Chi Lam Au Yeung

Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1

Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer.

Clitocine Reversal of P-Glycoprotein Associated Multi-Drug Resistance through Down-Regulation of Transcription Factor NF-κB in R-HepG2 Cell Line

Multidrug resistance(MDR)is one of the major reasons for failure in cancer chemotherapy and its suppression may increase the efficacy of therapy. The human multidrug resistance 1 (MDR1) gene encodes the plasma membrane P-glycoprotein (P-gp) that pumps various anti-cancer agents out of the cancer cell. R-HepG2 and MES-SA/Dx5 cells are doxorubicin induced P-gp over-expressed MDR sublines of human hepatocellular carcinoma HepG2 cells and human uterine carcinoma MES-SA cells respectively. Herein, we observed that clitocine, a natural compound extracted from Leucopaxillus giganteus, presented similar cytotoxicity in multidrug resistant cell lines compared with their parental cell lines and significantly suppressed the expression of P-gp in R-HepG2 and MES-SA/Dx5 cells. Further study showed that the clitocine increased the sensitivity and intracellular accumulation of doxorubicin in R-HepG2 cells accompanying down-regulated MDR1 mRNA level and promoter activity, indicating the reversal effect of MDR by clitocine. A 5′-serial truncation analysis of the MDR1 promoter defined a region from position −450 to −193 to be critical for clitocine suppression of MDR1. Mutation of a consensus NF-κB binding site in the defined region and overexpression of NF-κB p65 could offset the suppression effect of clitocine on MDR1 promoter. By immunohistochemistry, clitocine was confirmed to suppress the protein levels of both P-gp and NF-κB p65 in R-HepG2 cells and tumors. Clitocine also inhibited the expression of NF-κB p65 in MES-SA/Dx5. More importantly, clitocine could suppress the NF-κB activation even in presence of doxorubicin. Taken together; our results suggested that clitocine could reverse P-gp associated MDR via down-regulation of NF-κB.

3,5-Dimethyl-7H-Furo[3,2-g]Chromen-7-One as a Potential Anticancer Drug by Inducing p53-Dependent Apoptosis in Human Hepatoma HepG2 Cells

Background/Aims: Coumarins are natural compounds found in many plants that possess medical value by itself and its modified derivatives. Method: Six novel coumarin derivatives were synthesized and examined for their potential anticancer cytotoxicity. Result: Among the 6 derivatives, 3,5-dimethyl-7H-furo[3,2-g]chromen-7-one (DMFC) presented the strongest cytotoxicity against human hepatoma HepG2 cells in vitro with an IC50 value of 8.46 ± 0.28 µM in a 48-hour treatment. Further experiments revealed that DMFC induced apoptosis in HepG2 cells through both extrinsic and intrinsic apoptotic pathways in a p53-dependent manner. Mechanistically, DMFC activated caspases 3, 8 and 9, depolarized mitochondrial membrane potential and induced cytochrome c and apoptosis-inducing factor release. DMFC-induced apoptosis was also characterized by DNA fragmentation, phosphatidylserine externalization and sub-G1 peak in DNA histograms. Moreover, both caspase 8 and 9 inhibitors suppressed the apoptosis induced by DMFC. Western blot analyses revealed that DMFC also significantly increased the expression levels of p53, Fas death receptor, Fas-associated death domain protein and proapoptotic Bcl-2 family members such as Bax, Bad and tBid, as well as decreased the levels of pro-survival members such as Bcl-2 and Bcl-xl. Conclusion: DMFC is potentially an effective therapeutic agent in liver cancer therapy.

Human papillomavirus type 16 E6 suppresses microRNA-23b expression in human cervical cancer cells through DNA methylation of the host gene C9orf3

Oncogenic protein E6 of human papillomavirus type 16 (HPV-16) is believed to involve in the aberrant methylation in cervical cancer as it upregulates DNA methyltransferase 1 (DNMT1) through tumor suppressor p53. In addition, DNA demethylating agent induces the expression of one of the HPV-16 E6 regulated microRNAs (miRs), miR-23b, in human cervical carcinoma SiHa cells. Thus, the importance of DNA methylation and miR-23b in HPV-16 E6 associated cervical cancer development is investigated. In the present study, however, it is found that miR-23b is not embedded in any typical CpG island. Nevertheless, a functional CpG island is predicted in the promoter region of C9orf3, the host gene of miR-23b, and is validated by methylation-specific PCR and bisulfite genomic sequencing analyses. Besides, c-MET is confirmed to be a target gene of miR-23b. Silencing of HPV-16 E6 is found to increase the expression of miR-23b, decrease the expression of c-MET and thus induce the apoptosis of SiHa cells through the c-MET downstream signaling pathway. Taken together, the tumor suppressive miR-23b is epigenetically inactivated through its host gene C9orf3 and this is probably a critical pathway during HPV-16 E6 associated cervical cancer development.

Abstract 715: Adipose tissues derived exosomal microRNAs and their variants in ovarian cancer progression

Most ovarian cancers are diagnosed at an advanced stage when the tumor is widely metastatic. The 5-year survival drops to 50% for the cancer cases that spread beyond the pelvis to the omentum. However, the mechanisms underlying the effect of omental adipose tissue on ovarian cancer progression are poorly understood. Recent studies showed that exosomes also contain non-coding RNAs such as microRNAs (miRNAs). Thus, we hypothesize that the transfer of microRNAs and their variants from ovarian cancer-associated omental adipose tissues to ovarian cancer cells via exosomes may contribute to the nearby microenvironment for ovarian cancer metastasis and cancer progression. Ion Torrent next generation sequencing was performed on miRNAs isolated and enriched from exosomes and cell lysates of ovarian cancer cell lines (OVCA), the epithelial component of microdissected omental ovarian cancer tissues (CT), normal omental adipose tissues (OMN) and ovarian cancer-associated omental adipose tissues (OMT). By integrating the miRNA expression profiles, 65 miRNAs were expressed at significant higher levels in OMT-derived exosomes compared with those in OMN-derived exosomes and OVCA-derived exosomes. A set of miRNAs (miR-32a, miR-221 and miR320a), which had been implicated in controlling cell growth and chemoresistance, was identified. Also, the Ion Torrent results were validated and exosomal transfer of OMT-derived miRNAs was confirmed in vitro. The exosomal communication between adipose tissues and ovarian cancer cells in the omental tumor microenvironment is verified. The transferable miRNAs and their variants may remain functional in the recipient ovarian cancer cells and confer more aggressive phenotypes in these cells. Citation Format: Chi Lam Au Yeung, Tetsushi Tsuruga, Ngai Na Co, Tsz-Lun Yeung, Cecilia S. Leung, Kwong K. Wong, Samuel C. Mok. Adipose tissues derived exosomal microRNAs and their variants in ovarian cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 715.

Abstract 1535: Omentin: A novel adipokine linking visceral obesity to ovarian cancer progression

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Advanced stage serous ovarian cancer metastasizes preferentially to the omentum, which is a major site of intra-abdominal fat accumulation, suggesting that the omental microenvironment is a favorable niche for ovarian cancer cells. The 5-year survival drops to below 50% for the cancer cases that spread beyond the pelvis to the omentum. It has also become apparent that obesity contributes to a poor clinical outcome. The mechanisms by which omental adipose tissue promotes tumor growth and disease progression are not entirely clear. Using transcriptome profiling analysis on the microdissected adipose tissue from patients with benign gynecologic diseases and from patients with advanced high-grade serous ovarian cancer (HGSOC), we found that a novel adipokine called omentin (Intestinal Lactoferrin Receptor ITLN1) was significantly down-regulated in ovarian cancer associated adipose tissue compared with the normal adipose tissue. Survival correlation studies demonstrated that patients with serum ITLN1 levels of >350 ng/mL at the time of first treatment experienced longer survival times than those with lower levels of ITLN1. We showed that ITLN1 suppressed the ovarian cancer cell migration ability and invasion potential in vitro. To delineate the underlying molecular mechanisms, RNA sequencing and pathway analyses were performed on ITLN1 treated ovarian cancer cells and we identified MMP1 as one of the potential mediators. It has been shown that MMP1 expression was induced by lactoferrin, which is abundant in ascites. We hypothesized ITLN1 could abrogate the effect of lactoferrin on ovarian cancer motility and invasion potential. Our preliminary data suggested that secreted ITLN1 may sequester lactoferrin in the ascites, thereby preventing it from binding to the low-density-lipoprotein-receptor-related-protein-1 (LRP-1) on ovarian cancer cell surface and thus inactivate downstream signaling pathways that control MMP1 expression. The study provides the first evidence that ovarian cancer cells modify the visceral adipose tissue through down-regulation of omentin to facilitate their growth in the omental microenvironment. Citation Format: Chi Lam Au Yeung, Ngai Na Co, Michaela Onstad, Tsz-Lun Yeung, Cecilia S. Leung, Rosemarie Schmandt, Karen H. Lu, Samuel C. Mok. Omentin: A novel adipokine linking visceral obesity to ovarian cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1535. doi:10.1158/1538-7445.AM2015-1535

Abstract 4887: Omentin: A novel adipokine in the omental microenvironment associated with ovarian cancer progression

Advanced stage serous ovarian cancer metastasizes preferentially to the omentum, which is a well-vascularized fold of peritoneal tissue and is a major site of intra-abdominal fat accumulation, suggesting that the omental microenvironment is a favorable niche for ovarian cancer cells. The 5-year survival drops to below 50% for the cancer cases that spread beyond the pelvis to the omentum. It has also become apparent that obesity contributes to a poor clinical outcome. The mechanisms by which omental adipose tissue promotes tumor growth and disease progression are not entirely clear. Using transcriptome profiling analysis on the microdissected adipose tissue from patients with benign gynecologic diseases and from patients with advanced high-grade serous ovarian cancer (HGSOC), we identified a gene signature for ovarian cancer associated omental adipose tissue, suggesting that alteration of these genes in the ovarian cancer associated omental adipose tissue may generate a permissive microenvironment to support ovarian cancer growth. Among genes that are significantly up- or down-regulated in ovarian cancer associated adipose tissue compared with the normal adipose tissue, we seek to focus on evaluating the role of omentin (Intestinal Lactoferrin Receptor ITLN1) in ovarian cancer progression since it is a novel adipokine that is predominantly expressed and secreted by visceral adipose tissue and is barely detectable in subcutaneous fats. Our data showed for the first time that omentin was expressed predominantly by the mesothelial cells covering the visceral adipose tissue but not by other cell types in the omental adipose tissue. Interestingly, we showed that circulating omentin level is significantly lower in patients with HGSOC compared with those in the BMI matched healthy individuals. In addition, using monolayer culture models, we demonstrated that omentin suppressed ovarian cancer motility and invasion potential directly and ovarian cancer growth only in the presence of adipocytes. We also showed that omentin can increase insulin-dependent glucose up-take in adipocytes and omentin expression can be down-regulated by co-culturing with ovarian cancer cells and in the presence of TNF-α. The study provides the first evidence that ovarian cancer cells modify the visceral adipose tissue through down-regulation of omentin to facilitate their growth in the omental microenvironment. Citation Format: Chi Lam Au Yeung, Ngai Na Co, Michaela Onstad, Tsz-Lun Yeung, Cecilia S. Leung, Rosemarie Schmandt, Karen H. Lu, Samuel C. Mok. Omentin: A novel adipokine in the omental microenvironment associated with ovarian cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4887. doi:10.1158/1538-7445.AM2014-4887

Abstract 1939: Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Ovarian cancer is the most common cause of gynecologic malignancy related mortality in women. The combination of tumor reductive surgery and chemotherapy is the main therapeutic strategy for ovarian cancer. However, especially in the most of advanced cases, complete site reduction is impossible to achieve. In those cases, the sensitivity to chemotherapy strongly affects their prognoses. MicroRNAs (miRNAs), which are approximately 22-nucleotide non-coding RNAs, regulate protein expression by translational inhibition and degradation of their target mRNAs. The expression of miRNAs is dysregulated in various types of cancers, suggesting that miRNAs are involved in carcinogenesis and cancer progression. The purpose of present work was to identify miRNAs which are related to chemo-sensitivity in ovarian cancer. Using Next Generation Sequencing (NGS), miRNA profiling was performed in 38 advanced high-grade serous ovarian cancers (HGSOCs). RNAs including miRNAs were procured from the epithelial component of the laser microdissected tumor tissue removed from patients undergoing primary surgery without neo-adjuvant chemotherapy. A total of 1935 miRNAs and miRNA variants were detected by NGS and normalized expression data of each miRNAs and miRNA variants were used for further analyses. Comparing the copy numbers of each miRNAs between chemo-refractory group (patients with disease progression in the period of first line chemotherapy, n=7) and chemo-sensitive group (patients with disease progression in more than 6 months after completion of first line chemotherapy, n=13) by Mann-Whitney U test, 77 miRNAs and miRNA variants were found to be dysregulated with significant differences (p<0.05). The 10 most significant miRs identified by NGS were selected for further validation studies by real-time reverse transcription PCR (RT-PCR) analysis on the same sample set. To determine whether miRNAs could be used as a prognostic marker HGSOC, expression levels of those 10 miRNAs were quantified by RT-PCR using 95 HGSOC samples including the 38 samples used in the discovery set. The results showed that mir-625-3p expression levels were significantly reduced in the chemo-refractory group compared with the chemo-sensitive group as indicated by the NGS data (n=20, p=0.029), and by the RT-PCR data (n=63, p=0.004). Furthermore, Kaplan-Meier survival analysis with log-rank test indicated that low expression of mir-625-3p (less than mean expression) was significantly associated with shorter progression-free survival (n=95, p=0.007) and shorter over-all survival (n=95, p<0.001) as compared with the high expression group (more than mean expression). Our results strongly suggest that mir-625-3p might be a prognostic marker that can be used to predict chemo-sensitivity in patients with HGSOC. Further characterization of mir-625-3p as a therapeutic target for HGSOC is in progress. Citation Format: Tetsushi Tsuruga, Chi Lam Au Yeung, Cecilia S. Leung, Tsz-Lun Yeung, Kwong K. Wong, Rosemarie Schmandt, Ngai Na Co, Karen H. Lu, Samuel Mok. Identification of microRNAs related to chemosensitivity in ovarian cancer using Next Generation Sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1939. doi:10.1158/1538-7445.AM2013-1939