Elevated XPO6 expression as a potential prognostic biomarker for prostate cancer recurrence
Recurrence of localized prostate cancer following treatment can lead to lethal metastatic castration-resistant prostate cancer. Although numerous studies aimed at developing biomarkers for predicting recurrence of localized prostate cancer are promising, they have not yet led to useful applications. Dysregulation of exportins (XPOs, nucleocytoplasmic transporters) associated with subcellular mislocalization of proteins has been reported for various human cancers. However, most of the XPOs have not been studied in prostate cancer. In this study, we are the first to examine whether changes in expression of XPOs could be used as potential biomarkers for recurrence of localized prostate cancer. Using the oncomine database, gene expressions of 7 known XPOs by 1128 patient samples, obtained from 16 independent prostate cancer patient cohorts, were analyzed. Relatively highly elevated expression of XPO6 (compared to prostate cancer tissue) was found to be significantly associated with poor patient prognosis, in particular, with rapid recurrence in a clinical “low risk” group. As such, expression of XPO6 may be a potential prognostic biomarker for predicting prostate cancer recurrence.
Prostate cancer is the most commonly diagnosed non-cutaneous cancer and a leading cause of cancer death for North American men (1). When the malignancy is localized to the prostate, surgery and radiation therapy can be curative. However, many treated patients will experience local recurrence which can lead to metastatic cancer for which there is currently no cure (2). Currently, one of the most commonly used strategies for recurrence risk prediction is the D’Amico risk stratification which is based on the initial PSA level, biopsy Gleason score and clinical T stage (3-5). However, this method has limited predictive power leading to either overtreatment or undertreatment (6,7). Recently, studies aimed at developing biomarkers for predicting prostate cancer progression have made promising progress in the laboratory (8-21). However, very few biomarkers have proven useful in the clinic. Clearly, discovery and development of new reliable biomarkers to predict cancer recurrence is urgently required for improving disease management and patient survival.
In eukaryotic cells, proteins made in the cytoplasm need to be transported to various subcellular locations, such as the nucleus, to fulfill their particular functions. Proper localization of proteins is of major importance for normal functioning of cells. The transportation of the proteins is mediated by karyopherins. Proteins also need to be exported from the nucleus and, in such a case, their localization is mediated by karyopherins known as exportins (XPOs) (22). Exportins are proteins which can identify and bind to a cargo via recognition of a specific nuclear export signal (NES); they share a common N-terminal domain. To date, 7 XPOs have been identified in humans, each of them being responsible for exporting specific molecules from the nucleus to the cytoplasm (22). Dysregulation of XPOs associated with subcellular mislocalization of proteins has been reported for various types of human cancer (23). Upregulation of certain XPOs in particular has been associated with cancer progression (24-32).
The present study was aimed at (i) investigating changes in the gene expression of XPOs in prostate cancer, and (ii) determining whether the changes could be used as potential biomarkers for the recurrence of localized prostate cancer. Using the oncomine database, we examined the expressions of XPOs in data from a total of 1128 patient samples obtained from 16 independent clinical cohorts (8, 10, 33-46). We found that XPO6 was elevated in primary prostate cancer tissues as well as metastatic tissues and that its elevated expression correlated with increased prostate cancer aggressiveness, suggesting that the XPO6 protein can provide a novel, prognostic biomarker for prostate cancer recurrence.
3. Materials and methods
3.1. Oncomine database analysis
3.2. Prognostic value analysis
Gene expression data from 131 primary prostate cancer tissue specimens and the biochemical recurrence-free survival times of the patients (acquired from NCBI GEO under accession GSE21032) (42) were analyzed using Kaplan-Meier analysis and Cox proportional-hazards regression analysis. Clinical and pathologic data (patient age, tumor site, PSA level, T stage, Gleason score, metastasis, biochemical recurrence, time until biochemical recurrence) were also collected (Table 5) for validating the prognostic values of the XPOs.
3.3. Statistical analysis
P<0.0.5 was used as the significant threshold level unless otherwise mentioned. Significance comparisons between 2 different groups were calculated using the Student’s t test. GraphPad Prism software (Version 4.0.3, GraphPad Software Inc., La Jolla, CA) was used for Kaplan-Meier analysis and the log-rank test was used to determine the difference between curves. Univariate and multivariate Cox proportional-hazards regression models were analyzed by SigmaPlot software (Bersion 11.0., Systat Software Inc., San Jose, CA) and the significance values and odds ratios were calculated by the likelihood ratio test. Both analyses were used to evaluate the association of various factors with biochemical recurrence. The level of significance in the statistical analyses is indicated as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P<0.0001.
4.1. Elevated expression of XPO3 and XPO6 in prostate cancer
To reveal potential differential expressions of the XPOs in prostate cancer, we compared the expression of the XPOs in prostate cancer tissues and normal prostate tissues from 16 independent prostate cancer patient cohorts (8, 10, 33-46) using the same cut-offs (P value<0.0.1, gene rank top/bottom 10%, Table 1). In none of these cohorts a significant difference was found in XPO1 or XPO5 expression between normal and cancer tissues. Elevated expression of XPO4 and XPO2 was found only in 1 and 3 cohorts, respectively. There was no significant change in the expression of XPO7 as there were conflicting results between the various cohorts. The expressions of XPO3 and XPO6 were markedly and consistently upregulated in 8 and 7 cohorts, respectively (Figure 1A, B). XPO3 had a higher expression in prostate cancer tissues compared to normal tissues in 8 cohorts with fold changes varying between 1.34 to 1.96 and a significant P value (1.13E-9 to 6.00E-3). XPO6 expression was elevated in 7 different cohorts with fold changes between 1.14 to 2.28 and significant P value (4.01E-5 to 2.00E-3).
|Genes||Studies (up/total)||Studies (down/total)||Sample number||P value||Fold change||Gene rank(%)||Reference|
|XPO2||3/16||0/16||82||5.00E-5–7.09E-4||1.13–1.53||3.94–9.62||38, 45, 51|
|XPO3||8/15||0/15||495||1.13E-9–6.00E-3||1.34–1.96||0.80–7.59||37, 38, 42, 44, 45, 47, 49, 51|
|XPO6||7/15||0/15||461||4.01E-5–2.00E-3||1.14–2.28||3.48–9.84||37, 44, 45, 47, 48, 50, 51|
|XPO7||1/15||3/15||89/74||1.06E-4/(1.21E-4–1.00E-3)||3.10/(−1.23–−1.93)||1.21/(2.37–3.98)||45, 46, 48, 52|
|Study details included in the supplementary materials (Supplementary Table 5-10)|
4.2. Elevated XPO6 expression is correlated with poor patient prognosis
We determined whether there was a correlation between the expression of XPO3 and XPO6 and poor patient prognosis (Table 2). XPO6 expression was significantly higher in patients with elevated PSA levels (>20 ng/ml) prior to radical prostatectomy (P=0.02). Elevated XPO6 expression was also found in patients with higher combined Gleason score (≥8) in both biopsy and radical prostatectomy specimens (P=1.34E-3, P=3.09E-3, respectively). Furthermore, a positive association was found for the elevated expression of XPO6 and lymph node metastasis occurrence (P=0.01), biochemical recurrence (P=1.57E-3), and distant metastasis occurrence (P=3.27E-4), whereas there was no significant correlation between XPO3 expression and poor prognosis or poor patient outcomes. Some cohorts showed that the expression of XPO6 was higher in CRPC and metastatic prostate cancer compared to primary tumors (Figure 1C, D). Taken together, the results suggest that elevated expression of XPO6 is significantly correlated with more aggressive prostate cancers and poor clinical outcomes.
|Poor prognosis factor||P value|
|PSA<20vs. PSA≥20 ng/ml||0.39||0.02|
|Biopsy Gleason score<8vs. Gleasonscore≥8||0.47||1.34E-03|
|Radical prostatectomy Gleason score<8vs. Gleason score≥8||0.38||3.09E-03|
|N0vs. N1(Lymph node metastasis)||0.39||0.01|
|No recurrence after treatment vs. recurrence after treatment||0.45||1.57E-03|
|M0vs. M1(distant metastasis)||0.42||3.27E-04|
4.3. Elevated XPO6 expression is associated with biochemical recurrence of prostate cancer
We further focused on the correlation between the elevated expression of XPO3 and XPO6 and biochemical recurrence. Recurrence-free survival curves were calculated using the Kaplan-Meier analysis. The samples were grouped according to the expression levels, where high expression means samples with top 30% XPO3 expression among all primary tumors. The expression of XPO3 did not significantly correlate with biochemical recurrence (P=0.07, Figure 2A), as indicated by Kaplan-Meier analysis. The latter also indicates that patients bearing tumors with elevated expression of XPO6 had a significant shorter time until recurrence (P=0.04, Hazard Ratio=2.19, 95% CI between 1.07 and 5.635, Figure 2B). The mean recurrence-free survival time of patients with elevated XPO6 expression was 6.5. months shorter compared to other patients.
Using the Cox proportional-hazards regression method, we confirmed that the elevated expression of XPO3 did not correlate with recurrence-free survival time (OR=0.44, P=0.50, Table 3), whereas there was a significant correlation between the elevated expression of XPO6 and biochemical recurrence (Odds Ratio=7.32, P=6.81E-3, Table 3). The combination of using XPO6 expression as a predictive factor and the D’Amico stratification gave a better prognostic prediction value (OR=14.04, P=8.93E-4) than when these approaches were used on their own (OR=7.32, P=6.81E-3; OR=10.11, P=1.47E-3, respectively). Both XPO6 expression and D’Amico stratification contributed significantly to this combination (XPO6: HR=2.9.7, 95% CI between 1.04 and 8.51, P=0.04; D’Amico: HR=2.96, 95% CI between 1.34 and 6.53, P=7.35E-3, Table 4).
|1Odds ratio, 2degrees of freedom. Significance levels were calculated using the likelihood ratio test|
|B1||SE2||Wald3||P||HR4||95% CI-L5||95% CI-U6|
|1Coefficient, 2Standard error, 3Wald Chi-Square, 4Hazard ratio, 595%CI-L: 95% confidence interval lower limit, 695%CI-U: 95% confidence interval upper limit|
|Tumor samples from primary site||131||87.33|
|Tumor samples from metastatic site||19||12.67|
|Age at diagnosis/median(range)||58.00(37.30-83.00)|
|PSA at diagnosis/median(range)||6.30(1.09-506.00)|
|Biopsy Gleason score|
|Clinical T stage|
|PSA level prior to radical prostatectomy/median(range)||6.60(1.15-506.00)|
|Radical prostatectomy Gleason score|
|Pathology T stage|
|Time until biochemical recurrence(months)/median(range)||45.45(1.38-149.19)|
|Metastasis resulting from the primary tumor|
|XPO2||Study number||Study names||Sample number||P value||Fold change||Gene rank %||Reference|
|XPO3||Study number||Study names||Sample number||P value||Fold change||Gene rank %||Reference|
|XPO4||Study number||Study names||Sample number||P value||Fold change||Gene rank %||Reference|
|XPO6||Study number||Study names||Sample number||P value||Fold change||Gene rank %||Reference|
|XPO7||Study number||Study names||Sample number||P value||Fold change||Gene rank %||Reference|
4.4. XPO6 as a prognostic biomarker in low risk patient group
To explore whether the expression of XPO6 can benefit current prostate cancer risk stratification, we focused on patients who were grouped at the time of diagnosis as “low risk” based on D’Amico risk stratification. 60 primary samples were grouped as “low risk” and 8 of them were found to have a biochemical recurrence. Using the Cox proportional-hazards regression method, we found that elevated expression of XPO6 significantly correlated with biochemical recurrence (P=0.02, Odds Ratio=5.61). To confirm this, we separated patients into two groups according to “High XPO6” (XPO6 expression 1.5. fold higher than the median) and “Low XPO6” (the remaining samples), and subjected them to Kaplan-Meier analysis. As shown in Figure 3, the two groups of patients had significantly different times until recurrence (P=2.70E-7, Hazard ratio=15.79, 95% CI of the ratio 423.4.-7.26E5). These results indicate that XPO6 expression may be used as a novel biomarker for identification of potential “high risk” patients in a clinical low risk group.
Cancer recurrence following therapy of localized prostate cancer is a first indicator showing that a cancer may gradually develop into a lethal, metastatic CRPC. The risk stratification system currently used to predict cancer recurrence following therapy of localized prostate cancer lacks predictive ability and there is a critical need for reliable prognostic biomarkers (48). In developing a candidate biomarker, it is of paramount importance that it has clinical relevance. Thus the majority of biomarkers that successfully pass preclinical tests fail when they are used in clinical trials (49). Using gene expression data and clinical data from patient cohorts we have, in the present study, shown that the expression of XPO6 was significantly upregulated in prostate cancers. Furthermore, substantially elevated expression of XPO6 correlated with increased prostate cancer aggressiveness and poor patient prognosis, as indicated by elevated blood PSA levels, increased Gleason score, biochemical recurrence, and lymph node/distant metastases. This suggests that use of relatively highly elevated XPO6 expression (compared to prostate cancer tissue) as a potential biomarker for predicting poor prognosis, following therapy of localized prostate cancer, has clinical relevance.
Recently, various studies have suggested that the expression of both protein-coding and non-protein-coding genes can be used as potential biomarkers for the prediction of recurrence following therapy of localized prostate cancers (50-59). The majority of the studies, however, failed to discuss how such potential biomarkers could benefit the currently used clinical risk stratification system. In the present study, it is suggested that use of relatively highly elevated expression of XPO6 (compared to prostate cancer tissue) as a prognostic biomarker could be particularly useful for identification of cancer recurrence in “low risk” patients (see Figure 3). If a “low risk” patient is shown to have relatively highly elevated expression of XPO6, he could be recommended for more aggressive treatment than normally used for localized prostate cancer, such as androgen deprivation therapy. Thus, relatively highly elevated expression of XPO6 may be particularly useful as a biomarker in combination with the D’Amico risk stratification system to determine which treatment option should be selected for an individual patient. However, the Taylor patient cohort (42) used in the present study is the only publically available prostate cancer patient cohort with detailed patient clinical and pathologic information. Other cohorts are either limited in the number of patients or lack complete clinical and pathologic information. This obstacle weakens the findings since the clinical relevance was both found and validated using the same patient cohorts. To achieve better clinical relevance and strengthen the findings, more patient samples need to be analyzed when more patient cohorts are available.
Although the expression of XPO3 was also found to be elevated in prostate cancer, it had poor clinical relevance as there was no correlation with prostate cancer progression. In contrast to a report suggesting that XPO1 expression was elevated in prostate cancer cell lines (60), the present study did not show a statistically significant difference in XPO1 expression between prostate cancer and normal prostate tissue. This discrepancy may be due to an inability of in vitro systems to accurately reflect tumor physiology (61).
In conclusion, relatively highly elevated expression of XPO6 (compared to prostate cancer tissue) may provide a prognostic biomarker for identifying patients with high risk of developing recurrence following therapy of localized prostate cancer, in particular for patients with “low risk” of recurrence based on the D’Amico risk stratification system.
This study was supported by the Canadian Institutes of Health Research (YZW), BC Cancer Foundation (YZW), Prostate Cancer Canada (YZW). The authors thank Dr.Fang Zhang, Dr.Dong Lin and Sifeng Qu for stimulating discussions and valuable comments.
Abbreviations: PSA: prostate specific antigen; NPCs: nuclear pore complexes; XPOs: exportins; NES: nuclear export signal; AR: androgen receptor; TP53: tumor protein p53; BRCA1: breast cancer1; XPO1: exportin 1; XPO2: CSE1L, CSE1 chromosome seqregation 1-like; XPO3: XPOT, exportin tRNA; XPO4: exportin 4; XPO5: exportin 5; XPO6: exportin 6; XPO7: exportin 7; CRPC: castration-resistant prostate cancer; OR: Odds ratio; HR: Hazard ratio; CI: Confidence interval
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