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Checkpoint kinase 1 (Chk1) expression is enhanced in most cancers owing to oncogenic activation and constant replicative stress. Chk1 inactivation is a promising cancer therapy, as its inactivation leads to genomic instability, chromosomal catastrophe, and cancer cell death. Herein, we observed that miR-320c, downregulated in triple-negative breast cancer (TNBC) patients, can target Chk1. In addition, downregulated miR-320c expression was associated with poor overall survival in TNBC patients. As Chk1 was associated with the DNA damage response (DDR), we investigated the effect of miR-320c on DDR in TNBC cells. To induce DNA damage, we used platinum-based drugs, especially oxaliplatin, which is most effective with miR-320c. We observed that overexpression of miR-320c in TNBC regulated the oxaliplatin responsiveness by mediating DNA damage repair through the negative regulation of Chk1 in vitro. Furthermore, using a xenograft model, a combination of miR-320c mimic and oxaliplatin effectively inhibited tumor progression. These investigations indicate the potential of miR-320c as a marker of oxaliplatin responsiveness and a therapeutic target to increase the efficacy of chemotherapy in TNBC.

With aging, the skin becomes thin and drastically loses collagen. Extracellular superoxide dismutase (EC-SOD), also known as superoxide dismutase (SOD) 3, is the major SOD in the extracellular matrix of the tissues and is well-known to maintain the reduction‒oxidation homeostasis and matrix components of such tissues. However, the role of EC-SOD in aging-associated reductions of skin thickness and collagen production is not well-studied. In this study, we compared the histological differences in the dorsal skin of EC-SOD‒overexpressing transgenic mice (Sod3+/+) of different age groups with that in wild-type mice and also determined the underlying signaling mechanism. Our data showed that the skin thickness in Sod3+/+ mice significantly increased with aging compared with that in wild-type male mice. Furthermore, Sod3+/+ mice had promoted collagen production through the activation of adenosine monophosphate-activated protein kinase and Nrf2/HO-1 pathways in aged mice. Interestingly, subcutaneous injection of adeno-associated virus‒overexpressing EC-SOD exhibited increased skin thickness and collagen expression. Furthermore, combined recombinant EC-SOD and dihydrotestosterone treatment synergistically elevated collagen production through the activation of TGFβ in human dermal fibroblasts. Altogether, these results showed that EC-SOD prevents skin aging by promoting collagen production in vivo and in vitro. Therefore, we propose that EC-SOD may be a potential therapeutic target for antiaging in the skin.

E6 oncoprotein derived from high-risk human papillomavirus (HPV) drives the development of cervical cancer through p53 degradation. Because cervical cancer therapies to inactivate HPV or E6 protein are not available, alternative strategies are required. Here, we show that HPV-mediated nuclear export of human heterochromatin protein 1γ (HP1γ) reduces the stability of p53 through UBE2L3-mediated p53 polyubiquitination during cervical cancer progression. In general, HP1 plays a key role in heterochromatin formation and transcription in the nucleus. However, our immunostaining data showed that the majority of HP1γ is localized in the cytoplasm in HPV-mediated cervical cancer. We found that HPV E6 protein drives unusual nuclear export of HP1γ through the interaction between the NES sequence of HP1γ and exportin-1. The mutation of the NES sequence in HP1γ led to nuclear retention of HP1γ and reduced cervical cancer cell growth and tumor generation. We further discovered that HP1γ directly suppresses the expression of UBE2L3 which drives E6-mediated proteasomal degradation of p53 in cervical cancer. Downregulation of UBE2L3 by overexpression of HP1γ suppressed UBE2L3-dependent p53 degradation-promoting apoptosis of cervical cancer cells. Our findings propose a useful strategy to overcome p53 degradation in cervical cancer through the blockage of nuclear export of HP1γ.

The hyaluronan-mediated motility receptor (HMMR) is overexpressed in gastric cancer; however, the apparent role of HMMR has not been well defined owing to lack of detailed studies on gastric tumorigenesis. Therefore, we elucidated the functional and regulatory mechanisms of HMMR in gastric cancer. Using publicly available data, we confirmed HMMR overexpression in patients with gastric cancer. HMMR silencing decreased proliferation, migration, and invasion of gastric cancer cells, whereas HMMR overexpression reversed these effects. A gastric cancer xenograft mouse model showed statistically significant inhibition of tumor growth upon HMMR depletion. Previous data from cDNA microarray showed reduced HMMR expression upon inhibition of galectin-3. However, overexpression of galectin-3 increased HMMR expression, cell proliferation, and motility in gastric cancer cells, whereas HMMR silencing blocked these effects. Interestingly, galectin-3 interacted directly with C/EBPβ and bound to HMMR promoter to drive its transcription, and gastric cancer cell proliferation and motility. Altogether, high expression of HMMR promoted gastric cancer cell proliferation and motility and could be a prognostic factor in gastric cancer. In addition, HMMR expression was regulated by the interaction between C/EBPβ and galectin-3. Therefore, targeting HMMR along with galectin-3 and C/EBPβ complex could be a potential treatment strategy for inhibiting gastric cancer progression and metastasis. Implications: This study provides evidence that galectin-3 interacts with C/EBPβ in gastric cancer, and galectin-3 and C/EBPβ complex promotes gastric cancer cell progression and motility through upregulating HMMR expression.

Ovarian cancer is the most lethal gynecologic disease because usually, it is lately sensed, easily acquires chemoresistance, and has a high recurrence rate. Recent studies suggest that ovarian cancer stem cells (CSCs) are involved in these malignancies. Here, we demonstrated that galectin-3 maintains ovarian CSCs by activating the Notch1 intracellular domain (NICD1). The number and size of ovarian CSCs decreased in the absence of galectin-3, and overexpression of galectin-3 increased them. Overexpression of galectin-3 increased the resistance for cisplatin and paclitaxel-induced cell death. Silencing of galectin-3 decreased the migration and invasion of ovarian cancer cells, and overexpression of galectin-3 reversed these effects. The Notch signaling pathway was strongly activated by galectin-3 overexpression in A2780 cells. Silencing of galectin-3 reduced the levels of cleaved NICD1 and expression of the Notch target genes, Hes1 and Hey1. Overexpression of galectin-3 induced NICD1 cleavage and increased expression of Hes1 and Hey1. Moreover, overexpression of galectin-3 increased the nuclear translocation of NICD1. Interestingly, the carbohydrate recognition domain of galectin-3 interacted with NICD1. Overexpression of galectin-3 increased tumor burden in A2780 ovarian cancer xenografted mice. Increased expression of galectin-3 was detected in advanced stages, compared to stage 1 or 2 in ovarian cancer patients, suggesting that galectin-3 supports stemness of these cells. Based on these results, we suggest that targeting galectin-3 may be a potent approach for improving ovarian 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.

// Hyun Kyung Kong 1 , Sae Jeong Park 1 , Ye Sol Kim 1 , Kyoung Min Kim 2 , Hyun-Woo Lee 3, 4 , Hyeok-Gu Kang 3, 5 , Yu Mi Woo 1 , Eun Young Park 1 , Je Yeong Ko 1 , Hiromu Suzuki 6 , Kyung-Hee Chun 3, 5 , Erwei Song 7 , Kyu Yun Jang 2 , Jong Hoon Park 1 1 Department of Biological Science, Sookmyung Women’s University, Seoul, Republic of Korea 2 Department of Pathology, Chonbuk National University Medical School, Research Institute of Clinical Medicine and Research Institute for Endocrine Sciences, Jeonju, Republic of Korea 3 Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul, Republic of Korea 4 Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea 5 Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Republic of Korea 6 Department of Molecular Biology, Sapporo Medical University, Sapporo, Japan 7 Department of Breast Surgery, Sun Yat-Sen Memorial Hospital, Sun-Yat-Sen University, Guangzhou, Peoples Republic of China Correspondence to: Jong Hoon Park, email: parkjh@sookmyung.ac.kr Kyu Yun Jang, email: kyjang@chonbuk.ac.kr Erwei Song, email: songerwei02@yahoo.com.cn Keywords: breast cancer, LY6K, metastasis, DNA methylation, histone modification Received: December 06, 2015 Accepted: July 06, 2016 Published: August 01, 2016 ABSTRACT The role of lymphocyte antigen 6 complex, locus K (LY6K) in breast cancer has been studied, whereas the epigenetic control of LY6K transcription is not fully understood. Here, we report that breast cancer patients with increased LY6K expression had shorter disease-free and overall survival than the patients with low levels of LY6K by multivariate analysis. LY6K also was upregulated in breast cancer patients with distant metastases than those without distant metastases, downregulating E-cadherin expression. Furthermore, xenograft tumor volumes from LY6K knockdown nude mice were reduced than those of mice treated with control lentivirus. Interestingly, LY6K has a CpG island (CGI) around the transcription start site and non-CGI in its promoter, called a CGI shore. LY6K expression was inversely correlated with methylation in not only CGI but CGI shore, which are associated with histone modifications. Additionally, LY6K methylation was increased by the PAX3 transcription factor due to the SNP242 mutation in LY6K CGI shore. Taken together, breast cancer risk and metastasis were significantly associated with not only LY6K expression, but also methylation of CGI shore which induced by SNP242 mutation. Our results suggest that an understanding epigenetic mechanism of the LY6K gene may be useful to diagnose carcinogenic risk and predict outcomes of patients with metastatic breast cancer.

// Hye-Young Kim 1, 2 , Yunhee Cho 1, 3 , HyeokGu Kang 1, 3 , Ye-Seal Yim 1, 3 , Seok-Jun Kim 1, 3 , Jaewhan Song 2 , Kyung-Hee Chun 1, 3 1 Department of Biochemistry & Molecular Biology, Yonsei University College of Medicine, Seodaemun-gu, Seoul 03722, Korea 2 Department of Biochemistry, College of Life Science and Biotechnology, Seodaemun-gu, Seoul 03722, Korea 3 Brain Korea 21 PlusProject for Medical Science, Yonsei University, Seodaemun-gu, Seoul 03722, Korea Correspondence to: Kyung-Hee Chun, email: khchun@yuhs.ac Keywords: WEE1, AZD1775 (MK-1775), 5-FU, Paclitaxel, gastric cancer Received: September 07, 2015 Accepted: May 28, 2016 Published: June 23, 2016 ABSTRACT Wee1 is a member of the Serine/Threonine protein kinase family and is a key regulator of cell cycle progression. It has been known that WEE1 is highly expressed and has oncogenic functions in various cancers, but it is not yet studied in gastric cancers. In this study, we investigated the oncogenic role and therapeutic potency of targeting WEE1 in gastric cancer. At first, higher expression levels of WEE1 with lower survival probability were determined in stage 4 gastric cancer patients or male patients with accompanied lymph node metastasis. To determine the function of WEE1 in gastric cancer cells, we determined that WEE1 ablation decreased the proliferation, migration, and invasion, while overexpression of WEE1 increased these effects in gastric cancer cells. We also validated the clinical application of WEE1 targeting by a small molecule, AZD1775 (MK-1775), which is a WEE1 specific inhibitor undergoing clinical trials. AZD1775 significantly inhibited cell proliferation and induced apoptosis and cell cycle arrest in gastric cancer cells, which was more effective in WEE1 high-expressing gastric cancer cells. Moreover, we performed combination treatments with AZD1775 and anti-cancer agents, 5- fluorouracil or Paclitaxel in gastric cancer cells and in gastric cancer orthotopic-transplanted mice to maximize the therapeutic effect and safety of AZD1775. The combination treatments dramatically inhibited the proliferation of gastric cancer cells and tumor burdens in stomach orthotopic-transplanted mice. Taken together, we propose that WEE1 is over-expressed and could enhance gastric cancer cell proliferation and metastasis. Therefore, we suggest that WEE1 is a potent target for gastric cancer therapy.

Clear cell renal carcinoma (RCC), the most common malignancy arising in the adult kidney, exhibits increased aerobic glycolysis and low mitochondrial respiration due to von Hippel-Lindau gene defects and constitutive hypoxia-inducible factor-α expression. Sirt3 is a major mitochondrial deacetylase that mediates various types of energy metabolism. However, the role of Sirt3 as a tumor suppressor or oncogene in cancer depends on cell types. We show increased Sirt3 expression in the mitochondrial fraction of human RCC tissues. Sirt3 depletion by lentiviral short-hairpin RNA, as well as the stable expression of the inactive mutant of Sirt3, inhibited cell proliferation and tumor growth in xenograft nude mice, respectively. Furthermore, mitochondrial pyruvate, which was used for oxidation in RCC, might be derived from glutamine, but not from glucose and cytosolic pyruvate, due to depletion of mitochondrial pyruvate carrier and the relatively high expression of malic enzyme 2. Depletion of Sirt3 suppressed glutamate dehydrogenase activity, leading to impaired mitochondrial oxygen consumption. Our findings suggest that Sirt3 plays a tumor-progressive role in human RCC by regulating glutamine-derived mitochondrial respiration, particularly in cells where mitochondrial usage of cytosolic pyruvate is severely compromised.

Triple negative breast cancer (TNBC) has higher aggressiveness and poorer outcomes compared with other subtypes of breast cancer. However, the genomic and molecular aberrations of TNBC are largely unknown. In this study, miR-374a-5p was discovered as a novel TNBC-specific miRNA and its functions and the molecular mechanisms involved were investigated. Combined gene expression profiling of miRNA-microarray and human transcriptome dataset analysis revealed that miR-374a-5p is specifically upregulated in TNBC patients. Functional studies using in vitro and in vivo models indicated that upregulated miR-374a-5p promotes tumor progression in TNBC. miR-374a-5p was also found to directly target arrestin beta 1 (ARRB1) that is specifically downregulated in TNBC patients in several human genomic datasets. Overexpressed ARRB1 reduced TNBC cell growth and migration, and the ARRB1 expression level is inversely correlated with the histological grade of the breast cancer and positively associated with TNBC patient survival, suggestive of a tumor-suppressive function of ARRB1 in breast cancer. Interestingly, increased ARRB1 activates AMPK in TNBC cells, associated with the expression of miR-374a-5p. Taken together, the findings suggest that miR-374a-5p is a potential prognostic marker of TNBC.

The receptor Notch1 plays an important role in malignant progression of many cancers, but its regulation is not fully understood. In this study, we report that the kinase HIPK2 is responsible for facilitating the Fbw7-dependent proteasomal degradation of Notch1 by phosphorylating its intracellular domain (Notch1-IC) within the Cdc4 phosphodegron motif. Notch1-IC expression was higher in cancer cells than normal cells. Under genotoxic stress, Notch1-IC was phosphorylated constitutively by HIPK2 and was maintained at a low level through proteasomal degradation. HIPK2 phosphorylated the residue T2512 in Notch1-IC. Somatic mutations near this residue rendered Notch1-IC resistant to degradation, as induced either by HIPK2 overexpression or adriamycin treatment. In revealing an important mechanism of Notch1 stability, the results of this study could offer a therapeutic strategy to block Notch1-dependent progression in many types of cancer. Cancer Res; 76(16); 4728–40. ©2016 AACR.

One of the properties of human breast cancer cells is cancer stemness, which is characterized by self-renewal capability and drug resistance. Protein kinase D1 (PRKD1) functions as a key regulator of many cellular processes and is downregulated in invasive breast cancer cells. In this study, we found that PRKD1 was upregulated in MCF-7-ADR human breast cancer cells characterized by drug resistance. Additionally, we discovered that PRKD1 expression was negatively regulated by miR-34a binding to the PRKD1 3'-UTR. PRKD1 expression increased following performance of a tumorsphere formation assay in MCF-7-ADR cells. We also found that reduction of PRKD1 by ectopic miR-34a expression or PRKD1 siRNA treatment resulted in suppressed self-renewal ability in breast cancer stem cells. Furthermore, we confirmed that the PRKD1 inhibitor CRT0066101 reduced phosphorylated PKD/PKCμ, leading to suppression of breast cancer stemness through GSK3/β-catenin signaling. PRKD1 inhibition also influenced apoptosis initiation in MCF-7-ADR cells. Tumors from nude mice treated with miR-34a or CRT0066101 showed suppressed tumor growth, proliferation, and induced apoptosis. These results provide evidence that regulation of PRKD1, a novel miR-34a target, contributes to overcoming cancer stemness and drug resistance in human breast cancer.