|Year : 2016 | Volume
| Issue : 2 | Page : 37-40
The overexpression of leucine-rich repeat-containing g-protein-coupled receptor 5 in pituitary adenomas
Jiuzhou Li1, Yuqi Zhang2, Ningning Cheng1, Quancheng Zhao1, Hongen Liu1, Shaowei Song3
1 Department of Neurosurgery, Binzhou People's Hospital, Binzhou, Shandong, China
2 Department of Anesthesiology, Binzhou People's Hospital, Binzhou, Shandong, China
3 Department of Orthopedic Surgery, Tongan Hospital of Dongying, Shandong, China
|Date of Submission||09-Jun-2016|
|Date of Acceptance||23-Jun-2016|
|Date of Web Publication||1-Jul-2016|
Department of Neurosurgery, Binzhou People's Hospital, Binzhou, Shandong 256600
Source of Support: None, Conflict of Interest: None
Aim: To determine the leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) level in human pituitary adenomas and the role of LGR5 in the development of human pituitary adenomas. Methods: Immunohistochemistry is performed to determine the expression of LGR5 in pituitary adenomas and normal pituitaries. LGR5 expression level is determined by the criteria as follows. The one-way analysis of variance (ANOVA) was used to evaluate any significant differences of LGR5 among individual groups. The correlation between expression and the other clinical factors were evaluated by using the Mann-Whitney test and one-way ANOVA. Results: LGR5 is significantly overexpressed in pituitary adenomas compared to normal pituitaries. Conclusion: The overexpression of LGR5 may facilitate the growth of these tumors. Potentially, the finding in the study could be exploited to develop innovative molecular targeted diagnosis and treatment for human pituitary adenomas.
Keywords: Invasiveness, leucine-rich repeat-containing G-protein-coupled receptor 5, pituitary adenoma, target therapy
|How to cite this article:|
Li J, Zhang Y, Cheng N, Zhao Q, Liu H, Song S. The overexpression of leucine-rich repeat-containing g-protein-coupled receptor 5 in pituitary adenomas. Transl Surg 2016;1:37-40
|How to cite this URL:|
Li J, Zhang Y, Cheng N, Zhao Q, Liu H, Song S. The overexpression of leucine-rich repeat-containing g-protein-coupled receptor 5 in pituitary adenomas. Transl Surg [serial online] 2016 [cited 2020 Sep 30];1:37-40. Available from: http://www.translsurg.com/text.asp?2016/1/2/37/185197
| Introduction|| |
Human pituitary adenomas comprise 10% of all brain tumors and occur in about 20% of the population. Pituitary adenomas are classified into noninvasive pituitary adenoma and invasive pituitary adenoma. They present with symptoms as a result of their size and location, or the inappropriate expression of pituitary hormones, or a combination of both factors. , Clinically, functional pituitary tumors, such as growth hormone (GH), adrenocorticotropic hormone (ACTH), and prolactin (PRL) adenomas, generally secrete a significant amount of GH, PRL, or ACTH and therefore give rise to potentially life-threatening clinical syndromes, such as acromegaly or Cushing's disease or result in impaired reproduction.  Nonfunctional (NF) pituitary adenomas account for approximately 30% of all pituitary adenomas due to their lack of clinical hormone hypersecretion.  Clinically, NF pituitary adenomas are usually larger at the time of diagnosis than their functional counterparts because clinical features are not apparent until tumor mass effects occurs. There are 4 treatment options for pituitary adenomas: observation, neurosurgery, radiation therapy, and target therapy.  To improve the survival rate, it is important to identify potential biological markers as diagnostic and therapeutic targets of pituitary adenomas, particularly in the early stages of the disease.
Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) also termed G-protein-coupled receptor 49 or G-protein-coupled receptor 67 is a protein that is encoded by the LGR5 gene in the human genome. , LGR5 belongs to a member of the G-protein-coupled receptor class An orphan receptor proteins. LGR5 is expressed across a diverse range of tissues, including the muscle, placenta, spinal cord, and brain, and specifically serves as a biomarker of adult stem cells in certain tissues, such as adipose tissue and skeletal muscles.  LGR5 was first identified by Hsu et al.  in Drosophila and acts as a cancer stem cell (CSC) marker for colorectal carcinoma (CRC). In the case of colorectal CSC differentiation, LGR5 methylation regulates its expression and serves as prognosis predict of in CRC.  In basal cell carcinoma, LGR5 expression is controlled by IKKα through the activating signal transducer and activator of transcription 3 signaling pathway.  LGR5 and its related receptors LGR4 and LGR6 bind the R-spondin growth factors (RSPO1-4) and function to potentiate Wnt signaling. , Numerous studies have revealed that LGR5 serves as a CSC marker. , However, little is known for the role of LGR5 in the pathogenesis of pituitary adenomas.
In the present study, the expression of LGR5 was examined in human pituitary adenomas, and normal pituitary tissues using immunohistochemistry. The clinical significance and diagnostic value of this marker in pituitary was evaluated.
| Materials and Methods|| |
Patients and tissues
A total of 63 tumor tissue specimens from patients with pituitary adenomas were obtained from the affiliated hospital of Chongqing Medical University and Binzhou People's Hospital. Tumor samples were taken during the surgery and frozen in liquid nitrogen and then stored at − 80°C until use. The tumor samples embedded in paraffin were obtained from the Department of Pathology in Affiliated Hospital of Chongqing Medical University and Binzhou People's Hospital, and were subjected to immunohistochemistry staining. Three normal human pituitary tissue specimens were obtained through an organ donor program. Mean diameter of the was determined by magnetic resonance imaging. Tumors were classified as either invasive or noninvasive depending on clinical or imaging characteristics. Invasive adenomas were defined as Knosp classification grade III and IV. The infiltration of bones and cavernous sinus or encasement of sinus structures observed during surgery also defined invasive adenomas. Additionally, the patients were classified into two groups according to their ages (< 60 years, ≥ 60 years). All samples were collected and handled according to national guidelines. The study was approved by the Ethics Committee of both hospitals.
The paraffin-embedded specimens were sliced into 4 μm sections. The slides were dried at 37°C overnight, and the tissue sections were baked at 60°C for 2 h. The slides were then deparaffinized with xylene and rehydrated using ethanol and immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. An antigen retrieval process was accomplished by a subjecting the slides to a pressure of 103 kPa (15 psi) for 3 min in tris/EDTA (pH 8.0) at 120°C. The slides were then incubated with primary rabbit antihuman polyclonal antibody against LGR5 (cat no. 21833-1-AP; Protein Tech, Chicago, IL, USA) for 1 h at room temperature in a moist chamber with saturated humidity. The dilution used for the LGR5 antibody was 1:600. The specimens were stained with 3,3'-diaminobenzidine subsequent to incubation with the secondary antibody (goat anti-rabbit IgG; 1:400 dilution; cat no. sc-2040; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 30 min. Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted. Confirmed biopsy specimens were used as a positive control. The slides treated with nonspecific serum were used as a negative control.
Criteria for evaluation
The LGR5 protein is found in both the cytoplasm and nucleus. LGR5 expression was identified by brown staining. In total, five microscopic fields in each slide were randomly selected. Positive cells were counted under high magnification (×200) and the results were expressed according to the following criteria: (−) absent, no expression; (+) weak expression, < 50% of tumor cells stained pale yellow; and (++ to +++) mild to strong expression, > 50% tumor cells stained medium brown or brown. The slides were independently reviewed by an experienced pathologist and researcher, blinded to clinical data. Inconsistent cases were assessed jointly reviewed.
The one-way analysis of variance (ANOVA) was used to evaluate any significant differences of LGR5 between the individual groups. The correlation between LGR5 expression and the other clinical factors was evaluated by use of the Mann-Whitney test and one-way ANOVA. P < 0.05 were considered statistically significant. The statistical software SPSS 13.0 (SPSS Inc., Chicago, IL, USA) was used for analyzing experimental results.
| Results|| |
Leucine-rich repeat-containing G-protein-coupled receptor 5 is expressed in pituitary adenomas but not in normal glands
Immunostaining results showed no expression of LGR5 in normal glands [Table 1] but LGR5 was expressed in 18 of 63 (28.6%) pituitary adenomas. Mild to strong expression of LGR5 was observed in pituitary adenoma tissues 11 of 63 (17.5%). The representative pictures of immunostaining were shown in [Figure 1].
|Figure 1: Expression of leucine-rich repeat-containing G-protein-coupled receptor 5 in normal glands compared to pituitary adenomas. (a) Expression of leucine-rich repeat-containing G-protein-coupled receptor 5 in normal glands (−) (×200); (b) Expression of CD56 in pituitary adenomas as positive control (+++) (×200); (c) Expression of leucine-rich repeat-containing G-protein-coupled receptor 5 in noninvasive pituitary adenomas (+) (×200); (d) Expression of leucine-rich repeat-containing G-protein-coupled receptor 5 in noninvasive pituitary adenomas (++) (×200); (e) Expression of leucine-rich repeat-containing G-protein-coupled receptor 5 in invasive pituitary adenomas (+++) (×200)|
Click here to view
Association between the expression of leucine-rich repeat-containing G-protein-coupled receptor 5 and clinical features
To identify the association between LGR5 and clinical features of patients with pituitary adenoma, the Mann-Whitney test and one-way ANOVA were performed. We found that the expression of LGR5 had no correlation with age, gender, mean tumor diameter or functional status. However, LGR5 expression level was significantly higher in invasive tumors than in noninvasive tumors (P < 0.05, [Table 2]). The association is also reported in detail in [Table 2].
|Table 2: Correlation between LGR5 protein expression and the clinical features of patients with pituitary adenomas|
Click here to view
| Discussion|| |
In our study, LGR5 was found to be significantly overexpressed in pituitary adenomas compared to normal pituitary glands. In pituitary adenomas, LGR5 expression was much higher in the invasive group than in the noninvasive group. Therefore, it may play an important role in the development and progression of invasive pituitary adenomas. However, the expression of LGR5 had no significant association with age, gender, mean diameter of tumor, or functionality. Potentially, these results could be explored to develop innovative molecular targeted diagnosis and treatment for human pituitary adenomas.
Numerous studies support the hypothesis that cancer cells exhibit CSC markers, and certain CSC markers may demonstrate considerable clinical importance in cancer development. , LGR5 serves as a CSC marker and plays an important role in maintaining the undifferentiated state of cancer cells. In the previous studies, LGR5 has been demonstrated to have a role in the pathogenesis of various human cancers, including hepatocellular carcinoma,  basal cell carcinoma,  endometrial cancer,  colon cancer, and ovarian cancer.  These findings suggest that LGR5 may be a marker for carcinoma in humans. Our results are consistent with the previous reports and showed the involvement of LGR5 in the pathogenesis of pituitary adenomas. Moreover, the expression of LGR5 was significantly increased in the majority of the invasive pituitary adenomas. As we know, the majority of invasive tumors have a faster rate of cell division and growth rate.  Our data therefore indicate that overexpression of LGR5 in invasive pituitary tumors correlated with the lever of invasiveness of pituitary adenomas, and might be used for evaluation and categorizing of pituitary adenomas. However, the expression of LGR5 had no significant relationship with age, gender, whether the surgery was repeated or not, and mean diameter of the tumors.
The treatment of pituitary adenomas has reached a high level with improved surgical and radiotherapeutic strategies, which are less likely to cause toxicity to normal tissues and more effective. However, the greater promise will likely come from molecular target therapy. Therefore, it is particularly crucial to identify potential biological markers as diagnostic and therapeutic targets of NF pituitary tumors. The selective overexpression of LGR5 in pituitary adenomas provides a unique opportunity for targeted diagnosis and therapy for the patients. In future, a mix of well-established agents, such as siRNA or specific antibody, could be used to target pituitary adenomas' biomarkers, such as LGR5, and block the invasive capacity of tumors overexpressing these biomarkers. Such rational, biomarker-based therapies will expand the realm of pituitary adenoma treatment, as they now do for other areas of oncology. Once biomarkers such as LGR5 are well established, and the use of targeted inhibitors is further developed, pituitary adenomas also present one of the better opportunities for expanding the benefits of molecular targeted therapy for tumor control and cure.
| Conclusion|| |
LGR5 was significantly overexpressed in pituitary adenomas but not in normal pituitary glands. In pituitary adenomas, LGR5 expression was much higher in the invasive group than in the noninvasive group.
This study was supported by the Department of Pathology from the Affiliated Hospital of Chongqing Medical University and Binzhou People's Hospital.
Financial support and sponsorship
The study was supported by the Key Research Project Grant of Shandong Province (No. 2015WSA16004).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Asa SL, Ezzat S. The cytogenesis and pathogenesis of pituitary adenomas. Endocr Rev
Levy A, Lightman SL. Diagnosis and management of pituitary tumours. BMJ
McCutcheon IE. Pituitary adenomas: Surgery and radiotherapy in the age of molecular diagnostics and pathology. Curr Probl Cancer
Levy A. Molecular and trophic mechanisms of pituitary tumourigenesis. Horm Res Paediatr
2011;76 (Suppl 1):2-6.
McDonald T, Wang R, Bailey W, Xie G, Chen F, Caskey CT, Liu Q. Identification and cloning of an orphan G protein-coupled receptor of the glycoprotein hormone receptor subfamily. Biochem Biophys Res Commun
McClanahan T, Koseoglu S, Smith K, Grein J, Gustafson E, Black S, Kirschmeier P, Samatar AA. Identification of overexpression of orphan G protein-coupled receptor GPR49 in human colon and ovarian primary tumors. Cancer Biol Ther
Hsu SY, Liang SG, Hsueh AJ. Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol
Su S, Hong F, Liang Y, Zhou J, Liang Y, Chen K, Wang X, Wang Z, Wang Z, Chang C, Han W, Gong W, Qin H, Jiang B, Xiong H, Peng L. Lgr5 methylation in cancer stem cell differentiation and prognosis-prediction in colorectal cancer. PLoS One
Jia J, Shi Y, Yan B, Xiao D, Lai W, Pan Y, Jiang Y, Chen L, Mao C, Zhou J, Xi S, Cao Y, Liu S, Tao Y. LGR5 expression is controlled by IKKα in basal cell carcinoma through activating STAT3 signaling pathway. Oncotarget
2016;doi: 10.18632/oncotarget. 8465.
Carmon KS, Gong X, Lin Q, Thomas A, Liu Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling. Proc Natl Acad Sci U S A
de Lau W, Barker N, Low TY, Koo BK, Li VS, Teunissen H, Kujala P, Haegebarth A, Peters PJ, van de Wetering M, Stange DE, van Es JE, Guardavaccaro D, Schasfoort RB, Mohri Y, Nishimori K, Mohammed S, Heck AJ, Clevers H. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature
Vermeulen L, Todaro M, de Sousa Mello F, Sprick MR, Kemper K, Perez Alea M, Richel DJ, Stassi G, Medema JP. Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci U S A
Takashima S, Kadowaki M, Aoyama K, Koyama M, Oshima T, Tomizuka K, Akashi K, Teshima T. The Wnt agonist R-spondin1 regulates systemic graft-versus-host disease by protecting intestinal stem cells. J Exp Med
Visvader JE, Lindeman GJ. Cancer stem cells in solid tumours: Accumulating evidence and unresolved questions. Nat Rev Cancer
Barker N, Ridgway RA, van Es JH, van de Wetering M, Begthel H, van den Born M, Danenberg E, Clarke AR, Sansom OJ, Clevers H. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature
Yamamoto Y, Sakamoto M, Fujii G, Tsuiji H, Kenetaka K, Asaka M, Hirohashi S. Overexpression of orphan G-protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with beta-catenin mutations. Hepatology
Sun X, Jackson L, Dey SK, Daikoku T. In pursuit of leucine-rich repeat-containing G protein-coupled receptor-5 regulation and function in the uterus. Endocrinology
Ding Z, Li B, Wang Q, Miao Y, Lu X. Increase in folate receptor alpha expression in nonfunctional pituitary adenomas. Turk Neurosurg
[Table 1], [Table 2]
|This article has been cited by|
||LGR5 and Downstream Intracellular Signaling Proteins Play Critical Roles in the Cell Proliferation of Neuroblastoma, Meningioma and Pituitary Adenoma
| ||Mina Hwang,Myung-Hoon Han,Hyun-Hee Park,Hojin Choi,Kyu-Yong Lee,Young Joo Lee,Jae Min Kim,Jin Hwan Cheong,Je Il Ryu,Kyueng-Whan Min,Young-Ha Oh,Yong Ko,Seong-Ho Koh |
| ||Experimental Neurobiology. 2019; 28(5): 628 |
|[Pubmed] | [DOI]|