A multi-targeted approach to suppress tumor-promoting inflammation

Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes

The impact of endoplasmic reticulum stress on melanoma progression

Das Melanom stellt im Vergleich zum Basalzellkarzinom und Plattenepithelkarzinom die aggressivste Form von Hautkrebs dar und kann je nach Tumordicke, Befall von Lymphknoten und Metastasierungsgrad in verschiedene Stadien eingeteilt werden. Anhand dieser Einteilung kann eine spezifische Behandlung erfolgen, wobei für das metastatische Melanom im Stadium IV die größte Bandbreite an Therapieoptionen zur Verfügung steht. Jedoch zeigen die angewandten Therapien des metastatischen Melanoms bei vielen Patienten schwere Nebenwirkungen und haben nur mäßige, positive Effekte auf die Verlängerung der Lebenszeit. Die Überlebens-Prognose für metastatisches Melanom beträgt zwischen 6 bis 12 Monaten. Die größte Herausforderung in der Entwicklung von spezifischen Krebstherapien stellen die noch zu wenig erforschten Veränderungen dar, die bei der Progression von einer gutartigen zu einer bösartigen Krebszelle stattfinden. Eine wesentliche Rolle in der Entwicklung spielt Endoplasmatischer Retikulum (ER) Stress, der zunächst Signalwege der "Unfolded Protein Response" (UPR) anschaltet um den Anforderungen einer Krebszelle gerecht zu werden. Die UPR wird über drei Signalkaskaden übermittelt: den ATF6-, der PERK- und der IRE1-Signalweg. Kann aber beispielsweise der erhöhten Proteinmenge, falsch gefalteten Proteinen und Hypoxie nicht stand gehalten werden, leiten diese Signalwege Apoptose ein. Eine Krebszelle jedoch kann dies umgehen und sich stattdessen an chronischen Stress adaptieren. In humanen BRAFV600E mutierten, isogenen Melanomzelllinien wurde in-vitro eine erhöhte UPR Aktivität in den metastasierenden Zellen festgestellt. Aufgrund der Relevanz von ER Stress und der UPR in Krebserkrankungen wurden diese im Zusammenhang mit der Malignität des Melanoms untersucht. In der metastatischen BRAFV600E mutierten Melanomzelllinie konnte gezeigt werden, dass diese Zellen die Fähigkeit besitzen besser mit chemisch induziertem Stress umgehen und diesem standhalten zu können. Zusammen mit der Beobachtung, dass metastatische Melanomzellen erhöhten ER Stress aufweisen, repräsentieren sie ein gutes Modell um den Einfluss von chronischem ER Stress auf die Malignität des Melanoms zu untersuchen und damit auch andere Krebserkrankungen besser zu verstehen. Des weiteren konnte gezeigt werden, dass eine erhöhte Aktivität von zwei Signalwegen der UPR, der ATF6 und PERK Signalweg, jedoch nicht der IRE1 Signalweg, stark mit dem Überleben von Melanompatienten korreliert. In einer Genomexpressionsanalyse wurde eine Verbindung der UPR mit der Expression von Fibroblasten-Wachstumsfaktoren (FGFs) und Migration festgestellt. Die regulatorische Verknüpfung dieser Signalwege konnte in-vitro bestätigt werden, da die erhöhte Expression von FGF1 und FGF2-Wachstumsfaktoren und auch Migration in den metastatischen Melanomzellen durch die ER Stress Reduktion mit 4-Phenylbutyrat (4-PBA) vermindert wurden. Zusätzlich wurde mit der Behandlung von 4-PBA auch die Zellviabilität in metastatischen Melanomzellen erniedrigt. Diese regulatorische Verknüpfung der UPR mit den FGF Signalwegen konnte auch bei der Analyse von Patientendaten demonstriert werden. Die Expression der FGFs zeigten eine Korrelation mit der Aktivität der ATF6 und PERK Signalwege der UPR. Mit dieser Arbeit konnte eine Verbindung von chronischem ER Stress und der daraus resultierenden erhöhten UPR Aktivität, mit der Regulierung von FGFs und Migration im Melanom aufgedeckt werden. Aufgrund der starken Beeinflussung dieser Signalwege tragen sie zur Malignität des Melanoms bei. Deswegen ist es dringend notwendig Medikamente, wie zum Beispiel Chaperone, die diese Signalkaskaden antagonisieren, anzuwenden. Zusammen mit den bereits vorhandenen Behandlungen für fortgeschrittenes Melanom könnten diese kombiniert werden und einen vielversprechenden Therapieansatz darstellen.Melanoma is the most aggressive type of skin cancer and so far promising therapies for long time survival of patients with advanced stages of melanoma are limited. Unfortunately, the events contributing to the progression of melanoma towards a metastatic phenotype are poorly understood and treatment options remain insufficient and are often accompanied with severe side effects. In the last decade endoplasmic reticulum (ER) stress and the induction of the unfolded protein response (UPR) gained attention, due to its abnormal regulation in several cancer types. ER stress regulates ambiguous events during carcinogenesis by enabling the adaption to altered metabolic demands of a cancer cell as well as the induction of apoptosis. In this thesis the focus was directed on understanding the impact of ER stress and the subsequent unfolded protein response (UPR) in melanoma. To investigate the role of intrinsic UPR in the progression of melanoma we used isogenic, BRAFV600E-mutated melanoma cell lines. In the publication arising from this thesis we show that the three branches of the UPR (ATF6, PERK and IRE1) were elevated in metastatic compared to non-metastatic melanoma cells. Thus, these melanoma cell lines represent a good model for the investigation of chronic ER stress. In available datasets of human melanoma patients the ATF6 and PERK pathways of the UPR are associated with poor survival, further suggesting a role of the UPR in regulating the conversion towards a metastatic phenotype. Upon acute ER stress induction using thapsigargin, metastatic melanoma cells showed an increased ability to manage ER stress conditions in contrast to non-metastatic cells, providing a survival advantage for metastatic melanoma cells. A whole-genome expression array revealed a link between the UPR activity and the expression of genes involved in migration, invasion and fibroblast growth factors production. Elevated levels of FGF1 and FGF2 in metastatic melanoma cells were reduced after the chemical chaperone 4-PBA was used to ameliorate ER stress. Beneficial effects of 4-PBA-treatment were also observed for migration and cell viability, as both were reduced after ER stress reduction. Additionally, the expression of FGF was correlated with ATF6 and PERK activity in human melanoma patients, confirming the relevance of the in-vitro results. In this project we revealed that ER stress and the UPR are implicated in melanoma malignancy by up-regulating genes involved in migration, invasion and growth factor production. Therefore, the investigation of drugs intervening with the UPR represents a promising strategy in cancer therapy.submitted by Karin EignerAbweichender Titel laut Übersetzung der Verfasserin/des Verfassers2017, Dissertation(VLID)256407

Bile acids reduce endocytosis of high-density lipoprotein (HDL) in HepG2 cells.

High-density lipoprotein (HDL) transports lipids to hepatic cells and the majority of HDL-associated cholesterol is destined for biliary excretion. Cholesterol is excreted into the bile directly or after conversion to bile acids, which are also present in the plasma as they are effectively reabsorbed through the enterohepatic cycle. Here, we provide evidence that bile acids affect HDL endocytosis. Using fluorescent and radiolabeled HDL, we show that HDL endocytosis was reduced in the presence of high concentrations of taurocholate, a natural non-cell-permeable bile acid, in human hepatic HepG2 and HuH7 cells. In contrast, selective cholesteryl-ester (CE) uptake was increased. Taurocholate exerted these effects extracellularly and independently of HDL modification, cell membrane perturbation or blocking of endocytic trafficking. Instead, this reduction of endocytosis and increase in selective uptake was dependent on SR-BI. In addition, cell-permeable bile acids reduced HDL endocytosis by farnesoid X receptor (FXR) activation: chenodeoxycholate and the non-steroidal FXR agonist GW4064 reduced HDL endocytosis, whereas selective CE uptake was unaltered. Reduced HDL endocytosis by FXR activation was independent of SR-BI and was likely mediated by impaired expression of the scavenger receptor cluster of differentiation 36 (CD36). Taken together we have shown that bile acids reduce HDL endocytosis by transcriptional and non-transcriptional mechanisms. Further, we suggest that HDL endocytosis and selective lipid uptake are not necessarily tightly linked to each other

GW4064 and CDCA reduce CD36 expression and function.

<p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours and gene expression was analyzed by qRT-PCR (n = 3). (b) Cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hrs and protein expression was determined by western blot analysis and results were quantitated by densitometry (n = 3). (c) Fatty-acid uptake was determined after treatment with 10 µM GW4064 or 100 µM CDCA as described in the methods section (n = 3).</p

Taurocholate reduces HDL endocytosis SR-BI-dependently.

<p>(a) HepG2 cells were incubated with or without 1 mM taurocholate and ATP hydrolysis was measured as a decrease in extracellular ATP. One representative experiment out of three independent experiments is shown. (b) SR-BI knockdown efficiency in HepG2 cells transfected with scrambled shRNA and HepG2 cells transfected with SR-BI shRNA (n = 3). Selective lipid uptake analysis using double labeled ¹²⁵I/³H-CE-HDL in scrambled control (c) or SR-BI knockdown (d) HepG2 cells (n = 3). Selective cholesteryl-ester uptake was calculated by subtracting ¹²⁵I-HDL uptake from ³H-CE-HDL uptake.</p

Bile acids and a non-steroidal FXR agonist reduce HDL endocytosis.

<p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours. Gene expression was analyzed by qRT-PCR and expression levels were normalized to GAPDH expression (n = 2). The increase in SHP mRNA indicates FXR activation. (b) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 50 µg/ml HDL-Alexa⁴⁸⁸ for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = 10 µm. (c) Quantification of fluorescence intensities of (b). (d) HepG2 cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 µg/ml ¹²⁵I-HDL for 1 hour. Uptake was determined after displacing cell surface bound HDL by a 100-fold excess at 4°C for 1 hour (n = 3).</p

Taurocholate neither exerts cytotoxic effects, nor inhibits transferrin or LDL endocytosis in HepG2 cells.

<p>(a) Cells were incubated with the indicated concentrations of taurocholate for 1 hour. No release of LDH into the cell culture supernatant was detected. 0.1% Triton-X100 was used as a positive control. (b) Cells were incubated with 20 µg/ml transferrin-Alexa⁴⁸⁸ (b) or 50 µg/ml LDL-Alexa⁵⁶⁸ (c) with or without 1 mM taurocholate at 37°C for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: transferrin; red: LDL; blue: nucleus; bar = 10 µm. Neither transferrin nor LDL uptake were altered. Quantifications of fluorescent signals are depicted next to the images. (d) Cells were incubated with or without 1 mM taurocholate for 1 hour. Cells were fixed, stained with Filipin and imaged. Bar = 10 µm. Representative images of 3 independent experiments are shown.</p

GW4064 and CDCA reduce HDL endocytosis independently of SR-BI.

<p>(a) HepG2 cells were treated with the indicated concentrations of GW4064 or chenodeoxycholate (CDCA) in media containing lipoprotein-deficient serum (lpds) for 24 hours and gene expression was analyzed by qRT-PCR (n = 3). (b) Cells were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hrs and protein expression was determined by western blot analysis and results were quantitated by densitometry (n = 3). HepG2 cells transfected with scrambled shRNA (c) or SR-BI shRNA (d) were incubated with 10 µM GW4064 or 100 µM CDCA in media containing lpds for 24 hours. Cells were then incubated with 20 µg/ml double labeled ¹²⁵I/³H-CE-HDL for 1 hr. Selective cholesteryl-ester uptake was calculated by subtracting ¹²⁵I-HDL uptake from ³H-CE-HDL uptake (n = 3).</p

Modification of HDL by taurocholate does not alter endocytosis.

<p>(a) HDL was incubated with or without 1 mM taurocholate in media in the absence of cells for 1 hour. HDL size was then analyzed by size exclusion chromatography. HDL incubated with taurocholate is eluted earlier, indicating increased size. (b) HDL-Alexa⁴⁸⁸ was incubated with or without 1 mM taurocholate in media in the absence of cells for 1 hour. Free taurocholate was then removed using gel filtration and HepG2 cells were incubated with this modified HDL-Alexa⁴⁸⁸ for 1 hour. Cells were fixed, counterstained with DAPI and imaged. (c) Quantification of fluorescence intensities from (b); n = 3. Green: HDL; blue: nucleus; bar = 10 µm.</p

Bile acids reduce HDL endocytosis.

<p>HepG2 (a) and HuH7 (b) cells were incubated with 50 µg/ml HDL-Alexa⁴⁸⁸ with or without 1 mM taurocholate at 37°C for 1 hour. Cells were fixed, counterstained with DAPI and imaged. Green: HDL; blue: nucleus; bar = 10 µm. Representative images of 3 independent experiments are shown. (c) Quantification of fluorescence intensities of (a) and (b). (d) HepG2 cells were incubated in media containing 20 µg/ml ¹²⁵I-HDL with or without 1 mM taurocholate at 37°C for 1 hour. Uptake was determined after displacing cell surface bound HDL by a 100-fold excess at 4°C for 1 hour (n = 3). (e) Cells were incubated with 20 µg/ml ¹²⁵I-HDL with the indicated concentrations of taurocholate for 1 hour (n = 3). (f) Cells were incubated with 20 µg/ml ¹²⁵I-HDL together with different bile acids for 1 hour (n = 3). Of note taurodeoxycholate, deoxycholate and chenodeoxycholate were cytotoxic at 1 mM and were therefore used at 0.5 mM.</p