Several recent studies have shown the utility of miRs in the diagnosis and classification of CRC [
7,
8,
16‐
19]. There are ongoing prospective clinical trials evaluating miR based classifiers such as a 24-miR signature in lung cancer diagnosis [
20] (Gensignia) and a miR signature in prostate cancer screening [
21] (Exiqon). These trials highlight the feasibility and translational potential of miR-based classifiers. We identified a group of miRs that are responsive to radiation in mouse tumor models and focused on characterizing miR-451a as one of the robust early response miRs in CRC. Our data suggests that miR-451a behaves as an anti-proliferative miR in CRC cell lines in vitro. We also identify putative targets of miR-451a and show that expression of both the miR and two of the targets correlate with radiation responses in CRC. Taken together, our observations suggest that miR-451a modulation of CAB39 and EMSY target genes could alter radiation sensitivity of human CRC.
miR-451a has been found to inhibit cell proliferation and drug responses in other malignancies. For example, miR-451a was found to affect proliferation and sensitivity to tamoxifen in breast cancer via targeting of the macrophage migration inhibitory factor (MIF) [
22]. Similarly, miR-451a was shown to be tumor suppressive in gastric cancer by affecting the PI3K/mTOR pathway [
23]. In other tumor types, it has been shown that miR-451a expression is downregulated in the tumor cells in a manner consistent with a tumor suppressor function [
24,
25]. Interestingly, miR-451a appears to increase radiation responses in nasopharyngeal carcinoma cells [
26] and lung adenocarcinoma cells [
27]. Our observation in CRC cell lines suggests a function similar to the tumor suppressive role that has been documented in other cancers by these studies.
Using a bioinformatics approach, we narrowed down the targets of miR-451a to a group of 14 genes, which was further filtered to 4 genes –CAB39, EMSY, EREG and MEX3C based on either a known role in colorectal cancer or radiation responsiveness in other cancer types. Calcium binding protein (CAB) 39 has been previously shown to be a target of miR-451a in human glioma [
28] and in colorectal cancer [
29]. This protein is thought to affect STK11 activity and localization thereby influencing the PI3K/AKT signaling pathway. EMSY is a transcriptional repressor that associates with BRCA2 and is often amplified in breast and ovarian cancers [
30]. Functionally, EMSY colocalizes and forms foci with histone γH2AX in response to irradiation. Importantly, breast cancer patients with EMSY amplifications have poorer overall survival. Taken together, these functions suggest that modulation of EMSY by miR-451a may have significant impact on radiation responses and tumor cell survival. Indeed, consistent with the breast cancer dataset, our analysis of the TCGA colorectal cancer dataset (Additional file
2: Figure S9b-c) indicates that EMSY as well as CAB39 are increased at the protein level in a subset of patients and associated with worse outcome. Epiregulin (EREG) is a known ligand of the EGF family and regulates several key processes including cellular proliferation, inflammation, angiogenesis and wound healing [
31]. While it has been proposed as a biomarker for monitoring responses to cetuximab in colorectal cancer [
32], it is unclear whether there is a function for EREG specifically in the context of radiation responses in these tumors. MEX3C has been identified as a ubiquitin ligase as well as an RNA binding protein that modulates the levels of HLA-A allotypes [
33]. Interestingly, it is part of a group of genes that suppress chromosomal instability in colorectal cancer [
34] thereby likely contributing to tumor drug resistance. Our data suggests that miR-451a mRNA binds to all four of these mRNAs (Fig.
4a) and downregulates their expression at the RNA and protein levels. Given the low expression levels of the EREG and MEX3C in patient samples, and the discordance between our miR-Trap assay and qRT-PCR, we chose to focus on CAB39 and EMSY. CAB39 and EMSY upregulation worsened CRC survival in the TCGA. We also noticed a trend towards decreased miR expression in patients with more advanced disease compared to adjacent normal tissue (data not shown). We must emphasize that our small patient numbers preclude drawing stronger conclusions regarding the miR-451a and target levels in human disease, but rather lead us to hypothesize that tumors with increased miR-451a levels may respond better to CRT, improving survival.