Published OnlineFirst October 22, 2018; DOI: 10.1158/0008-5472.CAN-18-1662 Cancer Molecular Cell Biology Research The E2F1–miR-520/372/373–SPOP Axis Modulates Progression of Renal Carcinoma Meng Ding1,2, Xiaolan Lu1,2, Cheng Wang1,2, Quan Zhao2, Jingping Ge3, Qiuyuan Xia4, Junjun Wang1,KeZen2, Chen-Yu Zhang2, and Chunni Zhang1,2 Abstract Although renal cell carcinoma (RCC) is the most malignant nificantly decreased both tumor size and lung metastasis urologic cancer, its pathogenesis remains unclear, and effective ratio in mice bearing orthotopic xenograft tumors. Decreased treatments for advanced RCC are still lacking. Here, we report expression of miR-520/372/373 family was mediated by tran- that a novel E2F1–miR-520/372/373–SPOP axis controls RCC scription factor E2F1. In conclusion, our results demonstrate carcinogenesis. Speckle-type POZ protein (SPOP) was upre- that the E2F1-miR-520/372/373–SPOP axis functions as a gulated in over 90% of RCC tissues, whereas the miR-520/372/ key signaling pathway in RCC progression and metastasis and 373 family was downregulated and correlated inversely with represents a promising opportunity for targeted therapies. SPOP protein levels in RCC tissues. The miR-520/372/373 family targeted the SPOP 30-UTR and suppressed SPOP Significance: These findings show that the E2F1-miR-520/ protein expression, leading to elevation of PTEN and DUSP7 372/373 family–SPOP axis promotes RCC progression, levels and, consequently, decreased proliferation, invasion/ thereby contributing to our understanding of RCC patho- migration, and metastasis of RCC cells in vitro and in vivo. genesis and unveiling new avenues for more effective Tail-vein delivery of therapeutic miR-520/372/373 family sig- targeted therapies. Cancer Res; 78(24); 1–14. Ó2018 AACR. Introduction required (5, 6). Speckle-type POZ protein (SPOP) is a BTB/POZ domain protein that encodes an E3 ubiquitin ligase component Renal cell carcinoma (RCC) accounts for approximately 4% of (7). SPOP has been reported to participate in many biological all cancers and is the third most common urologic malignancy, functions, especially in regulating tumor growth including RCC which has the highest fatality rate among all urologic cancers (1, (6, 8–13). SPOP is highly expressed in RCC, in particular, in 2). RCC is notorious for its resistance to routine chemotherapy ccRCC (6, 8, 11, 12). It has been demonstrated that SPOP is and radiotherapy (3). When metastasis occurs, it is mostly incur- transcriptionally activated by hypoxia-inducible factor (HIF; able with a very low 5-year survival rate (3). The most commonly refs. 11, 14, 15) and induces RCC tumorigenesis through observed RCC subtype is clear-cell renal cell carcinoma (ccRCC), degradation of some tumor suppressors, such as PTEN, ERK which accounts for approximately 75%–85% of all cases of RCC, phosphatase, and DUSP7 in RCC (6, 11, 16, 17). Therefore, with the highest rates of local invasion, metastasis, mortality, and SPOP is regarded as an attractive target specifictoRCCthatmay refraction to current treatments (4, 5). Although intensive effort yield novel drug discovery efforts (11, 12). Recently, it has been has been put toward exploring the pathogenesis of this disease, shown that a structure-based design and subsequent hit opti- the mechanisms involved in the development and progression of mization yield small molecules that can inhibit the SPOP– RCC and more effective therapeutic approaches are urgently substrate protein interaction and thus suppress oncogenic SPOP signaling pathways in kidney cancer (12). However, 1 despite recent exploration of biological functions, studies on Department of Clinical Laboratory, Jinling Hospital, State Key Laboratory of fi Analytical Chemistry for Life Science, NJU Advanced Institute for Life Sciences the mechanism of SPOP expression in RCC are still insuf cient (NAILS), School of Life Sciences, Nanjing University, Nanjing, China. 2State Key (6, 18). Further investigation of the biological function, clinical Laboratory of Pharmaceutical Biotechnology and Jiangsu Engineering Research significance, and mechanism of SPOP is critical for an under- Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for standing of RCC pathogenesis. Life Sciences (NAILS), School of Life Science, Nanjing University, Nanjing, China. The discovery of miRNAs has added another layer of complex- 3Department of Urology, Jinling Hospital, Nanjing University School of Medicine, 4 ity to the control of gene expression. miRNAs are recently dis- Nanjing University, Nanjing, China. Department of Pathology, Jinling Hospital, – Nanjing University School of Medicine, Nanjing University, Nanjing, China. covered small (19 22 nucleotides) noncoding RNAs that func- tion as repressors of gene activity by binding to complementary Note: Supplementary data for this article are available at Cancer Research sequences in the 30 untranslated region (30-UTR) of target gene Online (http://cancerres.aacrjournals.org/). transcripts, leading to mRNA degradation and/or translational M. Ding, X. Lu, and C. Wang contributed equally to this article. repression (19, 20). Conservative estimates show that approxi- Corresponding Authors: Chunni Zhang, Department of Clinical Laboratory, mately 60% of all human mRNAs are regulated by miRNAs, Jinling Hospital, Nanjing University, 305 East Zhongshan Rd., Nanjing covering virtually all cellular functions (21). We and others have 210002, China. Phone: 8625-8086-3082; Fax: 8625-8368-6234; E-mail: shown that miRNAs are frequently aberrantly expressed in cancer [email protected]; and Chen-Yu Zhang, [email protected] tissues, have been referred to as "onco(genic)-miRNAs" or "tumor doi: 10.1158/0008-5472.CAN-18-1662 suppressor miRNAs," and play crucial roles in diverse regulation Ó2018 American Association for Cancer Research. pathways in multiple types of tumors, including RCC (22–24) www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst October 22, 2018; DOI: 10.1158/0008-5472.CAN-18-1662 Ding et al. (18)In this study, we found that SPOP was specifically targeted by (Gibco) and incubated in 5% CO2 at 37 C in a water-saturated the miR-520/372/373 family, which controlled the development atmosphere. All cell lines were confirmed to be Mycoplasma-free and progression of RCC both in vitro and in vivo, indicating a and authenticated by short tandem repeat DNA profiling by tumor suppressor role of this miRNA family in RCC. Furthermore, Genewiz Inc. we demonstrated the mechanism by which this miRNA family was regulated by the transcription factor E2F1. The newly iden- miRNA-related reagents, plasmid, and transfection tified E2F1-miR-520/372/373 family–SPOP axis functions as a miRNA mimics and negative controls were purchased from key pathway regulating tumorigenesis of RCC. GenePharma (GenePharma), and lentiviruses to overexpress miR- 520c-3p, miR-372-3p, and miR-373-3p were purchased from Materials and Methods Invitrogen. Cholesterol-conjugated miRNA mimics and siRNA sequences targeting different sites of gene open reading frames Tissue samples, IHC staining, and H&E staining (ORF) of SPOP and E2F1 were designed and synthesized by Tissue microarray (TMA) chips containing a total of 90 pairs of RiboBio Co., Ltd., and the sequences of the siRNAs were listed ccRCC samples and matched normal adjacent tissues (NAT) were in Supplementary Table S2. obtained from Shanghai Biochip Co., Ltd (Shanghai, China). For the protein overexpression assay, a mammalian expression Fresh human surgical specimens (paired normal and cancerous plasmid encoding the human SPOP ORF without the 30-UTR was tissues) were obtained from 30 patients undergoing surgery for purchased from GeneCopoeia. An empty plasmid (pReceiver- ccRCC at Jinling Hospital of Nanjing University (Nanjing, China). M02) served as the negative control. Transfection was performed All patients underwent a tumor resection prior to the receipt of any with Lipofectamine 2000 reagents (Invitrogen) according to the adjunctive therapy. The tissue collection and analyses were manufacturer's protocol. Cells were used for the experiments after approved by the Ethics Committee of Jinling Hospital (Nanjing, transfection for 24 or 48 hours. China), and all participants provided written informed consent. The studies were conducted in accordance with the International RNA isolation and qRT-PCR Ethical Guidelines for Biomedical Research Involving Human Total RNA was extracted from tissues and cells with TRIzol Subjects (CIOMS). All fresh tissues were immediately snap-frozen reagent (Invitrogen) following the manufacturer's protocol. in liquid nitrogen and stored at À80 C until analysis. IHC and Reverse transcription and qRT-PCR for miRNAs were performed hematoxylin and eosin (H&E) staining were performed using as described previously (25). qRT-PCR for SPOP and E2F1 paraffin-embedded sections of biopsies from ccRCC samples and mRNA were performed using a SYBR Premix E Â Taq Reverse matched NATs according to standard protocols by Cell Signaling Transcription PCR kit (Takara), and the expression levels were Technology. The antibodies used for IHC were as follows: anti- normalized to GAPDH. The sequences of the primers were SPOP antibody (ProteinTech; 16750-1-AP), anti-E2F1 antibody listed in Supplementary Table S3. The reactions were performed (Cell Signaling Technology; #3742), anti-Ki67 antibody (Cell using the LightCycler 480II Detection