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Supplementary Figure 1 from Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

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Cancer stem cells are responsible for tumor initiation and chemoresistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome and patient survival. The previous demonstration that the ovarian cancer stem cells are resistant to apoptotic cell death induced by conventional chemotherapy agents suggests that other forms of targeted therapy should be explored. We show in this study that targeting mitochondrial bioenergetics is a potent stimulus to induce caspase-independent cell death in a panel of ovarian cancer stem cells. Treatment of these cells with the novel isoflavone derivative, NV-128, significantly depressed mitochondrial function exhibited by decrease in ATP, Cox-I, and Cox-IV levels, and by increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of cellular starvation that activates two independent pathways: (i) AMPKα1 pathway leading to mTOR inhibition; and (ii) mitochondrial MAP/ERK kinase/extracellular signal-regulated kinase pathway leading to loss of mitochondrial membrane potential. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemoresistant cell population opens a new venue for treating ovarian cancer patients. Mol Cancer Ther; 10(8); 1385–93. ©2011 AACR.

Supplementary Figure 3 from Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

Supplementary Figure 2 from Targeting the Mitochondria Activates Two Independent Cell Death Pathways in Ovarian Cancer Stem Cells

Pathogenic mutations in the solute carrier family 7 member 5 (SLC7A5) gene, which encodes an amino acid transporter cause microcephaly and seizures, yet the mechanisms responsible for these phenotypes are unclear. Models have demonstrated that Slc7a5 deletion is embryonic lethal and that these embryos lack a fully formed telencephalon. This phenotype is similar to that of mammalian target of rapamycin (mTOR) protein kinase deletion or mTOR inhibition. Notably, in many cells, Slc7a5 import of amino acids is required to maintain mTOR activity. Slc7a5 is present within neurogenic regions during embryogenesis, is found in cultured neurons and can modulate neuronal electrophysiological properties. However, Slc7a5 is also highly expressed within endothelial cells of the blood–brain barrier where removal in conditional mice leads to severe behavioral defects and non-cell autonomous changes in neurons. Therefore, the extent that neural Slc7a5 is required for development is unclear. Here, subventricular zone neural stem cells that generate olfactory bulb granule cell neurons were electroporated with SLC7A5 or Slc7a5 short hairpin RNA encoding plasmids. Although early phases of neural development were unaltered, Slc7a5 knockdown effected late phases of GC dendrite maturation and survival. Slc7a5 knockdown also decreased mTOR pathway activity. Ras homolog enriched in brain, an mTOR activator, rescued the effect of Slc7a5 knockdown on mTOR pathway activity and dendrite arbors. The data presented here demonstrate that Slc7a5 is required for GC mTOR pathway activity, maturation and survival, which may help explain why Slc7a5 mutations prevent normal brain development and function.

Extracellular vesicles (EVs) are cellular derived particles found throughout the body in nearly all tissues and bodily fluids. EVs contain biological molecules including small RNAs and protein. EVs are proposed to be transferred between cells, notably, cells of the immune system. Tools that allow for in vivo EV labeling while retaining the ability to resolve cellular sources and timing of release are required for a full understanding of EV functions. Fluorescent EV fusion proteins are useful for the study of EV biogenesis, release, and identification of EV cellular recipients. Among the most plentiful and frequently identified EV proteins is CD9, a tetraspanin protein. A transgenic mouse containing a CRE-recombinase inducible CAG promoter driven CD9 protein fused to Turbo-GFP derived from the copepod Pontellina plumata was generated as an EV reporter. The transgenic inducible GFP EV reporter (TIGER) mouse was electroporated with CAG-CRE plasmids or crossed with tamoxifen inducible CAG-CRE-ERT2 or nestin-CRE-ERT2 mice. CD9-GFP labeled cells included glutamine synthetase and glial fibrillary acidic protein positive astrocytes. Cortical astrocytes released ~136 nm EVs that contained CD9. Intraventricular injected EVs were taken up by CD11b/IBA1 positive microglia surrounding the lateral ventricles. Neonatal electroporation and shRNA mediated knockdown of Rab27a in dorsal subventricular zone NSCs and astrocytes increased the number of CD11b/IBA1 positive rounded microglia. Neonatal astrocyte EVs had a unique small RNA signature comprised of morphogenic miRNAs that induce microglia cytokine release. The results from this study demonstrate that inducible CD9-GFP mice will provide the EV community with a tool that allows for EV labeling in a cell-type specific manner while simultaneously allowing in vivo experimentation and provides evidence that EVs are required immunomodulators of the developing nervous system.

High-grade serous ovarian carcinoma (HGSOC), the most lethal gynecological cancer, often leads to chemoresistant diseases. The p53 protein is a key transcriptional factor regulating cellular homeostasis. A majority of HGSOCs have inactive p53 because of genetic mutations. However, genetic mutation is not the only cause of p53 inactivation. The aggregation of p53 protein has been discovered in different types of cancers and may be responsible for impairing the normal transcriptional activation and pro-apoptotic functions of p53. We demonstrated that in a unique population of HGSOC cancer cells with cancer stem cell properties, p53 protein aggregation is associated with p53 inactivation and platinum resistance. When these cancer stem cells differentiated into their chemosensitive progeny, they lost tumor-initiating capacity and p53 aggregates. In addition to the association of p53 aggregation and chemoresistance in HGSOC cells, we further demonstrated that the overexpression of a p53-positive regulator, p14ARF, inhibited MDM2-mediated p53 degradation and led to the imbalance of p53 turnover that promoted the formation of p53 aggregates. With in vitro and in vivo models, we demonstrated that the inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment. Moreover, by two-dimensional gel electrophoresis and mass spectrometry we discovered that the aggregated p53 may function uniquely by interacting with proteins that are critical for cancer cell survival and tumor progression. Our findings help us understand the poor chemoresponse of a subset of HGSOC patients and suggest p53 aggregation as a new marker for chemoresistance. Our findings also suggest that inhibiting p53 aggregation can reactivate p53 pro-apoptotic function. Therefore, p53 aggregation is a potential therapeutic target for reversing chemoresistance. This is paramount for improving ovarian cancer patients' responses to chemotherapy, and thus increasing their survival rate.

Primary ovarian cancer is responsive to treatment, but chemoresistant recurrent disease ensues in majority of patients. Recent compelling evidence demonstrates that a specific population of cancer cells, the cancer stem cells, initiates and sustains tumors. It is therefore possible that this cell population is also responsible for recurrence. We have shown previously that CD44+/MyD88+ epithelial ovarian cancer stem cells (CD44+/MyD88+ EOC stem cells) are responsible for tumor initiation. In this study, we demonstrate that this population drives tumor repair following surgery- and chemotherapy-induced tumor injury. Using in vivo and in vitro models, we also demonstrate that during the process of tumor repair, CD44+/MyD88+ EOC stem cells undergo self-renewal as evidenced by upregulation of stemness-associated genes. More importantly, we show that a pro-inflammatory microenvironment created by the TLR2-MyD88-NFκB pathway supports EOC stem cell-driven repair and self-renewal. Overall, our findings point to a specific cancer cell population, the CD44+/MyD88+ EOC stem cells and a specific pro-inflammatory pathway, the TLR2-MyD88-NFκB pathway, as two of the required players promoting tumor repair, which is associated with enhanced cancer stem cell load. Identification of these key players is the first step in elucidating the steps necessary to prevent recurrence in EOC patients.

Citation Pizzonia J, Holmberg J, Orton S, Alvero A, Viteri O, Mclaughlin W, Feke G, Mor G. Multimodality animal rotation imaging system (MARS) for in vivo detection of intraperitoneal tumors. Am J Reprod Immunol 2012; 67: 84–90 Problem Ovarian cancer stem cells (OCSCs) have been postulated as the potential source of recurrence and chemoresistance. Therefore identification of OvCSC and their complete removal is a pivotal stage for the treatment of ovarian cancer. The objective of the following study was to develop a new in vivo imaging model that allows for the detection and monitoring of OCSCs. Method of Study OCSCs were labeled with X-Sight 761 Nanospheres and injected intra-peritoneally (i.p.) and sub-cutaneously (s.c.) to Athymic nude mice. The Carestream In-Vivo Imaging System FX was used to obtain X-ray and, concurrently, near-infrared fluorescence images. Tumor images in the mouse were observed from different angles by automatic rotation of the mouse. Results X-Sight 761 Nanospheres labeled almost 100% of the cells. No difference on growth rate was observed between labeled and unlabeled cells. Tumors were observed and monitoring revealed strong signaling up to 21 days. Conclusion We describe the use of near-infrared nanoparticle probes for in vivo imaging of metastatic ovarian cancer models. Visualization of multiple sites around the animals was enhanced with the use of the Carestream Multimodal Animal Rotation System.

Cancer stem cells are responsible for tumor initiation and chemoresistance. In ovarian cancer, the CD44+/MyD88+ ovarian cancer stem cells are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome and patient survival. The previous demonstration that the ovarian cancer stem cells are resistant to apoptotic cell death induced by conventional chemotherapy agents suggests that other forms of targeted therapy should be explored. We show in this study that targeting mitochondrial bioenergetics is a potent stimulus to induce caspase-independent cell death in a panel of ovarian cancer stem cells. Treatment of these cells with the novel isoflavone derivative, NV-128, significantly depressed mitochondrial function exhibited by decrease in ATP, Cox-I, and Cox-IV levels, and by increase in mitochondrial superoxide and hydrogen peroxide. This promotes a state of cellular starvation that activates two independent pathways: (i) AMPKα1 pathway leading to mTOR inhibition; and (ii) mitochondrial MAP/ERK kinase/extracellular signal-regulated kinase pathway leading to loss of mitochondrial membrane potential. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemoresistant cell population opens a new venue for treating ovarian cancer patients. Mol Cancer Ther; 10(8); 1385–93. ©2011 AACR.

Recurrent ovarian cancer is resistant to conventional chemotherapy. A sub-population of ovarian cancer cells, the epithelial ovarian cancer stem cells (EOC stem cells) have stemness properties, constitutive NFκB activity, and represent the chemoresistant population. Currently, there is no effective treatment that targets these cells. Aurora-A kinase (Aurora-A) is associated with tumor initiation and progression and is overexpressed in numerous malignancies. The aim of this study is to determine the effect of Aurora-A inhibition in EOC stem cells. EOC stem cells were treated with the Aurora-A inhibitor, MK-5108. Cell growth was monitored by Incucyte real-time imaging system, cell viability was measured using the Celltiter 96 assay and cytokine levels were quantified using xMAP technology. The intracellular changes associated with MK-5108 treatment are: (1) polyploidy and cell cycle arrest; (2) inhibition of NFκB activity; (3) decreased cytokine production; and (4) nuclear accumulation of IκBα. Thus, inhibition of Aurora-A decreases cell proliferation in the EOC stem cells by inducing cell cycle arrest and affecting the NFκB pathway. As EOC stem cells represent a source of recurrence and chemoresistance, these results suggest that Aurora-A inhibition may effectively target the cancer stem cell population in ovarian cancer.

Problem—The NFκB pathway is a major source of pro-inflammatory cytokines, which may contribute to cancer chemoresistance. We showed that constitutive NFκB activity is characteristic of the ovarian cancer stem cells (OCSCs). The aim of this study is to determine if the inhibition of NFκB by Eriocalyxin B (EriB) in the OCSCs may induce cell death in otherwise chemoresistant cells. Methods—OCSCs and mature ovarian cancer cells (mOCCs) were treated with increasing concentrations of EriB. Cell viability was measured using the Celltiter 96 assay and caspase activity was quantified using Caspase-Glo™ assay. Cytokine levels were quantified using xMAP technology. Results—EriB decreased the percent of viable cells in all cultures tested with GI50 of 0.5 −1µM after 48h of treatment. The intracellular changes associated with EriB-induced cell death are: 1) inhibition of NF-κB activity; 2) decreased cytokine production; 3) activation of caspases; and 4) down-regulation of XIAP. In addition, EriB is able to sensitize OCSCs to TNFα and FasLmediated cell death. Conclusions—Inhibition of the NFκB pathway induces cell death in the OCSCs. Since the OCSCs may represent the source of recurrence and chemoresistance, the use of NFκB inhibitors like EriB may prevent recurrence in ovarian cancer patients.

Cancer stem cells are responsible for sustaining the tumor and giving rise to proliferating and progressively differentiating cells. However, the molecular mechanisms regulating the process of cancer stem cell (CSC) differentiation is not clearly understood. Recently, we reported the isolation of the epithelial ovarian cancer (EOC) stem cells (type I/CD44+). In this study, we show that type I/CD44+ cells are characterized by low levels of both miR-199a and miR-214, whereas mature EOC cells (type II/CD44-) have higher levels of miR-199a and miR-214. Moreover, these two micro RNAs (miRNAs) are regulated as a cluster on pri-miR-199a2 within the human Dnm3os gene (GenBank FJ623959). This study identify Twist1 as a regulator of this unique miRNA cluster responsible for the regulation of the IKKbeta/NF-kappaB and PTEN/AKT pathways and its association of ovarian CSC differentiation. Our data suggest that Twist1 may be an important regulator of 'stemness' in EOC cells. The regulation of MIR199A2/214 expression may be used as a potential therapeutic approach in EOC patients.

Neovascularization is required for solid tumor maintenance, progression, and metastasis. The most described contribution of cancer cells in tumor neovascularization is the secretion of factors, which attract various cell types to establish a microenvironment that promotes blood vessel formation. The cancer stem cell hypothesis suggests that tumors are composed of cells that may share the differentiation capacity of normal stem cells. Similar to normal stem cells, cancer stem cells (CSCs) have the capacity to acquire different phenotypes. Thus, it is possible that CSCs have a bigger role in the process of tumor neovascularization. In this study, we show the capacity of a specific population of ovarian cancer cells with stem-like properties to give rise to xenograft tumors containing blood vessels, which are lined by human CD34+ cells. In addition, when cultured in high-density Matrigel, these cells mimic the behavior of normal endothelial cells and can form vessel-like structures in 24 hours. Microscopic analysis showed extensive branching and maturation of vessel-like structures in 7 days. Western blot and flow cytometry analysis showed that this process is accompanied by the acquisition of classic endothelial markers, CD34 and VE-cadherin. More importantly, we show that this process is vascular endothelial growth factor–independent, but IKKβ-dependent. Our findings suggest that anti-angiogenic therapies should take into consideration the inherent capacity of these cells to serve as vascular progenitors.

The mortality rate of ovarian cancer remains high due to late diagnosis and recurrence. A fundamental step toward improving detection and treatment of this lethal disease is to understand its origin. A growing number of studies have revealed that ovarian cancer can develop from multiple extra-ovarian origins, including fallopian tube, gastrointestinal tract, cervix and endometriosis. However, the mechanism leading to their ovarian localization is not understood. We utilized in vitro, ex vivo, and in vivo models to recapitulate the process of extra-ovarian malignant cells migrating to the ovaries and forming tumors. We provided experimental evidence to support that ovulation, by disrupting the ovarian surface epithelium and releasing chemokines/cytokines, promotes the migration and adhesion of malignant cells to the ovary. We identified the granulosa cell-secreted SDF-1 as a main chemoattractant that recruits malignant cells towards the ovary. Our findings revealed a potential molecular mechanism of how the extra-ovarian cells can be attracted by the ovary, migrate to and form tumors in the ovary. Our data also supports the association between increased ovulation and the risk of ovarian cancer. Understanding this association will lead us to the development of more specific markers for early detection and better prevention strategies.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Despite initial responsiveness, 80% of EOC patients recur and present with chemoresistant and a more aggressive disease. This suggests an underlying biology that results in a modified recurrent disease, which is distinct from the primary tumor. Unfortunately, the management of recurrent EOC is similar to primary disease and does not parallel the molecular changes that may have occurred during the process of rebuilding the tumor. We describe the characterization of unique in vitro and in vivo ovarian cancer models to study the process of recurrence. The in vitro model consists of GFP+/CD44+/MyD88+ EOC stem cells and mCherry+/CD44−/MyD88− EOC cells. The in vivo model consists of mCherry+/CD44+/MyD88+ EOC cells injected intraperitoneally. Animals received four doses of Paclitaxel and response to treatment was monitored by in vivo imaging. Phenotype of primary and recurrent disease was characterized by quantitative polymerase chain reaction (qPCR) and Western blot analysis. Using the in vivo and in vitro models, we confirmed that chemotherapy enriched for CD44+/MyD88+ EOC stem cells. However, we observed that the surviving CD44+/MyD88+ EOC stem cells acquire a more aggressive phenotype characterized by chemoresistance and migratory potential. Our results highlight the mechanisms that may explain the phenotypic heterogeneity of recurrent EOC and emphasize the significant plasticity of ovarian cancer stem cells. The significance of our findings is the possibility of developing new venues to target the surviving CD44+/MyD88+ EOC stem cells as part of maintenance therapy and therefore preventing recurrence and metastasis, which are the main causes of mortality in patients with ovarian cancer.

Epithelial ovarian cancer (EOC) cells with CD44 and CK19 coexpression may represent a subset of ovarian cancer stem cells (OCSCs). This study was conducted to evaluate the correlation of the frequency of putative OCSCs (CD44 + CK19 + OCSCs) with the clinicopathologic features and the prognostic value in patients with recurrent advanced stage EOC. A retrospective study was carried out on 33 patients with EOC and a uniformly treated tissue microarray was constructed. A multiplexed, immunofluorescence-based method of automated in situ quantitative measurement of protein analysis was used for evaluation of the frequency or density of CD44 + CK19 + OCSCs in EOC. The mean follow-up time was 42.8 ± 27.1 months. High frequency of EOC cells with CD44+ or CD44+/CK19+ was associated with chemoresistance (P = .033 and P = .02, respectively). Using K-M analysis with log-rank test, a high frequency of putative OCSCs was associated with short disease-free interval (7.9 months vs 20.9 months, P = .019). In univariable analysis, the frequency of OCSCs, International Federation of Gynecology and Obstetrics stage and residual tumor volume were significant predictor variables and were entered into multivariable analysis (P = .019, .037, and .005, respectively). Although no independent significant predictor was found, the frequency of putative OCSCs was the most promising predictor variable compared with the other 2 variables (hazard ratio = 2.344, P = .052). Our findings suggest that high frequency of OCSCs (CD44+ and CK19+) in epithelial ovarian tumors correlates with short progression-free intervals.

Epithelial-mesenchymal transition (EMT) is a critical process for embryogenesis but is abnormally activated during cancer metastasis and recurrence. This process enables epithelial cancer cells to acquire mobility and traits associated with stemness. It is unknown whether epithelial stem cells or epithelial cancer stem cells are able to undergo EMT, and what molecular mechanism regulates this process in these specific cell types. We found that epithelial-ovarian cancer stem cells (EOC stem cells) are the source of metastatic progenitor cells through a differentiation process involving EMT and mesenchymal-epithelial transition (MET). We demonstrate both in vivo and in vitro the differentiation of EOC stem cells into mesenchymal spheroid-forming cells (MSFCs) and their capacity to initiate an active carcinomatosis. Furthermore, we demonstrate that human EOC stem cells injected intraperitoneally in mice are able to form ovarian tumors, suggesting that the EOC stem cells have the ability to 'home' to the ovaries and establish tumors. Most interestingly, we found that TWIST-1 is constitutively degraded in EOC stem cells, and that the acquisition of TWIST-1 requires additional signals that will trigger the differentiation process. These findings are relevant for understanding the differentiation and metastasis process in EOC stem cells.

Epithelial ovarian cancer stem cells (EOC stem cells) have been associated with recurrence and chemoresistance. CD44 and CK18 are highly expressed in cancer stem cells and function as tools for their identification and characterization. We investigated the association between the number of CD44+ EOC stem cells in ovarian cancer tumors and progression-free survival. EOC stem cells exist as clusters located close to the stroma forming the cancer stem cell “niche”. 17.1% of the samples reveled high number of CD44+ EOC stem cells (>20% positive cells). In addition, the number of CD44+ EOC stem cells was significantly higher in patients with early-stage ovarian cancer (FIGO I/II), and it was associated with shorter progression-free survival (P=0.026). This study suggests that quantification of the number of EOC stem cells in the tumor can be used as a predictor of disease and could be applied for treatment selection in early-stage ovarian cancer.

The NFκB pathway is a major source of pro-inflammatory cytokines, which may contribute to cancer chemoresistance. We showed that constitutive NFκB activity is characteristic of the ovarian cancer stem cells (OCSCs). The aim of this study is to determine whether the inhibition of NFκB by Eriocalyxin B (EriB) in the OCSCs may induce cell death in otherwise chemoresistant cells.OCSCs and mature ovarian cancer cells (mOCCs) were treated with increasing concentrations of EriB. Cell viability was measured using the Celltiter 96 assay, and caspase activity was quantified using Caspase-Glo™ assay. Cytokine levels were quantified using xMAP technology.EriB decreased the percent of viable cells in all cultures tested with GI(50) of 0.5-1 μm after 48 hrs of treatment. The intracellular changes associated with EriB-induced cell death are: (i) inhibition of NF-κB activity; (ii) decreased cytokine production; (iii) activation of caspases; and (iv) down-regulation of XIAP. In addition, EriB is able to sensitize OCSCs to TNFα and FasL-mediated cell death.Inhibition of the NFκB pathway induces cell death in the OCSCs. Because the OCSCs may represent the source of recurrence and chemoresistance, the use of NFκB inhibitors like EriB may prevent recurrence in patients with ovarian cancer.

Background Development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4. Methods Because claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, in this study we evaluated the in vitro and in vivo bioactivity of the carboxy-terminal fragment of CPE (i.e., CPE290-319 binding peptide) as a carrier for tumor imaging agents and intracellular delivery of therapeutic drugs. Claudin-3 and -4 expression was examined with rt-PCR and flow cytometry in multiple primary ovarian carcinoma cell lines. Cell binding assays were used to assess the accuracy and specificity of the CPE peptide in vitro against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy and biodistribution assays were performed to evaluate the localization and uptake of the FITC-conjugated CPE peptide in established tumor tissue. Results Using a FITC-conjugated CPE peptide we show specific in vitro and in vivo binding to multiple primary chemotherapy resistant ovarian cancer cell lines. Bio-distribution studies in SCID mice harboring clinically relevant animal models of chemotherapy resistant ovarian carcinoma showed higher uptake of the peptide in tumor cells than in normal organs. Imunofluorescence was detectable within discrete accumulations (i.e., tumor spheroids) or even single chemotherapy resistant ovarian cancer cells floating in the ascites of xenografted animals while a time-dependent internalization of the FITC-conjugated CPE peptide was consistently noted in chemotherapy-resistant ovarian tumor cells by confocal microscopy. Conclusions Based on the high levels of claudin-3 and -4 expression in chemotherapy-resistant ovarian cancer and other highly aggressive human epithelial tumors including breast, prostate and pancreatic cancers, CPE peptide holds promise as a lead peptide for the development of new diagnostic tracers or alternative anticancer agents.

Background: New clinical and molecular studies suggest that HGSOC originates from the neighboring fallopian tubes, rather than the surface epithelial layer of the ovary. However, very little is known about the process of tubal malignant cells migrating and implanting into ovary. Our research focuses on understanding how the extraovarian malignant cells implant into the ovary and how ovulation affects the process of their migration and implantation. Method: Tumor initiating cells (TICs) isolated from human HGSOC were labeled by red fluorescent protein (RFP)-expressing lentivirus and subcutaneously or intrauterine injected to immunocompromised mice. Hormones (PMS and HCG) were interperitoneally injected to induce superovulation in mice. In the in vitro models, TICs were cultured with the granulosa cell conditional medium and the extra cellular matrix (ECM) extracted from ovarian stroma cells to evaluate their effects on the migration and attachment of TICs Result: Superovulation increased the ovarian tumor-forming rate from 38% (15/39 in control group) to 100% (24/24 in supervolation group). The increased frequency of ovulation enhances the formation of ovarian tumors in mouse models. In the intrauterine injection model, we were able to trace the migration of TICs towards ovaries and capture the early implantation of TICs inside ovaries in 5-10 days after the injection. We detected stage I ovarian tumors in mice. The tumors are encapsulated in the ovary and covered by the intact surface epithelial layer. These tumor cells closely interacted with the stroma of ovary, particularly the structure of corpus luteum. The granulosa cells secreted stromal cell-derived factor 1 (SDF-1, also known as CXCL12). SDF-1 attracts TICs that express CXCR4 (the receptor of SDF-1) to ovulatory wounds. Upon ovulation the ovulatory wounds expose the stroma of ovary, which provides a collagen enriched ECM for TICs to attach. Conclusion: The unique ovulatory wound microenvironment attracts the malignant cells to migrate towards the ovary and creates access for the malignant cells to implant in the collagen-enriched ECM in the stroma of ovary. The new mechanisms revealed by our data may explain why increased ovulation is associated with epithelial ovarian cancer. Our findings support the “extra-ovarian origin” theory and will help us better understand the initiation and progression of HGSOC. Citation Format: Yang Yang-Hartwich, Marta Gurrea, Natalia Sumi, Jennie Holmberg, Vinicius Craveiro, Ayesha Alvero, Gil Mor. Ovulatory wound: the site of origin for ovarian serous carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1974. doi:10.1158/1538-7445.AM2014-1974

Background: Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. The current standard of care in the treatment of EOC patients is surgical debulking and combination chemotherapy usually with Carboplatin and Paclitaxel. Although effective in majority of the cases, more than 20% of patients do not respond. Moreover, more than 80% of patients present with recurrent disease within 5 years. Therefore, there is an underlying biology that results in the differential response to treatment as well as the occurrence of recurrent disease. Tumors are heterogeneous and consist of multiple types of cancer cells, which exhibit different chemoresponsiveness. Our group previously described the characterization of CD44+/MyD88+ EOC stem cells and demonstrated their tumor-initiating properties. An important characteristic of these cells, which differentiate them from the CD44-/MyD88- EOC cells, is the presence of a functional TLR4-MyD88-NFkB pathway. This pathway confers Paclitaxel resistance to these cells. We hypothesize that a cause for recurrence following Paclitaxel treatment is the selective survival of cancer stem cells capable to recreate the tumor. In this study, we demonstrate that Paclitaxel enriches for the CD44+/MyD88+ EOC stem cells thus promoting recurrence. Methods: In vitro model: co-culture system consisting of (50%:50%) GFP+ CD44+/MyD88+ and RFP+ CD44-/MyD88- cells treated with 0.2 uM Paclitaxel. In vivo model: Ovarian tumors implanted s.c in Nude mice treated with 10 mg/kg Paclitaxel q3d for 21 days. Levels of GFP+ and CD44+ cells were determined by flow cytometry. Klf-4, Nanog, MyD88, and ALDH1 were determined by qPCR or western blot. Results: Control co-cultures were characterized by overgrowth of CD44-/MyD88- EOC cells while the Paclitaxel treated co-cultures had mainly CD44+/MyD88+ EOC stem cells. Similarly in vivo results show enrichment in CD44+ cells with Paclitaxel (49% CD44+ in control vs 88% CD44+ with treatment). Analysis of genes associated with stemness showed upregulation of Klf-4, Nanog ALDH1, and MyD88 in the cultures treated with Paclitaxel compared to control. Conclusion: We demonstrate that Paclitaxel selectively induce cell death in CD44-/MyD88- EOC cells but has a pro-survival effect and enhances self-renewal in the pleuripotent and chemoresistant CD44+/MyD88+ EOC stem cells. Based on these data, we propose that the mode of management for EOC patients should take into consideration the tumor's molecular phenotype. Our results highlight the need to identify patients that should not receive Paclitaxel - not only because they are resistant, but more importantly, because it can enrich for the more aggressive cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3471. doi:1538-7445.AM2012-3471

Myeloperoxidase (MPO) is an inflammatory heme protein present in myeloid cells- neutrophils, microglia, and macrophages. The enzyme activity of MPO utilizes hydrogen peroxide in the process of reactive oxygen species generation. Myeloperoxidase has been suggested recently as a likely marker in ovarian carcinoma. This relationship between the MPO and ovarian cancer however was deduced primarily from data obtained from in vitro studies using cancer cell lines and from immunostaining of ovarian carcinoma paraffin blocks. Therefore, a real-time determination of in vivo MPO activity in the tumor and during the progression of ovarian cancer will be valuable to assess the role of innate immune process in ovarian cancer progression, in cancer treatment, and the proper management of current therapies. Here, we developed non invasive methodologies to determine the MPO activity in intraperitoneal ovarian cancer tumor mouse model. Tri-modal non invasive imaging of the metastatic ovarian cancer in athymic mice model were performed to obtain fluorescence, luminescence, and X-ray images at different time points during the progression of the intraperitoneal tumor metastasis. Progression of the tumor metastasis was monitored using fluorescence; simultaneously MPO activity was obtained by luminescence imaging. The MPO activity was detected by i.p injection of Luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione) in physiological buffered saline (5 mg / 25gm mice). Luminol a redox-sensitive compound emits blue luminescence upon exposure to oxidizing agents, and in vivo, and has been established to have unique specificity to MPO activity. The fluorescence and luminescence images were overlaid on planar X-ray images for anatomical coregistration. The results show a robust increase in MPO activity in the mouse with the tumor development as compared to the control mice of the same cohort. Increase in MPO activity as a result of neutrophil activation was observed in the early phase of the ovarian cancer progression, and this increase subsides in the latter stages. However, the lumiscence signal intensities vary between the tumors, and near negligible MPO activity was observed in some tumors indicating a differential tumor microenvironment within the mouse. Further, the activity is far greater, at least in the initial stages, at the lung, nasal and other distal regions when compared to the intraperitoneal tumors. These real-time monitoring methodologies of MPO activity by in vivo imaging will greatly enhance the understanding of the tumor microenvironment during ovarian cancer progression, and contribute significantly in the development of better cancer treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5259. doi:1538-7445.AM2012-5259

Background: Cancer stem cells represent the cell population responsible for tumor initiation and progression. Resistance to cell death induced by cytotoxic agents is a characteristic shared by most identified cancer stem cells. In ovarian cancer, the CD44+/MyD88+ epithelial ovarian cancer (EOC) stem cells do not only represent the chemoresistant population, they are also able to repair the tumor and serve as tumor vascular progenitors. Targeting these cells is therefore necessary to improve treatment outcome. However, the previous demonstration that these cells are extremely chemoresistant, suggests that other forms of targeted therapy should be explored. We previously showed that the novel isoflavone derivative, NV-128, can induce caspase-independent cell death in ovarian cancer cells by inducing loss of mitochondrial membrane potential. We hypothesize that targeting mitochondrial bioenergetics may be a potent stimulus to induce cell death in the EOC stem cells. We demonstrate in this study that NV-128 is able to depress mitochondrial function leading to the activation of two independent pathways and cell death in these chemo-resistant cell population. Methods: A panel of CD44+/MyD88+ EOC stem cells was treated with NV-128 (1–10μg/ml). Inhibitory studies were done using the specific MEK inhibitor, U0126, or the ROS scavenger, MnTBAP. Mitochondrial integrity and function were assessed using Mitotracker Red, Mitotracker Green, MitoSox, and ApoSENSOR ADP/ATP kit. Cytoplasmic and mitochondrial fractions were prepared using the ApoAlertTM Cell Fractionation kit. Protein levels were determined using Western Blot. Results: NV-128 (10 μg/ml) significantly depressed mitochondrial function within 2h in all EOC stem cells tested. Treated cells had lower levels of ATP and Cox-IV, and higher levels of mitochondrial superoxide. Low cellular ATP led to mTOR inhibition through AMPKα1, while increased mitochondrial superoxide activated the mitochondrial MEK/ERK pathway and promoted Bax-dependent loss of mitochondrial membrane potential. Resulting cell death was characterized by Endo-G-induced but caspase-independent DNA fragmentation, as well as formation of autophagic vacuoles. Conclusion: We demonstrate that targeting the mitochondria in the EOC stem cells promotes a state of “cellular starvation” capable of activating two independent pathways: 1) AMPKα1 pathway leading to mTOR inhibition; and 2) mitochondrial MEK/ERK pathway leading to mitochondrial depolarization. The simultaneous activation of these pathways is a potent stimulus to induce cell death in cells. The demonstration that a compound can specifically target the mitochondria to induce cell death in this otherwise chemo-resistant cell population opens a new venue for treating ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-221. doi:10.1158/1538-7445.AM2011-LB-221

Background: Metastatic disease significantly contributes to mortality in ovarian cancer. Unfortunately, the source of metastatic cells and the processes involved in the generation of metastatic ovarian cancer is not well characterized. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial (MET) transitions have key roles in the process of tumor formation and metastasis. In this study we tested the hypothesis that epithelial ovarian cancer stem cells (EOC stem cells) are the source of metastasis. We demonstrate that the EOC stem cells can undergo both EMT and MET to yield cells with metastatic potential in vitro and create metastatic ovarian cancer in vivo. Methods: EOC stem cells were cultured in very confluent conditions for 30d. Changes in cellular morphology were monitored using the IncuCyte video imaging system. Levels of epithelial, mesenchymal, and stem cell markers were determined using RT-qPCR, Western blot analyses, and Flow cytometry. Gene expression profile was determined using EMT gene array. Results: I.p. injection of a pure culture of EOC stem cells suspended in matrigel in nude mice generated a solid tumor mass, whereas, injection of EOC stem cells without matrigel created carcinomatosis. Molecular characterization showed that the cells forming the solid tumor maintain an epithelial phenotype whereas cells forming the carcinomatosis acquire the mesenchymal markers Vimentin and Twist-1. In vitro, EOC stem cells grown in very confluent cultures formed viable mesenchymal-like spheroid cells (mspheroid cells) by day 30. EMT array results showed that the newly formed spheroids lost epithelial markers Ck18 and Ck19 but gained mesenchymal markers, Foxc2, Slug, Twist-1 and Vimentin. Moreover, the mspheroid cells exhibited a 4-fold increase in invasion capacity and significantly higher levels of MMP2 and MMP9 compared to the EOC stem cells. Finally, mspheroid cells plated in tissue culture flask re-attached and formed a monolayer of epithelial cells loosing expression of Foxc2 and Slug, suggesting MET. These results were not observed on parallel experiments performed on mature epithelial ovarian cancer cells (mOCCs), which do not possess stemness properties. Conclusion: We showed that the in vitro generation of mspheroid cells with highly metastatic potential and the creation of carcinomatosis in vivo occur only from a pure culture of EOC stem cells and not from cultures of mOCCs. Furthermore, we demonstrate the involvement of EMT and MET in this process. These results suggest that the EOC stem cells represent an early progenitor stage in the primary tumor, which have the capacity to differentiate, acquire the ability to detach and metastasize, and revert back to an epithelial phenotype at distant sites, thereby establishing metastatic disease. Acknowledgements: This study is supported in part by the Sands foundation, Brozman foundation, and grant from the NCI (RO1CA127913) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2011-3405

BACKGROUND: Coral reefs worldwide are in decline. Much of the mortality can be attributed to coral bleaching (loss of the coral's intracellular photosynthetic algal symbiont) associated with global warming. How corals will respond to increasing oceanic temperatures has been an area of extensive study and debate. Recovery after a bleaching event is dependent on regaining symbionts, but the source of repopulating symbionts is poorly understood. Possibilities include recovery from the proliferation of endogenous symbionts or recovery by uptake of exogenous stress-tolerant symbionts. METHODOLOGY/PRINCIPAL FINDINGS: To test one of these possibilities, the ability of corals to acquire exogenous symbionts, bleached colonies of Porites divaricata were exposed to symbiont types not normally found within this coral and symbiont acquisition was monitored. After three weeks exposure to exogenous symbionts, these novel symbionts were detected in some of the recovering corals, providing the first experimental evidence that scleractinian corals are capable of temporarily acquiring symbionts from the water column after bleaching. However, the acquisition was transient, indicating that the new symbioses were unstable. Only those symbiont types present before bleaching were stable upon recovery, demonstrating that recovery was from the resident in situ symbiont populations. CONCLUSIONS/SIGNIFICANCE: These findings suggest that some corals do not have the ability to adjust to climate warming by acquiring and maintaining exogenous, more stress-tolerant symbionts. This has serious ramifications for the success of coral reefs and surrounding ecosystems and suggests that unless actions are taken to reverse it, climate change will lead to decreases in biodiversity and a loss of coral reefs.

Background: In normal tissues following injury, there is an expansion of tissue specific stem cells prior to their differentiation to initiate injury repair. Once the tissue is repaired, the stem cells return to a quiescent state. Tumor tissue, might follow similar characteristics as in normal tissues, but the control of the expansion process may be significantly altered. Recently, ovarian cancer stem cells (ovCSC) were isolated from ovarian cancer tissue and ascites using CD44/CK18 as markers. We hypothesize that these cells play a critical role in tumor repair and renewal. To test this hypothesis, we used an in vitro wound/healing system. Methods: Cells were plated in an Essen ImageLock plate and wounds were made using an Essen WoundMaker. This sytem allows the generation of consistent wound locations and widths and precise imaging of the wound area. The healing process is monitored using an insitu imaging system (IncuCyte, Essen Instruments, NH) that records and quantifies the repair process in real time. Levels of cancer stem cell markers, CD44, Oct-4, and β-catenin were determined by Western blot analysis or Real Time PCR. Levels of cytokines and chemokines were determined using Luminex Technology. Results: Initially we tested whether an in vitro wound/healing system can be used as a renewal model. Interestingly, ovCSCs showed a well coordinated and organized response to the wound. First, the cells created a “straight line” to replace the irregular edge of the wound. Afterwards, the cells proliferated towards the wound, repairing the wound until confluence was reached. Analysis of cancer stem cell markers by Western blot revealed that the cells which repaired the wound maintained CD44, CK18, Oct4, and β-catenin, previously shown as ovCSC markers. Additionally, the process of repair was characterized by significant and differential up-regulation in IL-6, GRO-α, and MCP-1 expression and secretion. Interestingly, the CD44 expression was increased in wounded wells when compared to control. Moreover, using specific primers for CD44 variant isoforms we found that wound healing promotes expression of different CD44 forms. Conclusions: In this study we demonstrate ovCSCs have the capacity to repair an in vitro wound and that this process is accompanied by increased CD44 expression and cytokine secretion. This increased capacity of self renewal can lead to the accumulation of a large pool of progenitor cells, which has the potential to individually spread to other areas of the body as metastatic cells. 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 4229.

Introduction: Current studies show that the cancer stem cells represent the chemo-resistant phenotype and therefore may survive chemotherapy to eventually rebuild the tumor. Our group previously identified and characterized the ovarian cancer stem cells using CD44 and MyD88 as a marker and showed that these cells have a constitutively active IKKβ/NFκB axis. We hypothesize that specific targeting of this pathway may affect survival of the ovarian cancer stem cells. Methods: A panel of CD44+/MyD88+ ovarian cancer stem cells were treated with increasing concentrations of the specific IKKβ inhibitor, BAY 11-7082 (0.1, 0.5 1, 5, and 10 μg/ml). Effect on cell growth was monitored using the Incucyte real time imaging system and cell viability was determined using the Celltiter 96 assay. Specific inhibition of IKKβ was determined by: (a) the effect on the mTOR pathway by analyzing levels of phospho-S6 kinase and (b) by determining the effect on TNFα-induced NFκB activity by measuring levels of IκB. Activity of caspases- 3/7, −8, and −9 was measured using Caspase-Glo assay and levels of XIAP monitored using western blot analysis. Effect on mitochondrial membrane potential was measured using flow cytometry after staining with JC1 dye. In vivo activity was monitored using a s.c. ovarian cancer xenograft model in nude mice. The effect of BAY 11-7082 (10 mg/kg every other day given i.p.) was compared to carboplatin (40mg/kg q7d given i.p.) and paclitaxel (10mg/kg q3d given i.p.). Results: Treatment with BAY 11-7082 inhibited cell growth and decreased cell viability in all ovarian cancer stem cell cultures tested in a dose and time dependent manner with GI50 at ∼ 5μg/ml. Specific inhibition of IKKβ was observed at this dose by a decrease in phospho-p70S6 kinase and inhibition of TNFα-induced IκB degradation. Cell death was caspase-independent with no significant increase in caspases 3/7, 8, nor 9 and no effect on the levels of XIAP. Cell death was associated with loss of mitochondrial membrane potential with more than 90% of cells showing a shift in JC1 fluorescence from red to green in 4h. In vivo studies showed that BAY 11-7082 is able to inhibit tumor growth with T/C of 64%, significantly better than carboplatin and paclitaxel (T/C 87% and 74%, p=0.009 and 0.0025, respectively). Conclusion: The capacity of the cancer stem cells to survive chemotherapy and rebuild the tumor represent a major target to inhibit recurrence in ovarian cancer patients. The demonstration that specific targeting of IKKβ can induce cell death in the ovarian cancer stem cells opens new therapeutic options for ovarian cancer patients. 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 3342.