Renal cell carcinoma (RCC) associated with Xp11.2 translocation (TFE3-RCC) has been recently defined as a distinct subset of RCC classified by characteristic morphology and clinical presentation. The Xp11 translocations involve the TFE3 transcription factor and produce chimeric TFE3 proteins retaining the basic helix-loop-helix leucine zipper structure for dimerization and DNA binding suggesting that chimeric TFE3 proteins function as oncogenic transcription factors. Diagnostic biomarkers and effective forms of therapy for advanced cases of TFE3-RCC are as yet unavailable. To facilitate the development of molecular based diagnostic tools and targeted therapies for this aggressive kidney cancer, we generated a translocation RCC mouse model, in which the PRCC-TFE3 transgene is expressed specifically in kidneys leading to the development of RCC with characteristic histology. Expression of the receptor tyrosine kinase Ret was elevated in the kidneys of the TFE3-RCC mice, and treatment with RET inhibitor, vandetanib, significantly suppressed RCC growth. Moreover, we found that Gpnmb (Glycoprotein nonmetastatic B) expression was notably elevated in the TFE3-RCC mouse kidneys as seen in human TFE3-RCC tumors, and confirmed that GPNMB is the direct transcriptional target of TFE3 fusions. While GPNMB IHC staining was positive in 9/9 cases of TFE3-RCC, Cathepsin K, a conventional marker for TFE3-RCC, was positive in only 67% of cases. These data support RET as a potential target and GPNMB as a diagnostic marker for TFE3-RCC. The TFE3-RCC mouse provides a preclinical in vivo model for the development of new biomarkers and targeted therapeutics for patients affected with this aggressive form of RCC. Implications: Key findings from studies with this preclinical mouse model of TFE3-RCC underscore the potential for RET as a therapeutic target for treatment of patients with TFE3-RCC, and suggest that GPNMB may serve as diagnostic biomarker for TFE3 fusion RCC.

The tumor microenvironment promotes epigenetic changes in tumor cells associated with tumor aggressiveness. Here we report that in primary tumor cells, increased interleukin-6 (IL-6) expression brought on by DNA demethylation of its promoter by ten-eleven translocation 2 (TET2) promotes lung metastasis in osteosarcoma (OS). Xenograft experiments show increased IL-6 expression and decreased methylation of its promoter in OS cells after implantation relative to before implantation. In addition, changes in IL-6 methylation and expression seen in OS cells at the primary site were maintained at the metastatic site. TET2 knockdown in OS cells suppressed upregulation of IL-6 and demethylation of its promoter in xenograft tumors and decreased tumor metastasis. We also present evidence showing that tumor cell-derived IL-6 facilitates glycolytic metabolism in tumor cells by activating the MEK/ERK1/2/hypoxia-inducible transcription factor-1α (HIF-1α) pathway and increases lung colonization by OS cells by upregulating expression of intercellular adhesion molecule-1 (ICAM-1), enhancing tumor metastasis. Blocking IL-6 signaling with a humanized monoclonal antibody against the IL-6 receptor reduced lung metastasis and prolonged survival of xenografted mice. These findings suggest that TET2-dependent IL-6 induction enables acquisition of aggressive phenotypes in OS cells via the tumor microenvironment and that blocking IL-6 signaling could be serve as a potential therapy to antagonize metastasis.

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC ) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE 3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early‐stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC ‐ TFE 3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)‐204‐5p levels in urinary exosomes of 40‐week‐old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA‐204‐5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR‐204‐5p‐containing exosomes. Notably, we also observed increased miR‐204‐5p levels in urinary exosomes in 20‐week‐old renal PRCC ‐ TFE 3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40‐week‐old Tg mice, suggesting that miR‐204‐5p increases follow expression of constitutively active TFE 3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR‐204‐5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC ‐ TFE 3 fusion gene. These findings suggest that miR‐204‐5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC .

Purpose: Although cell-free DNA (cfDNA) testing is expected to drive cancer precision medicine, little is known about the significance of detecting low-frequency variants in circulating cell-free tumor DNA (ctDNA) in castration-resistant prostate cancer (CRPC). We aimed to identify genomic profile including low-frequency variants in ctDNA from patients with CRPC and investigate the clinical utility of detecting variants with variant allele frequency (VAF) below 1%. Experimental Design: This prospective, multicenter cohort study enrolled patients with CRPC eligible for treatment with abiraterone or enzalutamide. We performed targeted sequencing of pretreatment cfDNA and paired leukocyte DNA with molecular barcodes, and ctDNA variants with a VAF ≥0.1% were detected using an in-house pipeline. We investigated progression-free survival (PFS) and overall survival (OS) after different ctDNA fraction cutoffs were applied. Results: One hundred patients were analyzed (median follow-up 10.7 months). We detected deleterious ATM, BRCA2, and TP53 variants even in samples with ctDNA fraction below 2%. When the ctDNA fraction cutoff value of 0.4% was applied, significant differences in PFS and OS were found between patients with and without defects in ATM or BRCA2 [HR, 2.52; 95% confidence interval (CI), 1.24–5.11; P = 0.0091] and TP53 (HR, 3.74; 95% CI, 1.60–8.71; P = 0.0014). However, these differences were no longer observed when the ctDNA fraction cutoff value of 2% was applied, and approximately 50% of the samples were classified as ctDNA unquantifiable. Conclusions: Detecting low-frequency ctDNA variants with a VAF <1% is important to identify clinically informative genomic alterations in CRPC.

Angiopoietin-like protein 2 (ANGPTL2) is a secreted glycoprotein homologous to angiopoietins. Previous studies suggest that tumor cell-derived ANGPTL2 has tumor-promoting function. Here, we conducted mechanistic analysis comparing ANGPTL2 function in cancer progression in a murine syngeneic model of melanoma and a mouse model of translocation renal cell carcinoma (tRCC). ANGPTL2 deficiency in tumor cells slowed tRCC progression, supporting a tumor-promoting role. However, systemic ablation of ANGPTL2 accelerated tRCC progression, supporting a tumor-suppressing role. The syngeneic model also demonstrated a tumor-suppressing role of ANGPTL2 in host tumor microenvironmental cells. Furthermore, the syngeneic model showed that PDGFRα+ fibroblasts in the tumor microenvironment express abundant ANGPTL2 and contribute to tumor suppression. Moreover, host ANGPTL2 facilitates CD8+ T-cell cross-priming and enhances anti-tumor immune responses. Importantly, ANGPTL2 activates dendritic cells through PIR-B–NOTCH signaling and enhances tumor vaccine efficacy. Our study provides strong evidence that ANGPTL2 can function in either tumor promotion or suppression, depending on what cell type it is expressed in.