Wu et al. Cell Death and Disease (2021) 12:319 https://doi.org/10.1038/s41419-021-03617-8 Cell Death & Disease ARTICLE Open Access A Zic2/Runx2/NOLC1 signaling axis mediates tumor growth and metastasis in clear cell renal cell carcinoma Chen-Yan Wu 1,LeiLi2,Shi-LuChen1, Xia Yang1, Chris Zhiyi Zhang 3 and Yun Cao 1 Abstract Clear cell renal cell carcinoma (ccRCC) is one of the most common malignancies with rapid growth and high metastasis, but lacks effective therapeutic targets. Here, using public sequencing data analyses, quantitative real-time PCR assay, western blotting, and IHC staining, we characterized that runt-related transcription factor 2 (Runx2) was significantly upregulated in ccRCC tissues than that in normal renal tissues, which was associated with the worse survival of ccRCC patients. Overexpression of Runx2 promoted malignant proliferation and migration of ccRCC cells, and inversely, interfering Runx2 with siRNA attenuates its oncogenic ability. RNA sequencing and functional studies revealed that Runx2 enhanced ccRCC cell growth and metastasis via downregulation of tumor suppressor nucleolar and coiled-body phosphoprotein 1 (NOLC1). Moreover, increased Zic family member 2 (Zic2) was responsible for the upregulation of Runx2 and its oncogenic functions in ccRCC. Kaplan–Meier survival analyses indicated that ccRCC patients with high Zic2/Runx2 and low NOLC1 had the worst outcome. Therefore, our study demonstrates that Zic2/ Runx2/NOLC1 signaling axis promotes ccRCC progression, providing a set of potential targets and prognostic indicators for patients with ccRCC. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction molecular characteristics and histological classification, Renal cell carcinoma (RCC), also known as renal ade- clear cell renal cell carcinoma (ccRCC) is the most com- nocarcinoma, is a common malignant tumor arising from mon histological subtype in RCC accounting for nearly the renal tubular epithelial system. In 2019, the cancer 80% of RCC patients3. Although patients with early-stage statistics in United States showed an estimated 73,820 ccRCC can obtain a well therapeutic effect through new diagnoses and 14,770 deaths for RCC. The incidence nephrectomy or nephrectomy-preserving tumor resec- and mortality of RCC are increasing every year1. There are tion, there are still many cases with local recurrence or main causes of RCC, such as smoking, obesity, cadmium distant metastasis. Especially metastatic ccRCC, the effi- exposure, the use of chronic analgesics, cystic kidney cacy of traditional chemical drugs is insignificant because disease, and certain genetic syndromes2. According to of the high drug resistance of ccRCC cells4,5. Therefore, investigating the key oncogenes involved in the aberrant proliferation and metastasis of ccRCC is a valuable goal Correspondence: Chris Zhiyi Zhang ([email protected])or toward revealing the mechanisms of cancer progression Yun Cao ([email protected]) and identifying new therapeutic targets for ccRCC 1 Department of Pathology, State Key Laboratory of Oncology in South China treatment. and Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, 510060 Guangzhou, China Runt-related transcription factor (Runx) proteins, 2Department of Clinical Oncology, The University of Hong Kong, Hong Kong, including Runx1, Runx2, and Runx3, are known as tran- China scription factors in the physiological and pathological pro- Full list of author information is available at the end of the article These authors contributed equally: Chen-Yan Wu, Lei Li cesses of the body. Runx2 as a member of the Runx family is Edited by G.-Q. Chen © The Author(s) 2021 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 linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Wu et al. Cell Death and Disease (2021) 12:319 Page 2 of 11 widely involved in bone formation and osteoblast differ- University Cancer Center (Guangzhou, China). Human entiation6. Furthermore, Runx2 effectively prevented immortalized renal epithelial cell 293T and ccRCC cell apoptosis of intestinal epithelial cells during the develop- lines ACHN, 786-O, SKRC39, and CAKI-1 were pre- ment of Crohn’s disease7. In the last decade, Runx2 has served in our laboratory. All cells were cultured with been shown to be involved in the regulation of cancer DMEM (Gibco) supplemented with 10% fetal bovine progression with dual functions of oncogenic potential and serum (FBS, Gibco) at 37 °C with 5% CO2. When the cells tumor suppressor abilities8,9. Transcriptional inhibition of reached 80–90% confluence, digestion and passage were Runx2 was negatively correlated with aggressive clin- performed with trypsinization. icopathological outcomes, whereas nuclear location of Runx2 promoted metastasis in prostate cancer10.Over- Plasmid construction, RNA interference, and cell expression of Runx2 suppressed osteosarcoma cell growth transfection in vitro11. Runx2 could inhibit p53-dependent apoptosis The coding sequence of Runx2 was obtained by reverse through the functional collaboration with HDAC6 in transcription PCR (RT-PCR) and cloned into pZM02 response to DNA damage12. In addition, increasing evi- vector. The promoter of NOLC1 was also amplified by dences suggested an aggressive role of Runx2 in various RT-PCR and cloned into pGL4.10 luciferase reporter cancers. For instance, high expression of Runx2 was sig- vector. The expressions of Zic2, Runx2, and NOLC1 were nificantly correlated with larger tumor size, lymph node silenced with siRNA oligonucleotides (Table S1). Scram- metastasis, and shorter postoperative survival time of ble RNA was used as a negative control. The culture patients with non-small cell lung cancer13. Repression of medium was changed to fresh DMEM with 10% FBS 24 h Runx2 transcriptional activity blocked the proliferation, before transfection. The plasmids and siRNA oligonu- migration, and invasion of epithelial ovarian carcinoma cleotides were transfected into cancer cells with Lipo- cells14. In-depth studies revealed that Runx2 promoted fectamine 2000 (50 nM, Invitrogen). Transfected cells cancer progression by inducing the epithelial–mesenchymal were cultured at 37 °C with 5% CO2 for 6 h, and then the transition (EMT) progress in breast cancer15, thyroid car- culture medium was replaced by fresh normal medium. cinoma16, and hepatocellular carcinoma (HCC)17.There- The expression levels of target genes were confirmed with fore, dysregulation of Runx2 modulates tumor cell quantitative real-time PCR (qRT-PCR) and western blot proliferation and metastasis, and revealing the underlying at 24 or 36 h after transfection, respectively. mechanism is urgent in the future development of indivi- dual treatments against different types of cancer. However, RNA extraction and qRT-PCR the expression and role of Runx2 in ccRCC are, as yet, Total RNA from fresh tissues or cell lines was extracted unclear. with TRIzol Reagent (Invitrogen). Note that fresh ccRCC In the present study, we clearly characterized the expres- and normal renal tissues were frozen with liquid nitrogen sion and prognosis prediction of Runx2 in ccRCC, and found and grinded until powdered before lysis with TRIzol. that aberrant overexpression of Runx2 was significantly Complementary DNA was synthesized with reverse associated with poor survival of ccRCC patients. Functional transcriptase (Takara) according to the manufacturer’s and mechanism studies revealed that Runx2 was upregulated instruction. The mRNA expression levels of genes were by Zic family member 2 (Zic2) and enhanced ccRCC cell analyzed by qRT-PCR using FastStart Universal SYBR proliferation and migration via transcriptional inhibition of Green Master (Roche) and a Real-Time PCR Detection tumor suppressor nucleolar and coiled-body phosphoprotein System (Roche); 18S rRNA was tested as an internal 1 (NOLC1). Therefore, our findings clearly demonstrate that control. The relative expression level (defined as fold −ΔΔ dysregulation of Zic2/Runx2/NOLC1 signaling promotes change) of the target gene (2 Ct) was normalized to the ccRCC progression. endogenous 18S reference (ΔCt). The primers used were listed in Table S2. Materials and methods Clinical samples and cell lines Western blot Human ccRCC and normal renal tissues were obtained Western blot analysis was performed according to the from ccRCC patients without preoperative chemor- standard protocol18. Human fresh ccRCC/normal renal adiotherapy treatment at Sun Yat-sen University Cancer tissues and cells were lysed with RIPA lysis buffer (Cell Center (Guangzhou, China). Tissue microarray contain- Signaling Technology) supplemented with cocktail pro- ing 301 paired ccRCC and normal renal tissues was tease inhibitor (Roche)