Baptiste Gras

EMT Inducers Catalyze Malignant Transformation of Mammary Epithelial Cells and Drive Tumorigenesis towards Claudin-Low Tumors in Transgenic Mice

The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT–inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell–like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation.

Endoplasmic reticulum calcium release through ITPR2 channels leads to mitochondrial calcium accumulation and senescence

Senescence is involved in various pathophysiological conditions. Besides loss of retinoblastoma and p53 pathways, little is known about other pathways involved in senescence. Here we identify two calcium channels; inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) (also known as inositol 1,4,5-triphosphate receptor 2 (IP3R2)) and mitochondrial calcium uniporter (MCU) as new senescence regulators in a loss-of-function genetic screen. We show that loss of ITPR2, known to mediate endoplasmic reticulum (ER) calcium release, as well as loss of MCU, necessary for mitochondrial calcium uptake, enable escape from oncogene-induced senescence (OIS). During OIS, ITPR2 triggers calcium release from the ER, followed by mitochondrial calcium accumulation through MCU channels. Mitochondrial calcium accumulation leads to a subsequent decrease in mitochondrial membrane potential, reactive oxygen species accumulation and senescence. This ER-mitochondria calcium transport is not restricted to OIS, but is also involved in replicative senescence. Our results show a functional role of calcium release by the ITPR2 channel and its subsequent accumulation in the mitochondria.

PLA2R1 Mediates Tumor Suppression by Activating JAK2

Little is known about the physiological role of the phospholipase A2 receptor (PLA2R1). PLA2R1 has been described as regulating the replicative senescence, a telomerase-dependent proliferation arrest. The downstream PLA2R1 signaling and its role in cancer are currently unknown. Senescence induction in response to activated oncogenes is a failsafe program of tumor suppression that must be bypassed for tumorigenesis. We now present evidence that PLA2R1 functions in vitro as a tumor suppressor, the depletion of which is sufficient to escape oncogene-induced senescence (OIS), thereby facilitating oncogenic cell transformation. Furthermore, mice that are genetically deficient in PLA2R1 display increased sensitivity to RAS-induced tumorigenesis by facilitating OIS escape, highlighting its physiological role as a tumor suppressor. Unexpectedly, PLA2R1 activated JAK2 and its effector signaling, with PLA2R1-mediated inhibition of cell transformation largely reverted in JAK2-depleted cells. This finding was unexpected as the JAK2 pathway has been associated mainly with protumoral functions and several inhibitors are currently in clinical trials. Taken together, our findings uncover an unanticipated tumor suppressive role for PLA2R1 that is mediated by targeting downstream JAK2 effector signaling.

PLA2R1 kills cancer cells by inducing mitochondrial stress

Little is known about the biological functions of the phospholipase A2 receptor (PLA2R1) except that it has the ability to bind a few secreted phospholipases A2 (sPLA2s). We have previously shown that PLA2R1 regulates senescence in normal human cells. In this study, we investigated the ability of PLA2R1 to control cancer cell growth. Analysis of expression in cancer cells indicates a marked PLA2R1 decrease in breast cancer cell lines compared to normal or nontransformed human mammary epithelial cells. Accordingly, PLA2R1 ectopic expression in PLA2R1-negative breast cancer cell lines led to apoptosis, whereas a prosenescence response was predominantly triggered in normal cells. PLA2R1 structure-function studies and the use of chemical inhibitors of sPLA2-related signaling pathways suggest that the effect of PLA2R1 is sPLA2-independent. Functional experiments demonstrate that PLA2R1 regulation of cell death is driven by a reactive oxygen species (ROS)-dependent mechanism. While screening for ROS-producing complexes involved in PLA2R1 biological responses, we identified a critical role for the mitochondrial electron transport chain in PLA2R1-induced ROS production and cell death. Taken together, this set of data provides evidence for an important role of PLA2R1 in controlling cancer cell death by influencing mitochondrial biology.

Snail Family Members Unequally Trigger EMT and Thereby Differ in Their Ability to Promote the Neoplastic Transformation of Mammary Epithelial Cells

By fostering cell commitment to the epithelial-to-mesenchymal transition (EMT), SNAIL proteins endow cells with motility, thereby favoring the metastatic spread of tumor cells. Whether the phenotypic change additionally facilitates tumor initiation has never been addressed. Here we demonstrate that when a SNAIL protein is ectopically produced in non-transformed mammary epithelial cells, the cells are protected from anoikis and proliferate under low-adherence conditions: a hallmark of cancer cells. The three SNAIL proteins show unequal oncogenic potential, strictly correlating with their ability to promote EMT. SNAIL3 especially behaves as a poor EMT-inducer comforting the concept that the transcription factor functionally diverges from its two related proteins.

Multidrug resistance protein 3 loss promotes tumor formation by inducing senescence escape

Oncogenic-stress-induced senescence (OIS) is a stress response allowing normal cells, when receiving oncogenic signals, to stably arrest their proliferation. OIS thus acts to prevent aberrant cell proliferation and tumor formation. To identify novel tumor suppressive pathways, we have recently completed a loss-of-function genetic screen to identify novel genes promoting escape from OIS and thus, potentially, tumor formation when their functions are lost. Using this approach, we unexpectedly found that loss of function of the multidrug resistance protein 3 (MRP3 or ABCC3) promotes escape from OIS in human epithelial cells. Importantly, ABCC3 expression is reduced in human skin tumors, and ABCC3-knockout mice display increased sensitivity to RAS-induced skin carcinogenesis, concomitantly with decreased OIS. ABCC3 participates in resistance to chemotherapy via its transporter activity. Our data show that this transporter activity is involved in ABCC3-induced senescence, demonstrating that this protein has a complex role in cancer, since its loss of function may promote escape from OIS and tumor formation whereas its gain of function promotes resistance to chemotherapy.

The SCN9A channel and plasma membrane depolarization promote cellular senescence through Rb pathway

Oncogenic signals lead to premature senescence in normal human cells causing a proliferation arrest and the elimination of these defective cells by immune cells. Oncogene‐induced senescence (OIS) prevents aberrant cell division and tumor initiation. In order to identify new regulators of OIS, we performed a loss‐of‐function genetic screen and identified that the loss of SCN9A allowed cells to escape from OIS. The expression of this sodium channel increased in senescent cells during OIS. This upregulation was mediated by NF‐κB transcription factors, which are well‐known regulators of senescence. Importantly, the induction of SCN9A by an oncogenic signal or by p53 activation led to plasma membrane depolarization, which in turn, was able to induce premature senescence. Computational and experimental analyses revealed that SCN9A and plasma membrane depolarization mediated the repression of mitotic genes through a calcium/Rb/E2F pathway to promote senescence. Taken together, our work delineates a new pathway, which involves the NF‐κB transcription factor, SCN9A expression, plasma membrane depolarization, increased calcium, the Rb/E2F pathway and mitotic gene repression in the regulation of senescence. This work thus provides new insight into the involvement of ion channels and plasma membrane potential in the control of senescence.

Abstract 3066: Comprehensive interactome of embryonic transcription factors, mitogenic stresses and miRNAs involved in breast tumorigenesis.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epithelial-to-Mesenchymal Transition (EMT) is associated with tumour initiation and progression. EMT is associated with a profound genetic reprogramming including reactivation of embryonic transcription factors (ETFs) and severe changes in miRNA expression. Cross-regulation between EMT-inducers and miRNAs was previously described like the Zeb1-miR-200 feed-back loop. Our goal is to determine whether specific expression networks between ETFs, miRNAs and mitogenic stresses exist in tumors, and represents a driving force towards transformation in breast tumorigenesis. We performed oncogenic cooperation assays in immortalized human mammary epithelial cells (HMEC-hTert) by using an EMT-inducer expression library in combination with various mitogenic stresses (activated form of beta-catenin, EGFR, shPTEN, shP53). As recently demonstrated in the laboratory, these combinations were efficient in transforming human cells, as assessed by soft-agar colony assay. EMT-inducer expression was next analyzed in a large number of colonies to evaluate a potential specificity of EMT-inducers according to the mitogenic insult. We found that expression of Slug was specifically enriched in colonies generated when EGFR is highly expressed, whereas Zeb1 expression was selected following PTEN depletion, p53 inactivation and beta-catenin activation. FoxC2 expression was enriched in cells expressing the activated form of beta-catenin. The prediction of specificity of the identified combinations is currently being validated by bioinformatics tools. In parallel, we developed an in silico approach utilizing predictive algorithms to identify novel miRNAs targeting embryonic transcription factors. We have identified two novel miRNAs able to regulate EMT inducers and we have validated their down-regulation in a panel of basal B breast cancer cell lines. We have also found new feedback loops between these two miRNA and some ETFs. The interplay between miRNAs, mitogenic proteins and EMT inducers will be further examined by assessing their expression in a large cohort of Basal B/ Claudin-low human breast tumours, a recently identified subtype displaying a mammary stem cell signature and EMT features. Citation Format: Emmanuelle Ruiz, Stephanie Courtois-Cox, Baptiste Gras, Amelie Veron, Stephane Ansieau, Alain Puisieux, Caroline MOYRET-LALLE. Comprehensive interactome of embryonic transcription factors, mitogenic stresses and miRNAs involved in breast tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2013-3066

Abstract 2417: Oncogenic properties shared by all Snail family members

The epithelial to mesenchymal transition (EMT) is a latent embryonic process promoting cell migration by transiently affording epithelial cells a mesenchymal phenotype, motility and a survival advantage. The inappropriate activation of this program in adult cells is associated with several pathologies including tumor development where EMT promotes cancer cell dissemination and multidrug resistance development. We have recently demonstrated that EMT-inducers, beyond their prometastatic potential, facilitate the malignant transformation of human mammary epithelial cells in vitro and promote breast carcinogenesis in vivo. Although their ability to alleviate senescence and apoptosis failsafe program induction unquestionably contributes to their oncogenic potentials, we sought to investigate whether EMT inducers share additional properties. To this end, the activities of all three Snail members were examined. Likewise SNAI1 and SNAI2, SNAI3 expression is aberrantly activated in cancers, especially in mammary carcinomas. Ectopic expression of all three proteins in mammary epithelial cells induces their commitment into EMT, provides cells with a survival advantage in low adherent conditions and promotes their neoplastic transformation, with a similar gradient of efficiencies. Interestingly, this efficiency parallels with their ability to upregulate intracellular signaling pathways. We will update on the progress of our work aimed at understanding how these dysregulations impact on their oncogenic and prometastatic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2417. doi:1538-7445.AM2012-2417

Abstract 4813: EMT inducers catalyze malignant transformation of mammary epithelial cells and drive tumorigenesis towards claudin-low tumors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The malignant transformation of human epithelial cells is generally described as a multistep process resulting from the accumulation of five to seven rate-limiting changes. Here, we challenge this dogma and demonstrate that this number is radically reduced when cells undergo an epithelial-mesenchymal transition (EMT). EMT is an embryonic transdifferentiation process that consists of the conversion of polarized epithelial cells into motile mesenchymal ones. EMT-inducing transcription factors, including Twist and Zeb proteins, are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. We demonstrate that, beyond their prometastatic potential, EMT inducers also act as potent drivers of tumorigenesis. We indeed show that the Twist1 EMT-inducing transcription factor promotes breast and skin cancer development in vivo in cooperation with the K-RasG12D oncoprotein. Importantly, in the model of breast tumorigenesis, transgene expression in differentiated mammary epithelial cells leads to the development of undifferentiated tumors exhibiting all the characteristic features of the claudin-low subtype. These observations challenge the concept that this tumor subtype specifically arises from transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Consistently, oncogenic cooperation assays performed in human mammary epithelial cells with Twist or Zeb EMT-inducers in combination with H-RasG12V generate transformed cell lines displaying all characteristics of claudin-low tumors including mesenchymal features, undifferentiated traits, and stem-cell-like properties. EMT might thus drive the development of claudin-low tumors by exhibiting a dual role in cell transformation and dedifferentiation. In other terms, the claudin-low tumor subtype of breast cancers might thus constitute a first example of human adult malignancies driven by aberrant reactivation of an embryonic transdifferentiation program. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4813. doi:1538-7445.AM2012-4813