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Feline sarcoma-related protein (Fer) is a ubiquitously expressed non-receptor protein tyrosine kinase associated with proliferation in various cancer cells. However, no reports have described the pathological roles and prognostic value of Fer expression in renal cell carcinoma (RCC). We investigated Fer expression in three RCC cell lines (ACHN, Caki-1, and Caki-2) and in normal tubule cells (HK-2) by immunoblotting. Fer expression was highest in ACHN cells, with Caki-1 showing intermediate levels and Caki-2 showing low levels, and was undetectable in HK-2. RNA interference was therefore used to assess the effects of Fer knockdown in ACHN. Knockdown of Fer expression was found to inhibit RCC cell proliferation and colony formation. Immunohistochemical analysis of 131 human RCC tissues (110 conventional, 11 chromophobe, and 10 papillary) investigated relationships between Fer expression and clinicopathological features, including cancer cell proliferation, apoptosis, and prognostic value for survival. In human tissues, Fer expression was significantly higher in cancer cells than in normal tubules. In addition, expression levels correlated with cancer cell proliferation, but not with apoptosis. Multivariate analysis indicated associations of Fer expression with pT stage, tumor grade, and metastasis (P < 0.001). Fer expression was also prognostic for cause-specific survival according to multivariate analysis (hazard ratio, 3.89; 95% confidence interval, 1.02–14.84, P = 0.047). Fer expression correlates with RCC cell proliferation both in vitro and in vivo, and with tumor progression and survival. This represents useful information for discussing the pathological and clinical significance of Fer in RCC.
Protein-tyrosine kinases are important regulators of intracellular signal transduction pathways. Feline sarcoma (Fes) belongs to subgroup IV of the non-receptor protein tyrosine kinases. Fes-related protein (Fer) is a member of a distinct subgroup of Src homology 2-containing non-receptor tyrosine kinases.[1, 2] Fes is known to be expressed in hematopoietic cells, endothelial cells, and neuronal cells. Conversely, Fer is ubiquitously expressed, but its potential involvement in cancer remains enigmatic. Fer expression has been associated with proliferation of several cultured cancer cell lines and poor prognosis in prostate cancer has recently been correlated with increased Fer expression levels.[3-5] More generally, Fer is implicated in signaling downstream to a variety of receptor systems that regulate cell proliferation and invasion in various cell types.[3-8] For example, Fer can regulate both β-catenin, a key molecule in the cell proliferation and cell adhesion systems, and cortactin, which plays important roles in cell migration and invasion.[7, 8] These observations suggest potential roles for Fer in the regulation of proliferation as well as the metastatic potential of cancer cells.
Renal cell carcinoma (RCC) is a common urological malignancy with a high frequency of invasion and metastasis being apparent at the time of diagnosis. Actually, over 30% of patients show non-organ-confined tumor on diagnosis. Approximately one-third of patients with localized RCC will eventually develop metastasis despite various treatments.[9-11] Molecular targeting therapies have been used for patients with advanced RCC. These treatments represent a major breakthrough with well-established antitumoral effectiveness, because conventional therapies are generally ineffective. However, side-effects are occasionally observed and the effectiveness of such therapies is sometimes insufficient, because the pharmacotherapy must be abandoned. Various information regarding the pathological mechanisms and molecular basis of cancer cell progression thus needs to be determined to allow a proper discussion of new treatment strategies. We have previously reported the association between Fes expression and cancer cell proliferation and tumor growth in RCC cells, but no reports to date have described the expression of the related Fer in RCC. Because Fer has been implicated in many cellular functions associated with cancer, we reasoned that expression in RCC might provide predictive or prognostic value in RCC patients.
Here we describe in vitro and in vivo investigations of Fer expression in both RCC cell lines and human tissues. The results suggest that Fer expression might represent a valuable prognostic biomarker for RCC. Furthermore, the involvement of Fer in cell signaling processes linked to cancer cell progression also points to Fer as a potential therapeutic target for future treatments in RCC patients.
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This study indicated a highly significant correlation between Fer expression and tumor progression, grade, and survival in patients with RCC. In a previous report, we showed that Fes was closely associated with proliferation in RCC cell lines. In the present study, we also showed that Fer expression showed a positive correlation with cancer cell proliferation. Conversely, no such correlation was found between Fer expression and apoptosis. Fer expression levels have previously been associated with cell proliferation in cultured breast, colon, and prostate cancer cells,[3, 4] and elevated Gleason's scores have been correlated with Fer expression levels in human prostate cancer. Downregulation of Fer also correlated with decreased prostate cancer cell proliferation;[3, 4, 20] other researchers have reported no significant effects on apoptosis in prostate cancer (PC3) or breast cancer (MDA-MB-231) cell lines. These observations implicate Fer in tumor promotion at the level of proliferation and metastasis, rather than by affecting the survival of cancer cells.
One of the most important results of the present study is the potential role of Fer expression as a significant independent prognostic biomarker of survival in RCC patients, as shown by multivariate analysis. This is the first study to report the value of Fer expression in predicting cancer survival. In addition, Fer expression was positively associated with pT stage and metastasis. Regulation of cell proliferation has important clinical and pathological implications, although tumor progression and survival are affected by numerous other factors. The correlation between Fer expression and cancer cell proliferation, although statistically significant, was not overly strong, leading us to speculate that the roles of Fer in promoting invasion and metastasis may be more relevant to tumor progression than cancer cell proliferation. In support of this, immunohistochemical staining for Fer in cancer cells was strongest at the invasive front and within invading cancer cells. In addition, such findings were common in tumors with high pT stage compared to those with low pT stage. However, although Fer expression was significantly associated with postoperative incidence of metastasis in univariate analysis, this significance was not independent and significant in a multivariate analysis model including pathological features. Based on these results, further study of the pathological roles of Fer in cancer cell invasion and metastasis is necessary. Examination of the relationships between Fer and other invasion-related biomarkers was beyond the scope of this study, however, this issue should be pursued in future studies. Various adherens junction-, cell migration-, and invasion-related proteins, including p120 catenin, β-catenin, E-cadherin, PTP1B, and cortactin are regulated by Fer.[6-8, 21, 22] These proteins have also been associated with tumor progression in several cancers, including RCC,[22-26] suggesting that some may also be regulated by Fer and could play important roles in determining malignant aggressiveness in RCC.
Targeted disruption of fer in mice with a kinase-inactivating missense mutation correlated with enhanced inflammatory responses to endotoxin challenge,[27, 28] even though these mice were otherwise healthy and fertile. This suggests that systemic inhibition of Fer in a clinical setting using pharmacological inhibitors could be well tolerated. Cells from these Fer-deficient mice showed a number of defects, including reduced migration.[29, 30] Given these results, Fer inhibition might provide therapeutic benefits in RCC, and possibly in other malignancies.
Our in vitro study showed that Fer expression was highest in ACHN cells. In addition, although no significant difference was evident, Fer expression tended to be high in papillary RCC of human tissues. From these results, we speculated that Fer could play important roles in papillary RCC. Furthermore, given the results in RCC tissues, similar mechanisms may be present in other histological types. Unfortunately, survival analysis of Fer expression according to types of papillary RCC was not carried out in the present study because the number of patients was too low. It is an important question for further investigation.
In conclusion, we have shown an association between Fer expression and both cancer cell proliferation and tumor development in RCC. Expression of Fer in these patients was also related to malignant aggressiveness, including grade and metastasis. These results offer the first suggestion that Fer expression could be a potentially useful biomarker for prognosis in RCC. The results also suggest Fer as a possible therapeutic target as options develop for the treatment of patients with RCC in the future.