in vivo 35 : 2631-2640 (2021) doi:10.21873/invivo.12545 The Expression of NRIP1 and LCOR in Endometrioid Endometrial Cancer STEFANOS FLINDRIS 1, NIKOLAOS KATSOULAS 2, ANNA GOUSSIA 3, ANDREAS CHRISTOS LAZARIS 2, IORDANIS NAVROZOGLOU 1, MINAS PASCHOPOULOS 1 and IRENE THYMARA 2 1Department of Obstetrics and Gynecology, University Hospital of Ioannina, Ioannina, Greece; 2First Department of Pathology, School of Medicine, National and Kapodistrian University of Athens, Laiko General Hospital of Athens, Athens, Greece; 3Department of Pathology, University Hospital of Ioannina, Ioannina, Greece Abstract. Background: The aim of the study was to Endometrial cancer is the sixth most common cancer among analyze the expression of nuclear receptor interacting females worldwide and the fourth leading cause of cancer protein 1 (NRIP1) and its partner ligand-dependent nuclear (1). It is considered as the most common gynecological receptor co-repressor (LCOR) in endometrioid endometrial malignancy, accounting for 7% of all female cancer (1). Two cancer and to investigate their association with estrogen broad pathogenetic types of endometrial carcinoma have receptor (ER), progesterone receptor (PR), Ki-67, been recognized: Type I tumors are endometrioid carcinomas clinicopathological parameters and patient survival. and type II are non-endometrioid, including serous and clear- Materials and Methods: Immunohistochemical evaluation cell carcinomas (2). Most cases (90%) of endometrial was carried out to investigate the subcellular expression of carcinomas occur in women older than 50 years of age, with NRIP1 and LCOR in endometrioid endometrial cancer a median age of 62 years at diagnosis, and are detected in samples. Statistical analysis was used to identify the early stages [80% in International Federation of Obstetrics correlations of NRIP1 and LCOR expression with and Gynecology (FIGO) stage I] (2, 3). clinicopathological variables and to estimate the survival Recent studies have focused on the involvement of nuclear rates. Results: Endometrial cancer tissues exhibited higher receptor interacting protein 1 (NRIP1/RIP140) and Ligand- expression of NRIP1 and LCOR in comparison with the dependent nuclear receptor co-repressor (LCOR) in tumor normal tissues. Cytoplasmic LCOR expression was development through complex regulation of pathogenetic positively associated with ER and PR expression, while pathways (4). NRIP1 is a co-regulator which consists of four cytoplasmic NRIP1 expression was positively associated inhibitory domains that recruit C-terminal binding proteins, with ER expression. Moreover, cytoplasmic expression of and one of histone deacetylase (HDAC) (5-8). It acts NRIP1 was positively associated with Ki-67. Conclusion: primarily as a co-repressor through binding to nuclear Our study demonstrated that high cytoplasmic expression of receptors of other co-factors in a ligand-dependent manner LCOR may predict a longer overall survival of patients with to limit their transactivation (9-12). Intriguingly, NRIP1 endometrioid endometrial cancer. Patients with tumors interacts with E2F transcription factors, suppressing their expressing low levels of LCOR showed a worse survival transcriptional function and also suppresses proliferator compared to those expressing high levels. activated receptor gamma (PPARG) through receptor coactivator 1 (NCOA1), thereby inhibiting cell proliferation (13). This kind of dysregulation may affect other signaling pathways, such as NOTCH, p53, Hedgehog and Hippo, resulting in up-regulation of their expression on cancer cells This article is freely accessible online. (14). According to many studies, NRIP1 is engaged in the development and progression of solid tumors (15-18). Correspondence to: Stefanos Flindris (ORCID: 0000-0002-2361- Ferreira et al. showed that NRIP1 to be the most frequently 3470), MD, Resident in Obstetrics and Gynecology, University mutated gene in endometrial cancer cell lines and to be Hospital of Ioannina, Stavros Niarchos Avenue, 45500 Ioannina, Greece. Mobile: +30 6957342754, e-mail: [email protected] associated with microsatellite instability, dysregulating the gene expression of estrogen receptor (ER) pathway (19). Key Words: Endometrioid endometrial cancer, NRIP1, LCOR, ER, NRIP1 is considered an essential transcriptional co-factor for PR. estrogen signaling (11, 20). It has been found that it interacts 2631 in vivo 35 : 2631-2640 (2021) mainly with ER β and may also act as a tumor suppressor in Materials and Methods ovarian and colonic cancer (14, 21). Specifically, in ovarian cancer NRIP1 may be involved in the suppression of ER α Patient characteristics. Representative paraffin-embedded tissue activity through ER β (21). In colonic cancer, NRIP1 has a samples were selected from 93 patients with endometrioid endometrial carcinoma, who were treated with total or radical (when negative effect on the WNT signaling pathway by reducing appropriate) hysterectomy and bilateral salgingo-ophorectomy at the gene expression and thereby inhibiting colonic cancer cell Department of Obstetrics and Gynecology, University Hospital of proliferation (14). Similarly, high expression of NRIP1 in Ioannina, Ioannina Greece during the period of 2008-2015. All chronic lymphocytic leukemia has a favorable prognostic tumors were classified using the 2018 FIGO classification (IA-B II, significance (15). Importantly, the NRIP1 expression level IIIA-B/C1-2, IVA-B) (32), myometrial invasion and the presence or was higher in the nucleus of breast cancer cells, whereas it absence of metastases was determined by computed tomography was higher in the cytoplasm of stromal cells in benign (33). Patient data, such as age, pathology reports, status of metastasis, local recurrence, record of adjuvant therapy after tumors (10, 22). surgery, radiotherapy treatment and outcome were retrieved from Another transcriptional co-repressor, LCOR functions the clinical database of the Departments of Pathology and Obstetrics similarly to the aforementioned mechanisms of NRIP1, and Gynecology of University Hospital of Ioannina, Ioannina, using C-terminal binding proteins and HDAC domains (8, Greece. In order to compare our findings with normal tissues, 23). Several studies demonstrated that NRIP1 and LCOR samples were collected from 23 patients who had undergone total may play a key role in tumorigenesis (14, 24-26). It has hysterectomy for benign uterine tumors (leiomyomas). All patient been shown that NRIP1 directly interacts with LCOR, and data were fully anonymized, and all diagnostic procedures had already been fully completed when samples were collected for the that both proteins co-localize in the nucleus of human breast study. Authors were blinded from the clinical information during the cancer cells (24). The physiological role of LCOR is poorly experimental analysis. The study was approved by the Ethics understood, although it may participate in homeostasis of Committee of the National and Kapodistrian University of Athens, liver and in prostate cancer (27, 28). Of importance, LCOR Athens, Greece (approval number 095, 25/02/2019). interacts with ER α and drives repressive activity in breast cancer cells (8, 11). In breast cancer cells, proliferation is Immunohistochemical analysis. Formalin-fixed paraffin-embedded suppressed by LCOR through down-regulation of gene tissue sections were retrieved from the Department of Pathology, expression mediated by estrogens (22). This is the result of University of Ioannina, Ioannina, Greece. A hematoxylin-eosin the strong regulation of LCOR expression by NRIP1 (11). evaluation round was employed for obtaining representative tissue samples for immunohistochemical analysis. Immunohistochemistry Interestingly, LCOR interacts with a subunit of tripartite was performed on 2.5 μm-thick tissue sections, which were motif containing 28 (TRM28), kruppel-like factor 6 (KLF6) consecutively incubated with the following primary antibodies to and PPARG (29, 30). Moreover, NRIP1 and LCOR act NRIP1 (HPA046571, 1:400 in phosphate-buffered saline polyclonal synergistically, recruiting co-factors involved in rabbit IgG; Sigma Aldrich, St. Louis, MO, USA) and anti-LCOR (C- transcriptional co-repression or recognizing the same 6) sc-377019 (1:200 in phosphate-buffered saline mouse polyclonal coactivator binding domains in ligand binding domains of IgG; Santa Cruz Biotechnology, Dallas, TX, USA). The staining nuclear receptors (24, 26). In cervical cancer, low levels of procedures were performed by the avidin-biotin technique using LSAB2 System-HRP (Dako Omnis, Glostrup, Denmark). Appropriate NRIP1 and LCOR are associated with better survival tissues (placenta and salpinx) were used as positive controls, while outcome (25). Furthermore, LCOR was found to be an omission of primary antibodies was used as a negative control. For all attenuator of progesterone-regulated gene expression in tumor cases, immunostaining of ER, progesterone receptor (PR) and breast cancer, and inhibits prostate cancer growth through Ki-67 proteins was performed by using antibodies to ER (6F11, 1:40 co-repression of activated androgen receptor in murine in phosphate-buffered saline mouse monoclonal IgG1; Leica models (23, 27). Other studies demonstrated the relevance Biosystems Newcastle, Newcastle Upon Tyne, UK), PR (686, 1:150 of LCOR with inhibition of mammary cancer cell activity in phosphate-buffered saline mouse monoclonal IgG1; Dako Omnis) and anti-Ki-67 M7240 (1:150 mouse monoclonal IgG1; Dako Omnis). (23, 27, 31).