Masato Kitazawa

ASC plays a role in the priming phase of the immune response to type II collagen in collagen-induced arthritis

Although rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology, the role of IL-1β and IL-18 in the pathophysiology of RA has been well established. IL-1β and IL-18 are generated via cleavage of their pro-forms in the presence of the apoptosis-associated speck-like protein containing a caspase recruit domain (ASC), a known adaptor protein that activates procaspase-1. As such, we investigated the involvement of ASC in the progression of murine collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) using ASC-deficient (ASC−/−) and wild-type (ASC+/+) mice. Analyses were performed by immunohistochemistry for tissues and ELISA for sera. We observed an increase in the expression of ASC, as well as IL-1β and IL-18, in the joints of CIA DBA mice, which indicated that ASC is involved in disease development. Next, we demonstrated that the infiltration of inflammatory cells and cartilage/bone destruction in CIA knee joints were significantly increased in ASC+/+ mice compared with ASC−/− mice. No such differences were noted in ASC+/+ and ASC−/− CAIA mice. In terms of cytokine expression in knee joints, IL-1β and IL-18 were depressed in ASC-deficient CIA mice compared with wild-type mice, but were similarly expressed in CAIA joints in both mice groups. Taken together, we can conclude that ASC is involved in the development of CIA and plays a role in the priming phase of the immune response to type II collagen.

Killian-Jamieson diverticulitis with cervical cellulitis: Report of a case

A Killian-Jamieson (K-J) diverticulum is an uncommon hypopharyngeal diverticulum related to the better-recognized Zenker’s diverticulum. Cervical cellulitis due to K-J diverticulitis is also highly exceptional. We report the case of a 53-year-old woman with cervical cellulitis caused by K-J diverticulitis. The cellulitis was cured by the administration of an antibiotic agent. The patient underwent a resection of the K-J diverticulum 2 months after the cellulitis was cured. The cervical diverticulum was judged to be a K-J diverticulum because the diverticulum prolapsed laterally just below the cricopharyngeus muscle on the esophagogram. The left recurrent laryngeal nerve adhered to the proximity of the orifice of the diverticulum. The recurrent laryngeal nerve was carefully preserved before the resection of the diverticulum. Accurate differential diagnosis between K-J and Zenker’s diverticula is necessary before surgery to preserve the recurrent laryngeal nerve.

Gastric Composite Tumor of Alpha Fetoprotein-Producing Carcinoma/Hepatoid Adenocarcinoma and Endocrine Carcinoma with Reference to Cellular Phenotypes

Alpha-fetoprotein-producing carcinoma (AFPC)/hepatoid adenocarcinoma (HAC) and neuroendocrine carcinoma (NEC) are uncommon in the stomach. Composite tumors consisting of these carcinomas and their histologic phenotypes are not well known. Between 2002 and 2007, to estimate the prevalence of composite tumors consisting of tubular adenocarcinoma, AFPC/HAC and NEC, we reviewed specimens obtained from 294 consecutive patients treated surgically for gastric cancer. We examined histological phenotype of tumors of AFPC or NEC containing the composite tumor by evaluating immunohistochemical expressions of MUC2, MUC5AC, MUC6, CDX2, and SOX2. Immunohistochemically, AFPC/HAC dominantly showed the intestinal or mixed phenotype, and NEC frequently showed the gastric phenotype. In the composite tumor, the tubular and hepatoid components showed the gastric phenotype, and the neuroendocrine component showed the mixed type. The unique composite tumor predominantly showed the gastric phenotype, and the hepatoid and neuroendocrine components were considered to be differentiated from the tubular component.

Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells

BackgroundSince cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling.MethodsWe evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling.ResultsUpon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells.ConclusionIn RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.

Clinical significance of apoptosis-associated speck-like protein containing a caspase recruitment domain in oral squamous cell carcinoma

OBJECTIVES To assess apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) expression in oral squamous cell carcinoma (OSCC) and analyze its clinical and pathological significance. STUDY DESIGN ASC expression was studied using immunohistochemistry in 119 OSCCs patients. The relationships between ASC expression and clinical and pathological parameters were statistically analyzed. In addition, the relationships between ASC expression and cell differentiation [IVL (involcrin) expression] and apoptosis (TUNEL [TdT-mediated dUTP nick end labeling] positive cell number) were investigated. RESULTS ASC expression showed significant correlations with parameters including clinical tumor stage, mode of invasion, and histological differentiation, and had a significant impact on survival of OSCC. The distribution of ASC correlated well with that of IVL. ASC expression was significantly correlated with the TUNEL-positive cell number. CONCLUSIONS Lower ASC expression correlates with clinical and pathological malignancy and, consequently, poor prognosis of OSCC. ASC has a close association with cell differentiation and apoptosis.

ASC Induces Apoptosis via Activation of Caspase-9 by Enhancing Gap Junction-Mediated Intercellular Communication

ASC (apoptosis-associated speck-like protein containing a CARD) is a key adaptor molecule of inflammasomes that mediates inflammatory and apoptotic signals. Aberrant methylation-induced silencing of ASC has been observed in a variety of cancer cells, thus implicating ASC in tumor suppression, although this role remains incompletely defined especially in the context of closely neighboring cell proliferation. As ASC has been confirmed to be silenced by abnormal methylation in HT1080 fibrosarcoma cells as well, this cell line was investigated to characterize the precise role and mechanism of ASC in tumor progression. The effects of ASC were examined using in vitro cell cultures based on comparisons between low and high cell density conditions as well as in a xenograft murine model. ASC overexpression was established by insertion of the ASC gene into pcDNA3 and pMX-IRES-GFP vectors, the latter being packed into a retrovirus and subjected to reproducible competitive assays using parental cells as an internal control, for evaluation of cell viability. p21 and p53 were silenced using shRNA. Cell viability was suppressed in ASC-expressing transfectants as compared with control cells at high cell density conditions in in vitro culture and colony formation assays and in in vivo ectopic tumor formation trials. This suppression was not detected in low cell density conditions. Furthermore, remarkable progression of apoptosis was observed in ASC-introduced cells at a high cell density, but not at a low one. ASC-dependent apoptosis was mediated not by p21, p53, or caspase-1, but rather by cleavage of caspase-9 as well as by suppression of the NF-κB-related X-linked inhibitor-of-apoptosis protein. Caspase-9 cleavage was observed to be dependent on gap junction formation. The remarkable effect of ASC on the induction of apoptosis through caspase-9 and gap junctions revealed in this study may lead to promising new approaches in anticancer therapy.

Novel role of ASC as a regulator of metastatic phenotype.

Disorders of cytoskeletal remodeling and signal transduction are frequently involved in cancer progression. In particular, apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC) has been reported a proapoptotic molecule that is epigenetically silenced in several human cancers. ASC is a well‐characterized adaptor protein involved in the formation of multiprotein oligomers, called inflammasomes, and plays a crucial role in the activation and secretion of interleukin‐1β and interleukin‐18 in innate immune cells. However, the function of ASC in the regulation of tumor progression remains elusive. The present investigation examined the involvement of ASC in cancer progression and the acquisition of metastatic ability. To determine the effect of ASC depletion in in vitro and in vivo model systems, ASC was stably knocked down in B16 murine melanoma cell lines using retroviral transduction of shRNA. ASC suppression increased the motility of B16BL6 cells in scratch assays and augmented invasiveness as assessed by a Matrigel‐coated transwell system. Invadopodia formation and Src phosphorylation level were markedly enhanced in ASC‐knockdown cells as well. Since caspase‐8 has been reported to enhance cellular migration by Tyr380 phosphorylation via Src, we examined Tyr380 phosphorylation of caspase‐8 in ASC‐knockdown cells and found it to be elevated in ASC‐knockdown cells but attenuated by z‐VAD‐fmk or z‐IETD‐fmk. Moreover, ASC ablation increased pulmonary metastasis in mice after intravenous injection of B16BL6 cells. Our cumulative findings indicate that ASC suppresses cancer metastasis and progression via the modulation of cytoskeletal remodeling and the Src‐caspase‐8 signaling pathway.

How much length of the distal esophagus is removed by transhiatal approach for squamous cell carcinoma and Barrett's adenocarcinoma in Japanese patients?

BACKGROUND/AIMS The removed length of the esophagus by transhiatal distal esophagectomy (THDE) for patients with squamous cell carcinoma (SCC) and Barretts adenocarcinoma (BAC) in the distal/abdominal esophagus was investigated. METHODOLOGY Twenty six patients with carcinoma mainly located in the distal/ abdominal esophagus were treated by THDE: SCC (Group-S, n=17) and BAC (Group-A, n=9). The length of the mobilized esophagus (D1) and that of the removed esophagus (D2) were measured. After surgery, we measured the distance between the stapled line of the esophagogastrostomy and the diaphragmatic hiatus (D3). The ratio of D3 to the distance between the bronchial bifurcation and the hiatus (D3-ratio) was calculated. RESULTS The mean D1, D2 and D3 were 91.2mm, 65.2mm and 63.2mm, respectively. There were no differences among D1, D2 and D3 between Group-S and Group-A. The mean D3-ratio was 51.9%, and there was no difference between Group-S and Group-A. No postoperative mortality was observed, although postoperative complications were observed in 2 patients (7.7%). After THDE, no local recurrence was observed, node recurrence in 4 (15.4%) and distant metastasis in 7 (26.9%). Five-year survival rate after THDE was 70.1%. Seven patients (26.9%) complained of heartburn and only one of Group-S frequently felt heartburn. In endoscopic findings, most of the patients with heartburn were classified as Grade M. CONCLUSIONS Approximately 65mm of the distal esophagus and approximately 50% of the distance between the bronchial bifurcation and the hiatus was safely removed by THDE for SCC and BAC of the distal/abdominal esophagus, in spite of limited cases.

Survival pathway of cholangiocarcinoma via AKT/mTOR signaling to escape RAF/MEK/ERKpathway inhibition by sorafenib

Cholangiocarcinoma (CCC) is a strongly aggressive malignancy for which surgical resection is the only potential curative therapy. Sorafenib, a multikinase inhibitor of the RAF/MEK/ERK pathway, is a molecular-targeted drug that is approved for hepatocellular carcinoma (HCC) but not for CCC. The differences in signaling pathway characteristics under sorafenib treatment between HCC (HLF, Huh7, PLC/PRF/5) and CCC (RBE, YSCCC, Huh28) cell lines were therefore investigated using cell proliferation, western blotting, and apoptosis analyses. Sorafenib inhibited cell growth significantly less in CCC cells than in HCC cells, with lower suppression of ERK phosphorylation. Significantly decreased AKT Ser473 phosphorylation in HCC cells, and conversely enhanced phosphorylation of AKT Ser473 and mTORC2 in CCC cells, were observed with sorafenib treatment. Disassembly of the mTORC2 complex in RBE cells with siRNA targeting Rictor resulted in the downregulation of AKT Ser473 phosphorylation and enhanced apoptosis presumably via increased FOXO1, which consequently suppressed RBE cell proliferation. Phosphorylation of mTORC1 and autophagy were not influenced by sorafenib in CCC cells. Simultaneous administration of everolimus to suppress activated mTORC1 in RBE cells revealed that combined everolimus and sorafenib treatment under mTORC2 disassembly could enhance growth inhibition through the suppression of both sorafenib- and everolimus-dependent AKT Ser473 phosphorylation in addition to the inhibition of mTORC1 phosphorylation. Prevention of escape by AKT/mTOR signaling from the RAF/MEK/ERK pathway in sorafenib treatment by suppressing mTORC2 activity may lead to promising new approaches in CCC therapy.

High incidence of other primary malignancies in patients with synchronous multiple gastric cancers “a multi-center retrospective cohort study”

This study evaluated the relationship between synchronous multiple gastric cancer and other primary malignancies. During 2002–2013, 1094 consecutive surgically treated gastric cancer patients were enrolled. Preoperatively, we performed total colonoscopy and whole-body computed tomography. When malignancies in other organs were suspected, detailed organ-specific examinations were performed. Synchronous multiple gastric cancer occurred in 102 patients (9.3%)which was frequently observed in patients with preoperative other primary malignancies (p < 0.001). Preoperative other primary malignancy was an independent risk factor for synchronous multiple gastric cancer (p = 0.001; hazard ratio: 2.145, 95% confidence interval: 1.354–3.399) and an independent prognostic factor of overall survival in patients undergoing gastrectomy with curative intent (p = 0.021; hazard ratio: 1.481, 95% confidence interval: 1.060–2.070). Thus, patients with preoperative other primary malignancies have a high risk of synchronous multiple gastric cancer. Careful preoperative examination is recommended to improve survival.