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Previously, we reported that the overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, is correlated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer (NSCLC). In the present study, we further analyzed FER-overexpressed NSCLC cases and identified various patterns of chimeric mRNAs, composed of paraja ring finger 2 (PJA2) and FER. We detected no genomic rearrangements between PJA2 and FER and attributed these chimeric mRNAs to alterations at the transcriptome level: i.e., trans-splicing. Several chimeric patterns were detected concurrently in each patient, and the pattern sets varied among patients, although the pattern in which PJA2 exon 1 was fused to FER exon 3 (designated as Pe1-Fe3 mRNA) was detected constantly. Therefore, in a wide screening for PJA2-FER mRNAs in NSCLC, we focused on this chimeric pattern as a representative chimera. In analyses of 167 NSCLC samples, Pe1-Fe3 mRNA was identified in about 10% of the patients, and the presence of chimeric mRNA was significantly correlated with a high expression level of parental FER mRNA. Furthermore, we found that the detection of Pe1-Fe3 mRNA was correlated with poor postoperative survival periods in NSCLC, consistent with a previous finding in which FER overexpression was correlated with poor postoperative prognosis in NSCLC. This report is the first to suggest a correlation between chimeric mRNA and the expression level of parental mRNA. Furthermore, our findings may be clinically beneficial, suggesting that PJA2-FER mRNAs might serve as a novel prognostic biomarker in NSCLC.
Despite significant advances in diagnosis and treatment, lung cancer remains the leading cause of cancer-related mortality worldwide, with over 1 million deaths each year, and the 5-year survival rate of lung cancer is below 20%. The best chance of achieving long-term survival is in complete surgical resection; however, even among resected stage IA patients, approximately 30% succumb to their disease within 5 years. Although some recent trials have demonstrated that adjuvant chemotherapies improve the 5-year survival rate of patients with non-small cell lung cancer (NSCLC) who undergo complete surgical resection,[4-6] clear evidence of a clinical benefit of adjuvant chemotherapy has not yet been obtained. Given this situation, the ability to predict the clinical outcome of lung cancer after surgical resection and to identify patients who are at a high risk of recurrence and might benefit from further therapy after surgical resection is important. Some clinicopathological factors, such as tumor size, preoperative serum CEA level, visceral pleural invasion, vascular vessel invasion, and histological grade, are well-known predictors of clinical outcome in NSCLC patients after surgical resection.[7-11] In addition to these clinicopathological factors, to improve the clinical outcome of NSCLC, molecular prognostic biomarkers that can serve as more selective and creditable markers are needed.
Recent advances in high-throughput sequencing technology have provided novel insights into genomics; one of these insights is the discovery of chimeric mRNAs that are not caused by chromosomal rearrangements, but by transcription-induced mechanisms such as trans-splicing. Trans-splicing is a form of alternative splicing in which RNA exons from two separate primary transcripts are joined, and this form of alternative splicing is now thought to be more common than previously believed[13-15] especially in cancers,[16-18] where the quality control systems governing transcription are frequently disrupted. Although some mechanisms that cause trans-splicing have been suggested, such as the close proximity of nascent pre-mRNAs or the presence of complementary sequences between them,[19, 20] the exact cause and regulation of trans-splicing remains to be elucidated.
In the present study examining NSCLC specimens, we identified various chimeric mRNAs, composed of paraja ring finger 2 (PJA2) and fer tyrosine kinase (FER), that probably resulted from trans-splicing. In a wide screening of NSCLC specimens, we found that the presence of chimeric mRNAs was positively correlated with a high expression level of parental FER mRNA. We also found that the detection of chimeric mRNAs was correlated with poor postoperative survival periods in a manner that was independent of other clinicopathological factors, consistent with our previous finding that FER overexpression is correlated with poor postoperative prognosis in NSCLC.
To the best of our knowledge, this report is the first to suggest a correlation between chimeric mRNA and the expression level of parental mRNA; furthermore, because the RT-PCR method used here to detect the chimeric mRNAs is a simple method that can be easily performed at most institutions, PJA2-FER mRNAs may serve as a convenient prognostic biomarker in NSCLC.
- Top of page
- Materials and Methods
- Disclosure Statement
- Supporting Information
Here, we identified novel chimeric PJA2-FER mRNAs that were correlated with a high expression level of parental FER mRNA and served as a potential biomarker for the postoperative prognosis in NSCLC. PJA2 and FER are located next to each other, but in inverse orientations, on chromosome 5q. PJA2 encodes an E3 ubiquitin-protein ligase, and although the precise function of PJA2 has not been clarified, its expression level is moderately high in almost all normal tissues according to our present quantitative RT-PCR results and microarray analysis (RefExA, http://www.lsbm.org/). FER is a 94-kDa non-receptor tyrosine kinase involved in the reorganization of the actin cytoskeleton or the modulation of cell–cell and cell–substrate interactions through its association with adhesion molecules[24-31] and has been reported to play a role in the proliferation and growth of several cancers, such as prostate cancer,[33, 34] colon cancer, breast cancer, hepatocellular carcinoma, malignant mesothelioma, and gastric cancer. Especially, very recently, FER expression has been shown to be correlated with poor prognosis in NSCLC and renal cell carcinoma.[40, 41]
We attempted the PCR amplification of a putative recombined chromosome using several primers flanking the expected translocation site, but no genomic PJA2-FER fusion sequences were detected. Thus, we attributed these chimeric mRNAs to alterations at the transcriptome level: i.e., trans-splicing. Trans-splicing is a form of alternative splicing in which mRNA exons from two separate primary transcripts are joined. Since this phenomenon was first discovered in trypanosomes, several findings have demonstrated that it may also occur in mammalian cells, and this form of alternative splicing has now been shown to be more common than previously believed.[13-15] As for the mechanisms that cause trans-splicing, some reports suggest that trans-splicing occurs at high frequencies when the parental pre-mRNAs are closely positioned to each other or when there are complementary sequences between them.[14, 19, 20, 43]
In the case of PJA2 and FER, because the PJA2 gene is located next to the FER gene on the same chromosome, the two pre-mRNAs are thought to be present in very close proximity to each other, possibly creating a predisposition toward trans-splicing between them. In addition, interestingly, we found that there was a significant positive correlation between the presence of Pe1-Fe3 mRNA and a high expression level of parental FER mRNA in the present study. From a biochemical perspective, it seems reasonable to assume that a larger amount of material (pre-mRNAs) would be likely to produce products (chimeric transcripts) more easily and that an abundance of parental transcripts might be an accelerating factor contributing to trans-splicing. As for PJA2 and FER, because the expression level of PJA2 mRNA is invariably high among NSCLC cases, the likelihood of trans-splicing between them would depend on the expression level of FER mRNA.
Regarding biological function, trans-splicing is generally expected to increase proteomic diversity, but its exact significance remains controversial. In cancer, some trans-splicing products, such as CYCLIN D1-TRO2 mRNA in ovarian and mammary cancer or SLC45A3-ELK4 mRNA in prostate cancer,[17, 45] are reported to be involved in the oncogenic mechanism. In our study, we could not examine whether novel proteins were translated from chimeric PJA2-FER mRNAs, and as far as we were able to examine using in vitro assays, the PJA2-FER chimeras showed no transforming activities. Additionally, when we analyzed the amino acid sequences that were possibly translated from PJA2-FER mRNAs in silico, they were not predicted to have any domain that might be related to oncogenic function. However, further functional analyses are needed to examine whether they have any other biological functions.
On the other hand, because FER overexpression has been previously shown to be correlated with poor postoperative prognosis in NSCLC, we assumed that PJA2-FER mRNA, which was found to be correlated with a high expression level of FER mRNA, might also serve as a prognostic biomarker in NSCLC. As expected, the detection of Pe1-Fe3 mRNA was shown to be correlated with poor postoperative prognosis in patients with NSCLC independently of other clinical prognostic factors, although the correlation with further factors such as histological subtype, which is an established and convenient prognostic factor for stage I adenocarcinoma, should be analyzed in the future studies with a larger number of samples.
Reverse transcription-polymerase chain reaction was used in the present study to detect Pe1-Fe3 mRNA, and this method is relatively simple and can easily be performed at most institutions. As shown in Figure 1(c), FER itself was detected as a band using RT-PCR method in all the NSCLC samples, including those without FER overexpression. Therefore, to distinguish FER expression level, simple RT-PCR method is not sufficient, and further quantitative RT-PCR or IHC analyses are needed, which are sometimes difficult to perform and lead to controversial results because of technical inadequacies or cost problems in some institutions or because of the inadequate volume of resected samples. Considering such situations, the identification of Pe1-Fe3 mRNA as a biomarker that can be examined easily as the detection of a band using a simple RT-PCR method is likely to be clinically more beneficial.
In conclusion, we have identified novel chimeric PJA2-FER mRNAs that were correlated with a high expression level of parental FER mRNA and poor postoperative prognosis in NSCLC. Although further molecular studies or a larger epidemic study are needed, our findings might provide some insights into the mechanism of trans-splicing and might also be clinically beneficial by helping to easily identify NSCLC patients at risk after surgery who are likely to benefit from adjuvant chemotherapy, thereby improving the clinical outcome of NSCLC.