BET Protein Inhibition Evidently Enhances Sensitivity to PI3K/mTOR Dual Inhibition in Intrahepatic Cholangiocarcinoma

Background & Aims: Intrahepatic cholangiocarcinoma (ICC), the second most common primary liver cancer, is a fatal malignancy with a poor prognosis. Molecular targeted therapy is a promising treatment strategy. However, resistance to molecular-targeted therapy is inevitable and represents a major clinical challenge. Our objective was to provide a novel and efficient therapeutic strategy in treating primary ICC.Approach & Results: We generated an orthotopic ICC mouse model through hydrodynamic transfection of AKT and YAP into mouse liver. Genetic approaches were applied to study the mechanism that mediated resistance to a PI3K/mTOR dual inhibitor in mediating AKT/YapS127A-driven tumorigenesis. Haematoxylin and eosin staining, immunohistochemistry and immunoblotting were applied to assess the efficacy of ICC combination therapy in vitro and in vivo.Conclusions: We confirmed that mTOR signalling is the most significantly affected pathway in ICC. BEZ235, a novel PI3K/mTOR dual inhibitor, can suppress the proliferation, invasion and colony formation abilities of ICC cells in vitro but cannot effectively inhibit ICC progression in vivo. The inhibition of PI3K/mTOR allowed the upregulation of c-Myc and YAP through the suppression of the phosphorylation of LATS1. This is a novel mechanism that mediates resistance to PI3K/mTOR dual inhibitors. However, BET inhibition by JQ1 downregulates c-Myc and YAP transcription, which could enhance the efficacy of PI3K/mTOR inhibitors. The efficacy results of combination therapy exhibited effective treatment of ICC in vitro and in vivo. Our data further confirmed that the combination of a PI3K/mTOR dual inhibitor and BET inhibition induces M1 polarization and suppresses M2 polarization in macrophages by regulating the expression of HIF-1a.

Hyperfibrinolysis increases blood–brain barrier permeability by a plasmin- and bradykinin-dependent mechanism

Key Points Hydrodynamic transfection of plasmids encoding for plasminogen activators leads to a hyperfibrinolytic state in mice. Hyperfibrinolysis increases BBB permeability via a plasmin- and bradykinin-dependent mechanism.

Age-related immune clearance of hepatitis B virus infection requires the establishment of gut microbiota

A unique feature of hepatitis B virus (HBV) infection in humans is that viral clearance heavily depends on the age of exposure. However, the reason for this remains unclear. Here we show that gut microbiota contribute to the age dependence of HBV immunity in a hydrodynamic transfection mouse model. Although adult (12-wk-old) C3H/HeN mice cleared HBV within 6 wk postinjection (wpi), their young (6-wk-old) counterparts remained HBV-positive at 26 wpi. Sterilization of gut microbiota from 6 to 12 wk of age using antibiotics prevented adult mice from rapidly clearing HBV. Young mice with the Toll-like-receptor (TLR) 4 mutation (C3H/HeJ) exhibited rapid HBV clearance. The results suggest that an immuno-tolerating pathway to HBV prevailed in young mice, before the establishment of gut bacteria, through a TLR4-dependent pathway and that the maturation of gut microbiota in adult mice stimulated liver immunity, resulting in rapid HBV clearance.

Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-κB

Bruton tyrosine kinase (Btk) is critical for B-cell development. Btk regulates a plethora of signaling proteins, among them nuclear factor-[κ]B (NF-κB). Activation of NF-κB is a hallmark of B cells, and NF-κB signaling is severely compromised in Btk deficiency. We here present strong evidence indicating that NF-κB is required for efficient transcription of the Btk gene. First, we found that proteasome blockers and inhibitors of NF-κB signaling suppress Btk transcription and intracellular expression. Similar to Btk, proteasome inhibitors also reduced the expression of other members of this family of kinases, Itk, Bmx, and Tec. Second, 2 functional NF-κB–binding sites were found in the Btk promoter. Moreover, in live mice, by hydrodynamic transfection, we show that bortezomib (a blocker of proteasomes and NF-κB signaling), as well as NF-κB binding sequence-oligonucleotide decoys block Btk transcription. We also demonstrate that Btk induces NF-κB activity in mice. Collectively, we show that Btk uses a positive autoregulatory feedback mechanism to stimulate transcription from its own promoter via NF-κB.

Analysis of the Effect of Soluble Transferrin Receptor 1 (sTfR1) on Iron Metabolism in Mice.

Transferrin receptor 1 (TfR1) is a cell surface-expressed protein that mediates cellular uptake of iron. The TfR1 molecule may be shed from cells by proteolytic cleavage at Arg100 - Leu101, resulting in a cleaved form called soluble transferrin receptor 1 (sTfR1), which circulates in the blood as a 74 kDa monomer bound to transferrin. Measurement of soluble transferrin receptor has been widely used for the diagnosis of iron deficiency, but the levels of the receptor are also increased in patients with ineffective erythropoiesis, because the amount of circulating sTfR1 is directly proportional to the total amount of cell-associated TfR1. The key factors that stimulate increased sTfR1 levels are the integrated effects of reduced iron availability and increased erythropoietic stimulation. It is notable that both in iron deficiency and in diseases such as thalassemia, in which there is active ineffective erthropoiesis, iron absorption is increased This raises the question of whether sTfR1 is merely an intermediate in the degradation of the protein or whether it is a physiological regulator of iron absorption. To investigate this potential signaling function of sTfR1, a hydrodynamic gene transfer technique was established to express transfected plasmid constructs of human sTfR1 (hsTfR1, aa89–759) and murine sTfR1 (MsTfR1, 86–763) from the livers of C57Bl6 mice. To assess the effect that hsTfR1 and MsTfR1 might have on iron metabolism, iron absorption, serum iron and gene expression of hepcidin was measured in hydrodynamically transfected mice. The efficacy of the hydrodynamic technique was demonstrated by sustained expression of hsTfR1 in mice, at a level six fold higher than the normal level of hsTfR1 in humans (Fig. 1). Iron absorption was determined in transfected mice by measuring the retention of gavage fed 59Fe in a carrier iron solution, using a whole animal counting technique. Repeated experiments showed neither hsTfR1 nor MsTfR1 had any effect on the amount of iron absorbed. In agreement with these data, hepcidin levels were found by real time PCR to be unaltered in the same transfected mice. When serum iron levels were measured, expression of either hsTfR1 or MsTfR1 in mice resulted in significantly higher serum iron levels versus control mice (P=0.0141 and P=0.0192 respectively by t-test). We conclude that despite its attractiveness as a potential modifier of iron absorption, sTfR1 does not have any effect on hepcidin expression or iron absorption in mice. Mice transfected with hsTfR1 or MsTfR1 do have significantly higher serum iron levels, which may reflect an increased capability of these mice to retain transferrin in the serum. We have found that hydrodynamic transfection is a useful method to increase the levels of putative biologically active compounds in mice; this technique can be valuable in investigating the roles of serum or liver specific proteins in mice. Fig. 1 Sustained Expression of hsTfR1 in Mice Fig. 1. Sustained Expression of hsTfR1 in Mice