The microRNA modulation of chemosensitivity in adrenocortical carcinoma

Abstract

The initial aim of this work was narrow, with a translational focus foremost. It was, in brief, to determine if microRNAs (miRNAs), replaced in adrenocortical carcinoma (ACC), could mitigate chemoresistance to commonly used adjuvant treatments, including doxorubicin and mitotane. This was in recognition that complete surgical resection offers the best chance of a cure, with response to adjuvant treatment heterogeneous, at best. However, ACC often presents late with advanced disease, as symptoms may be non-specific, and diagnosis is often late. Additionally, ACC can rapidly grow and upstage from diagnosis to resection. Finally, as a rare cancer, expert knowledge in the management of ACC is centred at quaternary referral centres. To fulfil the original aim, it was necessary to review patients’ clinical files to correlate the course of their disease with adjuvant treatments given. It became apparent that there was a deficit in the Australasian literature detailing long-term management approaches and outcomes of ACC in quaternary referral centres. This was in stark contrast to publications from North American, European and Asian centres where ACC is recognised as a challenging rare cancer needing ongoing clinical and scientific study. With over 21 years of specialist multidisciplinary management, inclusive of 29 patients managed primarily and 7 other patients referred for quaternary care, outcomes were in line with those published from international centres. Low Ki-67 index (<10) was found to be a major prognosticator across all stages of disease. Impressive disease response and survivorship were seen in Stage 4 patients with low Ki-67, which indicates that this index may be a proxy for disease response. This finding deserves further exploration. In examining the outcomes of patients, especially those with Stage 4 disease, it became clear that there were, in some patients, molecular differences that conferred a better ability to respond to adjuvant therapy. This led back to the first intent of the thesis. MicroRNAs (miRNAs), short non-coding RNAs that are powerful post-translational modulators of activity, were investigated in a Stage 4 ACC cohort for their influence upon disease response. miR-34a, a well-studied tumour suppressor strongly implicated in cancer chemosensitivity, was found, significantly, to be 3.3-fold under-expressed in a group of Stage 4 ACC patients refractory to adjuvant treatment compared to a group responsive to treatment. miR-195, also with well-documented tumour suppressive roles, has already been identified as under-expressed in ACC compared with adrenocortical adenoma (ACA) and normal adrenal. These findings have been derived from studies utilising a microarray approach and validated in independent cohort samples. In concordance with these findings, serum miR-195 has been found to be under-expressed in ACC patients. Considered together, miR-34a and miR-195 were well-positioned as candidates for replacement of ACC cell lines. However, replacement of these miRNAs, followed by either doxorubicin or mitotane treatment, did not effect a decreased half maximal inhibitory concentration, indicating that the miR-34a/195-replaced cells exhibited no greater effectiveness for inhibition of cell proliferation when combined with doxorubicin/mitotane treatment. We proceeded to investigate for another miRNA candidate in a different fashion. Using a high throughput technique, where 384 miRNAs per patient tumour sample were quantified, miR-431 was shown to be 100-fold under-expressed in Stage 4 non-responsive ACC. This result was validated in a larger ACC cohort, which involved all stages of disease. ACC, replaced with miR-431, underwent doxorubicin/mitotane treatment. There was both significant cellular proliferation inhibition and a marked apoptotic effect in the miR-431 replaced group. ZEB1 was identified as a target of miR-431 in ACC, with evidence of epithelial mesenchymal transition reversal in miR-431 cells undergoing doxorubicin treatment. In conclusion, this thesis embarked upon several avenues. The first dedicated and wide-ranging review of ACC management and outcomes in an Australasian-dedicated endocrine cancer group is presented. Attempts were made to address an obvious and continual clinical problem of poor response and survivorship of Stage 4 ACC patients, by studying patients with remarkable clinical response and long survival. miR-431 was found, for the first time, to influence ACC response to conventional drug therapy. It is hoped that these findings may provide a basis for further in vivo work in orthotopic mouse models of ACC. With effective miRNA delivery well-established in pre-clinical human trials, the possibility for broadly applicable new therapy, in conjunction with already established and studied adjuvant therapy regimens, may be within reach.