Determining the role of γ-actin in cancer cells

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Copyright: Shum, Michael Sing Yeung
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Abstract
Anti-microtubule agents such as the vinca alkaloids and taxanes that target tubulin/microtubules and block cell division are highly effective in the treatment of both haematological and solid cancers. Resistance to these chemotherapeutic agents remains a major clinical issue to the successful treatment of both childhood and adult cancers. Despite considerable research into resistance mechanisms, clinical drug resistance mechanisms remain poorly defined. Recent studies from our laboratory have identified γ-actin as a novel biomarker in anti-microtubule resistant cell lines and clinical samples. In particular, mutations and/or reduced expression of γ-actin were shown to confer resistance to anti-microtubule agents. Despite strong functional evidence linking γ-actin and drug resistance, the mechanism by which γ-actin confers anti-microtubule drug resistance is not understood. Limited information on the normal physiological function of γ-actin hinders understanding of its role in mediating anti-microtubule drug resistance. Therefore, in order to investigate γ-actin’s role in anti-microtubule drug resistance, there first needs to be a clearer understanding of the functional role of γ-actin in cancer cells. The aim of this thesis was to examine the role of γ-actin in protein-protein interactions, cellular localisation and function, as well as through examination of its role in ovarian cancer and tumour metastases. To address the role of γ-actin mutations in mediating anti-microtubule drug resistance, protein interaction studies were performed to examine whether specific mutations associated with anti-microtubule drug resistance affected protein interactions. Pull down assays revealed eukaryotic elongation factor 1 α (eEF1A1) as a γ-actin interacting protein that has previously been demonstrated to bundle actin filaments and sever microtubules. eEF1A1 displayed reduced binding to both P98L and V103L mutant γ-actin. This was the first demonstration that mutations in γ-actin affect protein-protein interactions. Functional studies were carried out using siRNA knockdown of eEF1A1 to examine its role in mediating anti-microtubule drug resistance. Clonogenic and trypan blue exclusion assays showed that the knockdown of eEF1A1 in mouse fibroblast NIH-3T3 cells did not affect paclitaxel sensitivity. This result demonstrated that although eEF1A1 bound poorly to mutant γ-actin, the method of mimicking reduced protein interaction through eEF1A1 knockdown was not sufficient in producing a drug resistant phenotype. These results revealed a more complex mechanism of resistance mediated by γ-actin mutations. To determine the physiological function of γ-actin, siRNA knockdown studies were conducted in human neuroblastoma SH-EP cells. The knockdown of γ-actin showed no significant compensation in β-actin protein expression. Furthermore, the knockdown of γ-actin affected numerous cellular functions such as cell migration, focal adhesion, as well as cell polarity. Moreover, the regulation of cell migration was associated with the modulation of phosphorylated actin regulatory proteins such as cofilin and myosin light chain. The inhibition of the ROCK signalling pathway through the use of a specific ROCK inhibitor (Y-27632) restored cell migration in γ-actin knockdown cells, and demonstrated that γ-actin governs cell migration through the modulation of the ROCK signalling pathway. These results demonstrated a novel regulatory role for γ-actin in cell motility that is not shared with β-actin. The role of γ-actin expression in ovarian cancer was examined through the use of ovarian cancer cell lines, clinical patient samples and ovarian cancer tissue microarrays. Changes in actin isotype composition were observed in paclitaxel and epothilone B resistant ovarian cancer cells, when compared to drug sensitive parental cells. Real time PCR showed variation in γ-actin expression in a small cohort of patient samples and immunohistochemistry conducted on ovarian cancer tissue microarray showed significant changes in γ-actin expression between primary and metastatic tumours, highlighting a possible role for γ-actin in tumour metastases. In conclusion, this thesis has provided new insights into the protein interactions and functional role of γ-actin, in particular, the relationship between γ-actin and the Rho GTPase signalling pathway. γ-Actin’s role in cell motility regulation may potentially be linked to metastasis in ovarian cancer. The novel findings in this thesis have advanced some of the basic understandings of γ-actin and actin isotype functional differences.
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Author(s)
Shum, Michael Sing Yeung
Supervisor(s)
Kavallaris, Maria
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Publication Year
2011
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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