A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81–127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81–127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81–127–based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis

Biallelic disruption of DDX41 activity is associated with distinct genomic and immunophenotypic hallmarks in acute leukemia

IntroductionInherited DDX41 mutations cause familial predisposition to hematologic malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), with the majority of DDX41 mutated MDS/AMLs described to date harboring germline DDX41 and co-occurring somatic DDX41 variants. DDX41-AMLs were shown to share distinguishing clinical features such as a late AML onset and an indolent disease associated with a favorable outcome. However, genotype-phenotype correlation in DDX41-MDS/AMLs remain poorly understood.MethodsHere, we studied the genetic profile, bone marrow morphology and immunophenotype of 51 patients with DDX41 mutations. We further assessed the functional impact of ten previously uncharacterized DDX41 variants of uncertain significance.ResultsOur results demonstrate that MDS/AML cases harboring two DDX41 variants share specific clinicopathologic hallmarks that are not seen in other patients with monoallelic DDX41 related hematologic malignancies. We further showed that the features seen in these individuals with two DDX41 variants were concordant with biallelic DDX41 disruption.DiscussionHere, we expand on previous clinicopathologic findings on DDX41 mutated hematologic malignancies. Functional analyses conducted in this study unraveled previously uncharacterized DDX41 alleles and further illustrate the implication of biallelic disruption in the pathophysiology of this distinct AML entity

コウクウ ヘンペイ ジョウヒ ガン ノ ビョウタイ ニ オケル インターロイキン 6 IL-6 ノ カンヨ ト チリョウ エ ノ オウヨウ

近年、口腔癌においてインターロイキン6（IL-6)の発現が上昇し、その病態に関与していることが報告されている。しかし、IL-6 が口腔癌におよぼす影響と、IL-6 receptor(IL-6R) シグナリングが口腔癌治療の標的となりうるかについては不明である。そこで本研究では、口腔癌で最も頻度の高い口腔扁平上皮癌（OSCC）における IL-6R シグナリングの生物学的活性の解明および IL-6R シグナリングに対して阻害的に働くヒト化抗IL-6R抗体（トシリズマブ）の口腔癌治療における有用性を検討することを目的とした

CYLD: a critical regulator of hypoxia-mediated inflammation in tumors: DOI: 10.14800/ics.479

Cylindromatosis (CYLD) was originally identified as a tumor suppressor, because loss of its function causes a benign human tumor.  In the past, multitude of  efforts have been made toward elucidating the biological features of CYLD, and uncovered not only its multiple functions as deubiquitinase, but also the clinical significance of CYLD in a wide variety of diseases.  At present, dysregulation of CYLD by loss of its expression is believed to play key roles in a multiple of pathological processes, including tumor cell proliferation, survival, and inflammatory responses by regulating their specific cell signaling pathway.  Recently, we discovered that loss of CYLD expression in hypoxic regions of human glioblastoma multiforme (GBM), the most aggressive brain tumor, suggesting the clinical significance of CYLD in the pathogenesis of GBM.  Here, we reviewed the diverse biological features and clinical significance of CYLD, particularly focusing on the roles of CYLD as a critical regulator of hypoxia-mediated inflammation in GBM

The Tumour Suppressor CYLD Is Required for Clathrin-Mediated Endocytosis of EGFR and Cetuximab-Induced Apoptosis in Head and Neck Squamous Cell Carcinoma

Epidermal growth factor receptor (EGFR) is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a target for the therapeutic antibody cetuximab (CTX). However, because only some patients have a significant clinical response to CTX, identification of its predictive biomarkers and potentiation of CTX-based therapies are important. We have recently reported a frequent downregulation of cylindromatosis (CYLD) in primary HNSCC, which led to increased cell invasion and cisplatin resistance. Here, we show that CYLD located mainly in lipid rafts was required for clathrin-mediated endocytosis (CME) and degradation of the EGFR induced by EGF and CTX in HNSCC cells. The N-terminus containing the first cytoskeleton-associated protein-glycine domain of CYLD was responsible for this regulation. Loss of CYLD restricted EGFR to lipid rafts, which suppressed CTX-induced apoptosis without impeding CTX’s inhibitory activity against downstream signalling pathways. Disruption of the lipid rafts with cholesterol-removing agents overcame this resistance by restoring CME and the degradation of EGFR. Regulation of EGFR trafficking by CYLD is thus critical for the antitumour activity of CTX. Our findings suggest the usefulness of a combination of cholesterol-lowering drugs with anti-EGFR antibody therapy in HNSCC

The Distribution of Phosphatidylcholine Species in Superficial-Type Pharyngeal Carcinoma

Objectives. Superficial-type pharyngeal squamous cell carcinoma (STPSCC) is defined as carcinoma in situ or microinvasive squamous cell carcinoma without invasion to the muscular layer. An exploration of the biological characteristics of STPSCC could uncover the invasion mechanism of this carcinoma. Phosphatidylcholine (PC) in combination with fatty acids is considered to play an important role in cell motility. Imaging mass spectrometry (IMS) is especially suitable for phospholipid analysis because this technique can distinguish even fatty acid compositions. Study Design. IMS analysis of frozen human specimens. Methods. IMS analysis was conducted to elucidate the distribution of PC species in STPSCC tissues. STPSCC tissue sections from five patients were analyzed, and we identified the signals that showed significant increases in the subepithelial invasive region relative to the superficial region. Results. Three kinds of PC species containing arachidonic acid, that is, PC (16:0/20:4), PC (18:1/20:4), and PC (18:0/20:4), were increased in the subepithelial invasive region. Conclusion. These results may be associated with the invasion mechanism of hypopharyngeal carcinoma

Therapeutic approaches targeting midkine suppress tumor growth and lung metastasis in osteosarcoma

Midkine (MK) plays important roles in tumorigenesis, however, the biological function of MK and whether MK can be a therapeutic target in osteosarcoma are unclear. Here, we found that osteosarcoma tissues showed high MK expression. MK knockdown by small interfering RNA significantly induced apoptosis in osteosarcoma cells, whereas recombinant MK increased cell proliferation. Inhibition of MK signaling by anti-MK monoclonal antibody (anti-MK mAb) suppressed growth of osteosarcoma cells both in vitro and in vivo. Moreover, inhibition of MK function significantly suppressed lung metastasis in xenograft transplantation model. Targeting MK by anti-MK mAb may have value in the treatment of osteosarcoma

Loss of Tumor Suppressor CYLD Expression Triggers Cisplatin Resistance in Oral Squamous Cell Carcinoma

Cisplatin is one of the most effective chemotherapeutic agents commonly used for several malignancies including oral squamous cell carcinoma (OSCC). Although cisplatin resistance is a major obstacle to effective treatment and is associated with poor prognosis of OSCC patients, the molecular mechanisms by which it develops are largely unknown. Cylindromatosis (CYLD), a deubiquitinating enzyme, acts as a tumor suppressor in several malignancies. Our previous studies have shown that loss of CYLD expression in OSCC tissues is significantly associated with poor prognosis of OSCC patients. Here, we focused on CYLD expression in OSCC cells and determined whether loss of CYLD expression is involved in cisplatin resistance in OSCC and elucidated its molecular mechanism. In this study, to assess the effect of CYLD down-regulation on cisplatin resistance in human OSCC cell lines (SAS), we knocked-down the CYLD expression by using CYLD-specific siRNA. In cisplatin treatment, cell survival rates in CYLD knockdown SAS cells were significantly increased, indicating that CYLD down-regulation caused cisplatin resistance to SAS cells. Our results suggested that cisplatin resistance caused by CYLD down-regulation was associated with the mechanism through which both the reduction of intracellular cisplatin accumulation and the suppression of cisplatin-induced apoptosis via the NF-κB hyperactivation. Moreover, the combination of cisplatin and bortezomib treatment exhibited significant anti-tumor effects on cisplatin resistance caused by CYLD down-regulation in SAS cells. These findings suggest the possibility that loss of CYLD expression may cause cisplatin resistance in OSCC patients through NF-κB hyperactivation and may be associated with poor prognosis in OSCC patients

Association Midkine in plasma as a novel breast cancer marker

Midkine, a heparin-binding growth factor, is up-regulated in many types of cancer. The aim of this study was to measure plasma midkine levels in patients with breast cancer and to assess its clinical significance. We examined plasma midkine levels in 95 healthy volunteers, 11 patients with ductal carcinoma in situ (DCIS), 111 patients with primary invasive breast cancer without distant metastasis (PIBC), and 25 patients with distant metastatic breast cancer (MBC), using an automatic immunoasssay analyzer (TOSOH AIA system). In PIBC, we studied the correlation between plasma midkine levels and clinicopathological factors. Immunoreactive midkine was detectable in the plasma of healthy volunteers, and a cut-off level of 750 pg/mL was established. In breast cancer patients, plasma midkine levels were increased above normal values. These elevated levels of midkine were seen in one (9.1%) of 11 patients with DCIS, 36 (32.4%) of 111 patients with PIBC, and 16 (64.0%) of 25 patients with MBC. Increased levels of midkine were correlated with menopausal status (P = 0.0497) and nuclear grade (P = 0.0343) in PIBC. Cancer detection rates based on midkine levels were higher than those based on three conventional markers including CA15-3 (P < 0.0001), CEA (P = 0.0077), and NCCST-439 (P < 0.0001). Detection rates of breast cancer using a combination of two conventional tumor markers (CA15-3/CEA, CA15-3/NCCST-439, or CEA/NCCST-439) with midkine is significantly higher than those using combination of three conventional tumor markers. Midkine may be a useful novel tumor marker for detection of breast cancer, superior to conventional tumor markers. (Cancer Sci 2009; 100: 1735-1739