Johann Kern

GRP-78 secreted by tumor cells blocks the antiangiogenic activity of bortezomib.

Antiangiogenic effects of the proteasome inhibitor bortezomib were analyzed on tumor xenografts in vivo. Bortezomib strongly inhibited angiogenesis and vascularization in the chicken chorioallantoic membrane. Bortezomibs inhibitory effects on chorioallantoic membrane vascularization were abrogated in the presence of distinct tumor xenografts, thanks to a soluble factor secreted by tumor cells. Through size-exclusion and ion-exchange chromatography as well as mass spectroscopy, we identified GRP-78, a chaperone protein of the unfolded protein response, as being responsible for bortezomib resistance. Indeed, a variety of bortezomib-resistant solid tumor cell lines (PC-3, HRT-18), but not myeloma cell lines (U266, OPM-2), were able to secrete high amounts of GRP-78. Recombinant GRP-78 conferred bortezomib resistance to endothelial cells and OPM-2 myeloma cells. Knockdown of GRP78 gene expression in tumor cells and immunodepletion of GRP-78 protein from tumor cell supernatants restored bortezomib sensitivity. GRP-78 did not bind or complex bortezomib but induced prosurvival signals by phosphorylation of extracellular signal-related kinase and inhibited p53-mediated expression of proapoptotic Bok and Noxa proteins in endothelial cells. From our data, we conclude that distinct solid tumor cells are able to secrete GRP-78 into the tumor microenvironment, thus demonstrating a hitherto unknown mechanism of resistance to bortezomib.

The Dickkopf-homolog 3 is expressed in tumor endothelial cells and supports capillary formation.

A critical event in tumor growth and progression is the upregulation of angiogenesis. Thus, targeting angiogenesis has become an attractive treatment modality in cancer medicine. Our study analyzed various solid tumor types for the expression of Dkk‐3, a cystein‐rich, N‐glycosylated secreted member of the dickkopf protein family that has been proposed as a tumor suppressor gene. Tissue microarrays of gliomas (n = 30), high‐grade non‐Hodgkins lymphomas (NHL, n = 80), colorectal cancer (n = 35) and melanoma (n = 30) were immunohistochemically analyzed for Dkk‐3 and CD31 expression. Moreover, the effects of Dkk‐3 were studied in vitro in primary endothelial colony‐forming cells (ECFC) and in vivo in a mouse melanoma model. In comparison to normal tissue, the number of blood vessels expressing Dkk‐3 was increased in glioma, high‐grade NHL, melanoma and colorectal carcinoma. Confocal laser scanning microscopy revealed that Dkk‐3 vesicles localized also in Weibel Palade bodies. In vitro cell proliferation and migration of ECFC was not significantly affected by adenoviral overexpression or siRNA‐mediated downregulation of Dkk‐3. Interestingly, tube formation in matrigel decreased after downregulation of Dkk‐3 and increased after adenoviral overexpression. Stable overexpression of murine Dkk‐3 in B16F10 cells significantly increased microvessel density in the C57/BL6 melanoma model. Thus, we postulate a novel function of Dkk‐3 in endothelial cells as a differentiation factor involved in remodeling the tumor vasculature.

Quantification of circulating endothelial and progenitor cells: comparison of quantitative PCR and four-channel flow cytometry

BackgroundCirculating endothelial cells (CEC) and endothelial precursor cells (CEP) have been suggested as markers for angiogenesis in cancer. However, CEC/CEP represent a tiny and heterogeneous cell population, rendering a standardized monitoring in peripheral blood difficult. Thus, we investigated whether a PCR-based detection method of CEC/CEP might overcome the limitations of rare-event flow cytometry.FindingsTo test the sensitivity of both assays endothelial colony forming cell clones (ECFC) and cord blood derived CD45- CD34+ progenitor cells were spiked into peripheral blood mononuclear cells (PBMNC) of healthy volunteers. Samples were analyzed for the expression of CD45, CD31, CD34, KDR or CD133 by 4-color flow cytometry and for the expression of CD34, CD133, KDR and CD144 by qPCR. Applying flow cytometry, spiked ECFC and progenitor cells were detectable at frequencies ≥ 0.01%, whereas by qPCR a detection limit of 0.001% was achievable. Furthermore, PBMNC from healthy controls (n = 30), patients with locally advanced rectal cancer (n = 20) and metastatic non-small cell lung cancer (NSCLC, n = 25) were analyzed. No increase of CEC/CEP was detectable by flow cytometry. Furthermore, only CD34 and KDR gene expression was significantly elevated in patients with metastatic NSCLC. However, both markers are not specific for endothelial cells.ConclusionQPCR is more sensitive, but less specific than 4-channel flow cytometry for the detection of CEC/CEP cell types. However, both methods failed to reliably detect an increase of CEC/CEP in tumor patients. Thus, more specific CEC/CEP markers are needed to validate and improve the detection of these rare cell types by PCR-based assays.

Alternative Splicing of Vasohibin-1 Generates an Inhibitor of Endothelial Cell Proliferation, Migration, and Capillary Tube Formation

Objective—In this study, the alternative splicing product of vasohibin 1 (VASH1B) was analyzed in direct comparison to the major isoform (VASH1A) for antiangiogenic effects on endothelial colony forming cells (ECFCs) from peripheral blood and on human umbilical vein endothelial cells (HUVECs). Methods and Results—Expression studies in primary human endothelial cells revealed that both vasohibin proteins, hVASH1A and hVASH1B, localized in the nucleus and cytoplasm. Adenoviruses carrying the cDNA for VASH1A/B and purified recombinant proteins were used to study the function of both molecules in ECFCs and HUVECs. Recombinant VASH1A protein did not inhibit cell proliferation, tube formation, or vessel growth in vivo in the chick chorioallantoic membrane (CAM) assay, but promoted endothelial cell migration in vitro. The VASH1B protein had an inhibitory effect on cell proliferation, migration, tube formation, and inhibited blood vessel formation in the CAM assay. Adenoviral overexpression of VASH1B, but not of VASH1A, resulted in inhibition of endothelial cell growth, migration, and capillary formation. Interestingly, overexpression of VASH1A and B induced apoptosis in proliferating human fibroblasts, but did not affect cell growth of keratinocytes. Conclusion—Our data point out that alternative splicing of the VASH1 pre-mRNA transcript generates a potent antiangiogenic protein.

Vasohibin inhibits angiogenic sprouting in vitro and supports vascular maturation processes in vivo

BackgroundThe murine homologue of human vasohibin (mVASH1), a putative antiangiogenic protein, was investigated for its effects on in vitro and in vivo angiogenesis.MethodsCell growth and migration were analyzed in murine fibroblasts, smooth muscle cells and endothelial cells. Angiogenic sprouting was studied in human umbilical vein endothelial cells (HUVECs) in the spheroid sprouting assay. In vivo effects on blood vessel formation were investigated in the chorioallantoic membrane (CAM) assay and in the C57BL/6 melanoma xenograft model.ResultsPurified murine and human VASH1 protein induced apoptosis of murine fibroblasts in vitro, but not of vascular aortic smooth muscle cells (AoSMC) or endothelial cells. Adenoviral overexpression of murine and human VASH1 inhibited capillary sprouting of HUVECs in the spheroid assay. Administration of recombinant murine and human VASH1 inhibited growth of large vessels in the CAM assay and promoted the formation of a dense, fine vascular network. Murine VASH1-overexpressing B16F10 melanomas displayed a reduction in large vessels and vascular area. Moreover, tumors showed more microvessels that stained positive for the mural cell markers α-smooth muscle cell actin (ASMA) and proteoglycan (NG2).ConclusionOur data imply that murine VASH1 causes angiogenic remodelling by inhibiting angiogenic sprouting and large vessel growth, thereby supporting the formation of a vascular bed consisting predominantly of mature microvessels.

SOCS2 correlates with malignancy and exerts growth-promoting effects in prostate cancer.

Deregulation of cytokine and growth factor signaling due to an altered expression of endogenous regulators is well recognized in prostate cancer (PCa) and other cancers. Suppressor of cytokine signaling 2 (SOCS2) is a key regulator of the GH, IGF, and prolactin signaling pathways that have been implicated in carcinogenesis. In this study, we evaluated the expression patterns and functional significance of SOCS2 in PCa. Protein expression analysis employing tissue microarrays from two independent patient cohorts revealed a significantly enhanced expression in tumor tissue compared with benign tissue as well as association with Gleason score and disease progression. In vitro and in vivo assays uncovered the involvement of SOCS2 in the regulation of cell growth and apoptosis. Functionally, SOCS2 knockdown inhibited PCa cell proliferation and xenograft growth in a CAM assay. Decreased cell growth after SOCS2 downregulation was associated with cell-cycle arrest and apoptosis. In addition, we proved that SOCS2 expression is significantly elevated upon androgenic stimulation in androgen receptor (AR)-positive cell lines, providing a possible mechanistic explanation for high SOCS2 levels in PCa tissue. Consequently, SOCS2 expression correlated with AR expression in the malignant tissue of patients. On the whole, our study linked increased SOCS2 expression in PCa with a pro-proliferative role in vitro and in vivo.

Antimyeloma activity of the sesquiterpene lactone cnicin: impact on Pim-2 kinase as a novel therapeutic target

Despite recent advances in therapy, multiple myeloma, the second most common hematologic tumor in the Western world, is still incurable. Identification of substances that display a wide range of tumor-killing activities and target cancer-specific pathways constitute a basis for the development of novel therapies. In this study, we investigate the cytotoxic effect of the natural substance cnicin in multiple myeloma. Cnicin treatment reveals potent antiproliferative effects and induces cell death in cell lines and primary myeloma cells even in the presence of survival cytokines and the tumor microenvironment. Other cell lines of hematopoietic origin also succumb to cell death whereas stromal cells and endothelial cells are unaffected. We show that activation of caspases, accumulation of reactive oxygen species and downregulation of nuclear factor kappa-light-chain-enhancer of activated B cell contribute to the cytotoxic effects of cnicin. Microarray analysis reveals downregulation of Pim-2, a serine/threonine kinase. We provide evidence that Pim-2 constitutes a new survival kinase for myeloma cells in vitro and is highly expressed in malignant but not in normal plasma cells in vivo. Combining cnicin with current standard or experimental therapeutics leads to enhanced cell death. Thus, our data indicate that cnicin induces myeloma cell death via several pathways and reveals Pim-2 as a novel target. These findings provide a rational for further evaluation of cnicin as a new anti-tumor drug and underline the potential of sesquiterpene lactones in tumor therapy.

Increased Dkk3 protein expression in platelets and megakaryocytes of patients with myeloproliferative neoplasms.

Dickkopf-3 (Dkk3) has been proposed as tumour suppressor gene and a marker for tumour blood vessels. We analysed the expression and function of Dkk3 in platelets and megakaryocytes from healthy controls and patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN). Dkk3 protein and gene expression in platelets was compared with endothelial and other blood cell populations by ELISA, real-time PCR, and immunofluorescence. Moreover, megakaryocytes were isolated from bone marrow aspirates by CD61 microbeads. Immunohistochemical studies of Dkk3 expression were performed in essential thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF) and control reactive bone marrow cases (each n=10). Compared to all other blood cell populations platelets showed the highest concentration of Dkk3 protein (150 ± 19 ng/mg total protein). A strong DKK3 gene and protein expression was also observed in isolated megakaryocytes. Dkk3 co-localised with VEGF in α-granules of platelets and was released similar to VEGF upon stimulation. Addition of recombinant Dkk3 had no influence on blood coagulation (aPTT, INR) and platelet aggregation. Significantly more Dkk3+ megakaryocytes/mm2 could be found in bone marrow biopsies from patients with MPN (ET 40 ± 10, PV 31 ± 4, PMF 22 ± 3) than in controls (15 ± 3). The mean proportion of Dkk3+ megakaryocytes was increased in MPN as well (ET 83% ± 15%; PV 84% ± 12%; PMF 77% ± 8%) compared to controls (53% ± 11%). Dkk3+ megakaryocytes correlated with microvessel density in PV and PMF. We conclude that Dkk3 might be involved in the pathogenesis of MPN.

Biofunctionalization of scaffold material with nano-scaled diamond particles physisorbed with angiogenic factors enhances vessel growth after implantation

UNLABELLED Biofunctionalized scaffold facilitates complete healing of large defects. Biological constraints are induction and ingrowth of vessels. Angiogenic growth factors such as vascular endothelial growth factor or angiopoietin-1 can be bound to nano-scaled diamond particles. Corresponding bioactivities need to be examined after biofunctionalization. We therefore determined the physisorptive capacity of distinctly manufactured, differently sized nDP and the corresponding activities of bound factors. The properties of biofunctionalized nDPs were investigated on cultivated human mesenchymal stem cells and on the developing chicken embryo chorio-allantoic membrane. Eventually porous bone substitution material was coated with nDP to generate an interface that allows biofactor physisorption. Angiopoietin-1 was applied shortly before scaffold implantation into an osseous defect in sheep calvaria. Biofunctionalized scaffolds exhibited significantly increased rates of angiogenesis already one month after implantation. Conclusively, nDP can be used to ease functionalization of synthetic biomaterials. FROM THE CLINICAL EDITOR With the advances in nanotechnology, many nano-sized materials have been used in the biomedical field. This is also true for nano-diamond particles (nDP). In this article, the authors investigated the physical properties of functionalized nano-diamond particles in both in-vitro and in-vivo settings. The positive findings would help improve understanding of these nanomaterials in regenerative medicine.

Favorable prognosis of operable non-small cell lung cancer (NSCLC) patients harboring an increased expression of tumor endothelial markers (TEMs)

Genome analyses of endothelial cells identified genes specifically expressed by tumor endothelial cells, called tumor endothelial markers (TEMs). Currently there are no data available concerning the role of TEMs in non-small cell lung cancer (NSCLC). Therefore, the aim of this study was to investigate the role of TEMs in NSCLC in vitro and in vivo. First we evaluated the expression of various TEMs (Robo4, Clec14 and ECSCR) by qRT-PCR and Western blot analyses in three NSCLC cell lines (A549, Calu1, Colo699) and compared them to human umbilical vein endothelial cells (HUVECs), endothelial colony forming cells (ECFCs) and human bronchial epithelial cells (HBEpCs). Next the expression of TEMs was measured in resected tumor tissue of NSCLC patients (n = 63) by qRT-PCR and compared to adjacent non-cancerous lung tissue (n = 52). Further, immunohistochemical analysis of Robo4 expression in tumor tissue (n = 33) and adjacent non-cancerous tissue (n = 27) was performed. We found that NSCLC cell lines and HBEpC did not express TEMs on the mRNA level compared to HUVECs (p = 0.001). In the contrary, a significant up-regulation of Robo4 and Clec14 was found in tumor samples (Robo4 p = 0.03, Clec14 p = 0.002). Both facts clearly indicate that these proteins are allocated to the tumor stromal department. Correlation with clinical data showed that increased TEM expression correlated with prolonged overall survival of operated NSCLC patients (Robo4 high 120.5 vs. Robo4 low 47.6 months, Clec14 high 108.1 vs. Clec14 low 54.5 months and ECSCR high 120.5 vs. ECSCR low 42.2 months). In summary, we found that TEMs are overexpressed in NSCLC stromal tissue and that an increased TEM expression correlated with an increased overall survival in early stage NSCLC.