Gene Therapy (2010) 17, 532–540 r 2010 Nature America, Inc. All rights reserved 0929-1903/10 www.nature.com/cgt

ORIGINAL ARTICLE Antisense HIF-1a prevents acquired tumor resistance to gene therapy X Sun1,2, M Vale1, X Jiang2, R Gupta1 and GW Krissansen1 1Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand and 2Hepatosplenic Surgery Center, Department of General Surgery, First Affiliated Hospital of Harbin Medical University, Harbin, China

Angiostatin is a naturally occurring inhibitor of that is being developed as a to fight cancer. In this study we reveal that EL-4 tumors established in mice rapidly develop resistance to angiostatin gene therapy by upregulating hypoxia-inducible pathways. Angiostatin initially delayed tumor growth for 6 days by reducing blood vessel density. However, tumors quickly responded by upregulating the production of hypoxia-inducible factor-1a (HIF-1a) and its effector vascular endothelial growth factor (VEGF) in response to increasing tumor hypoxia, leading to restored angiogenesis and rapid tumor growth. Theoretically, blockade of HIF-1 should prevent resistance to anti-angiogenic therapy by preventing a tumor from responding to induced hypoxia. Antisense HIF-1a inhibited the expression of HIF-1a and of the HIF-1 effectors VEGF, glucose transporter-1 and lactate dehydrogenase. As a monotherapy, it was effective in eradicating small 0.1 cm diameter tumors, but only delayed the growth of large 0.4 cm diameter tumors. In contrast, timed injection of a combination of angiostatin and antisense HIF-1a plasmids completely eradicated large EL-4 tumors within 2 weeks, and prevented upregulation of hypoxia-inducible pathways induced by angiostatin. The data indicate that blocking hypoxia-inducible pathways by antisense HIF-1a can circumvent hypoxia-induced drug resistance and thereby augment the efficacy of anti-angiogenic therapies. Cancer Gene Therapy (2010) 17, 532–540; doi:10.1038/cgt.2010.7; published online 26 March 2010 Keywords: angiostatin; hypoxia-inducible factor-1; angiogenesis; vascular endothelial growth factor; drug resistance

Introduction -mediated anti-angiogenic therapy of murine mammary carcinomas caused a compensatory increase in 8 Solid tumors need to establish blood vessels to grow tumoral VEGF mRNA expression. Indeed, VEGF and beyond 1–2 mm and metastasize, which provides the other angiogenic factors are being considered as soluble rationale for anti-angiogenic approaches to cancer ther- markers for the detection of hypoxia as an indication of 9–11 apy.1 It had been believed that targeting tumor blood the apparent efficacy of anti-angiogenic treatment. vessels should not lead to drug resistance as endothelial Angiostatin, a 38 kDa protein containing several cells, unlike cancer cells, are genetically stable.2,3 How- internal kringle domains of plasminogen, inhibits en- ever, human clinical trials of anti-angiogenic agents to dothelial cell proliferation and migration, and suppresses 12–14 date have been disappointing as they show only modest tumor growth and . However, angiostatin effects on the overall survival of cancer patients.4 Tumors as a monotherapy to combat cancer has not progressed 15 respond and become resistant to anti-angiogenic therapies further than a phase 1 clinical trial. Human neuroblas- although in most cases the mechanisms have not been toma and breast xenografts were found to be resistant to 16,17 ascertained. Higher quantities of basic fibroblast growth the effects of adenovirally delivered angiostatin. An- factor were detected in mice, which had been treated with giostatin was apparently not able to counteract the very vascular endothelial factor (VEGF) receptor , high level of VEGF released by the tumors, suggesting accompanied by reactivation of tumor angiogenesis.5 that it was susceptible to drug resistance. It is important Increased plasma VEGF levels were observed in clinical to identify and successfully block the pathways respon- trials of anti-angiogenic tyrosine kinase inhibitors.6,7 sible for tumor resistance to angiostatin if this promising drug is to be of clinical benefit. Hypoxic microenvironments, which are a feature of Correspondence: Dr GW Krissansen, Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, many solid tumors, could theoretically be amplified University of Auckland, Auckland, New Zealand. by anti-angiogenic therapy as the latter suppresses E-mail: [email protected] blood supply and oxygen to the tumor. Hypoxia Received 7 April 2009; revised 17 August 2009; accepted 13 orchestrates a wide spectrum of molecular pathways, December 2009; published online 26 March 2010 and is a major cause of tumor resistance to radiotherapy Antisense HIF-1a prevents resistance to angiostatin X Sun et al 533 and chemotherapy.18,19 Hypoxia-inducible factor-1 (HIF-1), injection of 2 Â 105 EL-4 tumor cells into the right flank the ‘master regulator’ of classical hypoxia-inducible of mice, and growth was determined by measuring two pathways,20 mediates the adaptation of cancer cells to perpendicular diameters. Animals were killed when the hypoxic environment by controlling the expression of tumors reached more than 1 cm in diameter, in accord hundreds of genes,21,22 including VEGF, glycolytic with the Animal Ethics Approval (University of Auck- enzymes and glucose transporters. HIF-1, formed by the land). Tumors were injected with 100 mg (100 ml) of assembly of HIF-1a and HIF-1b, binds hypoxia-response expression plasmid upon reaching either 0.1 or 0.4 cm in elements in the promoters of the above genes.20 HIF-1b is diameter. For combinational treatment, reagents were constitutively expressed, whereas HIF-1a is rapidly delivered in a timed manner, in which the angiostatin degraded during normoxia, and dramatically stabilized plasmid was injected first, followed by antisense HIF-1a and activated during hypoxia, by oxygen sensing and plasmid after 24 h. Empty pcDNA3 vector served as a signaling processes.23 We have previously reported that plasmid control. All experiments included six mice per antisense HIF-1a gene therapy enhances immunotherapy group, and each experiment was repeated at least once. to eradicate EL-4 lymphomas in mice.24 In addition, we recently showed that antisense HIF-1a gene therapy Evaluation of tumor hypoxia enhances the efficacy of doxorubicin and transcatheter The EL-4 tumors in a group of three mice were injected arterial embolization to combat liver .25,26 In the with angiostatin plasmid or empty pcDNA3 vector when present study we examine tumor resistance to angiostatin the tumors reached 0.4 cm in diameter, as above. At 7 gene therapy and test the hypothesis that blockade of days after injection, the mice received an intravenous HIF-1a to inhibit hypoxia-inducible pathways will pre- injection of pimonidazole hydrochloride (Hypoxyprobe- vent acquired drug resistance. This is the first study to 1) dissolved in phosphate-buffered saline at a dose of analyze whether targeting HIF-1 has the potential to 120 mg kg–1. The mice were killed 24 h later and the improve the therapeutic efficacy of an anti-angiogenic tumors were harvested, sectioned and immunostained cancer drug. with an anti-Hypoxyprobe-1 Ab.

Immunohistochemistry Tumor cryosections (10 mm) were incubated overnight Materials and methods with primary , followed by subsequent incuba- Mice, cell lines, plasmids and antibodies tion with appropriate secondary antibodies (VECTAS- Male C57BL/6 mice, 6–8 weeks old, were obtained from TAIN Universal Quick kit; Vector Laboratories, the Animal Resource Unit, Faculty of Medical and Burlingame, CA), and developed with Sigma FAST Health Sciences, University of Auckland, Auckland, DAB (3,30-diaminobenzidine tetrahydrochloride) and New Zealand. The EL-4 thymic lymphoma, which is of CoCl2 enhancer tablets (Sigma-Aldrich New Zealand C57BL/6(H-2b) origin, was purchased from the American Ltd., Auckland, New Zealand). Sections were counter- Type Culture Collection (Rockville, MD). It was cultured stained with Mayer’s hematoxylin. at 37 1C in Dulbecco’s modified Eagle’s medium (Gibco BRL, Grand Island, NY) supplemented with 10% fetal Western blot analysis –1 calf serum, 50 U ml penicillin/streptomycin, 2 mM Tumors were excised, homogenized in a protein lysate L-glutamine and 1 mM pyruvate. The pcDNA3 expression buffer and protein samples (50 mg) were resolved by vectors encoding the kringle 1–4 form of mouse angios- sodium dodecyl sulfate-polyacrylamide gel electrophor- 27 tatin, and an antisense version of the 50 end of HIF-1a esis. Proteins were electrophoretically transferred to 24 have been described previously. The antibodies (Abs) nitrocellulose Hybond C extra membranes, and the used include those against plasminogen (recognizing membranes were incubated with primary antibodies and kringles 1–3 of angiostatin; Calbiochem-Novabiochem subsequently with horseradish peroxidase-conjugated Corp., San Diego, CA), VEGF (Ab-1; Lab Vision Corp., secondary antibodies. Immunoreactivity was developed Fremont, CA), mouse HIF-1a (H1a67; Novus Biologi- by enhanced chemiluminescence (Amersham Interna- cals, Inc., Littleton, CO), CD31 (MEC13.3 Ab; Pharmin- tional, Buckingham, UK) and exposure to X-ray gen, San Diego, CA), glucose transporter 1 and film. Band density was quantified using Sigma ScanPro lactate dehydrogenase A (Santa Cruz Biotechnology, software (Systat Software Inc., San Jose, CA). Santa Cruz, CA). Pimonidazole hydrochloride (Hypoxyp- robe-1) and an anti-Hypoxyprobe-1 Ab were obtained Assessment of tumor vascularity from Chemicon (Temecula, CA). Tumor vascularity was measured as described pre- viously.24,27 In brief, tumor sections (10 mm) prepared 4 Gene transfer of expression plasmids and measurement days after plasmid injection were immunostained with an of tumor growth anti-CD31 antibody. Stained blood vessels were counted Purified plasmids were diluted to 1 mg ml–1 in a solution in five blindly chosen random fields (0.155 mm2)at  40 of 5% glucose in 0.01% Triton X-100, and mixed in a magnification, and the mean of the highest three counts ratio of 1:3 (wt:wt) with DOTAP cationic liposomes was calculated. The concentric circles method was used to (Boehringer Mannheim, Mannheim, Germany), as des- assess vascularity,24,27 in which 5 to 6 tumor sections were cribed previously.24,27 Tumors were established by analyzed for each plasmid-injected tumor.

Cancer Gene Therapy Antisense HIF-1a prevents resistance to angiostatin X Sun et al 534 Detection of apoptotic cells in situ 7 days after gene transfer, respectively (Figure 1d). The Frozen tumor sections (6 mm) were fixed, permeabilized hypoxia marker pimonidazole hydrochloride was intrave- and incubated with the terminal deoxynucleotidyl trans- nously injected 7 days after gene transfer into mice bearing ferase dUTP nick end labeling reagent for 60 min at 37 1C EL-4 tumors to confirm that upregulated expression of and examined by fluorescence microscopy. Adjacent HIF-1a and VEGF was in response to angiostatin-induced sections were counterstained with hematoxylin and eosin. increases in tumor hypoxia. As shown in Figure 1e, the The total number of apoptotic cells in 10 randomly staining of pimonidazole hydrochloride was increased in selected fields was counted. The index was angiostatin gene-injected tumors compared with tumors calculated as the percentage of positive staining cells, injected with empty plasmid control. namely, AI ¼ number of apoptotic cells  100/total number of nucleated cells. Antisense HIF-1a downregulates hypoxia-inducible pathways, eradicates small tumors and suppresses the Statistical analysis growth of large tumors Results are expressed as mean values þ s.d. The Student’s Small EL-4 tumors, 0.1 cm in diameter, were established t-test was used for evaluating statistical significance, in as above, and injected with 100 mg of antisense HIF-1a which a P-value of o0.05 denotes statistical significance. plasmid or empty control vector. Tumors in the control group grew rapidly, reaching 0.9 cm in size 18 days after gene transfer. In contrast, tumors injected with the Results antisense HIF-1a plasmid rapidly regressed and comple- Hypoxia-inducible pathways are activated in response tely disappeared 10 days after gene transfer (Figure 2a). to treatment of tumors with angiostatin Antisense HIF-1 gene therapy was less effective against Small EL-4 tumors, 0.1 cm in diameter (Figure 1a), and large 0.4 cm in diameter tumors. It significantly (Po0.01) large EL-4 tumors, 0.4 cm in diameter (Figure 1b), were suppressed tumor growth for 10 days (Figure 2b), but establishedinC57BL/6mice,andinjectedwitheither eventually all tumors re-grew, reaching 0.9 cm in diameter 100 mg of angiostatin plasmid or empty vector control. within 3 weeks. The ineffectiveness of antisense HIF-1 Tumors grew rapidly in the control groups, reaching 0.9 cm therapy against large tumors was not the result of an in diameter in 12–18 days (depending on initial tumor size) inadequate dosage of plasmid, as increasing the plasmid after gene transfer. In contrast, angiostatin gene transfer dosage to 250 mg did not significantly increase the suppressed the growth of both small (Figure 1a) and large inhibition of tumor growth (data not shown), as reported 24 tumors (Figure 1b) for 6 days. Surprisingly, thereafter previously. Antisense HIF-1 therapy strongly inhibited tumors grew faster than those of control mice, reaching endogenous HIF-1a expression, and expression of the 1 cm in diameter on days 12 or 18 (depending on initial downstream effectors VEGF, glucose transporter 1 and tumor size). As expected, angiostatin gene therapy resulted lactate dehydrogenase A, as measured by western blot in increased tumoral expression of angiostatin, measured 4 analysis of homogenates of large tumors prepared 2 days and 7 days after gene transfer (Figure 1c, panels 1–3). after gene transfer (Figure 2c). Tumoral expression of both HIF-1a (Figure 1c, panels 4–6) and its effector VEGF (Figure 1c, panels 7–9) increased in Antisense HIF-1a prevents acquired tumor resistance to response to therapy. HIF-1a was detected in the cytoplasm angiostatin in addition to the nucleus, in which cytoplasmic HIF-1 Gene transfer of antisense HIF-1a and angiostatin was presumably results from increased synthesis of HIF-1a combined to determine whether antisense HIF-1a would needed to maintain or increase the levels of nuclear HIF- effectively prevent angiostatin-mediated activation of 1.28 Western blot analysis of tumor homogenates confirmed hypoxia-inducible pathways that promote tumor growth the latter findings. Thus, gene transfer of angiostatin and result in acquired resistance to angiostatin. Large EL-4 upregulated the expression of HIF-1a and VEGF by tumors, 0.4 cm in diameter, were first injected with 100 mg 1.0-fold in 4 days after gene transfer and by twofold in of angiostatin plasmid, and then with 100 mg of the

Figure 1 Hypoxia-inducible pathways are activated in response to treatment of tumors with angiostatin, resulting in uncontrolled tumor growth. (a, b) Established EL-4 tumors, approximately 0.1 cm (a) or 0.4 cm (b) in diameter, were injected at day 0 with a pcDNA3 expression plasmid encoding angiostatin, or an empty pcDNA3 control plasmid. Tumor size was recorded until tumors reached 1 cm in diameter, at which point mice were killed. (c) Immunohistochemistry of tumor sections. Tumors in (b) injected with empty control vector (images 1, 4 and 7) or with angiostatin plasmid (images 2, 3, 5, 6, 8 and 9) were excised 4 days (images 1, 2, 4, 5, 7 and 8) or 7 days (images 3, 6 and 9) after angiostatin gene transfer. Tumor sections were stained brown with antibodies against angiostatin (images 1–3), HIF-1a (images 4–6) and VEGF (images 7–9). Magnification  100. (d) Angiostatin gene therapy upregulates the expression of HIF-1a and VEGF. Tumors in (b) were excised 4 days (lanes 1 and 2), and 7 days (lane 3) after injection of empty vector (lane 1) or angiostatin plasmid (lanes 2 and 3), and tumor homogenates prepared. Homogenate proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and western blotted with antibodies against angiostatin, HIF-1a, VEGF and tubulin (internal control) as indicated (left panel). The density of each band was measured and compared with that of tubulin (right panel). (e) Mice bearing established EL-4 tumors of 0.4 cm diameter were injected intratumorally with empty pcDNA3 (image 1) or angiostatin expression plasmid (image 2). At 7 days after gene transfer, the mice were intravenously injected with pimonidazole hydrochloride and were killed 24 h later. The tumors were sectioned and immunostained with an anti-Hypoxyprobe-1 Ab.

Cancer Gene Therapy Antisense HIF-1a prevents resistance to angiostatin X Sun et al 535 1.2 1.2

1 pcDNA3 pcDNA3 1 angiostatin angiostatin 0.8 0.8 0.6 0.6 0.4 Tumour size (cm) Tumour size (cm) 0.2 0.4

0 0.2 0 2 4 6 8 10 12 18 0122 4 6 8 10 Days after gene injection Days after gene injection

6

123 3 1 2 3

Angiostatin 2 HIF-1α 1 Density VEGF Relative Band 0 Tubulin AngiostatinHIF-1α VEGF

Cancer Gene Therapy Antisense HIF-1a prevents resistance to angiostatin X Sun et al 536 1.2 1.2 pcDNA3 pcDNA3 12 1 1 aHIF aHIF HIF-1α 0.8 0.8 VEGF 0.6 0.6 Glut1 0.4 0.4 LDHA Tumour size (cm)

Tumour size (cm) 0.2 0.2 0 0 Tubulin 0182 4 6 8 10 12 0182 4 6 8 10 12 14 Days after gene injection Days after gene injection Figure 2 Intratumoral injection of antisense HIF-1a eradicates small tumors, suppresses the growth of large tumors and prevents the induction of hypoxia-inducible pathways. (a, b) EL-4 tumors, approximately 0.1 cm (a) and 0.4 cm (b) in diameter, were injected at day 0 with expression plasmids encoding antisense HIF-1a or empty pcDNA3 plasmid. Tumor size was recorded until tumors reached 1 cm in diameter. (c) Antisense HIF-1a therapy downregulates the expression of HIF-1a, VEGF, glucose transporter 1 (Glut1) and lactate dehydrogenase A (LDHA). Homogenates were prepared from tumors in (b) 2 days after injection of empty vector (lane 1) or angiostatin plasmid (lane 2). Homogenate proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and western blotted with antibodies against HIF-1a, VEGF, Glut1, LDHA and tubulin (internal control) as indicated.

antisense HIF-1a expression plasmid after 24 h. Tumors (Po0.01) reduced vessel density by day 4, with a further injected with either empty vector or angiostatin plasmid significant (Po0.001) reduction on day 10 (Figures 4a or antisense HIF-1a plasmid served as controls. The and b). The median distance to the nearest CD31-labeled combination of antisense HIF-1a and angiostatin plas- vessel from an array of points within tumors was mids caused tumors to regress over 2 weeks and significantly increased on both day 4 (Po0.01) and day completely disappear (Figure 3a). Mice remained tumor- 10 (Po0.001) compared with that for control tumors free for 2 months (data not shown). In contrast, control (Figure 4c). tumors grew unchecked. The growth of tumors treated with angiostatin was initially suppressed, but tumors Antisense HIF-1 a and angiostatin synergize to induce subsequently grew very rapidly as in Figure 1b. The tumor cell apoptosis growth of tumors treated with antisense HIF-1a was Sections prepared from the tumors described in Figure 3b significantly suppressed for at least 9 days, as described in were stained with the terminal deoxynucleotidyl transfer- Figure 2b. Combinational gene transfer increased the ase dUTP nick end labeling agent to determine the extent expression of angiostatin and decreased the expression of of programmed death of tumor cells. Only small numbers HIF-1a and its effectors VEGF, glucose transporter 1 and of apoptotic cells were detected in tumors injected with lactate dehydrogenase A, as measured by western blot empty plasmid (Figures 5a and b). In contrast, tumor analysis of tumor homogenates prepared 4 and 10 days apoptosis was almost doubled on day 4 after injection of after gene transfer (Figure 3b). Angiostatin expression angiostatin plasmid, but had declined significantly by day and silencing of HIF-1a and its effectors was maintained 10. The combination of angiostatin and antisense HIF-1a for at least 10 days after gene transfer. plasmids significantly increased tumor apoptosis on day 4 (Po0.01), with a further increase on day 10 (Po0.001; Antisense HIF-1a and angiostatin synergize to inhibit Figures 5a and b). tumor angiogenesis Sections prepared from the tumors described in Figure 3b were stained with an anti-CD31 Ab to visualize tumor blood vessels (Figure 4a). Angiostatin gene therapy Discussion significantly (Po0.01) reduced tumor vessel density 4 days after gene transfer (Figures 4a and b). In contrast, by Currently, dozens of promising anti-angiogenic that day 10 it was clear that the vessel density of tumors had target different facets of endothelial function are under- rebounded and was slightly increased compared with that going evaluation in clinical trials. The results of trials to of control tumors treated with empty vector. This result date have been disappointing, with only modest increases was in spite of the fact that tumoral expression of in overall patient survival being achieved, even when anti- angiostatin remained elevated at day 10 (Figure 3b), and angiogenic drugs have been combined with chemother- even at day 12 (data not shown). At day 4 the median apy. It has to be appreciated that anti-angiogenic therapy distance to the nearest CD31-labeled vessel from an array leads to hypoxia, which serves as a platform for drug of points within tumors treated with angiostatin was resistance. In this study we have shown that angiostatin significantly increased compared with that for tumors gene therapy initially delays the growth of tumors treated with empty vector, but by day 10 there was no accompanied by reduced tumoral blood vessel density, difference between angiostatin-treated tumors and con- but within 1 week of therapy tumors recover and re-grow trols (Figure 4c). Combinational therapy significantly more rapidly than control tumors. At first glance these

Cancer Gene Therapy Antisense HIF-1a prevents resistance to angiostatin X Sun et al 537 1.2 pcDNA3 123 angiostatin aHIF Angiostatin 1 angiostatin + aHIF 0.8 HIF1α

0.6 VEGF

0.4 Glut1 Tumour size (cm) 0.2 LDHA

0 Tubulin 0183 6 9 12 15 Days after gene injection Figure 3 Antisense HIF-1a synergizes with angiostatin to eradicate large tumors. (a) EL-4 tumors of approximately 0.4 cm in diameter were injected at day 0 with expression plasmids encoding angiostatin, antisense HIF-1a or a combination thereof. Tumors injected with empty pcDNA3 served as a control. Tumor size was recorded until tumors reached 1 cm in diameter, at which point mice were killed (denoted by vertical arrows). Complete tumor regression is denoted by stars. (b) Antisense HIF-1a blocks hypoxia-inducible pathways activated by angiostatin therapy. Homogenates were prepared from tumors in (a) 4 days (lanes 1 and 2) and 10 days (lane 3) after injection of empty vector (lane 1) or the combination of angiostatin and antisense HIF-1a plasmids (lanes 2 and 3). Homogenate proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and western blotted with antibodies against angiostatin, HIF-1a, VEGF, glucose transporter 1 (Glut1), lactate dehydrogenase A (LDHA) and tubulin (internal control) as indicated. results might seem inexplicable, given that angiostatin cytokines or chemoattractants involved in the mobiliza- expression was still highly elevated 12 days after gene tion of myeloid cells. therapy. The explanation seems to reside in the finding In contrast to the very modest affects of angiostatin, a that the expression of HIF-1a and VEGF was elevated single injection of antisense HIF-1a was able to com- within 4 days of angiostatin therapy and further increased pletely eradicate small tumors, due in part to the by day 7, accompanied by increased tumor blood vessel downregulation of expression of VEGF, glucose trans- density. Thus, tumors upregulated hypoxia-inducible porter 1 and lactate dehydrogenase A. The latter two pathways in response to angiostatin-imposed hypoxia, proteins are required for the high glycolytic rates of releasing pro-angiogenic factors that effectively competed cancer cells, and hence are important for the bioenergetics with angiostatin and promoted angiogenesis and acceler- of tumor growth. In accord, antisense HIF-1a was found ated tumor growth. We had earlier reported that VEGF to downregulate the expression of proliferating cell expression was elevated in response to angiostatin in a nuclear antigen, and inhibited cellular proliferation in study examining the ability of angiostatin to augment liver tumors.25 We have previously reported that antisense cancer immunotherapy.27 In accord, and as mentioned HIF-1a also induces natural killer cell-dependent rejec- earlier, basic and VEGF protein tion of small tumors.24 Antisense HIF-1a was not able to and/or mRNA levels increased in response to anti- eradicate larger tumors. It is plausible that the large angiogenic therapy using either antibodies and tyrosine tumors developed HIF-1a-independent pathways of kinase inhibitors against VEGF receptors or endostatin.5–7 angiogenesis,30 although this possibility was not analyzed. The treatment of rectal cancer patients with the anti- Oncogenes such as K-ras activate transcription pathways VEGF led to signifi- that upregulate pro-angiogenic factors independently of cant decreases in blood flow and microvessel density; HIF-1.31 however, plasma levels of VEGF and the angiogenic It is important that anti-angiogenic therapy involves factor placental growth factor markedly increased. Thus, combinations of anti-angiogenic agents, given that multi- hypoxia-induced resistance is a feature encountered with a ple HIF-dependent and HIF-independent angiogenic variety of anti-angiogenic agents. pathways exist. In this study we have shown that a Non-hypoxia pathways may also potentially contribute combination therapy involving the timed gene transfer of to tumor resistance to angiostatin. Thus, Shojaei et al.29 angiostatin and antisense HIF-1a is effective in eradicat- reported that EL-4 tumors were refractory to anti-VEGF ing large tumors, whereas the monotherapies are ineffec- Ab therapy because of their ability to recruit tive. Antisense HIF-1a prevents acquired resistance to CD11b þ Gr1 þ myeloid cells that confer resistance. angiostatin. If HIF-1-independent angiogenic pathways Although not analyzed in this study, CD11b þ Gr1 þ exist for EL-4 lymphomas, then they are not effective in myeloid cells may also contribute to the failure of competing with angiostatin to promote tumor angiogen- angiostatin gene therapy, although the likelihood is esis. Abdollahi et al.32 proposed the concept of combining lessened by the fact that angiostatin and anti-VEGF Ab ‘direct’ and ‘indirect’ angiogenesis inhibitors. They exert their anti-angiogenic effects through different classified direct angiogenesis inhibitors as those that mechanisms. Nevertheless, in this scenario, antisense directly target microvascular endothelial cells and prevent HIF-1a therapy may prevent tumor cells from expressing them from responding to pro-angiogenic stimuli. This

Cancer Gene Therapy Antisense HIF-1a prevents resistance to angiostatin X Sun et al 538

30 40 ** * * 30 20 * * ** 20

10 Vessel counts per surface area

Median distance to 10 CD31 labelled venules

0 0 Day4 Day10 Day4 Day10 Day4 Day10 Day4 Day10 Day4 Day10 Day4 Day10 Control Angiostatin Angiostatin Control Angiostatin Angiostatin + aHIF + aHIF Figure 4 Antisense HIF-1a synergizes with angiostatin in inhibiting tumor angiogenesis. (a) Illustrated are sections prepared from 0.4 cm tumors in Figure 3a excised 4 days (images 1, 2, 4 and 5) or 10 days (images 3 and 6) after injection of control pcDNA3 plasmid (images 1 and 4), angiostatin plasmid (images 2 and 3) or the combination of angiostatin and antisense HIF-1a plasmids (images 5 and 6). The sections were stained with the anti-CD31 antibody MEC13.3 to visualize blood vessels. The hue of the images was changed to better distinguish the vessels from background. (b, c) Measurement of tumor vascularity. (b) Tumor blood vessels stained with the anti-CD31 mAb were counted in five blindly chosen random fields to record mean blood vessel counts per section ( Â 40 magnification field). (c) Histograms showing the median centile distances (± s.d.) to the nearest CD31-labeled venules from an array of points within tumors 4 and 10 days after injection of either angiostatin plasmid or the combination of angiostatin and antisense HIF-1a plasmids. Tumors 4 days after injection of empty vector served as controls. Five tumors from each group of mice were assessed. A significant difference in mean vessel counts, or median distance to nearest CD31-stained vessels, compared with that of control groups is denoted by an asterisk (Po0.01) or two asterisks (Po0.001).

class presumably would also include vascular disrupting It is important to consider the in vitro effects of agents that can destroy established tumor vessels, such as angiostatin and HIF1a silencing on EL-4 cell viability and the tubulin-binding agent combretastatin, and the flavo- proliferation. Koshiji et al.34 took advantage of an noid 5,6-dimethylxanthenone-4-acetic acid.33 In contrast, oxygen-dependent degradation domain-deficient HIF-1a indirect angiogenesis inhibitors target pro-angiogenic (DODD), which is stable and functional in normoxia, and proteins and their receptors and/or their interaction. showed that HIF-1a, even in the absence of a hypoxic With them, the combination of endostatin (direct signal, induces cell cycle arrest by antagonizing Myc inhibitor) with SU5416 (indirect inhibitor; a vascular activity, resulting in derepression of p21cip1. Given this endothelial growth factor receptor 2 receptor tyrosine result, antisense HIF-1a would be expected to down- kinase inhibitor) was more effective than the respective regulate HIF-1, thereby releasing tumor cells from the monotherapies against human prostate, lung and glioma growth-inhibitory effects of HIF-1. In contrast, antisense cancer xenografts. In the present study angiostatin HIF-1a inhibits the growth of tumors, suggesting that represents the ‘direct’ inhibitor, and antisense HIF-1a tumor cells that are growth inhibited by HIF-1a never- could be considered to represent the ‘ultimate’ indirect theless provide important factors such as angiogenic inhibitor as it prevents the upregulation of hypoxia- factors to aid the survival and growth of tumors cells inducible pathways and pro-angiogenic proteins. presumably located in anoxic regions of the tumor. In Angiostatin seems to be able to compete effectively with accord, we have recently shown that antisense HIF-1a HIF-1-independent pathways that remain after antisense downregulated the expression of proliferating cell nuclear HIF-1a therapy. antigen and inhibited cellular proliferation in liver tumors

Cancer Gene Therapy Antisense HIF-1a prevents resistance to angiostatin X Sun et al 539

5 ** * 4 * 3 2 1 Apoptosis Index 0 ControlDay4 Day10 Day4 Day10 Angiostatin Angiostatin + aHIF Figure 5 Antisense HIF-1a synergizes with angiostatin to enhance tumor cell apoptosis. (a) Illustrated are sections prepared from 0.4 cm tumors in Figure 3a excised 4 days (images 1, 2, 4 and 5) or 10 days (images 3 and 6) after injection of control pcDNA3 plasmid (images 1 and 4), angiostatin plasmid (images 2 and 3) or the combination of angiostatin and antisense HIF-1a plasmids (images 5 and 6). Tumor sections were stained with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) agent to detect apoptotic cells (colored white). Magnification  100. (b) TUNEL-positive cells were counted to record the apoptosis index (AI;  40 magnification field). Five tumors were assessed per group. A significant difference in the AI compared with that of control tumors is donated by an asterisk (Po0.01) or two asterisks (Po0.001). in situ.25 Regarding the effects of angiostatin, we have Acknowledgements previously shown that EL-4 cells transfected with an angiostatin-expressing plasmid and empty vector control This work was supported by grants from the Wellcome had identical growth rates in vitro, indicating that Trust (UK), the World Health Organization, the Royal angiostatin has no detectable effect on the viability and Society of New Zealand, the Cancer Society of New proliferation of EL-4 cells.27 Zealand, the Health Research Council of New Zealand, the Gene therapy as used in this study provides a rapid Lottery Grants Board of New Zealand, the Maurice and means of assessing the utility of therapeutic targets, either Phyllis Paykel Trust and the National Natural Scientific alone or in combination, but is not necessarily the best Foundation of China (30872987 and 30973474). GWK was clinical approach. The appropriate approach for translat- supported by a James Cook Research Fellowship funded by ing the results to the clinic will require additional work, the Royal Society of New Zealand. XS was supported by a but one would imagine that the combination of recombi- Wellcome Trust Research Leave Fellowship. nant angiostatin15 with a specific small molecule inhibi- tor35,36 of HIF-1a would be an excellent first approach. In summary, the combination of anti-angiogenic agents such as angiostatin that directly inhibit endothelial cells, References with agents that antagonize HIF-1, overcomes compen- satory angiogenic responses responsible for drug resis- 1 Folkman J. Tumour angiogenesis: therapeutic implications. tance, and warrants investigation as a therapeutic N Engl J Med 1971; 285: 1182–1186. approach in the . 2 Boehm T, Folkman J, Browder T, O’Reilly MS. Antiangio- genic therapy of experimental cancer does not induce acquired drug resistance. Nature 1997; 390: 404–407. 3 Cao Y. Antiangiogenic cancer therapy. Semin Cancer Biol Conflict of interest 2004; 14: 139–145. 4 Garber K. Angiogenesis inhibitors suffer new setback. Nat The authors declare no conflict of interest. Biotechnol 2002; 20: 1067–1068.

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