Background
Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide [
1]. Screening, surgery, and medical therapies are usually effective for the management of early-stage CRC, however, these treatment options are far less effective for advanced-stage disease. Cetuximab is an epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) that binds the EGFR extracellular domain and enhances receptor internalization and degradation. This mAb targeting EGFR is a common targeted agent used to treat patients with metastatic CRC (mCRC) with wild-type KRAS status [
2]. However, patients frequently develop drug resistance. A number of genetic biomarkers, including RAS (KRAS exons 3 and 4 or NRAS exons 2, 3, and 4) mutations [
3,
4], BRAF (V600E) [
5], PIK3CA (exon 20) [
6] and HER2 and MET amplification [
7,
8], are robustly correlated with cetuximab resistance. Despite these genetic biomarkers, additional mechanisms of resistance to EGFR mAb are thought to be present in mCRC.
Currently, with advancements in global transcriptome profiling technique, the functional roles of long noncoding RNAs (lncRNAs) have received considerable attention in human cancer [
9]. LncRNAs are mRNA-like transcripts ranging in length from 200 nucleotides (nt) to over 100 kilobases (kb) lacking significant protein-coding ability [
10]. Emerging evidence has demonstrated that lncRNAs play significant roles in biological processing, including cell cycle regulation, apoptosis and tumor invasion [
11]. Additionally, other lncRNAs have been shown to play functional roles in resistance to targeted therapy [
12]. Some lncRNAs could be potentially used as early diagnostic, prognostic and drug response biomarkers in malignant tumors [
12‐
14].
Urothelial carcinoma-associated 1 (UCA1) is a lncRNA with three exons, and several recent studies have demonstrated oncogenic functions of UCA1 in various types of cancer, such as breast, bladder, colorectal, and gastric cancer [
15]. In our previous study, we elucidated that UCA1 expressions are significantly increased in CRC tissues and cells, and this high UCA1 expression level is significantly correlated with larger tumor size, greater tumor depth and less differentiated histology. Additionally, patients with high UCA1 level have a significantly poorer prognosis than those with low UCA1 level. Moreover, UCA1 influences the proliferation, cell cycle and apoptosis of CRC cells. These data indicate an important role for UCA1 in the molecular etiology of CRC and implicate a potential application for UCA1 in CRC diagnosis, prognosis of tumor progression, and therapy [
16]. In addition to its oncogenic function, UCA1 regulates drug resistance in some kinds of malignant tumors [
17]. However, the value of UCA1 levels in body fluid samples regarding the response of CRC patients to cetuximab remains to be confirmed.
Exosomes are lipid vesicles with a diameter of 30–100 nm that are secreted by the fusion of multivesicular bodies with the plasma membrane or by budding from the membrane [
18]. Nucleic acids, proteins and lipids are loaded in exosomes, thereby allowing the transfer of genetic material and enabling the exchange of information between cells. Emerging evidence has demonstrated that some specific exosomal lncRNAs expression is correlated with cancer patients’ clinicopathological characteristics and therefore may act as a potential biomarker [
18‐
20]. Based on these observations, we hypothesize that exosomal UCA1 might predict the response of CRC patients to cetuximab.
The existence and stability of exosomal UCA1 in serum were systematically investigated in this study. Additionally, we identified a novel role of UCA1-containing exosomes in predicting the response of CRC patients to cetuximab. Moreover, for the first time, we suggest that UCA1 may be transmitted via exosomes, thus affecting drug metabolism.
Methods
Patients and sample processing
Informed consent was obtained from each patient, and the protocols were approved by the Ethics Committee of Harbin Medical University Cancer Hospital. Serum samples were collected from Harbin Medical University Cancer Hospital to investigate the feasibility of detecting exosomal UCA1. Changes in serum exosomal UCA1 levels were validated in an independent cohort of 53 CRC patients treated with cetuximab at Harbin Medical University Cancer Hospital from 2015 to 2016. Blood samples from all CRC patients were collected by vena puncture. To extract exosome from human blood, a two-step centrifugation protocol—1600g for 10 min and 16,000g for 10 min at 4 °C was used to isolate serum, and then serum was stored at − 80 °C until exosome extraction. Blood samples with evidence of hemolysis were excluded. According to the RECIST criteria for a pathological response, these patients were divided into two groups: 30 patients responded to cetuximab therapy [complete response (CR) or partial response (PR)], and 23 patients did not respond [stable disease (SD) or progressive disease (PD)].
Cell lines and culture
The human Caco2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). We established cetuximab-resistant cell lines by chronically exposing cetuximab-sensitive Caco2 (Caco2-CS) cells to increasing cetuximab doses in medium over a period of 6 months. The final concentration of cetuximab for the cetuximab-resistant subclone Caco2-CR was 300 μg/ml. Caco2-CS and Caco2-CR cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Invitrogen, USA) containing 10% fetal bovine serum (FBS, Gibco, Invitrogen, USA) and 1% penicillin–streptomycin (Invitrogen, USA) at 37 °C in a humidified atmosphere of 95% air/5% CO2.
Cell proliferation assay and colony formation assay
For the cell proliferation assay, cell viability was determined by Cell Counting Kit 8 (CCK8, Dojindo, Japan) according to the manufacturer’s instructions. For the colony formation assay, about 1000 cells were placed in each well of a 6-well plate in 2 ml media containing cetuximab (300 μg/ml for Caco2-CR). The media were changed every 3 day. After 12–15 days, the colonies were fixed in 80% methanol and stained with 0.1% crystal violet for 20 min. The number of colonies was counted using an inverted microscope.
Isolation of exosomes
Medium and serum were filtered through a 0.45 μm polyvinylidene fluoride filter (Millipore, Billerica, MA, USA); ExoQuick solution (System Biosciences, Mountain View, CA, USA) was added to the serum, which was then incubated for 0.5 h at room temperature, and ExoQuick-TC solution was added to the medium, which was then incubated at 4 °C for 12 h. The mixture was centrifuged at 1500g for 30 min and supernatant was removed by aspiration. Pelleted fractions were resuspended in 25 μl phosphate-buffered saline (PBS).
Transmission electron microscopy (TEM)
A sample of exosomes was diluted to a final concentration of 0.5 mg/ml in PBS, spotted onto a glow-discharged copper grid on filter paper and dried for 10 min. Exosomes were stained with 1% aqueous phosphotungstic acid (PTA) for 5 min and dried for 20 min and then examined at 100 keV with TEM (JEM-1-11 microscope, Japan).
Total RNA was extracted from cells using TRIzol® Reagent (Invitrogen, CA, USA). Exosomal RNA was extracted using the Total Exosome RNA and Protein Isolation Kit (Invitrogen, USA). The concentration and quality of RNA were measured by UV absorbance at 260 and 280 nm (260/280 nm) using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA).
Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells and exosomes as described above. RNA templates were treated with DNase I to avoid genomic DNA contamination. The first strand of cDNA was synthesized using the SuperScript First-Strand Synthesis System (Invitrogen, CA). PCR amplification was performed using an Applied Biosystems 7500 Detection System (Applied Biosystems, CA) and primers for UCA1 (forward: 5′-ACGCTAACTGGCACCTTGTT-3′, reverse: 5′-TGGGGATTACTGGGGTAGGG-3′) and β-actin (forward, 5′-CACCTTCTACAATGAGCTGCGTGTG-3′; reverse, 5′-ATAGCACAGCCTGGATAGCAACGTAC-3′). Real-time PCR was performed on triplicate samples according to the instructions of the SYBR® Premix Ex Taq™ Kit (Takara, Japan). The expression level of UCA1 was normalized to that of β-actin using the comparative 2−ΔΔCt method.
Western blot analysis
Proteins were extracted from cells using RIPA lysis buffer (Biouniquer Technology, China). Exosomal proteins were extracted using the Total Exosome RNA and Protein Isolation Kit (Invitrogen, USA). Protein content was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific, USA). Equal amounts of protein from each sample were separated by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gels before being transferred onto polyvinylidene difluoride (PVDF) membranes, blocked for 2 h with 5% skim milk, incubated overnight at 4 °C with primary antibodies against TSG101, Alix and CD81 (Abcam, Cambridge, UK) and then incubated for 1 h with the secondary antibody (Kangwei Ltd., China). The signals were visualized with an ECL detection system.
Immunofluorescence assay
For exosomal labeling, Dil (Beyotime Biotechnology, China) was added to the exosome suspension at 1 μM for 20 min, and the exosome suspension was then washed through Exosome Spin Columns (MW3000) (Invitrogen, USA) to remove excess dye. Dil-labeled exosomes were incubated with CRC cells and visualized by fluorescence microscopy or EPICS XL flow cytometry (Beckman Coulter) after 24 h.
RNA interference
Caco2-CR cells were transfected with UCA1-small interfering RNA (siRNA) (RNAi: forward primer: 5′-GCACCUUGUUAGCUACAUAAA-3′, reverse primer: 5′-UAUGUAGCUAACAAGGUGCCA-3′) using Lipofectamine™ 2000. After 48 h of transfection, RNAi efficiency was assessed by qRT-PCR. Another siRNA was designed as a negative control (Ctrl siRNA): forward primer: 5′-UUCUCCGAACGUGUCACGUdTdT-3′, reverse primer: 5′-ACGUGACACGUUCGGAGAAdTdT-3′.
Electroporation of UCA1 into exosomes
UCA1 was electroporated into exosomes using a GenePulser Xcell™ electroporation system (Bio-Rad, USA) as previously described [
21]. Briefly, 2 μg exosomes and 400 nmol RNA were mixed in 400 μl electroporation buffer and electroporated at 350 V and 150 μF in a 4-mm cuvette. The mixture was incubated at 37 °C for 30 min to ensure that the exosome membrane fully recovered, and then, the mixture was treated with RNase to remove unincorporated RNA. Labeled RNA in exosomes was quantified by detecting fluorescence using a Fluorescence Spectrophotometer F-4600 (HITACHI, Japan).
Apoptosis assay
Twenty-four hours after 1 × 105 Caco2-CS cells were seeded in six-well plates, different amounts of CR-exo (1, 5, or 10 μg) were added to the supernatants. The samples were divided into the following four groups: CS, CS + 1 μg CR-exo, CS + 5 μg CR-exo, and CS + 10 μg CR-exo. After culturing for 24 h, the cells were treated with cetuximab (300 μg/ml) for 48 h. Then, all the cells were harvested by centrifugation, washed with PBS, stained using Annexin V fluorescein isothiocyanate (APC) and propidium iodide (PI) and analyzed using a flow cytometer (BD Biosciences, San Diego, CA).
Statistical analyses
All statistical analyses in this study were performed with SPSS 16.0 software. All experiments were carried out three times independently. Data are presented as the mean ± standard deviation. p values < 0.05 by Student’s t-test or one-way ANOVA indicated statistical significance. All statistical tests were two-sided.
Discussion
Cetuximab is one of the most widely used EGFR inhibitors in the treatment of mCRC patients with wild-type KRAS status. However, primary or acquired resistance to cetuximab often occurs during targeted therapy. To optimize individualized cetuximab therapy in CRC patients, it is essential to identify possible indicators of the response to cetuximab therapy.
The lncRNA UCA1 has been found to reactive and is a promising diagnostic and prognostic biomarker in various malignant tumors [
15]. Furthermore, many studies have shown that UCA1 induces drug resistance in various cancers [
17]. The role of UCA1 in CRC drug resistance has also been described [
24]. Bian et al. [
24] showed that ectopic expression of UCA1 increased the chemoresistance of CRC cells to 5-fluorouracil (5-FU), whereas knockdown of UCA1 enhanced apoptosis in HCT116 cells. Mechanistically, UCA1 acts as a sponge for miR-204-5p and upregulates the level of several target genes of miR-204-5p, suggesting the important role of UCA1 in chemoresistance. Therapeutic monitoring using human blood is considered a noninvasive and convenient method, and circulating lncRNAs as biomarkers for predicting therapeutic efficacy in cancer has been investigated for years [
19,
25]. However, the potential of using UCA1 levels in body fluid samples as a predictive biomarker of drug resistance remains to be confirmed.
Increasing evidence indicates that the tumor microenvironment can also contributes to changes in tumor characteristics [
26,
27], such as resistance to some anticancer drugs [
28,
29]. Exosomes are a promising tumor-derived material for the characterization of some tumor behaviors [
30,
31]. Sugimachi et al. [
32] showed that serum exosomal miRNAs could be potential novel biomarkers for predicting tumor recurrence and prognosis. Furthermore, Ma et al. [
33] demonstrated that TrpC5-containing extracellular vesicles might be a potential biomarker for chemoresistant breast cancer. Here, we sought to investigate whether exosomal UCA1 is involved in the drug resistance process and to determine the significance of the transfer of cetuximab-resistance properties in CRC. In the current study, we first clarified the upregulation of UCA1 levels in cetuximab-resistant cells and their exosomes. Interestingly, it was observed that UCA1 detectable in cells and serum is concentrated in exosomes. In fact, the increase in exosomes is more evident than in cells, and therefore, purifying exosomes could improve the sensitivity of circulating UCA1 detection. Therefore, we reason that exosome-mediated transfer of UCA1 might be an important mechanism of acquitted cetuximab resistance in CRC cells. We also systematically evaluated the stability of exosomal UCA1, and the results indicated that exosomal UCA1 is evidently stable when subjected to severe conditions, suggesting that the exosomal UCA1 can be protected by exosome membrane. Furthermore, we observed that circulating UCA1-containing exosomes could predict the clinical outcome of cetuximab therapy in CRC patients. Although the quantity of exosomes was nearly equal in the PD/SD and PR/CR groups, interestingly, UCA1 level was markedly higher in PD/SD patients than in PR/CR patients. As exosomes are important players in intercellular communication processes [
34‐
36], we further investigated whether exosomes are involved in drug resistance process and evaluated the significance of the transfer of drug-resistance properties in CRC. We observed that exosomes derived from cetuximab-resistant cells can transmit drug resistance to sensitive cells and cause changes in UCA1 level. These findings further support for the hypothesis that analyzing exosomal lncRNA expression in blood as a biomarker become ever more feasible and that UCA1-containing exosomes can predict cetuximab therapy efficacy in CRC patients.
The molecular mechanisms by which most lncRNAs function remain to be elucidated. LncRNAs play roles in signaling and can act as decoys, guides or scaffolds by specifically binding to target DNAs, RNAs and proteins. While these mechanisms are not mutually exclusive, some lncRNAs may have one or more roles. In different cancer cells, UCA1 has been demonstrated to bind to several miRNAs [
24,
37‐
43]. In addition to its oncogenic effect, UCA1 also exerts a regulatory effect on drug resistance in several types of cancer. For example, UCA1 can induce acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutant nonsmall cell lung cancer by activating the AKT/mTOR pathway [
44]. It can also increase the chemoresistance of bladder cancer cells by activating the Wnt signaling pathway in a Wnt6-dependent manner [
45]. Knockdown of UCA1 in adriamycin-resistant SGC7901/ADR cells can significantly decrease resistance [
46]. The major limitation of this study is that downstream regulation of UCA1 in cetuximab-resistant cells was not investigated. We hypothesized that some molecular effectors, such as miRNA, might be important targets of UCA1, leading to dysregulation of the signaling pathway in cetuximab resistance. However, further studies are required to validate our hypothesis.
Conclusions
In summary, this is the first demonstration that exosomal UCA1 has the key characteristics of a tumor marker: it can be assayed in a noninvasive manner, and it is relatively abundant and stable. Moreover, we explored exosomal UCA1 as a biomarker for predicting drug sensitivity in patients who were treated with cetuximab. UCA1-containing exosomes appear to be essential for conferring cetuximab resistance. Although some factors involving in drug resistance has been widely studied, our findings on UCA1 open important possibilities for future clinical application. Thus, both basic and clinical researchers should engage in multidisciplinary efforts monitoring of cetuximab resistance. As mentioned above, UCA1 is a potential good broad-spectrum biomarker for cancer diagnosis, prognosis or prediction of therapeutic response. However, no UCA1 products have been identified in clinical trials. Further preclinical and clinical studies will highlight and better elucidate the role of UCA1 in anticancer drug resistance and may lead to future investigations of the various clinical applications of UCA1.
Authors’ contributions
SJ and YH designed the experiments and edited the manuscript. YY and RZ conducted the experiments and wrote the manuscript. JD, HY, YL, XW and XD performed experiments and analysed data. All authors read and approved the final manuscript.