Targeted inhibition of the c-Jun N-terminal kinases ( JNKs) has shown therapeutic potential in intrahepatic cholangiocarcinoma (CCA)-related tumorigenesis. However, the cell-type-specific role and mechanisms triggered by JNK in liver parenchymal cells during CCA remain largely unknown. Here, we aimed to investigate the relevance of JNK1 and JNK2 function in hepatocytes in two different models of experimental carcinogenesis, the dethylnitrosamine (DEN) model and in nuclear factor kappa B essential modulator (NEMO) hepatocyte-specific knockout (Δhepa) mice, focusing on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development. Moreover, regulation of essential genes was assessed by reverse transcription polymerase chain reaction, immunoblottings, and immunostainings. Additionally, specific Jnk2 inhibition in hepatocytes of NEMO Δhepa /JNK1 Δhepa mice was performed using small interfering (si) RNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors. Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocellular carcinoma (HCC) in both the DEN model and in NEMO Δhepa mice but in contrast caused massive proliferation of the biliary ducts. Indeed, Jnk1/2 deficiency in hepatocytes of NEMO Δhepa (NEMO Δhepa /JNK Δhepa ) animals caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression but reduced HCC. Furthermore, siJnk2 treatment in NEMO Δhepa /JNK1 Δhepa mice recapitulated the phenotype of NEMO Δhepa /JNK Δhepa mice. Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NEMO Δhepa mice. We found that NEMO Δhepa /JNK Δhepa livers exhibited overexpression of the interleukin-6/signal transducer and activator of transcription 3 pathway in addition to epidermal growth factor receptor (EGFR)-rapidly accelerated fibrosarcoma (Raf )-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade. The functional relevance was tested by administering lapatinib, which is a dual tyrosine kinase inhibitor of erythroblastic oncogene B-2 (ErbB2) and EGFR signaling, to NEMO Δhepa /JNK Δhepa mice. Lapatinib effectively inhibited cystogenesis, improved transaminases, and effectively blocked EGFR-Raf-MEK-ERK signaling. Conclusion: We define a novel function of JNK1/2 in cholangiocyte hyperproliferation. This opens new therapeutic avenues devised to inhibit pathways of cholangiocarcinogenesis. (Hepatology Communications 2020;4:834-851). Bile duct hyperplasia and aberrant cholangiocyte growth can result in hepatic cystogenesis, differentially diagnosed on the basis of cholangioma, cholangiofibrosis, intrahepatic cholangiocarcinoma (CCA), and oval cell hyperplasia. (1,2) CCA, a malignancy that arises in the setting of chronic inflammation of biliary epithelium cells, has an increasing incidence and is the second most common primary Abbreviations: α-SMA, alpha smooth muscle actin; Δhepa, hepatocy...

Lipid-based RNA nanocarriers have been recently accepted as a novel therapeutic option in humans, thus increasing the therapeutic options for patients. Tailored nanomedicines will enable to treat chronic liver disease (CLD) and endstage liver cancer, disorders with high mortality and few treatment options. Here, we investigated the curative potential of gene therapy of a key molecule in CLD, the c-Jun N-terminal kinase-2 (Jnk2). Delivery to hepatocytes was achieved using a lipid-based clinically employable siRNA formulation that includes a cationic aminolipid to knockdown Jnk2 (named siJnk2). After assessing the therapeutic potential of siJnk2 treatment, non-invasive imaging demonstrated reduced apoptotic cell death and improved hepatocarcinogenesis was evidenced by improved liver parenchyma as well as ameliorated markers of hepatic damage, reduced fibrogenesis in 1-year-old mice. Strikingly, chronic siJnk2 treatment reduced premalignant nodules, indicative of tumor initiation. Furthermore, siJnk2 treatment led to a significant activation of the immune cell compartment. In conclusion, Jnk2 knockdown in hepatocytes ameliorated hepatitis, fibrogenesis, and initiation of hepatocellular carcinoma (HCC), and hence might be a suitable therapeutic option, to define novel molecular targets for precision medicine in CLD.

Hepatic clearance of lipid nanoparticles (LNP) with encapsulated nucleic acids restricts their therapeutic applicability. Therefore, tools for regulating hepatic clearance are of high interest for nucleic acid delivery. To this end, this work employs wild‐type (WT) and low‐density lipoprotein receptor (Ldlr)−/− mice pretreated with either a leukotriene B4 receptor inhibitor (BLT1i) or a high‐density lipoprotein receptor inhibitor (HDLRi) prior to the injection of siRNA‐LNP. This work is able to demonstrate significantly increased hepatic uptake of siRNA‐LNP by the BLT1i in Ldlr−/− mice by in vivo imaging and discover an induction of specific uptake‐related proteins. Irrespective of the inhibitors and Ldlr deficiency, the siRNA‐LNP induced RNA‐binding and transport‐related proteins in liver, including haptoglobin (HP) that is also identified as most upregulated serum protein. This work observes a downregulation of proteins functioning in hepatic detoxification and of serum opsonins. Most strikingly, the HDLRi reduces hepatic uptake and increases siRNA accumulation in spleen and myeloid immune cells of blood and liver. RNA sequencing demonstrates leukocyte recruitment by the siRNA‐LNP and the HDLRi through induction of chemokine ligands in liver tissue. The data provide insights into key mechanisms of siRNA‐LNP biodistribution and indicate that the HDLRi has potential for extrahepatic and leukocyte targeting.

Fibropolycystic liver disease is characterized by hyperproliferation of the biliary epithelium and the formation of multiple dilated cysts, a process associated with unfolded protein response (UPR). In the present study, we aimed to understand the mechanisms of cyst formation and UPR activation in hepatocytic c-Jun N-terminal kinase 1/2 (Jnk1/2) knockout mice. Floxed JNK1/2 (Jnkf/f) and Jnk∆hepa animals were sacrificed at different time points during progression of liver disease. Histological examination of specimens evidenced the presence of collagen fiber deposition, increased α-smooth muscle actin (αSMA), infiltration of CD45, CD11b and F4/80 cells and proinflammatory cytokines (Tnf, Tgfβ1) and liver injury (e.g., ALT, apoptosis and Ki67-positive cells) in Jnk∆hepa compared with Jnkf/f livers from 32 weeks of age. This was associated with activation of effectors of the UPR, including BiP/GRP78, CHOP and spliced XBP1. Tunicamycin (TM) challenge strongly induced ER stress and fibrosis in Jnk∆hepa animals compared with Jnkf/f littermates. Finally, thioacetamide (TAA) administration to Jnk∆hepa mice induced UPR activation, peribiliary fibrosis, liver injury and markers of biliary proliferation and cholangiocarcinoma (CCA). Orthoallografts of DEN/CCl4-treated Jnk∆hepa liver tissue triggered malignant CCA. Altogether, these results suggest that activation of the UPR in conjunction with fibrogenesis might trigger hepatic cystogenesis and early stages of CCA.