Background
Colorectal cancer is the third leading cause of cancer-related death in the Western world (National Cancer Institute; 2009). Over 50% of those diagnosed require systemic therapy at some point during the disease trajectory. Clinical resistance is almost inevitable for advanced colorectal cancer within 6-12 months of any given therapy. For example, clinical responses of metastatic cancers (
e.g. death and elimination of cancer cells) to the most advanced therapeutic agents range from 15 to 40%, indicating intrinsic resistance in a majority of colorectal cancer tissues [
1]. In addition, acquired resistance almost inevitably occurs in tumors that initially responded [
1]. One of the most effective regimens against colorectal cancers, either in adjuvant or in metastatic settings, is the combination of fluoropirimidine and oxaliplatin. Oxaliplatin is a third generation platinum analogue that kills cells by forming adducts on DNA, the most prevalent of which is intra-strand linkage of two adjacent guanines [
2]. Nevertheless, the majority of patients with colon cancer are either intrinsically resistant to this drug or become resistant during therapy. Oxaliplatin-resistant cells are characterized by decreased DNA adduct formation [
3]. The exact mechanisms responsible for this resistance are still elusive to this date.
Several studies have indicated that the overexpression of YB-1 (Y box-binding protein-1) is related with secondary resistance to cisplatin in melanomas, breast, ovarian, and bladder cancers [
4‐
7]. Furthermore, depletion of YB-1 expression protein with anti-sense RNA against YB-1 specific mRNA results in increased sensitivity to cisplatin [
8]. Interestingly, YB-1 is increased in cultured cell lines resistant to cisplatin. In fact, several studies have indicated that the level of nuclear expression of YB-1 is predictive of drug resistance and patient outcome in breast tumors, ovarian cancers, and synovial sarcomas [
5,
7,
9‐
11]. YB-1 preferentially binds to cisplatin-modified DNA [
12]. Further analyzes have indicated that YB-1 actively promotes strand separation of duplex DNA containing either mismatches or cisplatin modifications independently of the nucleotide sequence [
13] in addition to having an exonuclease activity [
14]. YB-1 was originally described as a transcription regulator that binds to inverted CCAAT box DNA sequences present in the control regions of several genes [
15]. In addition to the regulation of transcription, YB-1 is a multifunctional protein that also affects the splicing and the translation of specific mRNAs [
16‐
18]. Several mRNAs regulated by YB-1 are potentially important for chemoresistance [
18].
It has been reported that YB-1 expression is increased in colorectal carcinomas compared to normal colon tissues [
19]. Although overexpression of YB-1 confers cisplatin resistance in breast and ovarian cancers, it is unknown whether increase YB-1 expression would also confer oxaliplatin resistance in colorectal cancers [
20]. In this study, we investigated the impact of YB-1 on oxaliplatin resistance in two different colon adenocarcinoma cell lines. We show that overexpression of YB-1 confers oxaliplatin resistance and a depletion of YB-1 sensitizes cells to oxaliplatin treatments in culture. In addition, we identified by mass spectrometry analyses important YB-1 interactors required for such oxaliplatin resistance in these colorectal cancer cell lines. Knock down analyses of two of these proteins, NONO and RALY, increased oxaliplatin sensitivity in otherwise resistant colorectal cancer cells overexpressing YB-1.
Methods
Cell lines and drugs
The human HT29 colon adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC) and maintained in McCoy's 5A media supplemented with 2 mM L-Glutamine, 10% Fetal Bovine Serum (FBS), and 1% Antibiotic-Antimytotic (Invitrogen, Carlsbad, CA) at 37°C in atmosphere of 5% CO2. The human SW480 colon adenocarcinoma cell line (also obtained from ATCC) was maintained in RPMI supplemented with 10% Fetal Bovine Serum (FBS), and 1% Antibiotic-Antimytotic (Invitrogen, Carlsbad, CA) at 37°C in atmosphere of 5% CO2. Oxaliplatin was purchased from the Hôtel-Dieu Hospital in Quebec City (Qc, Canada).
Antibodies
A polyclonal antibody against the N-terminus portion of YB-1 (ab12148) was purchased from Abcam, Inc. (Cambridge, MA). The mouse monoclonal antibody against HNRPB
2A
1 (sc-32316) and a rabbit polyclonal antibody against HNRPL (sc-28726) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse polyclonal antibody against RALY (H00138046-B030), a mouse polyclonal antibody against MRPL13 (H00028998-B01), and a goat polyclonal antibody against NONO (PAB 7027) were purchased from Abnova Corp. (Taipei City, Taiwan). A rabbit polyclonal antibody against RIBC2 (HPA003210) was purchased from Sigma-Aldrich Inc. (St-Louis, MO). Finally, all horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG: NAV934V and anti-mouse IgG: NA931V) were purchased from GE Healthcare Limited (Piscataway, NJ). The above antibodies were used as indicated by the manufacturers. Western blotting analyses were performed as described [
13].
Plasmids and transfections
The YB-1 cDNA was cloned in the pNTAP-B vector in-frame with the TAP-epitope consisting of both calmodulin and streptavidin epitopes (Stratagene, LaJolla, CA). SW480 cells were transfected with Effectene (Qiagen, Inc., Mississauga, ON) and selected in 600 μg/ml of neomycin (Invitrogen, Carlsbad, CA) for two weeks. Colonies were expended and analyzed by Western blotting with an antibody against YB-1 to select TAP-YB-1 expressing clones. The pCMV-YB-1 construct was described before [
13]. This YB-1 expression vector was transfected into SW480 cells with the Amaxa nucleofector kit V as described by the manufacturer (Lonza Company, Basel, Switzerland). A transfection efficiency of 19.8% in SW480 cells was routinely obtained with a fluorescent GFP-YB-1 construct using this transfection protocol (data not shown).
Tandem affinity purification and mass spectrometry analyses
The TAP-YB-1 protein was purified from a stable SW480 clone expressing this protein construct with a TAP purification kit (Stratagene, LaJolla, CA) as described by the manufacturer. We also used large amounts of RNAse A (100 μg/mL) in our extraction buffers to eliminate contaminating ribonucleic acids that could be used as bridging molecules in YB-1 containing complexes. SW480 cells containing an empty TAP vector were used as a control. Eluted proteins were analyzed by SDS-PAGE and lanes corresponding to control TAP and TAP-YB-1 expressing cells were cut into small gel slices. Gel slices were sent to the Proteomics platform of the Quebec Genomic Center (Quebec City, Qc, Canada) for spectrometry analyses and protein identifications. Briefly, gel slices were disposed into 96-well plates and in-gel trypsin digestion was performed on a MassPrep™ liquid handling station (Waters, Mississauga, ON) according to the manufacturer's specifications. Peptide extracts were dried out using a SpeedVac™. Peptide extracts were separated by online reversed-phase nanoscale capillary LC and analyzed by electrospray MS (ES MS/MS). The experiments were performed on a Thermo Surveyor MS pump connected to a LTQ linear ion trap mass spectrometer (Thermo Electron, San Jose, CA) equipped with a nanoelectrospray ion source (Thermo Electron, San Jose, CA). Peptide separation took place within a PicoFrit column BioBasic C18, 10 cm × 0.075 mm internal diameter (New Objective, Woburn, MA) with a linear gradient from 2% to 50% solvent B (acetonitrile, 0.1% formic acid) in 30 min, at 200 nl/min. Mass spectra were acquired using data-dependent acquisition mode (Xcalibure software, version 2.0). Each full-scan mass spectrum (400-2000 m/z) was followed by collision-induced dissociation of the seven most intense ions. The dynamic exclusion function was enabled (30 s exclusion), and the relative collisional fragmentation energy was set to 35%.
All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2.0). Mascot was set up to search against human Uniref_100 protein database assuming a digestion with trypsin. Fragment and parent ion mass tolerance were, respectively, of 0.5 Da and 2.0 Da. Iodoacetamide derivative of cysteine was specified as a fixed modification. Deamidation of asparagine and glutamine, acetylation of lysine and arginine and oxidation of methionine were specified as variable modifications. Scaffold (version 01_07_00; Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at > 95.0% probability as specified by the Peptide Prophet algorithm [
21]. Protein probabilities were assigned by the Protein Prophet algorithm [
22]. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Using these stringent identification parameters, the rate of false positive identifications is < 1%.
To confirm protein interactions, TAP-YB-1 protein complexes were purified from a stable SW480 clone expressing the TAP-YB-1 construct with a TAP purification kit (Stratagene, LaJolla, CA) as described by the manufacturer. SW480 cells containing an empty TAP vector were used as a control. Eluted proteins were analyzed by SDS-PAGE followed by Western blotting with the appropriate antibodies.
Oncomine analyses
Oncomine is a cancer microarray database and web-based data-mining platform aimed at facilitating discovery from genome-wide expression analyses. Data can be queried and visualized for a selected gene across microarray analyses available to the public. The parameter for the research in Oncomine included as primary filters the official name of the gene (or protein interacting with YB-1) and the analysis type, which was colorectal cancer versus normal tissue analysis. The threshold used to obtain the most significant probes of the queried gene for each microarray data included a two-fold difference in expression between cancers and normal tissues with a P-value < 1 × 10-4.
siRNA screening
The antisense technology has been described as a potential anti-cancer therapy for the treatment of several clinical cancers and several molecules are in phase I and II trials [
23]. In general, cells were reverse-transfected with a custom arrayed set of 18 genes (4 siRNA sequences/gene) using RNAiMax (Invitrogen, Carlsbad, CA) for SW480 cells or DharmaFect 3 (Dharmacon, Lafayette, CO) for HT29 cells and incubated at 37°C for 24 hours followed by treatment with various concentrations of oxaliplatin and incubated for an additional 96 hours before measuring cell viability via the CellTiter-Glo luminescent assay kit according to the manufacturer's protocol (Promega, Madison, WI). All scramble siRNA controls (GFP, ASNS, and NS) as well as the positive transfection control (Allstar Cell Death Control or ACDC) were purchased from Qiagen (Valencia, CA).
One microliter of 0.667 μM siRNA was printed into bar-coded 384 well solid-white bottom plates (Corning 8749, Lowell, MA) using a Biomek FX Laboratory Automation Workstation for a final assay concentration of 13 nM per well. siRNA buffer [100 mM Potassium Acetate, 30 mM HEPES-KOH, 2 mM Magnesium Acetate (pH 7.6)] was printed into the first two columns of the plate to serve as a control. A transfection reagent solution of RNAiMax and serum free RPMI was added to the plates using a BIO-TEK μFill Microplate Dispenser at 20 μl per well (40 nl RNAiMax/well). The plates containing siRNA and transfection reagents were incubated at room temperature for 30 minutes prior to adding cells to allow complexes to form. Cells were trypsinized, counted, resuspended in 10% FBS assay media, and then seeded at a concentration of 1000 cells/20 μl per well for SW480 cells using the BIO-TEK μFill Microplate Dispenser for a final FBS concentration of 5% (20 μl serum free + 20 μl 10% FBS media). Plates were incubated at 37°C for 24 hours prior to dosing them with 10 μl/well of varying concentrations of oxaliplatin diluted in 5% FBS assay media using a μFill. Initially, twelve different drug concentrations, together with vehicle control, were applied for each control siRNA to obtain a drug dose response curve. Subsequently, six concentrations of oxaliplatin [200, 66.7, 22.2, 7.4, 2.47, 0.82, and 0 μM] spanning the entire response range of the drug were applied to SW480 cells during the siRNA screening process. The first column of every plate (containing siRNA buffer, no siRNA) was left untreated to serve as a control for plate-to-plate variation. Plates were incubated for an additional 96 hours post drug treatment and then read for cell viability. The screen in SW480 cells was performed in two independent runs.
siRNAs altering SW480 sensitivity to oxaliplatin were also tested in HT29 cells. Identical protocols were used for these cells, with the following exceptions: DharmaFECT 3 was delivered at a concentration of 200 nl/well and six concentrations of oxaliplatin [250, 100, 40, 16, 6.4, 2.56, and 0 μM] spanning the entire dose response range of HT29 cells were used for the siRNA screening process based on an initial test with twelve different oxaliplatin concentrations.
The siRNA sequences against YB-1 are 5'-AAGAAGAAAUAUGAAAUUCCA-3' for sequence siRNA-A, 5'-CUGCAAGCACCUGUUAAUAAA-3' for siRNA-B, and 5'-CAGGCGAAGGUUCCCACCUUA-3' for siRNA-C.
Growth curves and FACS analyses
For growth curves, 10 to 50 thousands cells were plated in 60 mm dishes (or 35 mm dishes when indicated) and counted with a hemacytometer by the trypan blue exclusion technique every other day. For FACS analyses, trypsinized cells were collected and centrifuged on a top bench centrifuge for 2 min. Cell were resuspended in PBS and an equal volume of 95% cold ethanol was added. The next day cells were centrifuged and the pellet was resuspended in a propidium iodide buffer (0.1% citrate; 0.3% NP-40; 0.002 mg/ml of RNAse A; 50 μg/ml propidium iodide at pH 7.4). Cells were incubated 30 min at 37°C and then analyzed on a Coulter® Epics XL-MCL™ Flow Cytometer (Beckman Coulter Canada, Inc., Mississauga, ON). Data were analyzed with the MultiCycle software (Phoenix Flow System, San Diego, CA, USA).
RT-PCR
The primers used to amplify a 116 base pairs fragment of the HPRT1 (hypoxanthine phosphoribosyltransferase 1) cDNA are HPRTRev 5'-GCACACAGAGGGCTACAATG-3' and HPRTFor 5'-TGAGGATTTGGAAAGGGTGT-3'. The primers used to amplify a 527 base pairs fragment of the YB-1 cDNA are Y159For 5'-CCAGCAAAATTACCAGAAT-3' and UNYB1Rev 5'-TGATGGTAGAGATGGTAAGC-3'. Reverse transcriptase was performed on 300 ng of total RNA with either the HPRTRev or the UNYB1Rev primer. The cDNA was then amplified with Taq DNA polymerase and the appropriate forward primers for each target mRNA. The PCR conditions for the HPRT1 cDNA were 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C for 30 cycles. The PCR conditions for the YB-1 cDNA were 1 min at 94°C, 1 min at 58°C, and 1 min at 72°C for 30 cycles. Amplified products were analyzed on a 2% agarose gel.
Discussion
Oxaliplatin has become an effective first-line therapy for colorectal cancer. One major problem with oxaliplatin regimen, however, is the appearance of resistant tumor cells during the course of the treatment. A second problem is the lack of knowledge on a common mechanism that confers oxaliplatin resistance in several colorectal adenocarcinomas. A number of microarray studies have indicated a significant increase in YB-1 expression in colon cancer tissues compared to normal colon tissues [
25‐
27] and one immunohistochemistry study had reached a similar conclusion [
19]. In this study, we examined whether overexpression of YB-1 could confer oxaliplatin resistance in two colorectal cancer cell lines. Transient transfection experiment in SW480 and HT29 colorectal cancer cells and stable SW480 clones overexpressing a TAP-YB-1 construct all demonstrated a significant resistance to the drug oxaliplatin. From this information, we then asked whether a specific YB-1 protein complex is required to confer oxaliplatin resistance in SW480 and HT29 cells. We first identified by mass spectrometry analyses proteins binding directly to a TAP-YB-1 construct in SW480 cells. Sixty-seven proteins were identified with at least two peptides. To focus on proteins with the highest physiological relevance to chemioresistance in colorectal cancer, we opted for an integrative approach by collecting information on each protein with regard to gene expression and status (which includes genetic rearrangements) in colon cancers. Based on different public databases, 23 of these YB-1 partners were overexpressed at the transcriptional level in colorectal tumors, in metastasis, and associated with increased genomic instability in colon cancers. The transcription of one protein was decreased in recurring colon cancer. These observations indicated that the transcription levels of such proteins could potentially be used as prognostic markers. Based on microarray studies of different cell lines, the proteins HNRPL, PABPC1, RPS4X, RPS7, and RALY are overexpressed in ovarian cancer cells resistant to oxaliplatin [
30]. From such information, we determined whether the depletion of these proteins up regulated in colorectal adenocarcinomas (Table
1) have an impact on oxaliplatin sensitivity on both SW480 and HT29 cancer cells. We used the knock down technology in this study as siRNA molecules can have potential therapeutic benefits. Importantly, the antisense technology has been described as a potential anti-cancer therapy for the treatment of several clinical cancers and several molecules are in phase I and II trials [
23]. We tested four siRNA sequences for each target proteins in the list of Table
1. Different siRNA sequences for the same target often gave inconsistent results in the two cells lines used in this study (Table
2). Such results are not surprising as it is now well accepted that a siRNA sequence may present off-target effects that may differ depending on the transcriptome of each cell line [
31‐
33]. We thus focused on proteins which showed at least two different siRNA sequences against the mRNAs corresponding to these proteins with the same phenotype in two different cancer cell lines. Based on these criteria, we observed that the depletion of RALY or NONO increased oxaliplatin sensitivity in both SW480 and HT29 cells. More importantly, the siRNA sequences showing increased oxaliplatin sensitivity efficiently depleted RALY or NONO proteins (Figure
6).
RALY (hnRNP-associated with lethal yellow homolog (mouse)) is a member of the heterogeneous nuclear ribonucleoprotein gene family. It is associated with the spliceosome complex [
34]. It is unknown, however, whether RALY also affects transcription and DNA repair like YB-1 in cells [
15,
35,
36]. One microarray study reported an increase of RALY expression in cells resistant to oxaliplatin along with the proteins HNRNPL, PABPC1, RPS4X, and RPS7 (Table
1) [
30]. Our results, however, indicate that RALY is the only protein that sensitizes colorectal cancer cells to oxaliplatin when depleted with siRNAs. It is possible that HNRNPL, PABPC1, RPS4X, and RPS7 correlate with oxaliplatin resistance but are not directly involved biochemically in the process of resistance in colorectal cancer cell types. We recently found that a depletion of RPS4X increased cisplatin resistance in YB-1 overexpressing breast cancer cell lines [
37]. We did not observe such cellular response in colorectal cancer cells upon oxaliplatin treatment in our study. This maybe due to the intrinsic difference between breast and colon cancer cell types, or it could be due to the different manner in which cells respond to cisplatin and oxaliplatin. For example, it has recently been reported that gamma irradiation of two teratoma cell lines
in vitro induced resistance to subsequent oxaliplatin treatment, but increased sensitivity to cisplatin [
38]. Notably, cisplatin and oxaliplatin DNA damage are not processed by the same DNA repair systems [
39,
40].
NONO (non-POU domain containing, octamer-binding) gene encodes an RNA-binding protein, which plays various roles in the nucleus, including transcriptional regulation and RNA processing [
41‐
44]. A study with a GFP-NONO has indicated that the protein localizes not only to the nucleolus but also in nuclear speckles rich in splicing factors [
41]. In addition to mRNA processing, NONO is involved in the repair of DNA double stranded breaks [
45‐
47]. It is believed to participate in both the non-homologous end joining and the homologous recombination repair pathways with its homologue SFPQ (splicing factor proline/glutamine-rich). We did not identify, however, SFPQ in our mass spectrometry analyses as a protein interacting with the TAP-YB-1 construct.
Messenger RNA processing (which includes splicing) is a common biological process shared by YB-1, NONO, and RALY proteins [
16,
34,
41]. Exon-array profiling is warranted in NONO, RALY, and YB-1 depleted cells to find common spliced mRNAs affected by these proteins during oxaliplatin response. Independent of the exact mechanisms involved in oxaliplatin resistance, NONO and RALY are good potential targets for the sensitization of otherwise oxaliplatin resistant YB-1 overexpressing colorectal cancer tumor cells (Figure
10). Interestingly, NONO or RALY knock down decreased endogenous YB-1 protein levels. The exact mechanism by which these proteins regulate YB-1 expression is unknown but our RT-PCR results indicate that a depletion of NONO also down regulates the level of YB-1 transcripts. In contrast, a depletion of RALY in SW480 cells did not significantly affect YB-1 mRNA levels. Thus, unlike NONO, RALY regulates the amount of YB-1 protein in SW480 cells at the post-transcriptional level. Additional experiments are required to determine which steps of YB-1 mRNA processing and transport NONO and RALY are involved in.
Finally, a depletion of YB-1 decreased NONO expression in SW480 cells but had no effect on RALY expression. This result is consistent with findings indicating that the promoter of NONO but not the promoter of RALY is a target of the YB-1 transcription factor [
48]. It is possible that YB-1 regulates the transcription of the NONO gene in colorectal cancer cells. Epidemiological studies on appropriate cohorts of patients with colorectal cancer are warranted to determine whether the expressions of YB-1, NONO, and RALY have predictive values for the response to oxaliplatin treatment.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SPT, HHY, and ML conceived and designed the experiments. SPT, CG, CS, DC, MA and DG performed the experiments. CG and DG generated the cells expressing the TAP-YB-1 constructs. SPT, CS, DC, MA HHY, and ML analyzed the data. ML and HHY wrote the paper. All authors read and approved the final manuscript.