SCIENCE CHINA Chemistry • REVIEWS • · SPECIAL TOPIC · The Frontiers of Chemical Biology and Synthesis

January 2011 Vol.54 No.1: 24–30 doi: 10.1007/s11426-010-4187-5

HIF-1 inhibitors as anti-cancer therapy MOORING Suazette Reid & WANG BingHe* Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, USA Received July 24, 2010; accepted September 24, 2010

Hypoxia is a hallmark of solid tumors. Hypoxia increases the progression of malignancy and metastasis by promoting angiogenesis and triggering the over-expression of various protein products critical for tumor growth. The transcription factor HIF-1 mediates cellular response to hypoxia by promoting processes, such as glycolysis and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Therefore, HIF-1 is a viable target for cancer therapy. This review summarizes agents that have been described in the literature as HIF-1 inhibitors. The majority of these compounds are indirect inhibitors of HIF-1. HIF-1, hypoxia, cancer therapy

1

Introduction

Hypoxia is characterized by a reduction in the partial oxygen pressure in cells or tissues and is characteristic of solid tumors. Tumor hypoxia results in reduced effectiveness of radiation and chemotherapy [1, 2]. In solid tumors, when the existing vascular system is unable to supply the growing tumor with adequate amounts of oxygen, it results in hypoxia, low pH and lack of sufficient nutrients [3, 4]. Hypoxia Inducible Factor (HIF) is the primary transcription factor activated by hypoxia and is responsible for orchestrating a number of cellular responses such as angiogenesis and glycolysis that are important to tumor cell survival under hypoxic conditions [5]. HIF-1 is a heterodimeric transcription factor with a helix-loop-helix motif. It is composed of two subunits – oxygen dependent HIF-1 and the constitutively expressed HIF-1. The levels of HIF-1 are determined by intracellular oxygen concentration (Figure 1). Under normoxic condi-

*Corresponding author (email: [email protected])

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tions, HIF-1 is continually degraded by ubiquitination and proteosomal degradation. In this process, HIF-1 is first hydroxylated by the prolyl hydroxylase enzyme (PHD) [6–8]. Then, HIF-1 binds to von Hippel-Lindau protein (VHL) and becomes a part of the E3-ubiqutin lipase complex. Ubiquitination of HIF-1 marks the protein for proteasomal degradation [9, 10]. Oxygen is required for the function of the prolyl hydroxylase enzyme. Therefore, under hypoxic conditions, HIF-1 is stabilized, accumulates and translocates to the nucleus where it interacts with HIF-1 to form the active transcription factor, HIF-1 [9, 10]. HIF-1 then activates a number of cellular genes by binding to hypoxia response element (HRE) [11, 12]. The genes activated include those for proteins that carry out anaerobic glycolysis, for erythropoietin (red blood cell production), and for vascular endothelial cell growth factor (VEGF). VEGF is believed to be a powerful stimulus to new capillary formation, is a major driver of tumor angiogenesis, and is the primary transcription factor activated by hypoxia [13]. The overexpression of HIF-1 is indicated in a number of human cancers [14–16] and is associated with poor response to treatment and patient mortality [14, 17–20]. As a result, HIF-1 chem.scichina.com

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Figure 1

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The HIF-1 pathway.

has been investigated for its potential as an anti-tumor therapeutic target with small molecule inhibitors. Various research groups have identified compounds (Figures 2 and 3) that inhibit the HIF-1 pathway through various mechanisms such as affecting HIF-1 synthesis and degradation, HIF-1HIF-1dimerization, DNA binding, and interactions with other proteins important for transcriptional activities.

Figure 2

Selected HIF-1 inhibitors.

2 Hsp90 inhibitors – Geldanamycin, 17AAG and 17-DMAG Hsp90 plays an important role in the stabilization of HIF-1 under hypoxic conditions [21–23]. Ansamycin derivative geldanamycin (GA) 1 [24] and its analogues 17-AAG 2 and 17-DMAG 3 [25] are Hsp90 inhibitors. These inhibitors act by binding to Hsp90 and interfering with its functions [26]. GA binds to the N-terminal ATP binding domain of Hsp90 and causes the destabilization and degradation of many Hsp90 client protein [27, 28], GA and 17-AAG induce proteasomal degradation of HIF-1 even in renal carcinoma cells that lack functional VHL [22, 29, 30]. 17-AAG and 17-DMAG has undergone phase I and phase II clinical trials [31, 32]. Clinical studies reported that 17-AAG in combination with trastuzmab was active in patients with Her 2-positive metastatic breast cancer who were not successful with trastuzmab therapy alone [33]. Though 17-AAG showed promising results, its poor pharmaceutical properties limited further development. The more water soluble derivative, 17-DMAG was also evaluated in clinical trials for oral and IV use and showed activity in patients with

Figure 3 Selected HIF-1 inhibitors.

acute myelogenous leukemia in combination with chemotherapy [34]. However, further clinical development was not pursued because of toxicity [35].

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3 Topoisomerase inhibitors – Topotecan and NSC 644221 Inhibitors of topoisomerase have also been identified as HIF inhibitors. Topoisomerases are enzymes that unwind and wind DNA, thereby controlling the synthesis of proteins. One such camptothecin analogue, topotecan (TPT) 4 [36–38], was shown to inhibit the accumulation of HIF-1. Exploration of the mechanism of action of topotecan revealed that its inhibitory activity is independent of proteosomal degradation. TPT inhibited HIF-1 even in the presence of protease inhibitors. To address that Top I is required for inhibitory activity of TPT, cells sensitive to camptothecins (CEM) and resistant to camptothecins (CEMC2) were tested for transcription and protein accumulation of HIF-1 in a luciferase assay. Addition of TPT to CEM cells showed a decrease in luciferase levels, whereas in CEM-C2 cells, TPT had no effect on luciferase levels. The investigators concluded that Top I was required for inhibition of HIF-1 activity by TPT. In addition, it was shown that RNA transcription, but not DNA replication was required for inhibition of HIF-1 protein accumulation by TPT. In essence, topotecan forms a stable covalent complex with the DNA/topoisomerase I complex, which leads to breaks in the DNA strand resulting in apoptosis. In 2007, hycamtin® by GlaxoSmithKline was approved by the FDA and TPT became the first Top I inhibitor for oral use. NSC 644221 5 is a Top II inhibitor that was selected from a screen of 140000 compounds from the open synthetic repository of the NCI [39]. NSC 644221 inhibited HIF-1 protein expression in a time and dose dependent manner. HIF-1 protein synthesis was affected by NSC 644211, but HIF-1 degradation was not. The alpha subunit of Top II (Top II) was required for the inhibition of HIF-1 by NSC 644221 but not for the hypoxic induction of HIF-1 protein. In addition, when Top I was silenced, inhibitory activity of NSC 644221 on HIF-1 was not affected. Collectively, these result indicated that the mechanism of inhibition of NSC 644221 involved Top II and also showed that Top I was not involved in the ability for NSC 644221 to inhibit HIF-1 expression. Interestingly, NSC 644221 showed cell line specificity in HIF-1 inhibition. A large number of human cancer cell lines were examined to determine if this phenomenon was a result of drug resistance. However, the results showed that cellular responses to NSC 644221, such as induction of p21 expression and G2-M arrest, were still present in cell lines in which HIF-1 was not inhibited even at concentrations as high as 50 mol/L. These results, rule out that the lack of HIF-1 inhibition was due to cellular resistance to NSC 644221 or more general resistance to top II targeting agents. In general, NSC 644221 had a significant cytostatic but not cytotoxic effect in several cancer cell lines irrespective of

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its ability to inhibit HIF-1. The low cytotoxicity of NSC 644221 makes it more desirable as a HIF-1 inhibitor. NSC 644221 may have a therapeutic effect on hypoxic areas of solid tumors that are usually resistant to chemotherapy.

4 Microtubule inhibitor – 2-Methoxyestradiol Also included in HIF-1 inhibitors is 2-methoxyestradiol (2ME2) 6 that affects microtubule formation [40–42]. 2ME2 inhibits tumor growth and angiogenesis by disrupting tumor microtubules (MTs) in vivo. 2ME2 also down-regulates HIF-1 at the posttranscriptional level and inhibits HIF-1induced transcriptional activation of VEGF expression. Inhibition of HIF-1 occurs downstream of the 2ME2/tubulin interaction, since disruption of interphase MTs is required for HIF-1 down-regulation. The effects of 2ME2 on human prostate cancer cells (PC-3) and human breast cancer cells (MDS-MB-231) were examined. Both PC-3 and MDA-MD-231 cells showed a reduction in nuclear and total HIF-1 protein. The inhibition of HIF-1 was dose dependent and seen under hypoxic and normoxic conditions. Also, VEGF protein levels were reduced by 2ME2 in a dose-dependent manner. To determine if the inhibition of VEGF was related to a direct effect on HIF-1, cells were transfected with luciferase gene and under the control of hypoxia response elements from the VEGF promoter. The result showed that 2ME2 treatment also blocked the hypoxiainduced transcriptional activity of HIF-1. Further examination of the effect of 2ME2 on HIF-1 showed that the decrease in HIF-1 levels in the presence of 2ME2 is due to degradation of HIF-1 protein. Lastly, the role of 2ME2 in microtubule depolymerization was also investigated. 2ME2 treated PC-3 cells were labeled with antibodies against tubulin and HIF-1. Using a laser scanning confocal microscope, dose dependent depolymerization of microtubules was observed in the 2ME2 treated cells compared to untreated control cells. 2ME2 displays a novel mechanism in which microtubules are depolymerized and HIF-1 protein levels as well as HIF transcriptional activity are downregulated independent of oxygen. Also, at concentrations of 2ME2 that inhibit tumor growth and vascularization, tumor microtubules are depolymerized. 2ME2 has been established as a small molecule inhibitor of HIF-1.

5 Thioredoxin inhibitors – PX-12 and pleurotin Thioredoxin inhibitors PX-12 7 and pleurotin 8 also have an effect on HIF-1 and Vascular Endothelial Growth Factor (VEGF). Thioredoxin is a small redox protein that is often overexpressed in human tumors [43]. Welsh and co-workers investigated the effect of PX-12 and pleurotin on HIF-1 [44]. These inhibitors decreased HIF-1 protein, HIF-1

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transactivation and expression of HIF-1, VEGF and inducible nitric oxide synthase (iNOS) in vitro. PX-12 and pleurotin inhibited the growth of MCF-7 cells (IC50 of 1.9 ± 0.8 and 0.9 ± 1.0 M respectively) and HT-29 cells (IC50 of 2.9 ± 2.0 and 0.9 ± 1.2 M respectively). Dose dependent decreases in HIF-1 protein in MCF-7 cells under hypoxic conditions when exposed to PX-12 and pleurotin were also observed. These inhibitors also decreased the expression of HIF-1 and VEGF proteins in vivo. The mechanism of the decrease in HIF-1 protein by these Trx-1 inhibitors is unknown. However, it is clear that the mechanism does not involve pVHL since PX-12 and pleurotin decreased HIF-1 in RCC4 cells that lack pVHL. The authors suggested that either an Hsp90 or PI3K/AKT pathway may be involved, but they were not investigated.

6 HIF-1 DNA interaction inhibitors – Echinomycin, DJ-12 Some inhibitors of HIF-1 do not affect the mRNA levels or protein levels but inhibit the binding of HIF-1 to DNA and prevent the activation of transcription. One such inhibitor – echinomycin 9 [45] has been shown to affect HIF-1 DNA binding. Echinomycin inhibits HIF-1 and HIF-1 DNA binding activity in a dose dependent manner and only inhibited DNA binding in HIF-1 dependent fashion. Echinomycin was previously determined to be a sequence-specific DNA binding agent [46]. That is, it binds to the sequence 5′-CGTG-3′ of HRE consensus sequence 5′-R(A/G)CGTG-3′. It was also shown that echinomycin inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Another inhibitor of HIF-1 DNA binding is DJ12 10, which was identified as a HIF-1 inhibitor by screening 15000 compounds [47]. The compounds were screened using Chinese hamster ovary cells, which were designed to stably express luciferase reporter construct under the control of a hypoxia response element. DJ12 inhibited VEGF in breast cancer cell lines MDA-468 and ZR-75, melanoma cell line MDA-435, and pVHL mutant renal cancer cell lines RCC4 and 786-0. DJ12 down-regulates mRNA of downstream targets of HIF-1, and significantly inhibited HIF-1 transactivation activity by blocking HIF-1 hypoxia response element-DNA binding. DJ12 may work by inhibition of HIF-1 transactivation through the blocking of HIF-1 HRE DNA binding. Direct addition of DJ12 to nuclear extracts containing constitutive expression of HIF-1 had no effect in blocking HIF-1 HRE-DNA binding, suggesting that DJ12 does not directly interfere with the formation of protein-DNA complex but may inhibit the formation of HIF-1, HIF-1, and CBP/p300 transcription complex or folding of HIF-1. Hence, DJ12 has been introduced as a novel HIF inhibitor that is effective against the HIF path-

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way for breast, renal and melanoma cancer cell lines. However, as with many HIF inhibitors, long term (16 hours) toxicity assay studies revealed that DJ12 was toxic to MDA-468 cells at IC50 of 191 mol/L in normoxic cells and greater than 250 mol/L in hypoxic cells. Toxicity was attributed to multiple HIF targets by these compounds. We have recently discovered a new class of fused benzopyran/furan compounds with HIF-1 inhibition activities [48–50]. These compounds do not change HIF-1 level, but show potent activities in HRE-based whole cell assays (luciferase).

7 HIF-1 transactivation inhibitor PX-478 11 is a HIF-1 transactivation inhibitor that reduces HIF-1 protein levels. PX-478 is an inhibitor of constitutive and hypoxia-induced HIF-1 levels and thus HIF-1 activity. Welsh et al initially reported that PX-478 suppresses hypoxia induction of HIF-1 in various cancer cell lines and suppresses constitutive HIF-1 in cells that have lost pVHL [51, 52]. PX-478 decreased hypoxia-induced HIF-1 protein levels in a number of cancer cell lines. The levels of constitutively elevated HIF-1 in Panc-1 (pancreatic cancer) and PC-3 (prostate cancer) and RCC4 (renal cancer) cells were also decreased. The elevated HIF in Panc-1 and PC-3 cells are due to increase in phosphatidylinositol 3-kinase/Akt signaling and HIF-1 gene amplification. In RCC4 cells, elevated HIF is a result of loss of pVHL. Therefore, the decrease in HIF-1 by PX-478 does not require oxygen or pVHL. The decrease in HIF-1 protein is accompanied by a decrease in HIF-1 transactivating activity and decrease in VEGF expression. More recent studies have shown that the inhibitory effect of PX-478 on HIF-1 level is primarily due to its inhibition of translation [53]. However other mechanisms such as inhibition of HIF-1 deubiquitination and reduction in HIF-1 mRNA levels also contribute to the activity of PX-478. Initially published results of a now completed Phase I clinical trial by the NCI (NCT00522652) showed that PX-478 enhanced the radiosensitivity of prostate carcinoma cells irradiated under normoxia and reduced cell survival [54].

8 HIF-1 mRNA expression inhibitor – EZN-2698 Among the more specific HIF-1 inhibitors are inhibitors of HIF-1 mRNA. EZN-2698 is a highly specific antagonist of HIF-1 mRNA, selectively reduces target mRNA and thus HIF-1 protein levels, and causes a reduction in HIF-1 regulated genes in vitro and in vivo [55]. EZN-2698 is composed of 16 nucleotide residues. The residues at posi-

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tions 1 to 3 and 13 to 15 are locked nucleic acid (LNA). LNA oligonucleotides are third-generation antisense technology that has a high target mRNA binding affinity, as well as tissue stability [56, 57]. EZN-2968 is completely complementary to residues in the mRNA coding sequence of HIF-1 derived from human or mouse tissue. In vitro, treatment of EZN-2968 results in the inhibition of tumor cell growth and down-regulation of HIF-1a regulated genes and abrogates the formation of tubes in human endothelial cells. In vivo, administration of EZN-2968 decreased endogenous HIF-1 mRNA expression in the liver of mice and also showed antitumor activity in xenograft models of human prostate cancer (DU145). Phase I clinical trials in patients with advanced solid tumors indicate that EZN-2968 can be given safely. Further clinical and animal studies are ongoing. Other mechanisms that decrease HIF-1 protein levels, include inhibitor of cyclin dependent kinase such as flavopiridol 12 [58], which has an effect on VEGF. As previously discussed, VEGF secretion is important for angiogenesis and vascular proliferation in cancer cells and VEGF is under the control of HIF-1 gene expression. Newcomb and co-workers have investigated the effect of flavopiridol on U87MG and T98G gliomas cells lines [58]. Flavopiridol has shown anti-angiogenic properties by inhibition of VEGF, decreased tumor cell migration, decreased hypoxia HIF-1 expression and reduced vascularity in Gl261 glioma cells in animals treated with flavopiridol.

9

p300 Inhibitor – Chetomin

Inhibition of p300 can indirectly lead to HIF-1 inhibition. Chetomin 13 is a member of the epidithiodiketopiperazine (ETP) family, specifically from the fungus Chaetominum species and was initially shown to have antimicrobial activity [59, 60]. CBP and p300 are required for the coactivation of HIF [61–64]. Chetomin has been shown to be a disrupter of HIF binding to p300 and works by disrupting the structure of the cysteine-histidine-rich domain 1 (CH1) domain of p300. Consequently, chetomin inhibits with the interactions between P300 and HIF and therefore HIF-1 transcription, leading to tumor growth inhibition [65]. More recently it was shown that chetomin reacts with p300, causing zinc ion ejection [66]. It was proposed that compounds such as chetomin cause zinc ion ejection via a mechanism related to other known zinc binding disrupting compounds [67] in which a zinc-binding cysteinyl thiol reacts with the torsionally strained disulfide of the ETP core to generate a transient protein-ETP disulfide. This disulfide can then rearrange to form an intramolecular protein disulfide with consequent reduction in zinc ion affinity. The ejected zinc ion (or zinc ETP complex) can then complex with a second ETP core to form a stable complex. Other compounds that affect HIF-1 indirectly include ty-

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rosine inhibitors such as transtuzumab/docatexel combination [68] and histone deacetylase (HDAC) inhibitor FK228 14 [69], which inhibits HIF-1 activity under hypoxic conditions and angiogenesis [70]. In addition, bortezomib 15 is a HIF inhibitor, which functions by interfering with the carboxyl-terminal transactivation domain of HIF-1 [68]. The antifungal drug amphoteric B also inhibits HIF-1 by inducing the interaction of HIF-1 with Factor Inhibiting HIF-1 (FIH-1), leading to decreased recruitment of p300 [71].

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Conclusions

Hypoxic tumors are resistant to radiotherapy and chemotherapy, which leads to increased aggressive and metastatic properties. The response of cells to hypoxia is primarily mediated by HIF-1. Therefore, targeting the HIF pathway can have therapeutic effect on hypoxic and angiogenic tumors. HIF-1 inhibitors can be especially effective in combination with existing therapies. The majority of the HIF-1 inhibitors mentioned in this review is indirect inhibitors of HIF-1 and does not specifically inhibit HIF-1. Obtaining HIF-1 inhibitors that are more specific is one of the major challenges of this field. Thus far, cell-based and in vitro high-throughput screening has been effective in indentifying new compounds that may inhibit HIF-1. Cell-based screenings that detect inhibition via HRE-induced expression of a reporter have certain disadvantages in that inhibitors of pathways in the upstream of HIF-1 may be detected. Therefore, the development of more specific HIF-1 agents will in part depend on more sensitive screening methods. Disruption of the binding of HIF-1 to HREs is especially promising for the development of more specific inhibitors. The Melillo group has developed a high-throughput screening that targets the Per-Arnt-Sim (PAS) domains of HIF-1 and HIF-1 that are important in heterodimer formation [72]. This screen shows potential for discovery of more specific inhibitors of HIF-1. In addition, the increasing availability of the structural complexes of HIF-1 can facilitate rational drug design approaches that can optimize existing compounds, as well as predict new inhibitors. Research in the authors’ lab has been supported by the National Institutes of Health (CA122536 and a minority supplement) and the GSU MBD program through a fellowship to SRM.

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