Expression of DNAJB1-PRKACA oncogene suppresses the differentiation potential of liver progenitor organoids towards a hepatocyte lineage

Abstract

Fibrolamellar carcinoma (FLC) is a type of primary liver cancer that predominantly affects healthy adolescents and young adults in a background of normal liver. The DNAJB1-PRKACA fusion gene is an oncogenic driver in FLC tumors. To investigate the oncogenic mechanisms of this fusion gene, we developed a model using human liver progenitor organoids engineered to express DNAJB1-PRKACA. Single-nucleus RNA sequencing of these organoids revealed an upregulation of genes that significantly overlap with those expressed in FLC epithelial cells. Additionally, the expression of DNAJB1-PRKACA led to the downregulation of genes coding for markers of mature epithelial cells, indicating a shift toward a less differentiated state. When compared to wild-type liver progenitor organoids, which exhibit a strong ability to differentiate into hepatocytes, the DNAJB1-PRKACA-expressing liver progenitor organoids displayed a markedly reduced capacity for hepatocyte differentiation. These findings suggest that the DNAJB1-PRKACA fusion gene disrupts the normal differentiation process of liver progenitor cells.

Fig. 1

WT-Org differentiation towards a hepatocytic lineage. (A) WT-Org morphology prior to initiation of differentiation protocol and 12 days following exposure to hepatocyte differentiation media. Images were taken on Invitrogen’s EVOS M5000 Imaging system at 2x magnification. (B) Fold change of ALB, CYP3A4, and HNF4A expression in undifferentiated (1) and 12-day differentiated (2665, 782, 2) WT-Org cells. (C) Fold change of ALB, CYP3A4, and HNF4A expression in undifferentiated (1) and 12-day differentiated (937, 2948, 3) WT-GFP-Org cells. All fold changes reflect a ratio of differentiated to undifferentiated transcript levels after normalization to LMNB2 as housekeeping gene. (D) Western blot analysis of WT-GFP-Org cells using Albumin antibody indicates higher albumin protein expression in differentiated cells compared to undifferentiated cells.

Fig. 2

DP-Org characterization. (A) Bright-field image of DP-Org cells taken on Invitrogen’s EVOS M5000 Imaging system at 4x magnification. (B) Western blot analysis of DP-Org cells using PKA antibody demonstrates expression of DNAJB1-PRKACA at 46 kD in DP-Org and tumor cells but not in normal organoids (WT-Org). FLC tumor tissue is included as positive control.

Fig. 3

FLC UMAP. Annotated cell clusters in FLC tumors.

Fig. 4

Comparison UMAP. (A) DP-Org and normal liver tissue (n = 4) plot demonstrating clustering of DP-Org cells with cholangiocyte cluster (black circle). (B) DP-Org cells cluster with FLC tumor cell population which has hepatocytic features (black circle).

Fig. 5

Comparison of expression of hepatocyte markers following differentiation of WT-GFP-Org and DP-Org cells. There is a statistically decreased expression of ALB and CYP3A4 in DP-Org cells following differentiation compared to WT-GFP-Org. Comparison of HNF4A reaches near significance at p = 0.0516. All fold changes reflect a ratio of differentiated to undifferentiated transcript levels after normalization to LMNB2 as housekeeping gene. Statistical analysis was performed using an unpaired t-test for ALB (n = 3) and CYP3A4 (n = 2) and a Welch’s t test for HNF4A (WT-GFP-ORG, n = 3; DP-ORG, n = 2).