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
] 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 ) 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 . Different siRNA sequences for the same target often gave inconsistent results in the two cells lines used in this study (Table ). 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
]. 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 ).
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
]. One microarray study reported an increase of RALY expression in cells resistant to oxaliplatin along with the proteins HNRNPL, PABPC1, RPS4X, and RPS7 (Table ) [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
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
]. 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
]. 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
]. 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 ). 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.