This is the first study to evaluate the status of GR, SgK1, and NDRG1 before and after NAC in ESCC. Notably, a high GR, Sgk1, and NDRG1 status in resected specimens was significantly associated with shorter OS of patients with ESCC undergoing NAC. A previous study reported no correlation of the GR status with the clinical outcomes of patients with ESCC [7]. In contrast, we comprehensively evaluated GR immunoreactivity by using the H-score. We also evaluated only NAC cases in which the GC-GR pathway could be more activated. This may explain the discrepancy between the two studies, although further investigations are required to confirm this.
Sgk1 has not been studied in ESCC. We found that the Sgk1 status in carcinoma cells was not only significantly associated with a shorter OS and DFS of patients, but also with more advanced pT, pN, and lymphatic vessel invasion. Sgk1 is well-known to activate beta-catenin/T cell factor signaling in human non-small cell lung cancer and is associated with tumor cell invasion and migration [38]. Therefore, Sgk1 may also enhance tumor cell invasion and migration in ESCC, as reported for other human malignancies such as esophagogastric junctional adenocarcinoma [17], colorectal cancer [18], and non-small cell lung carcinoma [38].
NDRG1 has been studied in ESCC by several investigators [31,32]. In our study, a high NDRG1 was associated with shorter OS and DFS, higher pT, and local progression factors such as venous invasion, which is consistent with the results of previous studies [32]. The Wnt pathway was also reported to be activated via NDRG1 and involved in epithelial-mesenchymal transition of ESCC tumor cells [32]. Our results indicate that NDRG1 was activated via Sgk1 in ESCC undergoing NAC and thus may be involved in the local progression of tumor cells.
We examined the correlation between GR, Sgk1, and NDRG1 status before and after NAC and clinicopathological factors of patients. Differences were found between post-NAC and pre-NAC. This discrepancy is considered as a limitations of the present study, as intratumoral heterogeneity was high and there was a small number of biopsy specimens available for examination.
In addition, a high GR status in resected specimens, but not biopsy specimens, was a poor prognostic factor. However, there were no significant changes in the GR score before and after chemotherapy. This may also be because of intratumoral heterogeneity; however, in patients with breast cancer, the profiles of steroid hormone receptors differed between pre- and post-chemotherapy [39]. In ESCC, similar changes in GR may occur.
Furthermore, GR, Sgk1, and NDRG1 were all significantly correlated with prognosis when evaluated by univariate analysis. However, results of multivariate analysis demonstrated that these factors were not independent predictors of patient outcomes. This discrepancy may be because GR, Sgk1, and NDRG1 were significantly correlated with each other in patients examined in this study. In addition, both Sgk1 and NDRG1 were significantly correlated with established clinicopathological factors such as pT and pN in patients.
In the present study, a significant positive correlation was detected among GR, Sgk1, and NDRG1 status in ESCC. Sgk1 is activated by growth factors, DNA damage, cell contraction, and oxidative stress, in addition to the GC-GR pathway [10-12]. NDRG1 is also activated by stress signals, oxidative balance [40], DNA damages, increased p53 expression [41], and hypoxia [42], in addition to the GC-GR pathway. Therefore, Sgk1 and NDRG1 in carcinoma cells of ESCC may be affected by various factors other than the GC-GR pathway, and further studies are required to clarify the GR-Sgk1-NDRG1 axis in ESCC.
In this study, we compared the GR, Sgk1, and NDRG1 status between ESCC before and after NAC to further clarify the significance of the GC-GR pathway in the therapeutic effects of NAC. A high NDRG1 status in carcinoma cells in pre-NAC biopsy specimens was significantly associated with lower NAC effects, and a high GR and Sgk1 status in carcinoma cells tended to be associated with lower NAC effect. Additionally, a lower NAC effect was significantly associated with the poor prognosis of patients with ESCC. Therefore, these results suggest that NAC sensitivity was decreased in tumors with high expression of GR, Sgk1, and NDRG1, resulting in higher residual tumor cells and subsequent adverse clinical outcome of patients. This indicates that the efficacy of NAC can be predicted by analyzing the GR-pathway.
One question in this study was whether a high NDRG1 status in biopsy specimens was significantly associated with decreased therapeutic effects of NAC in patients; no association was observed in resected specimens. This may be because of the relatively small number of biopsy specimens. However, carcinoma tissues were not available for examination among resected specimens of patients who completely responded to NAC.
In addition, the detailed molecular mechanisms underlying the GR-Sgk1-NDRG1 pathway-mediated resistance to NAC in patients with ESCC remains unknown. Cell cycle arrest via the GC-GR pathway contributes to chemoresistance in patients with cancer [43]. Factors including Sgk1 have been considered as the cause of decreased sensitivity of cytotoxic drug therapy by activating the GC-GR pathway in hepatocellular carcinoma and colon adenocarcinoma [44]. In addition, GR-mediated Sgk1 activation suppresses tumor cell apoptosis in breast carcinoma cells [45] and NDRG1 is induced by Sgk1 to suppress tumor cell apoptosis in ESCC tumor cells [46]. These results and our current results indicate that the GR-Sgk1-NDRG1 pathway in ESCC protects tumor cells from chemotherapy-induced apoptosis and mediates chemotherapy resistance.
We examined changes in various factors before and after NAC to explore the effects of NAC and synthetic steroids administered during NAC on GR, SgK1, and NDRG1 expression. Particularly, administration of synthetic steroids in NAC was also reported to induce chemotherapy resistance in patients with urological [47] and breast cancer [19]. Therefore, steroid administration may reduce treatment sensitivity. However, in this study, there were no significant differences in GR, Sgk1, and NDRG1 scores before and after NAC, although the Sgk1 score after NAC was significantly higher in patients with low NAC treatment effects than those without. Therefore, the therapeutic effects of chemotherapy may be reduced in the Sgk1 high group by stress stimulation or synthetic steroids administered during NAC administration. However, there were no significant associations between the changes in GR and NDRG1 scores before and after NAC and therapeutic effects. Therefore, further studies are required to clarify the effects of NAC and synthetic steroids administered during NAC on the GR, Sgk1, and NDRG1 status.