Abstract PR14: Mutations in microRNA processing genes dysregulate a miR-16/34-PLAG1-IGF2 axis in Wilms tumors

Abstract

Abstract Mutations in microRNA processing genes arise in ~20% of Wilms tumors, the most common pediatric kidney cancer. These mutations block microRNA production, yet the key microRNA target genes that drive tumorigenesis in this setting are unknown. To address this question, we performed whole-transcriptome and small RNA sequencing in Wilms tumors. In tumors with microRNA processing mutations, the most significantly overexpressed gene was Pleomorphic adenoma gene 1 (PLAG1). PLAG1 has known oncogenic activity in other cancers but has never been studied in Wilms tumor. This transcription factor is normally expressed only in the developing kidney and liver, where it transactivates insulin-like growth factor 2 (IGF2). Because IGF2 overexpression plays a major role in Wilms tumor pathogenesis, we hypothesized that microRNA pathway mutations contribute to Wilms tumor formation by de-repressing PLAG1, thus overexpressing IGF2. To determine the effect of ectopic PLAG1 expression in vivo, we generated mice that overexpress PLAG1 throughout the developing kidney (Wt1-Cre; LSL-PLAG1). These mice develop grossly enlarged kidneys and die by 4 weeks of age. Prenatally, nephron production is impaired, resulting in fewer glomeruli and dilated tubules. Importantly, PLAG1-overexpressing kidneys progressively acquire several neoplastic characteristics. The kidney parenchyma becomes replaced by cysts that develop polyps and dysplastic thickening. As these dysplastic cells continue to proliferate and fill the cyst lumen, they form nodules that appear precancerous and exhibit markers of active proliferation (Ki-67) and Igf2 signaling (phospho-Erk1/2, phospho-S6). Thus, ectopic PLAG1 expression in the developing kidney recapitulates the two key features of Wilms tumor: blocked differentiation and neoplastic proliferation. We next confirmed that PLAG1 is a microRNA target gene that promotes proliferation of Wilms tumor cells in vitro. Luciferase reporter assays demonstrated that highly conserved sequences in the PLAG1 3’UTR can be targeted by miR-16 and miR-34a. These two miRNAs are strongly downregulated in Wilms tumors with microRNA processing mutations. In addition, mimics and inhibitors of miR-16 and miR-34a modulate endogenous PLAG1 and IGF2 expression in the Wilms tumor cell line WiT49. Finally, PLAG1 overexpression in WiT49 produced increased IGF2 expression, increased activating phosphorylation of its receptor, and increased proliferation. Lastly, we interrogated PLAG1 levels in publicly available data from the TARGET consortium. In 75 relapsed favorable-histology Wilms tumors, microRNA pathway mutations were seen in 14 cases. However, other genomic aberrations were also associated with high PLAG1 expression. For instance, chromosome 8p12, containing the PLAG1 locus, is amplified in 15 cases. Additionally, nine cases show loss of chromosome 1p, which includes the miR-34a locus. These copy number changes rarely overlap with each other or with microRNA pathway mutations. Together, however, these three changes account for 35 of the 75 tumors, and all 35 display high PLAG1 expression. Regardless of mechanism, high levels of PLAG1 correlated with earlier relapse, implying that high PLAG1 expression drives more aggressive tumors. In sum, Wilms tumors can produce high levels of the oncogenic transcription factor PLAG1 through 1p loss, 8p12 gain, or microRNA processing blockade. PLAG1 activates IGF2 transcription and enhances proliferation of Wilms tumor cells in vitro. Overexpression of PLAG1 in developing mouse kidneys produces impaired differentiation and neoplastic growth. These data establish PLAG1 as a novel mediator of Wilms tumor formation and a potential therapeutic target. Citation Format: Kenneth S. Chen, Emily K. Stroup, Albert Budhipramono, Joshua T. Mendell, James F. Amatruda. Mutations in microRNA processing genes dysregulate a miR-16/34-PLAG1-IGF2 axis in Wilms tumors [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR14.