Targeting MYC and MYC target genes as therapeutic strategies in childhood medulloblastoma

MYC and its target genes have become attractive targets for cancer therapy, due to the correlations of the oncogene with high grade malignancy and poor prognosis in different types of tumors. My research investigations focused on novel strategies to target the oncogene MYC and/or its target genes, in the context of MYC-overexpressing embryonal tumors (ET), and in particular in medulloblastoma (MB). Although the MYC functions during normal development and oncogenesis in various systems have been extensively investigated, the transcriptional targets mediating MYC effects in MB are still elusive. A better knowledge about the MYC’s effector genes involved in MB onset and progression is of great relevance in order to find novel and suitable therapeutic strategies. Our experimental approach to investigate MYC-regulated genes consisted of using a model of MB-derived cells and profiling them by cDNA microarray upon genetic manipulation aiming at either over-expressing or down- regulating MYC. We defined a list of 209 candidates with potential relevance to MYC- dependent cellular responses in MB. The gene expression analysis we performed brought to our attention components of the bone morphogenetic protein (BMP) signaling pathway, which plays a crucial role during the development of the cerebellum. Our investigation shows for the first time the existence of a functional link between the over- expression of MYC and abnormal regulation of the BMP pathway in a model of MB. Taken together, the studies presented in this dissertation have investigated novel strategies to target the oncogene MYC and its target genes in ETs. Further validation, as well as translation to clinical studies, can, in the future, shed more light on the biology of these pediatric tumors and contribute to an improvement of therapies. Das Onkogen MYC und die dadurch regulierten Gene stehen seit geraumer Zeit im Forschungsinteresse für die Entwicklung neuer Krebstherapien, da MYC mit dem Auftreten hochgradig maligner Tumore und schlechter Prognose in unterschiedlichen Krebsarten korreliert. Im Mittelpunkt meiner Forschung stand die Untersuchung neuer „Targeting-Strategien“ für das Onkogen MYC in embryonalen Tumoren inklusive Medulloblastom, dem häufigsten kindlichen malignen Hirntumor. Obwohl die Funktionen von MYC sowohl in der normalen Entwicklung als auch in onkogenen Prozessen bereits in verschiedenen Modellsystemen intensiv untersucht wurden, sind die transkriptionellen Targets, welche die Effekte der Überexpression von MYC im Medulloblastom charakterisieren, weitgehend unbekannt. Um Medulloblastom- spezifische MYC-regulierte Gene zu finden, wurden MB Zellen mit einer MYC – Überexpression und MB Zellen, in denen MYC herunterreguliert war, einer cDNA Microarray Analyse unterzogen. Aus diesem Versuch konnten wir eine Liste von 209 Genen mit potentieller MYC-abhängigen Relevanz erstellen, die u. a. Gene des „Bone Morphogenetic Protein“ (BMP) -Signalwegs beinhaltete. Dieser Signalweg spielt eine wichtige Rolle in der Entwicklung des Kleinhirns. Unsere Forschung zeigte zum ersten Mal eine Verbindung zwischen der Überexpression von MYC und einer Dysregulierung des BMP-Signalwegs im Medulloblastom. Unsere Ergebnisse lassen darauf schliessen, dass der Wachstumsfaktor BMP7 MYC-abhängig induziert wird und es sich hierbei um ein direktes MYC-Target handelt. In der vorliegenden Dissertation wurden neue “Targeting-Strategien” für das Onkogen MYC in embryonalen Tumoren untersucht. Dadurch wurde das Verständnis der Biologie dieser Tumoren verbessert und es wurden Grundlagen geschaffen für die Entwicklung von zukünftigen Therapiemöglichkeiten

MicroRNA signatures as biomarkers and therapeutic target for CNS embryonal tumors: the pros and the cons

Embryonal tumors of the central nervous system represent a heterogeneous group of childhood cancers with an unknown pathogenesis; diagnosis, on the basis of histological appearance alone, is controversial and patients' response to therapy is difficult to predict. They encompass medulloblastoma, atypical teratoid/rhabdoid tumors and a group of primitive neuroectodermal tumors. All are aggressive tumors with the tendency to disseminate throughout the central nervous system. The large amount of genomic and molecular data generated over the last 5-10 years encourages optimism that new molecular targets will soon improve outcomes. Recent neurobiological studies have uncovered the key role of microRNAs (miRNAs) in embryonal tumors biology and their potential use as biomarkers is increasingly being recognized and investigated. However the successful use of microRNAs as reliable biomarkers for the detection and management of pediatric brain tumors represents a substantial challenge. This review debates the importance of miRNAs in the biology of central nervous systemembryonal tumors focusing on medulloblastoma and atypical teratoid/rhabdoid tumors and highlights the advantages as well as the limitations of their prospective application as biomarkers and candidates for molecular therapeutic targets

Detection and quantification of extracellular microRNAs in medulloblastoma

Aim: Medulloblastoma (MB) is the most common malignant brain tumor in children. The crucial role of extracellular-microRNAs (ex-miRNAs) in cancer has been widely recognized; however, their role in MB remains unknown. This study aimed to investigate MB-driven ex-miRNAs.Methods: Microarray analysis was used to disclose the identity and quantity of key miRNAs excreted in culture-medium (CM) of 3 human MB cell lines and cerebrospinal fluid (CSF) of brain tumors (including MB) and leukemia patients. MiRNA expression was validated by quantitative reverse transcription polymerase chain reaction.Results: We have demonstrated that the 3 MB cell lines tested commonly expressed 1,083 miRNAs in their spent CM. Among them, 57 miRNAs were specific to the CM of metastasis-related cell lines which represents the aggressive group 3 and group 4 MB subtypes. A significant number (1,254) of ex-miRNAs were identified in the CSF of a MB patient. Eighty-six of these miRNAs were found to be differentially expressed in this patient’s CSF compared with controls. Interestingly, 3 metastasis-associated miRNAs over-represented in CM of metastasis-related MB cell lines were found to be significantly enriched in the CSF of the MB patient.Conclusion: Although more samples are required to fully verify these results, our work provides the first evidence for the presence of a significant amount of miRNAs excreted extracellularly by MB cells and raises the possibility that, in the near future, miRNAs could be probed in CSF of MB patients and serve as novel biological markers

G-quadruplexes as potential therapeutic targets for embryonal tumors

Embryonal tumors include a heterogeneous group of highly malignant neoplasms that primarily affect infants and children and are characterized by a high rate of mortality and treatment-related morbidity, hence improved therapies are clearly needed. G-quadruplexes are special secondary structures adopted in guanine (G)-rich DNA sequences that are often present in biologically important regions, e.g. at the end of telomeres and in the regulatory regions of oncogenes such as MYC. Owing to the significant roles that both telomeres and MYC play in cancer cell biology, G-quadruplexes have been viewed as emerging therapeutic targets in oncology and as tools for novel anticancer drug design. Several compounds that target these structures have shown promising anticancer activity in tumor xenograft models and some of them have entered Phase II clinical trials. In this review we examine approaches to DNA targeted cancer therapy, summarize the recent developments of G-quadruplex ligands as anticancer drugs and speculate on the future direction of such structures as a potential novel therapeutic strategy for embryonal tumors of the nervous system

NOTCH ligands JAG1 and JAG2 as critical pro-survival factors in childhood medulloblastoma.

Medulloblastoma (MB), the most common pediatric malignant brain cancer, typically arises as pathological result of deregulated developmental pathways, including the NOTCH signaling cascade. Unlike the evidence supporting a role for NOTCH receptors in MB development, the pathological functions of NOTCH ligands remain largely unexplored. By examining the expression in large cohorts of MB primary tumors, and in established in vitro MB models, this research study demonstrates that MB cells bear abnormal levels of distinct NOTCH ligands. We explored the potential association between NOTCH ligands and the clinical outcome of MB patients, and investigated the rational of inhibiting NOTCH signaling by targeting specific ligands to ultimately provide therapeutic benefits in MB. The research revealed a significant over-expression of ligand JAG1 in the vast majority of MBs, and proved that JAG1 mediates pro-proliferative signals via activation of NOTCH2 receptor and induction of HES1 expression, thus representing an attractive therapeutic target. Furthermore, we could identify a clinically relevant association between ligand JAG2 and the oncogene MYC, specific for MYC-driven Group 3 MB cases. We describe for the first time a mechanistic link between the oncogene MYC and NOTCH pathway in MB, by identifying JAG2 as MYC target, and by showing that MB cells acquire induced expression of JAG2 through MYC-induced transcriptional activation. Finally, the positive correlation of MYC and JAG2 also with aggressive anaplastic tumors and highly metastatic MB stages suggested that high JAG2 expression may be useful as additional marker to identify aggressive MBs

The Ser/Thr kinase MAP4K4 drives c-Met-induced motility and invasiveness in a cell-based model of SHH medulloblastoma

Medulloblastoma (MB) comprises four molecularly and genetically distinct subgroups of embryonal brain tumors that develop in the cerebellum. MB mostly affects infants and children and is difficult to treat because of frequent dissemination of tumor cells within the leptomeningeal space. A potential promoter of cell dissemination is the c-Met proto-oncogene receptor tyrosine kinase, which is aberrantly expressed in many human tumors including MB. Database analysis showed that c-Met is highly expressed in the sonic hedgehog (SHH) subgroup and in a small subset of Group 3 and Group 4 MB tumors. Using a cell-based three-dimensional cell motility assay combined with live-cell imaging, we investigated whether the c-Met ligand HGF could drive dissemination of MB cells expressing high levels of c-Met, and determined downstream effector mechanisms of this process. We detected variable c-Met expression in different established human MB cell lines, and we found that in lines expressing high c-Met levels, HGF promoted cell dissemination and invasiveness. Specifically, HGF-induced c-Met activation enhanced the capability of the individual cells to migrate in a JNK-dependent manner. Additionally, we identified the Ser/Thr kinase MAP4K4 as a novel driver of c-Met-induced invasive cell dissemination. This increased invasive motility was due to MAP4K4 control of F-actin dynamics in structures required for migration and invasion. Thus, MAP4K4 couples growth factor signaling to actin cytoskeleton regulation in tumor cells, suggesting that MAP4K4 could present a promising novel target to be evaluated for treating growth factor-induced dissemination of MB tumors of different subgroups and of other human cancers

Disabling c-Myc in Childhood Medulloblastoma and Atypical Teratoid/Rhabdoid Tumor Cells by the Potent G-Quadruplex Interactive Agent S2T1-6OTD

We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex–forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a doseand time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC50, 0.25– 0.39 μmol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1- 6OTD resulted in a time-dependent (mainly c-Myc–dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors

Significance and Therapeutic Value of miRNAs in Embryonal Neural Tumors

Embryonal tumors of the nervous system are the leading cause of childhood cancer-related morbidity and mortality. Medulloblastoma, supratentorial primitive neuroectodermal tumors, atypical teratoid/rhabdoid tumor and neuroblastoma account for more than 20% of childhood malignancies and typify the current neural embryonal tumor model in pediatric oncology. Mechanisms driving the formation of these tumors point towards impaired differentiation of neuronal and neuron-associated cells during the development of the nervous system as an important factor. The importance of microRNAs (miRNAs) for proper embryonic cell function has been confirmed and their aberrant expressions have been linked to tumor development. The role of miRNAs in controlling essential regulators of key pathways implicated in tumor development makes their use in diagnostics a powerful tool to be used for early detection of cancer, risk assessment and prognosis, as well as for the design of innovative therapeutic strategies. In this review we focus on the significance of miRNAs involved in the biology of embryonal neural tumors, delineate their clinical significance and discuss their potential as a novel therapeutic target