Shubai Liu

Casein kinase I epsilon interacts with mitochondrial proteins for the growth and survival of human ovarian cancer cells

Epithelial ovarian cancer is the leading cause of death among gynaecologic cancers in Western countries. Our studies have shown that casein kinase I‐epsilon (CKIε), a Wnt pathway protein, is significantly overexpressed in ovarian cancer tissues and is associated with poor survival. Ectopic expression of CKIε in normal human ovarian surface epithelial cells and inhibition of CKIε in ovarian cancer cells and in xenografts demonstrated the importance of CKIε in regulating cell proliferation and migration. Interestingly, CKIε function did not seem to involve β‐catenin activity. Instead, CKIε was found to interact with several mitochondrial proteins including adenine nucleotide translocase 2 (ANT2). Inhibition of CKIε in ovarian cancer cells resulted in suppression of ANT2, downregulation of cellular ATP and the resulting cancer cells were more susceptible to chemotherapy. Our studies indicate that, in the context of ovarian cancer, the interaction between CKIε and ANT2 mediates pathogenic signalling that is distinct from the canonical Wnt/β‐catenin pathway and is essential for cell proliferation and is clinically associated with poor survival.

Characterization of aldehyde dehydrogenase isozymes in ovarian cancer tissues and sphere cultures

BackgroundAldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures.MethodsImmunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes.ResultsImmunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium.ConclusionsThe results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to cell states. Elucidating the function of the ALDH isozymes in lineage differentiation and pathogenesis may have significant implications for ovarian cancer pathophysiology.

Tolerance induction to human stem cell transplants with extension to their differentiated progeny

There is increasing interest in transplantation of human stem cells for therapeutic purposes. It would benefit future application if one could achieve their long-term acceptance and functional differentiation in allogeneic hosts using minimal immunosuppression. Allogeneic stem cell transplants differ from conventional tissue transplants insofar as not all alloantigens are revealed during tolerance induction. This risks that the immune system tolerized to antigens expressed by progenitors may still remain responsive to antigens expressed later during differentiation. Here we show that brief induction with monoclonal antibody-mediated coreceptor and costimulation blockade enables long-term engraftment and tolerance towards murine ESCs, hESCs, human induced pluripotent stem cells (iPSCs) and hESC-derived progenitors in outbred murine recipients. Tolerance induced to PSC-derived progenitors extends to their differentiated progenies, and sometimes even to different tissues derived from the same donor. Global gene expression profiling identifies clear features in T cells from tolerized grafts that are distinct from those involved in rejection.

Inference on differences between classes using cluster-specific contrasts of mixed effects

The detection of differentially expressed (DE) genes, that is, genes whose expression levels vary between two or more classes representing different experimental conditions (say, diseases), is one of the most commonly studied problems in bioinformatics. For example, the identification of DE genes between distinct disease phenotypes is an important first step in understanding and developing treatment drugs for the disease. We present a novel approach to the problem of detecting DE genes that is based on a test statistic formed as a weighted (normalized) cluster-specific contrast in the mixed effects of the mixture model used in the first instance to cluster the gene profiles into a manageable number of clusters. The key factor in the formation of our test statistic is the use of gene-specific mixed effects in the cluster-specific contrast. It thus means that the (soft) assignment of a given gene to a cluster is not crucial. This is because in addition to class differences between the (estimated) fixed effects terms for a cluster, gene-specific class differences also contribute to the cluster-specific contributions to the final form of the test statistic. The proposed test statistic can be used where the primary aim is to rank the genes in order of evidence against the null hypothesis of no DE. We also show how a P-value can be calculated for each gene for use in multiple hypothesis testing where the intent is to control the false discovery rate (FDR) at some desired level. With the use of publicly available and simulated datasets, we show that the proposed contrast-based approach outperforms other methods commonly used for the detection of DE genes both in a ranking context with lower proportion of false discoveries and in a multiple hypothesis testing context with higher power for a specified level of the FDR.

Abstract TMEM-032: MULTIDISCIPLINARY CHARACTERIZATION OF OVARIAN CANCER SPHERES

Sphere-forming cultures have been widely used in stem cell biology. Ovarian cancer sphere culture is also a good model to study ovarian cancer ascites spheroids, which propagate without attachment to a substratum. We studied non-adherent ovarian cancer sphere cultures derived from ovarian cancer cell lines and epithelial cancer cells isolated from clinical samples. Western blot analysis and immunofluorescence showed that ovarian cancer spheres expressed elevated levels of stem cell markers CD133, c-Myc, Nanog, and Oct4. Metabolically, ovarian cancer sphere cells showed decreased ATP levels and were under constant oxidative stress, with significant depletion of the endogenous antioxidant glutathione. Accordingly, supplementation with antioxidant supplement N-acetylcysteine (NAC) in cancer sphere cultures relieved oxidative stress and improved growth of the sphere cultures in a dose-dependent manner. In contrast, adherent cancer cell cultures did not show any significant growth enhancement. Furthermore, microarray gene expression profiling and activity-based proteomic profiling using a sulfonate ester chemical probe revealed that the ovarian cancer spheres demonstrated increased level of FOS/JUN expression, which activated the expression and activity of the omega class of glutathione-S transferase GSTO1, an enzyme involved in the metabolism of xenobiotics and cisplatin resistance. Knockdown of FOS expression using siRNA reduced the levels of GSTO1 and abrogated chemoresistance of the sphere cultures. Separately, gene set enrichment analysis of the cancer sphere expression profiles revealed elevated expression of endosomal pathway genes for exosome secretion in the sphere cultures. Knockdown of one elevated gene, Rab27B, significantly reduced the exosome number in the spent medium of ovarian cancer sphere cultures. Clinically, measurement of the exosome number in patient body fluids using the NanoSight instrument and a flow cytometry method indicated that plasma and urine samples from a panel of ovarian cancer patients had increased number of exosomes compared with samples derived from benign patients. CONCLUSIONS: Our multidisciplinary studies have discovered a plethora of properties of ovarian cancer spheres that may promote ovarian cancer growth. Elevated expression and activity of GSTO1 in ovarian cancer spheres may represent a novel mechanism by which ovarian cancer spheroids respond to heightened oxidative stress and chemotherapeutic agents. Antioxidant supplements may not have the intended benefits to cancer survivors. Instead, they may promote cancer growth by relieving the oxidative stress of ovarian cancer spheroids. In contrast, inhibitors that target GSTO1 may have better clinical value in prolonging survivorship of ovarian cancer patients with malignant ascites. Ovarian cancer spheres also have increased expression of endosomal pathway genes for exosome secretion, which may promote the establishment of tumor microenvironment for cancer propagation, and is consistent with the increased exosomes in the plasma and urine samples of ovarian cancer patients. Citation Format: Shubai Liu, Junzheng Yang, Pui-Wah Choi, Jamie Sui-Lam Kwok, Kathleen Hasselblatt, Daniel Nomura, Wing Ping Fong, Stephen KW Tsui, Allison Vitonis, Daniel Cramer, William R. Welch, Benjamin Cravatt, Ross Berkowitz, and Shu-Wing Ng. MULTIDISCIPLINARY CHARACTERIZATION OF OVARIAN CANCER SPHERES [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-032.

Abstract B29: The role of C-Terminal Binding Proteins in BRCA1 silencing and genetic instability of ovarian cancer cells

Objective: Ovarian cancer is the leading cause of mortality from gynecological malignancy in Western countries. High-grade serous ovarian carcinoma (HGSOC) is characterized by genetic instability and frequently harbors defects in genes such as BRCA1 for homologous recombination (HR) pathway. Our group recently demonstrated that the transcriptional co-repressors, C-Terminal Binding Proteins (CtBP1 and CtBP2), are overexpressed in epithelial ovarian carcinoma and CtBP2 overexpression is associated with poor patient survival. We sought to relate the expression of CtBP to genetic instability and chemoresponse in HGSOC cells. Methods: Gene expression profiling was performed for the control and CtBP2-knockdown ovarian cancer cell lines. Chromatin Immunoprecipitation (ChIP)-qPCR assays and a BRCA1 promoter-luciferase reporter assay were performed to compare the binding of CtBP complex to BRCA1 promoter and the activity of BRCA1 promoter in the ovarian cancer cells. Cell cycle status of the cell lines was determined by flow cytometry. DNA double-strand breaks (DSB) were determined by comet assay. esults: Analysis of gene expression profiles revealed potential functions of CtBP2 in silencing the expression of a panel of genes such as BRCA1 and Fanconi anemia complementation group gene FANCD2 that are involved in HR pathway, cell cycle checkpoints, and genetic instability. ChIP analyses and the luciferase assays strongly suggest that the CtBP2 complex binds to the BRCA1 promoter and represses BRCA1 transcription. Ovarian cancer cell lines with knockdown of either CtBP1 or CtBP2 showed increased expression of BRCA1 protein. CtBP2-knockdown ovarian cancer cells showed increased apoptosis and arrest in cell cycle. These cell lines also showed a significant increase in DSB after gamma irradiation than wild-type control cells in a comet assay. Conclusions: CtBP1 and CtBP2 function in the suppression of a panel of target genes such as BRCA1 and FANCD2 that are involved in cell cycle checkpoints and HR pathway. CtBP-mediated BRCA1 silencing may be a significant contributing mechanism for genetic instability and clinicopathologic development of HGSOC. Targeting this epigenetic mechanism may enhance the sensitivity of HGSOC to DNA damaging agents and contribute to improved outcome of ovarian cancer patients. Citation Format: Shubai Liu, Junzheng Yang, Taymaa May, Yuanyuan Hua, Shi Lu, Kevin Elias, Ronny Drapkin, William Welch, Christopher Crum, Ross S. Berkowitz, Shu-Wing Ng. The role of C-Terminal Binding Proteins in BRCA1 silencing and genetic instability of ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B29.