Kenta Mukaihara

Proteomics study of open biopsy samples identifies peroxiredoxin 2 as a predictive biomarker of response to induction chemotherapy in osteosarcoma

UNLABELLED We attempted to identify biomarkers that would predict responsiveness of osteosarcoma (OS) to induction chemotherapy. Tumor tissues obtained by open biopsy before induction chemotherapy were investigated. On the basis of histological observations at the time of surgery and the Huvos grading system, 7 patients were classified as good responders and the other 6 as poor responders. Protein expression profiling was performed by two-dimensional difference gel electrophoresis. Among 3494 protein spots observed, the intensity of 33 spots was found to differ significantly between the two patient groups. The proteins for these 33 protein spots were identified by mass spectrometry. The higher expression of peroxiredoxin 2 (PRDX2) in poor responders was confirmed by Western blotting. Gene silencing assay demonstrated that reduced expression of PRDX2 was associated with increased sensitivity of OS cells to chemotherapeutic drugs such as methotrexate, doxorubicin and cisplatin. Moreover, siRNA-induced silencing of PRDX2 resulted in a decrease of cell proliferation, invasion and migration. These findings indicated that PRDX2 would be a candidate biomarker of response to induction chemotherapy. Measurement of PRDX2 in open biopsy samples before treatment may contribute to risk stratification therapy for OS. BIOLOGICAL SIGNIFICANCE The response of osteosarcoma patients to induction chemotherapy is critical because the prognosis of responders is quite favorable, whereas that of non-responders is poor. Although there are many therapeutic options for osteosarcoma, no parameter for predicting the response to induction chemotherapy has been available. We conducted a proteomics study aimed at developing a biomarker that would predict the response of osteosarcoma to induction chemotherapy. Using open biopsy samples obtained before chemotherapy, we conducted 2D-DIGE with our originally devised large-format electrophoresis apparatus and identified peroxiredoxin 2 (PRDX2) as a novel predictive biomarker. The diagnostic performance of PRDX2 was confirmed by ROC analysis, and its functional properties were investigated in a series of in vitro functional assays. Our findings indicate the possible application of PRDX2 as a predictive biomarker in patients with osteosarcoma.

Proteomic approach toward personalized sarcoma treatment: Lessons from prognostic biomarker discovery in gastrointestinal stromal tumor

Sarcomas range from curable tumors to those causing death via metastasis and recurrence. Thus, there is an urgent need for biomarker identification in order to assess the degree of malignancy, predict prognosis, and evaluate possible therapies. Various proteomic approaches and different clinical materials have been used to this end, and candidate biomarkers have been reported for the different types of sarcomas. However, the sample size used in these biomarker studies was generally insufficient, and thus far, no biomarker has been proved useful in clinics. Given that sarcomas are rare, biomarker validation in this setting is more challenging than in other malignancies. In gastrointestinal stromal tumor, adjuvant therapy has proven to be effective. However, only 40% patients experience metastasis after curative surgery alone, and the rest of the patients may not need adjuvant therapy. Using a proteomic approach, we identified pfetin (potassium channel tetramerization domain containing 12, KCTD 12) as a novel prognostic biomarker for sarcoma, and immunohistochemically confirmed its clinical usefulness by a multiinstitutional validation study. Here, we describe our experience and discuss the critical points in the discovery of this biomarker.

Skeletal Metastasis of Unknown Primary Origin at the Initial Visit: A Retrospective Analysis of 286 Cases

Background Skeletal metastasis is a common metastatic event for several carcinomas, and the treatment for skeletal metastasis of unknown primary (SMUP) are a critical issue in cancer therapy. Making a diagnosis of the primary site is the most crucial step in the treatment of SMUP; however, the procedures are sometimes difficult and time-consuming, and the primary site often remains unknown. Therefore, to establish optimal diagnostic strategies and elucidate the overall survival rates of SMUP, we conducted this retrospective study. Methods We retrospectively analyzed the clinical data for 286 SMUP cases from a total of 2,641 patients with skeletal metastases who were treated between 2002 and 2014 at our initiations. Results The primary sites were identified in 254/286 patients (88.8%), while 32 (11.2%) primary sites were not detected by our diagnostic strategies. Lung cancer was identified in 72 (25.2%) cases, and was the most frequently observed primary lesion. The median survival time of the SMUP patients was 20.0 months, while the median survival times of solitary bone metastasis cases and multi-bone metastasis cases were 39.0 months and 16.0 months, respectively. The median survival times of prostate cancer cases was over 120 months, that of patients with primary lung cancers was 9.0 months and the median survival time of cases who were finally diagnosed with an unknown primary was 11.0 months. Conclusions We believe that our study would contribute to establishing an optimal strategy for diagnosing the primary site in SMUP patients, and our data provide definite indications for the survival times for different SMUP situations.

The Prognostic Value of Pfetin: A Validation Study in Gastrointestinal Stromal Tumors Using a Commercially Available Antibody

OBJECTIVE Adjuvant treatment with imatinib mesylate is an effective treatment for gastrointestinal stromal tumor. However, 50% of patients with gastrointestinal stromal tumor can be cured by surgery alone; hence, risk stratification for therapy with imatinib mesylate is the next challenge. Previously, using a proteomic approach, we discovered a potential prognostic biomarker for gastrointestinal stromal tumor, pfetin, and immunohistochemically validated its clinical utility using our original monoclonal antibody. In the present study, we examine the usefulness of a commercially available polyclonal antibody against pfetin. METHODS Western blotting and immunohistochemistry were performed using surgical specimens of primary tissues from gastrointestinal stromal tumor patients using a polyclonal antibody against pfetin and our original monoclonal antibody. Formalin-fixed and paraffin-embedded primary tissue sections from 112 gastrointestinal stromal tumor patients were subjected to immunohistochemistry. The immunohistochemistry results were integrated with the clinico-pathological observations. RESULTS Western blotting revealed that both antibodies recognized multiple post-translationally modified pfetin isoforms. The immunohistochemical study with the commercial antibody demonstrated that the disease-free survival rate was 88 and 56% for pfetin-positive and pfetin-negative patients, respectively. Univariate and multivariate analyses showed that pfetin expression as measured by the commercial antibody was a significant and independent prognostic factor among the clinico-pathological parameters examined. Of the 112 gastrointestinal stromal tumor cases examined, 13 yielded discordant results between the commercial antibody and our original antibody, and there were no significantly different clinical or pathological factors to account for this discrepancy. CONCLUSIONS Our observations suggest that the pfetin expression level assessed by the commercial antibody could be a prognostic biomarker in gastrointestinal stromal tumors.

Clinicopathological effects of protein phosphatase 2, regulatory subunit A, alpha mutations in gastrointestinal stromal tumors

Recently, several studies have reported that dysfunctions in protein phosphatase 2A (PP2A) caused by alterations in protein phosphatase 2 regulatory subunit A, alpha (PPP2R1A) are responsible for tumorigenesis and tumor progression in several types of cancers. The impact of PPP2R1A mutations remains unknown in gastrointestinal stromal tumors (GISTs), although mutations in KIT and PDGFRA, which result in constitutive activation of the receptor tyrosine kinase pathway, are important in GIST tumorigenesis. In this study, we performed mutation analysis of PPP2R1A to examine the frequency of PPP2R1A mutations and their clinicopathological correlation in 94 GIST cases. In addition, we performed an in vitro analysis to investigate the effects of PPP2R1A mutations on cell proliferation and kinase phosphorylation in GIST cells. Seventeen GIST cases (18%) harbored mutations in PPP2R1A. All but one of these 17 cases harbored a KIT, PDGFRA, HRAS, NRAS, or KRAS mutation as the oncogenic driver mutation, and the remaining case was immunohistochemically negative for succinate dehydrogenase B (SDHB). Multivariate analysis showed that larger tumor size, higher mitotic rate, and PPP2R1A mutation are independent prognostic factors for overall survival; however, PPP2R1A mutation was not an independent prognostic factor for disease-free survival. The transduction of GIST cells with mutant PPP2R1A induced an accelerated growth rate via increased phosphorylation of Akt1/2, ERK1/2, and WNK1, a kinase associated with angiogenesis. In addition, the transduction of GIST cells with mutant PPP2R1A caused increased c-kit phosphorylation, suggesting that c-kit is also a target of PP2A, reinforcing the tumorigenic capabilities of c-kit. Furthermore, the transducing GIST cells with wild-type PP2A dephosphorylated mutant c-kit. This study provides a new insight into the biology of GISTs and their phosphatase activity, and activated PP2A could be a therapeutic target in GISTs.

Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab

Giant cell tumors of bone (GCTB) are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the protein expression patterns and the results of the network analysis will provide a better understanding of the effects of denosumab administration in patients with GCTB.

Reduced argininosuccinate synthetase expression in refractory sarcomas: Impacts on therapeutic potential and drug resistance

Background Treating drug-resistant sarcomas remains a major challenge. The present study aimed to identify a novel therapy for drug-resistant sarcomas based on metabolic errors involving argininosuccinate synthetase1 (ASS1). Results ASS1 expression was reduced in Dox-resistant sarcoma cells. Immunohistochemistry and real-time PCR showed an inverse correlation between ASS1 and P-gp expressions. The inhibition of cellular proliferation with G1-arrest was shown to lead to autophagy with arginine deprivation. In addition, the combination of an autophagy inhibitor plus arginine deprivation was more effective than arginine deprivation alone. In cells with suppressed ASS1 expression, P-gp expression was upregulated as compared to that in negative controls. Discussion These results indicate that the reduced ASS1 expression in Dox-resistant sarcomas may contribute to drug resistance in association with the expression of P-gp. ASS1 deficiency is a potential target for novel drug therapies. The combination of arginine-deprivation therapy and an autophagy inhibitor may have anti-tumor effects in refractory sarcomas. Methods We assessed the expressions of ASS1 and P-glycoprotein (P-gp) in clinical specimens and cell lines of osteosarcoma (KHOS), doxorubicin (Dox)-resistant osteosarcoma (KHOSR2), epithelioid sarcomas (ES-X and VAESBJ) and alveolar soft part sarcoma (ASPS-KY). Each cell line was cultured in arginine-containing and arginine-free media. Cell growth was assessed using an XTT assay and flow cytometry. We analyzed the induction of autophagy in arginine-free medium. Moreover, we assessed the expression of P-gp after suppressing ASS1 in Dox-sensitive cells (MCF-7 and KHOS) and after transfecting ASS1 into Dox-resistant cells (ES-X, VAESBJ, ASPS-KY and KHOSR2).

Expression of F-actin-capping protein subunit beta, CAPZB, is associated with cell growth and motility in epithelioid sarcoma

BackgroundA previous proteomics study demonstrated the overexpression of F-actin capping protein subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS.MethodsCellular functional assays were performed in two EpiS cell lines using CAPZB siRNAs. In addition, comparative protein expression analyses using Isobaric Tags for Relative and Absolute Quantitation (i-TRAQ) method were performed to identify the specific proteins whose expression was dysregulated by CAPZB, and analysed the data with the Ingenuity Pathways Analysis (IPA) system using the obtained protein profiles to clarify the functional pathway networks associated with the oncogenic function of CAPZB in EpiS. Additionally, we performed functional assays of the INI1 protein using INI1-overexpressing EpiS cells.ResultsAll 15 EpiS cases showed an immunohistochemical expression of CAPZB, and two EpiS cell lines exhibited a strong CAPZB expression. Silencing of CAPZB inhibited the growth, invasion and migration of the EpiS cells. Analysis of protein profiles using the IPA system suggested that SWI/SNF chromatin-remodeling complexes including INI1 may function as a possible upstream regulator of CAPZB. Furthermore, silencing of CAPZB resulted in a decreased expression of INI1 proteins in the INI1-positive EpiS cells, whereas the induction of INI1 in the INI1-deficient EpiS cells resulted in an increased CAPZB mRNA expression.ConclusionsCAPZB is involved in tumor progression in cases of EpiS, irrespective of the INI1 expression, and may be a potential therapeutic target. The paradoxical relationship between the tumor suppressor INI1 and the oncoprotein CAPZB in the pathogenesis of EpiS remains to be clarified.

An Outpatient-based Survey about the Recognition of Locomotive Syndrome and the Results of the Loco-check at a University Hospital in Tokyo

Background: To prevent locomotor dysfunction, the Japanese Orthopaedic Association (JOA) proposed the concept of locomotive syndrome (LS) in 2007, and has carried out many campaigns to increase the awareness of LS. Recently, the JOA reported that 26.6% of people recognized LS in internet based surveys. However, a survey of the recognition of LS using outpatient (hospital-based) cohorts has never been performed. Therefore, we surveyed the recognition of LS, the prevalence of LS and any correlations between LS and Original Research Article British Journal of Medicine & Medical Research, 4(17): 3255-3268, 2014 3256 the clinical data in the outpatient cohort. Methods: To investigate the recognition of LS and prevalence of LS, we conducted a questionnaire survey including the “loco-check” in 1010 (429 male and 581 female) orthopaedic outpatients at Juntendo University Hospital (Tokyo, Japan) from April to June 2013. Results: Only 24.6% of the patients knew about the concept of LS. In terms of the media sources from which the patients had received information about LS, 49.5% received LS information from TV. With regard to the prevalence of LS, 43.8% was classified into the LS group. The prevalence of LS was 32.9% in males and 51.8% in females. When the relationship between LS and orthopaedic disease was examined, 50 of 55 (90.9%) knee osteoarthritis (OA) patients, 21 of 26 (80.8%) hip OA patients and 98 of 151 (64.9%) lumbar spondylosis patients had statistically significant correlations with LS (p<0.0001). Conclusion: This study confirmed the rate of recognition of LS indicated by the JOA survey. Our studies also revealed that LS score was related to orthopaedic disorders. Our outpatient-based surveys may help provide a better understanding of the effects and trends of the campaigns to promote the recognition of LS.

Cabozantinib and dastinib exert anti-tumor activity in alveolar soft part sarcoma

Background Alveolar soft part sarcoma (ASPS) is an extremely rare metastatic soft tissue tumor with a poor prognosis for which no effective systemic therapies have yet been established. Therefore, the development of novel effective treatment approaches is required. Tyrosine kinases (TKs) are being increasingly used as therapeutic targets in a variety of cancers. The purpose of this study was to identify novel therapeutic target TKs and to clarify the efficacy of TK inhibitors (TKIs) in the treatment of ASPS. Experimental design To identify novel therapeutic target TKs in ASPS, we evaluated the antitumor effects and kinase activity of three TKIs (pazopanib, dasatinib, and cabozantinib) against ASPS cells using an in vitro assay. Based on these results, we then investigated the phosphorylation activities of the identified targets using western blotting, in addition to examining antitumor activity through in vivo assays of several TKIs to determine both the efficacy of these substances and accurate targets. Results In cell proliferation and invasion assays using pazopanib, cabozantinib, and dasatinib, all three TKIs inhibited the cell growth in ASPS cells. Statistical analyses of the cell proliferation and invasion assays revealed that dasatinib had a significant inhibitory effect in cell proliferation assays, and cabozantinib exhibited marked inhibitory effects on cellular functions in both assays. Through western blotting, we also confirmed that cabozantinib inhibited c-MET phosphorylation and dasatinib inhibited SRC phosphorylation in dose-dependent fashion. Mice that received cabozantinib and dasatinib had significantly smaller tumor volumes than control animals, demonstrating the in vivo antitumor activity of, these substances. Conclusions Our findings suggest that cabozantinib and dasatinib may be more effective than pazopanib against ASPS cells. These in vitro and in vivo data suggest that c-MET may be a potential therapeutic target in ASPS, and cabozantinib may be a particularly useful therapeutic option for patients with ASPS, including those with pazopanib-resistant ASPS.