent, are major determinants of chemosensitivity in breast cancer cells.ConclusionTargeted therapy against Bcl-2 protein with the use of AS ODNs might enhance the effects of chemotherapy in patients with breast cancer.

Synergistic effects of docetaxel and S‐1 by modulating the expression of metabolic enzymes of 5‐fluorouracil in human gastric cancer cell lines

We have recently demonstrated in a Phase I/II study that combination chemotherapy with docetaxel (TXT) and S‐1 is active against metastatic gastric carcinomas. To elucidate the mechanisms underlying the synergistic effects of these drugs, both the growth inhibitory effects and the expression profiles of enzymes involved in fluorouracil (5‐FU) metabolism were examined in vitro and in vivo. TXT alone and in combination with 5‐FU inhibited the growth of each of the 5 gastric cancer cell lines that we examined (TMK‐1, and MKN‐1, ‐28, ‐45 and ‐74), in a time‐ and dose‐dependent manner. Moreover, striking synergistic effects were observed in TMK‐1 cells in vitro with IC50 values of between 4.73 and 0.61 nM 5‐FU. Furthermore, in TMK‐1 xenografts, 5‐FU/TXT cotreatments exhibited synergistic antitumor effects. The combination of S‐1 and TXT, however, exhibited greater growth‐inhibitory effects than the 5‐FU/TXT cotreatments. The mechanisms underlying these synergistic effects of S‐1 and TXT were examined by expression and activity analyses of the 5‐FU metabolic enzymes. The expression of thymidylate synthase (TS), and dihydropyrimidine dehydrogenase (DPD) were decreased 50 and 73% of control levels, respectively, and that of orotate phosphorybosyl transferase (OPRT) was increased by 3.9‐fold at the protein level. These findings suggested that biochemical modulation of the 2 drugs had occurred, which was further confirmed by the results of the activity assays. These data strongly indicate that a combination chemotherapy of TXT and S‐1 is effective against gastric carcinomas and is therefore a good candidate as a standard chemotherapeutic strategy in treating these tumors.

Cancer cell immune escape and tumor progression by exploitation of anti-inflammatory and pro-inflammatory responses

Apoptotic cells can be eliminated by phagocytosis, which is mediated by antigen-presenting cells (APCs), such as macrophages and dendritic cells (DCs), through phosphatidylserine (PS) on apoptotic cells and phosphatidylserine receptor (PSR) on APCs. The phagocytosis of apoptotic cells by macrophages is strictly regulated by not only the inflammatory reaction, but also by an increase in anti-inflammatory factors such as IL-10, TGF-_, and prostaglandin E2 (PGE2), leading to an anti-inflammatory situation, whereby apoptosis contributes to a non-inflammatory response. However, because PS and PSR are expressed in cancer cells, shed soluble phosphatidylserine (sPS) can interact with the PS receptor on macrophages, which promotes an anti-inflammatory response to macrophages that may lead to immune escape. The sPS derived from cancer cells also reacts with the PSR on the cancer cells to produce IL-10, TGF-_, and PGE2, which can cause suppression of antitumor immunity through the anti-inflammatory response to macrophages, which produces tumor-associated macrophages. Furthermore, sPS and TGF-_ inhibit the maturation of immature DCs, resulting in a functional inhibition of DCs. The potential roles of PS and PSR in cancer cells and macrophages in immune escape mediated by sPS and anti-inflammatory factors are discussed, which may explain their dual regulation of anti- and pro-inflammatory responses during tumor progression.

Sequential paclitaxel followed by tegafur and uracil (UFT) or S-1 versus UFT or S-1 monotherapy as adjuvant chemotherapy for T4a/b gastric cancer (SAMIT): a phase 3 factorial randomised controlled trial

BACKGROUND The prognosis for locally advanced gastric cancer is poor despite advances in adjuvant chemotherapy. We did the Stomach cancer Adjuvant Multi-Institutional group Trial (SAMIT) to assess the superiority of sequential treatment (paclitaxel then tegafur and uracil [UFT] or paclitaxel then S-1) compared with monotherapy (UFT or S-1) and also the non-inferiority of UFT compared with S-1. METHODS We did this randomised phase 3 trial with a two-by-two factorial design at 230 hospitals in Japan. We enrolled patients aged 20-80 years with T4a or T4b gastric cancer, who had had D2 dissection and a ECOG performance score of 0-1. Patients were randomly assigned to one of four treatment groups with minimisation for tumour size, lymph node metastasis, and study site. Patients received UFT only (267 mg/m(2) per day), S-1 only (80 mg/m(2) per day) for 14 days, with a 7-day rest period or three courses of intermittent weekly paclitaxel (80 mg/m(2)) followed by either UFT, or S-1. Treatment lasted 48 weeks in monotherapy groups and 49 weeks in the sequential treatment groups. The primary endpoint was disease-free survival assessed by intention to treat. We assessed whether UFT was non-inferior to S-1 with a non-inferiority margin of 1·33. This trial was registered at UMIN Clinical Trials Registry, number C000000082. FINDINGS We randomly assigned 1495 patients between Aug 3, 2004, and Sept 29, 2009. 374 patients were assigned to receive UFT alone, 374 to receive S-1 alone, 374 to received paclitaxel then UFT, and 373 to receive paclitaxel then S-1. We included 1433 patients in the primary analysis after at least 3 years of follow-up (359, 364, 355, and 355 in each group respectively). Protocol treatment was completed by 215 (60%) patients in the UFT group, 224 (62%) in the S-1 group, 242 (68%) in the paclitaxel then UFT group, and 250 (70%) in the paclitaxel then S-1 group. 3-year disease-free survival for monotherapy was 54·0% (95% CI 50·2-57·6) and that of sequential treatment was 57·2% (53·4-60·8; hazard ratio [HR] 0·92, 95% CI 0·80-1·07, p=0·273). 3-year disease-free survival for the UFT group was 53·0% (95% CI 49·2-56·6) and that of the S-1 group was 58·2% (54·4-61·8; HR 0·81, 95% CI 0·70-0·93, p=0·0048; pnon-inferiority=0·151). The most common grade 3-4 haematological adverse event was neutropenia (41 [11%] of 359 patients in the UFT group, 48 [13%] of 363 in the S-1 group, 46 [13%] of 355 in the paclitaxel then UFT group, and 83 [23%] of 356 in the paclitaxel then S-1 group). The most common grade 3-4 non-haematological adverse event was anorexia (21 [6%], 24 [7%], seven [2%], and 18 [5%], respectively). INTERPRETATION Sequential treatment did not improve disease-free survival, and UFT was not non-inferior to S-1 (and S-1 was superior to UFT), therefore S-1 monotherapy should remain the standard treatment for locally advanced gastric cancer in Japan. FUNDING Epidemiological and Clinical Research Information Network.