Modulation of the unfolded protein response by tauroursodeoxycholic acid counteracts apoptotic cell death and fibrosis in a mouse model for secondary biliary liver fibrosis

The role of endoplasmic reticulum stress and the unfolded protein response (UPR) in cholestatic liver disease and fibrosis is not fully unraveled. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, has been shown to reduce endoplasmic reticulum (ER) stress and counteract apoptosis in different pathologies. We aimed to investigate the therapeutic potential of TUDCA in experimental secondary biliary liver fibrosis in mice, induced by common bile duct ligation. The kinetics of the hepatic UPR and apoptosis during the development of biliary fibrosis was studied by measuring markers at six different timepoints post-surgery by qPCR and Western blot. Next, we investigated the therapeutic potential of TUDCA, 10 mg/kg/day in drinking water, on liver damage (AST/ALT levels) and fibrosis (Sirius red-staining), in both a preventive and therapeutic setting. Common bile duct ligation resulted in the increased protein expression of CCAAT/enhancer-binding protein homologous protein (CHOP) at all timepoints, along with upregulation of pro-apoptotic caspase 3 and 12, tumor necrosis factor receptor superfamily, member 1A (TNFRsf1a) and Fas-Associated protein with Death Domain (FADD) expression. Treatment with TUDCA led to a significant reduction of liver fibrosis, accompanied by a slight reduction of liver damage, decreased hepatic protein expression of CHOP and reduced gene and protein expression of pro-apoptotic markers. These data indicate that TUDCA exerts a beneficial effect on liver fibrosis in a model of cholestatic liver disease, and suggest that this effect might, at least in part, be attributed to decreased hepatic UPR signaling and apoptotic cell death

Effect of prolyl hydroxylase domain 2 haplodeficiency on liver progenitor cell characteristics in early mouse hepatocarcinogenesis

Activation of the hypoxia-inducible factor (HIF)-pathway in hepatocellular carcinoma (HCC) induces therapy resistant tumours, characterized by increased liver progenitor cell (LPCs) characteristics and poor prognosis. We previously reported corresponding results in mice with HCC in which hypoxia was mimicked by prolyl hydroxylase domain (PHD) inhibition. Here, we aimed at investigating whether induction of LPC characteristics occurs during the onset of hepatocarcinogenesis and if this is associated with activation of Notch signalling. Dietheylnitrosamine (DEN) was used to induce hepatic tumours in PHD2 haplodeficient (PHD2(+/-)) mice which were euthanized at 5, 10, 15 and 17 weeks following DEN during neoplastic transformation, before tumour formation. Neoplasia and mRNA expression of LPC and Notch markers were evaluated by histology and qPCR on isolated livers. PHD2 haplodeficiency resulted in enhanced expression of HIF target genes after 17 weeks of DEN compared to wild type (WT) littermates but had no effect on the onset of neoplastic transformation. The mRNA expression of Afp and Epcam was increased at all time points following DEN whereas CK19, Prom1 and Notch3 were increased after 17 weeks of DEN, without difference between PHD2(+/-) and WT mice. MDR1 mRNA expression was increased in all DEN treated mice compared to saline control with increased expression in PHD2(+/-) compared to WT from 15 weeks. These results indicate that the effects of PHD2 haplodeficiency on the expression of LPC and Notch markers manifest during tumour nodule formation and not early on during neoplastic transformatio

Next-generation proteasome inhibitor oprozomib synergizes with modulators of the unfolded protein response to suppress hepatocellular carcinoma

Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators

Placental growth factor inhibition modulates the interplay between hypoxia and unfolded protein response in hepatocellular carcinoma

Background: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality. We previously showed that the inhibition of placental growth factor (PlGF) exerts antitumour effects and induces vessel normalisation, possibly reducing hypoxia. However, the exact mechanism underlying these effects remains unclear. Because hypoxia and endoplasmic reticulum stress, which activates the unfolded protein response (UPR), have been implicated in HCC progression, we assessed the interactions between PlGF and these microenvironmental stresses. Methods: PlGF knockout mice and validated monoclonal anti-PlGF antibodies were used in a diethylnitrosamine-induced mouse model for HCC. We examined the interactions among hypoxia, UPR activation and PlGF induction in HCC cells. Results: Both the genetic and pharmacological inhibitions of PlGF reduced the chaperone levels and the activation of the PKR-like endoplasmic reticulum kinase (PERK) pathway of the UPR in diethylnitrosamine-induced HCC. Furthermore, we identified that tumour hypoxia was attenuated, as shown by reduced pimonidazole binding. Interestingly, hypoxic exposure markedly activated the PERK pathway in HCC cells in vitro, suggesting that PlGF inhibition may diminish PERK activation by improving oxygen delivery. We also found that PlGF expression is upregulated by different chemical UPR inducers via activation of the inositol-requiring enzyme 1 pathway in HCC cells. Conclusions: PlGF inhibition attenuates PERK activation, likely by tempering hypoxia in HCC via vessel normalisation. The UPR, in turn, is able to regulate PlGF expression, suggesting the existence of a feedback mechanism for hypoxia-mediated UPR that promotes the expression of the angiogenic factor PlGF. These findings have important implications for our understanding of the effect of therapies normalising tumour vasculature

Tauroursodeoxycholic acid dampens oncogenic apoptosis induced by endoplasmic reticulum stress during hepatocarcinogen exposure

Hepatocellular carcinoma (HCC) is characterized by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which activates the unfolded protein response (UPR). However, the role of ER stress in tumor initiation and progression is controversial. To determine the impact of ER stress, we applied tauroursodeoxycholic acid (TUDCA), a bile acid with chaperone properties. The effects of TUDCA were assessed using a diethylnitrosamine-induced mouse HCC model in preventive and therapeutic settings. Cell metabolic activity, proliferation and invasion were investigated in vitro. Tumor progression was assessed in the HepG2 xenograft model. Administration of TUDCA in the preventive setting reduced carcinogen-induced elevation of alanine and aspartate aminotransferase levels, apoptosis of hepatocytes and tumor burden. TUDCA also reduced eukaryotic initiation factor 2a (eIf2a) phosphorylation, C/EBP homologous protein expression and caspase-12 processing. Thus, TUDCA suppresses carcinogen-induced pro-apoptotic UPR. TUDCA alleviated hepatic inflammation by increasing NF-kappa B inhibitor I kappa Ba. Furthermore, TUDCA altered the invasive phenotype and enhanced metabolic activity but not proliferation in HCC cells. TUDCA administration after tumor development did not alter orthotopic tumor or xenograft growth. Taken together, TUDCA attenuates hepatocarcinogenesis by suppressing carcinogen-induced ER stress-mediated cell death and inflammation without stimulating tumor progression. Therefore, this chemical chaperone could represent a novel chemopreventive agent

Effective innovation strategies for incumbent firms

There is no doubt, firms acknowledge the importance of innovation for survival and growth. They spend up to 25% of their revenue on research and development (R&D) and R&D spending continues to increase in all regions and nearly all industries. Unfortunately, innovation excellence cannot just be bought by spending more dollars on R&D. Despite the enormous amounts of money involved and a rich source of managerial literature that asks firms to invest in innovation and promises success in return, firms continue to struggle in capturing value from their innovation efforts. Innovation remains a difficult and often frustrating task for many of them. Innovation requires strategy. This dissertation focuses on effective innovation strategies for incumbent firms or innovation strategies that pay off. Four studies focus on different aspects of firm’s innovation strategies. First, we apply a multilevel approach. Second, we look at four perspectives on effective innovation strategies: timing, location, technology and path to obtain in-depth insights into the complexity of managing and executing a firm’s innovation strategy. In sum, this dissertation concludes that in order to reach innovation strategy effectiveness, firms can engage in exploration and exploitation through a simultaneous or sequential way, should consider to opt for spatial proximity of their exploratory and exploitative activities, need to invest in technological development, and adjust these choices based on what happens during the execution of an innovation project