Joost A.P. Rens

Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41–42.5 °C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.

Differential effects of matrix and growth factors on endothelial and fibroblast motility : Application of a modified cell migration assay

Cell migration is crucial in virtually every biological process and strongly depends on the nature of the surrounding matrix. An assay that enables real‐time studies on the effects of defined matrix components and growth factors on cell migration is not available. We have set up a novel, quantitative migration assay, which enables unharmed cells to migrate along a defined matrix. Here, we used this so‐called barrier‐assay to define the contribution of fibronectin (FN) and Collagen‐I (Col‐I) to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and lysophosphatidic acid (LPA)‐induced cell migration of endothelial cells (EC) and fibroblasts. In EC, both FN and Col‐I stimulated migration, but FN‐induced motility was random, while net movement was inhibited. Addition of bFGF and VEGF overcame the effect of FN, with VEGF causing directional movement. In contrast, in 3T3 fibroblasts, FN stimulated motility and this effect was enhanced by bFGF. This motility was more efficient and morphologically completely different compared to LPA stimulation. Strikingly, directional migration of EC was not paralleled by higher amounts of stable microtubules (MT) or an increased reorientation of the microtubule‐organizing centre (MTOC). For EC, the FN effect appeared concentration dependent; high FN was able to induce migration, while for fibroblasts both low and high concentrations of FN induced motility. Besides showing distinct responses of the different cells to the same factors, these results address contradictive reports on FN and show that the interplay between matrix components and growth factors determines both pattern and regulation of cell migration. J. Cell. Biochem. 99: 1536–1552, 2006.

Histamine, a vasoactive agent with vascular disrupting potential, improves tumour response by enhancing local drug delivery

Tumour necrosis factor (TNF)-based isolated limb perfusion (ILP) is an approved and registered treatment for sarcomas confined to the limbs in Europe since 1998, with limb salvage indexes of 76%. TNF improves drug distribution in solid tumours and secondarily destroys the tumour-associated vasculature (TAV). Here we explore the synergistic antitumour effect of another vasoactive agent, histamine (Hi), in doxorubicin (DXR)-based ILP and evaluate its antivascular effects on TAV. We used our well-established rat ILP model for in vivo studies looking at tumour response, drug distribution and effects on tumour vessels. In vitro studies explored drug interactions at cellular level on tumour cells (BN-175) and Human umbilical vein endothelial cells (HUVEC). There was a 17% partial response and a 50% arrest in tumour growth when Hi was combined to DXR, without important side effects, against 100% progressive disease with DXR alone and 29% arrest in tumour growth for Hi alone. Histology documented an increased DXR leakage in tumour tissue combined to a destruction of the TAV, when Hi was added to the ILP. In vitro no synergy between the drugs was observed. In conclusion, Hi is a vasoactive drug, targeting primarily the TAV and synergises with different chemotherapeutic agents.

Differential TIMP3 expression affects tumor progression and angiogenesis in melanomas through regulation of directionally persistent endothelial cell migration

The angiogenic potential of solid tumors, or the ability to initiate neovasculature development from pre-existing host vessels, is facilitated by soluble factors secreted by tumor cells and involves breaching of extracellular matrix barriers, endothelial cell (EC) proliferation, migration and reassembly. We evaluated the angiogenic potential of human melanoma cell lines differing in their degree of aggressiveness, based on their ability to regulate directionally persistent EC migration. We observed that conditioned-medium (CM) of the aggressive melanoma cell line BLM induced a high effective migratory response in ECs, while CMs of Mel57 and 1F6 had an inhibitory effect. Further, the melanoma cell lines exhibited a varied expression profile of tissue inhibitor of metalloproteinase-3 (TIMP3), detectable in the CM. TIMP3 expression inversely correlated with aggressiveness of the melanoma cell line, and ability of the respective CMs to induce directed EC migration. Interestingly, TIMP3 expression was found to be silenced in the BLM cell line, concurrent with its role as a tumor suppressor. Treatment with recombinant human TIMP3 and CM of modified, TIMP3 expressing, BLM cells mitigated directional EC migration, while CM of TIMP3 silenced 1F6 cells induced directed EC migration. The functional implication of TIMP3 expression on tumor growth and angiogenic potential in melanoma was evaluated in vivo. We observed that TIMP3 expression reduced tumor growth, angiogenesis and macrophage infiltration of BLM tumors while silencing TIMP3 increased tumor growth and angiogenesis of 1F6 tumors. Taken together, our results demonstrate that TIMP3 expression correlates with inhibition of directionally persistent EC migration and adversely affects the angiogenic potential and growth of melanomas.

PDGF-Induced Migration of Vascular Smooth Muscle Cells Is Inhibited by Heme Oxygenase-1 Via VEGFR2 Upregulation and Subsequent Assembly of Inactive VEGFR2/PDGFRβ Heterodimers

Objective—In cardiovascular regulation, heme oxygenase-1 (HO-1) activity has been shown to inhibit vascular smooth muscle cell (VSMC) proliferation by promoting cell cycle arrest at the G1/S phase. However, the effect of HO-1 on VSMC migration remains unclear. We aim to elucidate the mechanism by which HO-1 regulates PDGFBB-induced VSMC migration. Methods and Results—Transduction of HO-1 cDNA adenoviral vector severely impeded human VSMC migration in a scratch, transmembrane, and directional migration assay in response to PDGFBB stimulation. Similarly, HO-1 overexpression in the remodeling process during murine retinal vasculature development attenuated VSMC coverage over the major arterial branches as compared with sham vector-transduced eyes. HO-1 expression in VSMCs significantly upregulated VEGFA and VEGFR2 expression, which subsequently promoted the formation of inactive PDGFR&bgr;/VEGFR2 complexes. This compromised PDGFR&bgr; phosphorylation and impeded the downstream cascade of FAK-p38 signaling. siRNA-mediated silencing of VEGFA or VEGFR2 could reverse the inhibitory effect of HO-1 on VSMC migration. Conclusion—These findings identify a potent antimigratory function of HO-1 in VSMCs, a mechanism that involves VEGFA and VEGFR2 upregulation, followed by assembly of inactive VEGFR2/PDGFR&bgr; complexes that attenuates effective PDGFR&bgr; signaling.

E-cadherin promotor methylation and mutation are inversely related to motility capacity of breast cancer cells

Inactivation of the tumor suppressor E-cadherin is an important event during breast tumorigenesis, as its decreased expression is linked to aggressiveness and metastasis. However, the relationship between the different modes of E-cadherin inactivation (mutation versus promotor hypermethylation) and breast cancer cell behavior is incompletely understood. The high correlation between E-cadherin inactivation status and cell morphology in vitro suggests different biological roles for the two inactivation modes during breast tumorigenesis. Because E-cadherin has been linked to cell invasion and metastasis, and cell motility is a crucial prerequisite to form metastases, we here compared the cell motility capacities of breast cancer cell lines with known E-cadherin status. Using barrier migration assays and time-lapse microscopy, we analyzed the migratory capacity of nine well-characterized human breast cancer cell lines (MDA-MB-231, MCF-7, T47D, BT549, MPE600, CAMA-1, SUM159PT, SUM52PE, and SK-BR-3). This subset was chosen based on E-cadherin gene status (wild-type, mutated, and promotor hypermethylated): three cell lines of each group. In addition, cell proliferation assays were performed for all conditions, to dissect migratory from proliferative effects. In this study, we demonstrate an overt association between the mode of E-cadherin inactivation and cell migration. Promotor hypermethylated E-cadherin cell lines showed a higher migration capacity, while cell lines with mutated E-cadherin were less motile compared to wild-type E-cadherin cell lines. Migration induction by fibronectin and basic fibroblast growth factor did not alter the cell motility association differences. Cell proliferation assays showed that the associations found were not caused by proliferation differences. Inhibition and overexpression of E-cadherin as well as DNA demethylation confirmed the relationship between E-cadherin and breast cancer cell motility. Our results demonstrate an association between the mode of E-cadherin inactivation and migration of breast cancer cells, which justifies more detailed research on the role of E-cadherin inactivation in cell migration and metastasis.

Intratumoural expression of TNF-R1 and EMAP-II in relation to response of patients treated with TNF-based isolated limb perfusion

Tumour necrosis factor‐alpha (TNF) has been used in the clinic for more than 10 years in an isolated limb perfusion (ILP). However, intra‐tumoural expression of TNF receptor‐1 (TNF‐R1) and TNF‐R1 upregulating factors are unknown. We determined the expression of TNF‐R1, proEMAP and endothelial monocyte‐activating polypeptide‐II (EMAP‐II) before and after ILP and evaluated this against clinical response. Tumour biopsies were taken before and after ILP of patients (n = 27) with advanced sarcoma or metastatic melanoma. Biopsies were randomly analysed by western blotting for proEMAP/EMAP‐II and TNF‐R1 expression. Appropriate melanoma biopsies were stained for EMAP‐II, TNF‐R1, CD31 and CD68. For melanomas we found that an up‐regulation of EMAP‐II, in contrast to proEMAP or TNF‐R1, directly after ILP significantly correlated with a complete tumour response. No correlation was found for sarcoma patients. In a comparative analysis we found that the overall proEMAP and EMAP‐II expression was higher in melanoma as compared to sarcoma cases and measurements in cell lines revealed high proEMAP expression by melanoma cells. We report high EMAP‐II expression by endothelial cells and association with macrophages. In addition, macrophages are recruited to vessel‐remnants after ILP. An upregulation of EMAP‐II directly after ILP of melanoma patients correlates with and might predict a complete response to TNF‐based ILP. The association of macrophages with EMAP‐II expression and vascular damage suggests a role for EMAP‐II in regulating the TNF‐based anti‐tumour effects observed with an ILP. Analysis of EMAP‐II expression in melanoma biopsies should be implemented in the ILP procedure.

EMAP-II facilitates TNF-R1 apoptotic signalling in endothelial cells and induces TRADD mobilization

Endothelial monocyte-activating polypeptide-II (EMAP-II), a proinflammatory cytokine with antiangiogenic properties, renders tumours sensitive to tumour necrosis factor-alpha (TNF) treatment. The exact mechanisms for this effect remain unclear. Here we show that human endothelial cells (EC) are insensitive to TNF-induced apoptosis but after a short pre-treatment with EMAP-II, EC quickly undergo TNF-induced apoptosis. We further analysed this EMAP-II pre-treatment effect and found no increase of TNF-R1 protein expression but rather an induction of TNF-R1 redistribution from Golgi storage pools to cell membranes. In addition, we observed EMAP-II induced mobilization and membrane expression of the TNF-R1-Associated Death Domain (TRADD) protein. Immunofluorescence co-staining experiments revealed that these two effects occurred at the same time in the same cell but TNF-R1 and TRADD were localized in different vesicles. These findings suggest that EMAP-II sensitises EC to apoptosis by facilitating TNF-R1 apoptotic signalling via TRADD mobilization and introduce a molecular and antiangiogenic explanation for the TNF sensitising properties of EMAP-II in tumours.

Exposing endothelial cells to tumor necrosis factor-α and peripheral blood mononuclear cells damage endothelial integrity via interleukin-1ß by degradation of vascular endothelial-cadherin

BACKGROUND AND PURPOSE We demonstrated previously that the administration of tumor necrosis factor alpha (TNF-α) for the treatment of solid tumors enhanced the response to chemotherapy by augmenting intratumoral drug accumulation. TNF-α changes the integrity of the endothelial cell monolayer in combination with interferon gamma (IFN-γ), which is further enhanced by the addition of peripheral blood mononuclear cells (PBMCs). The improved effect of PBMCs was mostly induced by the endogenous production of interleukin-1beta (IL-1ß) after TNF-α stimulation. In the current study, we demonstrate that exposing endothelial cells to TNF-α and PBMCs mediates the loss of vascular endothelial (VE)-cadherin, an important adherens junction protein for maintaining endothelial integrity, through endogenous IL-1ß. This loss increases permeability of the endothelial layer, thereby explaining the augmented passage of chemotherapeutics into the tumor. METHODS Human umbilical vein endothelial cells were exposed to TNF-α, IFN-γ, PBMCs, or IL-1ß, and the effects on the endothelial integrity were assessed by morphological changes and permeability changes with the use of fluorescein isothiocyanate-labeled bovine serum albumin flux. The loss of VE-cadherin was assessed using immunofluorescence, western blotting, and polymerase chain reaction. RESULTS Incubating endothelial cells with TNF-α, IFN-γ, and PBMCs increased cell elongation, gap formation, and subsequently the permeability of fluorescein isothiocyanate-labeled bovine serum albumin compared with control or TNF-α and IFN-γ-treated cells (P < .05). When PBMCs were replaced with IL-1ß, identical changes were observed. These changes in integrity were associated with a loss of VE-cadherin at the membrane. CONCLUSION We conclude that VE-cadherin is lost at the membrane when endothelial cells are exposed to TNF-α, IFN-γ, and PBMCs, which results in loss of integrity. IL-1ß can mimic the effects of PBMCs, indicating a dominant role of endogenously produced IL-1ß in this process.

Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody.

Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting.