Expression of E-Cadherin in Epithelial Cancer Cells Increases Cell Motility and Directionality through the Localization of ZO-1 during Collective Cell Migration

Collective cell migration of epithelial tumor cells is one of the important factors for elucidating cancer metastasis and developing novel drugs for cancer treatment. Especially, new roles of E-cadherin in cancer migration and metastasis, beyond the epithelial-mesenchymal transition, have recently been unveiled. Here, we quantitatively examined cell motility using micropatterned free edge migration model with E-cadherin re-expressing EC96 cells derived from adenocarcinoma gastric (AGS) cell line. EC96 cells showed increased migration features such as the expansion of cell islands and straightforward movement compared to AGS cells. The function of tight junction proteins known to E-cadherin expression were evaluated for cell migration by knockdown using sh-RNA. Cell migration and straight movement of EC96 cells were reduced by knockdown of ZO-1 and claudin-7, to a lesser degree. Analysis of the migratory activity of boundary cells and inner cells shows that EC96 cell migration was primarily conducted by boundary cells, similar to leader cells in collective migration. Immunofluorescence analysis showed that tight junctions (TJs) of EC96 cells might play important roles in intracellular communication among boundary cells. ZO-1 is localized to the base of protruding lamellipodia and cell contact sites at the rear of cells, indicating that ZO-1 might be important for the interaction between traction and tensile forces. Overall, dynamic regulation of E-cadherin expression and localization by interaction with ZO-1 protein is one of the targets for elucidating the mechanism of collective migration of cancer metastasis.

Role of RIP4 in lung adenocarcinoma differentiation

Jawahar Kopparam

Various studies have attributed NF-kB activation to promote KRAS mediated non-small cell lung cancer progression. In order to identify key genes regulating NF-kB activation and cell survival in lung adenocarcinoma, a synthetic lethal partner screen for KRAS was conducted in cell lines expressing mutant or wildtype KRAS. This screen identified an important role of Receptor-interacting serine/threonine protein kinase 4 (RIP4) in KRAS mediated oncogenesis. But altering RIP4 expression did not affect the viability of KRAS mutant cells, however a novel role of RIP4 in maintaining tumor differentiation was observed. Loss of epithelial differentiation and extracellular matrix remodeling are known to facilitate cancer progression and are associated with poor prognosis in patients with lung cancer. Bioinformatics analyses of human lung adenocarcinoma samples showed that poorly differentiated tumors express low levels of RIP4, whereas high levels are associated with better overall survival. Interleukin 6 (IL6) signaling is activated in lung adenocarcinoma expressing oncogenic KRAS and contributes to cancer invasiveness. Knockdown of RIP4 using shRNA in vitro enhanced activation of IL-6 signaling and also the ability of the cells to invade through collagen. In contrast, overexpression of RIP4 inhibited IL6-mediated Signal Transducer and activator of Transcription 3 (STAT3) activation, which abrogated IL6-dependent induction of lysyl oxidase, a collagen cross-linking enzyme. Tumor-specific Rip4 knockdown in an autochthonous mouse model of lung adenocarcinoma initiated by Kras(G12D) expression with loss of p53, progressed to a poorly differentiated state marked by an increase in Hmga2, reduced Ttf1 and enrichment of genes regulating extracellular matrix (ECM) remodeling and JAK-STAT signaling. Activated STAT3 can enhance NF-kB activation by interacting with NF-kB and promoting its nuclear retention. Tumors with RIP4 knockdown also exhibited enhanced STAT3- NF-kB interaction suggesting a cross talk between the two pathways. Tail vein injections of cells overexpressing RIP4 showed a reduced potential to invade and form tumors. Analysis of factors regulating RIP4 expression revealed that RIP4 is induced in a p53 dependent manner in response to chemotherapy. Altering the induced RIP4 levels in vitro had a modest effect on chemotherapy induced cell death. Our work has identified that loss of RIP4 enhances IL6 signaling in lung cancer cells, promoting the expression of ECM remodeling genes and cancer dedifferentiation.

EPFL2016

Hes1 as a Mediator of Notch Signalling in T-cell Development and Notch1- Induced T-ALL

Agnieszka Anna Wendorff

The evolutionarily conserved Notch signalling pathway controls a broad spectrum of cell fate decisions in organisms as diverse as fruit flies and mice. Whithin the murine haematopoietic system, Notch1 is indispensable for T cell development as conditional inactivation of Notch1 in bone marrow (BM) precursors results in a complete block in T-cell development and ectopic B-cell development in the thymus. Conversely, constitutive activation of Notch1 signalling plays a causative role in the development of acute T-cell lymphoblastic leukaemia (T-ALL). Numerous mutations within the Notch1 gene, which result in aberrent activation of Notch1 signalling, have been identified in > 50% of human T-ALL patients. Therefore, gain-of-function studies in murine models of T-ALL have been employed by many laboratories to elucidate the molecular mechanisms acting either cooperatively or downstream of oncogenic Notch1 signalling in development and progression of T-ALL. The best-characterized Notch1 target gene to date is a member of the bHLH family of transcriptional repressors, Hes1. This study focuses on the role of Hes1 as a mediator of Notch1 signalling in adult murine haematopoietic development, as well as the requirement for Hes1 in T-ALL development and maintenance. Conditional inactivation of Hes1 in adult murine BM cells results in severe thymic hypoplasia, while myeloid and B cell development remain unperturbed. Upon transplantation in congenic recipients, Hes1-/- progenitors give rise to only a small number of mature T cells. Hes1-inactivation does not however cause ectopic B-cell development in the thymus, and therefore does not phenocopy the loss of Notch1. Thus, while Notch1 signalling is indispensable for T versus B-cell fate specification, this function is not mediated exclusively via Hes1. Strikingly, in a competitive BM chimera setting, Hes1-/- progenitors completely fail to generate T cells. The mechanism by which Hes1 inactivation results in depleted T cell numbers remains to be elucidated, although we have shown that loss of Hes1 does not result in enhanced cell death. In addition, preliminary data are indicative of cell-cycle perturbation in Hes1-deficient immature T cells. Finally, we have demonstrated that upon IT transplantation, Hes1-deficient progenitors successfully give rise to T-cells at all stages of development. This data strongly indicates that Hes1 deficiency does not result in an intrinsic developmental defect but rather in the inability of T cell progenitors to efficiently home to the thymus from the BM. Upregulation of Hes1 expression has been observed in both human T-ALL cell lines as well as murine primary Notch1-induced T-ALL samples. However, the function of Hes1 as an effector of oncogenic Notch1 signalling in modulating disease onset or progression remained to be investigated. The requirement for Hes1 as a mediator of Notch1-induced T-ALL was addressed by inducing simultaneous deletion of Hes1 and expression of a dominant active form of Notch1 (NICD) in a murine model of T-ALL. Our data demonstrate that Hes1 is essential for the development of NICD-induced T-ALL. Loss of Hes1 prevents ectopic development of DP (CD4+CD8+) leukemic T cells in the BM and the subsequent accumulation of DP T cells in peripheral tissues, a hallmark of T-ALL, and consequently results in 10-fold increased life expectancy of experimental animals. We have further determined that Hes1 is required for the maintenance of T-ALL by inactivating Hes1 in a retrovirally pre-induced T-ALL model. Deletion of Hes1 in leukemic DP cells results in rapid cell death and enhanced survival. These data demonstrate that Hes1 is required as a mediator of Notch1 signalling in normal T cell development, and is indispensable to mediate the oncogenic effect of constitutive Notch1 activation in the induction of T-ALL.

EPFL2010

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.

EPFL2014