| Popis: |
Fundamental to eukaryotic cell function, trafficking into and out of the nucleus is conventionally mediated by the Importin (Imp) and Exportin (Exp) transporters respectively, of which there are multiple Impα, Impβ and Exp forms expressed in humans. Nuclear transport can be regulated at many levels, including through modulation of the Imp/Exp nuclear transport machinery itself. Interestingly, altered Imp/Exp levels are associated with cellular malignancy and tumour disease progression in terms of tumour grade, stage, aggressiveness and patient prognosis, but the functional relevance with respect to changes in Imp/Exp activity and nuclear transport has not been investigated. Through rigorous quantitative live cell imaging of a variety of isogenic non-transformed/transformed cell systems from various origins, including the MCF10A human breast tumour progression series, this thesis shows that nuclear accumulation of Impα/β1-recognised nuclear localisation signal (NLS)-containing proteins, but not their NLS-mutated derivatives, is up to 7-fold increased in malignant compared to non-transformed cell types. This is associated with a significantly faster rate of nuclear import in transformed cells, as revealed by analysis of an Impα/β1-recognised cargo using fluorescence recovery after photobleaching. Nuclear accumulation of NLS/nuclear export signal-containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp-1-mediated nuclear export is not impaired in transformed compared to non-transformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2- to-4-fold higher expression of Impα1, β1 and Exp-1 in the various transformed cell types, as indicated by quantitative Western analysis. Trafficking dependent on Impα/β1 was found to be selectively enhanced during the benign to malignant switch in the MCF10 model of human breast tumour progression (basal triple negative type, invasive ductal carcinoma), with the degree of enhancement correlating to advancing tumour disease state of the cells. In contrast, the nuclear accumulation of Impβ1-recognised cargoes was c. 2-fold decreased in malignant breast cells. Quantitative RT-qPCR and/or Western analysis indicate this is specifically associated with progressively increased expression of Impα1, α3, β1 and the Impα re-exporter CAS (Exp-2) in malignant compared to benign/non-transformed breast cell types, with RNAi/overexpression approaches establishing Impα1 levels to be the primary basis of elevated Impα/β1-dependent nuclear import efficiency and decreased Impβ1-dependent nuclear import efficiency. Underlining the physiological relevance of these observations, the degree of Imp alpha 1 expression was found to correlate with increasing tumour grade in clinical ductal carcinoma samples. Excitingly, the studies in this thesis show that targeting Impβ1 activity through RNAi is up to 33-fold more efficient in decreasing the viability of transformed/ductal carcinoma cell types compared to isogenic non-transformed counterparts, and is highly potent with tumour selective activity shown at subnanomolar siRNA concentrations. Flow cytometric analysis in malignant cells treated with Impβ1 siRNAs implied this effect was due to increased cell death. Importantly, the tumour selective killing activity was found to be specific to Impβ1 siRNA, and was not observed for siRNAs targeting other Imps such as Impα1, α3, CAS or Exp-1 that were also found to be overexpressed in malignant cell types. The results here established for the first time that enhanced Impα/β1-dependent nuclear import efficiency at a global scale is associated with malignant transformation and that transformed/tumour cell types display hypersensitivity to inhibition of Impβ1 through siRNA-mediated knockdown. The findings raise the exciting possibility that the enhanced Impα/β1-dependent nuclear import of transformed cells may be exploited to facilitate tumour cell-specific drug delivery, and have implications for development of tumour-selective anti cancer strategies through RNAi/drugs targeted specifically at Impβ1. |
