Characterization of the Role of HACE1 in Wilms' Tumours

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University of British Columbia (Canada), 2007 - 214 pages
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Our research group recently identified the novel ubiquitin-protein ligase gene, HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1), on Chr. 6 near a unique t(6;15)(q21;q21) translocation in a Wilms' tumour. HACE1 levels are reduced in ∼75% of Wilms' tumours and a number of other malignancies compared to patient-matched normal tissue. Furthermore, aberrant methylation patterns within CpG islands upstream of the HACE1 gene, in Wilms' tumours, correlate increased levels of methylation with lower expression (60%). This has led us to hypothesize that HACE1 functions as a tumour suppressor gene. To explore this hypothesis, I have established two retroviral model systems. First, I have observed that Wilms' tumour derived cell lines grown in soft agar show reduced colony number and size when cells were engineered to stably over-express HACE1 compared with empty vector or a non-functional HACE1 mutant. In vivo tumour formation in nude mice injected with a Wilms' tumour cell line overexpressing HACE1 is also highly attenuated compared with empty vector control. Second, I have identified a number of small interfering RNA (siRNA) sequences that functionally knock-down HACE1 expression. These siRNAs have been used in a stable lentiviral system to reduce HACE1 expression by as much as 80% in a number of cell lines, mimicking the low level of HACE1 observed in Wilms' tumours. Knocking down HACE1 levels in HEK293 cells resulted in an increase in both the number and size of soft agar colonies. HACE1 appears to affect several different growth and survival related pathways. Although direct targets of RACE 1 are not yet validated, changes in AKT and GSK3beta activation and the levels of cyclin D1 are consistently affected by HACE1 expression. Taken together, these data support a role for HACE1 as a novel tumour suppressor whose reduced expression can contribute to malignant progression.

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