Rui Chen

TLR-4 Signaling Promotes Tumor Growth and Paclitaxel Chemoresistance in Ovarian Cancer

Evidence suggests that an inflammatory profile of cytokines and chemokines persisting at a particular site would lead to the development of a chronic disease. Recent studies implicate bacterial infection as one possible link between inflammation and carcinogenesis; however, the crucial molecular pathways involved remain unknown. We hypothesized that one possible upstream signaling pathway leading to inflammation in carcinogenesis may be mediated by Toll-like receptors (TLR). We describe for the first time an adaptive mechanism acquired by ovarian cancer cells that allows them to promote a proinflammatory environment and develop chemoresistance. We propose that the TLR-4-MyD88 signaling pathway may be a risk factor for developing cancer and may represent a novel target for the development of biomodulators. Our work explains how bacterial products, such as lipopolysaccharide, can promote, directly from the tumor, the production of proinflammatory cytokines and the enhancement of tumor survival. In addition, we provide new evidence that links TLR-4 signaling, inflammation, and chemoresistance in ovarian cancer cells.

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.

Regulation of IKKβ by miR-199a affects NF-κB activity in ovarian cancer cells

Cancer progression is an abnormal form of tissue repair characterized by chronic inflammation. IκB kinase-β (IKKβ) required for nuclear factor-κB (NF-κB) activation plays a critical role in this process. Using EOC cells isolated from malignant ovarian cancer ascites and solid tumors, we identified IKKβ as a major factor promoting a functional TLR–MyD88–NF-κB pathway that confers to EOC cell the capacity to constitutively secrete proinflammatory/protumor cytokines and therefore promoting tumor progression and chemoresistance. Furthermore, we describe for the first time the identification of the microRNA hsa-miR-199a as a regulator of IKKβ expression. Our study describes the property of ovarian cancer cells to enhance the inflammatory microenvironment as a result of the expression of an active IKKβ pathway. Identification of these markers in patients tumor samples may facilitate the adequate selection of treatment and open new venues for the development of effective therapy for chemoresistant ovarian cancers.

Trophoblast-macrophage interactions: a regulatory network for the protection of pregnancy.

Problemu2002 Macrophages are one of the first immune cells observed at the implantation site. Their presence has been explained as the result of an immune response toward paternal antigens. The mechanisms regulating monocyte migration and differentiation at the implantation site are largely unknown. In the present study, we demonstrate that trophoblast cells regulate monocyte migration and differentiation. We propose that trophoblast cells ‘educate’ monocytes/macrophages to create an adequate environment that promote trophoblast survival.

Cancers take their Toll--the function and regulation of Toll-like receptors in cancer cells

Cancer could be deemed as an abnormal and uncontrolled tissue repair process. Therefore, it would not be surprising that factors that function in the tissue repair process, such as cytokines, chemokines, growth factors and Toll-like receptor (TLR) ligands, as well as growth signals for compensatory proliferation, would also be key factors in regulating and enhancing cancer progression. The TLR pathways, which play a critical role in tissue repair, are also key regulators in cancer progression as well as chemoresistance. TLRs serve as cell surface sensors that can initiate pathways leading to proliferation and chemoresistance; as well as mediators that are able to regulate the infiltrating immune cells to provide further support for cancer progression.

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.

Inflammation, cancer and chemoresistance: taking advantage of the toll-like receptor signaling pathway.

The association between chronic inflammation and cancer has long been observed. Furthermore, NF‐κB activation and the subsequent production of cytokines, chemokines, growth factors, and antiapoptotic proteins has been found to be involved in cancer progression and chemoresistance. However, the signals inducing NF‐κB in cancer cells are still not well understood. Here, we reviewed the association between chronic inflammation and cancer, the role of NF‐κB and its inhibitors as potential anticancer drugs, and Toll‐like receptors as possible signal initiators for NF‐κB activation and inflammation‐induced carcinogenesis and chemoresistance. Furthermore, we propose that, the stimulation of Toll‐like receptors by microbial components and/or endogenous ligands may represent the initial signal promoting a proinflammatory environment that will enhance tumor growth and chemoresistance.

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.

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 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.