| dc.description.abstract | Oesophago-gastric cancers are two of the most commonly diagnosed cancers and the majority of oesophageal adenocarcinoma (OAC) and gastric adenocarcinoma (GAC) patients are diagnosed at a late stage. The standard of care for locally advanced OAC is neoadjuvant chemoradiation therapy (neo-CRT) followed by surgery. The standard of care for locally advanced GAC is perioperative chemotherapy, which includes neoadjuvant chemotherapy (neo-CT), administered pre-surgery, and adjuvant chemotherapy, administered post-surgery. Unfortunately, radioresistance and chemoresistance remain a significant barrier to effective treatment and approximately 70% of OAC patients and 85% of GAC patients do not achieve a complete pathological response (pCR) following treatment. Therefore, there is a global unmet need to identify predictive biomarkers of response to neo-CRT and neo-CT. Furthermore, identification of therapeutic targets which enhance response to cytotoxic treatment would significantly improve quality of life and outcomes for OAC and GAC patients. Recent evidence has demonstrated that microRNAs (miRNAs) are widely dysregulated in cancer and play a crucial role in tumorigenesis and the response to treatment. In this thesis, the role of miRNAs in the radioresistance of OAC and the chemoresistance of GAC was investigated, to assess the potential for miRNAs as predictive biomarkers of treatment response, and as therapeutic targets to enhance treatment response.
miR-Predict (a 32-miRNA signature predictive of response to neo-CRT, identified in a pilot cohort of OAC patients) was profiled in TGCA OAC and GAC tumours and six key miRNAs (miR-320a, miR-200c-3p, miR-200c-5p, miR-34b-5p, miR-34a, and miR-31-5p) were identified based on their association with key clinicopathological factors and based on their predicted target genes and pathways. The radiosensitivity of a panel OAC cells was assessed, identifying Flo-1Par cells as significantly more radiosensitive than Flo-1LM cells, derived from Flo-1Par liver metastasis. OE33 R cells, an isogenic model of acquired radioresistance, were confirmed to be more radioresistant than parental OE33 P cells, and OE19 cells were confirmed as inherently more radioresistant than OE33 cells. Expression of the six key miR-Predict miRNAs were assessed in these cell line models and miR-34a expression was demonstrated to be decreased in all radiresistance OAC models. Decreased miR-34a expression was also associated with poor response to neo-CRT, advanced pathological T stage and poor prognosis in pre-treatment OAC tumour biopsies, supporting a role for miR-34a in the pathogenesis of OAC and as a potential novel biomarker of treatment response.
In radioresistant OE33 R cells, miR-34a was demonstrated to functionally modulate the response to radiation at a clinically relevant dose of 2 Gy. miR-34a overexpression significantly enhanced the radiosensitivity of OE33 R cells and was demonstrated to decrease proliferative capacity basally and following irradiation, impair DNA damage repair following irradiation, and resulted in accumulation of cells in the G0/G1 phase and an increased flux through the G2/M phase following irradiation. Altered expression of several DNA damage response and cell cycle-related genes were identified following miR-34a overexpression, including decreased expression of ATR and cyclin E2. Most notably, the cyclin E2 gene was demonstrated to contain a potential binding site for miR-34a, highlighting the potential of cyclin E2 as a direct target of miR-34a, which may provide a mechanism for miR-34a-mediated radiosensitisation in radioresistant OE33 R OAC cells.
The six key miR-Predict miRNAs were also investigated in GAC. The AGS GAC cell line was identified as inherently chemoresistant to oxaliplatin (OXA), 5-flurouracil (5-FU), and combination treatment of OXA and 5-FU, compared to MKN-45 GAC cells. Two novel in vitro isogenic models of chemoresistance were generated, one which was resistant to OXA (AGS OX R) and one which was resistant to 5-FU (AGS 5 R). These isogenic models of inherent and acquired chemoresistance were characterised in terms of pathways frequently implicated in chemoresistance. Additionally, increased miR-31 expression was significantly associated with inherent chemoresistance and with acquired OXA chemoresistance in these models, suggesting a potential role for miR-31 in determining chemoresistance in GAC.
miR-31 was demonstrated to functionally modulate the response to chemotherapy (OXA and 5-FU) in GAC cells. In chemoresistant AGS cells, miR-31 silencing significantly enhanced sensitivity to OXA and 5-FU, and reduced proliferative capacity, enhanced DNA damage induction, and increased ATOX1 expression. ATOX1 was demonstrated to contain predicted binding sites for miR-31, highlighting the potential of ATOX1 as a direct target of miR-31, which may provide a mechanism for miR-31-mediated chemoresistance in AGS GAC cells. Additionally, miR-31 suppression significantly reduced tumour formation and enhanced chemosensitivity in a novel GAC zebrafish cell line-derived xenograft (CDX) model, further supporting a role for miR-31 in the pathogenesis and treatment response of GAC.
Together, this thesis suggests that miR-34a is a potential biomarker of treatment response and functionally modulates radiosensitivity in OAC. This work also demonstrates that miR-31 functionally modulates chemosensitivity and tumour growth in GAC, suggesting the potential of these miRNAs as novel therapeutic targets to enhance treatment response in esophago-gastric cancer. | en |
