Neuroblastoma (NB) is one of the deadliest solid tumors in childhood, which remains fatal in almost a third of patients, despite intensive and multimodal therapies. This disease originates from aberrant and impaired differentiation of neural crest (NC)-derived sympathoadrenal progenitors. NB exhibits an extremely high heterogeneity, both genetically and biologically, which is reflected in the clinical outcome, ranging from spontaneous regression to an extremely aggressive and fatal disease.
The LHOP has a long-standing interest in the biology and genetics of NB. In past years, we have provided clues to the typical phenotypic properties of NB with specific markers expression (GD2, HNK-1), and typical loss of ubiquitous MHC-I, and CD44 cell surface molecules. Further identification of dysfunctions in the apoptotic response in high-risk NB revealed deregulation death receptors (FAS), and pro-apoptotic molecules (caspase-8/10). Our data have underlined essential interactions between the two apoptotic pathways by the demonstration that NB cells sensitivity to death-ligands could be enhanced by co-treatment with sub-toxic concentrations of chemotherapeutic drugs or of histone deacetylase inhibitors (HDACIs), through activation of the intrinsic apoptotic pathway. Recently, we have demonstrated a differential pro-apoptotic role of caspase-10A and –D, and anti-apoptotic role of caspase-10B and -10G in death receptor signaling, which may be relevant for fine tuning of apoptosis initiation.
Our laboratory has also been interested in the elucidation of the mechanisms involved in NB multi-drug resistance, which represents a major obstacle in the successful treatment of high- risk NB. We revealed that acquisition of multidrug resistance phenotype may results from strong amplification of the 7q21 region, which includes the multidrug resistance 1 (MDR1) locus. We further demonstrated the involvement of the Wnt1 receptor FZD1 in mediating NB multidrug-resistance through the activation of the Wnt/b-catenin pathway.
In collaboration with Dr J-M Joseph we expanded our research to include investigations on the role of the CXCR4/CXCR7/CXCL12 chemokine axis in NB tumor growth and selective metastatic dissemination using various in vivo models. We highlighted the crucial role of CXCR4 in NB primary and secondary tumor growth, while CXCR7 elicited anti-tumorigenic properties, particularly in presence of CXCR4. We also revealed that both CXCL12 receptors are involved in a complex and organ-dependent control of NB metastatic cell homing.
In recent years we were particularly interested in the identification and characterization of NB tumor initiating cells (TIC).By a microarray time course analysis of serial NB neurospheres passages, we identified a specific gene signature potentially associated with NB-TIC properties. We also investigated the particular contribution of the anaplastic lymphoma kinase gene (ALK) in NB oncogenesis and aggressiveness. By overexpression of activating ALK mutations, and ALK-wt in neural crest progenitor cells (NCPC), we demonstrated that ALK-wt, and activating mutants were sufficient to induce formation of neural crest cell-derived tumors, and to maintain them in highly undifferentiated state, but not to drive NB tumor development, highlighting a putative role of ALK in impairing NCPC differentiation potential in vivo (see ALK project).
ALK in the control of neural crest differentiation and neuroblastic tumor development
The anaplastic lymphoma kinase gene (ALK) is overexpressed, mutated or amplified in the majority of NB. The two most frequent activating mutations, ALK-F1174L and ALK-R1275Q, were demonstrated to contribute to NB tumorigenesis in transgenic and/or knock-in mouse models, and to cooperate with MYCN in the oncogenic process. We investigated the implication of ALK-wt and ALK-F1174L, and ALK-R1275Q mutants in neural crest (NC) progenitor fate and NB oncogenesis using immortalized murine NC progenitor cells (NCPC). In vivo studies, performed by orthotopic implantations, indicated that the expression of ALK-wt or the ALK activating mutations in NCPC was sufficient to induce the formation of highly undifferentiated NC cell-derived tumors, but not to drive NB development2. These results demonstrated for the first time the in vivo oncogenic activity of ALK-wt and suggested a putative role for ALK in the control of NCPC fate and tumor differentiation.
We are currently investigating the mechanisms responsible for ALK-mediated control of NCPC differentiation and neuroblastomagenesis. Involvement of ALK and its putative target genes in controlling NCPC differentiation capacity and fate is explored in vitro and in vivo using ALK-transduced NCPC cell lines and specific inhibitor or silencing strategies. The influence of ALK in NB cell neuronal differentiation mediated by retinoic acid is also examined in vitro. Moreover, ALK expression pattern in NB clinical tissue samples will be analyzed to establish a possible correlation between ALK expression and neuroblastic cell differentiation. These analyses should provide new insights into the molecular mechanisms responsible for dysfunctional NC cell maturation at the origin of NB development, and may contribute to identify new therapeutic targets.
Investigation of the role of TWIST1 in neuroblastoma aggressiveness and metastasis
The reactivation of the embryonic transcription factors TWIST1/2 is frequent in cancer and correlates with poor prognosis across many neoplasms. TWIST1/2 act as multifonctionnal oncogenes by promoting drug resistance, EMT, invasion, metastasis, and cancer stem cell properties. Although the role of TWIST1/2 has been extensively studied in various cancers, their implication in NB remains poorly understood. We are currently investigating the impact of TWIST1 expression on various NB cell properties, such as survival, drug resistance, as well as on the generation and maintenance of cancer stem cell properties, using primary and established NB cell lines expressing TWIST1 or silenced for TWIST1 through CRISPR/Cas9 technology. Particularly, the role of TWIST1 on NB cell tumorigenic and metastatic potential will be investigated by using established and novel in vivo preclinical models. The characterization of the molecular mechanisms and signaling cascade activated by TWIST1 will contribute to the identification of new therapeutic targets and may results in the design of alternative therapies that are urgently needed to cure this disease.