Takanao Sueyoshi

Interleukin‐6 signalling regulates vascular endothelial growth factor‐C synthesis and lymphangiogenesis in human oral squamous cell carcinoma

Lymph node metastasis is associated with resistance to conventional therapy and poor survival of patients with oral squamous cell carcinoma (OSCC). Although lymphangiogenesis is well known to be associated with the occurrence of lymph node metastasis in various cancers, the precise mechanisms of lymphangiogenesis in OSCC are largely unknown. IL‐6, a potent pro‐inflammatory cytokine, has been shown to play active roles in various cancers, including OSCC. This study aimed to investigate the involvement of IL‐6 signalling in lymphatic metastasis and to evaluate the efficacy of tocilizumab, a humanized anti‐human IL‐6 receptor antibody, as an anti‐lymphangiogenic agent for OSCC. This investigation confirmed that levels of expression of IL‐6 protein and VEGF‐C mRNA in OSCC tissues were significantly correlated with lymph node metastasis in patients with OSCC, as assessed by immunohistochemical analysis and real‐time quantitative RT–PCR. In vitro studies showed that IL‐6 regulated VEGF‐C mRNA expression in a human OSCC cell line, SAS cells, through the phosphoinositide 3‐kinase‐Akt pathway. In addition, treatment with tocilizumab led to markedly reduced VEGF‐C mRNA expression and OSCC‐related lymphangiogenesis in SAS xenografts. Together, these data suggest that tocilizumab acted as expected: it inhibited lymph node metastasis in OSCC by reducing tumour lymphangiogenesis. Copyright

Wild-type transthyretin-derived amyloidosis in various ligaments and tendons ☆

Transthyretin-derived amyloid deposition is commonly found in intercarpal ligaments of patients with senile systemic amyloidosis. However, the frequency of transthyretin-derived amyloid deposits in ligaments of other tissues remains to be elucidated. This study aimed to determine the frequency of amyloid deposition and the precursor proteins of amyloid found in orthopedic disorders. We studied 111 specimens from patients with carpal tunnel syndrome (flexor tenosynovium specimens), rotator cuff tears (rotator cuff tendon specimens), and lumbar canal stenosis (yellow ligament specimens). To identify amyloid precursor proteins, we used immunohistochemical staining with antibodies that react with transthyretin, immunoglobulin light chain, amyloid A protein, and β(2)-microglobulin. By means of Congo red staining, we identified 47 (42.3%) amyloid-positive samples, 39 of which contained transthyretin-derived amyloid (18 flexor tenosynovium specimens, 5 rotator cuff tendon specimens, and 16 yellow ligament specimens). Genetic testing and/or clinical findings suggested that all patients with transthyretin amyloid deposits did not have familial amyloidotic polyneuropathy. The occurrence of amyloid deposition in those tissues depended on age. These results suggest that transthyretin-derived amyloid deposits may occur more frequently in various ligaments and tendons than originally expected. In the future, such amyloid deposits may aid determination of the pathogenesis of ligament and tendon disorders in older patients.

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.

Amyloid deposits derived from transthyretin in the ligamentum flavum as related to lumbar spinal canal stenosis

Amyloidosis is a protein conformational disorder with the distinctive feature of extracellular accumulation of amyloid fibrils that come from different proteins. In the ligamentum flavum of the lumbar spine, amyloid deposits were frequently found in elderly patients with lumbar spinal canal stenosis and were at least partially formed by wild-type transthyretin. However, how amyloid deposits in the ligamentum flavum affect lumbar spinal canal stenosis has remained unclear. In this study, we analyzed clinical, pathologic, and radiologic findings of patients with lumbar spinal canal stenosis who had amyloid deposits in the ligamentum flavum. We studied 95 ligamentum flavum specimens obtained from 56 patients with lumbar spinal canal stenosis and 21 ligamentum flavum specimens obtained from 19 patients with lumbar disk herniation. We evaluated histopathologic findings and clinicoradiologic manifestations, such as thickness of the ligamentum flavum and lumbar spinal segmental instability. We found that all 95 ligamentum flavum specimens resected from patients with lumbar spinal canal stenosis had amyloid deposits, which we classified into two types, transthyretin-positive and transthyretin-negative, and that transthyretin amyloid formation in the ligamentum flavum of patients with lumbar spinal canal stenosis was an age-associated phenomenon. The amount of amyloid in the ligamentum flavum was related to clinical manifestations of lumbar spinal canal stenosis, such as thickness of the ligamentum flavum and lumbar spinal segmental instability, in the patients with lumbar spinal canal stenosis with transthyretin-positive amyloid deposits. To our knowledge, this report is the first to show clinicopathologic correlations in transthyretin amyloid deposits of the ligamentum flavum. In conclusion, transthyretin amyloid deposits in the ligamentum flavum may be related to the pathogenesis of lumbar spinal canal stenosis in elderly patients.

Midkine expression in malignant salivary gland tumors and its role in tumor angiogenesis

The aims of this study were to investigate midkine (MK) expression patterns in salivary gland tumors (SGTs) and to evaluate the correlation between MK expression and the degree of malignancy. We performed immunohistochemistry to examine MK expression in specimens of adenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), and pleomorphic adenoma (PA). In addition, we performed immunohistochemistry for CD31 and measured microvessel density (MVD), which is an indicator of angiogenesis. Immunohistochemistry showed that MK protein expression was significantly higher in specimens of malignant SGTs (ACC [P<0.01] and MEC [P<0.001]) than in benign SGT (PA) samples. Furthermore, MVD values tended to be higher in cases that exhibited high expression of MK, which indicated a significant correlation between the degree of MK expression and MVD (P<0.001). These results suggest that MK may play important roles in malignant transformation and tumor angiogenesis in SGTs.

Spinal multifocal amyloidosis derived from wild-type transthyretin

Abstract Spinal amyloidosis can occur as a part of systemic amyloidosis or as localized amyloidomas. However, the exact pathogenesis of the spinal amyloidosis remains to be fully understood. Transthyretin (TTR) is an amyloidogenic protein causing two kinds of amyloid diseases. One is senile systemic amyloidosis (SSA), which is caused by wild-type (WT) TTR and primarily affects cardiac functions. The other type is familial amyloidosis, which is mainly induced by mutated TTR. We report here the first case of multifocal spinal TTR amyloidosis derived from WT TTR with radiculomyelopathy and destructive spondylosis. The data and clinical manifestations suggest that the patient may develop SSA. Clinical manifestations of TTR-related amyloidosis may vary more than we previously thought. In spinal amyloidosis, WT TTR is one of the candidate precursor proteins for the disease.

Stromal expression of neutrophil gelatinase-associated lipocalin correlates with poor differentiation and adverse prognosis in oral squamous cell carcinoma

Neutrophil gelatinase‐associated lipocalin (NGAL) is a member of the lipocalin superfamily. Although its overexpression in various cancers has been reported, little is known about its expression and clinical significance in oral squamous cell carcinoma (OSCC). This study aimed to elucidate the clinical significance of NGAL in OSCC.

Knee osteoarthritis associated with different kinds of amyloid deposits and the impact of aging on type of amyloid

Abstract Amyloidosis is a protein conformational disorder in which amyloid fibrils accumulate in the extracellular space and induce organ dysfunction. Recently, two different amyloidogenic proteins, transthyretin (TTR) and apolipoprotein A-I (Apo A-I), were identified in amyloid deposits in knee joints in patients with knee osteoarthritis (OA). However, clinicopathological differences related to those two kinds of amyloid deposits in the knee joint remain to be clarified. Here, we investigated the clinicopathological features related to these knee amyloid deposits associated with knee OA and the biochemical characteristics of the amyloid deposits. We found that all of our patients with knee OA had amyloid deposits in the knee joints, especially in the meniscus, and those deposits were primarily derived from TTR and/or Apo A-I. Some patients with knee OA, however, had unclassified amyloid deposits. One of our interesting observations concerned the different effects of aging on each type of amyloid formed. The frequency of formation of ATTR deposits clearly increased with age, but that of AApo A-I deposits decreased. Furthermore, we found that ∼16% of patients with knee OA developed ATTR/AApo A-I double deposits in the meniscus. Amyloid deposition may therefore be a common histopathological feature associated with knee OA. Also, aging may induce ATTR formation in the knee joint in elderly patients with knee OA, whereas AApo A-I formation may be inversely correlated with age.

Abstract 16: Downregulation of CYLD leads to acquisition of mesenchymal state and increased migration via ligand-independent TGFβR1 activation in human oral squamous cell carcinoma cells

The 5-year survival of patients with oral squamous cell carcinoma (OSCC) has not changed apparently for the past 30 years. Although cylindromatosis (CYLD) is thought as a potent tumor suppressor, its biological significances in malignancies are largely unknown. The aim of this study was to clarify the role of CYLD in OSCC progression. We investigated expression of CYLD in OSCC including intraepithelial neoplasia (IEN; n = 48) and invasive carcinomas (n = 133) tissues and normal oral mucosal tissues (n = 35) by immunohistochemistry. In addition, effects of CYLD knockdown by siRNA transfection on OSCC progression using 5 OSCC cell lines (SAS, Tu4, HSC3, Ca9-22, and SCC-NA) and HaCaT keratinocytes. Our immunohistochemical analyses revealed that CYLD expression was significantly reduced at invasive lesions in OSCC tissues whereas it was conserved in normal epithelium and IEN. In addition, lower CYLD expression was associated with the correlation with the increased tumor size, advanced clinical stage, and poor overall survival in invasive OSCC. Accordingly, CYLD knockdown led to acquisition of mesenchymal state and increased migratory activity in all the OSCC cell lines as well as HaCaT keratinocytes. Notably, such EMT-like changes were completely blocked by a TGFβR1 inhibitor in all the OSCC cell lines, but not in HaCaT keratinocytes. Furthermore, treatment with excess amount of anti-TGFβ antibody (1D11) did not inhibit the EMT-like changes induced by CYLD repression. These findings suggest that downregulation of CYLD promotes invasion through EMT-like changes via ligand-independent TGFβR1 activation in OSCC cells in mechanisms distinct from normal epithelium. Further studies for these mechanisms probably provide new insights into the biology including “TGFβ switch” and development of a novel therapy in OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 16. doi:1538-7445.AM2012-16

Transthyretin-derived amyloidosis in musculoskeletal systems.

Transthyretin-derived amyloid deposition is commonly found in tenosynovium of senile systemic amyloidosis patients. However, that in ligaments of other organ sites remains to be elucidated. The purpose of this study was to determine the frequency and types of amyloid deposits found in musculoskeletal systems. We investigated patients with carpal tunnel syndrome, rotator cuff tears, and lumbar canal stenosis. We identified 39 cases with TTR amyloid deposits. The mean age in TTR positive cases was higher than non-TTR cases in tenosynovium and yellow ligaments, but not significantly in rotator cuff samples. In the elderly, TTR-derived amyloid deposits were frequently found in ligaments and tendons of various organ sites. Those amyloid deposits may be involved in the pathogenesis of the orthopedic disorders. Abbreviations: FAP1⁄4 familial amyloidotic polyneuropathy; SSA1⁄4 senile systemic amyloidosis; TTR1⁄4 transthyretin Introduction: Amyloidosis is a clinical disorder caused by extracellular deposition of insoluble abnormal fibrils, derived from aggregation of misfolded normally soluble protein [1]. So far, 27 different precursor proteins have been identified in different kinds of amyloidosis [1,2]. It has been well documented that mutated forms of amyloidogenic proteins are more prone to form amyloid fibrils [3]. Transthyretin (TTR) is one of amyloidogenic proteins causing two types of amyloid diseases because TTR itself has b-sheet rich structure and is highly amyloidogenic [1,4]. One is familial amyloid polyneuropathy (FAP), which is hereditary amyloidosis mainly derived from mutated-TTR [5] and the other is senile systemic amyloidosis (SSA). Recently, SSA has been focused in the recent attention [6]. SSA, non-hereditary form of amyloidosis in which wild-type (WT) TTR generates amyloid deposits especially in cardiac and plumonary tissues and occasionally in other systemic organs in the elderly [7]. It has been well documented that most of patients with SSA show a slowly progressive, infiltrative amyloid cardiomyopathy [7]. This disease is believed to be associated with the aging process. In addition, localized type of TTR amyloid has been sometimes reported [8]. However, it was not well known whether it became the pathogenesis of the disease. Accompanied with orthopedic disorders, amyloid deposition in the ligament and tendon has been reported [9], the relationship with disease and the pathogenesis remains to be elucidated. In this report, we examined the frequency of amyloid deposition in tissues resected by operations because of orthopedic disorders. In addition, relationship between amyloid deposition and clinical manifestations were also discussed. Patients and methods: Patients: We investigated 111 specimens of patients with carpal tunnel syndrome (54 specimens), rotator cuff tears (21 specimens), and lumbar canal stenosis (36 specimens). Those patients were diagnosed at the Department of Orthopaedic Surgery in Kumamoto University Hospital and its associated faculties based on clinical finding and radiological examinations from 2008 to 2009. Congo red staining and immunohistochemistry: Formalin-fixed, paraffin-embedded specimens were stained with hematoxylin–eosin and Congo red, as described previously [3]. Elucidation of FAP: To exclude TTR-related FAP, we performed genetic testing, such as a real time PCR or sequencing. Mass spectrometry using surface-enhanced laser desorption/ionization time-offlight mass spectrometry (SELDI/TOF MS) [10] was performed to confirm the patients who did not posses variant TTR in serum. Ethics: The study protocol was approved by Human Ethics Review Committee of Kumamoto University and a signed consent form was obtained from the family of subjects. All patients’ family members gave their informed consent for performing an autopsy. Results: The presence of amyloid deposits was determined by a positive reaction in paraffin sections with Congo red staining, and by an apple green birefringence in the same sections in polarized light. Histochemical analysis with Congo red staining revealed 47 (39%) amyloid positive samples (Figure 1A). Immunohistochemical examination was performed using anti-amyloid precursor proteins as mentioned in section ‘Materials and method’. AntiTTR antibody reacted with the lesions where amyloid deposition was positive in 39 cases (Figure 1B): 18 cases in flexor tenosynovium, 5 cases in rotator cuff tendons, and 16 cases in the yellow ligaments. In eight samples, any antibodies 163