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Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic progenitor cells that can undergo self-renewal and differentiate into multi-lineages. Bone marrow stromal stem cells (BMSCs) represent one of the most commonly-used MSCs. In order to overcome the technical challenge of maintaining primary BMSCs in long-term culture, here we seek to establish reversibly immortalized mouse BMSCs (imBMSCs). By exploiting CRISPR/Cas9-based homology-directed-repair (HDR) mechanism, we target SV40T to mouse Rosa26 locus and efficiently immortalize mouse BMSCs (i.e., imBMSCs). We also immortalize BMSCs with retroviral vector SSR #41 and establish imBMSC41 as a control line. Both imBMSCs and imBMSC41 exhibit long-term proliferative capability although imBMSC41 cells have a higher proliferation rate. SV40T mRNA expression is 130% higher in imBMSC41 than that in imBMSCs. However, FLP expression leads to 86% reduction of SV40T expression in imBMSCs, compared with 63% in imBMSC41 cells. Quantitative genomic PCR analysis indicates that the average copy number of SV40T and hygromycin is 1.05 for imBMSCs and 2.07 for imBMSC41, respectively. Moreover, FLP expression removes 92% of SV40T in imBMSCs at the genome DNA level, compared with 58% of that in imBMSC41 cells, indicating CRISPR/Cas9 HDR-mediated immortalization of BMSCs can be more effectively reversed than that of retrovirus-mediated random integrations. Nonetheless, both imBMSCs and imBMSC41 lines express MSC markers and are highly responsive to BMP9-induced osteogenic, chondrogenic and adipogenic differentiation in vitro and in vivo. Thus, the engineered imBMSCs can be used as a promising alternative source of primary MSCs for basic and translational research in the fields of MSC biology and regenerative medicine.

// Tingxiu Xiang 1 , Yichao Fan 2 , Chunhong Li 3 , Lili Li 2 , Ying Ying 2 , Junhao Mu 1 , Weiyan Peng 1 , Yixiao Feng 1 , Michael Oberst 4 , Kathleen Kelly 4 , Guosheng Ren 1 , Qian Tao 1, 2 1 Chongqing Key Laboratory of Molecular Oncology and Epigenetics, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China 2 Cancer Epigenetics Laboratory, Department of Clinical Oncology, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong and CUHK Shenzhen Research Institute, Hong Kong 3 Oncology Department, Suining Sichuan Center Hospital, Sichuan, China 4 Signal Transduction Section, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA Correspondence to: Guosheng Ren, email: rgs726@163.com Qian Tao, email: qtao@cuhk.edu.hk Keywords: DACT2, tumor suppressor, methylation, EMT, breast cancer Received: April 22, 2016 Accepted: September 14, 2016 Published: September 29, 2016 ABSTRACT Wnt signaling plays an important role in breast carcinogenesis. DAPPER2 (DACT2) functions as an inhibitor of canonical Wnt signaling and plays distinct roles in different cell contexts, with its role in breast tumorigenesis unclear. We investigated DACT2 expression in breast cancer cell lines and primary tumors, as well as its functions and molecular mechanisms. Results showed that DACT2 expression was silenced in 9/9 of cell lines. Promoter CpG methylation of DACT2 was detected in 89% (8/9) of cell lines, as well as in 73% (107/147) of primary tumors, but only in 20% (1/5) of surgical margin tissues and in none of normal breast tissues. Demethylation of BT549 and T47D cell lines with 5-aza-2’-deoxycytidine restored DACT2 expression along with promoter demethylation, suggesting that its downregulation in breast cancer is dependent on promoter methylation. Furthermore, ectopic expression of DACT2 induced breast cell apoptosis in vitro , and further inhibited breast tumor cell proliferation, migration and EMT, through antagonizing Wnt/β-catenin and Akt/GSK-3 signaling. Thus, these results demonstrate that DACT2 functions as a tumor suppressor for breast cancer but was frequently disrupted epigenetically in this cancer.

// Xuedong Yin 1, * , Tingxiu Xiang 1, * , Junhao Mu 1 , Haitao Mao 2 , Lili Li 2 , Xin Huang 2 , Chunhong Li 3 , Yixiao Feng 1 , Xinrong Luo 1 , Yuxian Wei 1 , Weiyan Peng 1 , Guosheng Ren 1 , Qian Tao 1, 2 1 Chongqing Key Laboratory of Molecular Oncology and Epigenetics, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China 2 Cancer Epigenetics Laboratory, Department of Clinical Oncology, State Key Laboratory of Oncology in South China, Sir YK Pao Center for Cancer and Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong and CUHK Shenzhen Research Institute, Hong Kong 3 Oncology Department, Suining Sichuan Center Hospital, Sichuan, China * These authors contributed equally to this work Correspondence to: Guosheng Ren, email: rengs726@126.com Qian Tao, email: qtao@cuhk.edu.hk Keywords: tumor suppressor, PCDH17, methylation, cancer, β-catenin Received: November 27, 2015 Accepted: May 28, 2016 Published: June 16, 2016 ABSTRACT Protocadherins play important roles in the regulation of cell adhesion and signaling transduction. Aberrant expression of protocadherins has been shown to be associated with multiple tumorigenesis. We previously identified PCDH17 , encoding protocadherin 17, as a frequently methylated and downregulated tumor suppressor gene (TSG) in gastric and colorectal cancers. Here, we examined the abnormalities and functions of PCDH17 in breast cancer pathogenesis. We used PCR and immunohistochemistry to check its expression pattern in breast tumor cell lines and primary tumors. Methylation-specific PCR (MSP) was applied to examine its promoter methylation status in breast tumor cell lines and primary tumors. The biological functions of PCDH17 in breast tumor cells were assessed using in vitro and in vivo assays. We found that PCDH17 was frequently downregulated or silenced in 78% (7/9) of breast tumor cell lines, as well as 89% (32/36) of primary tumors. Downregulation of PCDH17 in breast cancer was mainly due to the methylation of its promoter. Ectopic expression of PCDH17 in breast tumor cells inhibited cell proliferation and mobility through arresting cell cycle and inducing apoptosis. In breast tumor cells, PCDH17 significantly suppressed the active β-catenin level and its downstream target gene expression. Thus, we found that PCDH17 functions as a tumor suppressor inhibiting Wnt/β-catenin signaling and metastasis in breast cancer but is frequently methylated in primary tumors which could be a potential biomarker.