Kathleen Hasselblatt

Use of a Combination of Approaches to Identify and Validate Relevant Tumor-Associated Antigens and Their Corresponding Autoantibodies in Ovarian Cancer Patients

Purpose: Novel biomarkers are urgently needed to increase the sensitivity of CA125 for the early detection of ovarian cancer. Indeed, it has been shown that as much as 20% of early-stage patients do not express significant levels of this biomarker. Therefore, the possibility of using autoantibodies directed against tumor-associated antigens as putative cancer markers is being more examined. Indeed, many autoantibodies have recently been shown to correlate with cancer patient prognosis or to be suitable for detection of the disease. Experimental Design: In this study, we have used a new approach involving the use of proteomics, immunology, and ELISA methods to identify relevant autoantibodies in the plasma of ovarian cancer patients. To do so, we developed an innovative technique called two-dimensional differential gel electrophoresis analysis of immunoprecipitated tumor antigens. Results: This strategy allowed us to successfully identify novel circulating autoantibodies directed against the S100A7 protein in the plasma of ovarian cancer patients. Further real-time reverse transcription-PCR and immunohistochemical studies confirmed that the S100A7 mRNA and protein were highly expressed in ovarian tumors but absent in normal and benign tissues. Moreover, a preliminary study involving 138 patients confirmed that the plasma levels of anti-S100A7 antibodies are significantly elevated in early- and late-stage ovarian cancer patients compared with healthy controls and with patients with benign gynecologic diseases. Conclusions: This shows that our approach is a valuable tool to successfully identify autoantibodies and tumor-associated antigens in cancer patients and that future research assessing their putative clinical usefulness would be worthwhile.

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.

C-terminal binding protein-2 regulates response of epithelial ovarian cancer cells to histone deacetylase inhibitors

Ovarian cancer survival rates have stagnated in the last 20 years despite the development of novel chemotherapeutic agents. Modulators of gene expression, such as histone deacetylase (HDAC) inhibitors, are among the new agents being used in clinical trials. Predictors of sensitivity to chemotherapy have remained elusive. In this study, we show that the expression of the transcriptional corepressor C-terminal binding protein-2 (CtBP2) is elevated in human ovarian tumors. Downregulation of CtBP2 expression in ovarian cancer cell lines using short-hairpin RNA strategy suppressed the growth rate and migration of the resultant cancer cells. The knockdown cell lines also showed upregulation of HDAC activity and increased sensitivity to selected HDAC inhibitors. Conversely, forced expression of wild-type CtBP2 in the knockdown cell lines reversed HDAC activity and partially rescued cellular sensitivity to the HDAC inhibitors. We propose that CtBP2 is an ovarian cancer oncogene that regulates gene expression program by modulating HDAC activity. CtBP2 expression may be a surrogate indicator of cellular sensitivity to HDAC inhibitors.

Apoptosis in human term placenta : A morphological and gene expression study

The objective of this study was to examine placentas after delivery from normal, healthy patients at term gestation. The placentas were from elective cesarean sections (n = 10, prior to the onset of labor) and spontaneous vaginal delivery (n = 10, after labor). We found that deoxyribonucleic acid laddering was present in all placentas and consistent with the pattern found in tissues that undergo apoptosis. Paraffin-embedded sections of placental villi stained by the in situ terminal deoxynucleotidyl transferase mediated biotinylated dUTP nick end labeling method revealed positive apoptotic nuclei in the placental villi. Reverse-transcriptase polymerase chain reaction demonstrated expression of messenger RNA for testosterone-repressed prostate message 2 and B cell lymphoma/leukemia-2 in the placenta. Our data demonstrate that apoptosis occurs in human term placenta.

Loss of E-cadherin disrupts ovarian epithelial inclusion cyst formation and collective cell movement in ovarian cancer cells.

Increased inclusion cyst formation in the ovary is associated with ovarian cancer development. We employed in vitro three-dimensional (3D) organotypic models formed by normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells to study the morphologies of normal and cancerous ovarian cortical inclusion cysts and the molecular changes during their transitions into stromal microenvironment. When compared with normal cysts that expressed tenascin, the cancerous cysts expressed high levels of laminin V and demonstrated polarized structures in Matrigel; and the cancer cells migrated collectively when the cyst structures were positioned in a stromal-like collagen I matrix. The molecular markers identified in the in vitro 3D models were verified in clinical samples. Network analysis of gene expression of the 3D structures indicates concurrent downregulation of transforming growth factor beta pathway genes and high levels of E-cadherin and microRNA200 (miR200) expression in the cancerous cysts and the migrating cancer cells. Transient silencing of E-cadherin expression in ovarian cancer cells disrupted cyst structures and inhibited collective cell migration. Taken together, our studies employing 3D models have shown that E-cadherin is crucial for ovarian inclusion cyst formation and collective cancer cell migration.

Epigenetic Reprogramming Strategies to Reverse Global Loss of 5-Hydroxymethylcytosine, a Prognostic Factor for Poor Survival in High-grade Serous Ovarian Cancer

Purpose: A major challenge in platinum-based cancer therapy is the clinical management of chemoresistant tumors, which have a largely unknown pathogenesis at the level of epigenetic regulation. Experimental Design: We evaluated the potential of using global loss of 5-hydroxymethylcytosine (5-hmC) levels as a novel diagnostic and prognostic epigenetic marker to better assess platinum-based chemotherapy response and clinical outcome in high-grade serous tumors (HGSOC), the most common and deadliest subtype of ovarian cancer. Furthermore, we identified a targetable pathway to reverse these epigenetic changes, both genetically and pharmacologically. Results: This study shows that decreased 5-hmC levels are an epigenetic hallmark for malignancy and tumor progression in HGSOC. In addition, global 5-hmC loss is associated with a decreased response to platinum-based chemotherapy, shorter time to relapse, and poor overall survival in patients newly diagnosed with HGSOC. Interestingly, the rescue of 5-hmC loss restores sensitivity to platinum chemotherapy in vitro and in vivo, decreases the percentage of tumor cells with cancer stem cell markers, and increases overall survival in an aggressive animal model of platinum-resistant disease. Conclusions: Consequently, a global analysis of patient 5-hmC levels should be included in future clinical trials, which use pretreatment with epigenetic adjuvants to elevate 5-hmC levels and improve the efficacy of current chemotherapies. Identifying prognostic epigenetic markers and altering chemotherapeutic regimens to incorporate DNMTi pretreatment in tumors with low 5-hmC levels could have important clinical implications for newly diagnosed HGSOC disease. Clin Cancer Res; 24(6); 1389–401. ©2017 AACR.

Characterization of MicroRNA-200 pathway in ovarian cancer and serous intraepithelial carcinoma of fallopian tube

BackgroundOvarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors.MethodsRNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors.ResultsOvarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFβ1 and TGFβ2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection.ConclusionsThe activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

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 2054: A microRNA hypothesis for the precursor lesions of ovarian cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. Ovarian surface epithelial (OSE) cells were previously considered as the cells of origin of ovarian cancer. The ovarian surface epithelium is derived from the coelomic layer, which is consisted of peritoneal mesothelial cells. Malignant ovarian cancer cells, however, are found to be associated with epithelial phenotypes resembling cells from fallopian tube, uterus, and cervix. Recent studies have suggested that significant percentages of familial and sporadic high-grade serous ovarian carcinomas could be explained by an origin in the distal fallopian tube. We and others have identified overexpression of miR-200 family members in ovarian caner cells. In order to investigate whether the miR-200 pathway is involved in the early stages of ovarian pathogenesis, we have studied the expression levels of the pathway components in a panel of samples isolated from primary cultures and laser microdissection. The ovarian samples included OSEs, epithelial tumor cells from benign tumors, borderline tumors, nonserous invasive tumors, serous invasive tumors, ovarian cancer cell lines, and tumor-associated stromal fibroblasts. Fallopian tube samples included normal fallopian tubal epithelial (FTE) cells, tumor cells and stromal fibroblasts from frozen tubal intraepithelial carcinomas (TICs) of fallopian tube. Gene expression was determined using quantitative real-time polymerase chain reactions (qRT-PCRs). The reactions for the 4 target genes of miR-200s (ZEB1, ZEB2, TGFb1 and TGFb2), and 21 effector genes that were regulated by the target genes, were performed using a high-throughput BioMark array platform (Fluidigm). qRT-PCR of epithelial tumor cells from ovarian tumors and fallopian TICs showed concurrent up-regulation of miR-200s, down-regulation of the target genes, and up-regulation of several effector genes. To confirm the role of miR-200 family in regulating gene expression, synthetic miRNA precursors were transfected into normal OSE and FTE cells and qRT-PCRs were performed to determine the expression of target and effector genes. The results showed similar reduced expression of target genes and elevated levels of CDH1 and EpCAM effector genes in both normal OSE and FTE cells. However, the most interesting finding was the specific induction of tumor marker, CA125, in the normal FTE cells. Immunocytochemistry confirmed the induced CA125 proteins in FTE cells. In conclusion, the activation of the miR-200 pathway may be an early lesion that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian tumors usually express higher levels of CA125, the greater capacity of FTE cells to express CA125 by miR-200 induction is consistent with the notion that high-grade serous ovarian tumors have an origin in the distal fallopian tube. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2054.

Abstract 4789: Functional characterization and clinical correlation of the tumor antigen casein kinase 1-epsilon in ovarian cancer

OBJECTIVES: Prior studies have identified antibodies to casein kinase I epsilon (CKI-e) in the serum of patients with ovarian cancer. We investigated the expression pattern, functional roles, and clinical correlation of CKI-e in ovarian cancer. METHODS: The expression of CKI-e in 8 healthy ovaries and 68 ovarian tumor samples was determined by IHC and scored on intensity of antibody expression (scale 0-3) and the percentage of the area stained (scale 0-3). The average from both scores was assigned to each patient. Clinical characteristics for each patient were retrieved from electronic medical records. Statistical significance was determined using the Fisher exact test. Univariate analysis was performed by constructing probability curves according to the Kaplan-Meier method. Immortalized normal human ovarian surface epithelial (HOSE) cell lines with ectopic expression of CKI-e were established by transfection with a CKI-e expression construct. CKI-e-targeting shRNAs were used to generate ovarian cancer cell lines that have stably suppressed CKI-e expression. Protein expression was determined by Western blot. Growth rate was determined by cell counting. Three-dimensional spheroid culture was performed on Matrigel. The growth of ovarian cancer cell lines harboring either a control shRNA or a CKI-e-shRNA was evaluated in a mouse xenograft model. The effects of IC261, an inhibitor of CKI-e, on ovarian cancer cells were evaluated by MTT assay. RESULTS: CKI-e was overexpressed in 67% of invasive ovarian tumors (P-value=0.001) and in 14 of 17 ovarian cancer cell lines. Normal HOSE cells that have ectopic expression of CKI-e have accelerated growth rate and formed spheroids in Matrigel that were two to four times larger than those formed by control HOSE. In contrast, cancer cell lines harboring specific CKI-e shRNA had reduced growth rate and 6-fold reduced tumor weight in the xenograft model (P-value=0.007). The cancer cell lines also showed CKI-e-dependent sensitivity to IC261. The median CKI-e expression level among the ovarian cancer tissue samples was 3. For patients whose score was ≤ 3 and > 3, the mean overall survival was 91 months and 48 months, respectively. Similarly, the mean disease free survival interval was 43 months and 22 months for patients whose score was ≤ 3 and > 3, respectively. Patients who scored > 3 tended to have late stage disease (61%) compared to patients who scored ≤ 3 (39%), (p=0.06). CONCLUSIONS: CKI-e is highly expressed in ovarian tumors and cancer cell lines. Studies of cell lines with alterations in CKI-e expression showed that this signaling protein regulates cell growth and survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4789.