DNAJB1-PRKACA in HEK293T cells induces LINC00473 overexpression that depends on PKA signaling

PLoS One. 2022 Feb 15;17(2):e0263829. doi: 10.1371/journal.pone.0263829. eCollection 2022.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems
  • Carcinoma, Hepatocellular / genetics*
  • Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / genetics*
  • Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation, Neoplastic
  • HEK293 Cells
  • HSP40 Heat-Shock Proteins / genetics*
  • HSP40 Heat-Shock Proteins / metabolism
  • Humans
  • Mitochondria / metabolism
  • Models, Biological
  • Oncogene Proteins, Fusion / genetics*
  • Proteomics
  • RNA, Long Noncoding / genetics*
  • Up-Regulation*

Substances

  • DNAJB1 protein, human
  • HSP40 Heat-Shock Proteins
  • LINC00473 RNA, human
  • Oncogene Proteins, Fusion
  • RNA, Long Noncoding
  • Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
  • PRKACA protein, human

Supplementary concepts

  • Fibrolamellar hepatocellular carcinoma

Grant support

This work was supported by the Tisch Families’ Faculty Development Fund and CHMC Surgical Foundation, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.