Browsing by Subject "Medical Cell Biology"

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    The role of T-box transcription factor TBX3 in rhabdomyosarcoma
    (2016) Sims, Danica Anne; Prince, Sharon; Peres, Jade
    Cancer remains one of the leading causes of death worldwide due to late diagnosis and ineffective treatment options. To address this problem requires the elucidation of the molecular mechanisms, including the signaling pathways and transcription factors that drive cancer initiation and progression. In this regard, our laboratory has been particularly interested in the embryonically important T - box family of transcription factors which has been heavily implicated in promoting initiation and progression of a long list of cancers. For example, the overexpression of the T - box factor TBX3, has been reported to function in promoting immortalization, migration, invasion and tumour formation in a number of epithelial - derived malignancies. Furthermore, our laboratory recently reported that TB X3 is also overexpressed in a wide range of sarcoma subtypes including rhabdomyosarcomas. This suggests that TBX3 may also contribute to the development and/or progression of sarcomas and potentially may serve as a biomarker for their diagnosis and targete d therapy. This is exciting because sarcomas are diverse and heterogeneous cancers with varying clinical behaviours, high rates of metastasis and recurrence and are notoriously resistant to current chemotherapies. However, whether TBX3 is a molecular drive r of these mesenchymal - derived cancers remains to be determined. This project therefore aimed to elucidate the role of TBX3 overexpression in embryonal rhabdomyosarcomas (ERMS) which is the most common soft tissue sarcoma in children and adolescents. To this end, ERMS cell culture models were established in which TBX3 was either stably knocked down or stably overexpressed and the resulting cells were tested for several features of the cancer phenotype using in vitro and in vivo experiments. The results show that TBX3 promotes cell proliferation, anchorage independent growth and cell migration in vitro and tumour formation and invasion in vivo. This study also provides evidence that nucleolin binds to, and co - operates with, TBX3 to promote proliferation and migration of ERMS cells. Furthermore, data from initial experiments reveal that Hsc70 interacts with TBX3, to possibly increase its protein stability, and that oncogenic c - Myc and AKT 1 positively regulat e TBX3 levels in ERMS. This, albeit preliminary data, suggest that Hsc70, c - Myc and AKT1 are responsible, in part, for the overexpression of TBX3 in ERMS. Together findings from this study implicate TBX3 as an oncogene in ERMS and suggest that TBX3, nucleolin, Hsc70, c - Myc and AKT may be used in combination as biomarkers for the diagnosis and targeted therapy of ERMS.
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    The c-Myc/TBX3/nucleolin/Hsc70 signalling axis in breast cancer
    (2019) Ncube, Stephanie Maria; Prince, Sharon
    The T-box transcription factor TBX3, plays critical roles in development including the formation of the limbs, heart and mammary glands. While haploinsufficiency of TBX3 results in ulnar mammary syndrome, its overexpression is linked to several cancers. We and others have shown that TBX3 drives tumour formation, invasion and metastasis of several sarcoma subtypes as well as melanoma, cervical cancer and breast cancer. TBX3 has thus been proposed as a novel therapeutic target to treat these cancers. Direct targeting of transcription factors for therapies however continues to represent a serious challenge and therefore an understanding of the molecular mechanisms that regulate and mediate its oncogenic activity may reveal more amenable anti-cancer drug targets. This project therefore aimed to (1) identify signalling molecules that upregulate TBX3 expression in MCF-7 breast cancer cells as well as (2) identify and characterize protein partners that cooperate with TBX3 to drive its oncogenic functions in these cells. The overexpression of the basic helix-loop-helix oncogenic transcription factor c-Myc has been widely reported in breast cancer progression and c-Myc-driven pathways are elevated in aggressive drug resistant breast cancer cells and tumours. Our laboratory has previously shown that c-Myc directly binds and activates the TBX3 promoter in several sarcoma subtypes, and it was hypothesised that c-Myc may also activate TBX3 in breast cancer. To investigate this, the impact of transiently knocking down c-Myc on TBX3 mRNA and protein levels was firstly assessed by qRT-PCR and western blotting respectively. Results show that when c-Myc is depleted, TBX3 mRNA and protein levels decrease, suggesting that c-Myc may be transcriptionally upregulating TBX3. To confirm this, c-Myc was ectopically overexpressed in MCF-7 breast cancer cells in the presence or absence of Actinomycin D, an inhibitor of de novo transcription, and TBX3 mRNA and protein levels were measured by qRT-PCR and western blotting respectively. Indeed, results show that when de novo transcription is inhibited, the c-Myc mediated activation of TBX3 expression is abolished. To identify and characterize TBX3 protein partners, MCF-7 breast cancer cells that stably overexpress FLAG-TBX3 were firstly established to enable effective immunoprecipitation for mass spectrometry. The overexpression of FLAG-TBX3 was confirmed by western blotting and immunocytochemistry and the anti-proliferative and pro-migratory roles of TBX3 overexpression in breast cancer cells was confirmed using growth curves and scratch motility assays respectively. Through affinity purifications coupled with mass spectrometry a myriad of putative TBX3 protein co-factors were identified and from this list three partners viz nucleolin, Hsc70 and HnRNP K were validated by immunoprecipitation and colocalization experiments. Importantly, results show that the interaction of TBX3 with Hsc70 is required for TBX3 protein stability and that nucleolin and TBX3 cooperate to promote MCF-7 breast cancer cell migration. Furthermore, treatment of MCF-7 cells with the nucleolin targeting aptamer, AS1411, mis localizes TBX3 and nucleolin to the cytoplasm and causes a reduction in cell viability while having no effect on the viability of normal skin fibroblasts. Together the results from this study show that c-Myc/TBX3/nucleolin/Hsc70 may be an important oncogenic pathway in breast cancer and that AS1411 may be a potentially important aptamer for the treatment of TBX3-driven breast cancer.