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Title Phospholipase D2 Promotes Disease Progression Of View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Kyoto University Research Information Repository Phospholipase D2 promotes disease progression of renal cell Title carcinoma through the induction of angiogenin Kandori, Shuya; Kojima, Takahiro; Matsuoka, Taeko; Yoshino, Author(s) Takayuki; Sugiyama, Aiko; Nakamura, Eijiro; Shimazui, Toru; Funakoshi, Yuji; Kanaho, Yasunori; Nishiyama, Hiroyuki Citation Cancer Science (2018), 109(6): 1865-1875 Issue Date 2018-06 URL http://hdl.handle.net/2433/233106 © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. This is an open access article under the terms of Right the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. Type Journal Article Textversion publisher Kyoto University Received: 2 October 2017 | Revised: 1 March 2018 | Accepted: 4 April 2018 DOI: 10.1111/cas.13609 ORIGINAL ARTICLE Phospholipase D2 promotes disease progression of renal cell carcinoma through the induction of angiogenin Shuya Kandori1 | Takahiro Kojima1 | Taeko Matsuoka1 | Takayuki Yoshino1 | Aiko Sugiyama2 | Eijiro Nakamura2 | Toru Shimazui3,4 | Yuji Funakoshi5 | Yasunori Kanaho5 | Hiroyuki Nishiyama1 1Faculty of Medicine, Department of Urology, University of Tsukuba, Tsukuba, A hallmark of clear cell renal cell carcinoma (ccRCC) is the presence of intracellular Japan lipid droplets (LD) and it is assumed that phosphatidic acid (PA) produced by phos- 2 DSK Project, Medical Innovation Center, pholipase D (PLD) plays some role in the LD formation. However, little is known Kyoto University Graduate School of Medicine, Kyoto, Japan about the significance of PLD in ccRCC. In this study, we examined the expression 3Department of Urology, Ibaraki Prefectural levels of PLD in ccRCC. The classical mammalian isoforms of PLD are PLD1 and Central Hospital, Kasama, Japan PLD2, and the levels of both mRNA were higher at the primary tumor sites than in 4Faculty of Medicine, Department of Urology, Ibaraki Clinical Education and normal kidney tissues. Similarly, both PLD were significantly abundant in tumor cells Training Center, University of Tsukuba, as determined by analysis using immunohistochemical staining. Importantly, a higher Tsukuba, Japan level of PLD was significantly associated with a higher tumor stage and grade. 5Department of Physiological Chemistry, Faculty of Medicine and Graduate School Because PLD2 knockdown effectively suppressed the cell proliferation and invasion of Comprehensive Human Sciences, of ccRCC as compared with PLD1 in vitro, we examined the effect of PLD2 in vivo. University of Tsukuba, Tsukuba, Japan Notably, shRNA-mediated knockdown of PLD2 suppressed the growth and invasion Correspondence of tumors in nude mouse xenograft models. Moreover, the higher expression of Hiroyuki Nishiyama, Faculty of Medicine, Department of Urology, University of PLD2 was significantly associated with poorer prognosis in 67 patients. As for genes Tsukuba, Tsukuba, Ibaraki, Japan. relating to the tumor invasion of PLD2, we found that angiogenin (ANG) was posi- Email: [email protected] tively regulated by PLD2. In fact, the expression levels of ANG were elevated in Funding information tumor tissues as compared with normal kidney and the inhibition of ANG activity Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science with a neutralizing antibody significantly suppressed tumor invasion. Overall, we to TK (KAKENHI, No. 26462399) and EN revealed for the first time that PLD2-produced PA promoted cell invasion through (KAKENHI, No.17H04328). the expression of ANG in ccRCC cells. KEYWORDS angiogenin, cancer progression, phosphatidic acid, phospholipase D2, renal cell carcinoma 1 | INTRODUCTION those with confined primary tumors will develop metastatic disease.3 Although new target therapies and immunotherapies have emerged,1 Renal cell carcinoma (RCC) accounts for approximately 3% of all their efficacy is not sufficient to overcome advanced RCC. adult malignancies and there has been a continuous increase of Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidyl- new cases during the most recent 10-year period.1,2 Approximately choline into phosphatidic acid (PA) and choline. The classical mam- one-quarter of patients with RCC present with locally advanced or malian isoforms of PLD are PLD1 and PLD2.4,5 As a lipid second metastatic disease at diagnosis, and approximately 20%-40% of messenger, PA produced by PLD plays roles in numerous essential ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. | Cancer Science. 2018;109:1865–1875. wileyonlinelibrary.com/journal/cas 1865 1866 | KANDORI ET AL. cellular functions, such as vesicular trafficking, exocytosis, autophagy rehydrated. For antigen retrieval, the sections were pretreated by and the regulation of cellular metabolism.6 A hallmark of clear cell microwave for 21 minutes in a citric acid buffer. After the antigen RCC (ccRCC), the most common histological type of RCC, is the pres- retrieval procedure, endogenous peroxidase activity was blocked ence of intracellular lipid droplets (LD), which consist of a neutral lipid with 3% H2O2 for 25 minutes, and the slides were incubated with core containing triglycerides and cholesterol-esters surrounded by a the primary antibody at 4°C overnight. The immunohistochemical phospholipid monolayer.7 Although it has been reported that PA pro- reaction was visualized using the secondary antibody Histofine duced by PLD promotes the formation of intracellular LD,8-10 Wetter- Simple Stain MAX PO (Nichirei Bioscience, Tokyo, Japan) with sten et al11 showed that LD was lost in the high grade Fuhrman 4 or diaminobenzidine as the chromogen. The primary antibodies were spindle cell type. Interestingly, a previous report showed that the listed in Table S2. To evaluate the protein expression of PLD1 expression and the activity of PLD2 was elevated in ccRCC;12 how- and PLD2, the staining intensities of tumor cells were stratified ever, little is known about the roles of PLD in the tumor progression into high and low subgroups. When tumor cells were strongly of ccRCC. stained in not only cell membrane but also cytoplasm, we defined We previously reported the significant roles of PLD1 in the this as high expression. The other cases were defined as low tumor microenvironment of a mouse melanoma lung metastasis expression. model, showing that the administration of the PLD inhibitor led to a significant reduction of tumor angiogenesis and metastases.13 2.3 | Cell culture Importantly, the elevated expression and the activity of PLD were associated with cancer proliferation,14 survival,15 invasion and metas- 293T was purchased from RIKEN BioResource Center (Ibaraki, tasis15-18 in a series of human cancers.19 Japan). SKRC52 and SKRC59 cells were kindly gifted by Dr JG Old Based on the above findings, we have examined the roles of (Memorial Sloan Kettering Cancer Center, NY, USA). These cell lines PLD in the tumor progression of ccRCC. In this report, it was were cultured in RPMI 1640 medium or DMEM supplemented with revealed that PLD2 promoted tumor growth and the invasion of 10% FBS at 37°C and a 5% humidified CO2 atmosphere. ccRCC cells, and the level of PLD2 in a tumor specimen was signifi- cantly correlated with the prognoses of patients. Moreover, we clari- 2.4 | Quantitative RT-PCR fied a new mechanism of PLD2-mediated cell invasion through the induction of angiogenin (ANG). Our results suggest that PLD2 might Gene expression levels were quantitatively measured using a 7500 become a new therapeutic target in the treatment of advanced RCC. Fast Real-Time PCR machine with Fast SYBR Green Master Mix (Applied Biosystems). Hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as an internal control. The primer sequences were 2 | MATERIALS AND METHODS listed in Table S3. 2.1 | Patients and clear cell renal cell carcinoma samples 2.5 | Western blot analysis Samples of primary sites from 67 patients who received radical or par- Western blot analysis was carried out as described previously.22 tial nephrectomy were obtained at Tsukuba University Hospital The primary antibody concentrations used for western blot analysis between 2006 and 2015 under the protocols approved by the Ethics are listed in Table S4. As a secondary antibody, anti-rabbit or anti- Committee of Tsukuba University. These samples were used as forma- mouse immunoglobulin G, HRP-linked whole donkey Ab (GE lin-fixed and paraffin-embedded (FFPE) tissue sections of the primary Healthcare), was used at 1:10 000. Western blots were visualized sites. In 52 of 67 patients, frozen tissue samples of the cancer and with ImmunoStar Zeta (Wako, Tokyo, Japan) using a Fujifilm LAS- adjacent normal region were available for the primary sites. These 4000 imager (Fujifilm, Tokyo, Japan). b-actin was used as an
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