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Sustained activation of the Ca2+-release-activated Ca2+ (CRAC) channel is pivotal for effector Tcell responses. The mechanisms underlying this sustainability remain poorly understood. We find that plasma membrane localization of ORAI1, the pore subunit of CRAC channels, is limited in effector Tcells, with a significant fraction trapped in intracellular vesicles. From a targeted screen, we identify an essential component of ORAI1+ vesicles, naked cuticle homolog 2 (NKD2). Mechanistically, NKD2, an adaptor molecule activated by signaling pathways downstream of Tcell receptors, orchestrates trafficking and insertion of ORAI1+ vesicles to the plasma membrane. Together, our findings suggest that Tcell receptor (TCR)-stimulation-dependent insertion of ORAI1 into the plasma membrane is essential for sustained Ca2+ signaling and cytokine production in Tcells.

// Yan Jia 1, 2, * , Baoping Cao 1, 3, * , Yunsheng Yang 1 , Enqiang Linghu 1 , Qimin Zhan 4 , Youyong Lu 5 , Yingyan Yu 6 , James G. Herman 7 , Mingzhou Guo 1 1 Department of Gastroenterology & Hepatology, Chinese PLA General Hospital, Beijing 100853, China 2 Department of Breast Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, and Key Laboratory of Cancer Prevention and Therapy, Tianjin 300060, China 3 Medical College of NanKai University, Tianjin 300071, China 4 State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100021, China 5 Laboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital/Institute, Beijing 100142, China 6 Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China 7 The Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA * These authors have contributed equally to this work Correspondence to: Mingzhou Guo, e-mail: mzguo@hotmail.com James G. Herman, e-mail: Hermanj3@upmc.edu Keywords: NKD1, NKD2, SOX18, DNA methylation, gastric cancer Received: April 23, 2015 Accepted: September 02, 2015 Published: September 14, 2015 ABSTRACT Naked cuticle homolog2 ( NKD2 ) is located in chromosome 5p15.3, which is frequently loss of heterozygosity in human colorectal and gastric cancers. In order to understand the mechanism of NKD2 in gastric cancer development, 6 gastric cancer cell lines and 196 cases of human primary gastric cancer samples were involved. Methylation specific PCR (MSP), gene expression array, flow cytometry, transwell assay and xenograft mice model were employed in this study. The expression of NKD1 and NKD2 was silenced by promoter region hypermethylation. NKD1 and NKD2 were methylated in 11.7% (23/196) and 53.1% (104/196) in human primary gastric cancer samples. NKD2 methylation is associated with cell differentiation, TNM stage and distant metastasis significantly (all P < 0.05), and the overall survival time is longer in NKD2 unmethylated group compared to NKD2 methylated group ( P < 0.05). Restoration of NKD2 expression suppressed cell proliferation, colony formation, cell invasion and migration, induced G2/M phase arrest, and sensitized cancer cells to docetaxel. NKD2 inhibits SOX18 and MMP-2,7,9 expression and suppresses BGC823 cell xenograft growth. In conclusion, NKD2 methylation may serve as a poor prognostic and chemo-sensitive marker in human gastric cancer. NKD2 impedes gastric cancer metastasis by inhibiting SOX18.

// Yan Dong 1, 2 , Baoping Cao 1, 3 , Meiying Zhang 1, 3 , Weidong Han 2 , James G. Herman 4 , Francois Fuks 5 , Yali Zhao 6 , Mingzhou Guo 1 1 Department of Gastroenterology & Hepatology, Chinese PLA General Hospital, Beijing 100853, China 2 Department of Molecular Biology, Institute of Basic Medicine, School of Life Sciences, Chinese PLA General Hospital, Beijing 100853, China 3 Medical College of NanKai University, Tianjin 300071, China 4 The Hillman Cancer Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, U.S.A 5 Laboratory of Cancer Epigenetics, Free University of Brussels (U.L.B.), 808 route de Lennik, Brussels 1070, Belgium 6 Central Laboratory, The Affiliated Hainan Hospital of Chinese PLA General Hospital, Hai Tang Wan, Sanya 572013, China Correspondence to: Mingzhou Guo, e-mail: mzguo1@gmail.com Yali Zhao, e-mail: zhaoyl301@163.com Keywords: NKD2, DNA methylation, Wnt signaling, epigenetics, breast cancer Received: February 27, 2015 Accepted: June 08, 2015 Published: June 19, 2015 ABSTRACT Naked cuticle homolog 2 (NKD2) has been reported to antagonize Wnt signaling in zebrafish, mouse and mammals. The aim of this study is to investigate the epigenetic changes and mechanisms of NKD2 in human breast cancer development. Six breast cancer cell lines (MCF-7, ZR75–1, MDA-MB-468, MDA-MB-231, T47D and BT474) and 68 cases of primary human breast cancer were studied using methylation specific PCR, immunohistochemistry, western blot, flow cytometry techniques and a xenograft mouse model. The expression of NKD1 and NKD2 was regulated by promoter region methylation in breast cancer cells. No NKD1 methylation was found in primary human breast cancer. NKD2 was methylated in 51.4% (35/68) of human primary breast cancer samples. NKD2 methylation was significantly associated with reduction of NKD2 expression, and tumor stage ( p < 0.05). NKD2 suppressed breast cancer cell proliferation both in vitro and in vivo . NKD2 induced G1/S arrest and inhibited Wnt signaling in breast cancer cells. In conclusion, NKD2 is frequently methylated in human breast cancer, and the expression of NKD2 is regulated by promoter region methylation. NKD2 suppresses breast cancer proliferation by inhibiting Wnt signaling.

AIM: The present study was aimed to investigate NKD2 expression as well as promoter methylation and further analyze their clinical significance in patients with acute myeloid leukemia (AML). METHODS: Real-time quantitative PCR was carried out to detect the pattern of NKD2 expression in 113 AML patients and 24 controls. Real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP) were carried out to detect NKD2 promoter methylation in 101 AML patients and 24 controls with available DNA. RESULTS: The level of NKD2 transcript in AML patients was significantly down-regulated as compared with controls (P=0.039). NKD2 methylation level in AML patients was significantly higher than controls (P=0.044). Moreover, NKD2 methylation negatively correlated with NKD2 expression in AML patients (R=-0.218, P=0.029). Furthermore, demethylation of NKD2 could increase NKD2 expression in the leukemic cell line THP1 (P