Marina Poettler

Angiogenesis in cancer: anti-VEGF escape mechanisms

It is now widely accepted that tumor-angiogenesis plays a crucial role in tumor growth, tumor propagation and metastasis formation. Among several angiogenic activators, the vascular endothelial growth factor (VEGF) and its receptors represent one of the major inducers of tumor angiogenesis. Thus, this system has become the focus of therapeutic interventions, which led to the approval of the anti-VEGF blocking antibody bevacizumab and the VEGFR-2 pathway inhibitors pazopanib, sorafenib and sunitinib. However, not every cancer patient benefits from such treatment or finally becomes resistant to anti-VEGF approaches; others are suffering from adverse effects. Thus, there is an urgent need for a better understanding of VEGF-independent mechanisms leading to angiogenesis in cancer. This review focuses on anti-VEGF escape mechanisms of tumor cells and its microenvironment.

Soluble Carcinoembryonic Antigen Activates Endothelial Cells and Tumor Angiogenesis

Carcinoembryonic antigen (CEA, CD66e, CEACAM-5) is a cell-surface-bound glycoprotein overexpressed and released by many solid tumors that has an autocrine function in cancer cell survival and differentiation. Soluble CEA released by tumors is present in the circulation of patients with cancer, where it is used as a marker for cancer progression, but whether this form of CEA exerts any effects in the tumor microenvironment is unknown. Here, we present evidence that soluble CEA is sufficient to induce proangiogenic endothelial cell behaviors, including adhesion, spreading, proliferation, and migration in vitro and tumor microvascularization in vivo. CEA-induced activation of endothelial cells was dependent on integrin β-3 signals that activate the focal-adhesion kinase and c-Src kinase and their downstream MAP-ERK kinase/extracellular signal regulated kinase and phosphoinositide 3-kinase/Akt effector pathways. Notably, while interference with VEGF signaling had no effect on CEA-induced endothelial cell activation, downregulation with the CEA receptor in endothelial cells attenuated CEA-induced signaling and tumor angiogenesis. Corroborating these results clinically, we found that tumor microvascularization was higher in patients with colorectal cancer exhibiting higher serum levels of soluble CEA. Together, our results elucidate a novel function for soluble CEA in tumor angiogenesis.

Targeting of VEGF-dependent transendothelial migration of cancer cells by bevacizumab

Cancer progression is often associated with the formation of malignant effusions. Vascular endothelial growth factor (VEGF) is a major regulator of vascular permeability and has been implicated as mediator of tumor progression.

CD98hc (SLC3A2), a novel marker in renal cell cancer

Background  In a variety of malignant diseases, molecular targeting represents a therapeutic option, whereby, when compared with chemotherapy, fewer side effects are thought to be expected. Especially in renal cell cancer (RCC), tyrosine kinase‐inhibitors have been established as useful and highly effective therapy. However, tyrosine kinase‐inhibitors currently approved for RCC treatment lack single molecule specificity and bear a variety of side effects of the gastro‐intestinal tract, skin, heart and haematopoietic system. Therefore, the identification of novel cell surface markers is sought, which might lead to novel diagnostic and therapeutic strategies in cancer.

The urokinase receptor (CD87) represents a central mediator of growth factor-induced endothelial cell migration

Angiogenesis, the sprouting of blood vessels form pre-existing vasculature after injury or in neoplastic diseases, is initiated by growth factor-induced endothelial cell migration. Recently, the major angiogenic growth factor VEGF165 has become the target of therapeutic interventions. However, this approach has been clinically proven to be of limited efficacy, which might be due to the fact that tumour angiogenesis is not only induced by VEGF, but also by a variety of other growth factors. Thus, the identification of a common downstream mediator of growth-factor-induced endothelial cell migration is mandatory to effectively interfere with (tumour-) angiogenesis. We found that the urokinase-type plasminogen activator (uPA)-system, which affects proteolytic as well as adhesive capacities, represents an essential regulatory mechanism in growth factor-induced endothelial cell migration and invasion. This mechanism was not limited to VEGF165, but mediated pro-angiogenic endothelial cell behaviour induced by various growth factors. Thus, VEGF165, VEGF-E, FGF-2, EGF as well as HGF induced a PI3k-dependent activation of pro-uPA when bound to uPAR, which led to an increase in cell surface fibrinolytic activity. As a consequence, uPAR became internalised and redistributed via LDLR-proteins. Interference with these events led to a reduced migratory response of endothelial cells towards VEGF in vitro as well as endothelial cell invasion in vivo. These data give first evidence that the uPA-system, which represents the only level-of-evidence-1 cancer biomarker system for prognosis and/or prediction in node negative breast cancer, might directly affect (tumour-) angiogenesis.

CD98hc (SLC3A2) drives integrin-dependent renal cancer cell behavior

BackgroundOverexpression of CD98hc (SLC3A2) occurs in a variety of cancers and is suspected to contribute to tumor growth. CD98, a heterodimeric transmembrane protein, physically associates with certain integrin β subunit cytoplasmic domains via its heavy chain, CD98hc. CD98hc regulates adhesion-induced intracellular signal transduction via integrins, thereby, affecting cell proliferation and clonal expansion. Disruption of CD98hc led to embryonic lethality in mice (E 3.5 and E 9.5) and CD98hc −/− embryonic stem cell transplantation failed to form teratomas, while CD98hc over-expression in somatic cells resulted in anchorage-independent growth. However, it is unclear whether interference with CD98hc expression tumor cell behavior.MethodsRenal cell cancer cell lines have been used to determine the effect of CD98hc expression on cancer cell behavior using cell adhesion, cell trans-migration and cell spreading assays. Flow cytometric analysis was performed to study the rate of apoptosis after detachment or serum starvation. shRNA-lentiviral constructs were used to stably knockdown or reconstitute full length or mutated CD98hc. The role of CD98 as a promotor of tumorigenesis was evaluated using an in in vivo tumor transplantation animal model. Immunohistochemical analysis was performed to analyze cell proliferation and CD98 expression in tumors.ResultsThis report shows that CD98hc silencing in clear cell renal cancer cells reverts certain characteristics of tumorigenesis, including cell spreading, migration, proliferation and survival in vitro, and tumor growth in vivo. Acquisition of tumorigenic characteristics in clear cell renal cancer cells occurred through the integrin binding domain of CD98hc. A CD98hc/integrin interaction was required for adhesion-induced sustained FAK phosphorylation and activation of the major downstream signaling pathways PI3k/Akt and MEK/ERK, while overexpression of a constitutive active form of FAK rescued the CD98hc deficiency.ConclusionsIn this study we demonstrate that loss of CD98hc blocks tumorigenic potential in renal cell cancer.

Effects of cilengitide in osteoclast maturation and behavior.

Bone metastasis is a common burden in many types of cancer and has a severe impact on the quality of life in patients. Hence, specific therapeutic strategies inhibiting tumor induced osteolysis are urgently needed. In this study, we aimed to interfere with integrin adhesion receptors, which are central players of the bone resorption process. For this purpose, we used cilengitide, a cyclic RGD peptide, which blocks integrin αVβ3 and αVβ5-ligand binding. Our results revealed that cilengitide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with cilengitide exhibited reduced cell spreading, cell migration and cell adhesion on RGD-containing matrix proteins, which are ligands of integrin αV. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, FAK and c-Src, were consistently blocked by cilengitide. First evidence suggests that cilengitide might interfere with metastatic bone disease in vivo and this study describes a potential underlying mechanism of the inhibitory effect of cilengitide on αV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that cilengitide, which interferes with αV-integrins on osteoclasts, may represent a novel therapeutic strategy in the treatment of malignant bone disease.

Abstract 406: Effects of an RGD peptide in osteoclast maturation and behavior as a therapeutic option for metastatic bone disease

Metastatic bone disease is a common feature of many types of cancer and has a severe impact on the quality of life of patients. Hence, specific therapeutic strategies inhibiting tumor induced osteolysis are urgently needed. In this study, we aimed to interfere with integrin adhesion receptors, which are central players of the bone resorption process, including osteoclastogenesis as well as osteoclast/bone matrix interaction. For this purpose, we used a cyclic RGD peptide which blocks integrin aVâ3 and aVâ5-ligand binding. Our results revealed that the RGD peptide blocked osteoclast maturation in a dose-dependent manner. In detail, pre-osteoclasts treated with the RGD peptide exhibited reduced cell spreading, migration and adhesion on RGD-containing matrix proteins, such as osteopontin and fibrinogen, which are ligands of integrin aVâ3. The activation of the most upstream signal transduction molecules of the integrin receptor-initiated pathway, such as FAK and c-Src, were consistently blocked by the RGD peptide. First evidence has suggested that the RGD peptide might interfere with metastatic bone disease in vivo and the evidence presented herein describes the underlying mechanisms of the inhibitory effect of the RGD peptide on aV-integrin expressing pre-osteoclasts by blocking integrin ligand binding and interfering with osteoclast maturation and cell behavior. In conclusion, our findings suggest that using an RGD peptide to interfere with aV-integrins on osteoclasts may represent a novel therapeutic strategy in the treatment of malignant bone disease. Citation Format: Gerald Prager, Daniela Bianconi, Anastasia Chilla, Alexandra Dorda, Nisha Geetha, Matthias Unseld, Despoina Sykoutri, Marina Poettler, Kurt Redlich, Christoph Zielinski. Effects of an RGD peptide in osteoclast maturation and behavior as a therapeutic option for metastatic bone disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2015-406

Abstract C3: Tumor-derived soluble uPAR regulates tumor angiogenesis via PTEN.

The urokinase receptor uPAR (CD87), a GPI-anchored protein, is frequently over-expressed in tumor cells. uPAR provokes numerous cellular functions by focusing proteolytic activity on the cell surface, but also by inducing intracellular signal transduction via transmembrane interaction partners, thus inducing cell migration and invasion as well as cell survival. Soluble tumor-derived uPAR has recently been described to activate endothelial cells and was suggested to promote angiogenesis. Consistently, high uPAR expression in tumor cells or high serum levels of circulating uPAR predict worse prognosis of breast, lung or colorectal cancer patients. In this study, we found that tumor-derived soluble uPAR is a key regulator of PTEN expression in endothelial cells. HEK cells deficient in endogenous uPAR or uPAR deficient endothelial cells derived from uPAR -/- mice had high PTEN mRNA as well as protein levels, while reconstitution of endogenous as well as addition of soluble exogenous uPAR decreased PTEN levels. uPAR, thereby, led to an integrin-dependent activation of the NFkB pathway, a known transcription pathway inhibitor of PTEN. As a consequence of PTEN downregulation, the downstream PKB/Akt signalling pathway became activated, which led to an enhanced migratory as well as cell survival activity in endothelial cells. Cross-breeding of uPAR -/- mice with endothelial cell-specific PTEN +/- mice, thereby led to a rescue of the high migratory phenotype of PTEN heterozygous endothelial cells in vitro and angiogenesis formation in a directed in vivo angiogenesis assay (DIVAA). From our date we conclude that tumor-derived uPAR is a major regulator of PTEN expression in endothelial cells, which leads to the induction of the PI3K/Akt-pathway and an enhanced angiogenic phenotype. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C3. Citation Format: Gerald W. Prager, Matthias Unseld, Marina Poettler, Christoph Zielinski. Tumor-derived soluble uPAR regulates tumor angiogenesis via PTEN. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C3.

Abstract B6: CEA affects tumor- angiogenesis via integrin-dependent activation of endothelial cells.

CEA, first identified in 1965 by Phil Gold and Samuel O. Freedman, is aberrantly overexpressed by up to 70% of human cancers. In clinical use as extracellular marker for monitoring adenocarcinoma growth as well as treatment efficacy, CEA has become included into the routine practice of clinical oncology. In addition to its’ clinical relevance, CEA was so far only described to be pro-tumorigenic in an autocrine manner by inducing anti-apoptosis. Any potential paracrine effects of soluble CEA affecting tumor microenvironment including angiogenesis have not been described. Here, we report that CEA released by tumor cells induced angiogenesis by directly inducing pro-angiogenic endothelial cells behavior. As shown by gain and loss of function in vivo tumor transplant models, CEA directly stimulated endothelial cell proliferation and migration in vitro and endothelial cell invasion and functional active capillary-tube formation in directed in vivo angiogenesis assays. Thereby, CEA activated endothelial cells via integrin beta-3, leading to FAK phosphorylation and c-src activation as well as induction of MEK/ERK- and PI3kinase/Akt- pathways. Interfering with this chain of events at different steps, such as downregulation of its putative receptor for CEA (CEAR) by shRNA bearing lentivirus or blockage of integrin activity, abrogated CEA-induced endothelial cell activation and tumor-angiogenesis leading to tumor growth arrest. Notably, CEA acted independently and partially additive to the VEGF / VEGFR-2 (KDR or flk-1) system. The biological relevance was further verified by analysis of paraffin-embedded tumor-tissue samples derived from colon cancer patients, whereas vascularization of the tumor microenvironment was significantly higher whenever CEA plasma levels were high. Thus, our data indicate that the tumor marker CEA acts as a novel angiogenic molecule and might offer a potential therapeutic target in the treatment of tumor-angiogenesis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B6. Citation Format: Gerald W. Prager, Kira Braemswig, Marina Poettler, Christoph Zieleinski. CEA affects tumor- angiogenesis via integrin-dependent activation of endothelial cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B6.