Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide and in its advanced stage, has a poor clinical outcome. Resistance to chemotherapeutic agents, either intrinsic or acquired, is the primary cause of treatment failure in NSCLC. Tubulin binding agents (TBAs), such as paclitaxel and vinorelbine are important components in the treatment of NSCLC. Upregulation of the neuronal specific class III β-tubulin (β-III-tubulin) is frequently found in drug resistant cancer cell lines and human tumours, lending support that βIII-tubulin might play a role in the development of drug resistance in cancer cells. However, to date, compelling evidence supporting its direct role in drug resistance and response has been lacking. To address its role in NSCLC, RNA interference (RNAi) was employed to knock down βIII-tubulin expression in two drug naive NSCLC cell lines, Calu-6 and H460. Specific knockdown of βIII-tubulin resulted in increased sensitivity to TBAs and DNA damaging agents, two classes of agents that are commonly used in the treatment of NSCLC. Increased sensitivity to TBAs and DNA damaging agents in the βIII-tubulin knockdown cells was due to an increased propensity of the cells to undergo apoptosis, suggesting that this tubulin isotype may be a cellular survival factor. Interestingly, specific knockdown of βII- or βIVb-tubulin hypersensitised the cells to Vinca alkaloids but not taxanes, demonstrating that each isotype is unique in terms of drug-target interactions. Moreover, the β-tubulin isotype composition of a cell can influence response, and therefore resistance to TBAs. To determine whether βIII-tubulin differentially regulates microtubule behaviour and influences cell proliferation via an effect on microtubule dynamics, siRNAs were used to knockdown βIII-tubulin expression in H460 cells stably expressing GFP-βI-tubulin and the dynamic instability behaviour of individual microtubules was measured by time-lapse microscopy. In the absence of drug, silencing of βIII tubulin alone did not significantly affect the dynamic instability of interphase microtubules. However, at the IC50 for proliferation of either paclitaxel or vincristine, the overall dynamicity was suppressed significantly in the βIII-tubulin silenced cells as compared to the control, indicating that βIII-tubulin knockdown induces paclitaxel or vincristine sensitivity by enhancing the ability of these agents to suppress microtubule dynamics. At a concentration of drug that represented the IC50 for mitotic arrest, for either paclitaxel or vincristine, increased apoptosis induction was found to play a dominant role in βIII-tubulin knockdown, further supporting a role for βIII-tubulin as a cellular survival factor. Collectively, when βIII-tubulin is overexpressed in tumours cells, it is highly likely to be promoting cellular survival and resistance to TBAs. In addition to its proposed role in drug resistance, high expression of βIII-tubulin in tumours of non-neuronal origin such as NSCLC, has been positively correlated with the degree of tumour aggressiveness. H460 cells are known to display substrate- independent growth in soft agar and tumourigenicity in nude mice and provided an ideal model to investigate the role of βIII-tubulin in tumourigenesis. To address the role of βIII-tubulin, H460 cells stably expressing βIII-tubulin shRNA were generated, validated and examined using both in vitro and in vivo methods of tumourigenesis. Colony formation of H460 cells stably expressing βIII-tubulin shRNA was dramatically reduced in soft agar and significantly delayed tumour growth and reduced tumour incidence of subcutaneous xenografted tumours in nude mice when compared to respective controls. These results provide new insights into the function of βIII-tubulin and suggest that βIII-tubulin may play an important role in tumour development and progression in lung cancer. In conclusion, β-tubulin isotype status can serve as a valuable molecular marker capable of distinguishing patients with differential sensitivity to TBAs. These results not only shed new light on the role of specific β-tubulin isotypes in the response to TBAs, but also the role of βIII-tubulin in the biology of cancer that will lead to new treatment strategies for NSCLC.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--Northeastern University, 2008. / Title from title page (viewed April 3, 2009). Graduate School of Arts and Sciences, Dept. of Biology. Includes bibliographical references (p. 79-90).

Thesis (M. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 217-261) Also available in print.

Thesis (Ph. D.)--Lehigh University, 1999. / Includes vita. Bibliography: leaves 89-105.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2009. / Includes bibliographical references. Also available on the World Wide Web.

Thesis (Ph. D.) -- University of Maryland, College Park, 2005. / Thesis research directed by: Molecular and Cell Biology. Includes bibliographical references (p. 115-126).

Multidrug resistance has become a major issue in the treatment of both microbial infections and cancers. While genetically encoded drug resistance is fairly well understood, it cannot explain all observed cases of resistance, namely the ability of a subset of disease cells to persist in an otherwise susceptible population. This non-genetic resistance requires the heterogeneous expression of a drug resistance phenotype, which can be produced by certain gene regulatory network architectures. Two particular network motifs, the coherent feedforward loop (CFFL) and the positive feedback loop (PFL), have functional properties that implicate them in the development of non-genetic heterogeneity and response to changing conditions. Motivated by the observation that CFFL and PFL motifs are involved in the transcriptional regulation of multiple pleiotropic drug resistance (PDR) genes in yeast, it has been hypothesized that CFFLs and PFLs could contribute to the development of drug resistance. This hypothesis was based on model simulations and has not been tested experimentally. In this thesis, it is demonstrated experimentally that the PDR5 gene is indeed expressed heterogeneously within an isogenic population of yeast cells, and that this cell-to-cell variability enables a subset of cells to persist drug treatment. While these observations agree with model predictions, it is also observed that the resistant phenotype occurs within a subset of cells that are morphologically distinct. This subpopulation has previously been linked to abnormal mitochondrial function, which cannot be ruled out as a likely cause of the observed drug resistance. To validate the hypothesis that CFFLs and PFLs contribute to drug resistance, the expression of the PDR5 gene was placed under the control of synthetic gene regulatory networks constructed to contain different combinations of direct activation, indirect activation, and positive feedback. These networks are used to show that direct activation can provide a selective advantage enabling rapid responses, while indirect activation and positive feedback can provide a selective advantage by maintaining favourable gene expression states. These results demonstrate that a gene regulatory network combining CFFLs and PFLs can contribute to the development of drug resistance, and highlight plausible means by which cells can exploit certain network features to gain a fitness advantage.

M.Tech. (Medical Technology) / Please refer to full text to view abstract

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