Carman K.M. Ip

c-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/β-catenin–ATP-binding cassette G2 signaling

Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/β-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.

P70 S6 kinase in the control of actin cytoskeleton dynamics and directed migration of ovarian cancer cells

Ovarian cancer is highly metastatic with a poor prognosis. The serine/threonine kinase, p70 S6 kinase (p70S6K), which is a downstream effector of phosphatidylinositol 3-kinase/Akt pathway, is frequently activated in ovarian cancer. Here, we show that p70S6K is a critical regulator of the actin cytoskeleton in the acquisition of the metastatic phenotype. This regulation is through two important activities: p70S6K acts as an actin filament cross-linking protein and as a Rho family GTPase-activating protein. Ectopic expression of constitutively active p70S6K in ovarian cancer cells induced a marked reorganization of the actin cytoskeleton and promoted directional cell migration. Using cosedimentation and differential sedimentation assays, p70S6K was found to directly bind to and cross-link actin filaments. Immunofluorescence studies showed p70S6K colocalized with cytochalasin D-sensitive actin at the leading edge of motile cells. The p70S6K did not affect the kinetics of spontaneous actin polymerization, but could stabilize actin filaments by the inhibition of cofilin-induced actin depolymerization. In addition, we showed that p70S6K stimulated the rapid activation of both Rac1 and Cdc42, and their downstream effector p21-activated kinase (PAK1), but not RhoA. Depletion of p70S6K expression or inhibition of its activity resulted in significant inhibition of actin cytoskeleton reorganization and reduced migration, with a concomitant reduction in Rac1, Cdc42 and PAK1 activation, confirming that the effect was p70S6K specific. Similarly, the actin cytoskeleton reorganization/migratory phenotype could be reversed by expression of dominant negative Rac1 and Cdc42, or inhibition of PAK1. These results reveal a new direction for understanding the oncogenic roles of p70S6K in tumor progression.

Hepatocyte growth factor enhances proteolysis and invasiveness of human nasopharyngeal cancer cells through activation of PI3K and JNK

The hepatocyte growth factor (HGF) receptor, Met, is frequently overexpressed in nasopharyngeal cancer (NPC). Here, we showed for the first time that human NPC cells with high Met expression were more sensitive to the cell motility and invasion effect of HGF. The downregulation of Met by small interfering RNA decreased tumor cell invasion/migration. HGF significantly increased matrix metalloproteinase‐9 production. This was inhibited by blocking phosphatidylinositide 3‐kinase (PI3K) and c‐Jun N‐terminal kinase (JNK), but not extracellular signal‐regulated kinase 1/2 and p38 mitogen‐activated protein kinase signaling pathways. We also demonstrated that PI3K induced activation of JNK, with Akt as a potential point of this cross‐talk. These results provide new insights into the molecular mechanism responsible for NPC progression and metastasis.

Stemness and chemoresistance in epithelial ovarian carcinoma cells under shear stress

One of greatest challenges to the successful treatment of cancer is drug resistance. An exciting approach is the eradication of cancer stem cells (CSCs). However, little is known about key signals regulating the formation and expansion of CSCs. Moreover, lack of a reliable predictive preclinical model has been a major obstacle to discover new cancer drugs and predict their clinical activity. Here, in ovarian cancer, a highly chemoresistant tumor that is rapidly fatal, we provide the first evidence demonstrating the causal involvement of mechanical stimulus in the CSC phenotype using a customizable microfluidic platform and three-dimensional spheroids, which most closely mimic tumor behavior. We found that ovarian cancer cells significantly acquired the expression of epithelial-to-mesenchymal transition and CSC markers and a remarkable chemoresistance to clinically relevant doses of frontline chemotherapeutic drugs cisplatin and paclitaxel when grown under fluid shear stress, which corroborates with the physiological attainable levels in the malignant ascites, but not under static condition. Furthermore, we uncovered a new link of microRNA-199a-3p, phosphatidylinositol 3-kinase/Akt, and multidrug transporter activation in shear stress-induced CSC enrichment. Our findings shed new light on the significance of hydrodynamics in cancer progression, emphasizing the need of a flow-informed framework in the development of therapeutics.

A novel p70 S6 kinase-microRNA biogenesis axis mediates multicellular spheroid formation in ovarian cancer progression

Ovarian cancer is the leading cause of death of all gynecologic tumors, associated with widespread peritoneal dissemination and malignant ascites. Key to this is the ability to form multicellular spheroids (MCS); however, the tumor-specific factors that regulate MCS formation are unclear. p70 S6 kinase (p70S6K), which is a downstream effector of phosphatidylinositol 3-kinase/Akt, is frequently constitutively active in ovarian carcinoma. Here we identify p70S6K as a vital regulator of MCS formation. We also uncover a new mechanism of p70S6K function as a component of the microRNA biogenesis machinery in this process. We show that p70S6K phosphorylates, and inhibits the interaction of tristetraprolin (TTP) and Dicer that promotes the expression of a subset of miRNAs, including the maturation of miR-145. Twist and Sox9 are two divergent targets of miR-145, thereby enhancing N-cadherin, but not other cadherin, expression and MCS formation. Activating miR-145 suppresses ovarian tumor growth and metastasis in an orthotopic xenograft mouse model. Meta-analysis in the Oncomine database reveals that high p70S6K and low TTP levels are associated with ovarian tumor progression. These results define a critical link between p70S6K, miRNA maturation, and MCS formation that may underlie poor clinical outcome of ovarian cancer patients for developing novel therapeutic strategies.

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

Cadherin-Catenin Signaling in Ovarian Cancer Progression

Ovarian cancer is a highly metastatic disease and has the highest mortality rate of all gynecological tumors. In contrast to many other types of cancer that metastasize through lymphatics and/or hematogenous routes, ovarian cancer metastasizes by peritoneal dissemination, which relies on the ability of cancer cells to detach from the primary tumor, adhere to, and eventually invade through the peritoneum. This involves dynamic changes in cell-cell adhesion, which is primarily mediated by cell surface receptors known as cadherins. In this review, we will describe the unique profiles of cadherins with their associated signal molecules, catenins, in ovarian cancer and the roles of these adhesion molecules in disease development, tumor cell progression, and the formation of ascites. We will discuss how cadherins perform these functions and their link to a variety of signaling pathways. Finally, we will review the recent findings regarding the potential of cadherins as new therapeutic targets in the treatment of ovarian cancer.

Abstract 1449: p70 S6 kinase signals tristetraprolin/Dicer-mediated maturation of microRNA-145 to regulate tumor metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ovarian cancer is a highly metastatic tumor characterized by malignant ascites spheroids, which represent one of the most important prognostic factors of poor clinical outcome. Multicellular aggregates known as spheroids (MCS) are important for anchorage-independent growth and metastasis. MCS are also an important phenomenon of cancer stem cells. However, the factors that regulate MCS formation are largely unknown. MicroRNAs (miRNA) are short non-coding RNAs that are critically involved in different oncogenic events by actively modulating mRNA and protein synthesis. Despite their clinical significance, processes regulating miRNA biogenesis remain obscure. Here, we identify N-cadherin as a key regulator of MCS formation activated in response to p70 S6 kinase (p70S6K) signaling, a downstream effector of phosphatidylinositol 3-kinase/Akt which is hyperactive in human ovarian cancer. The results also identify a new mechanism of p70S6K function that mediates microRNA (miR)-145 coregulation of key transcription factors Twist and Sox-9, thereby enhancing N-cadherin and MCS formation. p70S6K regulates miR-145 by deactivating a distinct tristetraprolin (TPP)/Dicer program. The p70S6K phosphorylates TTP, leading to inhibition of interaction between TTP and Dicer and decreased TTP activity in miR-145 processing. Silencing of p70S6K enhances the TTP/Dicer interaction and its activating in regulating miR-145. These results providea novel regulatory mechanism of p70S6K involved in the formation and spread of MCS and insights on the development of new therapeutic targets (This work was supported by RGC grant HKU782111 and CUHK8/CRF/11R). Citation Format: Sophia S.N. Lam, Carman K.M. Ip, Alice S.T. Wong. p70 S6 kinase signals tristetraprolin/Dicer-mediated maturation of microRNA-145 to regulate tumor metastasis. [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 1449. doi:10.1158/1538-7445.AM2014-1449

Abstract 1527: Hepatocyte growth factor modulates adhesion of ovarian cancer cells to the peritoneum mesothelium through p70 S6 kinase

Ovarian cancer has the highest mortality of all gynecological cancers. These deaths are mainly due to the highly metastatic characteristic with already widespread peritoneal dissemination and malignant ascites at the time of diagnosis. However the molecular mechanisms that regulate ovarian cancer progression are poorly understood. A key step in this process is the adhesion of ovarian cancer cells to the peritoneal mesothelium. Hepatocyte growth factor (HGF) is present at high concentrations in ovarian cancer ascitic fluid. We show here a role for HGF signaling in the regulation of the peritoneal adhesion of ovarian cancer cells. HGF increased binding of ovarian cancer cells to peritoneal extracellular matrix (ECM) proteins fibronectin and laminin and their receptors α5, α6, and β1 integrin expression. Blockade of HGF resulted in reduced attachment of ovarian cancer cells to these ECM proteins, with a concomitant reduction of α5, α6, and β1 integrin expression, confirming that the effect was HGF specific. Similarly, the peritoneal ECM adhesion could be reversed by neutralizing antibodies to α5, α6, and β1 integrin. The adhesive phenotype and integrin expression activated by HGF could be blocked by specific inhibition of p70 S6 kinase (p70 S6K ) using short hairpin RNA or small molecule inhibitor, suggesting that HGF transmits the signal through p70 S6K . Importantly, it also inhibited adhesion to primary human peritoneal mesothelial cells. These results identify a potential novel mechanism for HGF-induced peritoneal adhesion, which is mediated through p70 S6K through regulation of α5, α6, and β1 integrin (This work is supported by the Hong Kong Research Grants Council Grant HKU7599/05M to A.S.T.W.). 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 1527. doi:10.1158/1538-7445.AM2011-1527

Abstract 5116: p70 S6 kinase regulates the actin cytoskeleton in ovarian cancer cell motility by binding and cross-linking actin filaments

p70 S6 kinase (p70 S6K ) is a downstream effector of phosphatidylinositol 3-kinase and is frequently activated in human ovarian cancer. Here we show for the first time that p70 S6K functions in actin cytoskeleton reorganization responsible for the acquisition of invasiveness during tumor progression. Ectopic expression of active p70 S6K in ovarian cancer cells induced dramatic reorganization of the actin cytoskeleton and promoted cell migration. Inhibition of p70 S6K by siRNA or small molecule inhibitor resulted in diminished cell migration and actin cytoskeleton reorganization, confirming that these effects were p70 S6K specific. By using cosedimentation and differential sedimentation assays, p70 S6K was found to bind actin filaments, and was more effective in the presence than in the absence of phosphorylation. Importantly, purified p70 S6K could be cosedimented with purified filamentous actin, indicating a direct association between p70 S6K and actin. The response to p70 S6K could be blocked with cytochalasin D, indicating that the binding of p70 S6K to the cytoskeleton is mediated by actin. In addition, our results suggest that this is a functional interaction, because p70 S6K was capable of generating specialized, actin-based cell morphologies via direct cross-linking activity. This bundling activity was confirmed by light scattering and electron microscopy. These results, in conjunction with our prior evidence that p70 S6K regulates Rac and Cdc42 GTPases, suggest that p70 S6K serves as an important regulator of the actin cytoskeleton, highlighting a role for p70 S6K in the regulation of tumor progression and metastasis in ovarian cancer (This work is supported by Hong Kong Research Grants Council Grant HKU7599/05M to A.S.T.W.). 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 5116.