Shoko Nakamura

Suppression of myeloid cell leukemia-1 (Mcl-1) enhances chemotherapy-associated apoptosis in gastric cancer cells.

BackgroundMyeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that regulates apoptosis sensitivity in a variety of cell types. Here we evaluate the roles of Mcl-1 in chemotherapy-associated apoptosis in gastric cancer cells. In addition, our study examined whether Mcl-1 contributed to apoptosis resistance in so-called cancer stem cell (CSC)-like populations in gastric cancer.MethodsSeven gastric cancer cell lines were used. The expression of Mcl-1 was assessed by either real-time polymerase chain reaction or Western blot analysis. Apoptosis was quantitated by morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knockdown the expression of Mcl-1. The release of cytochrome c was evaluated by subcellular fractionation and immunoblot analysis. To identify and isolate the CSC-like populations, we used the CSC-associated cell surface marker CD44 and flow cytometry.ResultsSix out of the 7 gastric cancer cell lines overexpressed Mcl-1 protein. These Mcl-1-expressing cell lines were relatively resistant to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin (CDDP). Depletion of Mcl-1 protein by RNAi technology effectively sensitized the cells to anticancer drug-induced mitochondrial cytochrome c release, caspase activation, and apoptosis. In addition, vast amounts of Mcl-1 mRNA were expressed in CD44-positive CSC-like cells. Mcl-1 suppression enhanced the apoptosis in CD44-positive cells to a level equivalent to that in CD44-negative cells, suggesting that Mcl-1 mediates chemotherapy resistance in CSC-like populations.ConclusionThese results suggest that Mcl-1 mediates the resistance to apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.

TGF-β1 in tumor microenvironments induces immunosuppression in the tumors and sentinel lymph nodes and promotes tumor progression.

In cancer patients, sentinel lymph nodes (SLNs) are crucial in the induction of antitumor T cells. However, in many cases, SLNs and tumors appear to be in immunosuppressive condition through mechanisms yet to be elucidated. In this study, the role of tumor-derived TGF-&bgr;1 in the generation of immunosuppressive microenvironments of tumors and SLNs was investigated. Murine colorectal carcinoma CT26 transduced with TGF-&bgr;1 cDNA (CT26-TGF-&bgr;1) showed enhanced tumor growth compared with mock-transduced CT26 (CT26-Mock) when implanted in syngeneic Balb/c mice, even though CT26-TGF-&bgr;1 shows slower growth in vitro. This enhanced growth was not observed when implanted in immunodeficient mice, suggesting that TGF-&bgr;1 enhanced tumor growth by suppressing antitumor T-cell responses. Analysis of immune cells in CT26-TGF-&bgr;1-implanted mice revealed impairment of dendritic cells (DCs), decrease of CD8+ T cells, and increase of MDSCs and Tregs in the tumors. Similarly, the SLNs of these mice showed an increase of MDSCs, Tregs, and PD-L1+ DCs, and decrease of T-cell stimulatory activity of DCs accompanied by decreased CD80 expression and TNF-&agr; production. In addition, induction of tumor antigen–specific T cells from SLNs of the CT26-TGF-&bgr;1–implanted mice was significantly reduced. These results demonstrate that overproduction of TGF-&bgr;1 is critical for the generation of immunosuppressive microenvironments in both tumors and SLNs, which may result in suppression of spontaneous antitumor CD8+ T-cell responses. Therefore, TGF-&bgr;1 is an attractive target for restoration of immunosuppressive condition in cancer patients.

Improvement of cancer immunotherapy by combining molecular targeted therapy.

In human cancer cells, a constitutive activation of MAPK, STAT3, β-catenin, and various other signaling pathways triggers multiple immunosuppressive cascades. These cascades result in the production of immunosuppressive molecules (e.g., TGF-β, IL-10, IL-6, VEGF, and CCL2) and induction of immunosuppressive immune cells (e.g., regulatory T cells, tolerogenic dendritic cells, and myeloid-derived suppressor cells). Consequently, immunosuppressive conditions are formed in tumor-associated microenvironments, including the tumor and sentinel lymph nodes. Some of these cancer-derived cytokines and chemokines impair immune cells and render them immunosuppressive via the activation of signaling molecules, such as STAT3, in the immune cells. Thus, administration of signal inhibitors may inhibit the multiple immunosuppressive cascades by acting simultaneously on both cancer and immune cells at the key regulatory points in the cancer-immune network. Since common signaling pathways are involved in manifestation of several hallmarks of cancer, including cancer cell proliferation/survival, invasion/metastasis, and immunosuppression, targeting these shared signaling pathways in combination with immunotherapy may be a promising strategy for cancer treatment.

Cancer-induced immunosuppressive cascades and their reversal by molecular-targeted therapy.

Immunological status in tumor tissues varies among patients. Infiltration of memory‐type CD8+ T cells into tumors correlates with prognosis of patients with various cancers. However, the mechanism of the differential CD8+ T cell infiltration has not been well investigated. In general, tumor‐associated microenvironments, including tumor and sentinel lymph nodes, are under immunosuppressive conditions such that the immune system is not able to eliminate cancer cells without immune‐activating interventions. Constitutive activation of various signaling pathways in human cancer cells triggers multiple immunosuppressive cascades that involve various cytokines, chemokines, and immunosuppressive cells. Signaling pathway inhibitors could inhibit these immunosuppressive cascades by acting on either cancer or immune cells, or both. In addition, common signaling mechanisms are often utilized for multiple hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression). Therefore, targeting these common signaling pathways may be an attractive strategy for cancer therapy including immunotherapy.

Roles of Signaling Pathways in Cancer Cells and Immune Cells in Generation of Immunosuppressive Tumor-Associated Microenvironments

Cancer cells trigger multiple immunosuppressive cascades and generate immunosuppressive tumor-associated microenvironments including tumor and sentinel lymph nodes. Constitutive activation of various signaling pathways (e.g., MAPK, STAT3, NF-κB, β-catenin) in human cancer cells was found to trigger the multiple immunosuppressive cascades through the production of immunosuppressive cytokines, such as TGF-β, IL-10, IL-6, and VEGF, and induction of immunosuppressive immune cells, such as regulatory T cells, tolerogenic dendritic cells, and myeloid derived suppressor cells. Some of these cancer-derived cytokines impair various immune cells through activation of their signaling molecules such as STAT3 and NF-κB. Inhibitors for these activated signals could inhibit the multiple immunosuppressive cascades by acting on both cancer cells and immune cells. Since common signaling mechanisms are often utilized for some of the hallmarks of cancer (e.g., cell proliferation/survival, invasion/metastasis, and immunosuppression), targeting these common signaling pathways may be an attractive strategy for cancer therapy, including immunotherapy.

Successful treatment of a large hyperplastic polyp in the jejunum by using single-balloon enteroscopy

S o t S E a An 82-year-old man with chronic kidney disease was referred for evaluation because of repeated melena with anemia. EGD and colonoscopy showed normal results. A small-bowel series showed a pedunculated polyp (A) that was about 5 cm in diameter in the upper jejunum. Single-balloon enteroscopy by mouth revealed a reddish, pedunculated polyp that was oozing (B). The polyp was resected endoscopically en bloc after we injected saline solution with a small amount of epinephrine into the stalk, and the resection site was closed by using hemoclips to prevent postpolypectomy bleeding (C). Histologic examination of the resected polyp showed hyperplastic changes of the epithelium and edema in the superficial layer of the lamina propria without the arborizing pattern of smooth-muscle proliferation and neoplastic lesions (D, H&E, orig. mag. 40). The final diagnosis was hyperplastic polyp of the M

Abstract 1017: Wnt signalings contributes to the maintenance of cancer stem cell-like populations in Huh7 hepatocellular carcinoma cell line: Role of hepatic stellate cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC BACKGROUND: Cancer stem cells play crucial roles in cancer development and progression. The function and survival of stem cells appears to be regulated by local microenvironment or niche. In many tumors, tumor cells actively recruit a variety type of cells including myofibroblasts and create a tumor niche that participates in the process of cancer progression, chemo-resistance, and metastasis. During liver injury, hepatic stellate cells (HSCs) transdifferentiate into myofibroblasts and the resulting fibrosis is associated with the development of hepatocellular carcinoma (HCC). However, the possible contribution of HSCs to tumor-stroma interactions is totally unknown. Therefore, our AIM is to investigate precise role of HSCs on interactions with tumor cells, especially with cancer stem cells. METHODS: Hepatoma cell line Huh7 was used in this study. FACS analysis was used to evaluate stem cell markers such as side population (SP) and CD133. Each subpopulation was isolated and stem cell-like properties were evaluated by in vivo tumorigenesity and in vitro sphere-forming activity. To evaluate the role of HSCs on tumor-stromal interactions, Huh7 cells (at lower dish) and immortalized HSC line hTERT HSCs (at upper dish) were co-cultured in Transwell culture dishes. In some experiments, Huh7 and hTERT HSCs were mix-cultured in the upper dish and Huh7 cells in the lower dish was analyzed by FACS. RESULTS: We identified a small percentage (approximately 0.6%) of Huh7 cells as a side population (SP) relative to the main population (MP) cells by Hoechst 33342-based FACS profiles. In SP subpopulation, CD133-positive cells were enriched as compared with MP subpopulation (60.3% in total cells, 81.3% in SP, and 58.0% in MP). Only CD133-positive SP cells, but not CD133-negative SP cells nor CD133-positive MP cells, displayed tumor forming capacity and sphere forming activity, suggesting that distinct CD133-positive SP subpopulation have stem cell-like properties in Huh7 cells. The SP subpopulation of Huh7 is increased after the co-culture with hTERT HSCs for 4 days (7.6 hold), suggesting that HSCs maintains self-renewal of cancer stem cells in Huh7. CD133-positive cells were not increased after the co-culture. An increased number of SP subpopulation was attenuated by the treatment with Wnt antagonist DKK-1. The SP subpopulation in the lower dish was further increased when cancer cells and HSCs were mix-cultured in the upper dish (13.5 hold v.s. single culture). CONCLUSION: CD133-positive SP cells are a distinct cancer stem cell population in Huh7 cells. HSCs play an important role in supporting cancer stem cells in the tumor niche via Wnt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1017.

Abstract 3325: Cancer stem cell like properties of 5-fluorouracil resistant Panc-1 cell

Cancer stem cells (CSCs), a small fraction of tumor-initiating cells, are known to be resistant to chemotherapy-induced apoptosis. CSCs are considered to be responsible for self-renewal/differentiation, invasion, and metastasis of malignant tumor. We have previously reported that pancreatic cancer derived side population (SP) cells, a cancer stem cell enriched fraction, were more resistant to chemotherapeutic agents and more likely to cause epithelial-to-mesenchymal transition (EMT) compare to major population (MP) cells. Therefore, we formulated the HYPOTHESIS that 5-fuluorouracil (5-FU) resistant population cells are enriched with CSCs. The AIM of this study is to evaluate whether apoptosis-resistant cancer cells have CSC-like properties, i.e., self-renewal, local invasion and EMT. METHODS: Seventy to eighty percent confluent pancreatic cancer cells, PANC-1, were incubated in the presence of 5-FU (30mg/ml) for 24 hours, and further incubated for one to five weeks without 5-FU. To assess the capacity of self-renewing, cells were planted using a method for anchorage-independent culturing to form spheroids. EMT was induced with TGF-beta (7.5 ng/ml, for 3 days), then evaluated mRNA expression by real time PCR for E-cadherin, Snail, and vimentin. E-cadherin protein level was also examined for immunoblot analysis. Activity of local invasion was analyzed by Matrigel invasion assay. To evaluate tumorigenicity, 5-FU resistant cells were injected into NOD/SCID mice. RESULTS: 5-FU induced significant apoptosis in PANC-1 cells, reducing the number of surviving cells to less than one percent. The survived cells showed a colony-forming activity as they started to re-proliferate at four weeks after 5-FU removal. There was a group of cells among the 5-FU resistant surviving cells, that gradually regains sensitivity to 5-FU by re-plating. The frequency of cells that were capable of initiating spheres was higher in 5-FU resistant cells and they over expressed stem cell marker genes, Oct 4 and Nanog. Incubation with TGF-beta induced EMT-associated gene alterations. These alterations were greater in 5-FU resistant surviving cells as compared to untreated cells. Matrigel invasion activity of apoptosis-resistant PANC-1 cells was greater by seven fold than controlled cells. In addition, in vivo tumor experiments showed 5-FU resistant cells to be more tumorigenic than conventional Panc-1 cells. In CONCLUSION, apoptosis-resistant cancer cells have CSC-like properties, i.e., initiating sphere formation, expressing stem cell genes, and respond to EMT stimulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3325.