Joanne L. Yu

A role for survivin in chemoresistance of endothelial cells mediated by VEGF.

Although standard anticancer chemotherapeutic drugs have been designed to inhibit the survival or growth of rapidly dividing tumor cells, it is possible to enhance the efficacy of such drugs by targeting the proliferating host endothelial cells (ECs) of the tumor vasculature. A theoretical advantage of this strategy lies in the possibility of circumventing, or significantly delaying, acquired drug resistance driven by the genetic instability of tumor cells. Here, we show that both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor significantly reduce the pro-apoptotic potency of chemotherapy on both micro- and macrovascular ECs. This cytoprotection to drug toxicity was found to be phosphatidylinositol 3-kinase-dependent and could be recapitulated in the absence of VEGF by overexpressing the dominant-active form of the serine/threonine kinase protein kinase B/Akt. Downstream of phosphatidylinositol 3-kinase, we also show that survivin plays a pivotal role in VEGF-mediated EC protection by preserving the microtubule network. In this respect, its induction effectively protects ECs against chemotherapeutic damage, whereas overexpression of its dominant-interfering mutant (C84A) abrogates the protective effects of VEGF. Accordingly, the potency of VEGF as a chemoprotectant was more pronounced with drugs that interfere with microtubule dynamics than those that damage DNA. These studies implicate a role for survivin up-regulation as a novel mechanism of EC drug “resistance” and support the notion that angiogenic factors that induce the expression of survivin may act to shield tumor ECs from the apoptotic effects of chemotherapy. Thus, exploiting chemotherapeutic drugs as antiangiogenics is likely to be compromised by the high concentrations of proangiogenic survival/growth factors present in the tumor microenvironment; targeting EC survival pathways should improve the antiangiogenic efficacy of antineoplastic agents, particularly microtubule-inhibitor drugs.

High-frequency 3-D color-flow imaging of the microcirculation.

Abstract The extension of ultrasound (US) color flow imaging (CFI) techniques to high frequencies (> 20 MHz) has the potential to provide valuable noninvasive tools for scientific and clinical investigations of blood flow in the microcirculation. We describe the development of a slow-scan CFI system operating in the 20–100-MHz range that has been optimized to image the microcirculation. The apparatus has incorporated elements of a previously reported pulsed-wave Doppler system and is capable of operating in either CFI or pulsed-wave mode. The performance of the CFI system was evaluated at a center frequency of 50 MHz using two PVDF transducers with −6-dB beam widths of 43 and 60 μm. The −6 dB-axial resolutions were estimated to be 66 and 72 μm, respectively. In vivo validation experiments conducted using the murine ear model demonstrated the detection of flow in vessels down to 15–20 μm in diameter with flow velocities on the order of mm per s. Further experiments examining experimental murine tumors confirmed the successful detection of flow in the tumor microcirculation.

Tissue Factor Regulation by Epidermal Growth Factor Receptor and Epithelial-to-Mesenchymal Transitions: Effect on Tumor Initiation and Angiogenesis

ErbB oncogenes drive the progression of several human cancers. Our study shows that in human carcinoma (A431) and glioma (U373) cells, the oncogenic forms of epidermal growth factor receptor (EGFR; including EGFRvIII) trigger the up-regulation of tissue factor (TF), the transmembrane protein responsible for initiating blood coagulation and signaling through interaction with coagulation factor VIIa. We show that A431 cancer cells in culture exhibit a uniform TF expression profile; however, these same cells in vivo exhibit a heterogeneous TF expression and show signs of E-cadherin inactivation, which is coupled with multilineage (epithelial and mesenchymal) differentiation. Blockade of E-cadherin in vitro, leads to the acquisition of spindle morphology and de novo expression of vimentin, features consistent with epithelial-to-mesenchymal transition. These changes were associated with an increase in EGFR-dependent TF expression, and with enhanced stimulation of vascular endothelial growth factor production, particularly following cancer cell treatment with coagulation factor VIIa. In vivo, cells undergoing epithelial-to-mesenchymal transition exhibited an increased metastatic potential. Furthermore, injections of the TF-blocking antibody (CNTO 859) delayed the initiation of A431 tumors in immunodeficient mice, and reduced tumor growth, vascularization, and vascular endothelial growth factor expression. Collectively, our data suggest that TF is regulated by both oncogenic and differentiation pathways, and that it functions in tumor initiation, tumor growth, angiogenesis, and metastasis. Thus, TF could serve as a therapeutic target in EGFR-dependent malignancies.

Contrasting effects of VEGF gene disruption in embryonic stem cell‐derived versus oncogene‐induced tumors

Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF‐deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF‐null tumors (528ras, 528neu) originated from VEGF−/− embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene‐driven tumor angiogenesis. Both ras and neu oncogenes down‐regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP‐1)]. This is functionally important as administration of an antiangiogenic TSP‐1 peptide (ABT‐526) markedly inhibited growth of VEGF−/− tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell‐derived VEGF in certain cases of ‘adult’ tumor angiogenesis, and thus highlight the importance of considering VEGF‐independent as well as VEGF‐dependent pathways when attempting to block this process pharmacologically.

Oncogenes and Angiogenesis: Down-regulation of Thrombospondin-1 in Normal Fibroblasts Exposed to Factors from Cancer Cells Harboring Mutant Ras

The onset of angiogenesis in cancer often involves down-regulation of endogenous angiogenesis inhibitors, of which thrombospondin-1 (TSP-1) is a paradigm. As this effect is thought to occur under the influence of transforming genetic lesions (e.g., expression of the mutant ras oncogene), its nature is regarded as intrinsic to cancer cells themselves. Here, we show that ras-transformed cancer cells can also induce TSP-1 down-regulation in their adjacent nontransformed stromal fibroblasts, but not in endothelial cells, in a paracrine and distance-dependent manner. Indeed, several H-ras-expressing fibrosarcoma (528ras1, B6ras, and NIH3T3Ras) and carcinoma (DLD-1 and IEC18Ras3) cells were found to release soluble factors capable of suppressing TSP-1 protein, mRNA, and promoter activity in nontumorigenic, immortalized dermal fibroblastic cell lines in culture (e.g., in fibroblasts expressing enhanced green fluorescent protein/TSP-1 reporter). This effect was abrogated in Id1-/- fibroblasts. At least two low molecular weight (<3 kDa), heat-labile, and trypsin-resistant mediators of TSP-1 suppression were found to be released from 528ras1 cells. Their effects on normal fibroblasts were inhibited (albeit to different extents) by pertussis toxin and, in one case, by dimethylsphingosine, none of which affected TSP-1 expression by 528ras1 cells. Collectively, our study suggests that the effect of mutant ras on tumor neovascularization is not limited to changes in angiogenic properties of cancer cells themselves. Rather, mutant ras, through a different signaling mechanism, may modulate the properties of the adjacent normal stroma, thus eliciting a proangiogenic field effect.

Angiogenesis and the role of epigenetics in metastasis

The major obstacle to devising effective ways to treat cancer is its heterogeneity and genetic instability. It was originally postulated that targeting the process of tumor angiogenesis could circumvent this problem, as it involves genetically stable epigenetically controlled host stroma. Thus, anti-angiogenic approaches should be applicable across various tumor types and organ sites, including metastases. However, early clinical experience with this therapy revealed unexpectedly distinct responses between different tumors and organ sites. Here we propose that the heterogeneity of pre-clinical and clinical results obtained with anti-angiogenic agents stems from the deep functional linkage that may exist between genetic and epigenetic tumor progression. Thus, epigenetic processes regulating tumor associated host blood vessels (such as tumor microenvironment) display unstable, heterogeneous and progressive characteristics to an extent comparable with (and causally linked to) the instability of the cancer cell genome. As well, many known epigenetic factors (such as hypoxia, inflammation, expression of growth factors, etc.) may have genetic causes and consequences (e.g., oncogene expression, loss of tumor suppressor genes). This reciprocal interrelationship and heterogeneity may translate into site and stage specific changes in angiogenesis regulation, and angiogenesis dependence, ultimately to changes in the metastatic ability/efficiency of cancer cells, even in the same patient. A better understanding of the linkage between genetic and epigenetic events in growth and metastasis of various cancers may result in more effective use of anti-angiogenic therapy in future.

Modulation of the oncogene-dependent tissue factor expression by kinase suppressor of ras 1

INTRODUCTION Tissue factor (TF) is the key trigger of the coagulation cascade and the membrane signalling receptor for coagulation protease, factor VIIa. In cancer, TF has been implicated in tumor cell survival, growth, and angiogenesis, and is upregulated as a result of oncogenic transformation. MATERIALS AND METHODS We assayed TF expression and tumourigenicity in mice in the case of human cancer cell lines expressing oncogenic receptor tyrosine kinases. These cells were also subjected to genetic modulation of the kinase suppressor of ras 1 (KSR1), and treated with oncoprotein inhibitors in vitro and in vivo. RESULTS Here we show that herceptin, AG1478 and CI-1033, inhibitors of two different members of the ErbB family of oncogenes (HER-2 and EGFR), reduce TF levels in epithelial cancer cells. In EGFR-driven A431 cells, TF upregulation is diminished upon genetic targeting of KSR1, the scaffolding protein involved in EGFR signalling. Conversely, upregulation of KSR1 in A431 cells increases their TF expression and tumourigenicity in mice. The latter property remains dependent on EGFR, as pan-Erb (EGFR) inhibitor, CI-1033, blocks TF promoter activity and inhibits tumour formation by the parental and KSR1 overexpressing A431 cells. CONCLUSIONS KSR1 emerges, as an important modulator of TF expression in EGFR-driven cancer cells, which also impacts their aggressiveness in vivo.

High frequency color flow imaging of the microcirculation

The extension of ultrasound color flow imaging (CFI) techniques to high frequencies (>30 MHz) has the potential to provide valuable non-invasive tools for clinical and scientific investigations of blood flow in the microcirculation. The authors report the development of a combined color flow mapping and pulsed wave system capable of operating in the 20 to 100 MHz range which has been optimized to image the microcirculation. The performance of the system was evaluated at a center frequency of 50 MHz for two PVDF transducers which provided lateral resolutions of 45 and 65 microns. The axial resolution was approximately 60 microns. In vivo validation experiments conducted using the mouse ear demonstrate the detection of flow in vessels down to 15-20 microns in diameter with flow velocities below 1 millimeter per second. Initial experiments examining experimental murine tumors indicate the successful detection of flow in the tumor microcirculation.

Monitoring antivascular therapy with high frequency ultrasound flow imaging

The effect of antivascular therapy on blood flow in superficial mouse tumors was monitored using high frequency Doppler flow imaging techniques (HFD). Volumetric flow imaging of established melanomas detected a significant reduction in blood flow 4 hours after injection of the tumor vascular targeting agent ZD6126 followed by a recovery of flow by 24 hours after injection. Measurements of tumor perfusion in situ by Hoechst perfusion staining correlated with the ultrasound results. This study demonstrates the feasibility of high frequency flow imaging as a quantitative tool for following longitudinally the effects of antivascular therapy on blood flow in superficial tumors.