Phosphorylation and Stabilization of PIN1 by JNK Promote Intrahepatic Cholangiocarcinoma Growth.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive type of liver cancer in urgent need of treatment options. Aberrant activation of the c-Jun N-terminal kinase (JNK) pathway is a key feature in ICC and an attractive candidate target for its treatment. However, the mechanisms by which constitutive JNK activation promotes ICC growth, and therefore the key downstream effectors of this pathway, remain unknown for their applicability as therapeutic targets. Our aim was to obtain a better mechanistic understanding of the role of JNK signaling in ICC that could open up therapeutic opportunities. APPROACH AND RESULTS: Using loss-of-function and gain-of-function studies in vitro and in vivo, we show that activation of the JNK pathway promotes ICC cell proliferation by affecting the protein stability of peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), a key driver of tumorigenesis. PIN1 is highly expressed in ICC primary tumors, and its expression positively correlates with active JNK. Mechanistically, the JNK kinases directly bind to and phosphorylate PIN1 at Ser115, and this phosphorylation prevents PIN1 mono-ubiquitination at Lys117 and its proteasomal degradation. Moreover, pharmacological inhibition of PIN1 through all-trans retinoic acid, a Food and Drug Administration-approved drug, impairs the growth of both cultured and xenografted ICC cells. CONCLUSIONS: Our findings implicate the JNK-PIN1 regulatory axis as a functionally important determinant for ICC growth, and provide a rationale for therapeutic targeting of JNK activation through PIN1 inhibition

Osteopontin is a proximal effector of leptin-mediated non-alcoholic steatohepatitis (NASH) fibrosis

AbstractIntroductionLiver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH.MethodsLeptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis.ResultsMCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFβ mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice.ConclusionOPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH

Cholangiocyte chemokine secretion and macrophage accumulation is mediated by osteopontin in murine liver models

Background and aims Progression of chronic liver disease involves accumulation of inflammatory cells towards the peri-portal regions during a ductular inflammatory response. Osteopontin (OPN), an effector of Hh signalling, contributes to liver fibrogenesis and ductular inflammation via activation of hepatic stellate and progenitor cells. In tissue injury, OPN regulates macrophage functions via pro-inflammatory chemoattractant properties. In liver, however, the role of OPN in macrophage activation and recruitment remains unclear. We investigated the role of OPN in cholangiocyte chemokine secretion and macrophage recruitment using in vivo, in vitro, and in silico approaches. Methods In MCD and DDC murine models of liver fibrosis, total liver chemokine expression was measured by qRTPCR and macrophages detected by FACS (CD 11b, F4/80, CCR2, Ly6C). Lentiviral-mediated shRNA (shOPN) was used for OPN knockdown in murine 603B cholangiocytes, and secreted OPN neutralized by specific aptamers. Cholangiocyte chemokine secretion was measured by cytometric bead array and mRNA by qRTPCR. Macrophage migration was assessed in transwells using RAW264.7 cells. To obtain a global perspective of genes affected by OPN, next-generation RNA sequencing was used to compare control and shOPN cholan- giocytes. Transcripts were assessed in DESeq and gene ontologies and pathways by GOrilla, DAVID, and ReviGO software. Results Liver fibrosis in MCD and DDC was accompanied by upregulated total liver OPN, TGF-β, Ccl2, Ccl5, and Cxcll mRNA, and accumulation of liver CDl1b/F4/80(+) CCR2(hi) macrophages. Mice treated with OPN-aptamers had less fibrosis, reduced Ccl2, Ccl5, and Cxcll mRNA, and reduced accumulation of liver CD11b/F4/80(+) CCR2(hi) macrophages and the Ly6C(hi) inflammatory monocyte subset. In shOPN cholangiocytes, RNAseq detected 670 affected genes (Ben- jamini-Hochsberg padj <0.05). Ten chemokines were significantly downregulated: Cxcl16, Cxcl11, Cxcl10, Ccl5, Ccl2, Ccl9, Ccl7 Cxcl1, Cx3cl1 and Cxcl5 (by a range of log2fold between 2.70 and 0.99). Enriched gene ontologies clustered around immunity, chemotaxis and cytokine and chemokine production Altered pathways involved chemokine production and NfKB signalling. Consistent reductions in chemokine secretion and mRNA were verified in shOPN cholangiocytes Additionally, RAW264.7 macrophages cultured with OPN-deficient 603B conditioned media exhibited decreased migration. Conclusions In progressive liver disease OPN promotes chol- angiocyte production of chemokines and the accumulation of macrophages, including a proinflammatory monocyte subset. These data support neutralization of OPN as an anti-inflammatory and anti-fibrotic strategy