Normal Ovarian Surface Epithelial Cells Research Articles

Overexpression and cytoplasmic localization of caspase-6 is associated with lamin A degradation in set of ovarian cancers

BackgroundIn most women with ovarian cancer, the diagnosis occurs after dissemination of tumor cells beyond ovaries. Several molecular perturbations occur ahead of tumor initiation including loss of lamin A/C. Our hypothesis was that the loss of nuclear structural proteins A type lamins (lamin A/C) transcribed from LMNA gene and substrate for active caspase-6 maybe one of the molecular perturbations. Our objective is to investigate the association between the loss of lamin A/C and the overexpression of caspase-6 in ovarian cancer cells.MethodWestern blotting and immunofluorescence were used to analyze the expression of lamin A/C and active caspase-6 in normal human ovarian surface epithelial (HOSE) cells, immortalized human ovarian surface epithelial cells and a set of seven ovarian cancer cell lines (including OVCAR3, OVCAR5, and A2780). The activity of caspase-6 was measured by densitometry, fluorescence and flow cytometry. Immunohistochemistry was used to evaluate the expression of caspase-6 in set of ovarian cancer tissues previously reported to have lost lamin A/C.ResultsThe results showed that HOSE cells expressed lamin A/C and no or low level of active caspase-6 while cancer cells highly expressed caspase-6 and no or low level of lamin A/C. The inhibition of caspase-6 activity in OVCAR3 cells increased lamin A but has no effect on lamin C; active caspase-6 was localized in the cytoplasm associated with the loss of lamin A.ConclusionOverexpression and cytoplasmic localization of caspase-6 in ovarian cancer cells may be involved in lamin A degradation and deficiency observed in some ovarian cancer cells.

Downregulation of miR-145-5p in cancer cellsand theirderived exosomes may contribute tothedevelopment of ovarian cancer by targeting CT.

The present study aimed to identify shared microRNAs (miRNAs) in ovarian cancer (OC) cells and their exosomes using microarray data (accession number GSE103708) available from the Gene Expression Omnibus database, including exosomal samples from 13 OC cell lines and 3 normal ovarian surface epithelial cell lines, and their original cell samples. Differentially expressed miRNAs (DE-miRNAs) were identified using the Linear Models for Microarray data method, and mRNA targets of DE-miRNAs were predicted using the miRWalk2 database. The potential functions of target genes were analyzed using Database for Annotation, Visualization and Integrated Discovery and intersected with known OC-associated pathways downloaded from the Comparative Toxicogenomics Database. The associations between crucial miRNAs and target genes, and their clinical associations, were validated using data from The Cancer Genome Atlas. As a result, 16 upregulated and 6 downregulated DE-miRNAs were shared in OC cell lines and their exosomes compared with normal controls. The target genes of 11 common DE-miRNAs were predicted. Among these DE-miRNAs, a low expression of homosapiens (hsa)-miR-145-5p was significantly correlated with a poor prognosis and higher stages. Although 91 target genes were predicted for hsa-miR-145-5p, only 4 genes [connective tissue growth factor (CTGF), myotubularin-related protein 14, protein phosphatase 3 catalytic subunit alpha and suppressor of cytokine signaling 7] were suggested as risk factors for prognosis. The subsequent Pearson’s correlation analysis validated a significant negative correlation between hsa-miR-145-5p and CTGF (r=−0.1126, P=0.02188). According to the results of the functional analysis, CTGF is involved in the Hippo signaling pathway (hsa04390). In conclusion, decreased expression of hsa-miR-145 in OC and OC-derived exosomes may be a crucial biomarker for the diagnosis and treatment of OC.

Abstract 1405: PAX2 induces tubular structure in ovarian cancer cells

Abstract In adult tissues, PAX2 protein is expressed in normal oviductal epithelial cells (OVE) but not in normal ovarian surface epithelial cells (OSE). Recent studies reported that PAX2 is expressed in serous ovarian carcinoma cases but the role of PAX2 in enhancing ovarian cancer is unrevealed yet. The aim of this study is to understand the biological consequences after Pax2 overexpression in mouse ovarian surface epithelial (MOSE) cells. We found that Pax2 overexpression in MOSE cells induced the formation of vascular channels both in vitro and in vivo, which indicate a possible contribution of PAX2 to ovarian cancer progression by increasing the vascular channels to supply nutrients to the tumor cells. Citation Format: Kholoud Alwosaibai. PAX2 induces tubular structure in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1405.

Identification of common differentially-expressed miRNAs in ovarian cancer cells and their exosomes compared with normal ovarian surface epithelial cell cells.

The aim of the present study was to identify common microRNAs (miRNAs) in ovarian cancer (OC) cells and their exosomes using microarray data (accession number GSE76449) available from the Gene Expression Omnibus database, including exosomal samples from 3 OC cell lines, 1 normal ovarian surface epithelial cell line and their original cell samples. Differentially-expressed miRNAs (DE-miRNAs) were identified using the Linear Models for Microarray data method, and mRNA targets of DE-miRNAs were predicted using the miRWalk2 database. The potential functions of the target genes of the DE-miRNAs were analyzed using the Database for Annotation, Visualization and Integrated Discovery tool. The association between crucial miRNAs and target genes, and their clinical associations, were validated using The Cancer Genome Atlas data. As a result, 12 upregulated and 12 downregulated DE-miRNAs were shared by the 3 OC cell lines compared with normal controls in the exosomal samples, while 5 upregulated and 65 downregulated DE-miRNAs were shared between the original cells. Among them, 9 downregulated DE-miRNAs were shared between exosomal and original cells. The target genes of 4 common DE-miRNAs between exosomal and original cells (miR-127-3p, miR-339-5p, miR-409-3p and miR-654-3p) were predicted. Functional enrichment analysis indicated that these target genes may be involved in the Wnt signaling pathway (miR-409-3p-CTBP1 and miR-339-5p-CHD8) and Proteoglycans in cancer (miR-127-3p-PPP1CA). The negative associations between these 3 miRNAs and target genes were confirmed by a Pearson's correlation analysis. miR-127 was negatively associated with tumor grade. In conclusion, our results describe a set of miRNAs involved in OC development, in exosomal and non-exosomal manners, by regulating their target genes. They may be potential targets for treatment of OC.

Low expression level of HMBOX1 in high-grade serous ovarian cancer accelerates cell proliferation by inhibiting cell apoptosis

Homeobox-containing 1 (HMBOX1) has been described as a transcription factor involved in the occurrence of some tumors, but its roles in ovarian cancer have never been reported. Here we aimed to investigate the roles of HMBOX1 on high-grade serous ovarian carcinoma (HGSOC). In this present study, HMBOX1 expression was decreased in HGSOC tissues and ovarian cancer cell lines (HO8910 and A2780) compared with ovarian surface epithelial tissues or normal human ovarian surface epithelial cell line (HOSEpiC). The cell proliferation of HOSEpiC was weaker than ovarian cancer cell lines. By altering the expression of HMBOX1 in A2780 and HOSEpiC, we demonstrated that HMBOX1 inhibited the cell proliferation and promoted the cell apoptosis. Furthermore, our study revealed that HMBOX1 downregulated the expression of anti-apoptotic proteins (Bcl-2, Bcl-xL), raised the expression of pro-apoptotic-regulated proteins (Bad, Bax), apoptotic executionior (Caspase3), and P53. In conclusion, HMBOX1 played important roles in occurrence of HGSOC through regulation of proliferation and apoptosis, which implied that HMBOX1 might serve as a new therapeutic target for HGSOC.

CTD-2020K17.1, a Novel Long Non-Coding RNA, Promotes Migration, Invasion, and Proliferation of Serous Ovarian Cancer Cells In Vitro.

BackgroundOvarian cancer is the most lethal malignant tumor of the female reproductive system, and the metastasis is one of the major factors that contribute to the poor outcome of patients with OC. Accumulating evidence indicates that lncRNAs are expressed and play important regulatory roles in ovarian cancer.Material/MethodsAberrant lncRNAs in primary ovarian cancer tissues (POCTs) and paired omental metastasis tissues (OMTs) of patients with HGSOC were studied via lncRNA microarray. Real-time PCR was performed to examine CTD-2020K17.1 expression in HGSOC tissues from 38 patients, a normal ovarian surface epithelium cell line, and 4 ovarian cancer cell lines. Additionally, Transwell assays, wound healing assays, CCK-8 proliferation assays, and flow cytometry were used to explore the biological function of CTD-2020K17.1 in ovarian cancer cells. Finally, Western blot analysis was used to verify the potential target gene of CTD-2020K17.1.ResultsA novel lncRNA named CTD-2020K17.1 was identified via microarray analysis. Expression of CTD-2020K17.1 was significantly increased in OMTs and in 4 ovarian cancer cell lines compared with POCTs (P<0.05) or normal ovarian surface epithelial cell line (P<0.05). Moreover, CTD-2020K17.1 overexpression promoted migration, invasion, and proliferation of ovarian cancer cells, and CTD-2020K17.1 regulated the expression of CARD11.ConclusionsCTD-2020K17.1 is significantly upregulated in OMTs and ovarian cancer cell lines. It can promote the migration, invasion, and proliferation of ovarian cancer cells, and CARD11 is regulated by CTD-2020K17.1.

Blocking Stemness and Metastatic Properties of Ovarian Cancer Cells by Targeting p70S6K with Dendrimer Nanovector-Based siRNA Delivery.

Metastasis is the cause of most (>90%) cancer deaths and currently lacks effective treatments. Approaches to understanding the biological process, unraveling the most effective molecular target(s), and implementing nanotechnology to increase the therapeutic index are expected to facilitate cancer therapy against metastasis. Here, we demonstrate the potential advantages of bringing these three approaches together through the rational design of a small interfering RNA (siRNA) that targets p70S6K in cancer stem cells (CSCs) in combinationwith dendrimer nanotechnology-based siRNA delivery. Ourresults demonstrated that the generation 6 (G6) poly(amidoamine) dendrimer can be used as a nanovector to effectively deliver p70S6K siRNA by forming uniform dendriplex nanoparticles that protect the siRNA from degradation. These nanoparticles were able to significantly knock down p70S6K in ovarian CSCs, leading to a marked reduction in CSC proliferation and expansion without obvious toxicity toward normal ovarian surface epithelial cells. Furthermore, treatment with the p70S6K siRNA/G6 dendriplexes substantially decreased mesothelial interaction, migration and invasion of CSCs invitro, as well as tumor growth and metastasis invivo. Collectively, these results suggest that p70S6K constitutes a promising therapeutic target, and the use of siRNA in combination with nanotechnology-based delivery may constitute a new approach for molecularly targeted cancer therapy to treat metastasis.

Ergosterol peroxide inhibits ovarian cancer cell growth through multiple pathways.

Ergosterol peroxide (EP), a sterol derived from medicinal mushrooms, has been reported to exert antitumor activity in several tumor types. However, the role of EP toward ovarian cancer cells has not been investigated. In this study, we analyzed the cytotoxicity of EP in various cell lines representing high-grade serous ovarian cancer and low-grade serous ovarian cancer, respectively. Although EP showed no significant inhibition of the viability of normal ovarian surface epithelial cells, it impaired the proliferation and invasion capacities of tumor cells in a dose-dependent manner. We further figured out key modulators involved in its antitumor effects by quantitative reverse transcription polymerase chain reaction, ELISA, and Western blot. The nuclear β-catenin was down-regulated upon EP treatment, subsequently reducing the Cyclin D1 and c-Myc expression levels. Meanwhile, the protein level of protein tyrosine phosphatase SHP2 was up-regulated in EP treated cells, whereas Src kinase activity was inhibited. Both activation of SHP2 phosphatase and inhibition of Src kinase decreased the phosphorylation level of transducer and activator of STAT3 protein, which was implicated in oncogenesis. On the other hand, EP remarkably inhibited the expression and secretion of VEGF-C, implying its involvement in counteracting tumor angiogenesis. Moreover, EP treatment showed comparable cytotoxic effect with β-catenin knock-down or STAT3 inhibition. Taken together, our results demonstrated that EP showed antitumor effects toward ovarian cancer cells through both β-catenin and STAT3 signaling pathways, making it a promising candidate for drug development.

Abstract 2417: Large scale integrated transcriptomic and epigenetic profiling defines the molecular hallmarks of HGSOC and disease origins

Abstract High-grade serous ovarian cancers (HGSOCs) are the most common subtype of epithelial ovarian cancer. Pathways driving HGSOC development are poorly understood, and the cellular origins are debated in the literature. Previously, ovarian surface epithelial cells (OSECs) were thought to be cellular precursors of HGSOC, but there is now strong evidence that at least half of all HGSOCs arise from fallopian tube secretory epithelial cells (FTSECs). We took a large-scale integrated transcriptomic-epigenomic profiling approach to defining the molecular hallmarks of HGSOC and to explore the disease cell-of-origin. RNA sequencing was performed on 121 OSEC cultures and 84 FTSEC cultures and integrated with 394 HGSOC transcriptomes profiled by TCGA. We identified around 100 genes that differentiate OSECs and FTSECs, and pathway analysis of this gene list identified ovarian cancer genes as highly enriched (p=2.2x10-8), with ZEB1 and estrogen the most significant predicted upstream regulators (P&amp;lt;3.7x10-7). We integrated gene expression data with clinical metadata to identify associations between gene expression signatures and clinical variables and found that in patients diagnosed with ovarian cancer, OSEC and FTSEC profiles converged, suggestive of a field cancerization phenotype occurring in the normal OSECs on the unaffected ovary contralateral to a clinically diagnosed ovarian carcinoma. Transcriptomic signatures for OSECs, FTSECs and HGSOCs were then merged with superenhancers defined by performing chromatin immunoprecipitation data for acetylated H3K27 profiled in primary HGSOC tissue specimens. We detected many more superenhancer-target gene relationships in the normal cells than in the tumor cells, suggesting superenhancer fingerprints define the cell of origin rather than determining upregulation of oncogenes driving cancer. To explore this further we determined the similarities of HGSOCs to OSEC and FTSEC using a machine learning model. Using a pre-defined gene signature of OSECs and FTSECs, our model (high specificity and sensitivity; 95% CI = 0.77-1, p-value=6.1-5) was applied to HGSOCs and we observed that the majority of HGSOCs were classified as FTSEC-like. Interestingly, the mesenchymal subtypes were enriched as FTSEC-like. Moreover, a subset of HGSOCs were characterized as OSEC-like, and these tumors were associated with poorer outcomes. Our data describe molecular subgroups within HGSOC precursor tissues as well as defining the transcriptional networks that define HGSOC precursor tissues, drive HGSOC development; furthermore we describe the interplay between the epigenetic landscape and the transcriptome in these cell types. In closing, these large-scale integrative analyses support a predominantly FTSEC origin for HGSOCs, but reinforce conclusions that OSECs cannot be excluded as origins of HGSOC, and may represent a subset of tumors with a different biology. Citation Format: Kate Lawrenson, Marcos Abraao, Felipe Segato, Janet M. Lee, Simon Coetzee, Ji-Heui Seo, Matthew L. Freedman, Dennis Hazelett, Simon Gayther, Houtan Noushmehr. Large scale integrated transcriptomic and epigenetic profiling defines the molecular hallmarks of HGSOC and disease origins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2417. doi:10.1158/1538-7445.AM2017-2417

Abstract 1501: Sideroflexin4: A novel regulator of iron metabolism in ovarian cancer

Abstract The five year survival rate for women with ovarian cancer is 9-34%. This high mortality is due to late stage diagnosis, marginally effective treatment and frequent disease recurrence. Alterations in metabolism have been widely recognized as an important hallmark of cancer. Metabolism of the micronutrient iron is significantly altered in many cancers, including ovarian cancers. The expression of genes governing iron transport (TFR1 and FPN), iron storage (FTL and FTH) and iron regulation (IRP1 and IRP2) are significantly altered in ovarian cancer compared to normal ovarian epithelial cells, resulting in higher intratumoral iron retention. However, specific pathways of intracellular iron utilization that are altered in cancer cells, and the functional consequences of disrupting these pathways, remain poorly understood. To identify pathways of iron utilization that are perturbed in ovarian cancer, we analyzed the expression of genes involved in iron metabolism using a publicly available microarray database of ovarian cancer and normal oviduct (GSE69428). We observed that sideroflexin4 (SFXN4), an inner mitochondrial membrane protein that is essential for mitochondrial respiratory homeostasis, was significantly upregulated (p= 0.002) in ovarian cancer patient samples. We screened several ovarian cancer cell lines and observed that SFXN4 was similarly up-regulated both at the mRNA and protein level when compared to normal human ovarian surface epithelial cells. To test the role of SFXN4 in ovarian cancer cells we performed knockdown and overexpression studies. Knockdown of SFXN4 induced S and G2-phase cell cycle arrest and reduced the ability of MDAH2774 and SKOV3 ovarian cancer cells to form colonies. Further investigation revealed that modulation of SFXN4 alters mitochondrial respiration by affecting iron-sulfur cluster biogenesis. Thus, knock-out cells exhibited impaired respiratory activity and a phenotype similar to cells with defects in iron-sulfur cluster biogenesis, including increased IRP-IRE binding and a decrease in IRP1/ACO1 aconitase activity. Furthermore, SFXN4 knock-out decreased the activity of iron-sulfur cluster-containing enzymes such as mitochondrial aconitase and succinate dehydrogenase. Based on these observations, we postulate that SFXN4 acts as a molecular mediator that channels the excess iron present in ovarian cancer cells to iron-sulfur cluster-dependent metabolic pathways that favor growth and metastasis. SFXN4 may be a potential druggable target in ovarian cancer. Citation Format: Bibbin Paul, Miranda Lynch, Frank Torti, Suzy Torti. Sideroflexin4: A novel regulator of iron metabolism in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1501. doi:10.1158/1538-7445.AM2017-1501

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 MIP-052: THE ROLES OF CD24 IN OVARIAN CANCER DEVELOPMENT

Abstract OBJECTIVES: CD24 is recently reported as ovarian cancer stem cell markers, and the expression of CD24 is increased from borderline tumors to invasive ovarian carcinomas. However, the underlying pathways controlled by CD24 in ovarian pathogenesis were poorly understood. We investigated the potential roles of CD24 in normal fallopian tube epithelial (FTE) cells and ovarian cancer development. METHODS: Lentiviral particles harboring CD24 shRNAs targeting CD24 expression were used to establish FTE cell lines and ovarian cancer cell lines with CD24 knockdown. The resulting cell lines were compared with the correspondent cells with non target shRNA control in colony formation assay, BrdU incorporation, cell growth and drug resistance. Western blot was used for analyzing downstream pathways affected by the knockdown of CD24. Expression of CD24 and its downstream effectors identified from western blot, including Pax 2 and Gli1 were studied in normal FTE, non–serous and serous tumor tissues by immunohistochemistry (IHC) to confirm whether the in vitro study is clinically relevant. RESULTS: Western blot analysis showed that CD24 expression was absent in normal ovarian surface epithelial (OSE) cells but was relatively high in normal FTE cells. In addition, serous cancer cell lines have a lower CD24 expression when compared to non–serous cancer cell lines. Knockdown of CD24 in FTE cells caused slower growth, lower ability in colony formation, reduced percentage of cells in S phase and decrease in expression of p–stat3 in JAK pathway and the JAK downstream targets Nanog and OCT3/4; and Pax2, which is normally expressed in FTE but lost in FTE secretory cell outgrowth (SCOUT). The loss of Pax2 in SCOUT is associated with serous carcinoma. IHC showed cytoplasmic co–localized expression of CD24 and Pax2 in the normal FTE tissue, suggesting the possibility of interaction between these two proteins in vivo. In contrast, knockdown of CD24 in ovarian cancer cells caused faster growth, higher ability in colony formation, increased percentage of cells in S phase, more resistant to carboplatin and higher expression of Shh and Gli1 in Sonic hedgehog (SHH) pathway. The above effects were more obvious in the non–serous cancer cell line. Activation of SHH pathway requires translocation of Gli1 into the nucleus. Concurrent with the in vitro results, IHC showed that the expression of Gli1 was found inside the nucleus in the non–serous tumor tissues but in the cytoplasm in serous–tissue, suggesting that the effect brought by SHH pathway is more prominent in the non–serous cancer cells. CONCLUSION: Our study is the first study revealing the linkage between CD24 and PAX2, and suggesting normal FTE cells depends on CD24/JAK pathway for growth and the loss of CD24 expression and hence, the loss of PAX2 may be associated with serous type tumor development, basing on other studies that SCOUTs were significantly associated with serous carcinoma. Moreover, the study also suggests CD24 pathway affects Type I and Type II (HGSOC) tumors differently. The CD24/SHH pathways may be responsible in controlling cell growth and drug resistance in ovarian cancer cells, hence, warrant further studies. Citation Format: Pui–Wah Choi, Brooke E. Howitt, Christopher P. Crum, Ross S. Berkowitz, and Shu–Wing Ng. THE ROLES OF CD24 IN OVARIAN CANCER DEVELOPMENT [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 MIP-052.

Abstract DPOC-003: DIFFERENTIAL PROTEIN EXPRESSION ANALYSIS OF SV40–IMMORTALIZED OVARIAN SURFACE, SV40–IMMORTALIZED FALLOPIAN TUBE AND OVARIAN CANCER SUBTYPE CELL LINE SECRETOMES BY ITRAQ®

Abstract BACKGROUND: In 2016, an estimated 22,280 new cases of ovarian cancer will be diagnosed with more than 14,000 women succumbing to the malignancy. These figures have not changed in the last decade. During this time new ovarian cancer models have been refined that involve either the fallopian tube or ovarian surface epithelial cells as the origin for tumorigenesis. With refined proteomics methodology, identification of new secreted biomarkers may be revealed that are specific to a primary fallopian tube or ovarian surface epithelium origin and may ultimately aid in the diagnosis, prognosis or therapy of ovarian cancer patients. EXPERIMENTAL PROCEDURES: The secretomes of eight cell lines, represented by SV40 immortalized fallopian tube (FT-194, FT-190), SV40 immortalized ovarian surface epithelium (1816-575), low-grade serous adenocarcinoma (HEY), high-grade serous adenocarcinoma (TOV-1946), clear cell adenocarcinoma (TOV21G), cisplatin-sensitive high-grade serous adenocarcinoma (A2780-S) and cisplatin-resistant high-grade serous adenocarcinoma (A2780-CP), were submitted for isobaric tagging for relative and absolute quantification (iTRAQ®) differential expression analysis. Briefly, each of the eight secretomes were trypsin-digested and labeled with one of eight iTRAQ® 8-plex reagents and subsequently submitted for either two-dimensional capillary liquid chromatography direct data dependent peptide tandem mass spectrometry on an Orbitrap Velos system or one-dimensional mass spectrometry analysis. RESULTS: 456 proteins were identified with the one-dimensional analysis with 2 distinct peptides. 2069 distinct differentially expressed proteins were identified in the two dimensional analysis. Initial relative differential expression rate comparisons of control cell lines, FT-194, FT-190 and 1816-575 versus ovarian cancer subtype and therapy-associated cell lines, HEY, TOV1946, TOV21G, A2780S and A2780CP, are promising. Four proteins were increased greater than three-fold across all ovarian cancer cell lines compared to normal fallopian tube and ovarian surface epithelial control cell lines: stromelysin-1, pigment epithelium-derived factor, matrix metalloproteinase-9 and receptor expression-enhancing protein 6. Large scale inferential statistical methodology will be used to differentiate cell lines. Citation Format: Yaling Zhou PhD, Daniel Brooks, PhD, Sumana Dey PhD, LeeAnn Higgins, PhD, Todd Markowski, BSc, Chad Hamilton MD, Robin Howard, MA, Sorana Raiciulescu MSc, Amy Skubitz PhD, John D. Andersen DO. DIFFERENTIAL PROTEIN EXPRESSION ANALYSIS OF SV40–IMMORTALIZED OVARIAN SURFACE, SV40–IMMORTALIZED FALLOPIAN TUBE AND OVARIAN CANCER SUBTYPE CELL LINE SECRETOMES BY ITRAQ® [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 DPOC-003.

Increased expression of protein kinase CK2α correlates with poor patient prognosis in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is one of the deadly gynecological malignancies. The function of protein kinase CK2α (CK2α) in EOC is still unknown. Our study aimed to investigate the relationship between the protein expression of CK2α and the tumor progression, the prognosis of human EOC. In this study, we analyzed the expression levels of CK2α through Western blot, using EOC cell lines like A2780, HO8910, COV644, OVCAR3, SKOV3, and the primary normal ovarian surface epithelial (NOSE) cells. Furthermore, OVCAR3 and SKOV3 EOC cells were employed as a cellular model to study the role of CK2α on cell growth, migration, invasion, apoptosis, and cell cycle distribution. In addition, we investigated CK2α protein expression in tumor tissues from patients with EOC by immunohistochemistry and analyzed the association between CK2α expression and clinicopathologic parameters and prognosis of EOC patients. And we found that compared with NOSE cells, CK2α protein expression was increased in A2780, HO8910, OVCAR3, and SKOV3 ovarian cancer cell lines. Decreased CK2α expression suppressed OVCAR3 and SKOV3 cell growth and induced more apoptosis. CK2α knockdown using specific siRNAs inhibited migration and invasion ability of OVCAR3 and SKOV3 cells. In addition, high CK2α protein expression was found in 68.4% (80/117) of EOC patients. Increased CK2α expression of was significantly correlated with FIGO staging and peritoneal cytology. Patients with higher CK2α expression had a significantly poorer overall survival compared with those with lower CK2α expression. Multi-variate Cox regression analysis proved that increased CK2α expression was an independent prognostic marker for EOC. Taken together, our data displayed that CK2α may play a role in tumor aggressive behavior of EOC and could be used as a marker for predicting prognosis of EOC patient. High CK2α expression might predict poor patient survival.

Cationic dendritic starch as a vehicle for photodynamic therapy and siRNA co-delivery

Cationic enzymatically synthesized glycogen (cESG) is a naturally-derived, nano-scale carbohydrate dendrite that has shown promise as a cellular delivery vehicle owing to its flexibility in chemical modifications, biocompatibility and relative low cost. In the present work, cESG was modified and evaluated as a vehicle for tetraphenylporphinesulfonate (TPPS) in order to improve cellular delivery of this photosensitizer and investigate the feasibility of co-delivery with short interfering ribonucleic acid (siRNA). TPPS was electrostatically condensed with cESG, resulting in a sub-50nm particle with a positive zeta potential of approximately 5mV. When tested in normal ovarian surface epithelial and ovarian clear cell carcinoma cell culture models, encapsulation of TPPS in cESG significantly improved cell death in response to light treatment compared to free drug alone. Dosages as low as 0.16μM TPPS resulted in cellular death upon illumination with a 4.8J/cm2 light dosage, decreasing viability by 96%. cESG-TPPS was then further evaluated as a co-delivery system with siRNA for potential combination therapy, by charge-based condensation of an siRNA directed at reducing expression of manganese superoxide dismutase (Sod2) as a proof of principle target. Simultaneous delivery of TPPS and siRNA was achieved, reducing Sod2 protein expression to 48%, while maintaining the photodynamic properties of TPPS under light exposure and maintaining low dark toxicity. This study demonstrates the versatility of cESG as a platform for dual delivery of small molecules and oligonucleotides, and the potential for further development of this system in combination therapy applications.

Correction: Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells.

// Yanli Zhang 1 , Jamie Sui-Lam Kwok 2 , Pui-Wah Choi 1 , Minghua Liu 2 , Junzheng Yang 1 , Margit Singh 1 , Shu-Kay Ng 4 , William R. Welch 5 , Michael G. Muto 1 , Stephen KW Tsui 2 , Stephen P. Sugrue 3 , Ross S. Berkowitz 1 , Shu-Wing Ng 1 1 Laboratory of Gynecologic Oncology, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School, Boston, MA, USA 2 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong 3 Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, USA 4 School of Medicine and Menzies Health Institute Queensland, Griffith University, Meadowbrook, Australia 5 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Correspondence to: Shu-Wing Ng, e-mail: sng@partners.org Keywords: ovarian cancer, tumorigenesis, RNA metabolism, cell adhesion, RNA sequencing Received: October 21, 2015 Accepted: January 24, 2016 Published: February 08, 2016 ABSTRACT Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Expression of Inorganic Pyrophosphatase (PPA1) Correlates with Poor Prognosis of Epithelial Ovarian Cancer

Ovarian serous carcinoma (OSC) is the most common epithelial ovarian cancer. Inorganic pyrophosphatase (PPA1) catalyzes the hydrolysis of pyrophosphate to inorganic phosphate, thereby providing extra energy for metabolism. The significance of PPA1 in the prognosis of OSC has not been investigated. Our study aimed to explore the expression and predictive role of PPA1 in OSC progression. We screened the expression of PPA1 protein in OSC tissues from 139 patients by immunohistochemistry, and evaluated its correlation with clinicopathological characteristics. PPA1 was categorized as high expression in 58 OSC cases (41.7%), which was correlated with poor differentiation, positive lymph node (LN) metastasis and advanced FIGO (The International Federation of Gynecology and Obstetrics) stages. Univariate and multivariate analyses identified PPA1 as a novel independent prognostic biomarker in OSC patients; meanwhile, conventional factors such as LN status and FIGO stages also showed statistical significance. Moreover, the expression levels of PPA1 protein were higher in A2780 and OVCAR3 human ovarian cancer cell lines than those in normal ovarian surface epithelial cells. Using these ovarian cancer cell lines, we showed that PPA1 overexpression caused the decrease in the expression level of p53, the tumor suppressor, with the increase in β-catenin level, as determined by Western blot analysis. Conversely, knockdown of PPAI expression was associated with the increase of p53 level and the decreased of β-catenin level. Consistently, the proliferation and invasion capacities of ovarian cancer cells were enhanced upon PPA1 overexpression. In conclusion, PPA1 serves as a potential prognostic biomarker for patients with OSC.

Long Noncoding RNA MALAT1 Functions as a Sponge of MiR-200c in Ovarian Cancer.

Previous researches have revealed that alteration of non-coding RNAs expression level in malignancies can significantlymodify the course of diseases. Current study was aimed to investigate the biological functions of lncRNA MALAT1 and miR-200c, as well as the interaction between them. Quantitative real-time polymerase chain reaction (qRT-PCR) showed that lncRNA MALAT1 was overexpressed in ovarian cancer tissues and cell lines in compare to adjacent normal tissue and normal human ovarian surface epithelial cells (HOSEPiCs). Contrarily, miR-200c expression was significantly decreased in ovarian cancer, which is negatively correlated with MALAT1 expression. In addition, overexpression of lncRNA MALAT1 appeared to be related to worse prognosis and higher metastasis. In consistent with clinical outcomes, down-regulation of lncRNA MALAT1 suppressed the cell viability, migration and invasion abilities of ovarian cancer cell lines. Moreover, bioinformatics analysis suggested that3'-UTR of lncRNA MALAT1 and miR-200c have a complementarity region. Rescue experiments confirmed that miR-200c could reverse the tumor-suppressive effect of knock-down of lncRNA MALAT1 on ovarian cancer cells. Nevertheless, luciferase assays verified the existence of direct binding between miR-200c and lncRNA MALAT1. In general, results of this study indicated that lncRNA MALAT1 is a oncogene in ovarian cancer, involved in the regulation of cell viability, migration and invasion abilities of ovarian cancer cells, which achieved its biological function by regulating miR-200c expression. Therefore, lncRNA MALAT1 could be a promising prognostic biomarker and therapeutic target for ovarian cancer and its relation with miR-200c might be a starting point for future researches.

Characterization of the Expression of the RNA Binding Protein eIF4G1 and Its Clinicopathological Correlation with Serous Ovarian Cancer.

BackgroundOvarian cancer is the most lethal type of malignant tumor in gynecological cancers and is associated with a high percentage of late diagnosis and chemotherapy resistance. Thus, it is urgent to identify a tumor marker or a molecular target that allows early detection and effective treatment. RNA-binding proteins (RBPs) are crucial in various cellular processes at the post-transcriptional level. The eukaryotic translation initiation factor 4 gamma, 1(eIF4G1), an RNA-binding protein, facilitates the recruitment of mRNA to the ribosome, which is a rate-limiting step during the initiation phase of protein synthesis. However, little is known regarding the characteristics of eIF4G1 expression and its clinical significance in ovarian cancer. Therefore, we propose to investigate the expression and clinicopathological significance of eIF4G1 in ovarian cancer patients.MethodsWe performed Real-time PCR in 40 fresh serous ovarian cancer tissues and 27 normal ovarian surface epithelial cell specimens to assess eIF4G1mRNA expression. Immunohistochemistry (IHC) was used to examine the expression of eIF4G1 at the protein level in 134 patients with serous ovarian cancer and 18 normal ovarian tissues. Statistical analysis was conducted to determine the correlation of the eIF4G1 protein levels with the clinicopathological characteristics and prognosis in ovarian cancer.ResultsThe expression of eIF4G1 was upregulated in serous ovarian cancer tissues at both the mRNA (P = 0.0375) and the protein (P = 0.0007) levels. The eIF4G1 expression was significantly correlated with the clinical tumor stage (P = 0.0004) and omentum metastasis (P = 0.024). Moreover, patients with low eIF4G1 protein expression had a longer overall survival time (P = 0.026).ConclusionsThese data revealed that eIF4G1 is markedly expressed in serous ovarian cancer and that upregulation of the eIF4G1 protein expression is significantly associated with an advanced tumor stage. Besides, the patients with lower expression of eIF4G1 tend to have a longer overall survival time. Thus, eIF4G1 may contribute to the occurrence and metastasis of ovarian cancer and can serve as a potential therapeutic target for the treatment of ovarian cancer.

Abstract 4998: Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer

Abstract Objective:Ovarian cancer is the most lethal gynecological malignancy. Five-year survival of advanced ovarian cancer patients remains less than 50% and the mortality rate has not changed in recent years. This study is aimed to screen for novel ovarian cancer therapeutic targets using quantitative proteomic approach and to develop antibody-based medicine targeting novel ovarian cancer antigens. Method:Exhaustive quantitative proteome analysis focused on cell surface membrane proteins were performed by isobaric tags for relative and absolute quantitation(iTRAQ) using a normal ovarian surface epithelial cell line and 7 ovarian cancer cell lines. By this approach, ovarian cancer-specific membrane proteins were identified. We confirmed the expression of lipolysis-stimulated lipoprotein receptor (LSR) using immunohistochemical staining, western blotting method, and flow cytometry. By modified MTT assay, cell growth inhibition was performed in LSR-knock down cells compared with control cells. Cell adhesion assay was also performed in vitro. Anti-LSR monoclonal antibody (anti-LSR mAb) was generated to evaluate the efficacy of the LSR targeted antibody therapy in vivo. For subcutaneous xenograft experiments, ovarian cancer cell line (RMG-1) was injected subcutaneously into the CB17/SCID mice. Mice were then randomly divided into two groups and administrated intraperitoneally anti-LSR mAb or control IgG antibody twice a week for 6 times. In addition, luciferase transfected ovarian cancer cell line (A2780-luc) was implanted intraperitoneally into the CB17/SCID mice. Mice were administrated anti-LSR mAb or control IgG antibody intraperitoneally twice a week for 5 times. Result:By iTRAQ analysis, we identified 1685 proteins and one of the ovarian cancer candidates protein, LSR was identified. We confirmed the expression of LSR in 8 out of 11 ovarian cancer cell lines and all of ovarian cancer clinical specimens. We performed proliferation assay using siRNA against ovarian cancer cell lines. When expression of LSR was suppressed, a tumor growth was significantly inhibited at day 4(p&amp;lt;0.01). In adhesion assay, we observed significant inhibition of cell adhesion in LSR knockdown cells. These results suggested that LSR is related to cell proliferation and adhesion. A significant anti-tumour effect with 78% was observed in the anti-LSR mAb antibody administrated group compared with control IgG antibody administrated group after 25 days (p &amp;lt;0.001). Furthermore, in intraperitoneal xenograft model, anti-LSR antibody administration group significantly decreased signal intensities compared with the control antibody administration group at day 22 after implantation. Conclusion:We showed that the LSR is related to cell proliferation and adhesion in vitro in ovarian cancer cells and proved the antitumor effect of anti-LSR mAb. These results suggested that LSR can be a new therapeutic target of ovarian cancer. Citation Format: Satoko Matsuzaki, Kosuke Hiramatsu, Satoshi Serada, Satoshi nakagawa, Shinya Matsuzaki, Yutaka Ueda, Minoru Fujimoto, Kiyoshi Yoshino, Tadashi Kimura, Tetsuji Naka. Lipolysis-stimulated lipoprotein receptor (LSR) can be a novel therapeutic target of ovarian cancer. [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 4998.