The overall premise of the Michael Cuccione Childhood Cancer Research Program is that by increasing our understanding of how tumour cells respond to extra- or intracellular signals, we will gain unique insights into the pathways that are specific for tumour cells. Targeting of these pathways can then be used as treatment strategies, while minimizing effects on normal growth.
It is evident that detailed analysis of cellular signal transduction pathways is central to understanding tumor cell biology. Moreover, the pharmaceutical industry is making an extraordinary effort to exploit molecular targets in signal transduction pathways for the development of new cancer therapeutics.
I would like to extend my deepest gratitude to the Michael Cuccione Foundation and its charitable partners for supporting the Michael Cuccione Childhood Cancer Research Program. If we are to remain at the forefront of cancer research, a steady flow of funding is critical. Only through research can we develop less invasive therapies and ultimately find a cure for cancer. Thank you for your unwavering support and generosity
Dr. Kirk R. Schultz
Director, Michael Cuccione Childhood Cancer Research Program at BC Children’s Hospital
One of the difficulties has been to decide which pathways to target. A major research strategy in our group is the characterization of non-random genetic alterations in human cancers as a means to more efficiently identify genes involved in tumorigenesis. We believe that analysis of tumour tissue, as opposed to model systems, is essential for initial identification and characterization of gene products involved in altered signal transduction in human malignancies. Then, once the involved proteins have been identified, model systems to further study their biology, such as transgenic or knock-out mice, can be invoked. For example, cloning of chromosomal translocation breakpoints allowed us to discover several previously unrecognized oncogenes or tumour suppressors in solid tumours. These include the t(21;22) associated EWS-ERG chimeric transcription factor in Ewing tumours (Sorensen et al, Nature Genetics, 1994), the t(12;15) associated ETV6-NTRK3 chimeric tyrosine kinase in pediatric sarcomas (Knezevich et al, Nature Genetics, 1998) and secretory breast carcinoma (Tognon et al, Cancer Cell, 2002), and the novel 6q21 HECT E3 protein-ubiquitin ligase, HACE1, in sporadic Wilms' tumour and neuroblastoma (Anglesio et al, Hum Mol Genet, 2004).
To understand how these proteins might contribute to oncogenesis requires extensive biochemical characterization of not only the proteins themselves but of their protein interactors, enzymatic substrates, or other molecules involved in common signal transduction pathways. Thus, analysis of signaling pathways activated or suppressed by translocation-associated oncoproteins or tumour suppressors forms another other major focus of our research group.
Our strategy has been to focus initially on specific recurrent genetic alterations in childhood cancers. In contrast to most adult malignant tumours, which appear to have complex genetic etiologies, many childhood cancers show recurrent chromosomal rearrangements including translocations. We have found that many of these translocations disrupt key genes regulating signal transduction pathways. For example, some chromosomal translocations lead to oncogenic gene fusions expressing chimeric oncoproteins.
After we identify genes altered by a particular genetic alteration, we quickly shift to biochemical studies to ascertain how the altered genes affect cellular signaling. In this way, we have been able to more efficiently single out those pathways that may be of relevance to particular childhood cancers. An additional strategy in my laboratory is to combine these studies with gene expression profiling using the Affymetrix platform. In fact, the gene expression patterns of a large series of childhood solid tumors are currently being generated through a collaborative study with other Children's Oncology Group institutions. We hope that this combination of cancer biology and cancer genetic studies will allow us to more readily elucidate those pathways that can be uniquely targeted in tumour cells while sparing normal cells.
Once we identify altered gene products and signaling pathways in childhood malignancies, we screen for potential relevance in adult malignancies. For example, we found that the t(12;15) associated ETV6-NTRK3 chimeric tyrosine kinase of pediatric sarcomas is also expressed in secretory breast carcinoma, a variant of (Tognon et al, Cancer Cell, 2002), This has lead to a number of studies focused on NTRK3 signaling in breast cancer. We have also started to screen for alterations HECT E3 protein-ubiquitin ligase, HACE1, in adult malignancies. Interestingly, targeted inactivation of the Hace1 gene in mice, which results in the development of spontaneous, late onset murine tumors. Gamma irradiation or inactivation of a single p53 allele on a Hace1-/- background dramatically increases tumor frequencies. Moreover, loss of Hace1 renders mice highly susceptible to lung cancer in response to alkylating agents. These studies confirm the tumor suppressor activity of Hace1, and suggest a role for this protein in cell stress.
A further strategy we have recently implemented for pathway dissection and analysis is to combine phenotypic screens at the cell level with libraries of reagents that target individual genes or combinations that target several components of a pathway. For cell level phenotypic analysis we have built up a high-content screening core which consists of a GE Incell 1000 analyser coupled to a liquid handling station, a thermo plate handling arm and a plate incubator hotel.
The ability to couple fluorescent tagging of proteins (whether by reporters or antibodies) with sub-cellular localization and morphology has proven to be a powerful phenotypic analysis tool. The high content screening platform (INcell) allows rapid fluorescent or brightfield imaging of multiwell plates (up to 384), followed by automated analysis of fluorescent/morphological phenotypes, for example to measure cell receptor internalisation, nuclear blebbing, cytoskeletal changes, cell division, apoptosis. The liquid handling stations and plate oven hotel allow for reagents to be added or the contents of medium in wells to be changed and for incubations to occur over a long period of time, with plates being moved to the INcell reader periodically for phenotypic assessment. The core is building up a modest library of small molecules and a genome wide library of RNAi and shRNA molecules as probe reagents to conduct genome wide screens for phenotypes at cell level.
The ultimate goal of such studies to use this information to develop novel molecular-based strategies for the treatment of human malignant diseases.