Abstract 653: Ovarian cancer persister cells: 2D and 3D in-depth characterization and analysis

Simple SummaryCancer drugs have the lowest success rate of approval in drug development programs. In order to address this, predictive preclinical assays that correctly reflect the clinical efficacy of a drug represent an urgent need in both clinical oncology and pharmaceutical research. To address this need, multiple tumor models have been developed, including tumor explant culture platforms, which are the only models that preserve the integrity of the tumor tissue. However, these models have not been fully characterized and multiple variables exist between studies. We investigated the effect of tissue size and culture vessel type on the survival of tumor explants by comparing micro-dissected tumor tissue with corresponding tumor slices. Our results show that the model geometry and culture vessel affect proliferation, apoptosis, and hypoxia in tissue cultures, and must be considered in designing tumor explant culture platforms.Predicting patient responses to anticancer drugs is a major challenge both at the drug development stage and during cancer treatment. Tumor explant culture platforms (TECPs) preserve the native tissue architecture and are well-suited for drug response assays. However, tissue longevity in these models is relatively low. Several methodologies have been developed to address this issue, although no study has compared their efficacy in a controlled fashion. We investigated the effect of two variables in TECPs, specifically, the tissue size and culture vessel on tissue survival using micro-dissected tumor tissue (MDT) and tissue slices which were cultured in microfluidic chips and plastic well plates. Tumor models were produced from ovarian and prostate cancer cell line xenografts and were matched in terms of the specimen, total volume of tissue, and respective volume of medium in each culture system. We examined morphology, viability, and hypoxia in the various tumor models. Our observations suggest that the viability and proliferative capacity of MDTs were not affected during the time course of the experiments. In contrast, tissue slices had reduced proliferation and showed increased cell death and hypoxia under both culture conditions. Tissue slices cultured in microfluidic devices had a lower degree of hypoxia compared to those in 96-well plates. Globally, our results show that tissue slices have lower survival rates compared to MDTs due to inherent diffusion limitations, and that microfluidic devices may decrease hypoxia in tumor models.

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

OTX1 silencing suppresses ovarian cancer progression through inhibiting the JAK/STAT signaling

Objective: Recent evidence suggests that the human omentum functions as a niche that promotes ovarian cancer metastasis. We investigated the role of the omentum in sequestration and enhancement of ovarian cancer stem cells. Materials and Methods: To further explore this unique phenomenon, we examined the ability of cells isolated from ovarian cancer implants at distinct anatomic sites to create multicellular aggregates known as spheroids. In ovarian and other cancers, spheroids promote a pluripotent phenotype and play a critical role in metastasis. Results: We found that ovarian cancer cells recovered from omentum express higher levels of the pluripotency-associated antigen CD133 and create larger numbers of more rapidly growing spheroids than cells recovered from the tubo-ovarian complex. Although CD133+ or CD44+ ovarian cancer cells are equally capable of creating large spheroids, spheroids self-assembled from CD133+ ovarian cancer cells are significantly more tumorigenic in vivo than CD44+, CD133−CD44− or unsorted cells. We also found that spheroids self-assembled from CD133+ ovarian cancer cells migrate to both the ovary and omentum of SCID mice and that CD133+ ovarian cancer cells induce intraperitoneal xenografts when inoculated subcutaneously. In contrast, our data indicate that spheroids self-assembled from CD44+ cells preferentially induce tumor adjacent to their inoculation site. Targeting CD133 expression inhibits the in vivo tumorigencity of spheroids assembled from A2780 ovarian cancer cells. Conclusions: In addition to delineating novel pathways by which the omental microenvironment promotes ovarian cancer metastasis, these findings establish a new paradigm for understanding how ovarian cancer stem cells can be potentially maintained or regenerated within the peritoneal cavity. Strategies to target the omental niche have enormous potential to improve ovarian cancer outcomes. Citation Format: Molly A. Brewer, Guigin Sun, Matthew Anderson, Fanliang Meng, Mei Zhong. The omental microenvironment promotes ovarian cancer metastasis and enhances CD-133 mediated self-assembly of ovarian cancer spheroids. [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 3953. doi:10.1158/1538-7445.AM2013-3953

The expression levels of estrogen receptor (ER α) are closely associated with estrogen-dependent growth, invasion and response to endocrine therapy in ERα-positive ovarian cancer. However, the underlying regulatory mechanisms remain to be fully understood. Previous studies have demonstrated that ERα is a direct target of microRNA (miR)-206. miR-206 has been found to be an important tumor suppressor in several cancer types, including ovarian, gastric and laryngeal cancer. However, the specific role of miR-206 in ovarian cancer remains unclear. The aim of the present study was to investigate the role of miR-206 in ER-a positive ovarian cancer in vitro. The present study demonstrated that miR-206 is significantly downregulated in ERα-positive but not ERα‑negative ovarian cancer tissues, compared with normal ovarian epithelium tissue. It was also found that the expression of miR-206 was decreased in ERα-positive ovarian cancer cell lines, CAOV-3 and BG-1, compared with normal ovarian epithelium tissues. This suggests that miR-206 may play a role in ERα-positive ovarian cancer cells via an estrogen-dependent mechanism. Further analysis revealed that 17β-E2 treatment significantly promoted cellular proliferation and invasion of estrogen-dependent CAOV-3 and BG-1 cells, which could be reversed by the introduction of miR-206 mimics. In conclusion, the present study suggests that miR-206 has an inhibitory role in estrogen-dependent ovarian cancer cells. Thus, miR-206 may be a promising candidate for the endocrine therapy of ERα-positive ovarian cancer.

Stanniocalcin 1 (STC1) is a secreted glycoprotein hormone. High expression of STC1 has been associated with several cancers including ovarian cancer, but its role in the development of ovarian cancer is not clear. We used five human ovarian epithelial cancer cell lines (OVCA420, OVCA432, OVCA433, SKOV3, and HEY), immortalized human ovarian surface epithelial cells (T29 and T80), ovarian cancer tissues from 342 patients, serum from 73 ovarian cancer patients and from58 control subjects, and 116 mice, with six or eight per group. Protein expression was assessed. Cells overexpressing STC1 protein were generated by ectopic expression of human STC1 cDNA. STC1 expression was silenced by using small interfering RNA against STC1. Cell proliferation, migration, colony formation, and apoptosis were assessed. Xenograft tumor growth in mice was studied. Neutralizing anti-STC1 antibody was used to inhibit STC1 function. All statistical tests were two-sided. STC1 protein expression was higher in all human ovarian cancer cell lines examined than in immortalized human ovarian epithelial cell lines, higher in ovarian cancer tissue than in normal ovarian tissue (P < .001), and higher in serum from ovarian cancer patients than from control subjects (P = .021). Ovarian cancer cells with STC1 overexpression, compared with corresponding control cells, had increased cell proliferation, migration, and colony formation in cell culture and increased growth of xenograft tumors in mice. These activities in normal or malignant ovarian cells with STC1 overexpression, compared with control cells, were also accompanied by increased expression of cell cycle regulatory proteins and antiapoptotic proteins but decreased cleavage of several caspases. Within 24 hours of treatment, apoptosis in cultures of HEY ovarian cancer cells treated with neutralizing anti-STC1 monoclonal antibody was higher (17.3% apoptotic cells) than that in cultures treated with mouse IgG control cells (4.4%) (12.9% difference, 95% confidence interval = 11.6% to 14.2%). STC1 protein may be involved in ovarian tumorigenesis.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that orchestrates cell signaling through integrins and growth factor receptors and plays important roles in tumorigenesis. Amplification and overexpression of FAK have been observed in aggressive human cancers including ovarian and breast cancers. VS-6063 and VS-4718 are potent, selective, and orally active FAK inhibitors. In a Phase 1 clinical trial, VS-6063 was well tolerated and demonstrated preliminary clinical activity. In addition, we have demonstrated that VS-6063 can be combined with paclitaxel and preliminary signs of clinical activity, including CA-125 reduction to normal levels, were observed with this combination in patients with ovarian cancer. VS-4718 was shown to inhibit tumor growth and metastasis in preclinical tumor models including an ovarian cancer xenograft model and is currently under evaluation in a Phase 1 clinical trial. We have shown previously that inhibition of FAK preferentially targets cancer stem cells in breast cancer models. We extended these studies and report here that the FAK inhibitors VS-6063 and VS-4718 effectively abrogate cancer stem cells (CSCs) in ovarian cancer models. VS-4718 and VS-6063 were first evaluated in several orthogonal cancer stem cell assays in vitro. Treatment of OVCAR-8 or TOV-21G human ovarian cancer cells with these FAK inhibitors in matrigel reduced the percentage of Aldefluor+ CSCs and attenuated tumorsphere forming efficiency. Accordingly, in a side population (SP) assay assessing CSCs by Hoechst dye exclusion, these FAK inhibitors reduced SP CSCs in the OVCAR-5 ovarian cancer cell line. Similar effects were observed with the breast cancer cell lines SUM159 and MDA-MB-231. In direct contrast, the standard-of-care cytotoxic agents, paclitaxel and carboplatin, increased the percentage of CSCs, indicating that these agents do not effectively target CSCs. Importantly, combination of VS-6063 or VS-4718 with cytotoxic agents attenuated the enrichment of CSCs caused by cytotoxic agents as measured by the Aldefluor assay and in vivo tumor initiating potential. To assess drug effects on tumor-initiating capability, the clearest arbiter of CSC activity, TOV-21G cells were pre-treated in vitro with various compounds and 1,000 of the resulting cells were injected into immunodeficient mice. VS-4718 alone or VS-4718 in combination with paclitaxel prevented tumor initiation, while paclitaxel alone did not reduce tumor initiation, suggesting that treatment with VS-4718 could effectively eliminate tumor initiating CSCs. Furthermore, ex vivo treatment of primary human ovarian cancer tissue with VS-4718 or VS-6063 also reduced the percentage of CSCs as assessed by CD24hi/CD117hi CSC markers and the Aldefluor assay. In addition to targeting CSCs, we also observed that FAK inhibitors enhanced the activity of paclitaxel to reduce bulk tumor cells both in vitro and in vivo. In summary, our data indicate that the FAK inhibitors VS-6063 and VS-4718 preferentially target cancer stem cells and enhance the activity of paclitaxel. These results provide rationale for the clinical development of Verastem's FAK inhibitors for the treatment of ovarian cancer. Citation Format: Vihren N. Kolev, Quentin G. Wright, Christian M. Vidal, Winnie F. Tam, Mahesh V. Pavdal, Qunli Xu, Jonathan A. Pachter. FAK inhibitors VS-6063 and VS-4718 preferentially target ovarian cancer stem 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 A39.

The therapeutic efficacy of antitumor drugs is often limited by non-specific, systematic delivery. Here we report a novel targeted delivery platform designed by coupling liposomal nanobins (NB) with the urokinase plasminogen activator receptor (u-PAR) antibody (ATN-658). The urokinase system is overexpressed in epithelial ovarian cancer (OvCa) cells and is expressed only at low levels in normal cells. Doxorubicin (Dox), a FDA approved antitumor drug, had been loaded into the nanobins as a payload. The size and surface charge of the nanobins were optimized to facilitate specific binding to u-PAR expressing OvCa cells. Confocal and transmission electron microscopy showed that ATN-658-NB(Dox) was internalized in OvCa cells in a receptor-dependent manner and was released from endosomes as a function of time. This uptake could be blocked by stably down-regulating u-PAR expression in the OvCa cells using shRNA. In an orthotopic ovarian cancer model, athymic mice treated with ATN-658-NB(Dox) had a significantly greater reduction in tumor burden (0.06±0.01g versus 0.1±0.01g; p&amp;lt;0.001) than mice treated with NB(Dox), which was corroborated by in vivo fluorescence imaging results. A survival study showed that targeted nanobins prolonged the survival of mice by 3 days (43 days versus 40 days, median survival) as compared to untargeted nanobins. Immunoblot analysis of tumor sections showed that targeted nanobins induced apoptosis (cleaved caspase 3) and inhibited proliferation (Ki-67) more efficiently when compared to untargeted nanobins. Furthermore, tumor-targeted delivery of ATN-658-ND(Dox) was confirmed by ex vivo fluorescence imaging, FACS analysis, and confocal microscopy. Taken together, our findings show that receptor-mediated delivery of targeted nanobins to cancer cells could serve as a novel approach to ovarian cancer treatment. Citation Format: Yilin Zhang, Elden P. Swindell, Patrick L. Hankins, Hilary A. Kenny, Thomas V. O’Halloran, Andrew P. Mazar, Ernst Lengyel. Targeted delivery of doxorubicin loaded nanobins to ovarian cancer cells through the urokinase plasminogen activator system [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1440.

Ovarian cancer is one of the three most common gynecological malignant tumors worldwide. The prognosis of patients suffering from this malignancy remains poor because of limited therapeutic strategies. Herein, we investigated the role of a long noncoding RNA named MIR4697 host gene (MIR4697HG) in the cell growth and metastasis of ovarian cancer. Results showed that the transcriptional level of MIR4697HG in cancerous tissues increased twofold compared with that in adjacent noncancerous tissues. MIR4697HG was differentially expressed in ovarian cancer cell lines, with the highest levels in OVCAR3 and SKOV3 cells. MIR4697HG knockdown by specific shRNA significantly inhibited cell proliferation and colony formation in both OVCAR3 and SKOC3 cells. Consistently, in a xenograft model of ovarian cancer, MIR4697HG depletion also significantly restricted tumor volumes and weights. Furthermore, MIR4697HG knockdown inhibited cell migration and invasion capacities. Invasion ability was inhibited by 58% in SKOV3 cells and 40% in OVCAR3 cells, and migration ability was inhibited by 73% in SKOV3 cells and 62% in OVCAR3 cells after MIR4697HG knockdown. MIR4697HG knockdown also caused a decrease in matrix metalloprotease-9, phosphorylated ERK, and phosphorylated AKT. These data suggested that MIR4697HG promoted ovarian cancer growth and metastasis. The aggressive role of MIR4697HG in ovarian cancer may be related to the ERK and AKT signaling pathways.

Ovarian cancer is one of the leading causes of gynaecological deaths in the United States and several other countries. Genetic alterations including overexpression of EGFR (about 70% of ovarian tumors) play a crucial role in the signal transduction pathways that regulate key cellular functions such as cell survival and proliferation and are responsible for comprising traditional chemotherapy. Several epidemiological studies suggested a decreased risk of ovarian cancer in the population consuming Brassica vegetables. 3,3′-diindolylmethane (DIM) is an indole compound present in Brassica vegetables. In our previous studies, we have demonstrated that BR-DIM, a formulated version of DIM suppress the growth of ovarian cancer cells by causing cell cycle arrest. However, the exact mechanism by which DIM suppress the growth of ovarian cancer cells was not clear. In the present study, we delineated the mechanism of DIM in SKOV3, OVCAR-3 and TOV-21G human ovarian cancer cells. Our results show that DIM treatment induces apoptosis in all the cell lines in a dose-dependent manner, as analyzed by flow cytometry. Our western blot analysis reveals that DIM treatment causes significant down regulation of constitutive EGFR and HER2 protein level as well as phosphorylation of EGFR at Tyr992, Tyr845 and Tyr1173 in SKOV-3 and OVCAR-3 cells. To determine whether DIM can suppress the activation of EGFR by activating phosphorylation, cells were treated with EGF after DIM treatment. Our results reveal that DIM treatment block the activation of EGFR induced by EGF in OVCAR-3 and TOV-21G cells. In addition, DIM treatment drastically reduces the phosphorylation of ERK without affecting the protein level. DIM treatment also inhibits the kinase activity of ERK as observed by the down regulation of p-ELK in all the three ovarian cancer cell lines. ERK is known to be regulated by EGFR. Taken together our results indicate that DIM induces apoptosis in ovarian cancer cells by inhibiting EGFR-ERK pathway. Further mechanistic studies are in progress. [Supported in part by RO1 grants CA 106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute]. 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 3781.

In search of new, potential therapeutic target proteins in primary ovarian cancer cells, we have performed in-tumor shRNA screens in an established, human ovarian cancer (ov ca) cell line growing either subcutaneously or intraperitoneally in immunologically incompetent mice. Validation tests were then performed on the confirmed hits in viability/proliferation assays performed, in parallel, in at least two, cultured primary papillary serous ov ca cell strains each of which was generated by direct intraperitoneal injection of primary, patient- derived ov ca cells. The lentiviral shRNA library used contained ~8,000 hairpins, collectively directed against all known human protein kinases and a number of established human oncoproteins. Approximately 40 hits were detected and confirmed in this study, in which the screen involved deep sequencing of DNA isolated from residual tumor material growing in multiple animals. Here the search was for any hairpins that were selectively and specifically depleted from these DNA samples. Currently, we are focusing on two, new hits in this effort with special attention to their effects on primary human ov ca cell tumor formation in nude mice. Citation Format: Maria Giuseppina Baratta, Maria Giuseppina Baratta, Joyce Liu, Joyce Liu, Yaara Zwang, Yaara Zwang, Yaara Zwang, Anna Schinzel, Anna Schinzel, Ronny Drapkin, Ronny Drapkin, William Hahn, William Hahn, William Hahn, David M. Livingston, David M. Livingston. In tumor screening for novel targets in papillary serous ovarian cancer. [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 IA3.

Isothiocyanate Iberin inhibits cell proliferation and induces cell apoptosis in the progression of ovarian cancer by mediating ROS accumulation and GPX1 expression

Objectives Existence of side population (SP) in human ovarian cancer cell line OVCAR3 and characteristics of both stem cells and cancer cells were detected. Relation between SP phenotype and ABCG2 protein( ATP-binding cassette superfamily G member 2/breast cancer resistance protein, BCRP) was examined,and the potential to use OVCAR3 as tools for investigating the characteristics of cancer stem cells (CSCs)was estimated as well. Methods Hoechst 33342 efflux SP cells and dye-uptaking non-SP cells were separated by flow cytometry from human serous ovarian adenocarcinoma cell line OVCAR3. Cell colonyforming efficiency (CFE) of both subgroups ware evaluated. Soft agar assay was performed to compare the growth capacity of SP cells and non-SP cells. ABCG2 expression was detected by Western blotting and immunofluoreacence using an anti-ABCG2 monoclonal antibody. Results Flow cytometric analysis confirmed the presence of small subpopulations of cells excluding Hoechst dye in OVCAP,3 cells (2. 0% ) in a verapamil-sensitive manner. Hoechst dye uptake in OVCAR3 cells was enhanced by addition of verapamil, consistent with activity of ABCG2 protein. CFE of both SP and non-SP was 46.17%±23.27% and 6.17%±3.06%, respectively(P<0. 05). The Hoechst dye excluding SP cells exhibited immunoreactivity to stem cell markers ABCG2, demonstrated by Western blotting. They could grow in soft agar plate and formed cell sphere. Conclusions Human epithelial ovarian carcinoma cell line OVCAR3 contains a small subpoptdation of cells that exhibits a cancer stem cell-like phenotype. Special significance is the expression of ABCG2 in human tumor cells, a calcium-sensitive cell surface protein that acts to exclude Hoechst dye. These cells play a principal role in maintaining cancer cell line. It can be seen that cancer cell lines are potentially useful tools for investigating the characteristics of CSCs under controlled experimental conditions. Key words: Ovarian neoplasms; Tumor stem cells; ATP-binding cassette superfamily G member 2; Side-population cells; Hoechst 33342

Histone acetyltransferase human males absent on the first (hMOF) is a member of MYST family which participates in posttranslational chromatin modification by controlling the acetylation level of histone H4K16. Abnormal activity of hMOF occurs in multiple cancers and biological alteration of hMOF expression can affect diverse cellular functions including cell proliferation, cell cycle progression and embryonic stem cells (ESCs) self-renewal. The relationship between hMOF and cisplatin resistance was investigated in The Cancer Genome Atlas (TCGA) and Genomics of Drug Sensitivity in Cancer (GDSC) database. Lentiviral-mediated hMOF-overexpressed cells or hMOF-knockdown cells were established to investigate its role on cisplatin-based chemotherapy resistance in vitro ovarian cancer cells and animal models. Furthermore, a whole transcriptome analysis with RNA sequencing was used to explore the underlying molecular mechanism of hMOF affecting cisplatin-resistance in ovarian cancer. The data from TCGA analysis and IHC identification demonstrated that hMOF expression was closely associated with cisplatin-resistance in ovarian cancer. The expression of hMOF and cell stemness characteristics increased significantly in cisplatin-resistant OVCAR3/DDP cells. In the low hMOF expressing ovarian cancer OVCAR3 cells, overexpression of hMOF improved the stemness characteristics, inhibited cisplatin-induced apoptosis and mitochondrial membrane potential impairment, as well as reduced the sensitivity of OVCAR3 cells to cisplatin treatment. Moreover, overexpression of hMOF diminished tumor sensitivity to cisplatin in a mouse xenograft tumor model, accompanied by decrease in the proportion of cisplatin-induced apoptosis and alteration of mitochondrial apoptosis proteins. In addition, opposite phenotype and protein alterations were observed when knockdown of hMOF in the high hMOF expressing ovarian cancer A2780 cells. Transcriptomic profiling analysis and biological experimental verification orientated that MDM2-p53 apoptosis pathway was related to hMOF-modulated cisplatin resistance of OVCAR3 cells. Furthermore, hMOF reduced cisplatin-induced p53 accumulation by stabilizing MDM2 expression. Mechanistically, the increased stability of MDM2 was due to the inhibition of ubiquitinated degradation, which resulted by increased of MDM2 acetylation levels by its direct interaction with hMOF. Finally, genetic inhibition MDM2 could reverse hMOF-mediated cisplatin resistance in OVCAR3 cells with up-regulated hMOF expression. Meanwhile, treatment with adenovirus expressing shRNA of hMOF improved OVCAR3/DDP cell xenograft sensitivity to cisplatin in mouse. Collectively, the results of the study confirm that MDM2 as a novel non-histone substrate of hMOF, participates in promoting hMOF-modulated cisplatin chemoresistance in ovarian cancer cells. hMOF/MDM2 axis might be a potential target for the treatment of chemotherapy-resistant ovarian cancer.

We have previously shown that MEF (myeloid ELF1-like factor, also known as ELF4) functions as a transcriptional activator of the interleukin (IL)-8, perforin, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-3 genes in hematopoietic cells. MEF is also expressed in non-hematopoietic tissues including certain ovarian cancer cells. To define the function of MEF in these cells, we examined primary human ovarian epithelial tumors and found that MEF is expressed in a significant proportion of ovarian carcinomas, and in the CAOV3 and SKOV3 ovarian cancer cell lines, but not in normal ovarian surface epithelium. Manipulating MEF levels in these cell lines altered their behavior; reducing MEF levels, using short hairpin RNA expressing vectors, significantly inhibited the proliferation of SKOV3 and CAOV3 cells in culture, and impaired the anchorage-independent growth of CAOV3 cells. Overexpression of MEF in SKOV3 cells (via retroviral transduction) significantly increased their growth rate, enhanced colony formation in soft agar and promoted tumor formation in nude mice. The oncogenic activity of MEF was further shown by the ability of MEF to transform NIH3T3 cells, and induce their tumor formation in nude mice. MEF is an important regulator of the tumorigenic properties of ovarian cancer cells and could be used a therapeutic target in ovarian cancer.