Sir Peter MacCallum Department of Oncology - Theses
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ItemDefining functional drivers of oesophageal tumourigenesis( 2019)The incidence of oesophageal adenocarcinoma (OAC) has risen rapidly over the last four decades and has a high overall mortality rate that has shown only incremental improvements over the same duration. OAC develops from the precursor intestinal metaplasia of the oesophageal mucosa known as Barrett’s oesophagus. However, limited knowledge of the molecular mechanisms driving disease progression makes effective clinical management of OAC challenging. One of the common genetic events associated with the progression from Barrett’s oesophagus to OAC is loss of the tumour suppressor, SMAD4 (mutated in 13% or loss of function in 34% of OAC cases). Herein, this thesis firstly investigated the effect of SMAD4 inactivation in Barrett’s carcinogenesis. Genetic knockdown or knockout of SMAD4 was sufficient to initiate tumourigenesis of dysplastic Barrett’s oesophagus cell line, CP-B, in vivo, establishing SMAD4 loss as a crucial event driving progression to OAC. Further, low coverage whole genome sequencing (LC-WGS) analysis revealed that tumourigenic SMAD4 knockdown/knockout CP-B cell lines exhibited distinctive and consistent copy number alterations (CNAs) compared to non-tumourigenic SMAD4 wild-type parental cells. Amongst the alterations we observed were loss of chromosome arm 14q, while amplified regions include chromosome arms 6q and 12p, consistent with common CNAs found in patient tumours. This high genomic instability, characterized by structural chromosomal rearrangements within the tumours following SMAD4 loss, implicates SMAD4 as a protector of genome integrity in OAC development and progression. Moreover, initial in vitro data shows that SMAD4 knockout in CP-B cell line, results in differential expression of transforming growth factor-beta (TGF-beta) pathway target genes (such as ACTA2, CRYAB, PTK2B, ATF3 and CDC6) and loss of cell cycle arrest in response to TGF-beta1 cytokine compared to SMAD4 wild-type parental cells. Furthermore, SMAD4 knockout negatively regulated transcript expression of the multifunctional tumour suppressor INK4/ARF locus, demonstrating the novel and complex network of SMAD4 tumour suppressive activity. This thesis also focused on deciphering the functional role of growth factor receptor bound protein 7 (GRB7) amplification and overexpression in OAC and its potential targeting. GRB7 gene is positioned within known 17q12 amplicon, together with HER2 gene encoding for human epidermal growth factor receptor 2 (HER2). GRB7 is an adaptor molecule that mediates networking of multiple cell surface receptors with downstream signalling pathways. GRB7 high expression was found to be associated with worse survival in OAC patient cohort. Further, genetic GRB7 knockdown (siRNA) inhibited cell proliferation and clonogenic survival and induced apoptosis in GRB7 amplified and overexpressing OAC cell lines (OE19 and Eso26), without altering proliferation of the cells with normal GRB7 expression. Furthermore, whilst HER2 amplification and overexpression was also observed in OE19 and Eso26 cells, they were not uniquely sensitive to trastuzumab (HER2 inhibitor), with Eso26 cells exhibiting resistance in vitro. Taken together, initial findings raise the possibility that GRB7 may be a better molecular therapeutic target than HER2 in OAC with the 17q12 amplicon. To address this possibility, OE19 and Eso26 cell line xenograft models with inducible expression of shRNA targeting GRB7 were used. Consistent with in vitro findings, HER2 expression did not predict sensitivity to trastuzumab, with Eso26 xenografts remaining refractory to trastuzumab treatment. Of high importance, mimicking GRB7 inhibition with inducible-shRNA significantly inhibited tumour growth in both OE19 and Eso26 xenografts. Thus, this part of the thesis demonstrates the functional role of GRB7 overexpression as an oncogenic driver independent of HER2. In summary, the identification of functional genetic drivers and a deeper understanding of the mechanisms that underlie tumour progression in Barrett’s carcinogenesis will lead to improved strategies for the clinical management of OAC patients. To this end, SMAD4 loss was sufficient for progression from dysplasia to OAC. Tumours driven by SMAD4 loss exhibit distinctive CNAs consistent with OAC and metastatic potential. In addition, GRB7 high expression predicts poor outcome in patients with OAC and as such, GRB7 represents an oncogenic driver that could be used as a therapeutic target. Crucially, this thesis provides in vitro and in vivo preclinical and molecular biology evidence for the potential therapeutic benefit of targeting GRB7 in cancer.