MicroRNA-330-5p as a Putative Modulator of Neoadjuvant Chemoradiotherapy Sensitivity in Oesophageal Adenocarcinoma

RESEARCH ARTICLE MicroRNA-330-5p as a Putative Modulator of Neoadjuvant Chemoradiotherapy Sensitivity in Oesophageal Adenocarcinoma Becky A. S. Bibby1, John V. Reynolds2, Stephen G. Maher1,2* 1 School of Biological, Biomedical and Environmental Sciences, University of Hull, Hull, United States of America, 2 Department of Surgery, Institute of Molecular Medicine, Trinity College Dublin, St. James Hospital, Dublin, Ireland * Abstract OPEN ACCESS Citation: Bibby BAS, Reynolds JV, Maher SG (2015) MicroRNA-330-5p as a Putative Modulator of Neoadjuvant Chemoradiotherapy Sensitivity in Oesophageal Adenocarcinoma. PLoS ONE 10(7): e0134180. doi:10.1371/journal.pone.0134180 Editor: Zheng Li, Peking Union Medical College Hospital, CHINA Received: April 29, 2015 Accepted: July 6, 2015 Published: July 29, 2015 Copyright: © 2015 Bibby et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Cancer and Polio Research Fund (www.cprf.org.uk) awarded to SGM and the Irish Cancer Society (www.cancer.ie) CRF10MAH awarded to SGM. BASB is supported by a University of Hull PhD scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Oesophageal adenocarcinoma (OAC) is the sixth most common cause of cancer deaths worldwide, and the 5-year survival rate for patients diagnosed with the disease is approximately 17%. The standard of care for locally advanced disease is neoadjuvant chemotherapy or, more commonly, combined neoadjuvant chemoradiation therapy (neo-CRT) prior to surgery. Unfortunately, ~60-70% of patients will fail to respond to neo-CRT. Therefore, the identification of biomarkers indicative of patient response to treatment has significant clinical implications in the stratification of patient treatment. Furthermore, understanding the molecular mechanisms underpinning tumour response and resistance to neo-CRT will contribute towards the identification of novel therapeutic targets for enhancing OAC sensitivity to CRT. MicroRNAs (miRNA/miR) function to regulate gene and protein expression and play a causal role in cancer development and progression. MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT. Here, to identify miRNAs associated with resistance to CRT, pre-treatment diagnostic biopsy specimens from patients with OAC were analysed using miRNA-profiling arrays. In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT. The role of miR-330 as a potential modulator of tumour response and sensitivity to CRT in OAC was further investigated in vitro. Through vector-based overexpression the E2F1/p-AKT survival pathway, as previously described, was confirmed as a target of miR-330 regulation. However, miR-330-mediated alterations to the E2F1/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil). In contrast, silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to clinically relevant doses of radiation. This study highlights the need for further investigation into the potential of miR-330-5p as a predictive biomarker of patient sensitivity to neo-CRT and as a novel therapeutic target for manipulating cellular sensitivity to neoCRT in patients with OAC. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0134180 July 29, 2015 1 / 15 MiR-330-5p Is Downregulated in Chemoradiation Resistant OAC Patients Introduction Globally, oesophageal adenocarcinoma (OAC) is the sixth most common cause of cancer death and is an epidemic in the West and developed world [1, 2]. Early diagnosis improves patient prognosis, however, the disease is relatively asymptomatic in the early stages and the majority of patients presenting with symptoms are diagnosed with advanced disease. The standard of care for OAC patients in the UK is currently neoadjuvant chemotherapy followed by surgical resection [3, 4]. Although surgery is generally successful for early stage disease, and mortality rates are low, as most patients present with late stage disease surgery alone is not sufficient to prevent disease recurrence in the long-term [5]. Patients typically receive a combination of epirubicin, oxaliplatin (or cisplatin) and capecitabine (5-fluorouracil) prior to surgery, to reduce locoregional recurrence and improve patient outcome [4, 6]. However, recent trials have highlighted the benefits of neoadjuvant combined chemoradiation therapy (neo-CRT), which is already the standard of care in most other European countries [4, 7]. Presently, there are a number of on-going definitive trials directly comparing neoadjuvant chemotherapy versus combined chemoradiotherapy for OAC, such as the neo-AEGIS trial (clinicaltrials.gov NCT01726452). This and other trials come as a result of previous reports suggesting that patients who receive neo-CRT may be up to ten times more likely to achieve a complete pathological response (pCR), compared to patients who receive only neoadjuvant chemotherapy [8, 9]. Of the patients who receive neo-CRT, 18–35% have a pCR, which is a proxy for improved prognosis and a reported increase in 5-year survival rate up to 50–60% [10, 11]. Ongoing UK clinical trials are in place to assess the use of neo-CRT as the future standard of care for UK patients [6, 12]. Unfortunately, however, the majority of patients (~60–70%) do not respond to neo-CRT, and the 5-year survival rate for OAC patients after treatment is 23% [13]. Consequently, the patients who fail to respond to neo-CRT are subject to an aggressive treatment regimen from which they gain little or no benefit. Additionally, in some cases the disease progresses during the neo-CRT regimen, which reduces the success of surgery and adversely affects patient prognosis [14, 15]. The identification of biomarkers, in a pre-treatment setting, which predict patient response to neo-CRT could aid treatment stratification for patients at the point of diagnosis. Furthermore, novel therapeutics agents, targeting functional regulators associated with response to treatment, could be exploited to enhance patient response to conventional CRT as part of multimodal treatment regimens. MicroRNAs (miRNA/miR) are a family of short non-coding RNA that repress the translation of mRNA targets [16]. Perfect Watson-Crick binding between the miRNA and its mRNA target is not essential for regulation, therefore a single miRNA can potentially target thousands of mRNA [17]. MiRNAs are predicted to regulate (...truncated)