High Expression of LGR6 is A Poor Prognostic Factor In Esophageal Carcinoma

Background: Leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6) promotes carcinogenesis and progression in some cancer types. However, there are few reports of LGR6 expression in esophageal squamous cell carcinoma (ESCC). LGR6 expression and clinicopathological features in ESCC were investigated by RNAscope, a high-sensitivity RNA in situ hybridization method. Methods: Appropriate tumors were selected from 41 cases of ESCC from which tissue microarrays were generated, and LGR6 expression was identied by RNAscope. Results: Thirty-seven patients had LGR6 expression. High LGR6 expression was observed in 17 cases and low LGR6 expression in 24 cases. LGR6 expression was signicantly higher in high histological grade ESCC than in low histological grade ESCC (P=0.0023). ESCC patients who received neoadjuvant chemotherapy had signicantly higher LGR6 expression than those without neoadjuvant chemotherapy (P=0.0109). Furthermore, high LGR6 expression showed a poorer prognosis than low LGR6 expression (log-rank test, P=0.0365). Conclusions: LGR6 may be a prognostic factor and a potential new therapeutic target in ESCC.

Results: Thirty-seven patients had LGR6 expression. High LGR6 expression was observed in 17 cases and low LGR6 expression in 24 cases. LGR6 expression was signi cantly higher in high histological grade ESCC than in low histological grade ESCC (P=0.0023). ESCC patients who received neoadjuvant chemotherapy had signi cantly higher LGR6 expression than those without neoadjuvant chemotherapy (P=0.0109). Furthermore, high LGR6 expression showed a poorer prognosis than low LGR6 expression (log-rank test, P=0.0365).

Conclusions:
LGR6 may be a prognostic factor and a potential new therapeutic target in ESCC.
LGR6 is involved in tissue development, regeneration, and injury repair.
Esophageal squamous cell carcinoma (ESCC) has a poor prognosis [7], and improvements are required in treatment outcome. To improve the survival rate of patients, the establishment of new biomarkers and targets is required. In this study, we analyzed the clinicopathological features of LGR6 and examined the possibility of their application to the treatment of ESCC.

Patients and materials
We identi ed 56 ESCC cases who underwent surgical resection between 2010 and 2019 at Shinshu University Hospital, Matsumoto, Japan, and evaluated their clinicopathological features. Among these patients, cases with invasion deeper than the submucosa were selected. Stage IV cases were excluded as well as those that were negative for the positive control (housekeeping gene). A re-evaluation of the cases was undertaken by two pathologists (T.U. and M.I.), and 41 ESCC cases remained as candidates for analysis. This study was approved by the ethics committee of Shinshu University, Japan (approval no. 4088).

Histopathology
Para n blocks xed with 8% formaldehyde containing su cient tumor for analysis were prepared for hematoxylin and eosin (HE) staining and tissue microarray analysis by extracting a core with a diameter of 3 mm from each case.
LGR6 RNA in situ hybridization LGR6 mRNA detection was performed using the RNAscope kit (Advanced Cell Diagnostics, Hayward, CA, USA), as described previously [8]. Additionally, a four-step evaluation method was used, as we previously reported [8].
LGR6 mRNA expression was categorized as low expression (grades 0, 1+, and 2+) and high expression (3+ and 4+). We analyzed the relationship between LGR6 expression and the clinicopathological data and prognosis of patients with ESCC, particularly regarding overall survival (OS). We also evaluated LGR6 expression in non-cancerous tissues of the background mucosa of ESCC.

Statistical analysis
Fisher's exact test was applied to assess statistical signi cance. A P-value of < 0.05 was considered statistically signi cant. The OS rates of ESCC patients were calculated using the Kaplan-Meier method, and differences were compared using the log-rank test. A P-value of <0.05 was considered to be statistically signi cant. Statistical analysis was performed using JMP version 13 (SAS Institute Japan, Tokyo, Japan).

Results
LGR6 expression in normal esophageal mucosa and ESCC LGR6 expression was identi ed in the basal cell layer of non-tumorous regions. In ESCC patients, 37 of 39 cases showed LGR6 expression. Among them, 17 cases were identi ed as having high LGR6 expression ( Fig. 1A and 1B), while LGR6 expression was completely absent in two cases ( Fig. 1D and 1E).
LGR6 expression varied from diffuse to scattered patterns.

Relationship between LGR6 expression and clinicopathological characteristics
LGR6 expression and clinicopathological data are shown in Table 1. LGR6 expression was signi cantly higher in high histological grade ESCC than in low histological grade ESCC (P=0.0023). ESCC patients with neoadjuvant chemotherapy had signi cantly higher LGR6 expression than ESCC patients without neoadjuvant chemotherapy (P=0.0109). No signi cant differences were identi ed between high LGR6 expression and low LGR6 expression regarding age, sex, vascular invasion, or TNM stage. The prognostic value of LGR6 expression in ESCC was analyzed by the Kaplan-Meier method and logrank test (Fig. 2)

Discussion
This is the rst report of RNAscope analysis of LGR6 expression in the esophagus. The correlation of poor prognosis with high LGR6 expression is consistent with previous immunohistochemical reports [9].
LGR6 expression is associated with ESCC differentiation in immunostaining studies [9]. However, maintenance of epithelial tissue is promoted by resident stem cells [10]. Given that LGR6 is used as a stem cell marker in the epidermis [2], identi cation of the position of stem cells exhibiting LGR6 expression in the esophageal epithelium, which is the same strati ed squamous epithelium in the skin, may lead to applications such as regenerative medicine.
In a murine study, it was reported that LGR6 has the characteristics of cancer stem cells (CSCs) in squamous cell carcinoma of the skin [11].
LGR6 interacts with R-spondin 1-4 and is involved in the Wnt/βcatenin signaling pathway via phosphorylation, exerting various biological actions [9] [12,13] [14] [15]. The Wnt/β-catenin signaling pathway plays an important role in the pathogenesis of various human diseases and tumors [13] LGR6 has been reported to promote the initiation and progression of cancers such as lung adenocarcinoma [5] and ovarian cancer including serous carcinoma [6].
LGR6 may contribute to the initiation and progression of ESCC by activating the Wnt/β-catenin signaling pathway, but further research is needed to con rm this hypothesis.
High LGR6 expression in neoadjuvant chemotherapy cases indicated that LGR6-expressing cells may have CSC-like characteristics with chemoresistance. In fact, LGR6 expression has also been reported to contribute to stem cell and chemoresistance in ovarian cancers, including serous carcinoma [6]. The function of chemoresistance is also considered to be in uenced by the Wnt/β-catenin pathway.
Nonetheless, it has been reported that LGR6 acts as a tumor suppressor gene in colorectal cancer [16]. These ndings may suggest that the functional control of LGR6 is complex, and its role depends on the tumor type. The clinical signi cance and function of LGR6 in ESCC has been rarely reported thus far, but on the basis of our current study, it seems to have a function as a CSC marker.
Therefore, the function of LGR6 needs to be veri ed in cultured cells. It is particularly interesting as to whether LGR6 is involved in tumor progression or tumor suppression. It is also necessary to search for differences between LGR6 and LGR5. Further analyses in additional cases are desired.

Conclusions
We have demonstrated that LGR6 may be an important prognostic factor in ESCC.
LGR6 may also be useful as a CSC marker and may be applicable in future cancer treatments.

Declarations
Ethics approval and consent to participate This study was approved by the ethics committee of Shinshu University School of Medicine (approval no. 4088). The requirement of informed consent was waived by the ethics committee of Shinshu University School of Medicine, and an opt-out method was used because of the retrospective design of the study. The investigation was conducted in compliance with the Helsinki Declaration.

Consent for publication
Not applicable.

Availability of data and materials
All data generated and analyzed in the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was partially supported by the Hokuto Foundation for Bioscience (grant awarded to TU). The funding body had no role in the study design, collection, analysis, or interpretation of the data or manuscript writing.
Authors' contributions TE participated in the design of the study, performed the pathological analysis, and drafted the manuscript. TU, SK, SA, and MI helped with the pathological analysis. TU performed the statistical analysis. TN and YK conducted the immunohistochemistry. TN and US examined the clinical data of cases. HO and TU critically revised the draft for important intellectual content.
1 . Gong X, Carmon KS, Lin Q, Thomas A, Yi J, Liu Q LGR6 is a high a nity receptor of R-spondins and potentially functions as a tumor suppressor. PLoS One. 2012;7:e37137.