5-FU is one of the main chemotherapeutic drugs used for treatment of CRC[6,7]. It exhibits its cytotoxicity by incorporating fluoronucleotides into RNA and DNA molecules, but its main toxic effects are mediated by inhibiting the nucleotide synthetic enzyme thymidylate synthase (TYMS)[7]. However, one of the major problems in managing progressed colorectal cancer is both the inherent and acquired failure of 5-FU-based therapy. Several analyses of 5-FU resistance in CRC have focused on genes involved in pharmacodynamic and pharmacokinetic pathways. One main point has been the activity of TYMS, a key therapeutic target of 5-FU. CRCs that are resistant to 5-FU-based chemotherapy have been shown to possess greater TYMS enzymatic activity than cancers that are sensitive to this therapy[8]. High levels of TYMS mRNA or protein expression in liver metastases have also been linked to lack of clinical response to 5-FU in vivo[9]. Moreover, a meta-analysis of 24 studies demonstrated that low expression levels of TYMS in metastatic colorectal tumors are associated with greater sensitivity to fluoropyrimidine-based chemotherapy[10]. Additionally, the activity of thymidine phosphorylase (TP), a key enzyme that catalyzes the transformation from 5-FU prodrugs of 5′-deoxy-5-fluorouridine (5′-DFUR) to 5-FU, is closely linked to failure of 5-FU and targeted therapy in CRC cells[11].

The epidermal growth factor receptor (EGFR) belongs to the ErbB family of receptors that are able to promote tumor cell proliferation in diverse epithelial malignancies[12]. For this reason, EGFR has become an important target in oncology. Monoclonal antibodies against EGFR are used as a standard therapy for some types of solid tumors. The chimeric IgG1 mouse/human antibody cetuximab and the human IgG2 antibody panitumumab are considered to be equally effective in mCRC. However, it has been extensively reported that primary resistance to these agents is mediated by mutations in downstream signaling molecules[13]. Misale et al[14] showed that the development of resistance to anti-EGFR treatment in CRC is associated with molecular alterations of KRAS. Although KRAS mutations confer strong resistance to anti-EGFR antibodies, not all CRC patients with KRAS wildtype (KRASwt) benefit from these agents. Therefore, there is a need for novel biomarkers to better identify which KRASwt patients would benefit from this therapy[15]. A few studies have shown that high EGFR gene copy number could be a potential favorable marker for anti-EGFR therapy in CRC, as patients with low gene copy number are unlikely to respond to anti-EGFR agents[16,17]. Very recently, rare mutations in exons 3 and 4 in the both the KRAS gene and NRAS gene have demonstrated predictive potential in regards to panitumumab treatment[18].

Angiogenesis is widely regarded as an important therapeutic target in many different types of cancer, including CRC[19]. It has been suggested that inhibition of angiogenesis in tumors can influence the development of new tumor blood vessels and probably lead to normalization of the existing tumor vasculature. The vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that plays a key role in tumor vascular development[20]. VEGF-neutralizing antibodies can prevent the binding to and activation of VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2 (vascular endothelial growth factor receptor 2) and the co-receptors NP1 (Neuropilin 1) and NP2 (Neuropilin 2). Bevacizumab, a humanized monoclonal antibody that binds to all isoforms of VEGF, is the first anti-angiogenic therapy approved for treatment of CRC[21,22]. It has also been used in combination with common chemotherapeutic agents, such as 5-FU and capecitabine as well as irinotecan and oxaliplatin[23]. Resistance to combinatorial therapy in CRC treatment may be due to resistance to bevacizumab, resistance to the chemotherapeutic with which bevacizumab was administered or a resistance to both. As observed, several pathways in addition to the VEGF pathway are involved in angiogenesis. Therefore, mechanisms of resistance to anti-angiogenic therapy may also include VEGF-independent anti-angiogenesis pathways[24].

Clinical trials have demonstrated that KRAS mutations are negative predictive biomarkers for EGFR-targeted therapy in CRC. Mechanisms of post-transcriptional downregulation of mutated KRAS might therefore be of clinical relevance in patients with mCRC[42,43]. The members of the let-7 family are known to target KRAS and their involvement in response to EGFR-targeted therapy has already been reported[44]. Upregulation of let-7 expression levels might provide a survival advantage by inhibiting mutated KRAS under EGFR-targeted therapy. In addition, let-7 may reveal further favorable effects by regulating other genes such as the cell cycle regulators, Myc, Bcl-2 (B-cell CLL/lymphoma 2), integrins and signal transducers. Ruzzo et al[45] analyzed the expression levels of let-7a in colorectal carcinomas with mutated KRAS and in mCRC patients treated with cetuximab and irinotecan. Their study revealed that intra-tumor expression of let-7a correlates with tumor response and overall survival in mCRC patients treated with anti-EGFR-based therapy in both KRAS mutant and wildtype populations. Moreover, they showed that downregulation of let-7e and let-7b can potentially be used to predict resistance to the monoclonal antibody cetuximab. Cappuzzo et al[46] investigated whether microRNAs can predict sensitivity to EGFR-targeting monoclonal antibodies in patients with mCRC. They identified the cluster Let-7c/miR-99a/miR-125b as a signature linked to an outcome different from that of EGFR targeting therapies. In the first cohort, patients with high-intensity signatures showed a significantly longer progression-free survival and longer overall survival. Moreover, in the KRAS wild-type population, high-intensity signature patients had a significantly longer progression-free survival, as demonstrated in the validation cohort. Therefore, the miR-99a/let-7c/miR-125b signature could improve the selection of patients with KRAS wild-type mCRC for treatment with EGFR targeting therapies.

Salendo et al[47] performed a genome-wide miRNA profiling in 12 colorectal cancer cell lines and established an individual in vitro signature for chemoradiosensitivity. Their study demonstrated that high expression of let-7g was linked with a good prognosis in rectal cancer patients. This finding suggests that let7g expression may serve as potential predictive biomarker.

An increasing number of studies proposed miR-126 as a player in the regulation of angiogenesis, a process that has already been considered as a target for development of novel drugs. High expression of miR-126 has already been associated with increased vascular endothelial growth factor A (VEGF-A) mediated signaling in endothelial cells and a higher blood vessel integrity[48]. Additionally, miR-126 has been identified as a putative tumor suppressor in primary tumors[49-51]. Hansen et al[48] investigated the role of miR-126 as a predictive marker in patients with CRC in relation to first-line treatment with capecitabine, a precursor of 5-FU, and oxaliplatin (XELOX). They demonstrated a significant relationship between expression of miR-126 in the primary tumor and sensitivity to first-line XELOX treatment. Low expression of miR-126 might therefore lead to tumor vessels with decreased integrity followed by an increase in interstitial pressure, which may explain the lower sensitivity in patients treated with XELOX. A recent study by Hansen et al[52] revealed that high expression of miR-126 in mCRC patients was strongly related to a longer progression-free survival. Their results confirm their previous findings on the prognostic value of miR-126 in mCRC. As VEGF-A may be a target of miR-126, the results of their study might provide predictive information in regards to next-generation anti-angiogenic therapy approaches.

Several studies revealed that miR-31 is upregulated in CRC, but there is little known about its role in modulating tumor cell response to chemotherapeutic drugs. Wang et al[53] showed that the treatment of HCT-116 colon cancer cells with an anti-miR-31 inhibitor increased the sensitivity to 5-FU as early as 24 hours after exposure without affecting cell proliferation. Combination of 5-FU treatment with a respective negative control did not lead to a reduction in cell viability. The apoptosis rate of HCT-116 cells treated with both 5-FU and the anti-miR-31 inhibitor was the highest among respective control groups, indicating that these agents inhibited proliferation via the apoptotic mechanism.

The expression levels of miR-192 and -215 were shown to be significantly reduced in CRC tissues compared to non-tumor counterparts. Furthermore, Chiang et al[54] demonstrated that low expression levels of miR-192 and -215 are related to increased tumor size in CRC. Thus, miR-129 and miR-215 could be useful biomarkers in the carcinogenesis of CRC. In addition, miR-192 and miR-215 were reported to negatively regulate CRC cell proliferation[55]. Boni et al[40] showed that miR-192 and -215 downregulate TYMS expression and thus increase resistance to 5-FU in CRC cell lines. TYMS plays a role in normal cell function and is a potential target for chemotherapeutic drugs such as 5-FU. Transcriptional and translational regulation of TYMS most likely affect cell chemosensitivity. Additionally, they demonstrated that miR-192 and miR-215 inhibit progression into the S phase, play a role in cell cycle control and prevent sensitivity to 5-FU. Zhang et al[56] identified a set of 6 miRNAs including miR-215 which could serve as an authentic prognostic and predictive tool for determination of disease recurrence in patients with stage II colon cancer. miR-215 could potentially predict which patients would benefit from adjuvant chemotherapy, which can in turn facilitate patient consultation and help individualize management of patients with this disease.

It has been shown that miR-148a is a pro-apoptotic miRNA in CRC which acts by targeting Bcl-2, a regulator of apoptosis. In addition, several in vitro studies have demonstrated that miR-148a functions as a tumor suppressor by targeting several genes such as PXR (nuclear receptor subfamily 1, group I, member 2), TGIF2 (TGFB-induced factor homeobox 2), MSX1 (msh homeobox 1), CDC25B (cell division cycle 25B), DNMT1 [DNA (cytosine-5-methyltransferase 1)] and DNMT3b [DNA (cytosine-5-methyltransferase 3 beta)][57]. Takahashi et al[58] showed that low expression of miR-148a is significantly linked to an unfavorable outcome in treatment of stage III CRC patients with 5-FU. They also demonstrated the link between decreased miR-148a expression and poorer sensitivity and survival rate in patients with stage IV CRC treated with oxaliplatin combined with 5-FU. Stage IV CRC patients that showed a high miR-148a expression level were shown to benefit from chemotherapeutic drugs, indicating that miR-148a may have predictive value in the assessment of response to CRC treatment. In addition, their data suggested that downregulation of miR-148a is mediated by DNA methylation. Takahashi et al[58] showed that there is a significant and independent association between miR-148a methylation and poor survival in stage IV patients. Therefore, the methylation status of miR-148a might be a potential prognostic marker in CRC.

Kjersem et al[59] investigated microRNAs in plasma as potential predictive markers for sensitivity to oxaliplatin-based chemotherapy. Their study revealed that a high expression level of miR-148a is associated with a decrease in progression-free survival. The results of their study suggest that miR-148 could serve as a non-invasive biomarker for predicting outcomes in mCRC patients treated with 5-FU and oxaliplatin-based chemotherapy.

Upregulated miR-21 expression is associated with some types of human cancer, including CRC. It has been demonstrated that miR-21 regulates several tumor suppressors such as p21, TGF-beta receptor II and B-cell leukemia/lymphoma 2-associated X protein. Moreover, overexpression of miR-21 is linked to poor response to 5-FU-based chemotherapy. Valeri et al[60] found an inverse expressional correlation of miR-21 and the tumor suppressor hMSH2. They demonstrated that miR-21 directly targets the 3’ UTR of hMSH2 mRNA and significantly reduces its protein expression. CRC cells that showed high expression levels of miR-21 have decreased hMSH2 protein expression and revealed significantly reduced 5-FU-induced G2/M damage arrest and apoptosis. This indicates a characteristic defect in the core mismatch repair system, suggesting that miR-21 might act as a regulator of genes associated with cell-cycle arrest and/or drug resistance. In addition, Deng et al[61] showed that forced miR-21 expression in HT-29 colon cancer cells significantly inhibited apoptosis, enhanced cell proliferation and invasion and increased resistance to the chemotherapeutic agent 5-FU. Moreover, they demonstrated that silencing of miR-21 inverted these effects on HT-29 cells and restored the sensitivity to 5-FU.

Reduced miR-129 expression levels have been reported in several tumor cell lines and primary tumors including CRC[62]. Karaayvaz et al[63] demonstrated that miR-129 expression was clearly lower in CRC patients. There was no significant difference between adenoma and stage I and II carcinomas. They observed that miR-129 expression was dramatically reduced in stage III and IV cancers. Hence, their results suggested that loss of miR-129 is linked to progression in CRC. Additionally, they identified miR-129 as a novel regulator of Bcl-2 expression. The expression of miR-129 promoted apoptosis, inhibited cell proliferation and caused cell-cycle arrest in CRC cells. miR-129-based therapies could help to achieve a multi-targeted anti-cancer therapeutic strategy. 5-FU-based chemotherapy still remains the main option for advanced mCRC treatment, but the possibility of miR-129 restoration may lead to a new strategy in reducing resistance to chemotherapeutic drugs. Furthermore, Karaayvaz et al[63] showed that miR-129 is a suppressor of the 5-FU target protein TYMS and therefore enhances chemosensitivity to 5-FU. In their in vivo tumor xenografts, they demonstrated that increasing miR-129 expression to normal levels using synthetic miRNAs made tumors more sensitive to 5-FU-based drugs.

MiR-19b is one of the 6 miRNAs which are encoded by the miR-17-92 cluster. The activation of this cluster is mediated by the transcription factor E2F1 which accumulates early in the G1 phase of the cell cycle, suggesting that miRNAs generated from this cluster might play a role in the G1 phase. Kurokawa and collegues found that miR-19b expression is upregulated in the DLD-1/R colon cancer cell line, but they did not observe any changes in the cell cycle profile after 5-FU treatment. In addition, they demonstrated that miR-19b expression correlated with response to the widely-used anti-cancer drug 5-FU[64]. Likewise, a recent study showed that miR-19a, a paralogue of miR-19b, is upregulated in HCT-119 and HT29 cells in response to 5-FU-based treatment[65].

MiR-34a is a member of the miR-34 family which also includes miR-34b and miR-34c. Low expression levels of miR-34a have been identified in various types of tumors, including CRC[66]. Moreover, miR-34a was observed as one of the most downregulated miRNAs in the 5-FU-resistant DLD-1 CRC cell line. Akao et al[67] showed that exposure to 5-FU activated the PI3K/Akt signaling pathway in the resistant DLD-1 cell line in comparison to the parental cells and led to an apparent increase in growth. In addition, miR-34a expression in the 5-FU-resistant cell line was prolonged at a low level, whereas it showed an upregulation in the parental cells after treatment with a 5-FU-based drug. They observed an upregulation of Sirt-1, a target gene of miR-34a which is associated with drug resistance, in the 5-FU-resistant cells and also that silencing of Sirt-1 significantly increased the sensitivity to 5-FU in the 5-FU-resistant cells. This suggested that miR-34a is a negative regulator of 5-FU resistance in the CRC cell line DLD-1.

A few studies showed that miR-143 expression is low in tumors compared to their normal counterparts, both at adenomatous and cancer stages of colorectal neoplasia, as well as in CRC cell lines[68]. In addition, miR-143 has been identified to directly target the KRAS mRNA[69]. Borralho et al[70] investigated the role of miR-143 in the HCT116 CRC cell line. They showed that transient overexpression of miR-143 led to a 60% reduction in cell viability and stable overexpression of miR-143 resulted in decreased viability and increased cell death after treatment with 5-FU. These changes were linked to increased nuclear fragmentation and caspase -3, -8 and -9 activities. Furthermore, they demonstrated that exposure of miR-143-overexpressing cells to 5-FU resulted in downregulation of the extracellular-regulated protein kinase 5 and Bcl-2 protein expression. In addition, miR-143 led to increased sensitivity to 5-FU- based drugs, suggesting that miR-143 is involved in CRC development and plays a role as a chemosensitizer to 5-FU.

Zhou et al[71] recently showed that miR-203 is upregulated in three oxaliplatin-resistant CRC cell lines. They demonstrated that exogenous expression of miR-203 in chemonaïve CRC cells reduced sensitivity of cells to oxaliplatin treatment. Silencing of miR-203 expression led to increased sensitivity of CRC cells to oxaliplatin. Furthermore, they performed an in-silico analysis and identified ataxia telangiectasia mutated (ATM), a primary mediator of the DNA damage response, as a potential target of miR-203. Their study showed that mutation of the putative miR-203 target region in the 3’ untranslated region of ATM mRNA eliminated the inhibitory effect of miR-203 on ATM. In addition, they demonstrated that stable knockdown of ATM led to resistance to oxaliplatin in chemo-naïve CRC cells.

Hur et al[72] reported that miR-200c expression modulates EMT in colorectal metastasis. In their study, they demonstrated for the first time that the miR-200c/429 cluster was significantly overexpressed in liver metastasis in comparison to primary CRC and that the expression of these miRNAs was specifically regulated by aberrant methylation of their promoter regions. A recent study by Toiyama et al[73] revealed that the serum levels of miR-200c might serve as potential prognostic and metastatic-predictive biomarkers in CRC patients. They showed that miR-200c expression in serum is strongly associated with a metastatic phenotype in CRC; in particular, expression of miR-200c in serum was a good predictive marker for lymph node metastasis. Moreover, they demonstrated that miR-200c expression in serum can be used as a prognostic and predictive marker of tumor recurrence in patients undergoing curative surgery.