Abstract
Background
Erythropoietin-producing hepatocellular (EPH) receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The Erythropoietin-producing hepatocellular receptor tyrosine kinase member EphB6 is a pseudokinase which has not attracted an equivalent amount of interest as its enzymatically-active counterparts. The aim of this study was to assess the expression of EphB6 in pituitary tumors.
Methods and Results
Human normal pituitaries and pituitary tumors were examined for EphB6 mRNA expression using real-time PCR and for EphB6 protein by immunohistochemistry and Western blotting. EphB6 was highly expressed in non-functioning pituitary neuroendocrine tumors (NF-PitNETs) versus the normal pituitary and GH-secreting PitNETs. EphB6 mRNA expression was correlated with tumor size.
Conclusions
Our results suggest EphB6 aberrant expression in NF-PitNETs. Future studies are warranted to determine the role and significance of EphB6 in NF-PitNETs tumorigenesis.
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Introduction
Ephs are the largest family of receptor tyrosine kinases (RTK). Ephs orchestrate cell positioning as well as tissue and organ patterning. Ephs also control cell survival during normal and neoplastic development and they have been implicated in cancer cell proliferation, adhesion, migration, tumor angiogenesis and invasion [1,2,3]. A unique feature of the Eph receptors is that their cognate ligands, the ephrins, are tethered to the cell surface, in contrast to other RTKs whose ligands are generally soluble [4]. Therefore, the resultant signaling is largely dependent on cell–cell contact and can occur in a bidirectional manner in neighboring cells [4]. Eph receptors are divided into two subfamilies, types A and B. Whereas EphA and EphB receptors have a similar structure, the structures of the ligand classes, ephrin-A and -B, are different. Ephrin-B are transmembrane ligands while ephrin-A ligands are smaller and tethered to the membrane only via a glycosylphosphatidylinositol (GPI) anchor [5].
Two members of the family, EphA10 and EphB6, are classified as pseudokinases due to the absence of key amino acids known to catalyze phosphoryl transfer from ATP in conventional kinases [6, 7]. Yet, these two receptors are able to function without tyrosine kinase activity. EphB6 was shown to maintain physiological homeostasis in kidney [8], vascular smooth muscle [9] and the immune system [10]. However, considerably more research was focused on EphB6 involvement in cancer. EphB6 was shown to reduce motility and invasion of breast [11,12,13] and lung [14, 15] cancer cells. In several malignancies, an inverse correlation between EphB6 expression and tumor aggressiveness was observed thereby suggesting that EphB6 may suppress invasive and metastatic phenotypes [16,17,18,19,20,21,22,23,24,25,26]. Interestingly though, despite its anti-invasive properties, EphB6 was found to promote tumor initiation in breast cancer xenografts [12] and in a colorectal cancer model [27, 28]. Consistent with this oncogenic potential of EphB6, its expression was positively correlated with tumor size and recurrence rate of malignant thyroid lesions [29] and was also coupled to poor outcome in breast cancer [30] and tongue squamous cell carcinoma [31].
Interestingly, Eph receptor/ephrin signaling is known to play important roles in various niches and was investigated also in the context of the normal physiology of the pituitary gland [32,33,34]. Prolactin secreting pituitary neuroendocrine tumors (PitNETs) are the most common pituitary tumors (50%) followed by non-functioning pituitary neuroendocrine tumors (NF-PitNETs) comprising ~ 30% of PitNETs [35]. Dysregulated expression of Eph family members in NF-PitNETs was reported or may be obtained from several reports as part of broad gene expression profiling of PitNETs [36,37,38,39,40,41,42,43,44,45]. In this study we aimed to examine the expression of EphB6 in PitNETs.
Materials and methods
Pituitary tumors
Samples of human pituitary tumors were obtained during transsphenoidal surgical resection with patients' informed consent in accordance with methods and conditions approved by the local institutional review board (approval number 0838-17). 30 NF-PitNETs and 17 GH-PitNETs were analyzed in this study. The clinical characteristics of the patients are presented in Table 1.
Mice
Pituitaries were extracted from GRIC-GFP or GRIC-tdTomato mice (a kind gift from Prof. Ulrich Boehm to Prof. Philippa Melamed) and gonadotropes were enriched by FACS (as in refs.[46, 47]). Animal experiments were performed after protocol approval by the Institutional Animal Care and Use Committee of Technion – Israel Institute of Technology (approval number IL0440415).
Gene expression
Total RNA was extracted from pituitary specimens and processed to cDNA with High Capacity cDNA Reverse Transcription kit (AB Applied Biosystems, Foster City, CA). Human normal adult pituitary RNA samples of two donors were purchased from BioChain. Human pituitary samples were analyzed in triplicates using Taqman gene expression assays (IDT, Coralville, Iowa, United States). Results were normalized to Cyclophilin B. qPCR reactions of mice samples were performed with PerfeCTa SYBR Green FastMix (Quanta) and normalized to Rplp0. EphB6 mouse primers were: Forward 5` CTAGGAAAGATCTGCGAGGTG 3`, Reverse 5` GTTTGCTCTCTTCATTTACTCTGC 3`.
Immunohistochemistry
Formalin-fixed, paraffin-embedded 5μ sections slides of pituitary tumors and normal pituitary (obtained from autopsy) were deparaffinized, rehydrated and boiled in citrate buffer. After washing and blocking slides were incubated at 4C with anti-EphB6 antibody (Bioss Antibodies Inc. (Woburn, MA, USA)) diluted 1:200 in PBS. Slides were washed and incubated with a secondary antibody Goat anti-rabbit Alexa Fluor 647 diluted 1:1000 in PBS. After additional washes, slides were stained with Dapi, washed and mounted. Images were obtained with Zeiss ApoTome.2 microscope and scored by ImageJ Fiji software. Setting was consistent for all samples in both image capture and analysis.
Protein extraction and western blotting
Frozen tissue specimens were minced in liquid nitrogen followed by homogenization in RIPA buffer together with protease and phosphatase inhibitors cocktails. Protein concentrations were quantified with Bradford protein assay (Bio-Rad Protein Assay Dye Reagent Concentrate, Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein extracts were loaded on 10% SDS-PAGE and Western blotting was conducted. Immunodetection was performed using Chemiluminescent Peroxidase Substrate. The optical density of the bands was measured and quantified employing the iBright Imaging System (ThermoFisher Scientific). Antibody against EphB6 was from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
Statistical analysis
For independent data Student’s t-test was performed. Correlations were calculated using Pearson test with the GraphPad software. P values < 0.05 were considered significant.
Results
Elevated EphB6 mRNA expression in NF-PitNETs
We first aimed to validate the high expression of EphB6 mRNA in NF-PitNETs that was found by Moreno CS et al. using gene arrays [39]. Quantitative PCR was performed and revealed that EphB6 mRNA level was significantly higher in all 17 NF-PitNETs samples compared to human normal pituitary and notably was correlated with the tumor size (Fig. 1a). We also examined GH-secreting PitNETs, which showed lower levels of EphB6 mRNA in 7 of 8 samples compared to the normal pituitary (Fig. 1b). Astonishingly, data obtained from Gene Expression Omnibus (GEO;[48], accession GSE147786) of PitNETs analyzed by Microarray GeneChips by Taniguchi‑Ponciano et al.[43], showed that all other types of PitNETs express low levels of EphB6 (Fig. 1c). Finally, since most NF-PitNETs are originated from gonadotropes cells, we aimed to examine if EphB6 dysregulated expression is a marker of normal gonadotropes or specific to tumors. For this purpose, we used genetically modified mice which express GFP in their gonadotropes (gonadotropin-releasing hormone receptor -IRES-Cre (GRIC)-GFP mice [49]). Gonadotropes were collected based on their GFP fluorescence by fluorescence-activated cell sorting at the neonatal period, after castration or when matured and primary cultures were prepared. Quantitative PCR was then performed using αT3 gonadotrope cell line as positive EphB6 control. EphB6 was not expressed in all these primary gonadotropes populations (Ct = O, data not shown). Together, these results suggest that EphB6 aberrant expression is unique to NF-PitNETs.
Elevated EphB6 protein expression in NF-PitNETs
We next sought to determine EphB6 protein expression. This was examined by immunohistochemistry of human tissue sections derived from normal pituitary and PitNETs specimens. While the normal pituitary and GH-secreting PitNETs showed low staining, NF-PitNETs exhibited high EphB6 expression (Table 2). Representative immunostained sections of NF-PitNET, GH-secreting PitNET and normal pituitary are shown in Fig. 2a. We also performed Western blot analysis of lysates derived from human PitNETs samples. NF-PitNETs showed significant higher expression levels of EphB6 protein expression compared to GH-secreting PitNETs (Fig. 2b, P = 0.043).
Discussion
Studies investigating the development of the rat pituitary gland have shown co-localization of ephrin B2 ligand (EFNB2) and EphB3 receptor in stem/progenitor cells in the two niches of the anterior lobe, the marginal cell layer and dense cell clusters in the parenchyma [32, 33]. Other EFNB2-candidate interacting receptors, EphB1, EphB2, and EphB4 were found specifically in the rat gonadotropes, corticotropes and endothelial cells, respectively [33]. Also, various ephrin ligands were found to be expressed in the rat anterior pituitary cells by single-cell RNA sequencing [50]. As for EphB6, data obtained from single-cell RNA sequencing studies [51, 52] suggest low EphB6 expression in human fetus gonadotropes which becomes undetectable in adult gonadotropes. In consistent with this, our results show undetectable EphB6 expression in mice gonadotropes populations at the neonatal period and when matured. Together it seems that EPH family members may play a role in the pituitary development and EphB6 is not a marker of the normal gonadotropes subtype.
Profiling studies of NF-PitNETs suggest molecular alterations in several EPH family members. EphB6 [39] and Ephrin-B3 ligand (EFNB3) [36, 39, 41] were found to be overexpressed in NF-PitNETs compared to normal pituitary in microarrays [39, 41] or the GEO database [36]. Moreover, data obtained from RNA sequencing analysis showed a 5.3 fold elevated expression of EphB6 in 43 NF-PitNETs samples compared to 22 functioning PitNETs [42] (GEO;[48], accession GSE209903). In-silico analysis of twenty-three microarray libraries also revealed high expression of EphB6, EFNB3 and other Ephrin receptors, EphA5, -A7, -A10 and -B1 in NF-PitNETs [44]. Quantitative proteomics using two dimensional liquid chromatography-tandem mass spectrometry revealed the expression of EphA10, EFNA5 and EFNB1 in NF-PitNETs [36]. Comparison of highly proliferative NF-PitNETs versus NF-PitNETs suggested differential gene expression of EPH receptor signaling pathway [45] which similarly was found hypomethylated in re-intervention versus stable NF-PitNET patients [37]. Transcriptome analysis of PitNETs identified upregulation of EphB6 in silent ACTH PitNETs, gonadotrophinomas and null cell PitNETs when clustered together and of EphA4 in ACTH-secreting PitNETs [43]. Taken together, these studies and our indicate that NF-PitNETs are characterized by aberrant expression of EphB6 and other EPH receptors and ligands.
In a manner similar to other pseudokinases, EphB6 was suggested to act as a molecular switch that is capable of modulating the signals generated by an Eph receptor cluster. By recruiting kinases, phosphatases, proteases or ubiquitinase ligases (directly or indirectly) EphB6 can modulate the phosphorylation state and thus kinase activity of individual members in the cluster [53]. For example, EphB6 interaction with EphA2 [54,55,56] and EphB2 [55] was shown to modulated their activities. EphB6 was shown to be phosphorylated by the EphB4 receptor and this tyrosine phosphorylation was crucial for EphB6 interaction with the ubiquitinase ligase c-Cbl and phosphorylation of c-Cbl partner, the Abl kinase [13]. EphB6 can be phosphorylated also by EphB1 [57] or by the Src family kinase Fyn [58]. EphB6 ligands are ephrin-B1 [57] and ephrin-B2 [59] and MAPK [12, 15, 60, 61] and Akt signaling pathways [27, 54, 62, 63] were shown to mediate EphB6 activities. A most recent study by Hanover et al. [64] which integrates bioinformatic analysis, proteomics and genomics reveals crosstalk of EphB6 and EGFR, enhancing the proliferation of cancer cells. Both PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways downstream to EGFR are activated in NF-PitNETs (reviewed in [65]), therefore a possible EphB6 and EGFR crosstalk in NF-PitNETs is appealing and yet to be investigated.
Conclusion
Our study show high expression of EphB6 mRNA and protein in NF-PitNETs compared to normal pituitary and GH-secreting PitNET. EphB6 mRNA level was correlated with tumor size. These findings suggest EphB6 as an attractive candidate for functional and clinical studies of NF-PitNETs.
Data availability
Not applicable.
Abbreviations
- NF-PitNETs:
-
Non-functioning pituitary neuroendocrine tumors
- GH-PitNETs:
-
Growth hormone secreting pituitary neuroendocrine tumors
- EPH:
-
Erythropoietin-producing hepatocellular
- EGF:
-
Epidermal growth factor
References
Hafner C, Schmitz G, Meyer S, Bataille F, Hau P, Langmann T, Dietmaier W, Landthaler M, Vogt T (2004) Differential gene expression of Eph receptors and ephrins in benign human tissues and cancers. Clin Chem 50(3):490–499. https://doi.org/10.1373/clinchem.2003.026849
Kania A, Klein R (2016) Mechanisms of ephrin-Eph signalling in development, physiology and disease. Nat Rev Mol Cell Biol 17(4):240–256. https://doi.org/10.1038/nrm.2015.16
Pasquale EB (2010) Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 10(3):165–180. https://doi.org/10.1038/nrc2806
Boyd AW, Bartlett PF, Lackmann M (2014) Therapeutic targeting of EPH receptors and their ligands. Nat Rev Drug Discovery 13(1):39–62. https://doi.org/10.1038/nrd4175
Taylor H, Campbell J, Nobes CD (2017) Ephs and ephrins. Curr Biol CB 27(3):R90–R95. https://doi.org/10.1016/j.cub.2017.01.003
Jacobsen AV, Murphy JM (2017) The secret life of kinases: insights into non-catalytic signalling functions from pseudokinases. Biochem Soc Trans 45(3):665–681. https://doi.org/10.1042/BST20160331
Shrestha S, Byrne DP, Harris JA, Kannan N, Eyers PA (2020) Cataloguing the dead: breathing new life into pseudokinase research. FEBS J 287(19):4150–4169. https://doi.org/10.1111/febs.15246
Ogawa K, Wada H, Okada N, Harada I, Nakajima T, Pasquale EB, Tsuyama S (2006) EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases. J Cell Sci 119(Pt 3):559–570. https://doi.org/10.1242/jcs.02777
Luo H, Wu Z, Tremblay J, Thorin E, Peng J, Lavoie JL, Hu B, Stoyanova E, Cloutier G, Qi S, Wu T, Cameron M, Wu J (2012) Receptor tyrosine kinase Ephb6 regulates vascular smooth muscle contractility and modulates blood pressure in concert with sex hormones. J Biol Chem 287(9):6819–6829. https://doi.org/10.1074/jbc.M111.293365
Freywald A, Sharfe N, Rashotte C, Grunberger T, Roifman CM (2003) The EphB6 receptor inhibits JNK activation in T lymphocytes and modulates T cell receptor-mediated responses. J Biol Chem 278(12):10150–10156. https://doi.org/10.1074/jbc.M208179200
Fox BP, Kandpal RP (2009) EphB6 receptor significantly alters invasiveness and other phenotypic characteristics of human breast carcinoma cells. Oncogene 28(14):1706–1713. https://doi.org/10.1038/onc.2009.18
Toosi BM, El Zawily A, Truitt L, Shannon M, Allonby O, Babu M, DeCoteau J, Mousseau D, Ali M, Freywald T, Gall A, Vizeacoumar FS, Kirzinger MW, Geyer CR, Anderson DH, Kim T, Welm AL, Siegel P, Vizeacoumar FJ, Kusalik A, Freywald A (2018) EPHB6 augments both development and drug sensitivity of triple-negative breast cancer tumours. Oncogene 37(30):4073–4093. https://doi.org/10.1038/s41388-018-0228-x
Truitt L, Freywald T, DeCoteau J, Sharfe N, Freywald A (2010) The EphB6 receptor cooperates with c-Cbl to regulate the behavior of breast cancer cells. Cancer Res 70(3):1141–1153. https://doi.org/10.1158/0008-5472.CAN-09-1710
Bulk E, Yu J, Hascher A, Koschmieder S, Wiewrodt R, Krug U, Timmermann B, Marra A, Hillejan L, Wiebe K, Berdel WE, Schwab A, Müller-Tidow C (2012) Mutations of the EPHB6 receptor tyrosine kinase induce a pro-metastatic phenotype in non-small cell lung cancer. PLoS One 7(12):e44591. https://doi.org/10.1371/journal.pone.0044591
Yu J, Bulk E, Ji P, Hascher A, Tang M, Metzger R, Marra A, Serve H, Berdel WE, Wiewroth R, Koschmieder S, Müller-Tidow C (2010) The EPHB6 receptor tyrosine kinase is a metastasis suppressor that is frequently silenced by promoter DNA hypermethylation in non-small cell lung cancer. Clin Cancer Res 16(8):2275–2283. https://doi.org/10.1158/1078-0432.CCR-09-2000
Bailey CM, Kulesa PM (2014) Dynamic interactions between cancer cells and the embryonic microenvironment regulate cell invasion and reveal EphB6 as a metastasis suppressor. Mol Cancer Res 12(9):1303–1313. https://doi.org/10.1158/1541-7786.MCR-13-0673
Fan YH, Ding HW, Kim D, Liu JY, Hong JY, Xu YN, Wang D, Yang XS, Lee SK (2020) The PI3Kα inhibitor DFX24 suppresses tumor growth and metastasis in non-small cell lung cancer via ERK inhibition and EPHB6 reactivation. Pharmacol Res 160:105147. https://doi.org/10.1016/j.phrs.2020.105147
Gu Y, Li F, Qian N, Chen X, Wang H, Wang J (2016) Expression of EphB6 in ovarian serous carcinoma is associated with grade, TNM stage and survival. J Clin Pathol 69(5):448–453. https://doi.org/10.1136/jclinpath-2015-203160
Kang M, Shi J, Li B, Luo M, Xu S, Liu X (2019) LncRNA DGCR5 regulates the non-small cell lung cancer cell growth, migration, and invasion through regulating miR-211-5p/EPHB6 axis. BioFactors 45(5):788–794. https://doi.org/10.1002/biof.1539
Liersch-Löhn B, Slavova N, Buhr HJ, Bennani-Baiti IM (2016) Differential protein expression and oncogenic gene network link tyrosine kinase ephrin B4 receptor to aggressive gastric and gastroesophageal junction cancers. Int J Cancer 138(5):1220–1231. https://doi.org/10.1002/ijc.29865
Liu J, Xu B, Xu G, Zhang X, Yang X, Wang J (2017) Reduced EphB6 protein in gastric carcinoma and associated lymph nodes suggests EphB6 as a gastric tumor and metastasis inhibitor. Cancer Biomarkers 19(3):241–248. https://doi.org/10.3233/CBM-160256
Yu H, Qin XK, Yin KW, Li ZM, Ni ED, Yang JM, Liu XH, Zhou AJ, Li SJ, Gao TM, Li Y, Li JM (2023) EphB6 deficiency in intestinal neurons promotes tumor growth in colorectal cancer by neurotransmitter GABA signaling. Carcinogenesis 44(8–9):682–694. https://doi.org/10.1093/carcin/bgad041
Mohamed ER, Noguchi M, Hamed AR, Eldahshoury MZ, Hammady AR, Salem EE, Itoh K (2015) Reduced expression of erythropoietin-producing hepatocyte B6 receptor tyrosine kinase in prostate cancer. Oncol Lett 9(4):1672–1676. https://doi.org/10.3892/ol.2015.2925
Tang XX, Evans AE, Zhao H, Cnaan A, London W, Cohn SL, Brodeur GM, Ikegaki N (1999) High-level expression of EPHB6, EFNB2, and EFNB3 is associated with low tumor stage and high TrkA expression in human neuroblastomas. Clin Cancer 5(6):1491-1496.25
Truitt L, Freywald A (2011) Dancing with the dead: Eph receptors and their kinase-null partners. Biochem. Cell Biol 89(2):115–129. https://doi.org/10.1139/o10-145
Xiang S, Wei M, Zhao L, Lin A, Xiong Z (2023) Integrated analyses of the expression and prognostic value of EPHB6 in cervical cancer and its correlation with immune infiltrates. J Oncol 2023:2258906. https://doi.org/10.1155/2023/2258906
Wang J, Zhang Y, Ma J, Yang C, Wang M, Lv J, Wang Y, Miao D, Wang Y, Li M, Chai C, Jiang S, Tong J, Li M, Yu Z (2021) Determining the effects of Ephrin Type B Receptor 6 and Type A Receptor 3 on facilitating colorectal epithelial cell malignant transformation. Neoplasma 68(5):955–964. https://doi.org/10.4149/neo_2021_210309N304
Xu D, Yuan L, Liu X, Li M, Zhang F, Gu XY, Zhang D, Yang Y, Cui B, Tong J, Zhou J, Yu Z (2016) EphB6 overexpression and Apc mutation together promote colorectal cancer. Oncotarget 7(21):31111–31121. https://doi.org/10.18632/oncotarget.9080
Giaginis C, Alexandrou P, Poulaki E, Delladetsima I, Troungos C, Patsouris E, Theocharis S (2016) Clinical significance of EphB4 and EphB6 expression in human malignant and benign thyroid lesions. Pathol Oncol Res POR 22(2):269–275. https://doi.org/10.1007/s12253-014-9879-2
Husa AM, Magić Ž, Larsson M, Fornander T, Pérez-Tenorio G (2016) EPH/ephrin profile and EPHB2 expression predicts patient survival in breast cancer. Oncotarget 7(16):21362–21380. https://doi.org/10.18632/oncotarget.7246
Dong Y, Pan J, Ni Y, Huang X, Chen X, Wang J (2015) High expression of EphB6 protein in tongue squamous cell carcinoma is associated with a poor outcome. Int J Clin Exp Pathol 8(9):11428–11433
Yoshida S, Kato T, Higuchi M, Chen M, Ueharu H, Nishimura N, Kato Y (2015) Localization of juxtacrine factor ephrin-B2 in pituitary stem/progenitor cell niches throughout life. Cell Tissue Res 359(3):755–766. https://doi.org/10.1007/s00441-014-2054-y
Yoshida S, Kato T, Kanno N, Nishimura N, Nishihara H, Horiguchi K, Kato Y (2017) Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland. Cell Tissue Res 370(1):99–112. https://doi.org/10.1007/s00441-017-2646-4
Zarbalis K, Wurst W (2000) Expression domains of murine ephrin-A5 in the pituitary and hypothalamus. Mech Dev 93(1–2):165–168. https://doi.org/10.1016/s0925-4773(00)00252-5
Daly AF, Beckers A (2020) The Epidemiology of Pituitary Adenomas. Endocrinol Metab Clin North Am 49(3):347–355. https://doi.org/10.1016/j.ecl.2020.04.002
Cheng T, Wang Y, Lu M, Zhan X, Zhou T, Li B, Zhan X (2019) Quantitative analysis of proteome in non-functional pituitary adenomas: clinical relevance and potential benefits for the patients. Front Endocrinol 10:854. https://doi.org/10.3389/fendo.2019.00854
Hallén T, Johannsson G, Dahlén R, Glad CAM, Örndal C, Engvall A, Carén H, Skoglund T, Olsson DS (2022) Genome-wide DNA methylation differences in nonfunctioning pituitary adenomas with and without postsurgical progression. J Clin Endocrinol Metab 107(8):2318–2328. https://doi.org/10.1210/clinem/dgac266
Michaelis KA, Knox AJ, Xu M, Kiseljak-Vassiliades K, Edwards MG, Geraci M, Kleinschmidt-DeMasters BK, Lillehei KO, Wierman ME (2011) Identification of growth arrest and DNA-damage-inducible gene beta (GADD45beta) as a novel tumor suppressor in pituitary gonadotrope tumors. Endocrinology 152(10):3603–3613. https://doi.org/10.1210/en.2011-0109
Moreno CS, Evans CO, Zhan X, Okor M, Desiderio DM, Oyesiku NM (2005) Novel molecular signaling and classification of human clinically nonfunctional pituitary adenomas identified by gene expression profiling and proteomic analyses. Can Res 65(22):10214–10222. https://doi.org/10.1158/0008-5472.CAN-05-0884
Morris DG, Musat M, Czirják S, Hanzély Z, Lillington DM, Korbonits M, Grossman AB (2005) Differential gene expression in pituitary adenomas by oligonucleotide array analysis. Eur J Endocrinol 153(1):143–151. https://doi.org/10.1530/eje.1.01937
Qiao X, Wang H, Wang X, Zhao B, Liu J (2017) Microarray technology reveals potentially novel genes and pathways involved in non-functioning pituitary adenomas. Balkan J Med Genet 19(2):5–16. https://doi.org/10.1515/bjmg-2016-0030
Silva-Júnior RMPD, Bueno AC, Martins CS, Coelli-Lacchini F, Ozaki JGO, Almeida-E-Silva DC, Marrero-Gutiérrez J, Santos ACD, Garcia-Peral C, Machado HR, Santos MVD, Elias PL, Moreira AC, Colli LM, Vêncio RZN, Antonini SR, de Castro M (2023) Integrating methylome and transcriptome signatures expands the molecular classification of the pituitary tumors. J Clin Endocrinol Metab 108(6):1452–1463. https://doi.org/10.1210/clinem/dgac703
Taniguchi-Ponciano K, Andonegui-Elguera S, Peña-Martínez E, Silva-Román G, Vela-Patiño S, Gomez-Apo E, Chavez-Macias L, Vargas-Ortega G, Espinosa-de-Los-Monteros L, Gonzalez-Virla B, Perez C, Ferreira-Hermosillo A, Espinosa-Cardenas E, Ramirez-Renteria C, Sosa E, Lopez-Felix B, Guinto G, Marrero-Rodríguez D, Mercado M (2020) Transcriptome and methylome analysis reveals three cellular origins of pituitary tumors. Sci Rep 10(1):19373. https://doi.org/10.1038/s41598-020-76555-8
Taniguchi-Ponciano K, Gomez-Apo E, Chavez-Macias L, Vargas G, Espinosa-Cardenas E, Ramirez-Renteria C, Ferreira-Hermosillo A, Sosa E, Silva-Román G, Peña-Martínez E, Andonegui-Elguera S, Vargas-Chavez S, Santiago-Andres Y, Peralta R, Marrero-Rodríguez D, Mercado M (2020) Molecular alterations in non-functioning pituitary adenomas. Cancer Biomarkers 28(2):193–199. https://doi.org/10.3233/CBM-191121
Wei Z, Zhou C, Li M, Huang R, Deng H, Shen S, Wang R (2021) Integrated multi-omics profiling of nonfunctioning pituitary adenomas. Pituitary 24(3):312–325. https://doi.org/10.1007/s11102-020-01109-0
Hoivik EA, Witsoe SL, Bergheim IR, Xu Y, Jakobsson I, Tengholm A, Doskeland SO, Bakke M (2013) DNA methylation of alternative promoters directs tissue specific expression of Epac2 isoforms. PLoS ONE 8(7):e67925. https://doi.org/10.1371/journal.pone.0067925
Wen S, Schwarz JR, Niculescu D, Dinu C, Bauer CK, Hirdes W, Boehm U (2008) Functional characterization of genetically labeled gonadotropes. Endocrinology 149(6):2701–2711. https://doi.org/10.1210/en.2007-1502
Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30(1):207–210. https://doi.org/10.1093/nar/30.1.207
Savulescu D, Feng J, Ping YS, Mai O, Boehm U, He B, O’Malley BW, Melamed P (2013) Gonadotropin-releasing hormone-regulated prohibitin mediates apoptosis of the gonadotrope cells. Molecular Endocrinol 27(11):1856–1870. https://doi.org/10.1210/me.2013-1210
Fletcher PA, Smiljanic K, Maso Prévide R, Iben JR, Li T, Rokic MB, Sherman A, Coon SL, Stojilkovic SS (2019) Cell Type- and sex-dependent transcriptome profiles of rat anterior pituitary cells. Front Endocrinol 10:623. https://doi.org/10.3389/fendo.2019.00623
Zhang Q, Yao B, Long X, Chen Z, He M, Wu Y, Qiao N, Ma Z, Ye Z, Zhang Y, Yao S, Wang Y, Cheng H, Chen H, Ye H, Wang Y, Li Y, Chen J, Zhang Z, Guo F, Zhao Y (2023) Single-cell sequencing identifies differentiation-related markers for molecular classification and recurrence prediction of PitNET. Cell Rep Med 4(2):100934. https://doi.org/10.1016/j.xcrm.2023.100934
Zhang S, Cui Y, Ma X, Yong J, Yan L, Yang M, Ren J, Tang F, Wen L, Qiao J (2020) Single-cell transcriptomics identifies divergent developmental lineage trajectories during human pituitary development. Nat Commun 11(1):5275. https://doi.org/10.1038/s41467-020-19012-4
Strozen TG, Sharpe JC, Harris ED, Uppalapati M, Toosi BM (2021) The EphB6 receptor: kinase-dead but very much alive. Int J Mol Sci 22(15):8211. https://doi.org/10.3390/ijms22158211
Akada M, Harada K, Negishi M, Katoh H (2014) EphB6 promotes anoikis by modulating EphA2 signaling. Cell Signal 26(12):2879–2884. https://doi.org/10.1016/j.cellsig.2014.08.031
Fox BP, Kandpal RP (2011) A paradigm shift in EPH receptor interaction: biological relevance of EPHB6 interaction with EPHA2 and EPHB2 in breast carcinoma cell lines. Cancer Genom Proteom 8(4):185–193
Yoon S, Choi JH, Kim SJ, Lee EJ, Shah M, Choi S, Woo HG (2019) EPHB6 mutation induces cell adhesion-mediated paclitaxel resistance via EPHA2 and CDH11 expression. Exp Mol Med 51(6):1–12. https://doi.org/10.1038/s12276-019-0261-z
Freywald A, Sharfe N, Roifman CM (2002) The kinase-null EphB6 receptor undergoes transphosphorylation in a complex with EphB1. J Biol Chem 277(6):3823–3828. https://doi.org/10.1074/jbc.M108011200
Matsuoka H, Obama H, Kelly ML, Matsui T, Nakamoto M (2005) Biphasic functions of the kinase-defective Ephb6 receptor in cell adhesion and migration. J Biol Chem 280(32):29355–29363. https://doi.org/10.1074/jbc.M500010200
Munthe E, Rian E, Holien T, Rasmussen A, Levy FO, Aasheim H (2000) Ephrin-B2 is a candidate ligand for the Eph receptor, EphB6. FEBS Lett 466(1):169–174. https://doi.org/10.1016/s0014-5793(99)01793-7
Bhushan L, Tavitian N, Dey D, Tumur Z, Parsa C, Kandpal RP (2014) Modulation of liver-intestine cadherin (Cadherin 17) expression, ERK phosphorylation and WNT signaling in EPHB6 receptor-expressing MDA-MB-231 cells. Cancer Genom Proteom 11(5):239–249
Guo H, Huang ZL, Wang W, Zhang SX, Li J, Cheng K, Xu K, He Y, Gui SW, Li PF, Wang HY, Dong ZF, Xie P (2017) iTRAQ-based proteomics suggests ephb6 as a potential regulator of the ERK Pathway in the prefrontal cortex of chronic social defeat stress model mice. Proteomics Clin App. https://doi.org/10.1002/prca.201700115.10.1002/prca.201700115
Maddigan A, Truitt L, Arsenault R, Freywald T, Allonby O, Dean J, Narendran A, Xiang J, Weng A, Napper S, Freywald A (2011) EphB receptors trigger Akt activation and suppress Fas receptor-induced apoptosis in malignant T lymphocytes. Journal of Immunology 187(11):5983–5994. https://doi.org/10.4049/jimmunol.1003482
Zangrossi M, Romani P, Chakravarty P, Ratcliffe CDH, Hooper S, Dori M, Forcato M, Bicciato S, Dupont S, Sahai E, Montagner M (2021) EphB6 regulates TFEB-lysosomal pathway and survival of disseminated indolent breast cancer cells. Cancers 13(5):1079. https://doi.org/10.3390/cancers13051079
Hanover G, Vizeacoumar FS, Banerjee SL, Nair R, Dahiya R, Osornio-Hernandez AI, Morales AM, Freywald T, Himanen JP, Toosi BM, Bisson N, Vizeacoumar FJ, Freywald A (2023) Integration of cancer-related genetic landscape of Eph receptors and ephrins with proteomics identifies a crosstalk between EPHB6 and EGFR. Cell Rep 42(7):112670. https://doi.org/10.1016/j.celrep.2023.112670
Rubinfeld H, Shimon I (2012) PI3K/Akt/mTOR and Raf/MEK/ERK signaling pathways perturbations in non-functioning pituitary adenomas. Endocrine 42(2):285–291. https://doi.org/10.1007/s12020-012-9682-3
Funding
Open access funding provided by Tel Aviv University. This work was partially supported by a grant from the Fingerhot Karol and Lionora Foundation, Faculty of Medicine, Tel-Aviv University (to I.S. 0601253951).
Author information
Authors and Affiliations
Contributions
HR: investigation; methodology; writing – original draft. ZR, UB, SFH, ALB: human samples providing. CD: performance of the qPCR reactions of mice samples. PM: supervision of mice experiments. IS: funding acquisition; supervision; writing –review and editing.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Ethical approval
Samples of human pituitary tumors were obtained during curative transsphenoidal surgical resection with patients' informed consent in accordance with methods and conditions approved by the local institutional review board. Animal experiments were performed after protocol approval by the Institutional Animal Care and Use Committee of Technion – Israel Institute of Technology.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Rubinfeld, H., Cohen, Z.R., Bendavid, U. et al. Erythropoietin-producing hepatocellular receptor B6 is highly expressed in non-functioning pituitary neuroendocrine tumors and its expression correlates with tumor size. Mol Biol Rep 51, 297 (2024). https://doi.org/10.1007/s11033-023-09186-7
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11033-023-09186-7