microRNAs: Key regulators of chemotherapy response and metastatic potential via complex control of target pathways in esophageal adenocarcinoma

© 2018 Elsevier. This manuscript version is made available under the CC-BY-NC-ND 4.0 license: http://creativecommons.org/licenses/by-nc-nd/4.0/ This author accepted manuscript is made available following 12 month embargo from date of publication (April 2018) in accordance with the publisher’s archiving policy.Introduction: Incidence of esophageal adenocarcinoma (EAC) increased significantly over the last decades. Lack of response to chemotherapy is a major problem in the treatment of this disease. This study aims to assess the biological relevance of characteristic microRNA profiles of chemotherapy resistant EAC cells with regards to response to chemotherapy and biological behavior. Methods: We selected 3 microRNAs from characteristic microRNA profiles of resistant EAC (miR-27b-3p, miR-200b-3p, and miR-148a-3p). Expression of microRNAs was modified in 6 EAC cell lines. Effects on chemotherapy, adhesion, migration, apoptosis and cell cycle were assessed using standard assays. Target analyses were performed using Western Blot and Luciferase techniques. Results: MiR-27b-3p significantly sensitized cells to 5FU and Cisplatin in 83% respectively in 33% of cell lines, miR-148a-3p in 67% respectively 33% of cases. MiR-200b-3p increased sensitivity only towards 5FU in 50% of cases. Co-transfections with miR-27b-3p/miR-148a-3p showed an additive effect on response to chemotherapy in 50% of cases. Upregulation of miR-148a-3p reduced protein expression levels of DNMT-1, MSK-1, Bcl-2 and Bim, and miR-27b upregulation led to downregulation of Sp1 and PPARy proteins implicating a potential negative post-transcriptional control via the respective microRNAs. Finally, we were able to confirm Bcl-2 for the first time as direct target of miR-148a-3p in EAC. Conclusion: This study demonstrates that specific microRNA profiles of chemotherapy resistant EAC in fact determine their response to chemotherapy and biological behavior. Our data further show that microRNA-mediated regulation of chemotherapy resistance is complex, and several microRNAs seem to “co-operate” at various steps within a broad number of pathways what fits very well to our recently proposed understanding of microRNA-mediated regulation as function of cellular functional complexes. These data highlight the promising potential of microRNAs to predict or monitor treatment response to chemotherapy in EAC, and to potentially modulate tumor biology in a therapeutic approach

MicroRNA Profiling in Oesophageal Adenocarcinoma Cell Lines and Patient Serum Samples Reveals a Role for miR-451a in Radiation Resistance

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, focusing on radiation. Intrinsic radiation resistance and chemotherapy drug resistance were assessed in eight OAC cell lines, and miRNA expression profiling was performed via TaqMan OpenArray qPCR. miRNAs discovered were either uniquely associated with resistance to radiation, cisplatin, or 5-FU, or were common to two or all three of the treatments. Target mRNA pathway analyses indicated several potential mechanisms of treatment resistance. miRNAs associated with the in vitro treatment responses were then investigated for association with pathologic response to neoadjuvant chemoradiotherapy (nCRT) in pre-treatment serums of patients with OAC. miR-451a was associated uniquely with resistance to radiation treatment in the cell lines, and with the response to nCRT in patient serums. Inhibition of miR-451a in the radiation resistant OAC cell line OE19 increased radiosensitivity (Survival Fraction 73% vs. 87%, p = 0.0003), and altered RNA expression. Pathway analysis of effected small non-coding RNAs and corresponding mRNA targets suggest potential mechanisms of radiation resistance in OAC

MicroRNA profiling in oesophageal adenocarcinoma cell lines and patient serum samples reveals a role for mir-451a in radiation resistance

Many patients with Oesophageal Adenocarcinoma (OAC) do not benefit from chemoradiotherapy treatment due to therapy resistance. To better understand the mechanisms involved in resistance and to find potential biomarkers, we investigated the association of microRNAs, which regulate gene expression, with the response to individual treatments, foc

Complex Epigenetic Regulation of Chemotherapy Resistance and Biology in Esophageal Squamous Cell Carcinoma via MicroRNAs

Background: Resistance towards chemotherapy is a major obstacle in the treatment of esophageal squamous cell carcinoma (ESCC). We investigated the role of specific microRNAs in chemotherapy resistance and tumor biology. Methods: We selected three microRNAs from characteristic microRNA signatures of resistant ESCC (hsa-miR-125a-5p, hsa-miR-130a-3p, hsa-miR-1226-3p), and hsa-miR-148a-3p. Effects on chemotherapy, adhesion, migration, apoptosis and cell cycle were assessed in six ESCC cell lines. Target analyses were performed using Western blotting and luciferase techniques. Results: MiR-130a-3p sensitized cells towards cisplatin in 100% of cell lines, miR-148a-3p in 83%, miR-125a-5p in 67%, miR-1226-3p in 50% (p ≤ 0.04). MiR-130a-3p sensitized 83% of cell lines towards 5-FU, miR-148a-3p/miR-125a-5p/miR-1226-3p only 33% (p ≤ 0.015). Several resistance-relevant pathways seem to be targeted on various levels. Bcl-2 was confirmed as a direct target of miR-130a-3p and miR-148a-3p, and p53 as a target of miR-125a-5p. All microRNAs decreased migration and adhesion, except miR-130a-3p, and increased apoptosis. Simultaneous manipulation of two microRNAs exhibited additive sensitizing effects towards cisplatin in 50% (miR-125a-5p/miR-148a-3p), and 75% (miR-148a-3p/miR-130a-3p) of cell lines (p ≤ 0.006). Conclusion: Our data present strong evidence that specific microRNA signatures are responsible for drug resistance and aggressiveness of ESCC. Final functional readout of these complex processes appears to be more important than single microRNA-target interactions

Oestrogen Receptor Isoforms May Represent a Therapeutic Target in Oesophageal Adenocarcinoma

Oesophageal adenocarcinoma is a rapidly increasing problem in which treatment options are limited. Previous studies have shown that oesophageal adenocarcinoma cells and tissues express oestrogen receptors (ERs) and show growth suppression and apoptosis in response to ER modulator agents such as tamoxifen. ERs are known to be expressed in a number of isoforms that act together to regulate cell growth and cell death. In this study, we used western blotting to profile the expression of ERα and ERβ isoforms, and expression of the oncologically related molecules p53, HER2, and EGFR, in a panel of oesophageal adenocarcinoma cell lines. The cytotoxicity of tamoxifen in the cell lines was determined with Annexin V-FITC flow cytometry, and correlations between cytotoxicity and receptor expression were assessed using Spearman’s rank-order correlation. Oesophageal adenocarcinoma cell lines showed varying cytotoxicity in response to tamoxifen. The ER species ERα90, ERα50, and ERα46, as well as p53, were positively associated with a cytotoxic response. Conversely, ERα74, ERα70, and ERβ54 were associated with a lack of cytotoxic response. The ER species detected in oesophageal adenocarcinoma cells may work together to confer sensitivity to ER modulators in this disease, which could open up a new avenue for therapy in selected patients

Correction: Complex Epigenetic Regulation of Chemotherapy Resistance and Biology in Esophageal Squamous Cell Carcinoma via MicroRNAs. <i>Int. J. Mol. Sci.</i> 2018, <i>19</i>, 499

We would like to submit a correction to the published paper [...

Economic Burden of Endoscopic Vacuum Therapy Compared to Alternative Therapy Methods in Patients with Anastomotic Leakage After Esophagectomy

Background!#!Endoscopic vacuum therapy (EVT) has become a promising option in the management of anastomotic leakage (AL) after esophagectomy. However, EVT is an effortful approach associated with multiple interventions. In this study, we conduct a comparative cost analysis for methods of management of AL.!##!Methods!#!All patients who experienced AL treated by EVT, stent, or reoperation following Ivor Lewis esophagectomy for esophageal cancer were included. Cases that were managed by more than one modality were excluded. For the remaining cases, in-patient treatment cost was collected for material, personnel, (par)enteral nutrition, intensive care, operating room, and imaging.!##!Results!#!42 patients were treated as follows: EVT n = 25, stent n = 13, and reoperation n = 4. The mean duration of therapy as well as length of overall hospital stay was significantly shorter in the stent than the EVT group (30 vs. 44d, p = 0.046; 34 vs. 53d, p = 0.02). The total mean cost for stent was €33.685, and the total cost for EVT was €46.136, resulting in a delta increase of 37% for EVT vs. stent cost. 75% (€34.320, EVT), respectively, 80% (€26.900, stent) of total costs were caused by ICU stay. Mean pure costs for endoscopic management were relatively low and comparable between both groups (EVT: €1.900, stent: €1.100, p = 0.28).!##!Conclusion!#!Management of AL represents an effortful approach that results in high overall costs. The expenses directly related to EVT and stent therapy were however comparatively low with more than 75% of costs being attributable to the ICU stay. Reduction of ICU care should be a central part of cost reduction strategies

Minor Sphincter Sparing Surgery for Successful Closure of Perianal Fistulas in Patients with Crohn’s Disease

The purpose of this study is to demonstrate that repetitive minor surgical procedures allow for a high rate of permanent closure of perianal fistulas in patients with Crohn’s disease (CD). Patients with perianal fistulizing CD (PFCD) who underwent perianal surgery at the University Hospital of Muenster between 2003 and 2018 were assessed for fistula characteristics and surgical procedures. We included 45 patients (m:f = 28:17) with a mean age of 27 years at first fistula appearance. Of these, 49% suffered from a complex fistula. An average of 4.2 (1–14) procedures were performed, abscess incisions and fistula seton drainages included. Draining setons were left in place for 5 (1–54) months, until fistula closure. Final surgical techniques were fistulotomy (31.1%), seton removal with sustained biological therapy (26.7%), Anal Fistula Plug (AFP) (17.8%), Over-The Scope-Clip proctology (OTSC) (11.1%), and mucosa advancement flap (4.4%). In 8.9% of cases, the seton was kept as permanent therapy. The time from first to last surgery was 18 (0–182) months and the median follow-up time after the last surgery was 90 (15–200) months. The recurrence rate was 15.5% after 45 (17–111) months. Recurrent fistulas healed after another 1.86 (1–2) surgical re-interventions. The final success rate was 80%. Despite biological treatment, PFCD management remains challenging. However, by repeating minor surgical interventions over a prolonged period of time, high permanent healing rates can be achieved

Complex Epigenetic Regulation of Chemotherapy Resistance and Biology in Esophageal Squamous Cell Carcinoma via MicroRNAs

Background: Resistance towards chemotherapy is a major obstacle in the treatment of esophageal squamous cell carcinoma (ESCC). We investigated the role of specific microRNAs in chemotherapy resistance and tumor biology. Methods: We selected three microRNAs from characteristic microRNA signatures of resistant ESCC (hsa-miR-125a-5p, hsa-miR-130a-3p, hsa-miR-1226-3p), and hsa-miR-148a-3p. Effects on chemotherapy, adhesion, migration, apoptosis and cell cycle were assessed in six ESCC cell lines. Target analyses were performed using Western blotting and luciferase techniques. Results: MiR-130a-3p sensitized cells towards cisplatin in 100% of cell lines, miR-148a-3p in 83%, miR-125a-5p in 67%, miR-1226-3p in 50% (p ≤ 0.04). MiR-130a-3p sensitized 83% of cell lines towards 5-FU, miR-148a-3p/miR-125a-5p/miR-1226-3p only 33% (p ≤ 0.015). Several resistance-relevant pathways seem to be targeted on various levels. Bcl-2 was confirmed as a direct target of miR-130a-3p and miR-148a-3p, and p53 as a target of miR-125a-5p. All microRNAs decreased migration and adhesion, except miR-130a-3p, and increased apoptosis. Simultaneous manipulation of two microRNAs exhibited additive sensitizing effects towards cisplatin in 50% (miR-125a-5p/miR-148a-3p), and 75% (miR-148a-3p/miR-130a-3p) of cell lines (p ≤ 0.006). Conclusion: Our data present strong evidence that specific microRNA signatures are responsible for drug resistance and aggressiveness of ESCC. Final functional readout of these complex processes appears to be more important than single microRNA-target interactions