You are here

  • Home
  • Archive
  • Volume 71, Issue 4
  • miR-194 as predictive biomarker of responsiveness to neoadjuvant chemoradiotherapy in patients with locally advanced rectal adenocarcinoma

Article Text

Article menu

Download PDFPDF

Original article
miR-194 as predictive biomarker of responsiveness to neoadjuvant chemoradiotherapy in patients with locally advanced rectal adenocarcinoma
  1. Edoardo D’Angelo1,2,
  2. Carlo Zanon3,
  3. Francesca Sensi2,
  4. Maura Digito2,
  5. Massimo Rugge4,
  6. Matteo Fassan4,
  7. Marco Scarpa5,
  8. Salvatore Pucciarelli1,
  9. Donato Nitti1,
  10. Marco Agostini1,2
  1. 1 Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Padua, Italy
  2. 2 Nanoinspired Biomedicine Lab, Pediatric Research Institute - Fondazione Città della Speranza, Padua, Italy
  3. 3 Neuroblastoma Laboratory, Pediatric Research Institute - Fondazione Città della Speranza, Padua, Italy
  4. 4 Department of Medicine (DIMED), University of Padua, Padua, Italy
  5. 5 Surgical Oncology Unit, Istituto Oncologico Veneto, IOV-IRCCS, Padua, Italy
  1. Correspondence to Dr Marco Agostini, Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Padua, Italy; m.agostini{at}unipd.it

Abstract

Aims Curative surgery remains the primary form of treatment for locally advanced rectal cancer (LARC). Recent data support the use of preoperative chemoradiotherapy (pCRT) to improve the prognosis of LARC with a significant reduction of local relapse and an increase of overall survival. Unfortunately, only 20% of the patients with LARC present complete pathological response after pCRT, whereas in 20%–40%, the response is poor or absent.

Methods We investigated the expression level of miR-194 in n=38 patients with LARC using our public microRNA (miRNA) expression dataset. miR-194 expression was further validated by real-time quantitative PCR (qRT-PCR) and in situ hybridisation (ISH). Protein–protein interaction network and pathway enrichment analysis were performed on miR-194 targets.

Results and discussion Using biopsy samples collected at diagnosis, mir-194 was significantly upregulated in patients responding to treatment (p value=0.016). The data was confirmed with qRT-PCR (p value=0.0587) and ISH (p value=0.026). Protein–protein interaction network and pathway enrichment analysis reveal a possible mechanism of susceptibility to pCRT involving Wnt pathway via its downstream mediator TRAF6. Finally, we interrogated the Comparative Toxicogenomics Database database in order to identify those chemical compounds able to mimic the biological effects of miR-194 as new possible therapeutic option in LARC treatment. The present study combining miRNA expression profiling with integrative computational biology identified miR-194 as predictive biomarker of response to pCRT. Using known and predicted drug mechanism of action, we then identified possible chemical compounds for further in vitro validation.

  • rectal cancer
  • tumour markers
  • oncology

https://doi.org/10.1136/jclinpath-2017-204690

Statistics from Altmetric.com

    Request Permissions

    If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.

    Introduction

    Colorectal cancer (CRC) is the third leading cause of cancer death in both sexes in the USA.1 The epidemiological data provided by the American Cancer Society estimated 59 000 deaths in 2016 due to CRC.1 Nearly one-third of CRCs are represented by rectal cancer (RC) and approximately 40% of RCs are locally advanced at diagnosis (locally advanced rectal cancer (LARC); T3, T4 or N+).2 Curative surgery remains the primary form of treatment for LARC. However, in the last two decades, new multimodality strategies have improved the prognosis of LARC with a significant reduction of local relapse and an increase in terms of overall survival. Recent data support the use of preoperative chemoradiotherapy (pCRT), with the aims to (i) reduce non-resectable tumours, (ii) achieve the tumour cell-free surgical margins and (iii) reduce tumour burden and increase the possibility for curative surgery.3 Unfortunately, only 20% of the patients with LARC present complete pathological response after pCRT, whereas in 20%–40%, the response is poor or absent.4 Thus, predictive signatures are urgently needed to identify patient groups that will gain maximum benefit from pCRT.

    MicroRNAs (miRNAs) are a class of small non-coding RNAs, ranging in size from 19 to 25 nucleotides that act as post-transcriptional regulators of genes expression, by silencing their 3′-UTR-mRNA targets.5 Several studies have demonstrated that miRNAs play a crucial role across diverse biological processes including proliferation, apoptosis and cell differentiation, which are frequently deregulated in human cancers.6 Based on these observations, we hypothesised that specific miRNAs may function as fine regulators of tumour response to pCRT and therefore may have predictive, prognostic and therapeutic potential in LARC.7 8 Recently, the expression pattern and function of miR-194 in various cancers has been reported. A series of studies reported a generally decreased expression of miR-194 in cancer, suggesting its tumour-suppressive function.9–11 In CRC, Chiang et al 12 showed that miR-194 was significantly downregulated compared with healthy mucosa. Furthermore, they found that the overexpression of miR-194 could significantly inhibit cell proliferation in HCT-116 cells. Another study revealed a pro-angiogenesis function of miR-194 in HCT-116 cells.13 To date, the function of miR-194 in CRC still remains largely unknown and controversial. Our study was designed (i) to evaluate whether miR-194 expression profile could be used as predictive biomarker in patients with LARC and (ii) to characterise the molecular mechanism that leads to responders phenotype by analysing miR-194 target genes using a systems biology approaches.

    Patients and methods

    Microarray analysis

    This study is based on the use of an RC patient cohort for a total of n=38 patients with LARC. The cohort comprises n=38 patients with primary adenocarcinoma of the rectum, who underwent pCRT followed by surgery at our institution (First Surgery Clinic, University of Padova), in which we reanalysed our public microarray miRNA expression profile dataset (GEO dataset: GSE68204).14 The microarray data platform was Agilent-021827 Human miRNA Microarray (V3). The endoscopic tissue samples were used to validate the expression profile of miR-194 using quantitative real-time PCR (qRT-PCR) after RNA extraction. Only few studies performed a complete miRNA expression profile using microarray technology on preoperative biopsy tissue samples in LARC. Online supplementary table S1 summarises the microarray studies for prediction of pCRT response in LARC with raw data available in GEO dataset and the reason for their exclusion from this study. Detailed information about the patients are listed in table 1. All patients fulfilled the following criteria: histological confirmed primary adenocarcinoma of the rectum, tumour within 11 cm from the anal verge by proctoscopic examination and/or endorectal ultrasonography, clinical stage cT3-4 and/or N-/+ and resectable disease. After pretherapeutically staging, all the patients were treated with pCRT. The detailed treatment protocols are listed in table 1.

    Supplementary file 1

    View this table:
    Table 1

    Clinical and pathological characteristics of n.38 patients with LARC enrolled in the study

    Pathological assessment and definition of tumour response

    Standardised histological examination of the surgical specimens was done according to the American Joint Committee on Cancer guidelines. The histological tumour response to pCRT was assessed according to the modified tumour regression classification of Mandard et al.15 Patients were subdivided into responders (R, TRG 1–2) and non-responders (NR, TRG 3–5).

    Tissue samples and RNA extraction

    Endoscopic tumour and normal adjacent rectal biopsies of n=29 patients with LARC enrolled in the study were tested using qRT-PCR analysis in order to confirm the microarray data (for 9 out of 38 patients, we did not have sufficient amount of biological material to perform the analysis). All biopsies underwent standardised histopathological examination based on haematoxylin–eosin staining of 5 µm frozen sections. Tumour specimens with ≥70% malignant cells were considered for the experiment. Total RNA extraction, from at least two micro-biopsies, was performed using TRIZOL Reagent (Invitrogen, Carlsbad, California) following standard procedures from each endoscopic biopsy using 20 µm thick sections. Total RNA was preserved in a final volume of 20 µl of DEPC water at −80°C with 1 µl RNase Inhibitor (RNaseOUT Recombinant, 40 U/µl, Invitrogen). RNA quantity was measured on an ND-1000 spectrophotometer (NanoDrop Technologies), and quality was assessed by capillary electrophoresis with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California). Samples with RIN >6.5 (RNA 6000 Series Nano Chips) and samples enriched for small nucleic acid fragments with a percentage <35% (Agilent Small RNA Kit) were selected for the qRT-PCR.

    Real-time quantitative PCR

    qRT–PCR was performed as follow: 10 ng of total RNA was reverse transcribed by using stem–loop RT-specific primers for miR-194 (ID: 000493) or small nuclear snRNA U6 (ID: 001973), following the instruction of TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, California, USA). miRNA expression was measured with a specific TaqMan MicroRNA Assay and assayed on an ABI Prism 7500 System (Applied Biosystems) with conditions of: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 60 s. The qRT-PCR reactions were conducted in duplicate. One random sample was used as internal calibrator.

    In situ RNA hybridisation

    Locked nucleic acid (LNA) probes with complementarity to 22 bp sections of hsa-miR-194 were labelled with 5′-digoxigenin and synthesised by Exiqon (Copenhagen, Denmark). Tissue sections were digested with in situ hybridisation (ISH) protease 1 (Ventana Medical Systems, Milan, Italy) and ISH performed as described, with minor modifications.16 Slides were counterstained in fast red solution. Positive (U6; Exiqon) and negative scrambled LNA probes were used as controls. A series of eight pretreatment endoscopy biopsies obtained from four responders and four non-responders RCs of our cohort were analysed. Only cytoplasmic miR-194 intensity was retained for scoring, and miRNA expression was quantified analysing chromogen-specific intensity by ImageJ in 5 HPF/case.

    Network analysis

    We performed in silico analyses on hsa-miR-194, to identify its gene targets. We selected ‘top third’ and ‘top 1%’ predicted targets of hsa-miR-194 from mirDIP 3.0 (http://ophid.utoronto.ca/mirDIP) and further selected only the ones that were predicted by at least six different algorithms. We then identified protein–protein interactions (PPIs) among miR-194 targets using IID database V.2016–03 (http://ophid.utoronto.ca/iid), creating a PPI network that we visualised and analysed in NAViGaTOR 3.0 (http://ophid.utoronto.ca/navigator). We then performed a functional annotation and comprehensive pathway enrichment analysis of all the proteins of the network using pathDIP V.2.5 pathway database17 (http://ophid.utoronto.ca/pathDIP), using the following settings: extended pathway associations; experimentally detected PPIs; minimum confidence level for predicted protein–pathway associations: 0.99. We considered significantly enriched only pathways with q-value (false discovery rate (FDR): Benjamini-Hochberh (BH) method) lower than 0.01. The PANTHER Classification System (accessed on 14 June 2016) was used to classify ‘top third’ and ‘top 1%’ predicted targets of hsa-miR-194 according to: molecular function and biological process. The study of the drugs targeting the same network was performed using the Comparative Toxicogenomics Database (CTD) V.14 736 (http://ctdbase.org/).

    Statistical analysis

    Relative expression of miR-194 was normalised to snRNA U6. Normalised expression was calculated using the comparative Ct method, and the fold change was expressed as 2−ΔΔCt. Differences in miR-194 expression were evaluated by Mann-Whitney test for microarray analysis and qRT-PCR analysis. Data were visualised using GraphPad Prism 5.0 software. Significance (Student’s t-test): *p<0.05. Data were expressed as mean values±SD.

    Results

    Patient, tumour and treatment characteristics

    Of the n=38 patients with LARC enrolled in the study, n=16 (42%) were classified as responders and n=22 (58%) were non-responders. There were no significant differences between the two cohorts regarding patients’ age, gender, treatment and tumour characteristics. Following the TRG classification, n=8 (21%) of them showed a pathological complete response (TRG 1).

    miR-194 is significantly upregulated in responders patients with LARC

    To test whether miR-194 could be used as predictive biomarker in patients with LARC, we evaluated its expression level in patients with LARC, reanalysing miRNA expression data of our public microarray platform. Our results showed that the expression level of miR-194 was significantly increased in R patients in comparison with NR (p value=0.016, Mann-Whitney test) (figure 1A). To confirm the expression profile of miR-194 obtained from the microarray analysis, we performed the validation qRT-PCR in n=29 patients with LARC of the same cohort. qRT-PCR corroborated the results from microarray analysis. We confirmed the overexpression of miR-194 in R patients compared with NR, although the result was marginally significant probably due to the reduced number of sample (p value=0.0587, Mann-Whitney test) (figure 1B). To further support the miR-194 expression results described above, we decided to perform tissue ISH analysis. Among all the 29 biopsies, only 8 samples (4 responders and 4 non-responders patients) were suitable for ISH studies. As highlighted in figure 1C, A consistent significant overexpression of miR-194 was observed in R patients compared with NR. miR-194 was highly expressed in the cytoplasm of cancerous epithelial cells and characterised by a granular pattern stained in blue.

    Figure 1
    Figure 1

    miR-194 is significantly upregulated in responders patients with LARC. (A) Tissue miR-194 expression level was evaluated by microarray reanalysis in our cohort of patients (box plot). normalised Relative Quantity (nRQ): responders, light grey; non-responders, dark grey; Mann-Whitney test. (B) Real-time quantitative PCR in the same samples (dot plot). nRQ: responders, green outline; non-responders, red outline; Mann-Whitney test. (C) Representative in situ hybridisation evaluation of miR-194 in R and NR rectal cancers performed on the same sample enrolled in microarray analysis (4 R vs 4 NR). The presence of miR-194 is shown by a grainy blue cytoplasmic stain; slides counterstained in fast red (Original magnifications 10× and 20×). RT-PCR, real-time PCR.

    PPI network and integrated network analysis

    Using mirDIP,18 we identified 507 predicted mRNA targeted by miR-194 that passed our selective filter (data not shown). In order to understand the molecular mechanism behind the observed clinical phenotype, we integrated the 507 identified gene targets, in a functional biological network with their protein–protein interactors retrieved from IID.19 As shown in figure 2, the most central proteins in this network include BMI1, IQGAP1, NCL and TRAF6.

    Figure 2
    Figure 2

    Functional interaction network of miR-194 predicted targets. NAViGaTOR PPI functional network generated with the interpolation of 507 mRNA targeted by miR-194. Node colour indicates the Gene Ontology Molecular function. Node size indicates the number of interaction with other proteins in the network (the bigger the node, the greater the number of interaction). Right: the most representative pathway identified using pathDIP.

    Analysis of the predicted target gene and pathway enrichment analysis

    To further investigate the biological role of miR-194, we performed a pathway enrichment analysis, using pathDIP. The ‘top six’ most significant pathways were: EGFR1, Interactome of polycomb repressive complex 2, Cell Cycle Checkpoints, the prc2 complex sets long-term gene silencing through modification of histone tails, WNT-Core and Oxidative Stress-Induced Senescence (for complete dataset see supplementary table S2). In addition, we performed a Panther Ontology (PO) clustering of the identified targets, considering molecular function and biological process (table 2A and B). The most represented PO molecular functions included ‘Organic cyclic compound binding’ (p value=1.1E-8) and ‘Protein binding’ (p value=1.15E-7), while the most enriched PO biological process included: ‘Macromolecule modification’, ‘Cellular protein modification process’ and ‘Protein modification process’ (p value<3.1E-8). Interestingly, in the top 10, biological processes included ‘Organic substances metabolic process’ (p value=9.46E-5).

    Supplementary file 2

    View this table:
    Table 2

    Panther Ontology (PO) clusterisation of 507 mRNA targeted by miR-194, based on: the (A) molecular function, (B) biological Process in which they are involved 

    View this table:

    The analysis of drug targets using CTD was performed in order to find those chemical compound that cause the 100% downmodulation of the miR-194 mRNAs target. We found 23 drugs targeting three or more proteins of the network with a 100% of downmodulation effect. The drugs targeting five or more proteins in the network (drug nodes with the highest degree) include gold, 4-aminophenylarsenoxide, 4-hydroxy-2-nonenal, PD0325901 (MEK inhibitor), salinomycin and lucanthone. In this network, the only chemotherapeutic agents conventionally used in LARC treatment is capecitabine with just two target downmodulated (online supplementary table S3).

    Supplementary file 3

    Discussion

    To date, the best treatment option for patients with LARC is represented by pCRT, followed by curative surgery. Unfortunately, after pCRT, the complete pathological response rate is approximately 20%, whereas in 20%–40% of patients, the response is poor or absent.20 Thus, the identification of predictive markers of pCRT response is one of the main priorities in management of patients with LARC. In this landscape, miRNA expression patterns have been successfully applied to predict the response to pCRT in CRC.21 Microarray studies for prediction of response to pCRT in LARC present heterogeneous pCRT regimen, different array platforms, different pCRT response evaluation system and often were focused on gene expression profile; therefore, they are difficult to integrate.

    In the present study, we demonstrated that miR-194 was upregulated in responders patients by analysing its expression profile in a cohort of patients with LARC, in which we performed a miRNA microarray expression profile analysis.14 Recent data have shown that miR-194 plays an important part in tumourigenesis. For example, miR-194 was found to act as a tumour suppressor in gastric cancer, endometrial cancer, renal cell carcinoma, lung cancer and breast cancer, while as an oncogene in pancreatic cancer.9 Our results obtained in patients with LARC suggest that miR-194 seems to function as tumour suppressor miRNA, in complete accordance with previous finding in CRC. In fact, Chiang et al 22 demonstrated that miR-192/194/215 cluster is frequently downregulated in CRC and associated to an increased cellular proliferation and tumour size. Zhao et al 23 functionally demonstrated that overexpression of miR-194 inhibited CRC cell viability and invasion in vitro and suppressed CRC xenograft tumour growth in vivo. Again, Li et al 24 observed that miR-194 inhibited the acquisition of the epithelial-mesenchymal transition phenotype in gastric cancer cells, inhibiting cell migration and invasion.

    In the current study, we demonstrated in endoscopic tumour samples collected before pCRT and before surgery that miR-194 is upregulated in responders patients and could be used as predictive biomarker of response to treatment. Furthermore, we investigated the molecular mechanisms downstream of miR-194 using a systems-biology-integrated approaches, aiming at the interpolation of data collected from genetic, protein and drug database in order to contextualise a single molecule into a complex network of molecular signals.

    First, to better link the biological role of this molecule to the observed responsive phenotype, we generated a functional interaction network with its 507 predicted mRNAs targets. Based on the number of interactions with other members, we extrapolated from the functional network the four proteins that could play a pivotal role in linking the observed phenotype with miR-194 deregulation: BMI1, NCL, IQGAP1 and TRAF6. BMI1 polycomb ring finger is a gene involved in the maintenance of stem cells, malignant transformation and biologic aggressiveness of several human carcinomas. The clinical significance of BMI1 in chemoresistance and its correlation with therapy failure in several cancer types has been established.25 However, the precise mechanism of BMI1 on the regulation of chemoresistance in tumour cells is not completely understood. In CRC, it has been reported that high expression of BMI1 was significantly associated with poor 5-year survival and correlated with TNM stage and histological grade. Nucleolin (NCL) is a highly conserved nucleocytoplasmic multifunctional protein, abundantly expressed in the nucleolus, with the main function of regulating mRNA translation and stability. Poor data exist about NCL and its role in antineoplastic treatment. Pichiorri et al reported that NCL promotes the maturation of a specific set of miRNAs that are implicated in the pathogenesis of several human cancers whose overexpression is often associated with greater aggressiveness and resistance to antineoplastic therapies.26 IQGAP1 is a scaffold protein that participates in PPIs and integrates diverse signalling pathways. The interacting partners of these IQGAP1 domains include actin, E-cadherin, b-catenin and members of the MAPK pathway.27 Through interaction with these proteins, IQGAP1 regulates various basic cellular activities such as cytoskeletal organisation, cell–cell adhesion, cell migration, transcription and signal transduction. Hayashi et al reported that IQGAP1 is involved in CRC cell invasion and may lead to vessel invasion and consequent poor prognosis.28 However, a clear evidence of IQGAP1 involvement in molecular mechanisms related to antineoplsatic therapies is still lacking. Interestingly, the core of this network is represented by TRAF6 that interacts with 57 different proteins. TRAF6 is a member of the TNF receptor-associated factor protein family able to transduce signals from members of the TNF receptor superfamily, such as Toll/IL-1 family, TNF receptor, FAS and TGFβ receptor.29 Furthermore, TRAF6 is a common downstream effector of a number of signalling pathways that are activated by the cell in case of DNA damage, inflammation, cytotoxic drug and irradiation exposure, in order to activate an antiapoptotic and proliferative response.30 Interestingly, in a similar independent study, this molecule resulted involved into a molecular mechanism of radio-chemosensitivity.4 In parallel, we performed a pathway enrichment analysis analysing miR-194 mRNAs targets using pathDIP. Surprisingly, we identified Wnt signalling pathway, among the most represented pathway, that has as downstream effector TRAF6, crucial for its role in the transmission of proliferative and antiapoptotic stimuli. Collectively, these data lead us to hypothesise a molecular mechanism triggered by miR-194 overexpression, in which occurs a global direct and indirect downmodulation of the Wnt signalling axis. Another clear suggestion about the crucial role of miR-194 in the response to radio-chemotherapy emerges from its involvement in the pathway of Oxidative Stress-Induced Senescence. In fact, the main citotoxic mechanism of many anticancer drugs acts by upregulating the level of ROS production and accumulation. In cancer cells, this factor enhances the anticancer activity of the agents and contributes to drug-induced apoptosis. Cancer cells with a high antioxidant capacity may be resistant to several anticancer drugs. Some tumour cells enhance total antioxidant capacity by upregulating their antioxidant enzymes, which allow them to bypass damage from chemotherapy or radiotherapy.31 Therefore, a miR-194 overexpression could lead to a fatal ROS accumulation favouring cancer cells apoptosis. In addition, PO classification of the 507 predicted mRNAs target of miR-194 highlighted that this small molecule affects the binding and metabolism of organic cyclic compound, such as 5-fluorouracil, capecitabine and oxaliplatin, three of the most used chemotherapeutics in pCRT of LARC.32 Finally, we identified a series of chemical compounds that might be used as miR-194 mimic, paving the way for a new therapeutic strategy based on molecular stratification of patients. The analysis of drug targets using CTD was performed in order to find those chemical compound that cause the 100% downmodulation of the miR-194 mRNA targets. We found 23 drugs targeting three or more proteins of the network with a 100% of downmodulation effect. The drugs targeting five or more proteins in the network (drug nodes with the highest degree) include gold, 4-aminophenylarsenoxide, 4-hydroxy-2-nonenal, PD 0325901 (MEK inhibitor), salinomycin and lucanthone. In this network, the only chemotherapeutic agent conventionally used in LARC treatment is capecitabine, with just two target downmodulated. In addition, analysing the CTD results, the identified compounds have to be subdivided between toxic and therapeutic because the database structure does not provide for a subdivision a priori. On this basis, one of the most interesting compounds was 4-aminophenylarsenoxide, an arsenic derivative, already used as a chemotherapeutic together with imatinib in the treatment of chronic myeloid leukaemia, that has been used alone with some success to treat multiple myeloma, myelodysplasia syndrome and non-Hodgkin’s lymphoma.33 Another intriguing compound was PD 0325901, a second-generation MEK inhibitor, currently in phase I trial for treating patients with solid tumours that have spread to other parts of the body or cannot be removed by surgery (National Cancer Institute, https://www.cancer.gov). Interestingly, Mohan et al 34 demonstrated that PD 0325901 was able to overcome resistance and enhance antitumour effects observed with the first generation of MEK/ERK/NF-κB inhibitors.

    In summary, we demonstrated that miR-194 is significantly deregulated in patients with LARC and its overexpression is associated to an effective clinical response to pCRT. Furthermore, using a systems biology approach, we delineated a possible molecular mechanism in which TRAF6, a direct target of miR-194, might act as indirect downmodulator of the Wnt signalling pathway. Further in vitro and in vivo analysis will be required to confirm an effective deregulation of those signalling pathway and their impact on cancer cells response to pCRT.

    Take home messages

    • Preoperative chemoradiotherapy is a strategy to improve the prognosis of patients with locally advanced rectal cancer.

    • miR-194 expression profile could be used as predictive biomarker in patients with locally advanced rectal cancer.

    • Systems biology identified TRAF6 as relevant druggable targets in patients with locally advanced rectal cancer.

    Acknowledgments

    The authors would like to thank Vincenza Guzzardo and Chiara Pastrello for their technical support. This research has been conducted using the resources of the Tissue Bank of the 1st Surgical Clinic, University Hospital of Padova.

    References

    1. ↵
      2. Siegel RL ,
      3. Miller KD ,
      4. Jemal A , et al
      . Cancer statistics, 2016. A cancer journal for clinicians 2016;66:7–30.doi:10.3322/caac.21332
      OpenUrl
    2. ↵
      2. Siegel R ,
      3. DeSantis C ,
      4. Jemal A , et al
      . Colorectal cancer statistics, 2014. A cancer journal for clinicians 2014;64:104–17.doi:10.3322/caac.21220
      OpenUrl
    3. ↵
      2. Agostini M
      . Predictive response biomarkers in rectal cancer neoadjuvant treatment. Frontiers in Bioscience 2014;S6:110–9.doi:10.2741/S418
      OpenUrl
    4. ↵
      2. Agostini M ,
      3. Zangrando A ,
      4. Pastrello C , et al
      . A functional biological network centered on XRCC3: a new possible marker of chemoradiotherapy resistance in rectal cancer patients. Cancer biology & therapy 2015;16:1160–71.doi:10.1080/15384047.2015.1046652
      OpenUrl
    5. ↵
      2. Bartel DP
      . MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97.
      OpenUrlCrossRefPubMedWeb of Science
    6. ↵
      2. Iorio MV ,
      3. Croce CM
      . MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol 2009;27:5848–56.doi:10.1200/JCO.2009.24.0317
      OpenUrlAbstract/FREE Full Text
    7. ↵
      2. D’Angelo E ,
      3. Fassan M ,
      4. Maretto I , et al
      . Serum miR-125b is a non-invasive predictive biomarker of the pre-operative chemoradiotherapy responsiveness in patients with rectal adenocarcinoma. Oncotarget 2016;7:28647–57.doi:10.18632/oncotarget.8725
      OpenUrl
    8. ↵
      2. Agostini M ,
      3. Janssen KP ,
      4. Kim LJ , et al
      . An integrative approach for the identification of prognostic and predictive biomarkers in rectal cancer. Oncotarget 2015;6:32561–74.doi:10.18632/oncotarget.4935
      OpenUrl
    9. ↵
      2. Wang B ,
      3. Shen ZL ,
      4. Gao ZD , et al
      . MiR-194, commonly repressed in colorectal cancer, suppresses tumor growth by regulating the MAP4K4/c-Jun/MDM2 signaling pathway. Cell Cycle 2015;14:1046–58.doi:10.1080/15384101.2015.1007767
      OpenUrl
    10. ↵
      2. Zhu X ,
      3. Li D ,
      4. Yu F , et al
      . miR-194 inhibits the proliferation, invasion, migration, and enhances the chemosensitivity of non-small cell lung cancer cells by targeting forkhead box A1 protein. Oncotarget 2016;7:13139–52.doi:10.18632/oncotarget.7545
      OpenUrl
    11. ↵
      2. Zhao Y ,
      3. Li F ,
      4. Zhang X , et al
      . MicroRNA-194 acts as a prognostic marker and inhibits proliferation in hepatocellular carcinoma by targeting MAP4K4. Int J Clin Exp Pathol 2015;8:12446–54.
      OpenUrl
    12. ↵
      2. Chiang YP ,
      3. Song YX ,
      4. Wang ZN , et al
      . microRNA-192, -194 and-215 are frequently downregulated in colorectal cancer. Experimental and Therapeutic Medicine 2012;3:560–6.
      OpenUrl
    13. ↵
      2. Sundaram P ,
      3. Hultine S ,
      4. Smith LM , et al
      . p53-responsive miR-194 inhibits thrombospondin-1 and promotes angiogenesis in colon cancers. Cancer Res 2011;71:7490–501.doi:10.1158/0008-5472.CAN-11-1124
      OpenUrlAbstract/FREE Full Text
    14. ↵
      2. Millino C ,
      3. Maretto I ,
      4. Pacchioni B , et al
      . Gene and MicroRNA expression are predictive of tumor response in rectal adenocarcinoma patients treated with preoperative chemoradiotherapy. J Cell Physiol 2016;232.doi:10.1002/jcp.25441
    15. ↵
      2. Mandard AM ,
      3. Dalibard F ,
      4. Mandard JC , et al
      . Pathologic assessment of tumor regression after preoperative chemoradiotherapy of esophageal carcinoma. Clinicopathologic correlations. Cancer 1994;73:2680–6.doi:10.1002/1097-0142(19940601)73:11<2680::AID-CNCR2820731105>3.0.CO;2-C
      OpenUrlCrossRefPubMedWeb of Science
    16. ↵
      2. Perilli L ,
      3. Vicentini C ,
      4. Agostini M , et al
      . Circulating miR-182 is a biomarker of colorectal adenocarcinoma progression. Oncotarget 2014;5:6611–9.doi:10.18632/oncotarget.2245
      OpenUrlCrossRefPubMed
    17. ↵
      2. Rahmati S ,
      3. Abovsky M ,
      4. Pastrello C , et al
      . pathDIP: an annotated resource for known and predicted human gene-pathway associations and pathway enrichment analysis. Nucleic Acids Res 2017;45:D419–D426.doi:10.1093/nar/gkw1082
      OpenUrlCrossRefPubMed
    18. ↵
      2. Shirdel EA ,
      3. Xie W ,
      4. Mak TW , et al
      . NAViGaTing the micronome—using multiple microRNA prediction databases to identify signalling pathway-associated microRNAs. PLoS One 2011;6:e17429.doi:10.1371/journal.pone.0017429
    19. ↵
      2. Kotlyar M ,
      3. Pastrello C ,
      4. Sheahan N , et al
      . Integrated interactions database: tissue-specific view of the human and model organism interactomes. Nucleic Acids Res 2016;44:D536–D541.doi:10.1093/nar/gkv1115
      OpenUrlCrossRefPubMed
    20. ↵
      2. Agostini M ,
      3. Pucciarelli S ,
      4. Calore F , et al
      . miRNAs in colon and rectal cancer: a consensus for their true clinical value. Clin Chim Acta 2010;411:1181–6.doi:10.1016/j.cca.2010.05.002
      OpenUrlCrossRefPubMedWeb of Science
    21. ↵
      2. Drebber U ,
      3. Lay M ,
      4. Wedemeyer I , et al
      . Altered levels of the onco-microRNA 21 and the tumor-supressor microRNAs 143 and 145 in advanced rectal cancer indicate successful neoadjuvant chemoradiotherapy. International Journal of Oncology 2011;39:409–15.doi:10.3892/ijo.2011.1036
      OpenUrlPubMed
    22. ↵
      2. Chiang Y ,
      3. Song Y ,
      4. Wang Z , et al
      . microRNA-192, -194 and -215 are frequently downregulated in colorectal cancer. Exp Ther Med 2012;3:560–6.doi:10.3892/etm.2011.436
      OpenUrlPubMed
    23. ↵
      2. Zhao HJ ,
      3. Ren LL ,
      4. Wang ZH , et al
      . MiR-194 deregulation contributes to colorectal carcinogenesis via targeting AKT2 pathway. Theranostics 2014;4:1193–208.doi:10.7150/thno.8712
      OpenUrl
    24. ↵
      2. Li Z ,
      3. Ying X ,
      4. Chen H , et al
      . MicroRNA-194 inhibits the epithelial-mesenchymal transition in gastric cancer cells by targeting FoxM1. Dig Dis Sci 2014;59:2145–52.doi:10.1007/s10620-014-3159-6
      OpenUrl
    25. ↵
      2. Siddique HR ,
      3. Saleem M
      . Role of BMI1, a stem cell factor, in cancer recurrence and chemoresistance: preclinical and clinical evidences. Stem Cells 2012;30:372–8.doi:10.1002/stem.1035
      OpenUrlCrossRefPubMedWeb of Science
    26. ↵
      2. Pichiorri F ,
      3. Palmieri D ,
      4. De Luca L , et al
      . In vivo NCL targeting affects breast cancer aggressiveness through miRNA regulation. Journal of Experimental Medicine 2013;210:951–68.doi:10.1084/jem.20120950
      OpenUrlAbstract/FREE Full Text
    27. ↵
      2. White CD ,
      3. Brown MD ,
      4. Sacks DB
      . IQGAPs in cancer: a family of scaffold proteins underlying tumorigenesis. FEBS Lett 2009;583:1817–24.doi:10.1016/j.febslet.2009.05.007
      OpenUrlCrossRefPubMedWeb of Science
    28. ↵
      2. Hayashi H ,
      3. Nabeshima K ,
      4. Aoki M , et al
      . Overexpression of IQGAP1 in advanced colorectal cancer correlates with poor prognosis-critical role in tumor invasion. Int J Cancer 2010;126:NA–74.doi:10.1002/ijc.24987
      OpenUrl
    29. ↵
      2. Ye H ,
      3. Arron JR ,
      4. Lamothe B , et al
      . Distinct molecular mechanism for initiating TRAF6 signalling. Nature 2002;418:443–7.doi:10.1038/nature00888
      OpenUrlCrossRefPubMedWeb of Science
    30. ↵
      2. Pommier Y ,
      3. Sordet O ,
      4. Antony S , et al
      . Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 2004;23:2934–49.doi:10.1038/sj.onc.1207515
      OpenUrlCrossRefPubMedWeb of Science
    31. ↵
      2. Pelicano H ,
      3. Carney D ,
      4. Huang P
      . ROS stress in cancer cells and therapeutic implications. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy 2004;7:97–110.doi:10.1016/j.drup.2004.01.004
      OpenUrl
    32. ↵
      2. Trakarnsanga A ,
      3. Ithimakin S ,
      4. Weiser MR
      . Treatment of locally advanced rectal cancer: controversies and questions. World J Gastroenterol 2012;18:5521–32.doi:10.3748/wjg.v18.i39.5521
      OpenUrlCrossRefPubMedWeb of Science
    33. ↵
      2. Zhang HN ,
      3. Yang LN ,
      4. Ling JY , et al
      . Systematic identification of arsenic-binding proteins reveals that hexokinase-2 is inhibited by arsenic. P Natl Acad Sci USA 2015;112:15084–9.doi:10.1073/pnas.1521316112
      OpenUrlAbstract/FREE Full Text
    34. ↵
      2. Mohan S ,
      3. Vander Broek R ,
      4. Shah S , et al
      . MEK inhibitor PD-0325901 overcomes resistance to PI3K/mTOR inhibitor PF-5212384 and potentiates antitumor effects in human head and neck squamous cell carcinoma. Clin Cancer Res 2015;21:3946–56.doi:10.1158/1078-0432.CCR-14-3377
      OpenUrlAbstract/FREE Full Text

    Footnotes

    • Handling editor Runjan Chetty.

    • Contributors ED ideated the study, performed experiments, analysed data and wrote manuscript; CZ performed statistical analysis; MD, MS, SP and DN provided biological sample; MF and MR performed pathological examinations, in situ hybridisation analysis and contributed to manuscript writing; FS contributed to data analysis; MA supervised this study.

    • Funding This work was supported by grants from the Ministry of Health RF-2011-02349645; AIRC Fellowship (No. 19486) and Investigator Grant (IG 2016 Id.19104); Fondazione CARIPARO-Fondazione Città della Speranza, Pediatric Research Grant.

    • Competing interests None declared.

    • Ethics approval The study was approved by the local ethics committee: Comitato Etico per la Sperimentazione—Azienda Ospedaliera di Padova.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Data sharing statement No additional data from the study are available.

    • Correction notice This article has been corrected since it was published Online First. Affiliations 2-4 have been corrected.