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Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor and third leading cause of cancer-related death worldwide. Most cases arise as a consequence of underlying liver disease, e.g. developed from chronic hepatitis B or C infectionsalcohol abuse or obesity, and are most often associated with liver cirrhosis. Hypoxiand the hypoxia inducible factors (HIF)-1α and -2α promote tumor progression of HCC, not only affecting tumor cell proliferation and invasion, but also angiogenesis and lymphangiogenesis and thus, increasing the risk of metastasis.
HCC is characterized as one of the most vascularized solid tumors. While HIF-1α and HIF-2α are frequently up-regulated in HCC only HIF-2α is correlated with high patientlethality. HIF-dependent regulation of HCC angiogenesis is controversially discussed.VEGFA, for example, as the most prominent factor inducing tumor angiogenesis represents not only a HIF-1 target, but also a HIF-2 target gene in HCC. This questions whether both isoforms have overlapping functions in regulating the angiogenic switch in HCC.
Besides angiogenesis also tumor-associated lymphangiogenesis significantly influences patient survival in HCC. Lymphatic spread is an important clinical determinant for the prognosis of HCC, but little is known how lymphangiogenesis is controlled in this context. To date, mainly HIF-1α was positively correlated with olymphatic invasion and metastasis in HCC, while a defined role of HIF-2α is missing. Thus, although HIF-1α and HIF-2α are structurally alike and regulate overlapping but not identical sets of target genes, they promote highly divergent outcomes in cancer progression and may even have counteracting roles. The aim of my work was to characterize the specific role of HIF-1α and HIF-2α in the angiogenic switch and lymphangiogenesis induction during HCC development.
Therefore, I created a stable knockdown of HIF-1α and HIF-2α in HepG2 cells and generated cocultures of HepG2 spheroids and embryonic bodies derived from embryonic mouse stem cells as an in vitro tumor model mimicking the cancer microenvironment to analyze which HIF isoform has key regulatory functions in HCC (lymph)angiogenesis. In cocultures with a HIF-2α knockdown angiogenesis was attenuated but lymphangiogenesis increased, while the knockdown of HIF-1α was without effect. Microarray analysis identified plasminogen activator inhibitor 1 (PAI-1)and insulin-like growth factor binding protein 1 (IGFBP1) as HIF-2 target genes.However, prominent angiogenic and lymphangiogenic factors such as VEGFs, PDGFB, ANG and their receptors were not regulated in a HIF-dependent manner. As PAI-1 was linked to angiogenesis in literature and IGF-signaling, which is negatively regulated by IGFBP-1, was correlated with lymphangiogenesis, I decided to investigate their HIF-2α-dependent influence on HCC (lymph)angiogenesis. The knockdown of PAI-1 in HepG2 cells also lowered angiogenesis in PAI-1k/d cocultures similar to the HIF-2α k/d phenotype. PAI-1 as the potent inhibitor of tPA and uPA, both inducing the conversion of plasminogen to plasmin, also inhibits plasmin directly. Therefore, I assumed an increase of plasmin in HIF-2α k/d and PAI-1 k/d cocultures as a result of the reduced PAI-1 levels. Blocking plasmin with aprotinin in HIF-2α k/d cocultures restored angioge nesis, suggesting that HIF-2α increases PAI-1 to lower concentrations of active plasmin, thereby supporting angiogenesis. In further experiments I could exclude PAI-1 to reduce angiogenesis by inducing plasmin-mediated apoptosis of differentiating stem cells in PAI-1 k/d and HIF-2α k/d cocultures, but demonstrated an increase of VEGFA165 degradation in these cocultures, suggesting plasmin-catalyzed proteolysis of VEGF as an additional layer of regulation required to explain the angiogenic phenotype. Besides the pivotal role of PAI-1 in angiogenesis I also investigated its potentialinfluence in lymphangiogenesis. Indeed, the knockdown of PAI-1 reduced lymphaticstructures and implied an important but opposing role in lymphangiogenesis comparedto induced lymphangiogenesis in HIF-2α k/d cocultures. However, blocking plasmin again with aprotinin in HIF-2α k/d cocultures restored lymphangiogenesis to the level of control virus, which indicates a divergent lymphangiogenic role of plasmin in PAI-1 k/d and HIF-2α k/d cocultures, possibly because of other essential pathways masking the lymphangiogenic effects of PAI-1 in HIF-2α k/d cocultures.
HIF-2α resulting in reduced IGFBP1 expression induced the differentiation of stem cells toward a lymphatic cell type and significantly enhanced the assembly of human dermal lymphatic endothelial cells into tubes. These data point the first time to an important impact of HIF-2 in the regulatin of lymphangiogenesis in vitro by inducing IGFBP1 and thus, scavenging IGF-1. Furthermore, matrigel plug assays to investigate the in vivorelevance of these observations confirmed HIF-2α as a crucial factor in the regulation of lymphangiogenesis in vivo
In conclusion, this work provides evidence that HIF-2α is a key regulator of angiogenesis and lymphangiogenesis in HCC by regulating PAI-1 and IGFBP1. HIF-2α positively influences the angiogenic switch via PAI-1 and negatively affects lymphangiogenesis via IGFBP1 expression. Targeting HIF-2α in HCC to reduce tumor angiogenesis should be approached carefully, as it might be overcome by induced lymphangiogenesis and metastasis.
Tumor development usually follows predictable paths where tumor cells acquire common characteristics and features known as the hallmarks of cancer. Recently, additional characteristics have been added to these hallmarks since solid tumors are composed of a very heterogeneous population of transformed, formerly normal tissue cells and stromal cells, e.g. immune cells and fibroblasts. Compelling evidence suggests that stromal cells and tumor cells maintain a symbiotic relationship to build up the tumor microenvironment and to fuel tumor growth. In cancer therapies, common features of tumors such as unrestricted cell growth, suppression of immunological responses, and the ability to form new blood vessels (angiogenesis) have emerged as the main targets of interest. The lipid mediator prostaglandin E2 (PGE2) is known to promote all these features and thus, is connected to cancer progression in general. Its synthesis is triggered in response to stress factors or during inflammation. Inducible PGE2 production relies on the enzymes cyclooxygenase 2 (COX-2) and microsomal prostanglandin E synthase 1 (mPGES-1), which are simultaneously expressed in response to a variety of different stimuli and are functionally coupled. Inhibition of COX-2 with non-steroidal antiinflammatory drugs (NSAIDs) for cancer treatment is, however, limited by cardiovascular risks, since selective COX-2 inhibition disrupts the prostacyclin/thromboxane balance. Therefore targeting mPGES-1 downstream of COX-2 for PGE2 inhibition was evaluated in this work in different steps of carcinogenesis. Knockdown of mPGES-1 in DU145 prostate cancer cells revealed that the mPGES-1 status did not affect growth of monolayer tumor cells, but significantly impaired 3D growth of multi-cellular tumor spheroids (MCTS). Spheroid formation induced COX-2 in DU145 and other prostate cancer spheroids. High levels of PGE2 were detected in supernatants of DU145 MCTS as opposed to monolayer DU145 cells. Pharmacological inhibition of COX-2 and mPGES-1 confirmed the pivotal role of PGE2 for DU145 MCTS growth. Besides promoting spheroid growth, MCTS-derived PGE2 also inhibited cytotoxic T lymphocyte (CTL) activation. When investigating the mechanisms of COX-2 induction during spheroid formation, the typical tumor microenvironmental factors such as glucose deprivation, hypoxia or tumor cell apoptosis failed to enhance COX-2. Interestingly, when interfering with apoptosis in DU145 spheroids, the pan-caspase inhibitor Z-VAD-FMK triggered a Summary 12 shift towards necrosis, thus enhancing COX-2 expression. Coculturing viable DU145 monolayer cells with isolated heat-shocked-treated necrotic DU145 cells, but not with necrotic cell supernatants, induced COX-2 and PGE2, confirming the impact of necrosis for MCTS growth and CTL inhibition. As mentioned, in vivo tumors are very heterogenous mixtures of tumor cells and stromal cells e.g. immune cells. Hence, the interaction of the immune system with tumors was investigated in further experiments. When coculturing MCF-7 breast cancer spheroids with human peripheral blood mononuclear cells (PBMCs), only low levels of PGE2 were detected, since MCF-7 cells did not upregulate COX-2 during spheroid formation and did not induce PGE2 production by PBMCs. Under inflammatory conditions, by adding the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) to cocultures, PGE2 production was triggered, spheroid sizes were reduced, and numbers of high levels of granzyme B expressing (GrBhi) CTLs were increased, while CD80 expression by tumor-associated phagocytes was also elevated. Inhibition of CD80 but not CD86 diminished numbers of GrBhi CTLs and attenuated spheroid lysis. To determine the role of ctivation-induced PGE2 production, use of the COX-2 inhibitor celecoxib and the experimental mPGES-1 inhibitor C3 further increased CD80 expression. Addition of PGE2, the prostaglandin E2 (EP2) receptor agonist butaprost, and the phosphodiesterase 4 (PDE4) inhibitor rolipram reduced LPS/C3-triggered CD80 expression, confirming the impact of COX- 2/mPGES-1-derived PGE2 on shaping phagocyte phenotypes in an EP2/cAMP-dependent manner. In a spontaneous breast cancer model (MMTV-PyMT), mPGES-1-deficiency significantly delayed tumor growth in mice, confirming an overall protumorigenic role of mPGES-1 in breast cancer development in vivo. However in tumors of mPGES-1-/- mice, tumor-infiltrating phagocytes expressed low levels of CD80 similar to their wildtype counterparts. These data suggest that the immunosuppressive microenvironment does not allow for immunostimulatory effects by mPGES-1 inhibition without an activating stimulus. Evidences in this study recommend the application of mPGES-1 inhibitors for treating cancer diseases, since mPGES-1 promotes tumor growth in multiple steps of carcinogenesis, ranging from well-characterized effects of tumor cell growth to immune suppression of CTL activity and phagocyte polarization. Regarding the latter, blunting PGE2 during immune activation may limit the tumor-favoring features of inflammation and improve the efficiency of TLR4 based immune therapies.
Die Wahrnehmung von Schmerzen ermöglicht es dem Organismus, auf noxische Reize zu reagieren. Der akute nozizeptive Schmerz hat somit eine natürliche Warnfunktion. Bei länger anhaltenden bzw. chronischen Schmerzen oder Nervenschädigungen kann es jedoch zu pathophysiologischen Veränderungen im Nervensystem kommen, die zur Verselbständigung des Schmerzes führen können. Unter diesen Umständen gilt der Schmerz nicht mehr als Warnsignal, sondern als eigenes Krankheitsbild. Die „International Association for the Study of Pain (IASP)“ definiert Schmerz als „ein unangenehmes Sinnes- und Gefühlserlebnis, das mit aktueller oder potenzieller Gewebsschädigung verknüpft ist oder mit Begriffen einer solchen Schädigung beschrieben wird“. Da bisher verfügbare Arzneimittel chronische Schmerzen in vielen Fällen nicht ausreichend reduzieren können und teilweise zu schwerwiegenden Nebenwirkungen führen, ist es unverzichtbar, an der Entwicklung neuer und noch spezifischer wirkenden Analgetika festzuhalten. Um Pharmaka zu entwickeln, die gezielt in den Mechanismus der Schmerzverarbeitung eingreifen können, ist es notwendig, diesen auf molekularer Ebene zu kennen und zu verstehen.
Immune cells are key players in several physiological and pathophysiological events such as acute and chronic inflammation, atherosclerosis and cancer. Especially in acute inflammation, macrophages are indispensable for the switch from the acute inflammatory phase to the resolution phase. Not only the phagocytosis of apoptotic cells, but especially the surrounding cytokines and mediators are able to switch macrophage polarization from inflammatory- to anti-inflammatory phenotypes. Within this cytokine environment, sphingosine-1-phosphate (S1P) plays an important role for immune cell activation, polarization and migration.