John F. Öhd

The inflammatory mediator leukotriene D4 induces β-catenin signaling and its association with antiapoptotic Bcl-2 in intestinal epithelial cells

Increased levels of the inflammatory mediator leukotriene D4 (LTD4) are present at sites of inflammatory bowel disease, and such areas also exhibit an increased risk for subsequent cancer development. It is known that LTD4 affects the expression of many proteins that influence survival and proliferation of intestinal epithelial cells. We demonstrate here that after LTD4 exposure, β-catenin translocates to the nucleus where it signals activation of the TCF/LEF family of transcription factors. These events are mediated via a phosphatidylinositol 3-kinase-dependent phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase 3β. We also show that in the presence of LTD4, free β-catenin translocates to the mitochondria where it associates with the cell survival protein Bcl-2. We hypothesize that LTD4 may enhance cell survival via activation of β-catenin signaling, in particular, by promoting the association of β-catenin with Bcl-2 in the mitochondria. Similar to Wnt-1 signaling, LTD4 signals an increased level of free β-catenin and elevated TCF/LEF promotor activity. This work in intestinal epithelial cells further lends credence to the idea that inflammatory signaling pathways are intrinsically linked with potential oncogenic signals involved in cell survival and apoptosis.

Regulation of leukotriene-dependent induction of cyclooxygenase-2 and Bcl-2.

Leukotrienes play an important pathophysiological role in chronic inflammatory states and, as previously shown, cause increased levels of cyclooxygenase-2 (COX-2) in intestinal epithelial cells. The anti-apoptotic protein Bcl-2 is also elevated by LTD(4) stimulation, and in colon cancer, so we studied the mechanisms of COX-2 and Bcl-2 regulation. We found that LTD(4) induced a 3-fold elevation of COX-2 transcription in Int 407 cells and a 2-fold equivalent in colon cancer cells, Caco-2. This was mediated through a pertussis toxin (PTX) sensitive G-protein and the MAP kinase Erk-1/2 pathway, and this was also shown to be the route to up-regulation of Bcl-2 by LTD(4). In good agreement with this, we detected a strong activation of Erk-1/2 that was further increased by COX-2 inhibition, pointing towards the existence of negative feedback regulation. Furthermore, COX-2 activity is responsible for the effects on Bcl-2, but this is not conveyed through the production of PGE(2).

The leukotriene receptor CysLT1 and 5-lipoxygenase are upregulated in colon cancer.

The metabolites of arachidonic acid are well connected to pathological situations such as inflammation, cancer and asthmA. Sheng et al. [7] found that COX-2 is upregulated in colon cancer tissue and tumor cell lines indicating that COX-2 is involved in colon cancer. This is supported by studies showing that patients treated with nonsteroidal anti-inflammatory drugs, inhibitors of COX-2, exhibit a lower frequency of colon cancer [8]. When the non-transformed intestinal epithelial cell line, Int 407 was stimulated with LTD4 or LTB4 we observed an accumulation of COX-2 in membrane fractions as well as an increased production of prostaglandin E2 [5]. Treatment of these cells with the COX-2 inhibitor NS-398 caused apoptosis and this effect could be prevented by LTD4 [5] or LTB4 [4]. Similar results were obtained when cell viability with LTD4 or LTB4 in the presence or absence of NS-398 was assayed [4,5]. The results demonstrate that these leukotrienes can suppress the NS-398 induced apoptosis in intestinal cells.

Pro-inflammatory mediator leukotriene D4 induces transcriptional activity of potentially oncogenic genes.

The inflammatory mediator LTD4 (leukotriene D4) is present at high levels in many inflammatory conditions, and areas of chronic inflammation have an increased risk for subsequent cancer development. We demonstrate here that following LTD4 stimulation, beta-catenin is translocated to the nucleus, triggering the transcriptional activity of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family of transcription factors. These events are dependent on phosphoinositide-3 kinase activation and glycogen synthase kinase inhibition. Our data suggest that, similar to Wnt signalling, LTD4 increases free beta-catenin and targets it to the nucleus.

Do Leukotrienes Increase Cell Viability in Human Intestinal Epithelial Cells

In this preliminary report we present data showing that leukotrienes increase the baseline cell viability in human intestinal epithelial cells and that LTB4 partially reverses the morphological hallmarks of non-necrotic cell death induced by the COX-2 specific inhibitor NS-398. The proposed signaling mechanisms regulating these events are summarized in fig. 3. Please view the work on LT signal transduction in these cells by Thodeti et al. in this volume.

Abstract 5101: Defining exposure-PD and efficacy relationships with the novel liver-targeting nucleotide prodrug MIV-818 for the treatment of liver cancers

Background: Many systemic chemotherapeutics have failed to show efficacy in hepatocellular carcinoma (HCC), often because systemic toxicity prevents efficacious liver levels of the drug from being reached. MIV-818, a nucleotide prodrug of troxacitabine-monophosphate (TRX-MP) has been designed as a novel approach to deliver high levels of the chain-terminating nucleotide troxacitabine-triphosphate (TRX-TP) to the liver after oral dosing while minimizing systemic exposure. We investigated MIV-818 and troxacitabine using in vivo models in order to identify therapeutic levels of TRX-TP required in the tumors. Methods: MIV-818 or troxacitabine were administered to nude mice with subcutaneous Hep3B or Huh7 xenografts. LC-MS/MS was used to assess MIV-818 and its metabolites. Effects on tumor growth, plasma AFP, inhibition of proliferation and induction of DNA damage were examined and correlated with exposures to TRX-TP in the tumor. Quantitative immuno-fluorescent histology was used to assess DNA damage (pH2AX) proliferation (BrdU), and hypoxia (pimonidazole). Results: Compared to the parent nucleoside troxacitabine, MIV-818 has increased potency of inhibition of HCC cell line growth, increased conversion to its active metabolite TRX-TP and in vitro properties optimized for oral bioavailability and liver targeting, including permeability and intestinal stability. MIV-818 also shows strong synergistic anti-proliferative activity with sorafenib in a number of HCC cell lines in vitro. Pronounced tumor growth inhibition of 70-100% and extensive tumor growth delays of up to 26 days were observed in the Hep3B xenograft model following a five day period of dosing. 12-32-fold induction of DNA damage was seen throughout the tumor sections, consistent with the expected mechanism of action, and with associated inhibition of proliferation. Clear PD responses were apparent even in hypoxic regions of the tumor, indicating effective distribution of TRX-TP even far from blood vessels. DNA damage persisted for up to 7 days after the final dose, demonstrating long-lasting effects and indicating that intermittent dosing is likely to be effective. Similar dosing regimens given to Huh7 xenograft models resulted in significant tumor growth inhibition and induction of DNA damage. Intratumoral TRX-TP exposures across both models were correlated with anti-tumor effects including DNA damage induction, proliferation inhibition and tumor growth inhibition. Conclusions: We have identified TRX-TP exposures required for pronounced anti-tumor effects to give a comprehensive understanding of PK-PD-efficacy relationships for the active metabolite of MIV-818. These data could be used to guide dosing and dose selection in clinical studies. MIV-818 is currently in preclinical development in preparation for the initiation of clinical trials in patients with advanced HCC and other liver cancers. Citation Format: Mark Albertella, Biljana Rizoska, Alastair Kyle, Andrew Minchinton, Annelie Linqvist, Sanja Juric, Susanne Sedig, Karin Tunblad, Fredrik Oberg, Bjorn Classon, Anders Eneroth, John Ohd, Richard Bethell. Defining exposure-PD and efficacy relationships with the novel liver-targeting nucleotide prodrug MIV-818 for the treatment of liver cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5101. doi:10.1158/1538-7445.AM2017-5101

Leukotriene D4-Induced Signalling Events in Human Epithelial Cells: Gαi3 Activation and Translocation

Our model of LTD4-induced signal transduction in epithelial cells is summarised in Figure 2. Extending what is already known about LTD4 signalling in epithelial cells, we identified the Gi3-protein as the crucial PTX sensitive G-protein and found that it is translocated to what might be a cytoskeletal fraction. This finding suggests a subtle response to LTD4, mediated via the bifurcation at the alpha/beta gamma junction. Although little is known about the role of epithelial cells in inflammation, it has been shown that such cells produce the potent chemoattractant LTB4 and the proinflammatory 5-HETE in response to intracellular accumulation of Ca2+ 24. The target protein(s) and the effect(s) of the translocation of the activated G alpha i3-proteins, as well as the possible role of the beta/gamma-subunits of Gi3, remain to be elucidated.