published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Medical Sciences

Breast cancer is the most common malignancy in women and represents one of the major causes of death worldwide. The PI3K-Akt-FOXO3a signalling pathway has been shown to play a crucial role in tumorigenesis and the development of drug resistance in many cancer types. However, previous studies on FOXO3a using breast cancer tissues were controversial. So this study aims at better understanding of the role of FOXO3a in the development of drug resistance, especially endocrine resistance and anthracycline resistance in breast cancer. Examination of FOXO3a and phosphorylated-Akt (P-Akt) expressions in breast cancer tissue microarrays revealed nuclear FOXO3a was significantly associated with poor prognosis (p=0.014) and lymph node positivity (p=0.052) in invasive ductal carcinoma. Using the tamoxifen and anthracycline-sensitive and -resistant breast cancer cell lines as models, we found that the nuclear accumulation of FOXO3a was associated with enhanced anthracycline-resistance but not tamoxifen-resistance. This was consistent with the finding that sustained nuclear FOXO3a was associated with poor prognosis, as cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis. We demonstrated a possible feedback mechanism in which induction of FOXO3a activity in the anthracycline-sensitive MCF-7 cells induced Akt phosphorylation and promoted cell proliferation arrest. Using MDA-MB-231-FOXO3a(A3):ER cells in which FOXO3a activity could be induced by 4-hydroxytamoxifen, we showed that FOXO3a induction could up-regulate PI3K-Akt activity but had little effect on cell proliferation, which indicates impaired Akt-FOXO3a axis in chemoresistant cell models. To further uncover the precise mechanism of Akt-FOXO3a deregulation in the development of chemoresistance, we have explored the post-translational regulation of FOXO3a by miRNAs. Through a series of Gain-and-Loss functional experiments and luciferase reporter assays in vitro, three miRNAs, including miR-222, miR-221 and miR-29a, were found to suppress FOXO3a protein expression through binding directly to FOXO3a 3’UTR. Moreover, the aberrant expressions of the miR-222/221 cluster and miR-29a in drug resistant cell lines could confer a proliferation advantage to cancer cells through suppressing FOXO3a expression. We further demonstrated that FOXO3a as a transcription factor could transactivate the oncogenic miR-222 and miR-221 expressions under certain chemotherapy stimulation. This suggests the existence of a feedback regulatory loop composed of the miR-222/221 cluster and FOXO3a which may not only play a self-protective role under drug treatment in chemosensitive cells, but also partially explain the tolerated nuclear FOXO3a in the breast cancer with poor prognosis. Taken together, our study suggested that lymph node metastasis and poor survival in invasive ductal breast carcinoma are linked to an uncoupling of the Akt-FOXO3a signalling axis, as in these breast cancers the nuclear-located FOXO3a was unable to induce cell death or cell cycle arrest. We also demonstrated post-translational regulation of FOXO3a by miR-222/221 and miR-29a, while aberrant expressions of miR-222/221 and miR-29a may promote cell resistance to therapy through directly suppressing FOXO3a. FOXO3a could further contribute to the deregulation of the miR-222/221 cluster as a transcription factor in breast cancer. Studying this Akt-FOXO3a-miRNAs signalling circuit will provide us better understanding in predicting and monitoring treatment response in breast cancer and other malignancies. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Tamoxifen is the most widely used endocrine therapy for both early and advanced estrogen receptor (ER) positive breast cancer patients. About half of the patients that initially respond to the antiestrogen become estrogen-independent and ultimately develop resistance to the treatment. The precise molecular mechanisms of tamoxifen resistance remain poorly understood. Dysregulation of microRNAs (miRNAs) has been frequently reported in breast cancer and linked to cancer development, progression and therapeutic response. To gain a more comprehensive picture of the miRNA regulatory network for modulating tamoxifen responsiveness, we examined global expression profiles of more than 600 miRNAs in a matched pair of tamoxifen-sensitive ZR75 and tamoxifen-resistant AK47 breast cancer cell lines using TaqMan Low Density Array (Applied Biosystems). Under 4-hydroxytamoxifen treatment, 102 miRNAs displayed differential responses between the sensitive cells and the resistant cells. At basal levels, upregulation of 32 miRNAs and downregulation of 75 miRNAs were observed in the resistant cells as compared to the sensitive cells. Among the 9 miRNAs of significant differential expression selected for validation, expression profiles of the 7 miRNAs could be reproduced. Of these, 4-hydroxytamoxifen treatment greatly increased miR-449a/b expression in sensitive ZR75 cells, whereas miR-449a/b expression was significantly reduced in resistant AK47 cells at basal levels, which was further confirmed in a panel of tamoxifen-resistant breast cancer cell lines. Such downregulation of miR-449a/b in the resistant cells was partially attributed to DNA methylation-mediated repression of miR-449a/b. Notably, miR-449a/b expression exhibited a significant positive correlation with ER-α status (miR-449a: P=0.006, miR-449b: P=0.013) and progesterone receptor (PR) status (miR-449a: P=0.010, miR-449b: P=0.021), and a prominent inverse association with tumor grade in 61 breast cancer tissues (miR-449a: P=0.001; miR-449b: P=0.009). Also, breast cancer patients with high miR-449a/b expression tended to have increased disease-free survival (miR-449a: P=0.019; miR-449b: P=0.117). To further support the tumor suppressor function of miR-449, stable miR-449b overexpression in the resistant cells reduced cell proliferation. More intriguingly, restoring miR-449b expression increased sensitivity to 4-hydroxytamoxifen-induced apoptosis via suppression of AKT activity without restoring ER-α expression. In contrast, miR-449a/b knockdown reduced ER-α and PR expression, but enhanced phosphorylation of AKT, extracellular signal-regulated kinase- 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and also ER-α at serine 167 and serine 118 residues. Furthermore, we demonstrated c-Myc is a target gene of miR-449 as confirmed by bioinformatics and experimental analyses. Computational algorithms predicted a highly conserved miR-449a/b binding site within C-MYC 3’untranslated region (3’UTR). Compared to the parental sensitive cells, c-Myc was overexpressed in the resistant cells. Forced expression of miR-449b suppressed c-Myc protein level. To further support the notion that c-Myc is a direct target of miR-449, interactions between miR-449b and C-MYC 3’UTR were confirmed by co-expression of miR-449b and c-Myc expression constructs and luciferase reporter assay. Taken together, our data strongly suggest the critical role of miR-449 in modulating altering response to tamoxifen via targeting c-Myc. Suppression of miR-449 repressed genomic ER action and concomitantly activated non-genomic ER pathways. These findings may provide insights to improve breast cancer management and open a wide avenue for therapeutic interventions for overcoming tamoxifen resistance. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy