In the EBP50 stained specimens, the average staining intensities were highest in the specimens of HGPIN, and lowest in the specimens of PCa and Mets. HGPIN had significantly higher staining than Mets, NAC, and PCa. Despite the fact that HGPIN was significantly different than the BPH and NDP groups but not the NAC group, the means were relatively similar between BPH, NDP, and NAC, suggesting that the staining intensity does not vary greatly between these classifications.
Previous work has shown that radixin, an ERM protein and binding partner of EBP50 that is responsible for linking F-actin to plasma membrane proteins[5, 18], also demonstrated higher absolute staining in specimens of HGPIN than in other prostatic tissue types, including PCa and NAC . This finding may indicate that the higher expression of both proteins in HGPIN may reflect a unique feature of the pre-cancerous tissue physiology, and that both may be down regulated in specimens of prostatic adenocarcinoma.
Mets tissue had significantly lower staining than all other tissue types, including PCa, indicating that loss of EPB50 expression may play a role in select cases of prostate cancer metastasis. This is further echoed by the fact that only 1/36 cases of metastatic tissue showed high intensity range staining for EBP50. Given that EBP50 has been previously shown in murine fibroblast models to promote adherens junction stabilization through mediating the interaction of β-catenin with E-cadherin, its loss of expression is plausible with tumor dissemination.
Despite this, however, it is important to note that these findings may also contain a correlative component that is not prognostic in nature. While all of the tumors in this study were primary tumors at the time of specimen retrieval, definitive follow-up information on these patients was not available. In this sense, from this study it is not possible to rule out that the decreased EBP50 expression, at least in part, may be due to the metastatic location itself. The current results, however, indicate and warrant later phase biomarker studies that will longitudinally correlate EBP50 expression directly with patient outcomes to further evaluate its potential to predict metastatic risk in prostate cancer.
A significant increase in EBP50 staining between Stage 2 and 3 PCa specimens was also observed. It is possible that this finding represents a change in tumor physiology between the two stages, however, given that this trend was not noted in Stage 4 PCa specimens, it is also possible that this represents a spurious finding, and should be further evaluated before definitive conclusions are reached. No differences were seen between the Gleason score classifications.
In general, EBP50's expression is increased in polarized epithelial cells, such as the liver, kidney, pancreas, small intestine, and the prostate. Considering the diverse intracellular roles that have been proposed for EBP50, it may be difficult to elucidate a clear, singular mechanism whereby it may promote oncogenesis or tumor suppression within individual tissue types.
Differing hypotheses to explain to the behavior of EBP50 in cancer have been proposed, especially given the multiple studies that seem to support two opposing functions for EBP50 in cancer [9–11, 21]. Zheng, et al have suggested that in many cases of breast cancer where EBP50 is expressed, it may not be expressed in sufficient quantities to halt tumor progression. Others have demonstrated that the hypoxia associated with tumor necrosis can increase EBP50 (NHERF1), which increases Na+/H+ activity, in turn decreasing local pH and promoting tumor dissemination[4, 21]. And while it has been shown that EBP50 can cluster with EGFR, PDGFR, and the tumor suppressor NF2 to halt cell signaling and hence cancer progression[11, 22–24], others have posited that EBP50's PDZ domains may actually allow for new tumor-specific interactions.
These different findings, in part, have been reconciled with the hypothesis that EBP50 may have different functions by cellular location, with a tumor suppressor function associated with a membranous/apical distribution, and an oncogenic function promoted by a cytoplasmic location. In support of this hypothesis, previous work has demonstrated a progression from luminal to cytoplasmic EBP50 expression occurs across normal to ductal carcinoma in-situ to invasive and metastatic breast cancer tissues. While membranous EBP50 has been shown to stabilize β-catenin at cell membranes, non-stabilized β-catenin is also capable of forming growth-promoting transcription complexes in the nucleus and has been prominently associated with hepatocellular carcinoma. Hence, it is interesting that an overexpression of EBP50 with a focal nuclear localization has been documented in hepatocellular carcinoma. A similar phenomenon has been noted in colorectal cancer, where membranous EBP50 loss and increased cytoplasmic expression has been noted in the colorectal adenoma-to-carcinoma transition, with subsequent increases in cellular invasion and epithelial-to-mesenchymal transition, processes that demonstrated reversibility when EBP50 was reexpressed at the apical membrane of intestinal epithelium. Additionally, in normal astrocytes, EBP50 has demonstrated a membranous distribution, while it has demonstrated a cytoplasmic distribution in many cases of glioblastoma multiforme. This corresponds with the absence of the EBP50-binding tumor suppressor PTEN and the activation of the growth-promoting Akt pathway, which is traditionally silenced by PTEN through recruitment to the plasma membrane by EBP50.
Relating these examples specifically to prostate cancer, it has been demonstrated that β-catenin can interact with androgen receptor and increase its transcriptional activity, hence contributing to prostate cancer progression[27, 28]. Moreover, the tumor suppressor PTEN is frequently found mutated in prostate cancer, with subsequent PI3K/Akt signaling shown to promote cell survival[27, 29]. Interestingly, the PI3K/Akt pathway also increases the stability of β-catenin in prostate cancer. Based on these findings, it is possible to suggest that EBP50, under the appropriate circumstances, may possess a tumor suppressor function in prostate cancer similar to those described above in other cancer types.
While a cytoplasmic staining pattern for EBP50 was noted across most specimens examined in this study, a membranous/apical staining pattern was clearly more prominent than cytoplasmic staining in many cores, most commonly in the benign and pre-neoplastic specimens (Figure 2). While cores with clearly more prominent membranous staining were found in 73.3% of BPH cases studied (Figure 2B), this finding was only noted in 9.7% of cases of PCa and not in any specimens of Mets. Although an overlap between the expression patterns still existed between many of the benign and cancerous specimens, this trend concurs with the above hypothesis regarding strong membranous expression and tumor suppression and warrants further study to determine its potential to assess metastatic risk.
As a final note of interest, in one model of EBP50 function, its PDZ-2 domain has been shown to bind to the C-terminal ERM-binding domain, inhibiting the binding of other proteins to the PDZ domains, such as PTEN and B-catenin. In this same model, when ezrin, an ERM protein, binds the C-terminal domain, the PDZ domains are freed up for additional binding partners. In the intestinal epithelium of ezrin knock-out mice, EBP50 has been shown to be displaced to the cytoplasm[5, 32]. In light of this, it is an interesting finding that ezrin expression has been inversely correlated with tumor differentiation in prostate cancer, and that moesin, another ERM protein, showed higher incidences of lymph node metastases when it was associated with a cytoplasmic distribution as opposed to a membranous one in oral squamous cell carcinoma. Hence, EBP50's location and function may also be directly linked to the location and presence of the ERM proteins that ultimately enable PDZ domain interactions.