International Journal of Molecular Sciences Article Hypomethylated RRBP1 Potentiates Tumor Malignancy and Chemoresistance in Upper Tract Urothelial Carcinoma Hao-Lun Luo 1 , Hui-Ying Liu 1, Yin-Lun Chang 1, Ming-Tse Sung 2, Po-Yen Chen 1 , Yu-Li Su 3 , Chun-Chieh Huang 4 and Jei-Ming Peng 5,* 1 Department of Urology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan; [email protected] (H.-L.L.); [email protected] (H.-Y.L.); [email protected] (Y.-L.C.); [email protected] (P.-Y.C.) 2 Department of Pathology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan; [email protected] 3 Department of Hematology and Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan; [email protected] 4 Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan; [email protected] 5 Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan * Correspondence: [email protected]; Tel.: +886-2-7317123-8597 Abstract: Ribosome-binding protein 1 (RRBP1) is a potential oncogene in several cancer types. However, the correlation between RRBP1 expression and the prognosis of patients with upper tract urothelial carcinoma (UTUC) remains unclear. In this study, we identified that RRBP1 is associated with carcinogenesis and metastasis in UTUC using a methylation profiling microarray. Citation: Luo, H.-L.; Liu, H.-Y.; High correlations between RRBP1 and cancer stages, nodal metastasis status, molecular subtypes, Chang, Y.-L.; Sung, M.-T.; Chen, P.-Y.; and prognosis in bladder urothelial cancer (BLCA) were found. Aberrant DNA methylation in Su, Y.-L.; Huang, C.-C.; Peng, J.-M. the gene body region of RRBP1 was determined in UTUC tissues by methylation-specific PCR. Hypomethylated RRBP1 Potentiates Tumor Malignancy and RRBP1 expression was significantly increased in UTUC tissues and cell lines, as determined by Chemoresistance in Upper Tract real-time PCR and immunohistochemistry. RRBP1 depletion significantly reduced BFTC909 cell Urothelial Carcinoma. Int. J. Mol. Sci. growth induced by specific shRNA. On the other hand, molecular subtype analysis showed that the 2021, 22, 8761. https://doi.org/ expression of RRBP1 was associated with genes related to cell proliferation, epithelial–mesenchymal 10.3390/ijms22168761 transition, and basal markers. A patient-derived organoid model was established to analyze patients’ responses to different drugs. The expression of RRBP1 was related to chemoresistance. Taken Academic Editor: Michael Welsh together, these results provide the first evidence that RRBP1 gene body hypomethylation predicts RRBP1 high expression in UTUC. The data highlight the importance of RRBP1 in UTUC malignancy Received: 19 July 2021 and chemotherapeutic tolerance. Accepted: 12 August 2021 Published: 16 August 2021 Keywords: upper tract urothelial carcinoma; hypomethylation; RRBP1; oncogene; patient-derived organoid; chemoresistance Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Upper tract urothelial carcinoma (UTUC) is a cancer that is difficult to detect at an early stage, when it involves the renal pelvis and ureter [1]. The incidence of UTUC accounts for approximately 5% of all urothelial cancers, and invasive UTUC accounts for 60% of Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. all UTUC patients. The 5-year cancer-specific survival (CSS) rate after the onset of early This article is an open access article UTUC is <50% [2]. Compared with bladder cancer, the difficulty of UTUC discovery is that distributed under the terms and it is more difficult to detect in the early stages. This reflects the relative rarity of UTUC and conditions of the Creative Commons the consequent lack of knowledge of the mechanism of its pathogenesis. Currently, radical Attribution (CC BY) license (https:// nephroureterectomy (RNU) is the standard treatment for local UTUC [2,3]. Regarding creativecommons.org/licenses/by/ prognostic effectiveness, 33% of RNU cases relapse within 5 years [4], indicating a high 4.0/). recurrence rate and poor prognosis [2,5], with a 5-year CSS rate of 73% [2,5]. Additional Int. J. Mol. Sci. 2021, 22, 8761. https://doi.org/10.3390/ijms22168761 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 8761 2 of 19 systemic treatment is required. However, the disadvantage of RNU is that it damages renal function and reduces eligibility for chemotherapy [6,7]. According to the treatment guidelines of the European Association of Urology, the chemotherapy method of UTUC is currently based on UCB-based treatment [8,9]. Cisplatin- based neoadjuvant chemotherapy (NAC) is regarded as the standard treatment for patients with locally advanced UTUC [10]. An increasing number of studies have shown that NAC and ACH have a certain effect [11,12]. However, due to the different responses of patients to chemotherapy, these cannot be used arbitrarily [13,14]. The advantage of URS is that the lesions can be directly observed, evaluated, and biopsied. Several factors that can predict poor prognosis include age, tumor grade, tumor stage, lymph node metastasis, CLS, lymphatic vessel invasion, multifocal tumor size, and tumor size [9,15–19]. As there are few biomarkers used to evaluate UTUC, finding more effective tumor markers for evaluating the prognosis of UTUC could help predict prognosis and promote effective treatment. Ribosome-binding protein 1 (RRBP1) is an important endoplasmic reticular protein that plays a key role in ribosomal binding and the termination of differentiation in secretory cells and tissues [20–22]. RRBP1 consists of 1410 amino acids, including a transmembrane domain that is essential for the transport and secretion of new proteins [23,24]. Although RRBP1 is mainly located in the endoplasmic reticulum (ER), it can also be detected in the nucleus and cytoplasm [25]. RRBP1 may be a potential oncogene. The RRBP1 is highly expressed in a variety of cancers, including lung [26], ovarian [27], prostatic [28], esophageal [22], rectal [29], and breast cancer [30,31]. RRBP1 is also a biomarker for poor prognosis in colorectal, prostate, and breast cancers. In addition, RRBP1 is associated with tumor cell growth and resistance to the unfolded protein response (UPR) [26], which might be an important milestone in tumor management and a potential prognostic biomarker for tumors. Even so, few studies have explored the mechanism by which RRBP1 regulates tumor cell growth, and its current functions are mainly related to ER pressure control. The role of RRBP1 in urothelial carcinoma remains unclear. Further studies on its expression, regulatory mechanisms, intracellular signaling pathways, cancerous tumors, degree of malignancy, and prognostic factors in urothelial carcinoma would inform further studies that could benefit clinical treatment of various cancers. UTUC is a cancer that is difficult to detect in the early stage, with a marked propensity to invade and metastasize. Using DNA methylation microarray chip analysis, we identified the membrane protein RRBP1 as a tumor oncogene in UTUC cells, and the potential of RRBP1 as a clinical prognostic marker from The Cancer Genome Atlas (TCGA) BLCA database was further confirmed. Especially in advanced and metastatic UTUC, the ex- pression of RRBP1 was significantly higher than that in normal tissues or early tumors. RRBP1 was highly expressed in both malignant tumors and cell lines in clinical specimens. Analysis of the expression of gene groups affected by RRBP1 in patients’ samples correlated increased RRBP1 expression with increased proliferation and metastasis-related proteins. Lee et al. established a patient-derived organoid (PDO) model for bladder cancer that could be used to simulate tumor response to drugs [32]. Using this method, we established PDO of UTUC and performed drug cytotoxicity analysis. The results showed that progressively lower expression levels of RRBP1 were associated with progressively increasing sensitivity of PDO to cytotoxicity due to cisplatin, gemcitabine, and epirubicin. Thus, high expres- sion of RRBP1 was implicated as a potential oncogene marker in UTUC with reduced susceptibility to drugs. 2. Results 2.1. RRBP1 Is a Hypomethylated and Highly Expressed Oncogene in UTUC UTUC is difficult to detect at an early stage and there are few predictive factors that can be used to evaluate UTUC. Analysis of the expression level of cancer cell membrane proteins is helpful to identify applicable marker proteins and for clinical detection [33]. Previously, we identified SPARCL1 as an oncogene that promotes carcinogenesis in a Int. J. Mol. Sci. 2021, 22, 8761 3 of 19 bioinformatics database of UTUC provided by the National Institutes of Health (NIH) in Taiwan [34]. However, the role of membrane proteins in lymphovascular invasion in UTUC has not been clearly discussed. Thus, we further investigated the potential oncogenic factors in membrane proteins of UTUC and re-explored the NIH bioinformatics database of UTUC. Normal urothelial tissues of three patients with low-stage/low-grade UTUC were compared with three patients with high-stage/high-grade UTUC tumors from the database. A methyl-CpG binding-domain-based (MBD) protein microarray was used to compare