Abstract
Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in adolescents and young adults in a background of normal liver tissue and has a poor prognosis due to lack of effective chemotherapeutic agents. The DNAJB1-PRKACA gene fusion (DP) has been reported in the majority of FLC tumors; however, its oncogenic mechanisms remain unclear. Given the paucity of cellular models, in particular FLC tumor cell lines, we hypothesized that engineering the DP fusion gene in HEK293T cells would provide insight into the cellular effects of the fusion gene. We used CRISPR/Cas9 to engineer HEK293T clones expressing DP fusion gene (HEK-DP) and performed transcriptomic, proteomic, and mitochondrial studies to characterize this cellular model. Proteomic analysis of DP interacting partners identified mitochondrial proteins as well as proteins in other subcellular compartments. HEK-DP cells demonstrated significantly elevated mitochondrial fission, which suggests a role for DP in altering mitochondrial dynamics. Transcriptomic analysis of HEK-DP cells revealed a significant increase in LINC00473 expression, similar to what has been observed in primary FLC samples. LINC00473 overexpression was reversible with siRNA targeting of PRKACA as well as pharmacologic targeting of PKA and Hsp40 in HEK-DP cells. Therefore, our model suggests that LINC00473 is a candidate marker for DP activity.
Highlights
Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in healthy adolescents and young adults in a background of normal liver tissue [1,2,3]
We identified 12 HEK293T clones (A1, A3, A5, A6, A9, A11, B2, B6, C2, C6, C10, C11) which contained the PRKACA-DNAJB1 fusion gene (HEK-DP) as determined by PCR analysis across the expected junction site (Figs 1 and S1)
Patient-derived FLC tumor sample was used as a positive control (Fig 1D)
Summary
Fibrolamellar carcinoma (FLC) is a primary liver cancer that most commonly arises in healthy adolescents and young adults in a background of normal liver tissue [1,2,3]. In 2014, the DNAJB1-PRKACA (DP) gene fusion was initially described in a cohort of FLC patients [5]. Subsequent studies have demonstrated the presence of this fusion gene in the majority of FLC tumors [6]. DNAJB1 gene codes for a heat shock protein (HSP40) and PRKACA gene codes for the Cα catalytic subunit of protein kinase A (PKA-Cα). PKA is a cyclic AMP (cAMP)-dependent protein kinase with its holoenzyme consisting of a tetramer of two regulatory (R) subunits and two catalytic (C) subunits that contain the active kinase site. An increase in the cytosolic second messenger cAMP results in binding to the regulatory subunits, with subsequent dissociation and activation of the catalytic subunits of PKA. The free catalytic subunits subsequently phosphorylate proteins involved in a number of cellular processes such as metabolism, synaptic transmission, differentiation, growth, and development [7,8,9]
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