Exportin-5 Functions as an Oncogene and a Potential Therapeutic Target in Colorectal Cancer

Colorectal cancer is the second leading cause of cancer-related deaths in the United States (1), and more than 1.2 million patients are diagnosed with colorectal cancer annually, with 5-year survival rates of approximately 64% (2, 3). Accumulating evidence indicates that epigenetic modifications play a critical role in the progression of colorectal cancer. In particular, over the last decade, dysregulated expression of miRNAs has emerged as a hallmark feature of human cancers, including colorectal cancer. miRNAs are small noncoding RNAs, 19–24 nucleotides in length, which regulate the expression of few to hundreds of downstream target genes posttranscriptionally (4, 5). In addition to their biological role, dysregulated expression of miRNAs is of clinical significance, as it often associates with key cancer-related clinicopathologic features (6, 7).

The biogenesis of miRNAs is mediated by a group of proteins, including Drosha, Dicer, TRBP, and exportin-5 (XPO5; ref. 8). Among these, XPO5, a member of the karyopherin family, is a key protein responsible for the transportation of precursor miRNAs (pre-miRNAs) from the nucleus to the cytoplasm (9). Although XPO5 is perhaps the most critical regulator of miRNA biogenesis, the clinical significance of altered XPO5 expression in colorectal cancer remains unclear. Furthermore, the functional role of altered XPO5 expression, its impact on downstream target miRNAs, and its influence on colorectal cancer biology have not been interrogated.

Although no comprehensive studies have been performed in colorectal cancer, limited evidence in previous studies has observed overexpression of XPO5 in urothelial (10) and breast cancer (11, 12). Another recent study showed that XPO5 is a critical regulator of cell cycle (13). These studies suggest the oncogenic potential of XPO5, but systematic studies are required to further elucidate its functional and clinical role as a biomarker and as a potential therapeutic target in colorectal cancer.

Herein, we systematically and comprehensively characterize the role of XPO5 in colorectal cancer by analyzing colorectal cancer cell lines, a xenograft animal model, and multiple cohorts of clinical specimens. Our data firstly demonstrate that XPO5 is upregulated in colorectal cancers and that patients with high XPO5 expression are associated with higher tumor burdens, worse clinicopathologic features and poor survival. Furthermore, we studied the functional role of altered XPO5 expression and demonstrated that it functions as an oncogene by upregulating the expression of important growth-promoting miRNAs in colorectal cancer. Taken together, our data highlighted the oncogenic role of XPO5, and high expression of XPO5 may serve as a potential therapeutic target in colorectal cancer.

This study included analysis of 661 colorectal tissues, which were analyzed in a three-phase study that included an initial discovery cohort and two subsequent validation cohorts. In the discovery cohort, we analyzed data from the Oncomine microarray database comprising 181 tissue specimens from normal and colorectal cancer tissues (14). This database included results from three independent gene expression profiles obtained from normal and colorectal cancer tissues (15–18). The Cancer Genome Atlas (TCGA) validation cohort included data from 234 tissue specimens in the TCGA dataset (19). The information on patient survival was obtained via cBioPortal (20). The clinical validation cohort included analysis of 246 matched normal and neoplastic tissues from patients enrolled at the Mie University (Mie, Japan). Patients with histologically confirmed stage I, II, III, or IV colorectal cancers who underwent surgical resection of their primary lesions were included in our study. Some stage IV colorectal cancer patients that were not candidates for surgical resection were excluded from our study due to unavailability of their clinical specimens. Treatments of patients followed guideline published by NCCN Clinical Practice Guidelines in Oncology. Written informed consent was obtained from each patient, and the study was approved by the institutional Review Boards of all involved institutions.

qRT-PCR was performed using the StepOne Real Time PCR System (Applied Biosystems). The mRNA expression levels were measured using predesigned XPO5 TaqMan probes (Life Technologies) and normalized to GAPDH. The relative expression of XPO5 was determined by calculating the differences between cancer and adjacent normal mucosa tissues. Reverse transcription and real-time PCR for miRNAs were performed using TaqMan probes for miR-10b, miR-21, miR-27a, miR-92a, miR-155, miR-182, pri-miR-21, pri-miR-10b, pri-miR-23a∼27a∼24-2 cluster, and pri-miR-182. A TaqMan primer set for the small nuclear RNA U6 (Life Technologies) was used as a normalization control for miRNAs, and GAPDH (Life Technologies) was used for normalization of pri-miRNA expression. The relative expression level of each miRNA was determined using the ΔΔC_t method. All assays were performed in duplicate.

Paraffin-embedded sections derived from 10 matched cancer/normal tissues were deparaffinized by xylene and ethanol. Following elimination of endogenous peroxidase activity by H₂O₂ and antigen retrieval by autoclaving at 121°C for 15 minutes, slides were incubated with anti-XPO5 antibody (EPR8452, Abcam, 1:400 dilution) for 1 hour. The color was developed using EnVision + Dual Link Kit (DAKO) and hematoxylin. The staining with an isotype antibody was used as the negative control. The staining levels of XPO5 were analyzed microscopically by two independent experts using staining score (from 1–5).

After the fixation of cells by methanol, cultured cells were reacted with anti-XPO5 antibody (EPR8452, Abcam, 1:400 dilution), followed by secondary fluorescence antibody (A-11034, Invitrogen, 10 μg/mL). Fluorescence signal was analyzed using a fluorescence microscope.

The microsatellite stable (MSS) colorectal cancer cell lines, SW480 and Caco-2, were purchased from the ATCC. All cell lines were cultured according to the manufacturer's specifications. Every few months, all cell lines were tested and authenticated using a panel of genetic and epigenetic markers.

XPO5-specific validated locked nucleic acid–modified Silencer Select siRNA (siXPO5; s33190; Ambion) and control siRNA (siControl; Ambion) were selected for transfection. All experiments were performed by forward transfection according to the manufacturer's protocol with a mixture of Optimem I (Invitrogen), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) and siRNA oligonucleotides (50 nmol/L for SW480, 100 nmol/L for Caco-2). Cells were incubated in culture media for 48 hours after transfection prior to harvesting for analyses. All experiments were conducted in triplicates, and at least three independent experiments were performed.

Western immunoblotting was performed as described previously (21). Anti-XPO5 antibody (EPR8452, Abcam, 1:1,000) was used for the XPO5 staining, and anti-β-actin antibody (A5441, Sigma-Aldrich, 1:5,000) and anti-vimentin antibody (H-84, Santa Cruz Biotechnology) was used as the protein-loading control.

NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher Scientific) was used for efficient cell lysis and extraction of cytoplasmic and nuclear protein fractions following the manufacturer's protocol. The localization of XPO5 was analyzed using Western blotting.

An MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay (Sigma-Aldrich) was performed to measure the growth proliferative effects of siXPO5 and siControl, using the methods as described previously (22).

A total of 500 cells transfected with siXPO5 or siControl were seeded into 6-well plates and cultured for 10 days in a humidified CO₂ incubator at 37°C. The numbers of colonies comprising of >50 cells were counted using GeneTools image analysis software (Syngene) after staining of colonies using crystal violet.

The invasiveness of cancer cells was evaluated using BioCoat Matrigel Invasion Chambers (Corning Life Sciences) as described previously (22). A total of 5 × 10⁵ cells transfected with siXPO5 or siControl were seeded into the invasion chambers in serum-free medium. The number of cells that invaded the underside of the membrane was measured.

The DNA content of siXPO5- and siControl-transfected colorectal cancer cells was evaluated using the Muse Cell Cycle Assay Kit and Muse Cell Analyzer (Millipore) according to the manufacturer's instructions. The proportions of G₀–G₁, S, and G₂–M cells were calculated.

Twelve male athymic nude mice were obtained from Harlan Laboratories at 5 weeks of age and kept under controlled conditions (12-hour light/dark cycles). The animal protocol was approved by the Institutional Animal Care and Use Committee of the Baylor Research Institute (Dallas, TX). Xenograft tumors were generated using SW480 cells with XPO5 siRNA or its controls, in which 3 × 10⁶ cells were subcutaneously injected in left and right flanks of the mice. The mice were monitored for 12 days following injection, the size of subcutaneous tumors were measured every 2 days by calipers. At 12 days postinjection, all animals were sacrificed. The expression of XPO5 and target miRNAs in xenograft tissues was confirmed by qRT-PCR.

Results are expressed as means ± SEs. JMP software (ver. 10.0, SAS Institute Inc.) was used to perform the statistical analyses. The Wilcoxon rank sum test was used to compare continuous variables, and Fisher exact test was used to analyze categorical variables. Wilcoxon signed rank test was used to compare matched continuous variables. Steel test was used for the multiple comparison of XPO5 expression level between normal and each stage. Overall survival (OS) was measured from the date of operation to the date of cancer-related death. Disease-free survival (DFS) was measured from the operation date to the date of recurrence or cancer-related death. The Kaplan–Meier method with Wilcoxon test was used to estimate distributions of OS and DFS in each patient group through univariate analyses. To determine the cut-off value for XPO5 expression, we first established ROC curves to discriminate patients with or without death. Youden index was then used to determine the optimal cut-off threshold for XPO5 expression from each cohort to predict the OS or DFS (22–25). Cox proportional hazards models were used to calculate HRs with corresponding 95% confidence intervals for each group in a multivariate analysis. All calculated P values are two-sided, and P < 0.05 was considered to indicate statistical significance. All data were represented as mean ± SEM.

In the discovery cohort, we evaluated the expression level of XPO5 in multiple gene expression databases from colorectal cancer patients (Fig. 1A). We observed that among the top 1% overexpressed genes, two genes, XPO5 and TKT, were among the most significantly upregulated (P < 0.0001; Fig. 1B and Supplementary Fig. S1A–S1C) in all three databases (15–17). As a previous study reported TKT as a potential oncogene (26, 27), we focused our attention on XPO5, considering its role in miRNA biogenesis and cancer. We discovered that XPO5 was not only overexpressed in colorectal cancers, but was significantly upregulated in colorectal adenomas (P < 0.0001; Supplementary Fig. S1D; ref. 18), highlighting its potential oncogenic role during the early steps of the normal–adenoma–carcinoma sequence.

To confirm the overexpression of XPO5 in colorectal cancer tissues, we analyzed a cohort of colorectal cancer patients enrolled in TCGA as our first validation cohort and assigned as the “TCGA validation cohort.” As expected, XPO5 expression levels were significantly higher in cancer tissues compared with matched adjacent normal mucosal tissues (P < 0.0001; Fig. 1C). Furthermore, XPO5 expression was significantly higher in cancer tissues at all stages (I through IV) relative to normal mucosa (P < 0.0001; Fig. 1D). In particular, XPO5 was significantly upregulated in stage III–IV cancers compared with stage I–II cancer (P = 0.0395; Supplementary Fig. S2A). In addition, the XPO5 expression levels were higher among patients with distant metastases than in those without (P = 0.0388; Supplementary Fig. S2B). These data support the potential involvement of XPO5 during the advanced stages of colorectal cancer progression. Next, we used an independent cohort, composed of 246 matched tissue pairs of cancer and adjacent normal mucosa (clinical validation cohort; Supplementary Table S1) to validate data obtained from the TCGA cohort by qRT-PCR. Consistent with our results from the discovery cohort and the TCGA validation cohort, we observed that XPO5 expression levels were significantly higher in neoplastic lesions compared with matched normal mucosa (P < 0.001; Fig. 1E). The expression levels of XPO5 at each cancer stage were higher than the normal mucosa (normal vs. stage I: P = 0.0026, normal vs. stage II, III, and IV: P < 0.0001; Fig. 1F). The XPO5 expression levels were significantly higher in T3 or T4 stages relative to T1 or T2 colorectal cancers (P = 0.0483; Supplementary Fig. S2C) and were significantly higher in venous invasion-positive compared with invasion-negative colorectal cancers (P = 0.0357; Supplementary Fig. S2D). We evaluated the relationship between XPO5 expression and colorectal cancer patient survival in the TCGA validation cohort. The high-XPO5 expression group yielded worse DFS (P = 0.0774; Fig. 2A) and OS (P = 0.0165; Fig. 2B) compared with the low-XPO5 group. Multivariate analysis demonstrated that high XPO5 expression was an independent prognostic factor in patients with colorectal cancer (Table 1). Likewise, the high-XPO5 group had a significantly worse DFS (P = 0.0426; Fig. 2C) and OS (P = 0.0461; Fig. 2D) compared with the low-XPO5 group in the clinical validation cohort. Cox proportional hazards analysis revealed that the tumors with high XPO5 expression was an independent prognostic factor in patients with colorectal cancer (P = 0.0152; Supplementary Table S2). Taken together, our data consistently demonstrate that the primary tumors with high XPO5 expression are associated with advanced pathologic staging and correlated with poor prognosis, suggesting that XPO5 has a functional role in colorectal cancer progression.

To further confirm the significance of our gene expression data, XPO5 expression was examined using immunohistochemical staining. We observed that XPO5 protein expression was primarily confined within the nuclear compartment, and its staining intensity was significantly higher compared with adjacent normal mucosa (staining score by experts; P = 0.038; Fig. 2E). This result confirms that alterations in XPO5 protein are manifested at the protein level, consistent with a putative oncogenic role in colorectal cancer.

Next, we analyzed the relationship between microsatellite instability (MSI) status and XPO5 expression in the TCGA validation cohort. Only 13.9% of all colorectal cancers were MSI, and the remaining 86.1% were MSS (Supplementary Fig. S3A). XPO5 expression was significantly lower in MSI versus MSS colorectal cancers (P = 0.0020; Supplementary Fig. S3B). In MSS colorectal cancers, XPO5 was upregulated in a stage-dependent manner (stage I/II vs. III/IV; P = 0.0118, Supplementary Fig. S3C), while no significant differences were observed in MSI-positive patients (Supplementary Fig. S3D). Patients with high XPO5 showed worse OS only in the MSS group (P = 0.0219; Supplementary Fig. S3E), highlighting the important role of XPO5 in MSS colorectal cancers.

We first determined the expression of XPO5 in several colorectal cancer cell lines, including Caco-2, LOVO, HCT116, SW480, SW620, and HT29, to clarify the role of XPO5 in cancer cell lines. As the relative expression levels of XPO5 did not differ significantly between cell lines (data not shown), we decided to use additional criteria for selecting cell lines. Because one of the aims of our study was also to clarify the significance of XPO5 expression in MSS colorectal cancer, we selected SW480 (MSS, KRAS G12V, BRAF wild type) and Caco2 (MSS, KRAS wild type, BRAF wild type) cells for further experiments.

To evaluate the functional consequences of altered XPO5 expression and to determine whether XPO5 modulates the biological characteristics of colorectal cancer cells, we performed siRNA transfection to knock down XPO5 expression in SW480 and Caco-2 cell lines. XPO5 was primarily detected in the nuclear compartment in SW480 and Caco-2 cells by immunofluorescence staining analysis (Fig. 3A). These results were further confirmed by performing Western immunoblotting using nuclear and cytoplasmic fractions (Supplementary Fig. S4). Both cell lines revealed effective inhibition in XPO5 mRNA expression posttransfection, with siXPO5-transfected cells displaying <30% expression versus siControl-transfected cells. These results were further validated at the protein level using Western blotting, where both colorectal cancer cell lines showed significant suppression of XPO5 protein expression following XPO5 knockdown (Fig. 3B).

To determine whether downregulation of XPO5 results in suppression of cell proliferation in human cancer cell lines, we analyzed cell proliferation rates by the MTT assay. Although the degree of proliferation inhibition varied between both cell lines, siXPO5-transfected cells showed a significant inhibition of cellular proliferation compared with siControl-transfected cells (P < 0.0001 SW480; P = 0.0489 Caco2; Fig. 3C). We performed colony formation assays to evaluate the effectiveness of siXPO5 and found that both SW480 and Caco-2 cells transfected with siXPO5 produced fewer colonies compared with siControl-transfected cells, and this effect was more pronounced in SW480 cells (P = 0.0039; Fig. 3D).

As a previous report suggested an important function of XPO5 in cell-cycle regulation (13), we investigated whether the inhibition of XPO5 alters cell-cycle dynamics in colorectal cancer cell lines. As expected, flow cytometry–based cell-cycle analyses revealed significant increases in the G₀–G₁ fraction after siXPO5 knockdown in both SW480 (P = 0.0090) and Caco-2 (P = 0.0090) cells (Fig. 3E). These data indicate that inhibition of XPO5 induces G₁–S cell-cycle arrest, which further supports our observations of reduced proliferation rates in colorectal cancer cells.

Our clinical data indicated that XPO5 overexpression significantly correlated with invasive and metastatic potential. Therefore, we tested the hypothesis that XPO5 knockdown would inhibit the invasive capacity of colorectal cancer cells using in vitro transwell invasion assays. The siXPO5 transfection resulted in significantly decreased invasiveness relative to siControl-transfected cells in both SW480 (P = 0.0088) and Caco-2 (P = 0.0088) colorectal cancer cells (Fig. 3F), supporting our observations in clinical specimens from colorectal cancer patients.

Many oncogenic miRNAs (oncomiRs) are overexpressed in several human cancers, including colorectal cancer. One of the critical functional roles of XPO5 is the regulation of miRNA biogenesis, and we hypothesized that XPO5 inhibition might result in suppressing the expression of critical oncomiRs in colorectal cancer. At first, we selected four overexpressed oncomiRs (miR-21, miR-10b, miR-92a, and miR-182) belonging to a panel of the top 20 most overexpressed miRNAs based upon more than 80% of read counts as reported in a next-generation sequencing database of colorectal cancers (28). In addition, two more oncomiRs, miR-27a and miR-155, which have been shown to play a key role in colorectal cancer, were also selected as the candidates for analysis (Fig. 4A). In support of our hypothesis, we observed that the expression of most oncomiRs was significantly downregulated in siXPO5 versus siControl-transfected SW480 and Caco-2 colorectal cancer cells (P < 0.05; Fig. 4B). On the other hand, pri-miR-21 was significantly overexpressed in siXPO5 SW480 cells (P < 0.05) compared with that of siControls. There were no significant differences in the expression levels of pri-miR-10b, pri-miR-23a∼27a∼24-2 cluster, and pri-miR-182 between siXPO5 cells and siControls. In Caco-2 cells, pri-miR-21 and pri-miR-182 were significantly overexpressed in siXPO5 cells (P < 0.05) compared with that of siControls. However, there were no differences in the expression levels of pri-miR-10b and pri-miR-23a∼27a∼24-2 cluster between Caco-2 siXPO5 cells and siControl cells (Supplementary Fig. S5). These results clearly highlight that the downregulation of miRNA by siXPO5 is not due to transcriptional suppression, but most likely because of the posttranscriptional interference of pre-miRNA transportation. Hence, oncogenic potential of XPO5 may be, at least in part, mediated through its ability to regulate the expression of important oncomiRs in colorectal cancer.

To confirm our in vitro findings, we generated xenograft tumors using SW480 cells transfected with either siXPO5 or siControl. Twelve days following the initial injection, the tumor volume and weight were significantly lower in animals that were implanted with siXPO5-transfected cells compared with siControl-transfected cells (P = 0.0002 volume; P = 0.0004 weight; Fig. 5A). To further confirm that the reduced tumor burden was as a result of lower XPO5 levels, we determined the XOP5 expression levels and found that they were significantly lower in siXPO5 tumors at the end of the experimental duration. The oncomiRs, including miR-21 and miR-182, were also downregulated in tumors (P < 0.05; Fig. 5B). Taken together, these results highlight the oncogenic role of XPO5 in colorectal cancer.

miRNAs are a class of small noncoding RNAs (18–25 nucleotides) that posttranscriptionally regulate gene expression through RNAi (29). Accumulating evidence indicates that dysregulation of miRNAs is associated with metastatic processes in colorectal cancer (25, 30). XPO5 mediates the transport of pre-miRNAs from the nucleus to the cytoplasm and the final processing into mature miRNA by Dicer (29, 31). Accordingly, XPO5 functions as a key regulator of miRNA biogenesis and facilitates the export of miRNAs from the nucleus to cytoplasm (29, 32–36). Despite its critical and well-recognized function, not much is known about its biological role or clinical significance in colorectal cancer. To fill this important gap in knowledge, the current study for the first time systematically interrogated the role of XPO5 in colorectal cancer by evaluating its expression in multiple independent cohorts of patients with colorectal cancer, followed by extensive analysis for its clinical significance in this cancer. Thereafter, we performed a series of functional validation studies in colorectal cancer cell lines and xenograft animal model and identified and validated that XPO5 acts like an oncogene in colorectal cancer.

In this report, we have made several important and previously unrecognized discoveries regarding the role of XPO5 in colorectal cancer. First, we observed that expression of XPO5 is frequently upregulated in colorectal cancer compared with normal mucosa by analyzing multiple independent clinical specimen cohorts of patients with colorectal cancer. Second, we identified that high expression of XPO5 correlated with advanced disease, increased invasion and metastasis, associated with poor OS and DFS in colorectal cancer patients, and is an independent prognostic factor. Third, we found that XPO5 protein expression is primarily confined within the nuclear compartment in the cells. Fourth, we demonstrated that dysregulated expression of XPO5 leads to altered expression of various oncomiRs in colorectal cancer. Fifth, we performed a series of functional studies to demonstrate that downregulation of XPO5 expression in colorectal cancer cells inhibited not only its invasive capacities, but also suppressed cell proliferation and induced cell-cycle arrest. Finally, we were able to validate our in vitro findings in a xenograft animal model, wherein we demonstrated that siXPO5 reduced the growth of tumors, further highlighting its oncogenic role in colorectal cancer.

In our original cohort of colorectal cancer patients, XPO5 expression was frequently upregulated, both at mRNA and protein levels, in tumors compared with adjacent normal mucosae. Furthermore, high XPO5 expression correlated with greater depth of tumor invasion and venous invasion, as well as worse DFS and OS. In a multivariate analysis, XPO5 expression remained as an independent prognostic factor for survival, highlighting an oncogenic role of XPO5 in colorectal cancer. In a recent study, mutated XPO5 was shown to have an oncogenic role in MSI-positive cell lines (37); however, as the majority of colorectal cancers are MSS, a systematic and comprehensive analysis for the clinical significance of XPO5 in colorectal cancer is warranted. To the best of our knowledge, this is the first study to systematically explore the role of XPO5 in colorectal cancer patients. Our results are consistent with previous data reported in breast (11) and urothelial cancers (10). From a functional standpoint, a prior study suggested that XPO5 plays a role during cell-cycle progression (13). Collectively, these data are consistent with our findings in colorectal cancer and support the oncogenic role of XPO5 in cancer.

We evaluated the relationship between XPO5 expression and colorectal cancer cells through in vitro and in vivo experimental models. The knockdown of XPO5 led to reduced invasiveness and suppression of proliferation in colorectal cancer cell lines, as well as suppressed tumor growth in the xenograft animal model. In line with a previous report (13), we observed G₁–S cell-cycle arrest following XPO5 inhibition in cancer cells, suggesting that XPO5 is a regulator of cell cycle in normal as well as cancer cells. Interestingly, many key oncomiRs were significantly suppressed by siXPO5, suggesting the importance of XPO5 in the maintenance of overexpressed oncomiRs in colorectal cancer. Even though knockdown of XPO5 induces inhibition of all miRNAs, most of the key tumor suppressor miRNAs, such as miR-34a (38) and miR-143/145 cluster (39), are known to be downregulated in cancers, indicating that further inhibition of these miRNAs is less relevant, and aberrantly overexpressed oncomiRs appear to be more important.

On the basis of these results, we conclude that XPO5 acts as an oncogene and may serve as a promising prognostic biomarker in colorectal cancer. Furthermore, as XPO5 expression is much higher in MSS compared with MSI colorectal cancers, overexpression of this oncogene is likely more critical in the background of MSS phenotype. Considering that XPO5 inhibition leads to reduced growth proliferation in cultured cancer cells and the xenograft model, this miRNA transporter may also be a potential therapeutic target in cancer. However, a previous study suggested that the underlying mechanisms for XPO5 in cancer may be more complex (37). There appears to be different affinity between XPO5 and each pre-miRNA, and further studies using immunoprecipitation and next-generation sequencing are needed to clearly establish the role of XPO5 in cancer.

One of the potential limitations with the invasion assays that we conducted was that we observed reduced proliferation in siXPO5 cells. Therefore, it is possible that the observed differences in proliferation may have affected the invasive potential in these cells. Nevertheless, even with the small differences in proliferation, siXPO5 cells had significantly reduced cellular invasiveness compared with the control cells.

In summary, this study provides a novel evidence for the oncogenic role of XPO5 in colorectal cancer. Our study highlighted the clinical and biological significance of XPO5 in colorectal cancer through a series of in vitro and in vivo experiments. In addition, analysis of multiple cohorts of clinical specimens demonstrated that high XPO5 expression correlated with worse clinicopathologic features and poor patient survival. Collectively, this is the first systematic and comprehensive demonstration for the biological and clinical significance of XPO5, and our data indicate that XPO5 may serve as a possible therapeutic target in colorectal cancer.

No potential conflicts of interest were disclosed.

Conception and design: K. Shigeyasu, S. Toden, A. Goel

Development of methodology: K. Shigeyasu, A. Goel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Shigeyasu, S. Toden, A. Goel

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Shigeyasu

Writing, review, and/or revision of the manuscript: K. Shigeyasu, S. Toden, C.R. Boland, A. Goel

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Okugawa, A. Goel

Study supervision: Y. Okugawa, A. Goel

The current work was supported by grants R01 CA72851, CA18172, CA184792, and U01 CA187956 from the NCI/NIH funds from the Baylor Research Institute, and a pilot grant from Charles A. Sammons Cancer Center. This work was also supported by a grant from the Uehara Memorial Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.