PLEKHA7 signaling is necessary for the growth of mutant KRAS driven colorectal cancer.

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a "sentry" role preventing PI(3,4)P2 and PI(3,4,5)P3 binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.

A new HIF-1α/RANTES-driven pathway to hepatocellular carcinoma mediated by germline haploinsufficiency of SART1/HAF in mice.

UNLABELLED: The hypoxia-inducible factor (HIF), HIF-1, is a central regulator of the response to low oxygen or inflammatory stress and plays an essential role in survival and function of immune cells. However, the mechanisms regulating nonhypoxic induction of HIF-1 remain unclear. Here, we assess the impact of germline heterozygosity of a novel, oxygen-independent ubiquitin ligase for HIF-1α: hypoxia-associated factor (HAF; encoded by SART1). SART1(-/-) mice were embryonic lethal, whereas male SART1(+/-) mice spontaneously recapitulated key features of nonalcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC), including steatosis, fibrosis, and inflammatory cytokine production. Male, but not female, SART1(+/-) mice showed significant up-regulation of HIF-1α in circulating and liver-infiltrating immune cells, but not in hepatocytes, before development of malignancy. Additionally, Kupffer cells derived from male, but not female, SART1(+/-) mice produced increased levels of the HIF-1-dependent chemokine, regulated on activation, normal T-cell expressed and secreted (RANTES), compared to wild type. This was associated with increased liver-neutrophilic infiltration, whereas infiltration of lymphocytes and macrophages were not significantly different. Neutralization of circulating RANTES decreased liver neutrophilic infiltration and attenuated HCC tumor initiation/growth in SART1(+/-) mice. CONCLUSION: This work establishes a new tumor-suppressor role for HAF in immune cell function by preventing inappropriate HIF-1 activation in male mice and identifies RANTES as a novel therapeutic target for NASH and NASH-driven HCC.

HNF4α is a therapeutic target that links AMPK to WNT signalling in early-stage gastric cancer.

BACKGROUND: Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. OBJECTIVE: To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. METHODS: We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. RESULTS: Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. CONCLUSIONS: Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate 'metabolic switch' characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.

Passing the baton: the HIF switch.

Hypoxia is an inadequate oxygen supply to tissues and cells, which can restrict their function. The hypoxic response is primarily mediated by the hypoxia-inducible transcription factors, HIF-1 and HIF-2, which have both overlapping and unique target genes. HIF target gene activation is highly context specific and is not a reliable indicator of which HIF-α isoform is active. For example, in some cell lines, the individual HIFs have specific temporal and functional roles: HIF-1 drives the initial response to hypoxia (<24h) and HIF-2 drives the chronic response (>24h). Here, we review the significance of the HIF switch and the relation between HIF-1 and HIF-2 under both physiological and pathophysiological conditions.

Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems.

BACKGROUND: "Biomarker-driven targeted therapy," the practice of tailoring patients' treatment to the expression/activity levels of disease-specific genes/proteins, remains challenging. For example, while the anti-ERBB2 monoclonal antibody, trastuzumab, was first developed using well-characterized, diverse in vitro breast cancer models (and is now a standard adjuvant therapy for ERBB2-positive breast cancer patients), trastuzumab approval for ERBB2-positive gastric cancer was largely based on preclinical studies of a single cell line, NCI-N87. Ensuing clinical trials revealed only modest patient efficacy, and many ERBB2-positive gastric cancer (GC) patients failed to respond at all (i.e., were inherently recalcitrant), or succumbed to acquired resistance. METHOD: To assess mechanisms underlying GC insensitivity to ERBB2 therapies, we established a diverse panel of GC cells, differing in ERBB2 expression levels, for comprehensive in vitro and in vivo characterization. For higher throughput assays of ERBB2 DNA and protein levels, we compared the concordance of various laboratory quantification methods, including those of in vitro and in vivo genetic anomalies (FISH and SISH) and xenograft protein expression (Western blot vs. IHC), of both cell and xenograft (tissue-sectioned) microarrays. RESULTS: The biomarker assessment methods strongly agreed, as did correlation between RNA and protein expression. However, although ERBB2 genomic anomalies showed good in vitro vs. in vivo correlation, we observed striking differences in protein expression between cultured cells and mouse xenografts (even within the same GC cell type). Via our unique pathway analysis, we delineated a signaling network, in addition to specific pathways/biological processes, emanating from the ERBB2 signaling cascade, as a potential useful target of clinical treatment. Integrated analysis of public data from gastric tumors revealed frequent (10 - 20 %) amplification of the genes NFKBIE, PTK2, and PIK3CA, each of which resides in an ERBB2-derived subpathway network. CONCLUSION: Our comprehensive bioinformatics analyses of highly heterogeneous cancer cells, combined with tumor "omics" profiles, can optimally characterize the expression patterns and activity of specific tumor biomarkers. Subsequent in vitro and in vivo validation, of specific disease biomarkers (using multiple methodologies), can improve prediction of patient stratification according to drug response or nonresponse.

FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

Improved synthesis of 17ß-hydroxy-16α-iodo-wortmannin, 17ß-hydroxy-16α-iodoPX866, and the [(131)I] analogue as useful PET tracers for PI3-kinase.

An improved method for the synthesis of 17ß-hydroxy-16α-iodo-wortmannin along with the first synthesis of 17ß-hydroxy-16α-iodoPX866 and [(131)I] radiolabeled 17ß-hydroxy-16α-[(131)I]iodo-wortmannin, as potential PET tracers for PI3K was also described. The differences between wortmannin and its iodo analogue were compared by covalently docking each structure to L833 in PI3K.

HIF-1 regulation: not so easy come, easy go.

The hypoxia-inducible factor-1 (HIF-1) is the master regulator of the cellular response to hypoxia and its expression levels are tightly controlled through synthesis and degradation. It is widely accepted that HIF-1alpha protein accumulation during hypoxia results from inhibition of its oxygen-dependent degradation by the von Hippel Lindau protein (pVHL) pathway. However, recent data describe new pVHL- or oxygen-independent mechanisms for HIF-1alpha degradation. Furthermore, the hypoxia-induced increase in HIF-1alpha levels is facilitated by the continued translation of HIF-1alpha during hypoxia despite the global inhibition of protein translation. Recent work has contributed to an increased understanding of the mechanisms that control the translation and degradation of HIF-1alpha under both normoxic and hypoxic conditions.

Pleckstrin Homology [PH] domain, structure, mechanism, and contribution to human disease.

The pleckstrin homology [PH] domain is a structural fold found in more than 250 proteins making it the 11th most common domain in the human proteome. 25% of family members have more than one PH domain and some PH domains are split by one, or several other, protein domains although still folding to give functioning PH domains. We review mechanisms of PH domain activity, the role PH domain mutation plays in human disease including cancer, hyperproliferation, neurodegeneration, inflammation, and infection, and discuss pharmacotherapeutic approaches to regulate PH domain activity for the treatment of human disease. Almost half PH domain family members bind phosphatidylinositols [PIs] that attach the host protein to cell membranes where they interact with other membrane proteins to give signaling complexes or cytoskeleton scaffold platforms. A PH domain in its native state may fold over other protein domains thereby preventing substrate access to a catalytic site or binding with other proteins. The resulting autoinhibition can be released by PI binding to the PH domain, or by protein phosphorylation thus providing fine tuning of the cellular control of PH domain protein activity. For many years the PH domain was thought to be undruggable until high-resolution structures of human PH domains allowed structure-based design of novel inhibitors that selectively bind the PH domain. Allosteric inhibitors of the Akt1 PH domain have already been tested in cancer patients and for proteus syndrome, with several other PH domain inhibitors in preclinical development for treatment of other human diseases.

Combined action of the dinuclear platinum compound BBR3610 with the PI3-K inhibitor PX-866 in glioblastoma.

Polynuclear platinum compounds are more effective at killing glioblastoma cells than cisplatin, work by a different mechanism, and typically do not induce high levels of apoptosis at early time points after exposure. Here, we tested the hypothesis that combining BBR3610, the most potent polynuclear platinum, with a phosphoinositide-3-kinase (PI3K) inhibitor would promote apoptosis and enhance the impact on glioblastoma cells. The PI3K pathway is commonly activated in glioblastoma and promotes tumor cell survival, suggesting that its inhibition would make cells more sensitive to cytotoxic agents. We chose PX-866 as a PI3K inhibitor as it is a clinically promising agent being evaluated for brain tumor therapy. Combining BBR3610 and PX-866 resulted in synergistic killing of cultured glioma cells and an extension of survival in an orthotopic xenograft animal model. Both agents alone induced autophagy, and this appeared to be saturated, because when they were combined no additional autophagy was observed. However, the combination of PX-866 and BBR3610 did induce statistically significant increases in the level of apoptosis, associated with a reduction in pAkt and pBad, as well as inhibition of transwell migration. We conclude that combining polynuclear platinums with PI3K inhibitors has translational potential and alters the cellular response to include early apoptosis.

Development of sulfonamide AKT PH domain inhibitors.

Disruption of the phosphatidylinositol 3-kinase/AKT signaling pathway can lead to apoptosis in cancer cells. Previously we identified a lead sulfonamide that selectively bound to the pleckstrin homology (PH) domain of AKT and induced apoptosis when present at low micromolar concentrations. To examine the effects of structural modification, a set of sulfonamides related to the lead compound was designed, synthesized, and tested for binding to the expressed PH domain of AKT using a surface plasmon resonance-based competitive binding assay. Cellular activity was determined by means of an assay for pAKT production and a cell killing assay using BxPC-3 cells. The most active compounds in the set are lipophilic and possess an aliphatic chain of the proper length. Results were interpreted with the aid of computational modeling. This paper represents the first structure-activity relationship (SAR) study of a large family of AKT PH domain inhibitors. Information obtained will be used in the design of the next generation of inhibitors of AKT PH domain function.

Structural basis for the association of PLEKHA7 with membrane-embedded phosphatidylinositol lipids.

PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.

IL-24 is expressed during wound repair and inhibits TGFalpha-induced migration and proliferation of keratinocytes.

Interleukin (IL)-24 is the protein product of melanoma differentiation-associated gene 7 (MDA-7). Originally identified as a tumor suppressor molecule, MDA-7 was renamed IL-24 and classified as a cytokine because of its chromosomal location in the IL-10 locus, its mRNA expression in leukocytes, and its secretory sequence elements. We previously reported that IL-24 is expressed by cytokine-activated monocytes and T lymphocytes. Here, we show that IL-24 is expressed in keratinocytes during wound repair. Paraffin-embedded tissues prepared from human skin sampled at days 2, 6, and 10 after wounding were examined by immunohistochemistry for the expression of IL-24. Protein expression was detected in the keratinocyte population with maximum expression at days 2 and 6, and no expression by day 10 (four of four subjects). In vitro studies showed that cytokines involved in wound repair, most notably transforming growth factor alpha (TGFalpha), TGFbeta, IFNgamma, and IFNbeta, upregulated IL-24 protein expression in normal human epidermal keratinocytes (NHEKs). Examination of the function of IL-24 in both in vitro wound repair and migration assays demonstrated that IL-24 inhibits TGFalpha-induced proliferation and migration of NHEKs. These data support the hypothesis that IL-24 functions during an inflammatory response in the skin by inhibiting the proliferation and migration of keratinocytes.

HIF-1alpha and cancer therapy.

Most solid tumors develop regions of hypoxia as they grow and outstrip their blood supply. In order to survive in the stressful hypoxic environment, tumor cells have developed a coordinated set of responses orchestrating their adaptation to hypoxia. The outcomes of the cellular responses to hypoxia are aggressive disease, resistance to therapy, and decreased patient survival. A critical mediator of the hypoxic response is the transcription factor hypoxia-inducible factor 1 (HIF-1) that upregulates expression of proteins that promote angiogenesis, anaerobic metabolism, and many other survival pathways. Regulation of HIF-1alpha, a component of the HIF-1 heterodimer, occurs at multiple levels including translation, degradation, and transcriptional activation, and serves as a testimony to the central role of HIF-1. Studies demonstrating the importance of HIF-1alpha expression for tumor survival have made HIF-1alpha an attractive target for cancer therapy. The growing l.ist of pharmacological inhibitors of HIF-1 and their varied targets mirrors the complex molecular mechanisms controlling HIF-1. In this chapter, we summarize recent findings regarding the regulation of HIF-1alpha and the progress made in identifying new therapeutic agents that inhibit HIF-1alpha.

Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is currently one of the most exciting drug targets in oncology. However, only a short time ago, the paradigm existed that drugs targeted to the four PI3K class I isoforms would be too toxic for use in cancer therapy due to effects on physiologic signaling. Since that time, studies have delineated the roles of these four isoforms in nonpathologic signaling as well as their roles in cancer. An extensive effort has gone into developing agents that inhibit one or more PI3K isoforms, as well as closely related proteins implicated in cancer. These agents have proved to be tolerable and therapeutically beneficial in animal studies, and a number are in clinical testing. The agents, their properties, and their molecular targets are discussed in this review.

Peroxisome proliferator-activated receptor gamma agonist pioglitazone prevents the hyperglycemia caused by phosphatidylinositol 3-kinase pathway inhibition by PX-866 without affecting antitumor activity.

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascade is an important component of the insulin signaling in normal tissues leading to glucose uptake and homeostasis and for cell survival signaling in cancer cells. Hyperglycemia is an on-target side effect of many inhibitors of PI3K/Akt signaling including the specific PI3K inhibitor PX-866. The peroxisome proliferator-activated receptor gamma agonist pioglitazone, used to treat type 2 diabetes, prevents a decrease in glucose tolerance caused by acute administration of PX-866. Our studies have shown that pioglitazone does not inhibit the antitumor activity of PX-866 in A-549 non-small cell lung cancer and HT-29 colon cancer xenografts. In vitro studies also showed that pioglitazone increases 2-[1-(14)C]deoxy-D-glucose uptake in L-6 muscle cells and prevents inhibition of 2-deoxyglucose uptake by PX-866. Neither pioglitazone nor PX-866 had an effect on 2-deoxyglucose uptake in A-549 lung cancer cells. In vivo imaging studies using [18F]2-deoxyglucose (FDG) positron emission tomography showed that pioglitazone increases FDG accumulation by normal tissue but does not significantly alter FDG uptake by A-549 xenografts. Thus, peroxisome proliferator-activated receptor gamma agonists may be useful in overcoming the increase in blood glucose caused by inhibitors of PI3K signaling by preventing the inhibition of normal tissue insulin-mediated glucose uptake without affecting antitumor activity.

The selective hypoxia inducible factor-1 inhibitor PX-478 provides in vivo radiosensitization through tumor stromal effects.

Hypoxia inducible factor-1 (HIF-1) promotes tumor cell adaptation to microenvironmental stress. HIF-1 is up-regulated in irradiated tumors and serves as a promising target for radiosensitization. We initially confirmed that the orally bioavailable HIF-1 inhibitor PX-478 reduces HIF-1 protein levels and signaling in vitro in a dose-dependent manner and provides direct radiosensitization of hypoxic cancer cells in clonogenic survival assays using C6 glioma, HN5 and UMSCCa10 squamous cells, and Panc-1 pancreatic adenocarcinoma cell lines. However, PX-478 yields striking in vivo tumor sensitization to single-dose irradiation, which cannot be explained by incremental improvement in direct tumor cell killing. We show that PX-478 prevents postradiation HIF-1 signaling and abrogates downstream stromal adaptation in C6 and HN5 reporter xenografts as measured by serial ultrasound, vascular magnetic resonance imaging, and hypoxia response element-specific micro-positron emission tomography imaging. The primacy of indirect PX-478 in vivo effects was corroborated by our findings that (a) either concurrent or early postradiation sequencing of PX-478 provides roughly equivalent sensitization and (b) constitutive vascular endothelial growth factor expression maintains refractory tumor vessel function and progression following combined radiation and PX-478. These results confirm that disruption of postradiation adaptive HIF-1 signaling by PX-478 imparts increased therapeutic efficacy through blockade of HIF-1-dependent reconstitution of tumor stromal function. Successful translation of targeted HIF-1 radiosensitization to the clinical setting will require specific consideration of tumor microenvironmental effects and mechanisms.

A Robust and Cost-Effective Luminescent-Based High-Throughput Assay for Fructose-1,6-Bisphosphate Aldolase A.

Hypoxic solid tumors induce the stabilization of hypoxia-inducible factor 1 alpha (HIF1α), which stimulates the expression of many glycolytic enzymes and hypoxia-responsive genes. A high rate of glycolysis supports the energetic and material needs for tumors to grow. Fructose-1,6-bisphosphate aldolase A (ALDOA) is an enzyme in the glycolytic pathway that promotes the expression of HIF1α. Therefore, inhibition of ALDOA activity represents a potential therapeutic approach for a range of cancers by blocking two critical cancer survival mechanisms. Here, we present a luminescence-based strategy to determine ALDOA activity. The assay platform was developed by integrating a previously established ALDOA activity assay with a commercial NAD/NADH detection kit, resulting in a significant (>12-fold) improvement in signal/background (S/B) compared with previous assay platforms. A screening campaign using a mixture-based compound library exhibited excellent statistical parameters of Z' (>0.8) and S/B (~20), confirming its robustness and readiness for high-throughput screening (HTS) application. This assay platform provides a cost-effective method for identifying ALDOA inhibitors using a large-scale HTS campaign.

Computational modeling of novel inhibitors targeting the Akt pleckstrin homology domain.

Computational modeling continues to play an important role in novel therapeutics discovery and development. In this study, we have investigated the use of in silico approaches to develop inhibitors of the pleckstrin homology (PH) domain of AKT (protein kinase B). Various docking/scoring schemes have been evaluated, and the best combination was selected to study the system. Using this strategy, two hits were identified and their binding behaviors were investigated. Robust and predictive QSAR models were built using the k nearest neighbor (kNN) method to study their cellular permeability. Based on our in silico results, long flexible aliphatic tails were proposed to improve the Caco-2 penetration without affecting the binding mode. The modifications enhanced the AKT inhibitory activity of the compounds in cell-based assays, and increased their activity as in vivo antitumor testing.

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1alpha.

We have reported previously that PX-478 (S-2-amino-3-[4'-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1alpha (HIF-1alpha) within the tumor. We now report that PX-478 inhibits HIF-1alpha protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1alpha inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1alpha mRNA and inhibits translation as determined by 35S labeling experiments and reporter assays using the 5' untranslated region of HIF-1alpha. Moreover, to a lesser extent, PX-478 also inhibits HIF-1alpha deubiquitination resulting in increased levels of polyubiquitinated HIF-1alpha. The inhibitory effect of PX-478 on HIF-1alpha levels is primarily due to its inhibition of translation because HIF-1alpha translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1alpha at multiple levels that together or individually may contribute to its antitumor activity against HIF-1alpha-expressing tumors.