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 have identified a specific gene signature potentially associated with NB-TIC properties, which included ALDH1A2 and ALDH1A3 genes.
We further demonstrated the involvement of ALDH enzymatic activity on the aggressive properties and 4-hydroxycyclophosphamide resistance of NB. In addition, we showed that the specific knock-out of ALDH1A3 through CRISPR/Cas9 technology reduced NB 3D-anchorage independent growth and mediated a cell type-dependent reduction of TIC self-renewal capacities.
We have also recently investigated the particular implication of ALK-wt and the ALK-F1174L, and ALK-R1275Q activating mutations 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 development. These results demonstrated for the first time the in vivo oncogenic activity of ALK-wt and ALK putative role in the control of NCPC fate and tumor differentiation (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. Recent data from our laboratory suggest that ALK activation may impair NCPC differentiation potential.
As a blockage of early sympathetic lineage differentiation may represent a potential crucial initiating event during NB oncogenesis, we are currently elucidating the impact of ALK deregulation in the development and differentiation of the sympathetic lineage using diverse ALK mutant mouse models. Specifically, the transition from NCPC to differentiated sympathoadrenergic neuroblats is investigated in details in the developing sympathetic chain and the adrenal medulla of ALK-wt and mutant embryos by IF and ISH (RNAscope® technology).
To expend our knowledge on ALK target genes and signaling pathways activated in vivo and potentially related to NB aggressive phenotype and/or differentiation, we generated orthotopic NB xenografts from cells overexpressing ALK-wt, ALK-F1174L or ALK-R1275Q with the aim to uncover specific ALK-status-driven transcriptomic signature and phenotype.
These analyses should provide new insights into the molecular mechanisms responsible for dysfunctional neuroblastic development underlying NB oncogenesis, and may contribute to the identification of new therapeutic targets by dissecting the downstream effects of ALK-wt and ALK activating mutations.
Investigation of the role of TWIST1 in neuroblastoma aggressive phenotype 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 multifunctional 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 proliferation, survival, drug resistance, as well as on cancer stem cell properties, using primary and established NB cell lines TWIST1-positive or TWIST1-knocked-out through CRISPR/Cas9 technology. Particularly, to investigate in vivo the involvement of TWIST1 on NB cell tumorigenic and metastatic potential, we are using established and we will develop novel NB preclinical models.
Furthermore, we intend to meticulously dissect at a molecular level the mechanisms and signaling cascades activated by TWIST1 that contribute to the aggressive nature of NB, and to elucidate TWIST1 interacting partners. This project should contribute to improve our understanding on NB biology and to identify new key players of NB progression, which may serve as alternative drug targets.