Clemens Pausz

Integrin beta-3 genetic variants and risk of venous thromboembolism in colorectal cancer patients

Background Integrin β3 is involved in tumor and endothelial cell biology as well as in platelet aggregation. Herein, we evaluated the predictive potential of three germline single nucleotide polymorphisms (SNPs) in the integrin β3 gene (rs3809865, rs5918 and rs4642) to predict the risk of venous thromboembolism (VTE) in colorectal cancer (CRC) patients, which is one of the leading causes of death among cancer patients. Methods 112 patients diagnosed with CRC enrolled in the prospective Vienna Cancer and Thrombosis Study (CATS) were assessed with a median follow-up of 46 months. DNA was isolated from venous blood samples and SNPs were analyzed by the PCR-RFLP method. Results VTE occurred in 12% (n = 13) of all patients. The SNPs rs5918 and rs4642 were not associated with VTE risk. For rs3809565, 23% (n = 11) of patients had the A/A genotype, 4% (n = 2) had the A/T genotype, but none (0%) had the T/T genotype. In the univariate analysis, patients with the A/A genotype had a significantly higher risk to develop VTE compared to the other polymorphisms (P = 0.0005 after Fine and Gray). In the multivariable analysis, the predictive value remained significant. Conclusions This study identified the rs3809865 A/A genotype as an independent risk factor for VTE in CRC patients. Our findings would help identify high risk patients and would be essential for tailored anticoagulant prophylaxis.

PTEN expression in endothelial cells is down-regulated by uPAR to promote angiogenesis

The tumour suppressor phosphatase and tensin homologue (PTEN), mutated or lost in many human cancers, is a major regulator of angiogenesis. However, the cellular mechanism of PTEN regulation in endothelial cells so far remains elusive. Here, we characterise the urokinase receptor (uPAR, CD87) and its tumour-derived soluble form, suPAR, as a key molecule of regulating PTEN in endothelial cells. We observed uPAR-deficient endothelial cells to express enhanced PTEN mRNA- and protein levels. Consistently, uPAR expression in endogenous negative uPAR cells, down-regulated PTEN and activated the PI3K/Akt pathway. Additionally, we found that integrin adhesion receptors act as trans-membrane signaling partners for uPAR to repress PTEN transcription in a NF-κB-dependent manner. Functional in vitro assays with endothelial cells, derived from uPAR-deficient and PTEN heterozygous crossbred mice, demonstrated the impact of uPAR-dependent PTEN regulation on cell motility and survival. In an in vivo murine angiogenesis model uPAR-deficient PTEN heterozygous animals increased the impaired angiogenic phenotype of uPAR knockout mice and were able to reverse the high invasive potential of PTEN heterozygots. Our data provide first evidence that endogenous as well as exogenous soluble uPAR down-regulated PTEN in endothelial cells to support angiogenesis. The uPAR-induced PTEN regulation might represent a novel target for drug interference, and may lead to the development of new therapeutic strategies in anti-angiogenic treatment.

Cannabinoid Receptors Are Overexpressed in CLL but of Limited Potential for Therapeutic Exploitation.

The cannabinoid receptors 1 and 2 (CNR1&2) are overexpressed in a variety of malignant diseases and cannabinoids can have noteworthy impact on tumor cell viability and tumor growth. Patients diagnosed with chronic lymphocytic leukemia (CLL) present with very heterogeneous disease characteristics translating into highly differential risk properties. To meet the urgent need for refinement in risk stratification at diagnosis and the search for novel therapies we studied CNR expression and response to cannabinoid treatment in CLL. Expression levels of CNR1&2 were determined in 107 CLL patients by real-time PCR and analyzed with regard to prognostic markers and survival. Cell viability of primary CLL cells was determined in suspension and co-culture after incubation in increasing cannabinoid concentrations under normal and reduced serum conditions and in combination with fludarabine. Impact of cannabinoids on migration of CLL cells towards CXCL12 was determined in transwell plates. We found CNR1&2 to be overexpressed in CLL compared to healthy B-cells. Discriminating between high and low expressing subgroups, only high CNR1 expression was associated with two established high risk markers and conferred significantly shorter overall and treatment free survival. Viability of CLL primary cells was reduced in a dose dependent fashion upon incubation with cannabinoids, however, healthy cells were similarly affected. Under serum reduced conditions, no significant differences were observed within suspension and co-culture, respectively, however, the feeder layer contributed significantly to the survival of CLL cells compared to suspension culture conditions. No significant differences were observed when treating CLL cells with cannabinoids in combination with fludarabine. Interestingly, biologic activity of cannabinoids was independent of both CNR1&2 expression. Finally, we did not observe an inhibition of CXCL12-induced migration by cannabinoids. In contrast to other tumor entities, our data suggest a limited usability of cannabinoids for CLL therapy. Nonetheless, we could define CNR1 mRNA expression as novel prognostic marker.

SAR-guided development and characterization of a potent antitumor compound toward B-cell neoplasms with no detectable cytotoxicity toward healthy cells.

Acute hematological diseases (leukemias and aggressive lymphomas) can be cured in approximately half of the patients, while the other patients die from their disease. Chronic leukemias and indolent lymphomas can be well controlled for years in most cases. However, the cure rate of these patients is low and the course of the disease is characterized by frequent recurrence. Therefore, novel agents for monotherapies or combination therapies still need to be explored. The presented study describes the identification of the chalcone derivative 15 on different types of human malignant cells of the lymphoid and myeloid lineage. Further experiments performed with compound 15 on peripheral blood mononuclear cells (PBMCs) of chronic lymphocytic leukemia (CLL) patients clearly stated a higher cytotoxicity in PBMC from CLL patients compared to healthy donors (HD). The newly identified chalcone derivative 15 showed a higher therapeutic potential than fludarabine, a drug already in use in lymphoma treatment.

Abstract 3602: PTEN dependent angiogenesis is mainly regulated by (tumor secreted-) uPAR

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tumor stroma crucially influences the growth of most epithelial tumors and is in focus of many therapeutic approaches. Several oncogenes and tumor suppressors coordinate complex signaling cascades in the tumor and its microenvironment. In this context, downregulation of the tumor suppressor PTEN was shown to be indispensable for (tumor-)angiogenesis and cancer outgrowth. However, the essential process leading to PTEN downregulation, favouring a pro-angiogenic phenotype, in tumor vasculature is unknown so far. Here we show that PTEN-dependent angiogenesis is centrally affected by tumor- secreted soluble uPAR. In detail, the presence of uPAR significantly influenced the levels of PTEN in vitro and in vivo. Endothelial cell stimulation with tumor- derived soluble uPAR remarkable decreased PTEN levels, which led to increased cell migration, enhanced wound healing as well as capillary-sprouting in vitro and enhanced endothelial cell invasion and functional vessel formation in a directed-in vivo angiogenesis assay. uPAR- derived PTEN regulation was not mediated via mRNA stability or decrease in PTEN degradation, but via regulation of PTEN transcription as revealed by actinomycin D experiments. uPAR thereby interacted with αv integrins, which enhanced FAK activation and - as a consequence - led to NFkB-dependent PTEN repression. Finally, the biological relevance of uPAR- dependent PTEN regulation was evaluated and confirmed via uPAR -/- crossbreds with either PTEN +/- or PTEN +/+ mice. Our results provide first evidence of the role of tumor-derived uPAR in regulation of the central phosphatase PTEN in angiogenic cell behaviour. Our findings give novel insights into the complex mechanism of the tumor- microenvironmental interaction and might lead to new therapeutic strategies in cancer. Citation Format: Matthias Unseld, Anastasia Chilla, Clemens Pausz, Johannes Breuss, Gernot Schabbauer, Gerald Prager. PTEN dependent angiogenesis is mainly regulated by (tumor secreted-) uPAR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2014-3602

In vivo Tube Assay: An Optimised Protocol of the Directed in vivo Angiogenesis Assay by Implementing Immunohistochemistry.

Background: Angiogenesis, the formation of new blood vessels, is an essential process under physiological and pathological conditions. Method: Here, we improved the directed in vivo angiogenesis assay (DIVAA®) test, which is based on the usage of small Matrigel-filled tubes that are implanted into mice subcutaneously for a period of up to 15 days. The subsequent ex vivo assessment of neoangiogenesis within the silicon tubes is then achieved by fluorometry. Results: We showed that the immunohistochemical quantification of the ingrowth of endothelial cells, based on CD31, was superior to the fluorometric quantification advised in the manufacturers instructions. We optimised the explantation procedure, ensuring the complete recovery of the ingrown vessels. Using this modified protocol, we investigated the effect of the length of stay of the implanted tubes as well as of the concentration of the growth factors VEGF and FGF on the assay. Conclusion: Our improved protocol offered an effective and reliable alternative to the original assay, which is expected to facilitate in vivo research on angiogenesis and, thus, might drive the development of novel therapeutic agents.

Abstract 2306: Quantitative assessment of one-side directed in vivo angiogenesis

Angiogenesis, the sprouting of new blood vessels from pre-existing vasculature, is an essential process under physiologic and pathological conditions, and is in focus of therapeutic intervention. Reliable in vivo models to study angiogenesis are a pre-requisite for the characterization of novel therapeutic targets. Several methods like the matrigel plug assay, the chick chorioallantoic membrane assay or the cornea assay have addressed the objective to analyse angiogenesis in vivo, but a precise method to quantify one side-directed angiogenesis is lacking so far. Here, we describe an assay, which is based on the use of small matrigel-filled silicone cylinders that are implanted subcutaneously into mice for a period of up to 15 days. In C57BL/6J mice, the effect of the incubation period as well as the dose of the growth factors VEGF and FGF on angiogenic ingrowth was investigated. We describe the appropriate explantation procedure of the silicon tubes, ensuring the complete recovery of the in-grown vessels. The subsequent ex vivo assessment of neo-angiogenesis was then achieved by anti-CD31 as well as anti-endomucin immuno-histochemical staining of ingrown endothelial cells. Additionally, we show that vessel perfusion can be assessed by intravenous injection of a cell staining dye. Altogether, the elaborated protocol offers an effective and reliable assay which facilitates in vivo research on angiogenesis thus might support the development of novel therapeutic agents. Citation Format: Gerald Prager, Matthias Unseld, Johannes Breuss, Clemens Pausz, Gernot Schabbauer, Christoph Zielinski, Pavel Uhrin. Quantitative assessment of one-side directed in vivo angiogenesis. [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 2306. doi:10.1158/1538-7445.AM2015-2306

556PINTEGRIN BETA-3 GENETIC VARIANTS PREDICT THE RISK OF THROMBO-EMBOLIC EVENTS IN PATIENTS WITH COLORECTAL CANCER

ABSTRACT Aim: Colorectal cancer patients are at increased risk for venous thromboembolism (VTE). Beta-3 integrin adhesion receptors play a central role in tumor cell biology, platelet aggregation and endothelial cell behavior. Therefore, we hypothesized that the germline single nucleotide polymorphisms (SNPs) rs3809865, which is thought to affect integrin expression, might predict the risk of VTE in colorectal cancer patients. Methods: 114/139 colorectal cancer patients were assessable for integrin beta-3 germline SNPs rs3809865 characterization within the study population recruited into the Vienna Cancer and Thrombosis Study (CATS) (Ay, C et al; JCO 2011 vol. 29 no. 15), an ongoing prospective observational cohort study initiated in October 2003 at the Medical University of Vienna. Whole blood samples were analyzed using PCR-RFLP or direct DNA-sequencing. VTE events were statistical analyzed using one-way Anova testing. Results: The patients demographics and tumor characteristics were balanced between groups. In 14 patients (12.28%) VTE occurred. 12 (25%) of 48 colorectal cancer patients with an rs3809865 A/A allele profile experienced a VTE, representing a statistical significant (p = 0.0015) increased risk of VTE when compared to other subgroups. Only 2 of 52 patients (3.85%) harboring an A/T allele VTE was of diagnosed and none (0%) of the T/T subgroup had any VTE. Other SNPs of the integrin beta-3 gene revealed no predictive value for VTE. In multivariable analysis including age, sex, chemotherapy, and anti-VEGF therapy rs3809865 A/A allele profile remained a statistical significant risk factor for VTE. Conclusions: This study identifies the germline polymorphisms in integrin beta-3 gene rs3809865 as independent prognostic markers for VTE in colorectal cancer. These data may help to select subgroups of patients who may benefit from an enforced prophylaxis of venous thromboembolism (VTE). Disclosure: All authors have declared no conflicts of interest.