Fibrolamellar carcinoma (FLC) is a rare form of liver cancer that occurs mostly in adolescents without preexisting liver conditions. A genomic identifier of FLC is a ~400kb deletion on chromosome 19 which results in a fusion of two genes, DNAJB1 and PRKACA (referred to as “DP”). The DP chimera is a functional kinase, the over-expression of which is tumorigenic in mice. Targeting DP pharmacologically has proven challenging; therefore, it is of critical importance to define downstream mediators of tumor phenotypes in order to identify candidate druggable targets. Genome-scale analysis of FLC tumors shows reproducible changes in gene expression. DP is likely to mediate gene expression through both transcriptional and post-transcriptional mechanisms, the latter of which includes microRNA mediated gene silencing. In order to define aberrant microRNAs in FLC tumors, we performed small RNA-sequencing (small RNA-seq) in >25 tumors and 3 non-malignant liver samples. We identified multiple miRNAs significantly upregulated in FLC, including miR-182, miR-10b, miR-21, miR-449a, and miR-92b. We then compared the expression levels of these miRNAs to the levels reported in other cancers and identified the upregulation of miR-10b in FLC to be greater than any other tumor type. We also found that only miR-10b expression is further elevated in metastatic samples relative to primary tumor tissue. Finally, in a cell culture model of FLC, we found that only miR-10b expression is induced (greater than 10-fold) by DP activity. We hypothesized that miR-10b may function as a pro-survival, oncogenic factor in FLC. To test this hypothesis, we performed loss-of-function experiments using miR-10b locked nucleic acid (LNA) inhibitors in a cell culture model generated from a patient-derived xenograft tumor. We found that suppression of miR-10b significantly lowered FLC cell viability and proliferation. We then performed an integrative analysis of chromatin run-on sequencing (ChRO-seq) and RNA-sequencing (RNA-seq) data to identify genes that are likely regulated by miR-10b in the FLC tumor tissue. This analysis led to the discovery of TRIM35, or tripartite motif-containing protein family member 35, which we validated in the FLC cell line. TRIM35 is known to regulate the function of pyruvate kinase, which may explain the reduction of cellular viability when miR-10b is inhibited. Our future directions include resolving the direct relationship between miR-10b and TRIM35, performing cellular migration and invasion assays after miR-10b inhibition, and developing and analyzing a miR-10b null FLC cell line. Additionally, we are identifying other oncogenic miRNAs with which miR-10b coordinates and directs FLC cell survival.
Citation Format: Adam Francisco, Andrew Massa, Matt Kanke, Praveen Sethupathy. MicroRNA-10b is a regulator of cellular viability and proliferation in fibrolamellar carcinoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-048.