Background
RAF family kinases, including BRAF and RAF1, function downstream of RAS as critical regulators of the MEK-ERK MAP kinase signaling pathway [
1]. This RAS-RAF-MEK-ERK cascade is a key pathway, which contributes to human oncogenesis controlling the cell cycle, proliferation, differentiation, angiogenesis, apoptosis, migration, and metastasis [
2‐
4]. Since the first identification of the
BRAF gene mutation in human cancer [
5], accumulating evidence has shown that a considerable proportion of various human malignancies, such as malignant melanoma (~50%) and other solid cancers including thyroid cancer (~60%), colorectal cancer (~10%), and lung cancer (~6%) carry the activated
BRAF mutations, which leads to constitutive activation of downstream RAF, MEK and ERK [
1].
In colorectal cancer, mutations of
BRAF predominantly occur in codon 600, particularly leading to p.V600E mutation [
6]. Recently, several lines of evidence suggest that colorectal cancer containing a
BRAF p.V600E mutation possesses more malignant potential when compared with other genotypes of colorectal cancer, including the
KRAS/
BRAF-wild-type tumor and the
KRAS-mutated tumor. In resectable colon cancer patients treated with adjuvant drug therapy, the
BRAF mutation has been associated with poor survival compared to wild-type
BRAF [
7,
8]. A similar tendency has been observed in studies of metastatic or recurrent colorectal cancer, where
BRAF mutations are associated with worse overall survival (OS) [
9,
10]. In addition to the negative prognostic impact, a
BRAF mutation is likely to have a predictive value for resistance to anti-EGFR therapies, such as monoclonal antibodies cetuximab and panitumumab [
11,
12]. However, its mutational status as a predictive marker has not been well established in clinical use.
Earlier studies have also shown that
BRAF mutations substantially overlap with other genetic and epigenetic subtypes of colorectal cancer, such as the microsatellite instability (MSI) phenotype characterized by change in the length of simple nucleotide repeats resulting from mismatch repair deficiency, and the CpG island methylator phenotype (CIMP) characterized by widespread hypermethylation of CpG islands [
13,
14]. These molecular subgroups, particularly
BRAF-mutant tumors and CIMP-positive tumors, are associated with the serrated pathway that plays a role in colorectal tumorigenesis, which is distinct from the well-characterized chromosomal instability pathway [
15]. Patients with colorectal cancer containing a
BRAF mutation or those with CIMP-positive tend to be older, smokers, women, right-sided, and have a higher-grade histology [
16,
17]. In light of the aforementioned evidence and other findings that patients with
BRAF-mutant and/or CIMP-positive colorectal cancer (particularly without MSI or impaired mismatch repair) exhibit worse clinical outcomes [
7,
10,
18], this subtype should be regarded as distinct from other molecular subtypes of colorectal cancer and should be treated using different strategies. The reason why this molecular subtype exhibits more malignant potential still remains to be elucidated, and promising molecular targets for therapy against this subtype remain to be identified.
microRNAs (miRNAs) are a class of small non-coding RNA, which exert their tumor suppressive and/or oncogenic functions primarily by binding to the 3′-untranslated region of the mRNA of target genes. The binding of miRNA to each mRNA leads to the inhibition of translation and/or enhanced degradation of the corresponding transcripts. Alteration of miRNA expression has been implicated in oncogenesis from early to late stages in various human cancers including colorectal cancer [
19,
20]. There are an increasing number of studies including ours that have analyzed the functional role of miRNAs in colorectal cancer, such as miRNA-21 (miR-21), miR-31, miR-34b/c, miR-135b, miR-137, miR-143, miR-145, miR-148a, miR-200, and miR-203 [
21‐
29]. Moreover, a few reports have focused on the relationship between
BRAF mutations and some miRNA alterations in other cancers, although their miRNA expression profiles were not similar [
30‐
32]. However, whether the
BRAF-mutant-specific miRNAs can contribute to oncogenesis of this more malignant subtype of colorectal cancer, remains unclear. If the miRNA-dependent mechanisms underlying the oncogenesis of
BRAF-mutant colorectal cancer are identified, this could lead to discovering novel molecular targets for therapy to improve the outcome of patients with colorectal cancer and even other cancers harboring
BRAF mutations.
In this study, we aimed to identify miRNAs that are specifically dysregulated in BRAF-mutant colorectal cancer using a genome-wide miRNA expression analysis, and to clarify whether these miRNAs play a role in colorectal tumorigenesis as an oncogene or a tumor-suppressor through functional assays using colorectal cancer cell lines. Moreover, we investigated whether the expression of the candidate BRAF-related miRNA, miR-193a-3p, was associated with the clinical outcome of patients with metastatic colorectal cancer treated with anti-EGFR therapy.
Methods
Patients
A total of 314 patients with colorectal cancer, comprising 255 patients who underwent drug therapy including cytotoxic agents and anti-EGFR antibody, and/or surgery in the Tohoku University Hospital (TUH) between 2004 and 2013, and 59 patients who received anti-EGFR antibody in the National Cancer Center Hospital (NCCH) between 2003 and 2012, were recruited in this study. The clinical information regarding clinical characteristics of patients and tumors, OS, progression-free survival (PFS) after initiations of drug therapies, and response rate (RR), was retrospectively analyzed through reviews of clinical records. As listed in Additional file
1: Table S1, clinical characteristics of patients within the TUH cohort are associated with earlier clinical stage and proximal location compared to those within the NCCH cohort.
DNA and RNA extraction
DNA was extracted from a 5 μm- or 10 μm-thick formalin-fixed paraffin-embedded (FFPE) tissue of each patient with colorectal cancer through the use of QIAmp DNA FFPE tissue kit (Qiagen, Valencia, CA, USA). Total RNA including miRNA fraction was extracted from the FFPE tissue of each colorectal cancer by using the Ambion RecoverAll Total Nucleic Acid Isolation Kit (Life Technologies Corporation, Carlsbad, CA, USA). Total RNA was also extracted from normal adjacent colonic mucosa of 11 patients with colorectal cancer from the cohort.
KRAS and BRAF sequencing
The mutational status of codon 12 and 13 of KRAS gene and the codon 600 of BRAF gene were analyzed by direct DNA sequencing through the use of CEQ2000EX automated DNA sequencer (Beckman Coulter, Brea, CA, USA). The accession number of cDNAs of KRAS, wild-type and p.V600E BRAF, were NM_033360.3, NM_004333.5 and HQ224878.1, respectively. Primers used for the amplification of fragments were 5′-accttatgtgtgacatgttc (forward) and 5′-atggtcctgcaccagtaata (reverse) for KRAS codons 12 and 13, and 5′-ataatgcttgctctgatagg (forward) and 5′-gtaactcagcagcatctcag (reverse) for BRAF codon 600.
Screening of miRNAs that are dysregulated in BRAF-mutant tumors by using miRNA microarray
The genome-wide miRNA expression levels of the 30 colorectal cancers from the screening set were analyzed by the SurePrint G3 Human miRNA Rel. 16.0 microarray (Agilent Technologies, Santa Clara, CA, USA), which covers 1222 human miRNAs, according to the manufacturer’s protocol. The microarray data were extracted using the GeneSpring ver. 12.5 (Agilent Technologies). The raw data was normalized by using the 90-percentile shift method, and the acquired data of each miRNA were compared between wild-type KRAS/BRAF tumors and mutant-BRAF tumors using Mann-Whitney U test. The microarray data has been deposited in the Gene Expression Omnibus database (accession No. GSE66548).
Quantification of miRNA expression levels by quantitative real-time RT-PCR
The miRNA expressions of colorectal tissues and colon cancer cell lines were quantified by Taqman real-time RT-PCR (qRT-PCR) using a CFX96 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). The relative expression of each miRNA was calculated by the delta CT value method, through the use of miR-16 expression for human colon samples [
27,
33] and RNU48 for colorectal cancer cell lines [
34] as a normalizer. At least two independent samples were loaded as an internal control in each PCR plate for miR-193a-3p analysis for colorectal tumors, to keep consistency of measurements throughout all plates. Each sample was amplified in triplicate and the results obtained from each run were normalized according to the data of internal controls.
Cell lines
Human colorectal cancer cell lines RKO (CRL-2577) and HCT116 (CCL-247) were purchased from the American Type Culture Collection in 2011. Human colorectal cancer cell lines DiFi, HCT8, LIM2405, and SW48 were kindly provided along with appropriate ethics rules and consents of both institutions by Dr. Mariadason in Ludwig Institute for Cancer Research, Australia. The cell lines were regularly authentificated by short tandem repeat analysis. RKO was cultured in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, St.Louis, MO, USA) with 10% fetal bovine serum and the other four cell lines were grown in Roswell Park Memorial Institute Medium 1640 (Sigma-Aldrich) with 10% fetal bovine serum at 37 °C.
Pre-miR-193a-3p and anti- miR-193a-3p transfection
The cells were transfected with precursor of miR-193a-3p or precursor of negative control (PM11123 or AM17110, Applied Biosystems), or anti-miR-193a-3p or anti-negative control (AM17000 or AM17010, Applied Biosystems) at a final concentration of 33–67 nM using Lipofectamine 2000 (Life Technologies Corporation), according to the manufacturer’s protocol.
Cell growth assay
The cells were seeded onto 96-well plates with the different number of cells (RKO, 7 × 103; HCT116, 5 × 103; SW48, 1.5 × 104). When attached, cells were transfected with precursors of miR-193a-3p or negative control as mentioned above. The cell viability was measured after 48 h in RKO or after 72 h in HCT116 and SW48 using the Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer’s protocol. Each experiment was performed in quadruplicate and data were obtained from three or more independent experiments.
Invasion assay
Invasion activities of RKO and HCT116 cells were analyzed using Boyden chambers with 8-mm pore membranes coated with matrigel (BD Biosciences, San Jose, CA, USA) following the standard protocol. In six-well plates, 3 × 105 of RKO cells and 1 × 105 of HCT116 cells were transfected with precursors of miR-193a-3p or negative control. After 24 h, the transfected cells (3 × 105 of the RKO cells and 1.5 × 105 of the HCT116 cells) were re-suspended in 500 μl of serum-free medium in the upper wells. Medium containing 10% fetal bovine serum was then added into the bottom wells. After incubation for 48 h for RKO or 24 h for HCT116, the invaded cells were stained with 0.2% crystal violet solutions. The number of cells was counted from four representative fields of each membrane, and the results were obtained from three independent experiments.
Quantification of gene expression levels by qRT–PCR
Total RNA was extracted using the RNeasy Mini Kit (Qiagen), and cDNA was synthesized from mRNA using the iScript advanced cDNA Synthesis Kit (Bio-Rad Laboratories) following the manufacturer’s protocol. To quantify gene expression levels of ZEB1, ZEB2, SNAI1, and SNAI2, qRT–PCR was performed on the CFX96 real-time PCR detection system using SYBR® Green PCR Master Mix (Applied Biosystems) following the manufacturer’s protocol. Each gene expression level was normalized to an expression level of GAPDH. The primers used were as follows: ZEB1 forward; 5-ttcaaacccatagtggttgct, ZEB1 reverse; 5-tgggagataccaaaccaactg, ZEB2 forward; 5-caagaggcgcaaacaagc, ZEB2 reverse; 5-ggttggcaataccgtcatcc, SNAI1 forward; 5-gctgcaggactctaatccaga, SNAI1 reverse; 5-gctgcaggactctaatccaga, SNAI2 forward; 5-tggttgcttcaaggacacat, SNAI2 reverse; 5-gttgcagtgagggcaagaa, GAPDH forward; 5-acccagaagactgtggatgg, GAPDH reverse; 5-cagtgagcttcccgttcag.
BRAF transfection
Human wild-type BRAF cDNA was prepared by PCR using the primers (forward; 5′-gtggaattctgcagatataagatggcggcgctgagcggtgg, reverse; 5′-gccactgtgctggatcctttgttgctactctcctgaactctctcactc), which cover full lengths of the coding region of the gene, from a human cDNA library. The amplified fragments were cloned into the pcDNA 3.1(+) vector (Life Technologies) using the In-Fusion HD Cloning Kit (Takara Bio, Shiga, Japan). Human mutant BRAF cDNA containing p.V600E mutation was constructed by site-directed mutagenesis using the wild-type BRAF expression vector as a template. The insert fragments containing wild-type or mutant BRAF was sequenced by ABI Prism 3130 (Life Technologies), to confirm that the fragments has no sequence variation in the coding region of BRAF other than p.V600E.
Western blot analysis
Western blot analysis was performed following a standard protocol [
35]. Anti-BRAF rabbit monoclonal antibody (#9433, Cell Signaling Technology, Danvers, MA, USA), anti-p-BRAF (Ser445) rabbit monoclonal antibody (#2696, Cell Signaling Technology), anti-p-MEK1/2 (Ser217/221) rabbit polyclonal antibody (#9121, Cell Signaling Technology), anti-p-ERK1/2 (Ser217/221) rabbit monoclonal antibody (#4094, Cell Signaling Technology), and anti-α-tubulin mouse monoclonal antibody (Sigma-Aldrich) were used as primary antibodies for detection of the specific proteins.
Statistical analysis
Statistical analyses were performed with JMP Pro ver. 11.0 (SAS Institute, Cary, NC, USA). The differences between two groups were analyzed by chi-square test, Fisher’s exact test, Student’s t test or Mann-Whitney U-test, depending on each parameter. Correlation analysis was performed by using Spearmann’s rank correlation method. Kaplan-Meier analysis was conducted to estimate distributions of OS, or PFS after the beginning of the first-line chemotherapy or after anti-EGFR therapies, and a log-rank test was utilized to analyze the statistical difference in the survival. Each difference was regarded as statistically significant when P < 0.05.
Ethics statement
This study was performed in accordance with the Declaration of Helsinki and was approved by the Ethical Committee of TUH and NCCH. A written informed consent was obtained from all patients.
Discussion
The purpose of this study was to identify miRNAs that were specifically dysregulated in human BRAF-mutant colorectal cancer, a molecular subtype of colorectal cancer with a higher malignant potential. We also sought to elucidate the functional significance of the miRNAs in colorectal cancer. We demonstrated a novel role of miR-193a-3p in colorectal cancer. First, using genome-wide miRNA expression analysis for a set of patients with colorectal cancer followed by the validation analysis for another set of patients, we identified miR-193a-3p as a down-regulated miRNA in BRAF-mutant colorectal cancer. Second, miR-193a-3p functioned as a tumor suppressor in a panel of colorectal cancer cell lines. Third, miR-193a-3p was partly affected by overexpression of mutant BRAF proteins. Finally, low miR-193a-3p expression status correlated with a worse clinical outcome to anti-EGFR therapy, independent of the BRAF mutation status. Taken together, our results indicate that the dysregulation of miR-193a-3p is involved in the tumorigenesis of colorectal cancer, particularly BRAF-mutant cancer, and is likely to affect drug sensitivities to anti-EGFR therapy in colorectal cancer regardless of BRAF mutational status. These data provide new insights into the molecular mechanisms underlying the oncogenesis of colorectal cancer.
Few studies have investigated the relationship between
BRAF mutations and altered miRNA expression in colorectal cancer. In contrast, a handful of studies have focused on the specific alterations of miRNA expressions in
BRAF-mutant cancer of other organs. Cahill et al. reported that 15 miRNAs were up-regulated and 23 miRNAs including miR-193a were down-regulated in
BRAF-mutant thyroid cancer cell lines compared to normal thyroid cells [
30]. Caramuta et al. reported that miR-193a-3p, miR-338 and miR-565 were under-expressed in melanomas with
BRAF mutations compared to those without
BRAF or
NRAS mutations [
31]. Our result of the specific down-regulation of miR-193a-3p in
BRAF-mutant colorectal tumors is in line with these previous reports, suggesting that miR-193a-3p may be involved in oncogenesis of various malignancies with mutated
BRAF.
More recently, Nosho et al. found that miR-31 is the most overexpressed miRNA in
BRAF-mutant colorectal cancers. This is the first study that analyzed the association between altered miRNA expressions and
BRAF mutations in colorectal cancer [
45]. Through a global miRNA expression analysis covering 760 miRNAs, they have identified 33 dysregulated miRNAs, all of which were up-regulated in
BRAF-mutant colorectal cancer. Our result that miR-31 was one of the most up-regulated miRNA in
BRAF-mutant colorectal cancers is consistent with their report [
45]. In addition, we found that miR-135b was also up-regulated, although it was not identified as among the 33 up-regulated miRNAs in their study. A more recent study also found miR-31 was the most up-regulated miRNA in
BRAF-mutant tumors through screening the miRNA expression profiles of seven patients with
BRAF-mutant tumors [
46]; however, the other nine miRNAs dysregulated in their study did not include miR-7, miR-135b, miR-148b, or miR-193a-3p that were detected as dysregulated in our study. We identified miR-193a-3p as a novel down-regulated miRNA in
BRAF-mutant colorectal cancer, which offers more information on the significance of the dysregulation of multiple miRNAs in colorectal tumorigenesis of this molecular subtype.
Our results obtained from the functional assays using a panel of colorectal cancer cell lines also support a possible role of miR-193a-3p as a tumor suppressor in colorectal cancer. To the best of our knowledge, our study is the first to demonstrate the tumor suppressive role of miR-193a-3p in colorectal cancer. The precise molecular mechanisms by which miR-193a-3p inhibits cellular oncogenic process, that is, the genes or pathways that this miRNA directly dictates in colorectal cancer, remains to be elucidated. However, recent evidence from the studies on other cancers offers some clues to this question. Uhlmann et al. demonstrated that three miRNAs, miR-124, miR-147, and miR-193a-3p, co-target EGFR-related pathway proteins, leading to an inhibition of cell-cycle progression and cell proliferation in breast cancer cell models through global miRNA screening using a high-throughput proteomic analysis [
36]. miR-193a-3p has been shown to directly target
JNK1, and overexpression of miR-193a-3p decreased the protein levels of JNK1 as well as CDK4, cyclin D1, and PIK3CA, which are all involved in the EGFR/cell cycle network pathways [
36]. A more recent study by
Yu et al. supported the role of miR-193a-3p in the EGFR-related signaling pathway [
37]. Using human non-small cell lung cancer samples and cell lines, they showed that miR-193a-3p and miR-193a-5p are under-expressed in non-small cell lung cancer, particularly in metastatic tumors of lung cancer, and that the overexpression of miR-193a-3p and miR-193a-5p leads to the inhibition of lung cancer metastasis in vitro and in vivo. The inhibitory effect of miR-193a-3p and miR-193a-5p on metastasis may be due to the down-regulation of the ERBB4/PIK3R2/mTOR/S6 K2 signaling pathway through direct targeting of
ERBB4 and
S6 K2 by miR-193a-3p, and
PIK3R3 and mTOR by miR-193a-5p [
37]. Their findings were supported by another recent study that confirmed that miR-193a-3p directly targets ERBB4 in lung cancer [
47]. Furthermore, surprisingly, a more recent report has revealed that miR-193a-3p directly targets
KRAS and inhibits tumor growth and metastasis in an ex vivo and in vivo lung cancer model [
48]. These lines of evidence, together with our results, support a notion that miR-193a-3p acts as a negative regulator for the EGFR/ERBB-related pathways in various cancers, although the full mechanisms underlying the miR-193a-3p-EGFR/ERBB regulatory network in colorectal cancer require further elucidation.
Another question is how miR-193a-3p is initially down-regulated in cancer cells. Our results that miR-193a-3p expression is modestly, but significantly, decreased by the overexpression of BRAF mutant protein in non-
BRAF-mutant cell lines (Fig.
3d) suggest the possibility that miR-193a-3p may directly regulate the EGFR-related pathway and also be regulated, at least in part, directly by BRAF or other downstream EGFR-related pathways within a feedback loop. However, both BRAF and MEK inhibition did not significantly affect miR-193a-3p expression in cells with already activated BRAF or KRAS. The possible mechanism of miR-193a-3p down-regulation via BRAF is likely more complicated. Furthermore, the luciferase assay has shown that the only modest decrease in miR-193a-3p expression induced by transient BRAF overexpression (by about 20%), which is a minor difference compared with the 2–3-fold difference observed between patients with
BRAF mutations and those without
KRAS/BRAF mutations (Figs.
1 and
2), did not lead to the recovery of expression of target genes (Additional file
4: Figure S3). Together, our results suggest that mutant BRAF may partly affect miR-193a-3p expression, but is unlikely to be a main direct cause of alterations in miR-193a-3p and its target gene expressions that are involved in tumorigenesis of colorectal cancer. Several earlier reports have raised another explanation for the mechanism for the down-regulation of miR-193a-3p. miR-193a is located in 17q11.2, and this miRNA and its promoter region are located within CpG islands. The promoter DNA methylation of miR-193a has been implicated in oral cancer [
40], acute myeloid leukemia [
41], and non-small cell lung cancer [
42]. The precise mechanism underlying this miRNA down-regulation, particularly in
BRAF-mutated colorectal cancer, should be further elucidated in future studies.
In addition, a clinically important novel finding of this study was that miR-193a-3p expression status was associated with the clinical outcome of colorectal cancer patients treated with anti-EGFR therapy. The miR-193a-3p expression status did not correlate with OS or PFS for cytotoxic drug therapies, but low expression status was associated with a worse PFS for anti-EGFR therapies among all patients analyzed (Fig.
4). Even when only patients without
KRAS/
BRAF mutations were analyzed, a similar trend was observed (Fig.
4), although it should be noted that the difference did not reach significance due to the limited number of patients. Our study was a retrospective setting, and the sample size used in the survival analysis was relatively small to draw a robust conclusion on the effect of miR-193a-3p dysregulation on the sensitivity to anti-EGFR therapy. Therefore, the association between miR-193a-3p and the outcomes of anti-EGFR therapy should be confirmed in larger, prospective studies. Nevertheless, the possible correlation between miR-193a-3p expression and the clinical outcome from anti-EGFR therapy in patients with colorectal cancer further support a role of miR-193a-3p in the EGFR-related signaling pathway.
Acknowledgments
We thank Dr. John M. Mariadason at Ludwig Institute for Cancer Research, Australia, for kindly providing the colorectal cancer cell lines. We also thank Hiromi Nakano for her technical assistance.