Han-Hsuan Fu

Molecular phenotyping of human ovarian cancer stem cells unravels the mechanisms for repair and chemoresistance

A major burden in the treatment of ovarian cancer is the high percentage of recurrence and chemoresistance. Cancer stem cells (CSCs) provide a reservoir of cells that can self-renew, can maintain the tumor by generating differentiated cells [non-stem cells (non-CSCs)] which make up the bulk of the tumor and may be the primary source of recurrence. We describe the characterization of human ovarian cancer stem cells (OCSCs). These cells have a distinctive genetic profile that confers them with the capacity to recapitulate the original tumor, proliferate with chemotherapy, and promote recurrence. CSC identified in EOC cells isolated form ascites and solid tumors are characterized by: CD44+, MyD88+, constitutive NFκB activity and cytokine and chemokine production, high capacity for repair, chemoresistance to conventional chemotherapies, resistance to TNFα-mediated apoptosis, capacity to form spheroids in suspension, and the ability to recapitulate in vivo the original tumor. Chemotherapy eliminates the bulk of the tumor but it leaves a core of cancer cells with high capacity for repair and renewal. The molecular properties identified in these cells may explain some of the unique characteristics of CSCs that control self-renewal and drive metastasis. The identification and cloning of human OCSCs can aid in the development of better therapeutic approaches for ovarian cancer patients.

TWISTing stemness, inflammation and proliferation of epithelial ovarian cancer cells through MIR199A2/214

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 IKKβ/NF-κB 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.

Stem-Like Ovarian Cancer Cells Can Serve as Tumor Vascular Progenitors†‡§

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. STEM CELLS 2009;27:2405–2413

Constitutive proteasomal degradation of TWIST-1 in epithelial–ovarian cancer stem cells impacts differentiation and metastatic potential

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.

NV-128, a novel isoflavone derivative, induces caspase-independent cell death through the Akt/mammalian target of rapamycin pathway.

Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase‐independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV‐128 as an inducer of cell death through a caspase‐independent pathway.

The Placental Syncytium and the Pathophysiology of Preeclampsia and Intrauterine Growth Restriction

Preeclampsia is associated with an increased release of factors from the placental syncytium into maternal blood, including the antiangiogenic factors soluble fms‐like tyrosine kinase‐1 and soluable endoglin, the antifibrinolytic factor plasminogen activator inhibitor‐1, prostanoids, lipoperoxides, cytokines, and microparticles. These factors are suggested to promote maternal endothelium dysfunction and are associated with placental damage in pregnancies also complicated with intrauterine growth restriction (IUGR). In this report, we briefly describe the interaction of syncytial factors with hypoxia, reactive oxygen species, and apoptosis in the pathophysiology of preeclampsia and IUGR. Given the critical role of the syncytium in these complications of pregnancy, we also present a novel methodology in which laser capture microdissection followed by Western blotting is used to assess levels of syncytial Fas ligand, a key protein in the apoptotic cascade.

Detection of cancer-related proteins in fresh-frozen ovarian cancer samples using laser capture microdissection.

Tumors are heterogeneous structures that contain different cell populations. Laser capture microdissection (LCM) can be used to obtain pure cancer cells from fresh-frozen cancer tissue and the surrounded environment, thus providing an accurate snapshot of the tumor and its microenvironment in vivo. We describe a new approach to isolate pure cancer cell population and evaluate protein expression. The process includes immunocytochemistry, laser microdissection, and western blot analysis. Using this technique, we can detect proteins such as X-linked inhibitor of apoptosis protein (XIAP) and Fas ligand (FasL) with as little as 1000 cells.

Abstract 3405: Epithelial ovarian cancer stem cells are the source of metastatic progenitor cells

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

Abstract B75: TWISTing Stemness, Inflammation, and Proliferation of Epithelial Ovarian Cancer Cells through MIR199A2/214

Introduction: Twist1, a basic helix‐loop‐helix (bHLH) transcription factor, has an important role in tumor biology by regulating epithelial‐mesenchymal transition. It also functions as a transcription repressor in NFκB‐dependent cytokine expression. Recently, we reported the identification and characterization of epithelial ovarian cancer (EOC) cells with stem‐like properties (Type I EOC cells). At the microRNA level, Type I EOC cells are characterized by low levels of hsa‐miR‐199a2 and hsa‐miR‐214. We also demonstrated the capacity of Type I EOC cells to differentiate into mature ovarian cancer cells (Type II EOC cells), which has lost stemness potential and express high levels of both hsa‐miR‐199a2 and hsa‐miR‐214. We also showed previously that hsa‐miR‐199a2 is able to regulate the levels of IKKβ and therefore have a direct effect on the NFκB pathway. Still, another group reported the regulatory control of hsa‐miR‐214 on the Akt pathway. In this study, we show that Twist1 is able to regulate the miR‐199a2/214 cluster in EOC cells and can therefore control both NFκB and Akt pathways. Methods: 5′‐RACE and 3′‐RACE was used to clone the full length pre‐miR‐199a gene. Real‐time PCR was used to determine the levels of Twist1 and miR‐199a2/214. Twist1 was knocked‐down in Type II EOC cells using siRNA. The effect of Twist1 knockdown on levels of IKKβ was determined by Western blot analysis. Cytokines were quantified using Luminex technology. Results: Characterization of full length of pre‐miR‐199a2 transcript reveals that the MIR199A2 gene contains a human microRNA cluster, miR‐199a2/214, and pre‐miR‐199a2 within the human Dnm3os gene (NCBI GeneID 474332). PCR analysis showed low levels of Twist1 and miR‐199a2/214 in Type I EOC cells but high levels of expression in Type II EOC cells. Knockdown of Twist1 in Type II cells induce a significant decrease in the levels both hsa‐miR‐199a2 and hsa‐miR‐214, and significant increase in IKKβ expression. Ectopic expression of hsa‐miR‐199a2 partially reverse the effect of Twist‐1 knockdown on the levels of IKKβ. The combination of knockdown Twist1 and TNFα stimulation in Type II EOC cells significantly increase Rantes, an NFκB‐dependent cytokine. Conclusion: We demonstrate for the first time that Twist1 plays a feedback role for the NFkB pathway by repressing IKKβ expression through hsa‐miR‐199a2. Twist1 inhibition of IKKβ expression through regulating hsa‐miR‐199a2, represents a novel negative feedback loop for the NFκB pathway. The demonstration that Twist1 can regulate the miR‐199a2/214 cluster and therefore both the IKKβ and Akt survival pathways, suggests the potential role of Twist1 in EOC differentiation. Furthermore it opens the opportunity to develop new approaches for targeting the ovarian cancer stem cells. Citation Information: Cancer Res 2009;69(23 Suppl):B75.