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Recurrence and drug resistance of breast cancer are still the main reasons for breast cancer-associated deaths. Cancer stem cell (CSC) model has been proposed as a hypothesis for the lethality of breast cancer. Molecular mechanisms underlying CSC maintenance are still unclear. In this study, we generated mammospheres derived from breast cancer MDA-MB231 cells and MCF7 cells to enrich CSCs and performed DNA microarray analysis. We found that the expression of carboxy terminus of HSP70-interacting protein (CHIP) E3 ubiquitin ligase was significantly downregulated in breast CSCs. CHIP depletion increased mammosphere formation, whereas CHIP overexpression reversed this effect. We identified interactomes by mass spectrometry and detected CHIP directly interacted with OCT4, a stemness factor. CHIP overexpression decreased OCT4 stability through proteasomal degradation. CHIP induced OCT4 ubiquitination, whereas H260Q, a catalytic CHIP mutant, did not. Interestingly, we determined that OCT4 was ubiquitinated at lysine 284, and CHIP overexpression did not degrade K284R mutant OCT4. CHIP overexpression decreased the proliferation and side population of breast cancer cells, but these were not occurred in K284R mutant OCT4 overexpressed cells. Only 1000 cells showing CHIP depletion or OCT4 overexpression sufficiently generated breast tumors and lung metastases in xenografted mice. Ubiquitination-defective mutant of OCT4(K284R) overexpressed cells drastically generated tumor burdens in mice. Patients with breast cancer who showed low CHIP expression had poor survival probability. Taken together, we suggest that CHIP-induced OCT4 ubiquitination is important in breast CSCs. Regulation of CHIP expression and OCT4 protein stability is a considerable approach for breast cancer therapy.

// Sun-Hyuk La 1, 2 , Seok-Jun Kim 1, 3 , Hyeok-Gu Kang 1, 3 , Han-Woong Lee 2 , Kyung-Hee Chun 1, 3 1 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seodaemun-gu, Seoul 03722, Republic of Korea 2 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seodaemun-gu, Seoul 03722, Republic of Korea 3 Brain Korea 21 PLUS Project for Medical Science, Yonsei University, Seodaemun-gu, Seoul 03722, Republic of Korea Correspondence to: Kyung-Hee Chun, email: khchun@yuhs.ac Keywords : telomerase, hTERT, galectin-3, cellular senescence, gastric cancer Received: May 23, 2016 Accepted: July 19, 2016 Published: August 1, 2016 ABSTRACT The human Telomerase Reverse Transcriptase (hTERT) gene encodes a rate-limiting catalytic subunit of telomerase that maintains genomic integrity. Suppression of hTERT expression could induce cellular senescence and is considered a potent approach for gastric cancer therapy. However, control of hTERT expression and function remains poorly understood in gastric cancer. In this study, we demonstrated that high expression levels of hTERT in malignant tissues are correlated with poor survival probability in gastric cancer patients. Knockdown of hTERT expression retarded cell proliferation and cellular senescence, which was confirmed by increased protein expression levels of p21 cip1 and p27 kip1 , and decreased phosphorylation of Rb. In contrast, overexpression of hTERT increased cell proliferation and decreased cellular senescence. Remarkably, the down-regulation of hTERT expression was detected in lgals3 -/- mouse embryo fibroblasts (MEFs). Knockdown of galectin-3 decreased the expression of hTERT in gastric cancer cells. Galectin-3 ablation-induced cellular senescence was rescued by concomitant overexpression of hTERT. hTERT ablation-induced cellular senescence and p21 cip1 and p27 kip1 expression was rescued by concomitant overexpression of galectin-3. The size of tumor burdens was increased in hTERT-overexpressed gastric cancer cells xenografted mice, whereas it was repressed by concomitant depletion of galectin-3. Additionally, we determined that the N-terminal domain of galectin-3 directly interacted with hTERT. The telomeric activity of hTERT was also decreased by galectin-3 ablation. Taken together, ablation of hTERT induces cellular senescence and inhibits the growth of gastric cancer cells, suggesting that it could be a potent target in gastric cancer therapy. We also propose that galectin-3 is an important regulator of hTERT expression and telomeric activity in gastric tumorigenesis.

// Seok-Jun Kim 1,8 , Yuan-Guo Wang 2 , Hyun-Woo Lee 1,3 , Hyeok Gu Kang 1,8 , Sun-Hyuk La 1,3 , Il Ju Choi 4 , Tatsuro Irimura 5 , Jae Y. Ro 6 , Robert S. Bresalier 7 and Kyung-Hee Chun 1,8 1 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul, Republic of Korea. 2 Minimally Invasive Tumor Therapy Laboratory, Radiology Department, Beth Israel Deaconess Medical Center, Boston, MA, USA. 3 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea. 4 Center for Gastric Cancer, Division of Translational & Clinical Research I, National Cancer Center, Gyeonggi-do, Republic of Korea. 5 Institute of medical innovation, St. Luke’s international Hospital, Chuo-ku, Tokyo, Japan. 6 The Methodist Hospital, Department of Pathology and Genomic Medicine, Weil Medical College of Cornell University, Houston, TX, USA. 7 Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA. 8 Brain Korea 21 PLUS Project for Medical Science, Yonsei University. Correspondence: Kyung-Hee Chun, email: // Keywords : Neogenin-1, Galectin-3, Heat shock factor (HSF)-1, Cancer metastasis, Gastric cancer Received : March 5, 2014 Accepted : May 12, 2014 Published : May 13, 2014 Abstract Although elevated expression of neogenin-1 has been detected in human gastric cancer tissue, its role in gastric tumorigenesis remains unclear due to the lack of neogenin-1 studies in cancer. Therefore, we demonstrated here the function and regulatory mechanism of neogenin-1 in gastric cancer. Neogenin-1 ablation decreased proliferation and migration of gastric cancer cells, whereas its over-expression reversed these effects. Xenografted analyses using gastric cancer cells displayed statistically significant inhibition of tumor growth by neogenin-1 depletion. Interestingly, galectin-3 interacted with HSF-1 directly, which facilitated nuclear-localization and binding on neogenin-1 promoter to drive its transcription and gastric cancer cell motility. The galectin-3-increased gastric cancer cell motility was down-regulated by HSF-1 depletion. Moreover, the parallel expression patterns of galectin-3 and neogenin-1, as well as those of HSF-1 and neogenin-1, were detected in the malignant tissues of gastric cancer patients. Taken together, high-expression of neogenin-1 promotes gastric cancer proliferation and motility and its expression is regulated by HSF-1 and galectin-3 interaction. In addition, we propose further studies for neogenin-1 and its associated pathways to provide them as a proper target for gastric cancer therapy.