miR-330-5p expression in pre-treatment diagnostic OAC tumour biopsies from responders vs. non-responders to neo-CRT.

<p>(<b>A</b>) MiR-330-5p expression is significantly lower in patients who do not respond to neo-CRT (TRG 4 and 5) when compared with responders (TRG 1 and 2). The two outlier values in the responder data set came from the two patients who were not clinical stage TNM T3. These two outlier values were biopsy specimens derived from tumours graded T<i>is</i> and T2. Analysis was performed using the Mann Whitney U-test; **<i>p</i><0.01. (<b>B</b>) MiR-330-5p expression is significantly lower in patients with TRG 4 (non-responders) compared to patients with TRG 1 and 2 (responders). Analysis was performed using the Mann Whitney U-test; *<i>p</i> < 0.05. Data are presented as the mean ± SEM.</p

MiR-330 overexpression induces a downregulation in the levels of p-Akt.

<p>Transient miR-330 overexpression induced a decrease in the levels of p-Akt, 72 h post-transfection, in concordance with a decrease in E2F1 protein expression. Analysis was performed using one-way ANOVA and Tukey post-test; *<i>p</i> < 0.05. Data are presented as the mean ± SEM.</p

miR-330 overexpression does not alter chemosensitivity.

<p>The clonogenic survival assay was used to assess alterations in cellular sensitivity to cisplatin and 5-FU with miR-330 overexpression. The approximate IC₅₀ doses of cisplatin and 5-FU were used to treat the cells for 24 h. The overexpression of miR-330 did not significantly alter cellular sensitivity to cisplatin or 5-FU. Analysis was performed using paired t-test. Data are presented as the mean ± SEM.</p

Alterations in chemo- and radiotherapy sensitivity with miR-330-5p silencing.

<p>(<b>A</b>) Silencing miR-330-5p (miRZIP-330-5p) in the OE33 cell line did not significantly alter cellular sensitivity to cisplatin or 5-FU compared to the control (miRZIP-VC). However, there was a significant increase in resistance to radiotherapy with miR-330 silencing. Analysis was performed using paired t-test; *<i>p</i> < 0.05. (<b>B</b>) Silencing miR-330-5p in the OE19 cell line did not significantly alter cellular sensitivity to cisplatin, 5-FU or radiation. Analysis was performed using paired t-test. Data are presented as the mean ± SEM.</p

Alterations in E2F1 protein expression with miR-330 overexpression and silencing.

<p>Transient miR-330 overexpression (<b>A</b> and <b>B</b>) significantly decreased E2F1 protein expression when compared to the miR-VC (vector control). Densitometry was used to analyse western blot images. Analysis was performed using the one sample t-test; miR-VC 72 h vs. miR-330 72 h ***<i>p</i> < 0.001. Silencing miR-330-5p (miRZIP-330-5p) (<b>C</b> and <b>D</b>) did not alter E2F1 protein expression when compared to the miRZIP-VC (vector control). Data are presented as the mean ± SEM.</p

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

<div><p>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 <i>in vitro</i>. 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 neo-CRT in patients with OAC.</p></div

OE33 R cells are more metabolically robust.

<p>(<b>A</b>) OE33 P and OE33 R cells display similar sensitivities to the glycolytic inhibitor 2-deoxyglucose (55 µg/mL). (<b>B</b>) ECAR is significantly increased in OE33 R cells following oligomycin treatment (2 µg/mL), when compared to OE33 P. (<b>C</b>) OE33 R cells have significantly enhanced clonogenic survival following treatment with oligomycin (2 µg/mL) for 1.5 h, when compared to OE33 P cells. Data are presented as mean ± SEM from at least 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05.</p

Radioresistant OE33 R cells have increased mitochondrial mutagenesis and altered mitochondrial function.

<p>(<b>A</b>) OE33 R cells demonstrate significantly elevated basal levels of random mitochondrial mutations, when compared to OE33 P. (<b>B</b>) OE33 R cells have significantly increased basal ROS levels, when compared to OE33 P. ROS levels are significantly increased in OE33 P cells at 24 h post irradiation with 2 Gy. (<b>C</b>) Mitochondrial mass is significantly increased in OE33 P cells at 24 h post irradiation with 2 Gy, this effect is not seen in OE33 R. Data are presented as mean±SEM from 3 independent experiments. Statistical analysis was performed by 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05.</p

Patient cohort characteristics

<p>Abbreviations:^aValues given are mean (range); TNM, Tumour-node-metastasis clinical staging classification; NS, not specified; TRG, Tumour regression grade; N0, indicates lymph node metastasis negative; N1, lymph node metastasis positive.</p

OE33 R cells display alterations in mitochondrial-associated gene expression, total cellular energy and energy metabolism pathways.

<p>(A) Basal expression of 15 mitochondrial function and energy metabolism-associated genes were altered 1.5-fold in OE33 R cells, when compared to OE33 P. Data are presented as mean ± SEM from 2 independent experiments. (B) OE33 R cells have significantly increased basal intracellular ATP levels, when compared to OE33 P. ATP levels significantly decrease in OE33 P cells at 24 h post irradiation with 2 Gy, when compared to basal levels. Data are presented as mean ± SEM from 1 independent experiment. (C) Basal oxygen consumption rates (OCR) are significantly increased in OE33 R cells, when compared to OE33 P. Data are presented as mean ± SEM from 4 independent experiments. (D) OE33 R and OE33 P cells demonstrate similar extracellular acidification rates (ECAR). Data are presented as mean ± SEM from 4 independent experiments. Statistical analysis was performed by 2-tailed Student’s <i>t</i>-test, *<i>P</i><0.05, **<i>P</i><0.01.</p