Publications by authors named "Yoshiyasu Nakamura"

48 Publications

Tissue factor pathway inhibitor‑2 is specifically expressed in ovarian clear cell carcinoma tissues in the nucleus, cytoplasm and extracellular matrix.

Oncol Rep 2021 03 20;45(3):1023-1032. Epub 2021 Jan 20.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama 241‑8515, Japan.

Tissue factor pathway inhibitor‑2 (TFPI‑2) is a promising candidate as a serum biomarker of ovarian clear cell carcinoma (OCCC), a lethal histological subtype of epithelial ovarian cancer (EOC). TFPI‑2 is a secreted serine protease inhibitor that suppresses cancer progression through the inhibition of matrix protease activities. Previous studies have also identified TFPI‑2 in the nucleus, and a possible function of nuclear TFPI‑2 as a transcriptional repressor of matrix metalloproteinase‑2 (MMP‑2) was recently demonstrated. We are currently establishing TFPI‑2 as a serum biomarker for OCCC patients; however, TFPI‑2 expression in OCCC tissues has not been previously investigated. In the present study, we examined TFPI‑2 expression and its localization in 11 OCCC cell lines by western blotting and enzyme‑linked immune assay. Four cell lines expressed TFPI‑2 in the nucleus, cytoplasm and culture plate-attached extracellular fraction, while four other cell lines expressed TFPI‑2 only in the extracellular fraction. In the remaining three cell lines, TFPI‑2 was not identified in any fraction. The amount of secreted soluble TFPI‑2 showed similar trends to that of the plate‑attached fraction. We next investigated the expression levels and distribution of TFPI‑2 in surgically resected EOC tissues by immunohistochemistry. In 52 of the 77 (67.5%) OCCC tumors, TFPI‑2 expression was detected in at least one of the nuclear, cytoplasmic and extracellular matrix fractions. In contrast, we did not identify TFPI‑2 in the other EOC subtypes (n=65). TFPI‑2‑positive expression distinguished CCC from the other EOC tissues with a sensitivity of 67.5% and specificity of 100%. Although the inherent tumor suppressor function, statistical analyses failed to demonstrate correlations between TFPI‑2 expression and clinical parameters, including 5‑year overall survival, except for the patient age. In conclusion, we identified TFPI‑2 expression in the nucleus, cytoplasm and extracellular matrix in OCCC tissues. The high specificity of TFPI‑2 may support its use for diagnosis of OCCC in combination with existing markers.
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http://dx.doi.org/10.3892/or.2021.7944DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859994PMC
March 2021

Galectin-9 expression as a poor prognostic factor in patients with renal cell carcinoma.

Cancer Immunol Immunother 2020 Oct 18;69(10):2041-2051. Epub 2020 May 18.

Research Institute, Kanagawa Cancer Center, 2-3-2 Nakao, Asahi-ku, Yokohama, Kanagawa, 241-8515, Japan.

Recently, the effectiveness of anti-programmed death 1 (PD-1) antibody therapy in the treatment of renal cell carcinoma (RCC) has been established. Nevertheless, efficacy has been reported to be limited to only 10-30% of patients. To develop more effective immunotherapy for RCC, we analyzed the immunological characteristics in RCC tissues by immunohistochemistry (IHC). We prepared a tissue microarray that consisted of tumor tissue sections (1 mm in diameter) from 83 RCC patients in Kanagawa Cancer Center between 2006 and 2015. IHC analysis was performed with antibodies specific to immune-related (CD8 and Foxp3) and immune checkpoint (programmed death ligand 1 (PD-L1) and 2 (PD-L2), B7-H4 and galectin-9) molecules. The numbers and proportions of positively stained tumor cells or immune cells were determined in each section. From multivariate analysis of all 83 patients, higher galectin-9 expression was detected as a factor associated with worse overall survival (OS) (P = 0.029) and that higher stage and higher B7-H4 expression were associated with worse progression-free survival (PFS) (P < 0.001 and P = 0.021, respectively). Similarly, in multivariate analysis of 69 patients with clear cell RCC, though not statistically significant, there was a trend for association between higher galectin-9 expression and worse OS (P = 0.067), while higher stage was associated with worse PFS (P < 0.001). This study suggests that higher galectin-9 expression is an independent adverse prognostic factor of OS in RCC patients. Therefore, to develop more effective personalized immunotherapy to treat RCC, it may be important to target not only PD-1/PD-L1, but also other immune checkpoint molecules such as galectin-9.
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http://dx.doi.org/10.1007/s00262-020-02608-6DOI Listing
October 2020

EZH2 and MMSET Were Identified as Potentially Useful Therapeutic Targets in Metaplastic Breast Carcinoma.

Anticancer Res 2020 Apr;40(4):2133-2139

Department of Surgery, Yokohama City University, Yokohama, Japan.

Background/aim: Metaplastic breast carcinoma (MBC) is a rare malignancy, which is often triple-negative for the hormone receptors and human epidermal growth factor receptor 2, and thus, does not benefit from targeted therapy. In this study, we examined the expression of methylation and demethylation enzymes by immunostaining MBC and the adjacent normal tissues or triple-negative ductal carcinoma (TNDC), and identified alterations that may be used as therapeutic targets.

Materials And Methods: We retrospectively studied surgical specimens from 15 patients who underwent surgery for MBC at Kanagawa Cancer Center between 2005 and 2016, and similarly from 14 patients with TNDC. The frequencies of high methylation/demethylation enzyme expression were compared among them.

Results: The frequencies of high enhancer of zeste homolog 2 (EZH2) and multiple myeloma SET domain (MMSET) expression were significantly higher in both MBC and TNDC than in normal tissue.

Conclusion: EZH2 and MMSET may be useful therapeutic targets in MBC.
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http://dx.doi.org/10.21873/anticanres.14172DOI Listing
April 2020

Induction of tryptophan hydroxylase in the liver of s.c. tumor model of prostate cancer.

Cancer Sci 2020 Apr 27;111(4):1218-1227. Epub 2020 Feb 27.

Molecular Pathology and Genetics Divisiosn, Kanagawa Cancer Center Research Institute, Kanagawa Cancer Center, Yokohama City, Japan.

Enhanced degradation of tryptophan (Trp) and thus decreased plasma Trp levels are common in several types of cancers. Although it is well known that Trp catabolism is induced in the tumor microenvironment by the enzymes expressed in cancer cells, immune cells, or both, few studies have examined systemic Trp catabolism in cancer pathophysiology. The present study aimed to evaluate Trp catabolism in both tumor and peripheral tissues using tumor-engrafted Copenhagen rats that were s.c. inoculated with AT-2 rat prostate cancer cells negative for expression of Trp catabolic enzymes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics showed significantly decreased plasma Trp levels in AT-2 engrafted rats, accompanied by increased kynurenine/Trp ratios in spleen and thymus and serotonin levels in liver and thymus. Quantitative PCR and enzymatic activity assays showed indoleamine-2, 3-dioxygenase, an inducible enzyme that catalyzes Trp to kynurenine, was increased in tumor tissues, whereas tryptophan-2,3-dioxygenase, a major Trp catabolic enzyme that regulates systemic level of Trp, tended to be increased in the liver of AT-2 engrafted rats. Furthermore, tryptophan hydroxylase-1 (TPH1), an enzyme that catalyzes the reaction of Trp to serotonin, was significantly increased in liver and spleen of AT-2 engrafted rats. Further histochemical analysis revealed that the induction of TPH1 in the liver could be attributed to infiltration of mast cells. A similar phenomenon was observed with nonneoplastic liver samples from colorectal cancer patients. These results suggested that Trp catabolism toward serotonin synthesis might be induced in peripheral remote tissues in cancer, which could have a pathophysiological effect on cancer.
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http://dx.doi.org/10.1111/cas.14333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156786PMC
April 2020

GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer.

Int J Cancer 2020 03 22;146(5):1369-1382. Epub 2019 Jul 22.

Department of Pathophysiology, Ehime University Graduate School of Medicine, Toon, Japan.

The prognosis of patients with progressive prostate cancers that are hormone refractory and/or have bone metastasis is poor. Multiple therapeutic targets to improve prostate cancer patient survival have been investigated, including orphan GPCRs. In our study, we identified G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5A) as a candidate therapeutic molecule using integrative gene expression analyses of registered data sets for prostate cancer cell lines. Kaplan-Meier analysis of TCGA data sets revealed that patients who have high GPRC5A expression had significantly shorter overall survival. PC3 prostate cancer cells with CRISPR/Cas9-mediated GPRC5A knockout exhibited significantly reduced cell proliferation both in vitro and in vivo. RNA-seq revealed that GPRC5A KO PC3 cells had dysregulated expression of cell cycle-related genes, leading to cell cycle arrest at the G2/M phase. Furthermore, the registered gene expression profile data set showed that the expression level of GPRC5A in original lesions of prostate cancer patients with bone metastasis was higher than that without bone metastasis. In fact, GPRC5A KO PC3 cells failed to establish bone metastasis in xenograft mice models. In addition, our clinical study revealed that GPRC5A expression levels in prostate cancer patient samples were significantly correlated with bone metastasis as well as the patient's Gleason score (GS). Combined assessment with the immunoreactivity of GPRC5A and GS displayed higher specificity for predicting the occurrence of bone metastasis. Together, our findings indicate that GPRC5A can be a possible therapeutic target and prognostic marker molecule for progressive prostate cancer.
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http://dx.doi.org/10.1002/ijc.32554DOI Listing
March 2020

Cholesterol Starvation and Hypoxia Activate the FVII Gene via the SREBP1-GILZ Pathway in Ovarian Cancer Cells to Produce Procoagulant Microvesicles.

Thromb Haemost 2019 Jul 5;119(7):1058-1071. Epub 2019 May 5.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Japan.

Interaction between the transcription factors, hypoxia-inducible factor (HIF1α and HIF2α) and Sp1, mediates hypoxia-driven expression of gene encoding coagulation factor VII (fVII) in ovarian clear cell carcinoma (CCC) cells. This mechanism is synergistically enhanced in response to serum starvation, a condition possibly associated with tumor hypoxia. This transcriptional response potentially results in venous thromboembolism, a common complication in cancer patients by producing procoagulant extracellular vesicles (EVs). However, which deficient serum factors are responsible for this characteristic transcriptional mechanism is unknown. Here, we report that cholesterol deficiency mediates synergistic expression under serum starvation and hypoxia (SSH) via novel sterol regulatory element binding protein-1 (SREBP1)-driven mechanisms. Unlike conventional mechanisms, SREBP1 indirectly enhances transcription through the induction of a new target, glucocorticoid-induced leucine zipper (GILZ) protein. GILZ expression induced in response to hypoxia by a HIF1α-dependent mechanism activates SREBP1 under SSH, suggesting reciprocal regulation between SREBP1 and GILZ. Furthermore, GILZ binds to the locus. Xenograft tumor samples analyzed by chromatin immunoprecipitation confirmed that HIF1α-aryl hydrocarbon nuclear translocator and GILZ bind to the (GILZ) and gene loci, respectively, thereby potentially modulating chromatin function to augment transcription. Thus, deficiency of both O and cholesterol, followed by interplay between HIFs, Sp1, and SREBP1-GILZ pathways synergistically induce fVII synthesis, resulting in the shedding of procoagulant EVs.
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http://dx.doi.org/10.1055/s-0039-1687876DOI Listing
July 2019

Size Distribution Analysis with On-Chip Multi-Imaging Cell Sorter for Unlabeled Identification of Circulating Tumor Cells in Blood.

Micromachines (Basel) 2019 Feb 25;10(2). Epub 2019 Feb 25.

Organization for University Research Initiatives, Waseda University, 3-14-9 Okubo, Shinjuku, Tokyo 169-0072, Japan.

We report a change of the imaging biomarker distribution of circulating tumor cell (CTC) clusters in blood over time using an on-chip multi-imaging flow cytometry system, which can obtain morphometric parameters of cells and those clusters, such as cell number, perimeter, total cross-sectional area, aspect ratio, number of nuclei, and size of nuclei, as "imaging biomarkers". Both bright-field (BF) and fluorescent (FL) images were acquired at 200 frames per second and analyzed within the intervals for real-time cell sorting. A green fluorescent protein-transfected prostate cancer cell line (MAT-LyLu-GFP) was implanted into Copenhagen rats, and the blood samples of these rats were collected 2 to 11 days later and measured using the system. The results showed that cells having BF area of 90 μm² or larger increased in number seven days after the cancer cell implantation, which was specifically detected as a shift of the cell size distribution for blood samples of implanted rats, in comparison with that for control blood. All cells with BF area of 150 μm² or larger were arranged in cell clusters composed of at least two cells, as confirmed by FL nucleus number and area measurements, and they constituted more than 1% of all white blood cells. These results indicate that the mapping of cell size distribution is useful for identifying an increase of irregular cells such as cell clusters in blood, and show that CTC clusters become more abundant in blood over time after malignant tumor formation. The results also reveal that a blood sample of only 50 μL is sufficient to acquire a stable size distribution map of all blood cells to predict the presence of CTC clusters.
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http://dx.doi.org/10.3390/mi10020154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413132PMC
February 2019

Predictive value of ERCC1, ERCC2, ERCC4, and glutathione S-Transferase Pi expression for the efficacy and safety of FOLFIRINOX in patients with unresectable pancreatic cancer.

Am J Cancer Res 2018 1;8(10):2096-2105. Epub 2018 Oct 1.

Department of Gastroenterology, Yokohama City University Graduate School of Medicine Yokohama, Japan.

The platinum-based chemotherapy regimen FOLFIRINOX (leucovorin, fluorouracil, irinotecan, and oxaliplatin) is currently used as a standard treatment for patients with unresectable pancreatic cancer. FOLFIRINOX is associated with severe toxicities, including neutropenia, febrile neutropenia, and anorexia; however, there are currently no reliable biomarkers to predict its efficacy and safety. Several studies of patients with various cancers have shown that tumor expression of excision repair cross-complementing (ERCC) proteins and glutathione S-transferase Pi (GSTPi) correlates with the response to platinum-based chemotherapies. Therefore, in this study, we examined the associations between expression of ERCC proteins and GSTPi and the safety and efficacy of FOLFIRINOX in 34 patients with unresectable pancreatic cancer. ERCC1, ERCC2, ERCC4, and GSTPi expression were examined by immunohistochemical staining of tumor specimens and the results were correlated with overall survival, progression-free survival, response rate, disease control rate, and the frequency of grade 3-4 neutropenia and non-hematologic toxicities. We found that ERCC1, ERCC2, ERCC4, and GSTPi were expressed in tumor samples from 64%, 24%, 18%, and 64% of patients, respectively. Notably, there were no statistically significant associations between the expression pattern of any of the proteins and either the clinical outcomes or the frequency of grade 3-4 neutropenia or grade 3-4 anorexia. Collectively, these data indicate that tumor expression of ERCC1, ERCC2, ERCC4, and GSTPi does not predict the safety or efficacy of FOLFIRINOX in patients with pancreatic cancer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6220150PMC
October 2018

Enhanced expression of PD-L1 in non-muscle-invasive bladder cancer after treatment with Bacillus Calmette-Guerin.

Oncotarget 2018 Sep 25;9(75):34066-34078. Epub 2018 Sep 25.

Kanagawa Cancer Center Research Institute, Yokohama, Japan.

Immune checkpoint molecules, such as PD-1/PD-L1, are reported to be closely associated with suppression of antitumor immunity, and their inhibitors have been used to treat various cancers including bladder cancer. However, there have been only a few studies investigating the effects of Bacillus Calmette-Guerin (BCG) administration on expression of the immune checkpoint molecules in bladder cancer. The current study examined the expression of PD-L1 and PD-L2 before and after BCG in non-muscle-invasive bladder cancer (NMIBC) patients. Tissue microarrays of 22 BCG-resistant NMIBC patients were stained by immunohistochemistry with antibodies against PD-L1, PD-L2, and CD8, and were compared between before and after BCG. The expression levels of PD-L1, but not of PD-L2, were significantly increased after BCG treatment on tumor cells (p < 0.001) and tumor-infiltrating inflammatory cells (p = 0.030) within tumor tissues, as well as on inflammatory cells within non-tumor normal tissues (p = 0.003). Although CD8 T cells were significantly increased within tumor tissues (p = 0.005) and non-tumor normal tissues (p = 0.007) after BCG treatment, they might be not effective for anti-tumor immunity. This study demonstrated for the first time that expression of PD-L1, which might contribute to the immune escape mechanism, was enhanced on tumor tissue after BCG treatment in BCG-resistant NMIBC patients. Our finding thus propose that immunotherapy with anti-PD-1/PD-L1 antibodies could be feasible as combination treatment with BCG or as secondary treatment at relapse after BCG in NMIBC patients.
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http://dx.doi.org/10.18632/oncotarget.26122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6183350PMC
September 2018

EZH2 Overexpression as a Useful Prognostic Marker for Aggressive Behaviour in Thyroid Cancer.

In Vivo 2018 Jan-Feb;32(1):25-31

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Japan.

Background/aim: Enhancer of zeste homolog 2 (EZH2) is a member of the polycomb group of genes, which are key factors in the regulation of cell proliferation and differentiation. EZH2 is overexpressed in many malignancies. We analyzed EZH2 protein expression levels in different histological subtypes of thyroid cancer to examine its utility as a prognostic factor.

Materials And Methods: We examined EZH2 protein expression by immunohistochemistry in tissue samples from 67 cases of poorly differentiated (PDTC) and 48 cases of anaplastic thyroid carcinoma (ATC), and in samples of adjacent normal and differentiated thyroid carcinoma (DTC). We examined differences in expression of EZH2 among various histological types of thyroid cancer, and the relationship between EZH2 expression and patient outcome.

Results: EZH2 protein was expressed in PDTC and ATC, but not in normal thyroid gland or DTC. EZH-positivity increased in the order of DTC, PDTC, and ATC (p<0.01). Higher EZH2 expression correlated with poorer survival in PDTC (p=0.004), and a similar but non-significant trend was observed in ATC (p=0.166). Multivariate analysis identified EZH2 as an independent prognostic factor similar to metastatic status in the Japanese Society of Thyroid Surgery (JSTS) classification of PDTC.

Conclusion: EZH2 overexpression is associated with malignant potential in thyroid cancer, and may thus be a useful prognostic marker of aggressive thyroid cancer.
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http://dx.doi.org/10.21873/invivo.11200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892628PMC
August 2018

Clinicopathological and prognostic significance of Ki-67 immunohistochemical expression of distant metastatic lesions in patients with metastatic breast cancer.

Breast Cancer 2017 Nov 19;24(6):748-755. Epub 2017 Apr 19.

Department of Surgery, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.

Background: Surgical biopsy of metastatic lesions followed by pathological confirmation for the investigation of biomarkers is occasionally proposed as an effective strategy in the treatment of metastatic breast cancer. However, few reports have examined Ki-67 immunohistochemical expression in distant metastatic lesions of breast cancer patients. This study aimed to investigate the clinicopathological significance of subtypes and Ki-67 immunohistochemical expression in metastatic breast cancer lesions.

Methods: We retrospectively studied surgical specimens of primary breast cancer tumors and their corresponding metastatic lesions from patients (n = 68) who underwent surgery for primary breast cancer tumors between December 1977 and March 2013. Tissue microarrays were constructed using primary and metastatic lesions, and were stained with antibodies against estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and Ki-67. We also examined the clinicopathological characteristics and outcome measures of patients with metastatic breast cancer using primary and paired metastatic lesions.

Results: Compared with the primary lesions, there was no significant difference in subtypes in the metastatic lesions according to metastatic sites. Metastatic lesions of the brain, viscera, and bone exhibited slightly higher levels of Ki-67 immunohistochemical expression compared with primary lesions. A Cox proportional hazards model using multivariate analysis demonstrated that high Ki-67 immunohistochemical expression in distant metastatic lesions was associated with poorer overall survival outcomes after biopsy of recurrence lesion (hazard ratio 2.307; 95% confidence interval 1.207-4.407, P = 0.011).

Conclusions: High Ki-67 immunohistochemical expression levels in distant metastatic lesions were independently associated with poorer overall survival outcomes after biopsy of recurrence lesion in breast cancer patients.
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http://dx.doi.org/10.1007/s12282-017-0774-zDOI Listing
November 2017

Expression of enhancer of zeste homolog 2 correlates with survival outcome in patients with metastatic breast cancer: exploratory study using primary and paired metastatic lesions.

BMC Cancer 2017 02 27;17(1):160. Epub 2017 Feb 27.

Department of Surgery, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan.

Background: In metastatic breast cancer, the status of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), as well as the Ki-67 index sometimes change between primary and metastatic lesions. However, the change in expression levels of enhancer of zeste homolog 2 (EZH2) between primary and metastatic lesions has not been determined in metastatic breast cancer.

Methods: Ninety-six metastatic breast cancer patients had biopsies or resections of metastatic lesions between September 1990 and February 2014 at the Kanagawa Cancer Center. We evaluated ER, PR, HER2, Ki-67, and EZH2 in primary lesions and their corresponding metastatic lesions using immunohistochemistry. We examined the change in expression of EZH2 between primary and metastatic lesions, the correlation between the expression of EZH2 and the expression of other biomarkers, and the relationship between EZH2 expression and patient outcome in metastatic breast cancer.

Results: EZH2 expression was significantly higher in metastatic lesions compared with primary lesions. EZH2 expression was highly correlated with Ki-67 expression in primary and metastatic lesions. High-level expression of EZH2 was associated with poorer disease-free survival (DFS) outcomes in patients with primary lesions (P < 0.001); however, high-level expression of EZH2 was not associated with poorer DFS outcomes in patients with metastatic lesions (P = 0.063). High-level expression of EZH2 was associated with poorer overall survival (OS) postoperatively in patients with primary (P = 0.001) or metastatic lesions (P = 0.005). High-level expression of EZH2 was associated with poorer OS outcomes after recurrence in patients with metastatic lesions (P = 0.014); however, high-level expression of EZH2 was not associated with poorer OS outcomes after recurrence in patients with primary lesions (P = 0.096). High-level expression of EZH2 in metastatic lesions was independently associated with poorer OS outcomes after recurrence.

Conclusions: EZH2 expression was significantly increased in metastatic lesions compared with primary lesions. High-level expression of EZH2 in metastatic lesions was associated with poorer OS outcomes after primary surgery and recurrence.
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http://dx.doi.org/10.1186/s12885-017-3154-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330119PMC
February 2017

Locked Nucleic Acid In Situ Hybridization Analysis of MicroRNA-21 Predicts Clinical Outcome in Patients After Resection for Pancreatic Cancer Treated with Adjuvant Gemcitabine Monotherapy.

Anticancer Res 2016 Mar;36(3):1083-8

Department of Molecular Pathology, Kanagawa Cancer Center, Asahiku, Yokohama, Japan.

Background: The overexpression of microRNA-21 (miR-21) in pancreatic cancer has been implicated in drug resistance to gemcitabine. Thus far, miR-21 has gained wide attention as a potential biomarker to predict the clinical response in patients with pancreatic cancer receiving gemcitabine. The aim of this study was to evaluate the predictive value of miR-21 expression, determined by locked nucleic acid in situ hybridization (LNA-ISH), in patients with pancreatic cancer who underwent adjuvant gemcitabine after curative surgery.

Materials And Methods: Tumor miR-21 expression was analyzed via LNA-ISH and correlated with the clinical outcomes of the patients treated with adjuvant gemcitabine.

Results: The overexpression of miR-21 in pancreatic cancer, determined by LNA-ISH, was significantly and independently associated with a shorter disease-free survival in patients who received adjuvant gemcitabine after curative resection.

Conclusion: The LNA-ISH analysis of miR-21 may serve as a significant predictor for gemcitabine resistance in patients with pancreatic cancer undergoing adjuvant gemcitabine after curative resection.
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March 2016

High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors.

Thromb Haemost 2016 Jan 8;115(2):299-310. Epub 2015 Oct 8.

Yohei Miyagi, 2-3-2 Nakao, Asahi-ku, Yokohama 241-8515, Japan, Tel.: +81 45 391 5761, E-mail:

Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.
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http://dx.doi.org/10.1160/TH15-03-0213DOI Listing
January 2016

Establishment of patient-derived cancer xenografts in immunodeficient NOG mice.

Int J Oncol 2015 Jul 11;47(1):61-70. Epub 2015 May 11.

Central Institute for Experimental Animals, Tonomachi, Kawasaki-ku, Kawasaki, Kanagawa 210-0821, Japan.

Viable and stable human cancer cell lines and animal models combined with adequate clinical information are essential for future advances in cancer research and patient care. Conventional in vitro cancer cell lines are commonly available; however, they lack detailed information on the patient from which they originate, including disease phenotype and drug sensitivity. Patient-derived xenografts (PDX) with clinical information (so-called 'cancer xenopatients') are a promising advance that may accelerate the development of anticancer therapies. We established 61 PDX lines from 116 surgically removed tumor tissues inoculated subcutaneously into NOG mice (53% success rate). PDX lines were established from various types of epithelial tumors and also from sarcomas, including gastrointestinal stromal tumors and Ewing/PNET sarcomas. The metastatic tumors yielded PDX lines more effectively (65%) than the primary tumors (27%, P<0.001). In our PDX models, morphological characteristics, gene expression profiles, and genetic alteration patterns were all well preserved. In eight cases (7%), the transplantable xenografts for several generations were composed of large monotonous nonepithelial cells of human origin, revealed to be Epstein-Barr virus infection-associated lympho-proliferative lesions. Despite this, PDX linked with clinical information offer many advantages for preclinical studies investigating new anticancer drugs. The fast and efficient establishment of individual PDX may also contribute to future personalized anticancer therapies.
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http://dx.doi.org/10.3892/ijo.2015.2997DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485657PMC
July 2015

Lipid starvation and hypoxia synergistically activate ICAM1 and multiple genes in an Sp1-dependent manner to promote the growth of ovarian cancer.

Mol Cancer 2015 Apr 8;14:77. Epub 2015 Apr 8.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, 2-3-2 Nakao, Asahi-ku, Yokohama, 241-8515, Japan.

Background: Elucidation of the molecular mechanisms by which cancer cells overcome hypoxia is potentially important for targeted therapy. Complexation of hypoxia-inducible factors (HIFs) with aryl hydrocarbon receptor nuclear translocators can enhance gene expression and initiate cellular responses to hypoxia. However, multiple molecular mechanisms may be required for cancer cells to adapt to diverse microenvironments. We previously demonstrated that a physical interaction between the ubiquitously expressed transcription factor Sp1 and HIF2 is a major cause of FVII gene activation in poor prognostic ovarian clear cell carcinoma (CCC) cells under hypoxia. Furthermore, it was found that FVII activation is synergistically enhanced when serum-starved cells are cultured under hypoxic conditions. In this study, we investigated whether HIFs and transcription factor Sp1 cooperate to activate multiple genes in CCC cells under conditions of serum starvation and hypoxia (SSH) and then contribute to malignant phenotypes.

Methods: To identify genes activated under hypoxic conditions in an Sp1-dependent manner, we first performed cDNA microarray analyses. We further investigated the molecular mechanisms of synergistic gene activations including the associated serum factors by various experiments such as real-time RT-PCR, western blotting and chromatin immunoprecipitation. The study was further extended to animal experiments to investigate how it contributes to CCC progression in vivo.

Results: ICAM1 is one such gene dramatically induced by SSH and is highly induced by SSH and its synergistic activation involves both the mTOR and autonomously activated TNFα-NFκB axes. We identified long chain fatty acids (LCFA) as a major class of lipids that is associated with albumin, a serum factor responsible for synergistic gene activation under SSH. Furthermore, we found that ICAM1 can be induced in vivo to promote tumor growth.

Conclusion: Sp1 and HIFs collaborate to activate genes required for the adaptation of CCC cells to severe microenvironments, such as LCFA starvation and hypoxia. This study highlights the importance of transcriptional regulation under lipid starvation and hypoxia in the promotion of CCC tumor growth.
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http://dx.doi.org/10.1186/s12943-015-0351-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396815PMC
April 2015

Development of on-chip multi-imaging flow cytometry for identification of imaging biomarkers of clustered circulating tumor cells.

PLoS One 2014 20;9(8):e104372. Epub 2014 Aug 20.

Kanagawa Academy of Science and Technology, Takatsu, Kawasaki, Japan; Department of Biomedical Information, Division of Biosystems, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo, Japan.

An on-chip multi-imaging flow cytometry system has been developed to obtain morphometric parameters of cell clusters such as cell number, perimeter, total cross-sectional area, number of nuclei and size of clusters as "imaging biomarkers", with simultaneous acquisition and analysis of both bright-field (BF) and fluorescent (FL) images at 200 frames per second (fps); by using this system, we examined the effectiveness of using imaging biomarkers for the identification of clustered circulating tumor cells (CTCs). Sample blood of rats in which a prostate cancer cell line (MAT-LyLu) had been pre-implanted was applied to a microchannel on a disposable microchip after staining the nuclei using fluorescent dye for their visualization, and the acquired images were measured and compared with those of healthy rats. In terms of the results, clustered cells having (1) cell area larger than 200 µm2 and (2) nucleus area larger than 90 µm2 were specifically observed in cancer cell-implanted blood, but were not observed in healthy rats. In addition, (3) clusters having more than 3 nuclei were specific for cancer-implanted blood and (4) a ratio between the actual perimeter and the perimeter calculated from the obtained area, which reflects a shape distorted from ideal roundness, of less than 0.90 was specific for all clusters having more than 3 nuclei and was also specific for cancer-implanted blood. The collected clusters larger than 300 µm2 were examined by quantitative gene copy number assay, and were identified as being CTCs. These results indicate the usefulness of the imaging biomarkers for characterizing clusters, and all of the four examined imaging biomarkers-cluster area, nuclei area, nuclei number, and ratio of perimeter-can identify clustered CTCs in blood with the same level of preciseness using multi-imaging cytometry.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104372PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139271PMC
May 2015

Proteomic analysis of proteins related to prognosis of lung adenocarcinoma.

J Proteome Res 2014 Nov 8;13(11):4686-94. Epub 2014 Jul 8.

Graduate School of Medical Life Science and Advanced Medical Research Center, Yokohama City University , 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan.

We attempted to identify prognosis-related proteins expressed in early resection lung adenocarcinomas that had higher metastatic potential. Early resection of lung adenocarcinoma tissues were collected from patients who experienced recurrence within 5 years after surgery; these patients are defined here as the poor prognosis group. From these samples, we prepared frozen tissue sections and then isolated cancerous areas by laser capture microdissection to allow extraction of cancer tissue-derived soluble proteins. Shotgun LC-MS/MS analysis detected and identified a total of 875 proteins in these cancer tissues. Relative quantitative analysis revealed that 17 proteins were preferentially expressed in the poor prognosis group relative to the good prognosis group, which consisted of patients who did not exhibit recurrence. Among them, 14-3-3 beta/alpha and calnexin were reported to be potentially involved in tumor recurrence and the malignant properties of lung cancer. Here immunological analyses confirmed disease-associated expression of these proteins. In a cell-culture model using A549, targeted depletion of either 14-3-3 beta/alpha or calnexin reduced proliferation, invasion, and migration, suggesting that both proteins are involved in determining the malignant properties of lung cancer that contribute to poor prognosis.
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http://dx.doi.org/10.1021/pr4012969DOI Listing
November 2014

Annexin A4 is involved in proliferation, chemo-resistance and migration and invasion in ovarian clear cell adenocarcinoma cells.

PLoS One 2013 11;8(11):e80359. Epub 2013 Nov 11.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Japan ; Department of Obstetrics and Gynecology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Ovarian clear cell adenocarcinoma (CCC) is the second most common subtype of ovarian cancer after high-grade serous adenocarcinomas. CCC tends to develop resistance to the standard platinum-based chemotherapy, and has a poor prognosis when diagnosed in advanced stages. The ANXA4 gene, along with its product, a Ca(++)-binding annexin A4 (ANXA4) protein, has been identified as the CCC signature gene. We reported two subtypes of ANXA4 with different isoelectric points (IEPs) that are upregulated in CCC cell lines. Although several in vitro investigations have shown ANXA4 to be involved in cancer cell proliferation, chemoresistance, and migration, these studies were generally based on its overexpression in cells other than CCC. To elucidate the function of the ANXA4 in CCC cells, we established CCC cell lines whose ANXA4 expressions are stably knocked down. Two parental cells were used: OVTOKO contains almost exclusively an acidic subtype of ANXA4, and OVISE contains predominantly a basic subtype but also a detectable acidic subtype. ANXA4 knockdown (KO) resulted in significant growth retardation and greater sensitivity to carboplatin in OVTOKO cells. ANXA4-KO caused significant loss of migration and invasion capability in OVISE cells, but this effect was not seen in OVTOKO cells. We failed to find the cause of the different IEPs of ANXA4, but confirmed that the two subtypes are found in clinical CCC samples in ratios that vary by patient. Further investigation to clarify the mechanism that produces the subtypes is needed to clarify the function of ANXA4 in CCC, and might allow stratification and improved treatment strategies for patients with CCC.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080359PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823662PMC
February 2015

Pulmonary tumors associated with the JC virus T-antigen in a transgenic mouse model.

Oncol Rep 2013 Dec 2;30(6):2603-8. Epub 2013 Oct 2.

Kanagawa Cancer Center Research Institute, Yokohama, Kanagawa 241-0815, Japan.

Many attempts to demonstrate the oncogenic role of the JC virus (JCV) have been partially successful in producing brain tumors, either by direct inoculation of JCV into the brain or in transgenic models in rodents. We previously reported the presence of JCV DNA with a relatively high incidence in pulmonary and digestive organs. However, we could not prove the oncogenic role of JCV. We prepared a transgene composed of the K19 promoter, specific to bronchial epithelium with the JCV T-antigen and established transgenic (TG) mice. Pulmonary tumors were detected without any metastasis in 2 out of 15 (13.3%) 16-month-old K19/JCV T-antigen TG mice. Using immunohistochemistry (IHC), these tumors showed JCV T-antigen, p53 and CK 19 expression, but not expression of nuclear and cytoplasmic β-catenin and insulin receptor substrate 1 (IRS1). IHC revealed the same expression pattern as in the bronchial epithelium of the TG mice. One tumor, which was examined with laser capture microdissection and molecular biological tools, demonstrated an EGFR mutation but not a K-ras mutation. We propose that the pulmonary tumors were derived from the JCV T-antigen in a TG mouse model. These findings shed light on pulmonary carcinogenesis.
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http://dx.doi.org/10.3892/or.2013.2782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839992PMC
December 2013

Enhanced autophagy is required for survival in EGFR-independent EGFR-mutant lung adenocarcinoma cells.

Lab Invest 2013 Oct 12;93(10):1137-46. Epub 2013 Aug 12.

1] Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Japan [2] Laboratory for Molecular Diagnostics, Kanagawa Cancer Center Hospital, Yokohama, Japan [3] Department of Pathology, Jichi Medical University, Tochigi, Japan.

Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.
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http://dx.doi.org/10.1038/labinvest.2013.102DOI Listing
October 2013

Pilot research for the correlation between the expression pattern of E-cadherin-β-catenin complex and lymph node metastasis in early gastric cancer.

Tumori 2013 Mar-Apr;99(2):234-8

Division of Gastroenterology, Kanagawa Cancer Center, Yokohama, Japan.

Aims And Background: Early gastric cancer without lymph node metastasis can be treated with minimally invasive endoscopic surgery. Hence, a better modality for predicting lymph node metastasis should be beneficial to early gastric cancer patients who may only require minimally invasive treatment. In vitro, phosphorylation of β-catenin induces the loss of membranous β-catenin and E-cadherin, subsequently increasing the potential for metastasis. We investigated the behavior of these molecules comparing lymph node metastasis-positive and lymph node metastasis-negative groups, using the specimens from the patients with early gastric cancer. This was a pilot research evaluating the usefulness of combined analysis of these molecules in predicting lymph node metastasis in early gastric cancer.

Methods: The clinicopathological features and immunohistochemical expression patterns of E-cadherin and β-catenin in the primary lesion were studied retrospectively in 28 patients (lymph node metastasis-positive versus lymph node metastasis-negative: 14 vs 14) selected from 272 patients. These patients underwent radical surgery for the early gastric cancer treatment from April 2000 to March 2004 at our hospital. All patients gave written informed consent to use their tissues for the clinical study. Statistical analyses were performed by the chi-square test and Mann-Whitney test.

Results: More loss of membranous E-cadherin was observed in the lymph node metastasis-positive group than in the lymph node metastasis-negative group. Although the finding was slightly more marked in the intestinal than in the diffuse type early gastric cancer, there was no statistical significance. Loss of membranous β-catenin showed a similar trend and no statistical significance. When we evaluated the expression patterns of both molecules, dual loss of membranous E-cadherin and β-catenin significantly correlated with lymph node metastasis [dual loss in lymph node metastasis-positive versus lymph node metastasis-negative patients: 12 (86%) vs 6 (43%), P = 0.046]. Additionally, corresponding proportions in intestinal type early gastric cancer were 5 of 6 (83%) vs 0 of 6 (0%), P = 0.015.

Conclusions: Based on our results, the combined analysis of E-cadherin and β-catenin localizations may be helpful to accurately predict lymph node metastasis in intestinal type early gastric cancer.
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http://dx.doi.org/10.1700/1283.14198DOI Listing
August 2013

Inflammation in background cirrhosis evokes malignant progression in HCC development from HCV-associated liver cirrhosis.

Scand J Gastroenterol 2013 Jun 5;48(6):729-35. Epub 2013 Apr 5.

Tarao's Gastroenterological Clinic, Yokohama, Japan.

Objective: It is accepted that inflammation promotes malignant progression in the development of cancers. Whether, this is true for hepatocellular carcinoma (HCC) remains as an open question. We examined the relationship between the inflammatory histology activity index (HAI) in the background liver cirrhosis (LC) and the histological grading of the HCC in the hepatectomized HCC patients with HCV-associated LC.

Material And Methods: Out of 264 HCC patients who underwent curative hepatic resection, 197 had HCV-associated LC. Among them, 52 patients with a small solitary HCC nodule (< 5 cm in diameter) were studied. Inflammation in the background LC was evaluated by modified Knodell's HAI. To evaluate the inflammation, piece meal necrosis, intra lobular cellular degeneration and focal necrosis, portal cellular inflammation (0-4, each) were estimated. The average HAI was calculated. The grade of malignancy of HCC was determined by WHO classification.

Results: The average HAI in the 15 patients with moderately differentiated HCC (4.3 ± 0.8, mean ± SD) was significantly larger than that in 11 patients with well differentiated HCC (3.5 ± 0.6, p = 0.036). The HAI in the 24 patients whose HCC nodules contained poorly differentiated HCC (5.2 ± 1.1) was significantly larger than that in patients with moderately differentiated HCC (p = 0.025). Thus, the HAI order was well differentiated group < moderately differentiated group < poorly differentiated group.

Conclusions: Inflammation in the background non-cancerous cirrhotic portion would evoke malignant progression in HCC development from HCV-associated LC.
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http://dx.doi.org/10.3109/00365521.2013.782064DOI Listing
June 2013

The clinical significance of SWI/SNF complex in pancreatic cancer.

Int J Oncol 2013 Feb 30;42(2):403-10. Epub 2012 Nov 30.

Department of Gastroenterological Surgery, Kanagawa Cancer Center, Asahi-ku, Yokohama, Kanagawa 241-0815, Japan.

Chromatin remodeling factors have been the subject of great interest in oncology. However, little is known about their role in pancreatic cancer. The objective of this study was to clarify the clinical significance of the SWItch/sucrose non-fermentable (SWI/SNF) complex in patients with pancreatic cancer. A total of 68 patients with pancreatic cancer who underwent R0, 1 resection were enrolled. Cancer tissues were processed to tissue microarray, then stained immunohistochemically by using antibody of SWI/SNF components; BRM, BRG1, BAF250a, BAF180 and BAF47. The correlation of expression levels and clinicopathological outcomes were analyzed, followed by the multivariate analysis of prognostic factors for overall survival. The expression levels of the SWI/SNF components were categorized as low or high according to the median value of Histoscore. Statistical analysis revealed that BRM expression was related to tumor size, T factor, M factor, lymphatic invasion and stage BRG1 expression to histology and stage BAF180 expression to tumor size and BAF47 expression to lymphatic invasion, respectively. Multivariate Cox proportional hazard analysis showed that high BRM and low BAF180 expression levels were independent predictors of worse survival in patients with pancreatic cancer. High BRM, and low BAF180 were also independent prognostic factors for poor survival in the subgroup with adjuvant gemcitabine. These results suggest that the specific cofactors of SWI/SNF chromatin remodeling complex certainly have roles in pancreatic cancer. High BRM, and low BAF180 are useful biomarkers for poor prognosis in pancreatic cancer.
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http://dx.doi.org/10.3892/ijo.2012.1723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3583622PMC
February 2013

Severe inflammation in the background liver cirrhosis correlates with the development of poorly differentiated HCC in HCV-associated liver cirrhosis.

Intern Med 2012 15;51(18):2495-501. Epub 2012 Sep 15.

Tarao's Gastroenterological Clinic, Japan.

Objective: Whether severe inflammation in the background liver cirrhosis might correlate with the development of poorly differentiated human hepatocellular carcinoma (HCC) was studied in hepatitis C virus (HCV)-associated liver cirrhosis.

Methods: Out of 214 HCC patients who underwent curative hepatic resection, 148 patients were HCV-associated liver cirrhosis (LC) patients. Out of these 148, 31 patients with small solitary HCC nodule (diameter ≤ 3 cm) were included in this study. Inflammation in the background LC was evaluated by modified histology activity index (HAI). To evaluate the inflammation, piece meal necrosis, intra lobular cellular degeneration and focal necrosis, portal cellular inflammation (each 0-4) were estimated. In each case, the average HAI was calculated. The grade of malignancy of HCC was determined by World Health Organization (WHO) classification.

Results: The average HAI score in the cirrhotic portion in 17 patients with poorly differentiated HCC (5.21 ± 1.15, mean ± standard deviation (SD)) was significantly larger than that in 14 patients without poorly differentiated HCC (4.05 ± 0.83, p<0.005). The occurrence rate of HCC containing poorly differentiated HCC component in the patients whose HAI was more than 5.0 was 80.0% (12 out of 15), and was significantly higher compared with those in patients whose HAI was less than 5.0 (5 out of 16, 31.3%, p<0.025). In univariate and multivariate analyses for contribution to poorly differentiated HCC development, HAI was the only significant contributor (p=0.011, p=0.012 respectively).

Conclusion: It is suggested that severe inflammation in the background cirrhosis accelerates the promotion in the HCC development from HCV-associated LC.
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http://dx.doi.org/10.2169/internalmedicine.51.7744DOI Listing
April 2013

NF-κB signaling is activated and confers resistance to apoptosis in three-dimensionally cultured EGFR-mutant lung adenocarcinoma cells.

Biochem Biophys Res Commun 2012 Jul 10;423(4):667-71. Epub 2012 Jun 10.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Yokohama, Japan.

Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cells cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of IκBα, the inhibitor of nuclear factor (NF)-κB, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-κB. Moreover, the inhibition of NF-κB with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-κB signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.
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http://dx.doi.org/10.1016/j.bbrc.2012.06.009DOI Listing
July 2012

HIF2α-Sp1 interaction mediates a deacetylation-dependent FVII-gene activation under hypoxic conditions in ovarian cancer cells.

Nucleic Acids Res 2012 Jul 8;40(12):5389-401. Epub 2012 Mar 8.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, 1-1-2 Nakao, Asahi-ku, Yokohama 241-0815, Japan.

Hypoxia-inducible factors (HIF)-1α and HIF2α are major transcription factors required for adaptive responses to hypoxia. HIFs form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) to bind to the regulatory regions of target genes. The acetylation of histones by histone acetyltransferases (HATs) is one of the epigenetic marks associated with active chromatin. Indeed, HIFs recruit p300 HAT to hypoxia response elements (HREs) within gene regulatory regions. Here, we report an unusual HIF-mediated transcriptional activation in ovarian clear cell carcinoma (CCC). While characterizing coagulation factor VII (FVII) gene induction during hypoxic conditions, we observed that the interaction of HIF2α with Sp1, but not with ARNT, could induce transcription of FVII in a HRE-independent manner. Unexpectedly, this gene activation is associated with histone deacetylation. We found that a class II HDAC, HDAC4, is recruited with HIF2α to the FVII promoter as a co-activator, while p300 HAT negatively regulated this process. Furthermore, this mechanism can be synergistically enhanced via a deacetylation-dependent pathway when cells are simultaneously exposed to hypoxic and serum-free conditions. These results suggest the presence of a stress-responsive transcription mediated by the HIF2α/Sp1/HDAC4 network and explain how CCC shed their procoagulant activity under hypoxia.
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http://dx.doi.org/10.1093/nar/gks201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3384323PMC
July 2012

WZ4002, a third-generation EGFR inhibitor, can overcome anoikis resistance in EGFR-mutant lung adenocarcinomas more efficiently than Src inhibitors.

Lab Invest 2012 Mar 12;92(3):371-83. Epub 2011 Dec 12.

Molecular Pathology and Genetics Division, Kanagawa Cancer Center Research Institute, Asahi-ku, Yokohama, Japan.

Src has a role in the anoikis resistance in lung adenocarcinomas. We focused on two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R+T790M), in suspension to elucidate whether suspended lung adenocarcinoma cells are eradicated by long-term treatment with Src tyrosine kinase inhibitors (TKIs). We also examined metastasis-positive lymph nodes from 16 EGFR-mutant lung adenocarcinoma patients for immunohistochemical expression of mutant-specific EGFR. Almost all suspended HCC827 cells underwent apoptosis after 144 h of combination treatment with AZD0530, trichostatin A (TSA), and ABT-263, whereas many suspended H1975 cells survived the treatment. AZD0530 is a Src TKI, TSA is a histone deacetylase inhibitor, and ABT-263 is a Bcl-2 inhibitor. During the therapy, the phosphorylation of EGFR decreased in HCC827 cells and remained stable in H1975 cells. The phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, was completely suppressed by the third generation EGFR TKI, WZ4002. Consequently, both the suspended cell lines were almost completely eradicated within 144 h, with the combined therapy of WZ4002, ABT-263, and TSA. Interestingly, treated suspended cells underwent apoptosis to a greater extent than did adherent cells. Intrasinus floating lung adenocarcinoma cells in the lymph nodes expressed a mutant-specific EGFR. These findings suggest that suspended EGFR-mutant lung adenocarcinoma cells depend significantly more on EGFR activation for survival than attached cells do. The tumor cells circulating in vessels, which express mutant-specific EGFR, would be highly susceptible to the combination therapy of WZ4002, ABT-263, and TSA.
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http://dx.doi.org/10.1038/labinvest.2011.187DOI Listing
March 2012

The global histone modification pattern correlates with overall survival in metachronous liver metastasis of colorectal cancer.

Oncol Rep 2012 Mar 10;27(3):637-42. Epub 2011 Nov 10.

Department of Gastro-intestinal Surgery, Kanagawa Cancer Center, 1-1-2 Nakao, Asahi-ku, Yokohama 241-0815, Japan.

Post-translational histone modifications are known to be altered in cancer tissues, and differences in the histone modification levels have recently been used to predict the clinical outcome in patients with certain types of cancer. In this study, we evaluated the immunohistochemical staining patterns of histone H3 dimethylation and acetylation in metachronous liver metastasis of colorectal carcinomas and examined its correlation with patient prognosis. Double 2 mm core tissue microarrays were made from 54 paraffin-embedded samples of liver metastasis from colorectal adenocarcinoma, and were examined by an immunohistochemical analysis of histone H3 lysine 4 (H3K4) dimethylation, histone, H3 lysine 9 (H3K9) dimethylation and histone H3 lysine 9 (H3K9) acetylation. Positive tumor cell staining for each histone modification was used to classify patients into low- and high-staining groups, which were then examined for correlations with the clinicopathological parameters and clinical outcome. Dimethylation of H3K4 correlated with the tumor histological type (P=0.043), and acetylation of H3K9 correlated with the tumor histological type (P=0.016). In addition, lower levels of H3K4 dimethylation correlated with a poor survival rate (P=0.035). The multivariate survival analysis showed that the H3K4 dimethylation status is an independent prognostic factor for colorectal cancer patients (P=0.011). We suggest that the pattern of histone modification as detected by immunohistochemistry may be an independent prognostic factor for metachronous liver metastasis of colorectal carcinomas.
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http://dx.doi.org/10.3892/or.2011.1547DOI Listing
March 2012

Immunohistochemical analysis of human equilibrative nucleoside transporter-1 (hENT1) predicts survival in resected pancreatic cancer patients treated with adjuvant gemcitabine monotherapy.

Ann Surg Oncol 2012 Jul 13;19 Suppl 3:S558-64. Epub 2011 Sep 13.

Department of Gastrointestinal Surgery, Kanagawa Cancer Center, Yokohama, Japan.

Background: Gemcitabine is a promising adjuvant treatment for patients with resected pancreatic cancer. Human equilibrative nucleoside transporter-1 (hENT1) is the major transporter responsible for gemcitabine uptake into cells. The aim of this study was to retrospectively determine the relationship between the outcome of pancreatic cancer after surgery followed by postoperative gemcitabine monotherapy and the expression of hENT1.

Methods: A total of 27 resected pancreatic cancer patients treated with adjuvant gemcitabine were analyzed for tumor hENT1 expression via an immunohistochemical analysis. The staining intensity and the percentage of positive tumor cells were scored, and the composite score (hENT1 score) was obtained by obtaining the sum of these two scores.

Results: There were 11 patients assigned to the low hENT1 expression group, and 16 patients to the high hENT1 group. The patients with tumors that had higher hENT1 expression had a significantly longer disease-free survival (DFS) (log rank, P = 0.022) and overall survival (OS) (P = 0.024). The hENT1 expression was indicated to be a significant and independent prognostic factor for OS by the univariate (P = 0.030) and multivariate analyses (P = 0.019).

Conclusions: A high expression of hENT1 in pancreatic cancer was found to be significantly associated with a longer survival in patients who received adjuvant gemcitabine monotherapy after curative resection, and hENT1 immunohistochemistry may well serve as a significant prognostic factor for these patients.
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http://dx.doi.org/10.1245/s10434-011-2054-zDOI Listing
July 2012
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