Loss of c‐Jun N‐terminal Kinase 1 and 2 Function in Liver Epithelial Cells Triggers Biliary Hyperproliferation Resembling Cholangiocarcinoma

Abstract

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...

JNK-mediated disruption of bile acid homeostasis promotes intrahepatic cholangiocarcinoma

“…Previous studies have established that hepatic JNK deficiency can suppress cholangiocyte proliferation and oncogenic transformation in a p53/Kras-induced model of cholangiosarcoma (35) and promotes cholangiocarcinoma in dethylnitrosamine and NEMO deficiency models of liver cancer (36). The results of the present study demonstrate that hepatic JNK deficiency is sufficient for the development of cholangiocyte malignancy (Fig.…”

Dual Targeting of G9a and DNA Methyltransferase‐1 for the Treatment of Experimental Cholangiocarcinoma

“…( 20 ) However, when these mice are treated with a carcinogen (i.e., DEN), the process can be accelerated, and histological and molecular features consistent with human iCCA are observable much earlier. ( 19 ) Therefore, we treated 2‐week‐old Jnk Δhepa mice with a single dose of DEN, and at 8 weeks started to receive CCl 4 twice per week until 22 weeks of age ( Jnk Δhepa + DEN + CCl 4 mice; Fig. 6A).…”

“…Besides finding reduced growth of xenografted CCA cells, and a strong growth inhibitory effect in a human CCA PDX mouse model, we observed a remarkable effect in a recently developed mouse model reproducing early stages of cholangiocarcinogenesis. Mice with hepatocellular deletion of Jnk1/2 develop hepatic lesions morphologically and molecularly resembling human CCA progression when subjected to liver injury, or spontaneously at later time points (19,20). Expression of the CM272 targets G9a and DNMT1 was readily detected in cyst-like structures compatible with cholangioma or malignant CCA compared with normal cholangiocytes.…”

“…The Jnk hepa mice, in a C57BL/6J background, were generated as previously described (19,20). At 14 days of age mice received 25mg/kg (i.p.)…”

c-Jun N-terminal kinase (JNK) signaling contributes to cystic burden in polycystic kidney disease

“…A previous study demonstrated a requirement of Jnk for hepatic cyst formation in models of mitochondrial redox stress-induced cholangiocarcinoma [73]. However, this role of JNK to promote cystogenesis appears to be dependent on physiological context because Jnk deletion alone was sufficient to cause hepatic cyst formation in another study of aged mice [74][75][76]. JNK signaling may have tissue-specific roles in ADPKD that will be important to evaluate for therapeutic development.…”