Adeline Ledoux

Sphingosine kinase 1 (SphK1) promotes cell proliferation and survival, and its abundance is often increased in tumors. SphK1 produces the signaling lipid sphingosine 1-phosphate (S1P), which activates signaling cascades downstream five G protein–coupled receptors (S1P_1–5) to modulate vascular and immune system function and promote proliferation. We identified a new function of the SphK1-S1P pathway specifically in the control of mitosis. SphK1 depletion in HeLa cells caused prometaphase arrest, whereas its overexpression or activation accelerated mitosis. Increasing the abundance of S1P promoted mitotic progression, overrode the spindle assembly checkpoint (SAC), and led to chromosome segregation defects. S1P was secreted through the transporter SPNS2 and stimulated mitosis by binding to and activating S1P₅ on the extracellular side, which then activated the intracellular phosphatidylinositol 3-kinase (PI3K)–AKT pathway. Knockdown of S1P₅ prevented the S1P-induced spindle defect phenotype. RNA interference assays revealed that the mitotic kinase Polo-like kinase 1 (PLK1) was an important effector of S1P-S1P₅ signaling-induced mitosis in HeLa cells. Our findings identify an extracellular signal and the downstream pathway that promotes mitotic progression and may indicate potential therapeutic targets to inhibit the proliferation of cancer cells.

There are two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-κB2(p52)/RelB containing complexes, and is dependent on IKKα and NF-κB inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-κB pathway subunits NF-κB2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-κB2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-κB target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-κB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-κB also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-κB can inhibit tumor suppressor function and promote tumorigenesis. Although the classical NF-κB pathway is frequently associated with the induction of cellular senescence and the senescence associated secretory phenotype (SASP), the role of the alternative NF-κB pathway, which is frequently activated in hematological malignancies as well as some solid tumors, has not been defined. We therefore investigated the role of the alternative NF-κB pathway in this process. Here we report that NF-κB2 and RelB, the effectors of the alternative NF-κB pathway, suppress senescence through inhibition of p53 activity. Using primary human fibroblasts, we demonstrate that this is accomplished through NF-κB2/RelB dependent control of a previously unknown pathway, incorporating regulation of CDK4 and 6 expression as well as regulators of p21WAF1 and p53 protein stability. Loss of NF-κB2/RelB results in suppression of retinoblastoma (Rb) tumour suppressor phosphorylation, which in turn leads to inhibition of EZH2 expression and de-repression of p53 activity. Interestingly, we find that CD40 ligand stimulation of cells from Chronic Lymphocytic Leukemia patients, which strongly induces the alternative NF-κB pathway, also induces EZH2 expression. We propose that the alternative NF-κB pathway can promote tumorigenesis through suppression of p53 dependent senescence, a process that may have relevance to cancer cells retaining wild type p53.

The tumour microenvironment can influence its response to anticancer therapies; here, the enzyme FAK in endothelial cells is shown to have a role in the induction of a number of cytokines during chemotherapy or irradiation, which in turn protect tumours from DNA-damaging agents. The tumour microenvironment can influence the response to anti-cancer therapies, and for example render cancers resistant to chemotherapy. Kairbaan Hodivala-Dilke and colleagues now show that in endothelial cells of the tumour vasculature, the enzyme known as focal adhesion kinase (FAK) has a role in the induction of a number of cytokines during chemotherapy or irradiation, which in turn protect tumours from DNA-damaging agents. The authors show that tumours can be sensitized to chemotherapy and radiotherapy when FAK is inhibited in endothelial cells, offering a possible avenue to novel therapeutic combinations that can overcome chemoresistance. Chemoresistance is a serious limitation of cancer treatment1. Until recently, almost all the work done to study this limitation has been restricted to tumour cells2. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

The NF-κB (nuclear factor κB) transcription factor family is a pleiotropic regulator of many cellular pathways, providing a mechanism for the cell to respond to a wide variety of stimuli and environmental challenges. It is not surprising therefore that an important component of NF-κB's function includes regulation of the cell cycle. However, this aspect of its behaviour is often overlooked and receives less attention than its ability to induce inflammatory gene expression. In the present article, we provide an updated review of the current state of our knowledge about integration of NF-κB activity with cell cycle regulation, including newly characterized direct and indirect target genes in addition to the mechanisms through which NF-κB itself can be regulated by the cell cycle.

Activation of the NFκB signaling pathway allows the cell to respond to infection and stress and can affect many cellular processes. As a consequence, NFκB activity must be integrated with a wide variety of parallel signaling pathways. One mechanism through which NFκB can exert widespread effects is through controlling the expression of key regulatory kinases. Here we report that NFκB regulates the expression of genes required for centrosome duplication, and that Polo-like kinase 4 (PLK4) is a direct NFκB target gene. RNA interference, chromatin immunoprecipitation, and analysis of the PLK4 promoter in a luciferase reporter assay revealed that all NFκB subunits participate in its regulation. Moreover, we demonstrate that NFκB regulation of PLK4 expression is seen in multiple cell types. Significantly long-term deletion of the NFκB2 (p100/p52) subunit leads to defects in centrosome structure. This data reveals a new component of cell cycle regulation by NFκB and suggests a mechanism through which deregulate...

Lung cancer is the leading cause of cancer-related death worldwide, mainly due to its highly metastatic properties. Previously, we reported an inverse correlation between RhoB expression and the progression of the lung cancer, occurring between preinvasive and invasive tumors. Herein, we mimicked the loss of RhoB observed throughout lung oncogenesis with RNA interference in nontumoral bronchial cell lines and analyzed the consequences on both cell transformation and invasion. Down-regulation of RhoB did not modify the cell growth properties but did promote migration and invasiveness. Furthermore, RhoB depletion was accompanied by modifications of actin and cell adhesion. The specific activation of the Akt1 isoform and Rac1 was found to be critical for this RhoB-mediated regulation of migration. Lastly, we showed that RhoB down-regulation consecutive to K-RasV12 cell transformation is critical for cell motility but not for cell proliferation. We propose that RhoB loss during lung cancer progression relates to the acquisition of invasiveness mediated by the phosphatidylinositol 3-kinase (PI3K)/AKT and Rac1 pathways rather than to tumor initiation. [Cancer Res 2009;69(15):6092–99]

RNA interference (RNAi)-mediated gene silencing approaches appear very promising for therapies based on the targeted inhibition of disease-relevant genes. The major hurdle to the therapeutic development of RNAi strategies remains, however, the efficient delivery of the RNAi-inducing molecules, the short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to the target tissue. With respect to cancer treatment the development of efficient delivery methods into solid tumors appears as a critical issue. However, very few studies have addressed this problem. In this study we have investigated the contribution of electrically mediated delivery of siRNA into murine tumors stably expressing an enhanced green fluorescent protein (EGFP) target reporter gene. The silencing of EGFP gene expression was quantified over time by fluorescence imaging in the living animal. Our study indicates that electric field can be used as an efficient method for siRNA delivery and associated gene silencing into cells of solid tumors in vivo.