Expression and Cytoplasmic Localization of SAM68 Is a Significant and Independent Prognostic Marker for Renal Cell Carcinoma Free

Renal cell carcinoma is the most lethal of the common urologic cancers, accounting for about 3% of all adult malignancies. More than 40% of renal cell carcinoma patients have died of the cancer, whereas the mortality rates associated with other urologic cancers, such as prostate cancer and bladder cancer, have been about 20% for last two decades (1, 2). It is estimated that the incidence of renal cell carcinoma in the United States has increased by an average of 3% per year for whites and 4% per year for African Americans since the 1970s, with an incidence of 36,160 new cases and 12,660 deaths in 2005 (3, 4). Approximately 30% of renal cell carcinoma patients are diagnosed with late-stage disease (stage III-IV), and even with radical nephrectomy, or combined with immunotherapy and targeted therapy as adjunctive therapy, the prognosis has not been significantly improved (5). Therefore, finding specific biomarkers for the diagnosis and prognosis of renal cell carcinoma, as well as novel therapeutic targets, is a priority.

SAM68 (Src-associated in mitosis, 68 kDa), originally identified as a Src-associated substrate in mitosis, belongs to the STAR (signal transduction and activation of RNA) family of RNA-binding proteins, characterized by a hnRNP K homology domain (KH domain) that locates within a larger GSG (GRP33-SAM68-GLD1) domain that is required for specificity and high-affinity binding to RNA (6-10). SAM68 regulates the RNA metabolism by modulating the nuclear export and cytoplasmic function of RNA, either binds to the poly(U) and poly(A) of RNA nonspecifically, or binds to UAAA or UUUA as determined by SELEX (systematic evolution of ligands by exponential enrichment) specifically (10). Posttranslational modifications affect the ability of SAM68 to regulate RNA metabolism. For instance, phosphorylation of SAM68 by Src family kinase or breast tumor kinase reduces its RNA-binding activity (11, 12), whereas acetylation of SAM68 by histone acetyltransferases enhances its RNA-binding activity (13). Methylation of SAM68 by methyltransferases is essential for RNA export and gene expression (14), and sumoylation of SAM68 represses cyclin D1 expression (13).

Previous studies have documented SAM68 participating in the regulation of cell cycle, cell proliferation, and apoptosis. It has been shown that deactivating SAM68 by random homozygous knockout in NIH3T3 cells could promote cells' anchorage-independent growth, reduce contact inhibition, and increase the metastatic ability of cells in nude mice (15), whereas conditional overexpression of SAM68 in fibroblasts could induce G₁ growth arrest and apoptosis by down-regulating cyclin D1 and cyclin E, suggesting that SAM68 could be a tumor suppressor (15, 16). On the other hand, knockout SAM68 in DT40 cells markedly decreased cell growth and prolonged the G₂-M phase (17), and ectopic expression of the dominant-negative form of SAM68 inhibited serum-induced DNA synthesis and cyclin D1 expression, which suggested that SAM68 might be a pro-oncogene (18).

In the current study, we report for the first time the characterization of SAM68 expression and localization in human renal cell carcinoma tissues, and its correlation with clinicopathologic grading of disease. The expression and localization of SAM68 could be an independent biomarker for predicting the prognosis of patients with renal cell carcinoma, making SAM68 a clinically valuable target for renal cell carcinoma therapy.

Normal renal tubular epithelial cells were established from tissue obtained during a nephrectomy for a benign lesion and cultured as described previously (19). The renal cell carcinoma cell lines 769-P, SN12C, SKRC-39, and ACHN were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (HyClone).

This study was conducted on a total of 241 paraffin-embedded renal cell carcinoma samples that were histopathologically and clinically diagnosed at the Sun Yat-sen University Cancer Center from 1999 to 2007. Prior patients' consent and approval from the Institutional Research Ethics Committee were obtained for the use of these clinical materials for research purposes. Clinical information on the samples is summarized in Supplementary Table S1. Tumor-node-metastasis (TNM) staging was determined according to the 2002 American Joint Committee on Cancer TNM classification of renal tumors (20). Tumor histology was classified according to the 2004 WHO classification (21), and tumors were graded according to the Fuhrman grading scheme (22). Nine pairs of renal cell carcinoma tissues and the matched tumor-adjacent morphologically normal renal tissues were frozen and stored in liquid nitrogen until further use.

Total RNA from cell lines and primary tumor tissues was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The extracted RNA was pretreated with RNAase-free DNase, and 2 μg RNA from each sample were used for cDNA synthesis primed with random hexamers. For PCR-mediated amplification of SAM68 cDNA, an initial amplification using SAM68 specific primers was done with denaturation at 95°C for 10 min, followed by 30 denaturation cycles at 95°C for 60 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 30 s. Upon completion of the cycling steps, a final extension at 72°C for 5 min was carried out before the reaction mixture was stored at 4°C. Real-time PCR was employed to determine the fold increase of SAM68 mRNA in each of the primary renal cell carcinoma tumors relative to the paired normal renal tissue taken from the same patient. Expression data were normalized to the geometric mean of the housekeeping gene glyceraldehydes 3-phosphate dehydrogenase (GAPDH). Reverse transcription-PCR (RT-PCR) and real time PCR primers were designed using Primer Express Software v. 2.0 (Applied Biosystems). Primer sequences are shown in Supplementary Table S2.

Western blotting was done according to standard methods as described previously (23), using anti-SAM68 rabbit polyclonal antibody (1:500; Santa Cruz). Anti-α-tubulin antibody (1:2,000 dilution; Sigma) was used as the loading control.

Immunohistochemical analysis was done to study altered protein expression in 10 normal human kidney tissues and 241 human renal cell carcinoma tissues. The immunohistochemistry procedure and the scores of SAM68 expression were done using previously described methods (23). In brief, paraffin-embedded specimens were cut into 4-μm sections and baked at 60°C for 2 h. The sections were deparaffinized with xylene and rehydrated, then submerged into EDTA antigenic retrieval buffer and microwaved for antigenic retrieval. After this, the sections were treated with 3% hydrogen peroxide in methanol to quench endogenous peroxidase activity, followed by incubation with 1% bovine serum albumin to block nonspecific binding. Sections were incubated with rabbit anti-SAM68 (1:200; Abgent) overnight at 4°C. Normal goat serum was used as a negative control. After washing, tissue sections were treated with biotinylated anti-rabbit secondary antibody (Zymed), followed by further incubation with streptavidin-horseradish peroxidase complex (Zymed). Tissue sections were then immersed in 3.3′-diaminobenzidine and counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted.

The degree of immunostaining of formalin-fixed, paraffin-embedded sections was reviewed and scored independently by two independent observers based on the proportion of positively stained tumor cells and intensity of staining (23-25). Tumor cell proportion was scored as follows: 0, no positive tumor cells; 1, <10% positive tumor cells; 2, 10% to 35% positive tumor cells; 3, 35% to 70% positive tumor cells; and 4, >70% positive tumor cells. Staining intensity was graded according to the method of mean optical density (26-28): 0, no staining; 1, weak staining (light yellow); 2, moderate staining (yellow brown); and 3, strong staining (brown). Staining index was calculated as the product of staining intensity score and the proportion of positive tumor cells. Using this method of assessment, we evaluated SAM68 expression in benign kidney tissue and malignant lesions by determining the staining index, with scores of 0, 1, 2, 3, 4, 6, 8, 9, and 12. SAM68 cutoff values were chosen on the basis of a measure of heterogeneity with the log-rank test statistical analysis with respect to overall survival. An optimal cutoff value was identified: a staining index score >6 was considered high SAM68 expression, whereas a staining index score <4 was considered low SAM68 expression.

All statistical analyses were carried out using the SPSS v. 13.0 statistical software packages. The relationship between SAM68 expression and clinicopathologic characteristics was analyzed by the χ² test. Bivariate correlations between study variables were calculated by Spearman's rank correlation coefficients. Survival curves were plotted by the Kaplan-Meier method and compared using the log-rank test. Survival data were evaluated using univariate and multivariate Cox regression analyses. In all cases P < 0.05 was considered statistically significant.

As shown in Fig. 1A, SAM68 protein expression was markedly higher in all cancer cell lines than in the normal human renal tubular epithelial cells. RT-PCR and real-time RT-PCR analysis revealed that SAM68 transcription was significantly up-regulated, by up to 27.5-fold, in renal cell carcinoma cell lines compared with normal renal tubular epithelial cells (Fig. 1B).

To examine further whether SAM68 up-regulation in renal cell carcinoma cell lines correlates with clinical progression, the expression of SAM68 was determined in nine pairs of renal cell carcinoma and adjacent normal renal tissues obtained from the same patient, using RT-PCR and Western blotting analyses. As shown in Fig. 2, SAM68 was overexpressed differentially in all nine human primary renal cell carcinoma samples at both the mRNA and protein levels. Real-time RT-PCR analysis revealed that the tumor/normal (T/N) ratio of SAM68 mRNA expression was as high as 9.2-fold in one case of paired primary renal cell carcinoma tissues (Fig. 2A). Importantly, all nine tumors displayed a >2-fold increase in SAM68 protein expression compared with tissues adjacent to the tumors (Fig. 2B), which was confirmed by immunohistochemical analysis (Fig. 2C). These results showed that SAM68 is up-regulated, at both the mRNA and protein levels, in renal cell carcinoma cell lines and clinically obtained renal cell carcinoma tissues.

To further investigate whether SAM68 protein overexpression is associated with clinicopathologic characteristics of renal cell carcinoma, 10 normal human paraffin-embedded, archived kidney tissues, and 241 paraffin-embedded, archived renal cell carcinoma tissues, including 119 cases of stage I, 45 cases of stage II, 45 cases of stage III, and 32 cases of stage IV tumors, were subjected to immunohistochemical staining with a human SAM68 antibody. Immunohistochemical results are summarized in Table 1. SAM68 protein was detected in 229 of the 241 (95%) human renal cell carcinoma cases. SAM68 was undetectable or only slightly detectable in normal kidney tissue and in the adjacent noncancerous tissues sections (Fig. 3A and B). In contrast, SAM68 was found to be overexpressed in renal cell carcinoma tissues (Fig. 3C-F). Taken together, these observations suggest that high levels of SAM68 expression are a common feature of primary renal cell carcinoma.

Statistical analyses were conducted to inspect the association between SAM68 expression, as detected by immunohistochemical staining, and the clinicopathologic characteristics of renal cell carcinoma. As shown in Table 1, there are no correlations between age and gender and the expression level of SAM68 protein. In contrast, the expression level of SAM68 is strongly associated with pathologic stage (P < 0.001), T classification (P = 0.003), N classification (P = 0.001), M classification (P = 0.006), histologic classification (P = 0.005), and Fuhrman grade (P < 0.001). Spearman correlation analysis was further applied to confirm the correlation between SAM68 expression level and clinicopathologic features. As shown in Supplementary Table S3, Spearman correlations of SAM68 expression levels to pathologic stage, T classification, regional lymph node metastasis, adjacent metastasis, histological classification, and Fuhrman grade were 0.268 (P < 0.001), 0.232 (P < 0.001), 0.227 (P < 0.001), 0.180 (P = 0.005), 0.192 (P = 0.003), and 0.307 (P < 0.001), respectively. Taken as a whole, SAM68 protein expression level positively correlated with pathologic stage, TNM classifications, histologic classification, and Fuhrman grade.

The SAM68 protein expression level in renal cell carcinoma significantly linked to patients' overall survival time (P = 0.013), with a correlation coefficient of -0.159, signifying that higher level of SAM68 expression was correlated with shorter survival time. Moreover, survival analysis was done to examine the effects of classic clinicopathologic characteristics (including age, gender, pathologic stage, T classification, N classification, distant metastasis, histologic classification, and Fuhrman grade) and SAM68 protein expression on survival, using Kaplan-Meier analysis and the log-rank test. As shown in Fig. 4A, the survival time was significantly different between the patients with low and high SAM68 expression (P < 0.001), with the low SAM68 expression group having a longer survival time and the high SAM68 expression group having a shorter survival time. The low SAM68 expression group had a cumulative 5-year survival rate of 96.1% (95% confidence interval, 0.9251-0.9969), whereas the high SAM68 expression group only had 63.8% (95% confidence interval, 0.5551-0.7209). Furthermore, prognostic value of SAM68 expression was determined in two subgroups of the 241 patients, divided by pathologic stage. Patients with tumors exhibiting low SAM68 expression had significantly longer overall survival than those with high expression of SAM68 either in the stage I plus II subgroup (n = 164; log-rank, P = 0.001; Fig. 4B, left) and in the stage III plus IV subgroup (n = 77; log-rank, P = 0.029; Fig. 4B, right), indicating that SAM68 could be a valuable prognostic marker for renal cell carcinoma patients at all disease stages. Moreover, statistical analysis revealed that SAM68 overexpression was associated with poor prognosis of patients with clear cell renal cell carcinoma and papillary renal cell carcinoma, which were sub-grouped according to histological classification (Fig. 4C, left and right).

Notably, 144 cases out of 241 renal cell carcinoma specimens showed SAM68 nuclear staining in the present study (Fig. 3C and D). However, we also observed 85 cases with cytoplasm-localization of SAM68 (Fig. 3E and F). Statistical analyses revealed that cytoplasm localization of SAM68 was significantly associated with pathologic stage (P = 0.003), T classification (P = 0.020), N classification (P = 0.023), M classification (P = 0.035), histologic classification (P < 0.001), and Fuhrman grade (P < 0.001), which were further confirmed by Spearman correlation analysis (Supplementary Table S3). Moreover, the effects of clinicopathologic characteristics of renal cell carcinoma, in conjunction with SAM68 protein localization, on patient survival, were evaluated with Kaplan-Meier analysis and the log-rank test. As shown in Fig. 5A, patients with cytoplasmic SAM68 had a lower cumulative 5-year survival rate than those with SAM68 expressed in nucleus (54.4% versus 90.1%, P < 0.001). Moreover, statistical analysis revealed a significant difference between curves of SAM68 nuclear-expressing and cytoplasma-expressing patients grouped according to pathologic classification: I-II (n = 152; log-rank, P = 0.026; Fig. 5B, left) and III-IV (n = 77; log-rank, P < 0.001; Fig. 5B, right), and that SAM68 overexpression was associated with poor prognosis of patients with clear cell renal cell carcinoma and papillary renal cell carcinoma, who were sub-grouped according to histologic classification (Fig. 5C, left and right). Taken together, these results suggest that cytoplasmic localization of SAM68 might be a potential marker for the prognosis of renal cell carcinoma patients.

In addition, univariate and multivariate analyses were further done to determine whether SAM68 expression level and cytoplasmic localization are independent prognostic factors of patient outcomes. As Supplementary Table S4 shows, the pathologic stage, M classification, SAM68 expression level, and SAM68 cytoplasmic localization were each recognized as independent prognostic factors. Taken together, our results suggest SAM68 might be a valuable marker for renal cell carcinoma patient prognosis.

The key finding from this study is that we reported, for the first time, the clinical significance of SAM68 in renal cell carcinoma. This is also the first study aimed at evaluating the possibility of using SAM68 as a clinically potential indicator for disease progression, as well as a prognostic marker for patient survival in cancer. We found that expression of SAM68 was up-regulated in renal cell carcinoma cell lines and renal cell carcinoma tissue, at both the mRNA and protein levels. Furthermore, we showed that the expression level of SAM68 protein significantly correlated with renal cell carcinoma clinical characteristics, including high pathologic stage, TNM classification, and Fuhrman grade, as well as prognosis. Moreover, patients with high SAM68 expression had a poorer prognosis than those with low SAM68 expression, making SAM68 a potential independent prognostic factor for renal cell carcinoma.

Recently, several molecular markers have been shown to be associated with the prognosis of renal cell carcinoma (29, 30). Bui et al. found that low CA IX staining was an independent prognostic indicator of poor survival in patients with metastatic renal cell carcinoma and nonmetastatic renal cell carcinoma (P < 0.001 and P = 0.085), using immunohistochemical analysis in 321 cases of renal cell carcinoma patients, which was further confirmed by SandLund's study (n = 288, P = 0.001; ref. 31). Furthermore, Gilbert et al. reported that CA IX expression in the peripheral blood was associated with recurrence in patients with renal cortical tumors (32). The tumor suppressor gene p53, frequently mutant in various types of cancer, has been found up-regulated in metastatic specimens of renal cell carcinoma and shown to be an independent prognostic marker for disease progression for clear cell renal cell carcinoma (n = 240, P < 0.0001; n = 240, P < 0.0001; ref. 33). Other studies further confirmed that increased staining for p53 was correlated with poorer survival in patients with clear cell renal cell carcinoma (34, 35). Moreover, Klatte and colleagues reported that CXCR3 was an independent prognostic factor in patients with localized clear cell renal cell carcinoma, and patients with low CXCR3 expression had a significantly worse prognosis than patients with high CXCR3 expression (n = 154, P = 0.009; ref. 36). In the current study, we found a significant correlation among SAM68 expression, cytoplasm localization of SAM68, pathologic stage, TNM classification, and Fuhrman grade. Renal cell carcinoma patients with higher SAM68 expression and cytoplasmic localization of SAM68 had shorter overall survival time than patients with lower SAM68 expression (P < 0.001 and P < 0.001). Multivariate analysis indicated that SAM68 protein overexpression and cytoplasmic localization of SAM68 were independent predictors for poor survival of renal cell carcinoma patients. Apparently, the correlations of SAM68 with the abovementioned molecular markers needed to be further investigated.

Previous studies have clearly documented that SAM68 was associated with various biological processes, such as signal transduction, RNA metabolism, cell cycle regulation, and apoptosis. However, the biological significance of SAM68 in cancer development and progression remains unclear. It has been reported that knockout of SAM68 in NIH3T3 fibroblasts could promote neoplastic transformation, and up-regulation of SAM68 could suppress cell cycle progression and induce apoptosis in both mammalian fibroblasts and Drosophila Schneider cells, which suggested that SAM68 has tumor suppressor property (15). However, mounting evidence has shown that SAM68 was associated with the pathogenesis of human cancer by which SAM68 could positively effect cancer cell proliferation and cell cycle progression. Busà R and colleagues have shown that SAM68, at both protein and mRNA levels, was up-regulated in prostate cancer, and down-regulation of SAM68 in prostate cancer cells could inhibit cell proliferation and sensitize cells to apoptosis induced by DNA-damaging agents (37). Recently, the evidence that the association of SAM68 with Vav1 contributed to tumorigenesis revealed the oncogenic potential of SAM68 (38). Moreover, disruption of SAM68 in MMTV-PyMT mice that could rapidly develop multifocal mammary adenocarcinomas with 100% penetrance and short latency, showed delayed onset of mammary tumoriformation and metastasis, suggesting that SAM68 was required for mammary tumorigenesis in vivo (39).

Our study represents the strong evidence that SAM68 overexpression might play a vital role in the development and progression of renal cell carcinoma. Up-regulation of SAM68 in renal cell carcinoma was confirmed from several aspects, including examination of SAM68 mRNA and protein expression in renal cell carcinoma cell lines in comparison with those in normal rental epithelial cells, comparative analysis of SAM68 expressions in paired renal cell carcinoma tissues and noncancerous renal tissues, and a clear showing of high expression level of SAM68 in a relatively large number of renal cell carcinoma specimens. The clinical significance of SAM68 overexpression in renal cell carcinoma is further revealed by its strong correlation with the advanced staging of the disease and poorer patient prognosis. These results not only suggest a potentially promising usefulness of SAM68 as a prognostic and survival indicator, but also warrant further studies on a possible link between the biological function of SAM68 and the pathogenesis of renal cell carcinoma, which might eventually lead to the development of a new anti–renal cell carcinoma strategy.

It is particularly noteworthy that SAM68 has been found, in our study, not only localized in the nucleus of cancer cells, but also in 35.3% of renal cell carcinoma lesions, in the cytoplasm. With two nonconventional nuclear localization signals in the COOH-terminal, SAM68 predominantly localizes in the nucleus through its RNA-binding domain, and interacts with Src kinases upon nuclear envelope breakdown during mitosis (6, 7). In the cell nucleus, SAM68 is concentrated in dots structures called SAM68 nuclear bodies, which contain nucleic acids visualized by electron microscopy (40). The subcellular localization of SAM68 could be regulated by posttranslational modifications, such as phosphorylation and methylation (11, 14, 41, 42). Activation of various receptor systems, such as leptin and insulin receptors, results in tyrosine phosphorylation of SAM68 and relocalization to the cytoplasm, where SAM68 interacts with signaling molecules including phospholipase C γ-1, STAT3, or p120 GTPase activating protein (43-45). Stimulation of oncogenic pathways, including the epidermal growth factor pathway in breast cancer and the RET pathway in thyroid carcinomas, also induces SAM68 phosphorylation, thereby inhibiting the RNA-binding capacity of SAM68 (31, 46). Hypomethylated SAM68 localizes to the cytoplasm, which might be a maturation process required for the proper localization and function of the protein (14). In the present study, our results provide strong evidence that SAM68 overexpression has a vital role in the development and progression of renal cell carcinoma. These results not only suggest a potentially promising use of SAM68 as a prognostic and survival indicator, but also warrant further studies on a possible link between the biological function of SAM68 and the pathogenesis of renal cell carcinoma, which might eventually lead to the development of a new anti–renal cell carcinoma strategy. In addition, the cytoplasmic localization of SAM68 correlated significantly with more malignant clinical features, including a lower 5-year survival rate. Therefore cytoplasmic SAM68 might play an important role in the development and progression of renal cell carcinoma.

In the current study, we reported for the first time that SAM68 expression was up-regulated in clinical renal cell carcinoma tissues, and high SAM68 expression levels were associated with more malignant clinical feature and poor patient prognosis. Our results suggest that SAM68 overexpression might be useful as a prognostic factor for renal cell carcinoma patients. Moreover, the cytoplasmic localization of SAM68 is shown to be an independent predictor for poor prognosis. Apparently, a further understanding of the molecular mechanism by which SAM68 is involved in cancer cell initiation, proliferation, and transformation in human renal cell carcinoma would help in the discovery of novel targeted agents, and may also lead to the development of new approaches for effective therapy.

No potential conflicts of interest were disclosed.

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