Jianying Guo

Clinical significance of CYLD downregulation in breast cancer

Abstract Cylindromatosis (CYLD) is a tumor suppressor gene that is mutated in familial cylindromatosis, a rare autosomal dominant disorder associated with numerous benign skin adnexal tumors. CYLD is now known to regulate various signaling pathways, including transforming growth factor-β signaling, Wnt/β-catenin signaling, and NF-κB signaling by deubiquitinating upstream regulatory factors. Downregulation of CYLD has been reported in several malignancies; however, the clinical significance of CYLD expression in many malignancies, including breast cancer, remains to be elucidated. This study investigated the clinical significance of CYLD in breast cancer and its roles in tumor progression. We evaluated CYLD expression in matched normal breast tissue samples and tumor breast tissue samples from 26 patients with breast cancer and in a series of breast cancer cell lines. In addition, by means of immunohistochemistry, we investigated CYLD protein expression and its clinical significance in 244 breast cancer cases. We also analyzed the effects of CYLD repression or overexpression on breast cancer cell viability, cell migration, and NF-κB activity with or without receptor activator of NF-κB ligand (RANKL) stimulation. Breast cancer tissues demonstrated significantly reduced CYLD mRNA expression compared with normal breast tissues. Downregulation of CYLD promoted cell survival and migratory activities through NF-κB activation, whereas CYLD overexpression inhibited those activities in MDA-MB-231 cells. As an important finding, CYLD overexpression also inhibited RANKL-induced NF-κB activation. Our immunohistochemical analysis revealed that reduced CYLD protein expression was significantly correlated with estrogen receptor negativity, high Ki-67 index, high nuclear grade, decreased disease-free survival, and reduced breast cancer-specific survival in primary breast cancer. Moreover, reduced CYLD expression was an independent factor for poor prognosis in breast cancer. CYLD downregulation may promote breast cancer metastasis via NF-κB activation, including RANKL signaling.

Novel function of transthyretin in pancreatic alpha cells

Although transthyretin (TTR) is expressed in pancreatic alpha (glucagon) cells in the islets of Langerhans, the function of TTR in pancreatic alpha cells remains unknown. In this study, by using TTR knockout (TTR KO) mice, we determined the novel role of TTR in glucose homeostasis. We demonstrated that TTR KO mice evidenced impaired recovery of blood glucose and glucagon levels. Lack of TTR induced significantly lower levels of glucagon in the islets of Langerhans. These results suggest that TTR expressed in pancreatic alpha cells may play important roles in glucose homeostasis via regulating the expression of glucagon.

Novel Antibody for the Treatment of Transthyretin Amyloidosis

Familial amyloidotic polyneuropathy (FAP) is a systemic amyloidosis mainly caused by amyloidogenic transthyretin (ATTR). This incurable disease causes death ∼10 years after onset. Although it has been widely accepted that conformational change of the monomeric form of transthyretin (TTR) is very important for amyloid formation and deposition in the organs, no effective therapy targeting this step is available. In this study, we generated a mouse monoclonal antibody, T24, that recognized the cryptic epitope of conformationally changed TTR. T24 inhibited TTR accumulation in FAP model rats, which expressed human ATTR V30M in various tissues and exhibited non-fibrillar deposits of ATTR in the gastrointestinal tracts. Additionally, humanized T24 (RT24) inhibited TTR fibrillation and promoted macrophage phagocytosis of aggregated TTR. This antibody did not recognize normal serum TTR functioning properly in the blood. These results demonstrate that RT24 would be an effective novel therapeutic antibody for FAP.

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.

Antibody therapy for familial amyloidotic polyneuropathy

Although it is believed that altered conformations exposing cryptic regions are intermediary and critical steps in the mechanism of transthyretin (TTR) amyloid formation, no effective therapy targeting this step is available. In this study, to establish the antibody therapy for familial amyloidotic polyneuropathy (FAP), we generated a monoclonal anti-TTR antibody, which specifically reacts with surface epitopes of TTR (MAb ATTR) and evaluated its binding affinity and specificity for TTR amyloid fibrils. MAb ATTR showed specific binding affinity for TTR amyloid fibrils, but not for native form of TTR. Moreover, MAb ATTR indeed showed the high consistency with Congo red positive areas in tissue specimens from FAP ATTR V30M patients, indicating that MAb ATTR showed binding affinity and specificity for TTR amyloid fibrils in vitro and in vivo. MAb ATTR may have a potential to suppress TTR amyloid deposition and become a candidate for the antibody therapy for FAP.

The occurrence of islet amyloid polypeptide amyloidosis in Japanese subjects.

Autoimmune chronic pancreatitis. Am J Gastroenterol. 2004;99:1605Y1616. 6. Finkelberg D, Sahani D, Deshpande V, et al. Autoimmune pancreatitis. N Engl J Med. 2006;355:2670Y2676. 7. Sahani D, Kalva SP, Farrell J, et al. Autoimmune pancreatitis: imaging features. Radiology. 2004;233:345Y352. 8. Choi EK, Kim MH, Lee TY, et al. The sensitivity and specificity of serum immunoglobulin G and IgG4 levels in the diagnosis of autoimmune pancreatitis: Korean experience. Pancreas. 2007;35: 156Y161. 9. Japan Pancreas Society. Diagnostic criteria for autoimmune pancreatitis. J Jpn Pancreas Soc. 2002;17:585Y587. 10. Kamisawa T, Okazaki K, Kawa S, et al. Diagnostic criteria for autoimmune pancreatitis. J Clin Gastroenterol. 2008;42(4):404Y407. 11. Chari S, Longnecker D, Kloppel G. The diagnosis of autoimmune pancreatitis: A western perspective. Pancreas. 2009;38(8):846Y848. 12. Ooi CY, Dorfman R, Cipolli M, et al. Type of CFTR mutation determines the risk of pancreatitis in patients with cystic fibrosis. Gastroenterology. 2011;140: 153Y161. 13. Schneider A, Larusch J, Sun X, et al. Combined bicarbonate conductance-impairing variants in CFTR and SPINK1 variants are associated with chronic pancreatitis in patients without cystic fibrosis. Gastroenterology. 2011;140:162Y171.

Transthyretin-derived amyloid deposition in the heart of an elderly Japanese population.

Although in Western countries the prevalence of senile systemic amyloidosis (SSA) in the elderly (480 years) was found to be about 25% based on examination of autopsy-derived cardiac specimens, prevalence in Asian countries is still unclear. The aim of the study was to identify clinicopathological features in Japanese patients with transthyretin (TTR)-derived amyloid deposition. Autopsy-derived cardiac specimens were examined. Pathological analyses revealed that TTR-derived amyloid deposition was found in 11% of the patients over 80 years of age. Although the prevalence of TTR-derived amyloid deposition was lower in a Japanese population than that in a Western population, we must consider SSA in elderly patients if the clinical manifestations cannot be explained by other pathologies. Introduction: Amyloidosis is a clinical disorder caused by extracellular deposition of insoluble abnormal fibrils, derived from normally soluble proteins. So far, 27 different precursor proteins have been identified in different kinds of amyloidosis [1]. Transthyretin (TTR) is one of the amyloidogenic proteins and causes two types of amyloid diseases. One is familial amyloid polyneuropathy (FAP), which is hereditary amyloidosis caused by mutatedTTR [2]. The other is non-hereditary senile systemic amyloidosis (SSA) in which wild-type (WT) TTR forms amyloid deposits especially in cardiac and pulmonary tissues, and occasionally in other systemic organs in the elderly [3]. Several reports showed that the prevalence of SSA was up to 25% in the elderly over the age of 80, based on examination of autopsy-derived cardiac specimens in the United State and Europe [4–6]. It has been well documented that most patients with SSA show a slowly progressive amyloid cardiomyopathy which causes congestive heart failure and arrhythmia. However, clinicopathological features of SSA in Asian countries have still not been clearly identified. In this report, we examined the frequency of TTRderived amyloid deposition using autopsy specimens of patients who had not been diagnosed with amyloidosis before their death and the incidence was compared to aging. In addition, the relationship between amyloid deposition and clinical manifestations was also discussed. Materials and methods: Materials: We examined autopsy-derived cardiac specimens of 175 consecutive individuals over 40 years of age at Kumamoto University Hospital, Japan between January 2001 and March 2008. FAP patients were excluded by genetic testing and/or clinical findings in medical records. Concerning age, 10 cases were in their forties, 33 in their fifties, 43 in their sixties, 63 in their seventies, 20 in their eighties, and 5 in their nineties; one case was over 100 years old. In cases with TTR amyloid deposits in cardiac specimens, other tissues were also examined. For immunohistochemical staining, an antibody for human TTR was purchased from Dako (Glostrup, Denmark). Other chemicals used in the study were purchased from Nakarai tesque (Kyoto, Japan). Congo red staining: Tissue samples were fixed with 10% formalin, embedded in paraffin, serially sectioned at a thickness of 4 mm, and placed onto microscopic slides. Sections were stained with alkaline Congo red and hematoxylin. Amyloid deposits were confirmed under polarized light for the presence of green birefringence. Immunohistochemical stainings: The specimens with amyloid deposits were used for immunohistochemical staining. The specimens were deparaffinated, dehydrated in a modified alcohol series, and incubated in blocking buffer (1% bovine serum albumin (BSA), and 5% goat serum in PBS). A polyclonal rabbit anti-human TTR antibody diluted 1:100 in blocking buffer was used as the primary antibody. A horseradish peroxidase-conjugated goat anti-rabbit IgG antibody diluted 1:100 in blocking buffer was used as the secondary antibody. Reactivity was visualized with the DAB Liquid System (DAKO), according to the manufacturer’s instructions. Sections were counterstained with hematoxylin. For parallel control sections, primary antibody was replaced by blocking buffer. Other immunohistochemical stainings with antibodies for other amyloid precursor proteins were not examined. Results: Congo red staining for the specimen revealed Congo red positive deposition in 17 of 175 cases (9.7%). In the 17 amyloid positive cases, we found TTR-derived amyloid deposition in 5 (2.9%). Immunohistochemical staining with antiTTR antibody was negative in another 12 cases. The incidence of cardiac amyloidosis increased with aging. The incidence of TTR-derived amyloid deposition increased with age: 2 of 63 cases in their seventies, one of 20 cases in their eighties, one of 5 cases in their nineties, and one of one case aged over 100 (Figure 1). In 175 autopsy cases, TTR-derived amyloid deposition was not detected under the age of 69 years. 180

Osteopontin‐integrin αvβ3 axis is crucial for 5‐fluorouracil resistance in oral squamous cell carcinoma

Clinical applications of a chemotherapeutic agent, 5‐fluorouracil (5‐FU) in oral squamous cell carcinoma (OSCC) have been limited because of drug resistance. This study aimed to identify novel mechanisms of 5‐FU resistance. Here we found increased osteopontin (OPN) gene expression in OSCC tissues with resistance to 5‐FU‐based chemoradiotherapy. OPN overexpression in OSCC cells led to 5‐FU resistance and abrogated the prosurvival effect of the drug in a mouse xenograft model. OPN‐induced 5‐FU resistance required integrin αvβ3. Targeting integrin αvβ3 reversed the resistance in a 5‐FU‐resistant clone highly expressing OPN. Our data suggest that the OPN‐integrin αvβ3 axis is crucial for 5‐FU resistance in OSCC.

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

Antioxidative effect of albumin on amyloid fibril formation in transthyretin-related amyloidosis.

Transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP) is characterized by systemic accumulation of amyloid fibrils caused by a point mutation in the TTR gene. Despite the urgent need for alternative therapeutic strategies, the pathogenesis of FAP still remains unclear. In this study, we focused on albumin, the most abundant protein in plasma, and reported the role of albumin in the TTR amyloid formation process. Serum albumin levels were significantly decreased in FAP patients as the disease progressed. N-ethylmaleimide-albumin, diminishing the antioxidant effect, abolished its inhibitory effect on amyloid formation, suggesting that albumin as an antioxidant effectively suppressed TTR amyloid formation. In FAP patients, albumin was significantly oxidized as the disease progressed. Moreover, loss of functional albumin accelerated TTR deposition in analbuminemia rats with a human variant TTR gene. Taken together, these results indicate that albumin, as an antioxidant, may play an inhibitory role in the process of TTR amyloid formation. Introduction: Familial amyloidotic polyneuropathy (FAP), which is induced by amyloidogenic transthyretin (ATTR), is characterized by systemic accumulation of amyloid fibrils in the peripheral nerves and other organs [1]. Although many studies have been carried out to identify various types of ATTR-related FAP, the precise mechanism of TTR amyloid formation remains to be clarified. TTR normally behaves as a soluble tetramer and binds to retinol binding protein and thyroxine (T4) in plasma. It has been proposed that tetrameric TTR is not itself amyloidogenic, but dissociation of the tetramer into a compact non-native monomer with low conformational stability can lead to amyloid fibril formation. In addition, post-translational modification of TTR is also recognized as playing a key role in amyloid fibril formation. The involvement of oxidative stress in amyloid fibril formation in tissues of FAP patients has been reported [2]. Our previous in vitro study showed that nitric oxide-mediated modification of TTR may play an important role in amyloid formation, indicating that oxidative stress facilitates amyloid formation [3]. Albumin is the most abundant protein in plasma. It serves as a transport carrier for various ligands and represents the major and predominant antioxidant in plasma [4]. Previous studies have shown that total reactive antioxidant potential in plasma, considered as an index of the level of antioxidants, was decreased in patients with FAP [5]. In addition, more recent studies have demonstrated that albumin suppressed amyloid formation of amyloid-b (Ab), a component of amyloid fibril in Alzheimer’s disease, through decreasing oxidative stress [6]. This evidence suggests that albumin as an antioxidant may play a crucial role in amyloid formation in FAP. In this study, we clarified the role of the antioxidative property of albumin in FAP disease progression. Methods: Both wild type (WT)-TTR and ATTRV30M were purified from serum samples obtained from healthy volunteers and homozygotic FAP ATTR V30M patients. Albumin was isolated from serum samples from healthy volunteers by ionexchange chromatography. Function of albumin isolated from FAP ATTR V30M patients was analyzed quantitatively and qualitatively. To evaluate the effect of albumin on amyloid formation, sandwich ELISA was performed by using monoclonal anti-TTR115-124 antibody, which specifically reacts with amyloid fibrils and preamyloid deposits [7]. Nethylmaleimide (NEM)-albumin was prepared by treatment with charcoal and NEM. To evaluate the effect of albumin on TTR deposition in vivo, analbuminemic transgenic rats possessing a human ATTR V30M gene (V30M Tg NAR) were analyzed. Results and discussion: The serum albumin levels were significantly decreased in FAP patients as the disease progressed. The effect of albumin on amyloid formation was assessed by sandwich ELISA. Of various serum proteins, albumin showed potent inhibition of amyloid formation, compared with alpha1-acid glycoprotein and transferrin. The amyloid formation of both WT-TTR and ATTR-V30M was suppressed in the presence of albumin in a dosedependent manner. These data suggest that albumin may be closely associated with the process of TTR amyloid formation. To clarify the antioxidant effect of albumin on TTR amyloid formation, we next assessed the effect of modified albumin, which was modified in its antioxidant effect. NEM-albumin, diminishing the antioxidant effect, abolished its inhibitory effect on amyloid formation, suggesting that the antioxidative 17