Genotoxic stress induced by anticancer drugs can lead to apoptosis of both angiogenic endothelial cells (ECs) and proliferating tumor cells. However, growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) present within the tumor microenvironment can promote chemoresistance by suppressing apoptotic mechanisms in these cells. Here, we have identified apoptosis signal-regulating kinase 1 (ASK1), a proapoptotic member of the MAP3K family, as a target of bFGF-mediated survival signaling in ECs. Evidence is provided that ASK1 is required for EC apoptosis in response to the genotoxic chemotherapeutic agent doxorubicin, and that bFGF, but not VEGF, neutralizes the death-promoting activity of ASK1. Specifically, bFGF stimulation promotes the formation of a Raf-1/ASK1 complex at the mitochondria, inhibits ASK1 kinase activity, and protects ECs from genotoxic stress. Mutation of the Raf-1 activation domain (SS338/9AA) not only prevents Raf-1/ASK1 complex formation but abolishes bFGF-mediated EC protection from genotoxic stress. In line with these observations, bFGF, but not VEGF, neutralizes the antiangiogenic effects of doxorubicin in vivo. These findings reveal a new pathway of EC survival signaling and define a molecular mechanism for chemoresistance induced by bFGF. [Cancer Res 2007;67(6):2766–72]
- Growth Factors
Anticancer treatments have relied in part on ionizing radiation or systemic administration of genotoxic chemotherapeutics designed to promote stress-mediated apoptosis of tumor cells as well as tumor-associated endothelium in response to DNA damage ( 1). In fact, proliferating vascular cells within the tumor microenvironment are likely to be the first targets of chemotherapeutic agents ( 2). Although growth factors present within the tumor can provide an epigenetic mechanism by which tumor cells as well as the genetically stable endothelium may acquire chemoresistance, little is known about specific mediators of apoptosis that are targeted by growth factor-induced survival signals.
Previous studies suggest that cell death in response to genotoxic stress is associated with the activation of apoptosis signal-regulating kinase 1 (ASK1) a serine/threonine kinase member of the MAP3K family ( 3, 4). ASK1 is a key mediator of a redox-sensitive signaling pathway leading to the initiation of cellular apoptosis. It is activated in response to a variety of apoptotic stimuli, including chemotherapeutic agents ( 3, 5), serum withdrawal ( 6), oxidative ( 4, 7), and endoplasmic reticulum stress ( 8, 9), as well as treatment with tumor necrosis factor (TNF)-α and Fas ( 10, 11). Mice with targeted disruption of ASK1 are phenotypically normal; however, retinal ganglion cells and embryonic fibroblasts have been shown to be partially resistant to cell death induced by ischemic injury ( 12), hydrogen peroxide, or TNF-α ( 13). ASK1 is present in both the cytoplasmic and mitochondrial fractions ( 14) and activates the MKK6/MKK7–c-jun-NH2-kinase and MKK3/MKK6-p38 kinase pathways ( 11), leading to mitochondrial cytochrome c release and activation of the caspase-9–dependent or intrinsic pathway of apoptosis ( 15). Mice with targeted disruption of ASK1 are phenotypically normal; however, retinal ganglion cells and embryonic fibroblasts have been shown to be partially resistant to cell death induced by ischemic injury ( 12), hydrogen peroxide, or TNF-α ( 13). These observations suggest an important role for ASK1 in mediating cell death that proceeds via the intrinsic or mitochondrial pathway of apoptosis.
In quiescent cells, ASK1 kinase activity is tightly regulated. Association of ASK1 with reduced thioredoxin ( 7) and other proteins such as glutaredoxin ( 16), 14-3-3 ( 17), and SOCS1 ( 18) has been shown to regulate its activity. Recent reports have shown that proapoptotic ASK1 activity is also regulated by Raf-1. Specifically, when experimentally overexpressed in cells, Raf-1 was shown to bind to the NH2-terminal negative regulatory domain of ASK1, suppressing its death-promoting activity ( 19, 20). In this respect, we have previously shown that differential activation of Raf-1 by basic fibroblast growth factor (bFGF) or vascular endothelial cell growth factor (VEGF) acted to promote endothelial cell (EC) protection from distinct apoptotic stimuli ( 21, 22). bFGF promoted translocation of Raf-1 to the mitochondria and preferentially protected ECs from the intrinsic or stress-mediated apoptosis. In contrast, VEGF protected cells against apoptosis induced by death ligands but had little protective effect on stress-mediated death. These findings provide a molecular basis to explain how distinct angiogenic growth factors activate common pathways of cell proliferation and invasion leading to angiogenesis, yet promote survival in response to distinct apoptotic stimuli. To further delineate the molecular basis underlying the chemoprotective function of growth factors, we examined the role of bFGF or VEGF stimulation of ECs in regulation of proapoptotic activity of ASK1 by Raf-1. Evidence is provided that ASK1 is required for stress-mediated apoptosis in ECs and that bFGF, but not VEGF, stimulation promotes formation of a complex between Raf-1 and ASK1 at the mitochondria, suppresses ASK1 activity, and provides chemoresistance to ECs. These findings suggest that the chemoprotective function of bFGF is linked to inhibition of proapoptotic ASK1 activity in ECs.
Materials and Methods
Electroporation and RNAi-mediated knockdown. Human umbilical vein endothelial cells (HUVEC) were electroporated using an Amaxa Nucleofector (Amaxa Biosystems, Cologne, Germany). For RNAi-mediated knockdown, 5 × 105 cells in 100 μL EBM-2 (Clonetics, Walkersville, MD) were electroporated with 2 μmol/L (200 pmol) control or ASK1 small interfering RNA (Santa Cruz Biotechnology, Santa Cruz, CA) using setting U-001. Cells were immediately transferred to 400 μL RPMI 1640 and incubated for 15 min at 37°C. Following incubation, cells were added to complete MCDB131 medium (described below), and the medium was changed after 2 h. For ASK1 and Raf-1 mutants, 6 μg of each expression construct was electroporated into 2 × 106 cells, and analysis was done after 48 h. We routinely obtain >70% expression efficiency using these conditions.
Reagents, antibodies, and constructs. ASK1 siRNA and antibodies for immunoprecipitation (H-300) and blotting (F-9) of ASK1, Raf-1 (sc-133) were from Santa Cruz Biotechnology. Phospho-ERK1/2 antibody was from Cell Signaling Technology, Inc. (Danvers, MA). Etoposide, doxorubicin, anti–β-actin and anti–FLAG-M2 antibodies were obtained from Sigma (St. Louis, MO). Anti-HA antibody was obtained from Upstate Biotechnology (Lake Placid, NY). Raf-1 was immunoprecipitated with polyclonal sc-133 antibody (Santa Cruz Biotechnology) and immunoblotted with a monoclonal antibody (BD Biosciences, San Jose, CA). Growth factor stimulations were done using indicated concentrations of human recombinant VEGF (Peprotech, Rocky Hill, NJ) and bFGF (National Cancer Institute).
Cell culture and fractionation. Passage 4-6 human umbilical vein ECs were maintained in complete MCDB131 medium containing 20% fetal bovine serum (FBS), endothelial cell growth supplement (Upstate), 5 units/mL Heparin (Sigma), 2 mmol/L glutamine and penicillin/streptomycin/fungizone (Invitrogen, Carlsbad, CA). Cellular fractionation was done using the Pierce Mitochondria Isolation System (Pierce, Rockford, IL) according to manufacturer's instructions.
Immunoprecipitation and immunoblotting. Cells were lysed in lysis buffer containing 1% Triton X-100, 150 mmol/L NaCl, 20 mmol/L Tris-HCl (pH, 7.5), 20 mmol/L β-glycerophosphate, 2 mmol/L sodium PPi, 2 mmol/L EDTA, 2 mmol/L sodium orthovanadate, 50 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 10% glycerol and complete protease inhibitor cocktail (Roche, Indianapolis, IN) for 20 min and cleared by centrifugation for 15 min. Protein concentrations were determined by Pierce BCA (bicinchoninic acid) Protein Assay. For immunoprecipitations, 1 mg HUVEC lysates was incubated with 2 μg primary antibody overnight followed by 2 h incubation with 20 μL protein A/G bead slurry (Pierce). Immunoprecipitates were washed thrice with lysis buffer before immunoblotting or kinase assays. Whole cell lysates or immunoprecipitates were separated using SDS-PAGE, transferred to nitrocellulose, blocked with 5% nonfat dry milk in TBST, and exposed to the described primary antibodies. Antibody binding was detected using horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence (Amersham, Arlington Heights, IL).
In vitro kinase assay. Following immunoprecipitation, beads were washed once in kinase buffer containing 20 mmol/L HEPES (pH, 7.6), 20 mmol/L MgCl2, 20 mmol/L β-glycerophosphate, 100 μmol/L sodium orthovanadate, 2 mmol/L DTT, and 20 μmol/L ATP. A total of 30 μL of kinase buffer containing 0.5 μg GST-MKK6 (Upstate) and 10 μCi [γ-32P]-ATP at 3,000 Ci/mmol (Perkin-Elmer, Shelton, CT) was added to the beads and incubated for 20 min at 30°C. The kinase reaction was terminated by the addition of the Laemmli buffer, and proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Phosphorylated MKK6 was detected by storage phosphor autoradiography and scanning using a STORM 860 Phosphoimager (Molecular Dynamics, Piscataway, NJ). Quantitation of radiolabeled protein was done using ImageJ software.
Analysis of cell death. Cell death was scored by analyzing binding of FITC-conjugated annexin V to externalized membrane phosphatidylserine (BD PharMingen, Palo Alto, CA) or by detection of active caspases using carboxyfluorescein-labeled fluoromethyl ketone peptide inhibitor of caspase, FAM-VAD-FMK (CaspaTag; Chemicon, Temecula, CA) by flow cytometry according to manufacturer's instructions. A total of 10,000 cells were analyzed for each condition. Plasma membrane integrity was analyzed by staining with 5 μg/mL propidium iodide.
Mouse Matrigel angiogenesis assay. Adult 6- to 8-week-old C57Bl/6J (The Jackson Laboratory, Bar Harbor, ME) mice were used in this study. The Matrigel angiogenesis assay is approved by the University of California, San Diego Institutional Animal Care and Use Committee, and is done according to the NIH Guide for the Care and Use of Laboratory Animals. Mice were injected with 400 μL of ice-cold growth factor–depleted Matrigel (Becton Dickinson, Bedford, MA) mixed with 400 ng/mL of each growth factor and indicated amounts of doxorubicin. After 5 days, mice were i.v. injected with 50 μg of fluorescein-conjugated endothelium-specific GSL I–isolectin B4 (Vector Laboratories, Burlingame, CA) in 100 μL of PBS. Thirty minutes after injection, mice were euthanized, the Matrigel plug was surgically resected and washed in ice-cold PBS. Matrigel plugs were homogenized and the extent of EC staining by FITC was quantitated by spectrophotometric analysis.
Statistical analysis. Each experiment was repeated 2 to 4 times. Values represent means ± SE from separate experiments. P values were determined using Student's t test assuming populations with equal variance and two-tailed distribution.
Role of ASK1 in EC apoptosis. Genotoxic chemotherapeutic agents such as doxorubicin and etoposide activate the stress or intrinsic pathway of apoptosis by inhibiting topoisomerase II leading to DNA damage. Cell death mediated by these agents has been linked to the activation of the proapoptotic kinase ASK1 leading to mitochondrial cytochrome c release and activation of caspases ( 3, 5). To determine whether endogenous ASK1 in ECs is required for stress-mediated death, we knocked down ASK1 expression by RNAi and monitored cell death in response to the DNA-damaging agent etoposide. Treatment of ECs with etoposide led to the induction of EC apoptosis, and knockdown of ASK1 resulted in a significant reduction in this death response ( Fig. 1A ). These findings suggest that endogenous ASK1 is required for apoptosis in response to genotoxic stress. To assess whether ASK1 expression was sufficient to induce death, ECs were transduced with adenovirus encoding wild-type (WT) HA-ASK1, and these cells were monitored for apoptosis. Exogenously expressed ASK1 produced a dose-dependent apoptotic response, suggesting that its expression in ECs was sufficient to induce apoptosis of these cells ( Fig. 1B). To establish a role for ASK1 kinase activity in stress-mediated apoptosis in ECs, we examined the ability of WT or kinase-inactive ASK1 (K709M) to induce cell death. Expression of WT ASK1 induced apoptosis, whereas expression of kinase-inactive ASK1 did not ( Fig. 1C). These findings show that ASK1 kinase activity is sufficient to induce EC apoptosis, and that it seems required to induce death in response to genotoxic stress.
bFGF selectively inhibits ASK1 kinase activity and protects ECs from ASK1-induced apoptosis. bFGF and VEGF act as survival factors for ECs and can produce chemoresistance to genotoxic drugs. To assess whether bFGF or VEGF promote EC survival based on their ability to regulate ASK1, ECs were transduced with adenovirus encoding HA-tagged WT ASK1 and then exposed to varying concentrations of bFGF or VEGF. Although both growth factors produced a maximal level of extracellular signal-regulated kinase stimulation (data not shown), exposure of cells to bFGF, but not VEGF, led to the protection of ECs from ASK1-induced cell death ( Fig. 2A ), demonstrating that the survival pathway induced by bFGF protects cells from ASK1-mediated death, whereas that induced by VEGF does not. However, in other experiments, VEGF was able to protect ECs from death induced by activators of caspase-8 ( 21). Next, we asked whether bFGF-mediated protection from ASK1-induced EC death occurs through inhibition of ASK1 kinase activity. We treated ECs overnight with doxorubicin and assessed the effect of bFGF or VEGF stimulation on ASK1 kinase activity as measured by phosphorylation of its substrate MKK6 in an in vitro kinase assay. Treatment of ECs with doxorubicin led to the activation of ASK1, and bFGF efficiently inhibited this activity to below baseline levels, whereas VEGF had no effect on this activity ( Fig. 2B). Selective inhibition of ASK1 by bFGF, but not VEGF, was associated with bFGF-mediated protection against apoptosis induced by treatment with doxorubicin ( Fig. 2C). These results show that genotoxic drugs such as doxorubicin induce ASK1 activity leading to EC death, and that bFGF preferentially suppresses ASK1 activity and the associated death response.
bFGF, but not VEGF, promotes Raf-1 interaction with ASK1. Previous studies have shown that the binding of Raf-1 to the NH2-terminal negative regulatory domain of ASK1 suppresses ASK1-induced cell death ( 19, 20). Although both bFGF and VEGF activate Raf-1, they do so via distinct mechanisms ( 21). We therefore considered whether the inhibition of ASK1 kinase activity selectively by bFGF in ECs is linked to the ability of bFGF-stimulated Raf-1 to form a complex with ASK1. To test this, lysates from ECs stimulated with bFGF or VEGF were subjected to immunoprecipitation of endogenous ASK1 followed by immunoblotting for Raf-1. Stimulation of cells with bFGF led to the formation of a Raf-1/ASK1 complex ( Fig. 3A ). Importantly, whereas VEGF leads to Raf-1 activation in ECs as detected by analysis of phospho-extracellular signal-regulated kinase (ERK) levels ( Fig. 3A), it did not induce the formation of a Raf-1/ASK1 complex ( Fig. 3A). To substantiate these findings, lysates from ECs expressing HA-tagged ASK1 were subjected to immunoprecipitation with anti-HA antibody followed by blotting for Raf-1. Once again, bFGF induced a complex between Raf-1 and ASK1, whereas VEGF did not. We obtained similar results by performing a reciprocal immunoprecipitation with anti–Raf-1 antibody and blotting with anti-HA ( Fig. 3B). These findings reveal that Raf-1 activation due to bFGF, but not VEGF, induces the formation of a Raf-1/ASK1 complex in ECs and may account for selective protection of ECs from stress-mediated death by bFGF.
Previous studies have shown that bFGF, but not VEGF, stimulation leads to the phosphorylation of Raf-1 SS338/9 ( 4). Interestingly, these sites were found to be required for bFGF-mediated protection of ECs against stress-mediated apoptosis. We therefore determined whether the mutation of SS338/9 of Raf-1 to alanines might impact bFGF-induced formation of the Raf-1/ASK1 complex, a process associated with the protection of cells from stress. To test this, HUVECs electroporated with expression constructs encoding FLAG-tagged WT, SS338/9AA, or SS497/9AA Raf-1 (a protein kinase C regulatory site mutant) were transduced with adenovirus encoding HA-tagged WT ASK1. Cells were starved overnight and stimulated with bFGF, and Raf-1 variants were detected in anti-HA immune complexes resolved by SDS-PAGE. Mutation of SS338/9 to alanines significantly reduced bFGF-induced Raf-1 ASK1 complex formation, whereas WT and SS497/9AA Raf-1 showed bFGF-dependent formation of a Raf-1/ASK1 complex ( Fig. 3C). Importantly, the SS338/9AA Raf-1 mutant reduced basal levels of Raf-1/ASK1 complex in starved ECs, suggesting that this mutant of Raf-1 not only prevents growth factor–induced Raf-1/ASK1 complex formation but may actually dissociate this complex in quiescent cells. These results provide a molecular basis to explain the requirement of SS338/9 sites of Raf-1 in protection against stress-mediated apoptosis ( 21, 23).
Subcellular localization of the Raf-1/ASK1 complex. bFGF promotes translocation of Raf-1 to mitochondria leading to protection against stress-mediated apoptosis that is independent of mitogen-activated protein (MAP)/ERK kinase/ERK signaling ( 21). Interestingly, p21-activated protein kinase (PAK)–mediated activation of Raf-1 and phosphorylation of SS338/9 sites have been shown to potentiate mitochondrial translocation of Raf-1 and provide protection from stress ( 21, 23– 25). Because ASK1 is associated with both cytoplasmic and mitochondrial compartments ( 14), we determined whether bFGF-induced translocation of Raf-1 to the mitochondria facilitates association of Raf-1 with the mitochondrial pool of ASK1. Untreated or doxorubicin-treated cells were stimulated by bFGF and subjected to subcellular fractionation. Endogenous ASK1 immune complexes from the cytoplasmic and mitochondrial fractions were resolved by SDS-PAGE and probed by immunoblotting for endogenous Raf-1 to detect Raf-1/ASK1 complexes. bFGF stimulation of ECs led to significant increase in levels of Raf-1/ASK1 complex at the mitochondria with no significant change in the level of this complex in the fraction containing the cytoplasm and plasma membrane (CT + PM in Fig. 4 ). Furthermore, the activation state of ASK1 did not impact the basal capacity of ASK1 to form a complex with Raf-1. These results suggest that bFGF-mediated translocation of Raf-1 to the mitochondria promotes the formation of a mitochondrial Raf-1/ASK1 complex leading to the protection of ECs from genotoxic stress. We observed minimal plasma membrane contamination in our mitochondrial preparations. However, this is unlikely to account for enrichment of Raf-1 with immunoprecipitated ASK1 because we did not observe an enrichment of the Raf-1/ASK1 complex in the fraction containing the cytoplasm and plasma membrane.
Stimulation with bFGF, but not VEGF, enhances chemoresistance of angiogenic ECs. In addition to stimulating proliferation and migration of ECs, bFGF and VEGF promote angiogenesis in part by acting as EC survival factors that protect against distinct apoptotic stimuli. Previous studies have suggested that these growth factors differentially influence the survival of ECs ( 21, 26). In this study, we have provided evidence suggesting an important role for bFGF, but not VEGF, in protection of ECs against cytotoxic chemotherapeutic agents. To substantiate our results in vivo, we determined whether bFGF and VEGF differentially impact the chemosensitivity of invasive angiogenic ECs in a mouse model of angiogenesis. Mice were injected with growth factor-depleted Matrigel containing increasing concentrations of the genotoxic stress-inducing drug doxorubicin in the presence or absence of each growth factor at a concentration to provide maximal angiogenic stimulation ( 21). Although bFGF and VEGF efficiently induced angiogenesis in the absence of doxorubicin, EC cell survival and angiogenesis was only observed in animals treated with bFGF. Increasing concentrations of doxorubicin selectively inhibited VEGF-mediated angiogenesis ( Fig. 5 ). These findings suggest that bFGF, but not VEGF, protects angiogenic ECs against the genotoxic effects of doxorubicin.
In this study, we provide evidence that bFGF selectively inhibits the proapoptotic activity of ASK1 in ECs and, in so doing, blocks apoptosis in response to genotoxic stress or exogenous expression of ASK1. Stimulation of ECs with bFGF, but not VEGF, promotes the formation of a complex between Raf-1 and ASK1 localized at the mitochondria, a critical site involved in the regulation of the intrinsic apoptosis pathway. The significance of these studies is underscored by the observation that ASK1 not only is sufficient to induce apoptosis, but is required for the induction of EC apoptosis in response to genotoxic stress. Thus, our studies summarized in Fig. 6 identify ASK1 as a target of antiapoptotic signaling by bFGF and provide evidence for an important role of Raf-1 activation in this response.
Accumulating evidence suggests that a critical function of Raf-1 is to protect cells from apoptosis. Targeted disruption of Raf-1 in mice leads to increased levels of cell death and is associated with elevated basal ASK1 kinase activity ( 27– 29). Importantly, the apoptotic phenotypes observed in cardiac-specific Raf-1 knock-out animals are rescued when ASK1 expression is also disrupted ( 29). These findings provide significant genetic evidence that the prosurvival function of Raf-1 is linked to its ability to block ASK1-mediated apoptosis. In support of these observations, exogenously expressed Raf-1 was shown to interact with and inhibit ASK1 in a manner that was not dependent on Raf-1 or MEK kinase activities ( 19). The NH2-terminal negative regulatory domain of ASK1 was shown to be required for the formation of this complex and subsequent inhibition of ASK1-induced apoptosis. To our knowledge, this is the first report of a physiologic stimulus leading to formation of an endogenous Raf-1/ASK1 complex leading to cell survival.
We have previously shown that Raf-1 plays an important role in EC survival and angiogenesis ( 21, 22, 30). Raf-1 was shown to be a point of convergence for growth factor–mediated survival signals where differential phosphorylation of the activation loop of Raf-1 mediated divergent protection mechanisms. Specifically, VEGF activated Raf-1 via Src kinase leading to phosphorylation of YY340/1 and preferential protection against the extrinsic or death receptor–mediated apoptosis in a manner that was dependent on ERK signaling. In contrast, bFGF activated Raf-1 via PAK and promoted the phosphorylation of SS338/9, promoted Raf-1 translocation to the mitochondria, and protected against stress-mediated or intrinsic pathway of apoptosis in a manner that did not require MEK1/2 activation ( 21, 23). These findings are consistent with previous observations showing that mitochondria-targeted Raf-1 protects cells against the stress- or caspase-9–dependent pathway of apoptosis ( 24, 25, 31). Given the subcellular localization of ASK1 in both the mitochondrial and cytoplasmic compartments, as well as the role of Raf-1 in regulation of ASK1-mediated apoptosis, we considered whether growth factor–mediated survival signaling depends on the regulation of mitochondrial ASK1 by Raf-1 through the formation of an inhibitory complex. In this report, we provide evidence that bFGF promotes the formation of an endogenous Raf-1/ASK1 complex at the mitochondria, inhibits ASK1 kinase activity, and prevents the activation of the downstream proapoptotic cascade. Therefore, inhibition of mitochondrial ASK1 may play an important role in bFGF-mediated protection of ECs against stress-mediated apoptosis. In support of these observations, expression of H-Ras, a critical mediator of bFGF signaling in cells, has been shown to inhibit proapoptotic signaling by ASK1 in a Raf-1–dependent but MEK1/2- and ERK1/2-independent manner ( 20).
Tumor growth depends on the production of growth factors such as bFGF and VEGF that stimulate quiescent endothelium to undergo angiogenesis, promoting increased EC proliferation and tissue invasion. However, the cytoprotective effect of these growth factors has been shown to provide chemoresistance to ECs, thus attenuating the efficacy of anticancer therapies designed to promote apoptosis. The observation that bFGF, but not VEGF, preferentially protects ECs from apoptosis in response to genotoxic stress (intrinsic pathway) may have significant ramifications because conventional anticancer chemotherapeutic agents function, in part, as angiogenesis inhibitors by causing DNA damage and promoting stress-mediated apoptosis of proliferating ECs within the tumor ( 2). In fact, tumor-derived bFGF has been shown to promote chemoresistance of both tumor cells as well as ECs ( 32– 37). Interestingly, inhibition of FGF receptor signaling has been shown to enhance cellular susceptibility to genotoxic agents such as doxorubicin and etoposide ( 32, 38). These observations support the notion that administration of genotoxic drugs or ionizing radiation, combined with agents that block growth factor–mediated protection from stress may synergize to enhance the efficacy anticancer therapy. Therefore, identification of apoptotic signaling cascades targeted by angiogenic growth factors present within the tumor microenvironment may enhance the design of effective therapies that use antiangiogenics as chemosensitizing agents.
Grant support: NIH grants CA095262, CA104898, CA50286, and CA78045 (D.A. Cheresh) and NIH grants R01 HL-65978-5 and P01 HL070295-6 (W. Min). W. Min is an established investigator of the American Heart Association (0440172N).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Yong Lee for WT HA-ASK1 adenovirus and Jacques Landry for kinase dead HA-ASK1 expression constructs.
Note: There are no conflicts of interest with the material presented in this article.
- Received October 9, 2006.
- Revision received December 6, 2006.
- Accepted January 11, 2007.
- ©2007 American Association for Cancer Research.