Student Name: Adam Francisco

Student Concentration: Molecular and Cellular Medicine

Committee Member 1: Robert Weiss

Committee Member 2: Scott Coonrod

Committee Member 3: Claudia Fischbach

Abstract: Fibrolamellar carcinoma (FLC) is a rare liver cancer predominantly affecting adolescents and young adults. FLC presents in patients lacking underlying liver disease and surgical resection is the primary intervention. Tumor burden is often identified at an advanced stage and many patients are therefore ineligible for surgery. FLC patients are not responsive to general chemotherapeutics, radiotherapeutics, or drugs shown to be effective for patients of other types of liver cancer. FLC tumorigenesis is driven by a characteristic deletion of ~400 kilobases on chromosome 19 resulting in the fusion of the first exon of DNAJB1 with exons 2-10 of PRKACA. The resulting chimera gene, DNAJB1-PRKACA (DP), is found in >80% of FLC patients and is oncogenic in mice. Pharmacological inhibition of DP is an attractive therapeutic avenue, but inhibitors designed thus far are not specific to DP because they also target PKA enzymatic activity, resulting in deleterious side effects since PKA is critical for normal physiology. In the absence of anti-DP therapy, alternate molecular targets need to be identified. Prior studies identified consistent changes in gene transcription and expression profiles in FLC patient samples. Genes are also under post-transcriptional regulation by microRNA (miR) molecules and a putative tumor suppressor, miR-375, was identified in a small patient cohort. However, analysis of miRs that may promote FLC progression has not been performed. In my thesis work, miR sequencing of an expanded FLC patient sample set was performed to identify highly expressed miRs. Transcriptional analysis identified potential factors promoting miR expression. Gene transcriptional and expression data were integrated to identify genes subject primarily to post-transcriptional regulation by miRs. MiR-10b exhibited the greatest fold-change and highest expression in FLC, was identified as a potential master regulator of post-transcriptionally suppressed genes, was responsive to DP activity, and affected FLC cell growth in part by regulating TRIM35, SUN2, and FANCC (genes shown to be tumor suppressors in other contexts). In other cancers, miR-10b promotes cancer metastasis by targeting components of the extracellular matrix (ECM). Analysis of genes associated with ECM biosynthesis revealed a significant up-regulation of chondroitin sulfate biosynthesis pathways. Further chemical and molecular analysis of FLC tumor samples confirmed an abundance of CS and the CS proteoglycan versican (VCAN). Single cell analysis of FLC tumor tissue by the assay for transposase accessible chromatin (scATAC) identified activated hepatic stellate cells as both a major component of FLC tumors and likely contributors of VCAN. Taken together, my thesis studies have made multiple contributions to extend the molecular characterization of FLC. The miRNA expression prolife unique to FLC was identified and a role for miR-10b to influence tumor cell proliferation was established. Additionally, chemical constituents and cellular populations of FLC tumors were characterized, revealing that chondroitin sulfate, VCAN, and hepatic stellate cells are likely relevant to FLC progression

Publications: Robinette TM, Nicholatos JW, Francisco AB, Brooks KE, Diao RY, Sorbi S, Ricca V, Nacmias B, Brieño-Enríquez MA, Libert S. SIRT1 accelerates the progression of activity-based anorexia. Nat Commun. 2020 Jun 4;11(1):2814. doi: 10.1038/s41467-020-16348-9. PMID: 32499508; PMCID: PMC7272424.