Abstract

   Although treatment of colorectal cancer with 5-florouracil and
   oxaliplatin is widely used, it is frequently followed by a relapse.
   Therefore, there is an urgent need for profound understanding of
   chemotherapy resistance mechanisms as well as the profiling of
   predictive markers for individualized treatment. In this study, we
   identified the changes in 14 miRNAs in 5-fluouracil and 40 miRNAs in
   oxaliplatin-resistant cell lines by miRNA sequencing. The decrease in
   miR-224-5p expression in the 5-fluorouracil-resistant cells correlated
   with drug insensitivity due to its overexpression-induced
   drug-dependent apoptosis. On the other hand, the miR-23b/27b/24-1
   cluster was overexpressed in oxaliplatin-resistant cells. The knockout
   of miR-23b led to the partial restoration of oxaliplatin
   susceptibility, showing the essential role of miR-23b in the
   development of drug resistance by this cluster. Proteomic analysis
   identified target genes of miR-23b and showed that
   endothelial–mesenchymal transition (EMT) was implicated in oxaliplatin
   insensibility. Data revealed that EMT markers, such as vimentin and
   SNAI2, were expressed moderately higher in the oxaliplatin-resistant
   cells and their expression increased further in the less drug-resistant
   cells, which had miR-23b knockout. This establishes that the balance of
   EMT contributes to the drug resistance, showing the importance of the
   miR-23b-mediated fine-tuning of EMT in oxaliplatin-resistant cancer
   cells.

   Keywords: oxaliplatin, 5-fluorouracil, chemoresistance, microRNA,
   miRNome, proteome, epithelial–mesenchymal transition, drug resistance

1. Introduction

   The majority of chemotherapeutic drugs do not kill a whole pool of
   heterogenic cancerous cells. Some of them survive and acquire
   resistance to therapy treatment, thus leading to the recurrence of
   malignance. Unfortunately, data obtained from cancer tissues before
   treatment do not always lead to the prediction of drug tolerance
   mechanisms, since the data from minor drug-resistant population are
   masked by background species from a pool of non-resistant cells
   [[42]1]. One way to overcome this limitation and to discover relevant
   predictive markers is to generate and analyze sublines of tumor
   cultures in vitro, which are more homogeneous with respect to genetic
   population and enable the determination of drug-resistance mechanisms
   under the more controlled in vitro conditions.

   The combinations of 5-fluorouracil (5-FU) and oxaliplatin (Oxa) drugs
   often supplemented with synergizing medications, such as leucovorin in
   the case of FOLFOX regimen, or folinic acid in FLOX regimen, are
   adjuvant chemotherapy schemes commonly used for treatment of patients
   with surgically resected colon cancer (CRC) [[43]2,[44]3]. The
   combination of Oxa and capecitabine represents an alternative CAPOX
   regimen, which could be used for colon cancer treatment [[45]4]. Many
   different biological processes as well as their combinations can be
   involved in the acquisition of 5-FU- or Oxa-resistance. These include
   drug metabolism, restriction of drug entrance, increase in drug efflux,
   DNA damage repair, changes in autophagy flux, cancer stem cells
   proliferation, epigenetic effects, and endothelial–mesenchymal
   transition (EMT) [[46]5,[47]6,[48]7]. Most of these mechanisms can be
   triggered or at least influenced by small non-coding RNAs, such as
   microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) via the
   involvement of massive alterations in gene expression [[49]7].

   The 5-FU anticancer action mainly depends on the thymidylate synthase
   (TS) and dihydropyrimidine dehydrogenase (DPYD) pathways. Therefore,
   miR-197, -203, and -218 that effect TS expression and miR-27a, -27b,
   -200c, and -494 that regulate DPYD activity stringently control
   cytotoxicity of the drug [[50]5,[51]8]. However, there is evidence that
   let-7, miR-20b, -135b, -182, -200c, -204, -224, and -587 miRNAs impair
   5-FU sensitivity by regulating proteins related to the PI3K/AKT
   signaling pathway. On the other hand, miR-125b, -149, -320, -181a-5p
   and lncRNA CRNDE change cancer cell resistance to 5-FU by controlling
   the Wnt/β-catenin signaling pathway [[52]5].

   Much less is known about miRNA-driven molecular mechanisms responsible
   for the development of resistance to the Oxa treatment. Perturbations
   in miR-520g expression affects the efficiency of the treatment via the
   cyclin-dependent kinase inhibitor p21-dependent pathway [[53]9]. On the
   other hand, miR-20a regulates Bcl-2-dependent apoptosis via BNIP2
   [[54]10]. Changes in miR-153 expression mediate Oxa resistance by
   inhibiting the transcription factor Forkhead box O3a (FOXO3a) [[55]11],
   and miR-203 modulates DNA damage response via Ataxia Telangiectasia
   Mutated (ATM) regulation [[56]12]. Notably, some miRNA-mediated effects
   require changes in the genetic background, such as KRAS or TP53
   mutations [[57]9,[58]13], once again suggesting multi-variant
   mechanisms of cancer cell drug-resistance acquisition.

   EMT in cancerous cells emerges among the major hallmarks for
   cancerogenesis and drug-resistance. Cells undergoing EMT show a feature
   similar to cancer stem cells, such as an increase in the drug efflux
   and anti-apoptotic properties [[59]14]. On the other hand, the
   plasticity of EMT as recently recognized mesenchymal–endothelial
   transition (MET) reversal of the EMT phenotype associated with the lack
   of stringent and widely recognized markers of those cell states creates
   a complicated picture of EMT–MET phenomena. The picture becomes even
   more complex after acknowledgment that an intermediate state of EMT