Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer

    Review: Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer Caroline Wigerup, Sven P˚ahlman, Daniel Bexell PII: DOI: Reference:

S0163-7258(16)30055-9 doi: 10.1016/j.pharmthera.2016.04.009 JPT 6901

To appear in:

Pharmacology and Therapeutics

Please cite this article as: Wigerup, C., P˚ ahlman, S. & Bexell, D., Review: Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer, Pharmacology and Therapeutics (2016), doi: 10.1016/j.pharmthera.2016.04.009

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ACCEPTED MANUSCRIPT P&T 22787

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Review: Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer

Translational Cancer Research, Medicon Village 404:C3, Lund University, Lund, Sweden

cancer,

hypoxia,

hypoxia-inducible

(HIF),

neuroblastoma,

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pheochromocytoma, paraganglioma

factor

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Keywords:

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Caroline Wigerup1, Sven Påhlman1 and Daniel Bexell1

Corresponding author: Sven Påhlman Translational Cancer Research Medicon Village 404:C3 Lund University SE-223 81 Lund, Sweden E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract Insufficient tissue oxygenation, or hypoxia, contributes to tumor aggressiveness and has a

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profound impact on clinical outcomes in cancer patients. At decreased oxygen tensions, hypoxia-inducible factors (HIFs) 1 and 2 are stabilized and mediate a hypoxic response,

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primarily by acting as transcription factors. HIFs exert differential effects on tumor growth

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and affect important cancer hallmarks including cell proliferation, apoptosis, differentiation,

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vascularization/angiogenesis, genetic instability, tumor metabolism, tumor immune responses, and invasion and metastasis. As a consequence, HIFs mediate resistance to chemo- and

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radiotherapy and are associated with poor prognosis in cancer patients. Intriguingly, perivascular tumor cells can also express HIF-2α, thereby forming a “pseudohypoxic”

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phenotype that further contributes to tumor aggressiveness. Therefore, therapeutic targeting of

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HIFs in cancer has the potential to improve treatment efficacy. Different strategies to target hypoxic cancer cells and/or HIFs include hypoxia-activated prodrugs and inhibition of HIF

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dimerization, mRNA or protein expression, DNA binding capacity, and transcriptional activity. Here we review the functions of HIFs in the progression and treatment of malignant solid tumors. We also highlight how HIFs may be targeted to improve the management of patients with therapy-resistant and metastatic cancer.

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ACCEPTED MANUSCRIPT Abbreviations 2-methoxyestradiol (2ME2); amphotericin B (AmB); cancer stem cell (CSC); carbonic

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anhydrase IX (CAIX); clear cell renal cell carcinoma (ccRCC); dichloroacetic acid (DCA); erythropoietin (EPO); geldanamycin (GA); genetically engineered mouse models (GEMMs);

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factor inhibiting HIF-1 (FIH-1); heat shock protein (Hsp); histone deacetylase (HDAC);

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hypoxia-inducible factor (HIF); hypoxia responsive element (HRE); ionizing radiation, (IR); iron-responsive element (IRE); patient-derived xenografts (PDXs); polyethylene glycol prolyl-4-hydroxylases

(PHDs);

reactive

oxygen

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(PEG);

species

(ROS);

succinate

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dehydrogenase (SDH); sympathetic nervous system (SNS); transactivation domains (TADs); vascular endothelial growth factor (VEGF); von Hippel Lindau (VHL) Contents

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1. Introduction 2. HIF regulation and function 3. Hypoxia and HIFs in cancer 3.1 Measuring tumor hypoxia 3.2 Patient prognosis 3.3 Tumor cell viability 3.4 Tumor angiogenesis 3.5 Cancer stem cells 3.6 Metabolic reprogramming 3.7 Tumor immune response 3.8 Genomic instability 3.9 Tumor cell invasion and metastasis 3.10 Treatment resistance 4. Targeting hypoxia and HIFs in cancer 4.1 Hypoxia-activated prodrugs 4.2 Drugs targeting hypoxic signaling 4.2.1 Inhibitors of HIF mRNA or protein expression 4.2.2 Inhibitors of HIF dimerization 4.2.3 Inhibitors of HIF-DNA binding 4.2.4 Inhibitors of HIF transcriptional activity 5. Neural crest-derived tumors as model systems for targeting HIF-2 and the pseudohypoxic phenotype 6. Summary and future perspectives Acknowledgments References 3

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1. Introduction An adequate oxygen supply to cells and tissues is crucial for the function and survival of

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aerobic organisms. Hypoxia occurs when oxygen supply fails to meet demand. The definition

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of hypoxia is somewhat ambiguous since normal oxygen pressure (PO2) varies between different tissues. For instance, the normal PO2 in arterial blood is approximately 100 mmHg (~

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13%) but around 40 mmHg (~5%) in the liver (Koh & Powis, 2012). No clear-cut threshold separates normoxia from hypoxia, so oxygen levels must be regarded in context. Likewise,

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definitions of mild, moderate, and severe hypoxia are not clearly defined. Nevertheless, 8-10 mmHg (~1%) is estimated to represent a critical PO2 since lower PO2 values are associated

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with adverse effects caused by reduced O2 consumption (Hockel & Vaupel, 2001). For in

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vitro studies, 1% oxygen is commonly used to mimic a hypoxic environment. Solid malignant

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tumors commonly contain hypoxic areas, and direct measurement of tissue oxygenation using O2-sensitive microsensors has shown that the tumor microenvironment is characterized by much lower PO2 values than their normal counterparts (Vaupel, Mayer, & Hockel, 2004). A major cause of tumor hypoxia is the formation of non-functional blood vessels in neoplastic tissue, particularly in rapidly growing tumors. Hypoxia is not merely epiphenomenal but is of clinical importance since tumor hypoxia is associated with aggressive tumor phenotypes, treatment resistance, and poor clinical prognosis (reviewed in Bertout et al. (Bertout, Patel, & Simon, 2008)). The cellular response to hypoxia is mainly mediated by the hypoxia-inducible factor (HIF) family of transcription factors, which regulate the expression of multiple genes involved in processes that drive the adaptation and progression of cancer cells. Therefore, tumor hypoxia and HIFs affect most of the cancer “hallmarks” including cell proliferation, apoptosis, metabolism, immune responses, genomic instability, vascularization, and invasion

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ACCEPTED MANUSCRIPT and metastasis. HIFs also appear to contribute to chemo- and radiotherapy resistance via multiple mechanisms. In clinical tumor samples, HIF expression is associated with poor

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prognoses and relapse on treatment. HIFs appear to be crucial molecular targets that can be exploited to improve on the current treatment of metastatic and treatment-resistant cancer.

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However, HIF biology is complex. Alternative HIF isoforms differentially regulate tumor

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growth; consequently, optimal HIF targeting depends on the specific tumor type and HIF-1

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and HIF-2 selectivity. Strategies to inhibit HIFs in cancer include drugs with indirect effects on HIF signaling, but direct HIF inhibitors also exist. In this review we describe HIF

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regulation and function, their role as master regulators of solid tumor hallmarks (Figure 1), the pseudohypoxic phenotype, and the different therapeutic strategies that have been

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developed to inhibit HIF activity (Figure 2).

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2. HIF regulation and function

HIFs are proteins that sense and respond to oxygen deficiency by acting as transcription factors (Figure 2). Each HIF transcription factor is composed of two subunits: the α-subunit and the β-subunit, both of which belong to the basic helix-loop-helix (HLH)-PER-ARNT-SIM (bHLH-PAS) protein family (Bersten, Sullivan, Peet, & Whitelaw, 2013). α-subunit stability is oxygen sensitive, while the β-subunit (HIF-1β or ARNT) is ubiquitously expressed. In the presence of oxygen, conserved proline residues on the α-subunit are hydroxylated, promoting its degradation (Ivan, et al., 2001; Jaakkola, et al., 2001). This hydroxylation is mediated by prolyl-4-hydroxylases (PHDs), a set of oxygen-, iron-, and ascorbate-dependent enzymes belonging to the 2-oxoglutarate-dependent oxygenase superfamily (Schofield & Ratcliffe, 2004). α-subunit hydroxylation serves as a recognition signal for the von Hippel-Lindau (pVHL) tumor suppressor protein, which subsequently targets the α-subunit for degradation

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ACCEPTED MANUSCRIPT by adding ubiquitin. However, under hypoxic conditions, PHDs cannot hydroxylate the αsubunit. This results in HIF-α protein stabilization, nuclear translocation, and dimerization to

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HIF-1β to form the HIF transcription factor. In the nucleus, HIFs bind to the A/GCGTG consensus motif in target gene promoter regions, known as hypoxia-responsive elements

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(HREs). By recruiting transcriptional co-activators, HIFs regulate the expression of numerous

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genes involved in different processes including angiogenesis, metabolism, erythropoiesis, apoptosis, pH regulation, metastasis, and cellular differentiation (Keith, Johnson, & Simon,

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2012; Semenza, 2003).

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There are three different α-subunit isoforms: HIF-1α, HIF-2α, and HIF-3α. HIF-1α and HIF2α have been most comprehensively studied, with less known about HIF-3α. Semenza and

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colleagues first described HIF-1α in the nineties (G. L. Wang, Jiang, Rue, & Semenza, 1995),

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while Tian et al. (Tian, McKnight, & Russell, 1997) described the second isoform (initially called EPAS1; endothelial PAS protein 1) soon after, findings that were quickly confirmed by

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several other groups. The α-subunits show conserved sequence homology (Figure 3), mostly in the bHLH- and PAS-A and PAS-B domain-containing N-terminal region that mediates DNA binding and interaction with HIF-1β. Two transactivation domains (TADs) located in the α-subunit N- and C-termini regulate HIF transcriptional activity: the N-TAD is located in the region where hydroxylation takes place, known as the oxygen-dependent degradation (ODD) domain. HIF transcriptional activity is also regulated by a second oxygen-sensitive hydroxylation event mediated by factor inhibiting HIF-1 (FIH-1) (Lando, et al., 2002; Mahon, Hirota, & Semenza, 2001). FIH-1 is a 2-oxoglutarate-dependent oxygenase (similar to the PHDs), and it catalyzes hydroxylation of an asparagine residue in the C-TAD of HIF-α, thereby preventing interaction with the p300/CBP co-activator. Of note, HIF-1α is more sensitive to FIH-1-mediated inhibition than HIF-2α (Khan, et al., 2011) due to a specific amino acid difference between the two HIFs (Bracken, et al., 2006). The affinity of PHDs and 6

ACCEPTED MANUSCRIPT FIH-1 for oxygen differs and, since FIH-1 has a higher affinity (lower Km) for oxygen, PHDs are thought to be more rapidly inhibited in low oxygen conditions (Pouyssegur, Dayan, &

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Mazure, 2006). Thus, when PHDs are inactive, the HIF-1α subunit is stabilized but FIH-1 can still inhibit transcription of genes that require HIF-1α C-TAD activity. Depending on the

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oxygen tension, different gene sets might be regulated according to their N-TAD or C-TAD

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requirement. Hypoxia-associated factor (HAF) is an E3 ubiquitin ligase that switches cells from HIF-1- to HIF-2-dependent signaling by targeting HIF-1α for degradation and increasing

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HIF-2α transactivation (Koh, Lemos, Liu, & Powis, 2011).

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The fact that there is less sequence homology in the HIF-1α and HIF-2α transactivating domains than the DNA-binding and heterodimerization domains suggests that HIF-1 and HIF-

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2 have unique target genes. Early studies showed that HIF-1 (but not HIF-2) induced

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expression of several glycolytic genes in various cell types, while unique HIF-2 target genes seemed to be cell type specific (C. J. Hu, Wang, Chodosh, Keith, & Simon, 2003; Raval, et

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al., 2005). The studies were mainly performed in VHL-defective renal cell carcinoma (RCC) cell lines with unique HIF expression patterns, i.e., expressing only HIF-2α or both HIF-1α and HIF-2α. HIF transcriptional target specificity was strikingly different in VHL-defective RCC cells compared to other cell types, and the HIF isoforms also showed a mutually dependent suppressive function in RCC (Raval, et al., 2005). Several investigators have used chromatin immunoprecipitation together with microarrays (ChIP-chip) to directly identify HIF target genes (Mole, et al., 2009; Xia & Kung, 2009; Xia, et al., 2009). These studies confirmed the core A/GCGTG HIF-binding motif and, in agreement with earlier findings, showed that HIF-1 targets genes important in glycolytic metabolism. Furthermore, genes encoding histone demethylases were identified as HIF-1 target genes, suggesting that HIFs participate in epigenetic homeostasis during hypoxia. These studies also identified considerable overlap between HIF-1 and HIF-2 binding sites, yet HIF-2 seemed to contribute 7

ACCEPTED MANUSCRIPT very little to the transcriptional changes during hypoxia (Mole, et al., 2009). Integrating data from ChIP-chip analyses with gene expression profiles over a hypoxia time course showed

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that HIF-1 stabilization causes HIF-1 to preferentially bind to loci already transcriptionally active prior to the onset of hypoxia, partially explaining why most hypoxia-induced

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transcriptional changes are cell type specific (Xia & Kung, 2009). A study using chromatin

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immunoprecipitation followed by next-generation high-throughput sequencing (ChIP-seq) showed that HIF binding sites exist outside of promoter sequences and that HIF binds to other

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sites as well as the core A/GCGTG motif (Schodel, et al., 2011). This was particularly true for

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HIF-2α, for which as many as 70% of the identified HIF-2 binding sites lay outside promoter regions. Consistent with other studies, glucose metabolism pathways were targeted by HIF-1, while HIF-2 targeted genes were involved in Oct4-regulated stem cell pluripotency. A

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representative list of shared and unique target genes regulated by HIF-1 and HIF-2 can be found in Keith et al. (Keith, et al., 2012). Moreover, while HIF-1 is thought to mediate the

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acute response to hypoxia, HIF-2 stabilizes over longer time frames and also at normal physiological oxygen levels (Holmquist-Mengelbier, et al., 2006). In addition to its role as a transcription factor subunit, HIF-2α is also part of a hypoxiaregulated translation initiation complex. Hypoxia induces the formation of a HIF-2α complex with the RNA-binding protein RBM4 and cap-binding protein eIF4E2, which is then recruited to a wide variety of mRNAs to promote active translation at polysomes (Uniacke, et al., 2012). This function is independent of HIF-1β dimerization. As well as direct gene targeting via HRE binding, both HIF-1α and HIF-2α can indirectly influence gene expression by interfering with the activity of other transcription factors such as MYC (Gordan, Bertout, Hu, Diehl, & Simon, 2007; Koshiji, et al., 2004), p53 (Bertout, et al., 2009; Chen, Li, Luo, & Gu, 2003), and Notch (Gustafsson, et al., 2005; Y. Y. Hu, et al., 2014). Importantly, for many of these interactions, HIF-1α and HIF-2α seem to have 8

ACCEPTED MANUSCRIPT completely opposing effects, a difference that needs to be considered when targeting HIFs for therapeutic benefit. Further adding to HIF complexity, α-subunits are differentially regulated the

transcriptional,

translational,

and

posttranslational

modification

levels.

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comprehensive description of HIF-α regulation and the conflicting roles of HIF-1α and HIF-

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2α are given in Keith et al. (Keith, et al., 2012).

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Although a major mode of HIF stabilization is via proline hydroxylation and VHL-mediated

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degradation, it is important to mention that several non-hypoxia-driven stimuli also regulate HIFs such as growth factors, cytokines, hormones, and various stressors. Several growth

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factors and their cognate receptors that signal through the phosphoinositide 3-kinase (PI3K) or Ras/MAPK pathways induce HIF expression, and both basal and mitogen-induced HIF

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expression is abrogated upon PI3K pathway inhibition (Beppu, Nakamura, Linehan,

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Rapisarda, & Thiele, 2005; Calvani, Trisciuoglio, Bergamaschi, Shoemaker, & Melillo, 2008; Mohlin, et al., 2015; Zhong, et al., 2000). HIFs are direct substrates of a variety of protein

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kinases that regulate HIF stabilization, nuclear translocation, and activation (Kietzmann, Mennerich, & Dimova, 2016). Interestingly, several factors also use reactive oxygen species (ROS) as intermediate signaling molecules, and it has been shown that ROS directly regulate HIFs at different levels (Gorlach & Kietzmann, 2007).

3. Hypoxia and HIFs in cancer 3.1 Measuring tumor hypoxia In early key findings, O2-sensitive microsensors were used to show that tumor hypoxia was associated with tumor progression (Vaupel & Mayer, 2007). Patients with hypoxic head and neck tumors (Gatenby, et al., 1988; Nordsmark, et al., 2005), cervical cancer (Hockel, et al., 1993; Hockel, et al., 1996), and soft tissue sarcoma (Nordsmark, et al., 2001) had a worse

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ACCEPTED MANUSCRIPT prognosis, although some results are conflicting (Nordsmark, et al., 2006). Endogenous biomarkers associated with hypoxia in vitro (e.g., HIF-1α, glucose transporters, and carbonic

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anhydrase IX (CAIX)) have also been used in tumor hypoxia experiments; perhaps surprisingly, low O2 measured by microsensors did not correlate with expression of these

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biomarkers in patient tumors (Mayer, Hockel, & Vaupel, 2008). Thus, even if endogenous

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“hypoxic” biomarkers are associated with poor clinical prognosis (as described below), it is worth remembering that a direct association between biomarker expression and intratumoral

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hypoxia is unproven, and tumor cells can also express “hypoxia” markers such as HIF-2α

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under normoxic conditions (discussed below and reviewed in Pietras et al. (Pietras, Johnsson, & Pahlman, 2010)). Different methods for measuring hypoxia in patients are reviewed in

3.2 Patient prognosis

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Vaupel et al. (Vaupel, et al., 2004).

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The HIF-1α protein is overexpressed in various common solid malignant tumors including breast, colon, gastric, lung, skin, ovarian, pancreatic, prostate, and renal carcinomas compared to their respective normal tissues. In contrast, many benign tumors show very little or no HIF1α expression (Daponte, et al., 2008; Mayer, Hockel, Wree, et al., 2008; Simiantonaki, Taxeidis, Jayasinghe, Kurzik-Dumke, & Kirkpatrick, 2008; Zhong, et al., 1999). Importantly, HIF-1α or HIF-2α overexpression is associated with poor clinical outcomes in patients with various cancers. HIF-1α expression is associated with poor survival in cervical cancer (Birner, et al., 2000), endometrial carcinoma (Sivridis, et al., 2002), oligodendroglioma (Birner, Gatterbauer, et al., 2001), ovarian cancer (Birner, Schindl, Obermair, Breitenecker, & Oberhuber, 2001), and different breast cancer subtypes (Bos, et al., 2003; Bos, et al., 2001; Generali, et al., 2006; Kronblad, Jirstrom, Ryden, Nordenskjold, & Landberg, 2006; Schindl, et al., 2002). The prognostic value of HIF-1α in breast cancer has, however, been questioned 10

ACCEPTED MANUSCRIPT in conflicting follow-up studies, but HIF-2α appears to be an independent prognostic factor in breast cancer (Helczynska, et al., 2008). HIF-2α expression is also a marker of poor prognosis

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in glioblastoma (Z. Li, et al., 2009), neuroblastoma (Holmquist-Mengelbier, et al., 2006), head and neck squamous carcinoma (Koukourakis, et al., 2002), and non-small cell lung

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cancer (Giatromanolaki, et al., 2001). In ccRCC, VHL loss impairs degradation of HIF-α

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proteins, leading to HIF accumulation irrespective of oxygen levels (Gnarra, et al., 1994; Krieg, et al., 2000). The pathways involved in the pseudo-hypoxic phenotype in ccRCC are

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further reviewed in Bratslavsky et al. (Bratslavsky, Sudarshan, Neckers, & Linehan, 2007).

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High nuclear expression of HIF-1α and high cytoplasmic expression of HIF-2α indicate poor prognosis in ccRCC patients (Fan, et al., 2015). Thus, while intratumoral hypoxia in general is associated with poor prognosis, the prognostic role of HIF-1α and HIF-2α differs between

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tumor types. In addition, HIF-1α overexpression is associated with improved prognosis in patients with head and neck cancer (Beasley, et al., 2002), non-small cell lung cancer (Volm

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& Koomagi, 2000), and favorable outcomes in neuroblastoma (Noguera, et al., 2009). Thus, similar to their molecular functions, HIF-1α and HIF-2α can have opposite clinical effects depending on the tumor type.

3.3 Tumor cell viability Hypoxia can result in decreased cell proliferation, cell cycle arrest, and/or apoptosis. Giaccia and co-workers demonstrated that hypoxia can induce tumor cell apoptosis, an effect that was reduced by loss of p53 tumor suppressor function or overexpression of anti-apoptotic Bcl-2 (Graeber, et al., 1996). Therefore, hypoxia can exert a selective pressure that leads to expansion of tumor cells with reduced apoptotic capacity due to, for example, TP53 mutations (Graeber, et al., 1996). It has been suggested that hypoxia induces a physical interaction between HIF-1α and p53, resulting in stabilization of p53 and increased apoptosis (An, et al., 11

ACCEPTED MANUSCRIPT 1998). The interaction between HIF-1α and p53 is, however, also mediated by Mdm2, and HIF-1α can increase p53 levels by inhibition of Mdm2-mediated degradation (Chen, et al.,

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2003). Intriguingly, p53 can also promote Mdm2-mediated degradation of HIF-1α (Ravi, et al., 2000). HIF-2α seems to have an opposing effect on p53 (at least in ccRCC) since HIF-2α

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inhibits p53 in an Mdm2-independent manner, thereby providing therapeutic opportunities via

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HIF-2α inhibition (Bertout, et al., 2009; Roberts, et al., 2009). The interplay between HIFs

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and the p53 family is reviewed in Amelio et al. (Amelio & Melino, 2015). c-Myc is an essential cell cycle regulator and oncogene, and HIFs can either interfere with or

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promote c-Myc activity. Hypoxia-induced HIF-1α inhibits c-Myc transcription and suppresses proliferation (Koshiji, et al., 2004), and HIF-1α can also promote c-Myc degradation (H.

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Zhang, et al., 2007). In contrast, HIF-2α can enhance c-Myc activity and promote cell cycle

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progression (Gordan, Bertout, et al., 2007). HIF-1α can further inhibit Myc by interfering with its partner protein Myc-associated protein X (Max), while HIF-2α can stabilize the Myc-Max

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complex and increase Myc activity. The complex interplay between HIFs and Myc is reviewed in Dang et al. (Dang, Kim, Gao, & Yustein, 2008). These opposing effects on Myc function further highlight the need to develop specific inhibitors that target either HIF-1α or HIF-2α. The effects of hypoxia and HIFs on tumor cell proliferation, survival, and apoptosis are reviewed in Gordan et al. and Keith et al. (Gordan, Thompson, & Simon, 2007; Keith, et al., 2012). In response to stressors such as hypoxia or nutrient deprivation, cells become dependent on increased autophagy for their survival. In autophagy - literally “eating of self” – autophagosomes are formed that sequester intracellular components prior to digestion by fusion with lysosomes. Basal levels of autophagy are needed for cellular homeostasis, but from the cancer perspective, autophagy is considered a double-edged sword since it can either be tumor suppressive or tumor promoting. Its suppressor effects arise because it prevents cells 12

ACCEPTED MANUSCRIPT from being damaged by unfolded proteins or damaged organelles. However, it can also promote tumor growth by providing tumor cells with metabolites and by preventing the

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accumulation of malfunctioning mitochondria that would otherwise lead to increased ROS and cell death. HIF-1 is directly involved in regulating mitochondrial autophagy by inducing

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expression of the BH3-only proteins BNIP3 and BNIP3L, which promotes release of Beclin-

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1, a major autophagy regulator (Bellot, et al., 2009; H. Zhang, Bosch-Marce, et al., 2008). For a more detailed review on autophagy in cancer we refer interested readers to White (White,

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2012).

3.4 Tumor angiogenesis

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A key feature of the hypoxic response is the upregulation of multiple genes that promote

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angiogenesis/vascularization to increase oxygen delivery. Cells grown under hypoxic

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conditions activate transcription of vascular endothelial growth factor (VEGF) (Forsythe, et al., 1996; Shweiki, Itin, Soffer, & Keshet, 1992), which is important for endothelial cell activation and proliferation. Other hypoxia-induced pro-angiogenic factors include plateletderived growth factor-β (PDGF-β) (Kelly, et al., 2003), angiopoietin-2 (ANGPT2) (Kelly, et al., 2003), and stromal-derived factor 1α (SDF-1α) (Ceradini, et al., 2004), all of which promote neoangiogenesis. In neuroblastoma, HIF-1 mediates acute hypoxia-induced VEGF expression, while HIF-2 regulates VEGF expression during prolonged hypoxia (HolmquistMengelbier, et al., 2006). Furthermore, HIF-1α loss decreases tumor vascularization and impairs vascular function (Carmeliet, et al., 1998), and HIF-2α knockdown in glioblastoma and neuroblastoma results in poorly vascularized tumors (Z. Li, et al., 2009; Pietras, et al., 2009). Vascularization is severely impaired in HIF1A-/- (Carmeliet, et al., 1998; Ryan, Lo, & Johnson, 1998) and HIF2A-/- (Peng, Zhang, Drysdale, & Fong, 2000) mice. Thus, both HIF-1

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ACCEPTED MANUSCRIPT and HIF-2 play crucial roles in tumor vascularization, although their effect may vary depending on the temporal and cellular context.

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3.5 Cancer stem cells

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Hypoxia and HIFs regulate the proliferation and differentiation of different stem cell

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populations including embryonic, neural, and hematopoietic stem cells. The stem cell-like state is mediated by upregulation of OCT4, Notch, c-Myc, and telomerase (Keith & Simon,

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2007). Certain tumor cells are known to possess cancer stem cell (CSC)-like features, although the presence of true CSCs in solid tumors remains to be definitively proven.

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Irrespective of semantics and whether or not CSCs exist as a distinct sub-population of the cancer cell population, it is clear that hypoxia and HIFs regulate stem cell-like features in a

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proportion of cancer cells and make them more aggressive. In neuroblastoma, hypoxia

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downregulates neuronal and neuroendocrine differentiation markers and upregulates genes

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expressed during normal neural crest development (Jogi, et al., 2002). HIF-2α is co-expressed with putative CSC markers in neuroblastoma and glioblastoma (Z. Li, et al., 2009; Pietras, et al., 2008). Furthermore, HIF-2α silencing in glioblastoma decreases self-renewal, tumor cell proliferation in vitro, and tumor-initiating capacity in vivo (Z. Li, et al., 2009). We and others have hypothesized that HIF inhibition promotes tumor cell differentiation and reduces tumor relapse after conventional therapy (Keith & Simon, 2007; Lofstedt, et al., 2007).

3.6 Metabolic reprogramming Reprogramming of energy metabolism is another hallmark of cancer. In 1924, Otto Warburg observed that cancer cells metabolize glucose differently than untransformed cells. Instead of metabolizing glucose via the tricarboxylic acid (TCA) cycle and producing maximum amounts of ATP via oxidative phosphorylation, cancer cells redirect glucose metabolism 14

ACCEPTED MANUSCRIPT away from these circuits. Instead of converting pyruvate generated from glycolysis into acetyl-CoA (for the TCA cycle), cancer cells convert pyruvate into lactate, the latter reaction

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only generating two ATP molecules per glucose molecule compared to 36 ATP molecules per glucose molecule during normal TCA cycling and oxidative phosphorylation. Under oxygen

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deprived conditions, untransformed cells also convert pyruvate into lactate, a process known

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as “anaerobic glycolysis”. However, cancer cells convert pyruvate into lactate even when oxygen is available, so their metabolism is often referred to as “aerobic glycolysis”, or “the

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Warburg effect” (Vander Heiden, Cantley, & Thompson, 2009). Warburg proposed that

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cancer cells use aerobic glycolysis due to impaired mitochondrial function; however, later studies have shown that most cancer cells have functional mitochondria. Furthermore, studies on proliferating primary lymphocytes have shown that glucose is primarily converted to

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lactate, demonstrating that aerobic glycolysis is not only restricted to cancer cells (DeBerardinis, Lum, Hatzivassiliou, & Thompson, 2008). This raises the question: why

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would proliferating cells, including cancer cells, choose a pathway that produces less ATP? Even though this question has not been fully answered, one explanation could be that proliferating cells do not need optimized metabolism for maximal ATP production because the nutrient supply is not limited, so aerobic glycolysis still produces sufficient ATP. Another reason might be that cells require other molecules in addition to ATP to divide. Macromolecules such as amino acids, nucleotides, and fatty acids are needed for cellular replication and, consequently, pathways that support synthesis of these biomolecules are preferentially selected (DeBerardinis, et al., 2008; Vander Heiden, et al., 2009). Lactate is a byproduct of aerobic glycolysis and is considered a major player in tumor acidification. However, recent studies suggest that lactate also plays a role as a signaling molecule and can modulate the tumor microenvironment, providing a rationale to target lactic acid transporter

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ACCEPTED MANUSCRIPT complexes. Further reading on the therapeutic benefit of targeting lactate transporters can be found in Marchiq & Pouyssegur (Marchiq & Pouyssegur, 2016).

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Hypoxia promotes aerobic glycolysis by increasing both glucose uptake and expression of glycolytic enzymes (Iyer, et al., 1998). Furthermore, Kim et al. provided the first evidence

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that HIF-1 actively participates in metabolic reprogramming from the TCA cycle to glycolysis

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by showing that HIF-1 directly targets the gene encoding pyruvate dehydrogenase kinase 1

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(PDK1) (Kim, Tchernyshyov, Semenza, & Dang, 2006). PDK1 inactivates pyruvate dehydrogenase (PDH), which catalyzes pyruvate into actetyl-CoA; therefore, by regulating

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PDK1 expression, HIF-1 steers cells toward glycolysis and away from the TCA cycle. This knowledge has led to the testing of drugs that inhibit glycolysis such as dichloroacetic acid

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(DCA), which inhibits PDK activity (Dunbar, et al., 2014).

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Although hypoxia and HIF signaling are important for glycolysis, other mechanisms regulate glycolytic metabolism. For instance, the oncogenic PI3K signaling pathway also promotes

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glucose uptake and metabolism. HIFs regulate a switch from glucose to glutamine metabolism to produce acetyl-CoA in fatty acid synthesis (Semenza, 2013). Cancer cells often consume glutamine, and those dependent on Myc signaling are highly sensitive to glutamine withdrawal (Yuneva, Zamboni, Oefner, Sachidanandam, & Lazebnik, 2007). Furthermore, tumor suppressor pathways such as p53 and LKB1/AMPK signaling also regulate cellular metabolism (Vander Heiden, et al., 2009). For further information on the role of hypoxia in metabolic reprogramming we refer readers to Masson et al. (Masson & Ratcliffe, 2014) and Semenza (Semenza, 2013).

3.7 Tumor immune response Inflammation is another cancer hallmark, and the anti-tumor immune response plays a crucial role in many cancer types (Hanahan & Weinberg, 2011). Intratumoral hypoxia modulates the 16

ACCEPTED MANUSCRIPT tumoral

immune

response

in

many

ways,

collectively

pointing

to

an

overall

immunosuppressive effect (Palazon, Aragones, Morales-Kastresana, de Landazuri, & Melero,

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2012). For instance, tumor hypoxia and/or HIFs can attract myeloid-derived suppressor cells (Corzo, et al., 2010), regulatory T-cells (Clambey, et al., 2012), and tumor-associated

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macrophages (Doedens, et al., 2010; Imtiyaz, et al., 2010) with immunosuppressive functions.

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Hypoxia can also suppress dendritic cell function and inhibit infiltrating cytotoxic Tlymphocyte activity in a HIF-1α-dependent manner (Barsoum, Smallwood, Siemens, &

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Graham, 2014; Palazon, et al., 2012). Adenosine accumulates in tumors during hypoxic stress

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and contributes to tumor progression via immune suppression and a direct effect on cancer cells. Adenosine is thus a crucial factor mediating the hypoxic inhibition of the anti–tumor immune response (Antonioli, Blandizzi, Pacher, & Hasko, 2013). In addition, hypoxia-

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induced HIF-1α drives expression of the immune checkpoint protein PD-L1, which contributes to immune suppression and evasion (Noman, et al., 2014). Therefore, most

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evidence suggests that HIFs exert a tumor-promoting effect by immunosuppression. Immunotherapy has recently shown great promise in cancer patients, especially for the treatment of metastatic melanoma, and the combination of HIF inhibitors and immunotherapy may be a useful approach for clinical testing to further improve outcomes.

3.8 Genomic instability Intratumoral hypoxia promotes genomic instability, another hallmark of most cancers (Bristow & Hill, 2008). Early studies demonstrated that cancer cells cultured under hypoxic conditions contain an increased number of mutations and exhibit diminished DNA repair following UV irradiation-induced DNA damage compared to normoxic cells (Reynolds, Rockwell, & Glazer, 1996; Yuan, Narayanan, Rockwell, & Glazer, 2000). Furthermore, hypoxia downregulates DNA repair gene expression, and hypoxia/reoxygenation cycles can 17

ACCEPTED MANUSCRIPT induce DNA strand breaks and oxidative base damage, gene amplifications, and DNA overreplication (Bristow & Hill, 2008). Hypoxia can also lead to the epigenetic regulation and

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silencing of the DNA repair gene BRCA1, thereby promoting genomic instability (Y. Lu, Chu, Turker, & Glazer, 2011). HIF-1α is responsible for genetic instability in cancer cells by

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inhibiting the DNA mismatch repair genes MSH2 and MSH6, which leads to decreased levels

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of the MSH2-MSH6 (MutSα) base mismatch recognition complex (Koshiji, et al., 2005). Hypoxia-induced HIF-1 upregulates microRNAs miR-201 and miR-373, thereby suppressing

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DNA repair pathways and causing additional genetic instability (Crosby, Kulshreshtha, Ivan,

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& Glazer, 2009). Conversely, HIF-2 does not have the same effects on genomic stability, in part due to Thr-324 phosphorylation in the PAS-B domain, which prevents HIF-2 from repressing DNA repair genes (To, Sedelnikova, Samons, Bonner, & Huang, 2006).

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Furthermore, HIF-2 regulates the expression of genes involved in protecting cells from DNA

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damaging ROS (Scortegagna, et al., 2003).

3.9 Tumor cell invasion and metastasis Metastasis is the main cause of cancer-related deaths. Intratumoral hypoxia is associated with metastasis in experimental tumors (Brizel, et al., 1996; Cairns, Kalliomaki, & Hill, 2001; De Jaeger, Kavanagh, & Hill, 2001; Young, Marshall, & Hill, 1988), and HIFs have been implicated in metastatic disease in various clinical studies (Dales, et al., 2005; Helczynska, et al., 2008; Holmquist-Mengelbier, et al., 2006; Zhong, et al., 1998; Zhong, et al., 1999). Interestingly, HIF-1α was overexpressed in 29% of primary breast cancers but 69% of breast cancer metastases (Zhong, et al., 1999). Insights from a transgenic mouse model of metastatic breast cancer (Liao, Corle, Seagroves, & Johnson, 2007) and xenograft models of breast cancer (Hiraga, Kizaka-Kondoh, Hirota, Hiraoka, & Yoneda, 2007; X. Lu, et al., 2010) also point to HIF-1α as a critical regulator of metastasis. 18

ACCEPTED MANUSCRIPT Mechanistically, hypoxia and HIFs are involved in many different steps of the metastatic process. Epithelial-to-mesenchymal transition (EMT) is an initial step in which tumor cells

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lose expression of the intercellular adhesion molecule E-cadherin (encoded by CDH1) and acquire a motile phenotype. Hypoxia and HIFs contribute to EMT by upregulating

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transcriptional repressors such as SNAIL (Imai, et al., 2003), TWIST (M. H. Yang, et al.,

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2008), TCF3, ZFHX1A, and ZFHX1B (Krishnamachary, et al., 2006), whose gene products in

dependent manner (Esteban, et al., 2006).

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turn repress CDH1. Loss of VHL in ccRCC also downregulates E-cadherin in a HIF-

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Hypoxia and HIFs can also induce tumor cell invasion and degradation of the extracellular matrix via various mechanisms including upregulation of cathepsin D, urokinase-type

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plasminogen activator receptor, and matrix metalloproteinases-1 and -2 (Krishnamachary, et

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al., 2003; Munoz-Najar, Neurath, Vumbaca, & Claffey, 2006) and activation of Met (Pennacchietti, et al., 2003). The RIOK3 kinase promotes re-organization of the actin

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cytoskeleton in a HIF-1α-dependent manner, which has been shown to increase breast cancer cell migration and invasion/metastasis (Singleton, et al., 2015). HIF-1 activates transcription of collagen prolyl hydroxylases, which promote collagen deposition and breast cancer cell migration along collagen fibers, thereby enhancing invasion and metastasis (Gilkes, et al., 2013). HIF regulates lysyl oxidase, a critical mediator of hypoxia-induced breast cancer cell migration and metastasis, which mediates its action through increased focal adhesion kinase activity and extracellular matrix modulation (Erler, et al., 2006). Hypoxia-induced VEGF production promotes lymph- and angiogenesis, which facilitate tumor cell intravasation and dissemination (Claffey, et al., 1996; Schoppmann, et al., 2006). VEGF can also contribute to systemic intravasation by increasing microvascular permeability (Claffey, et al., 1996; Dvorak, Nagy, Feng, Brown, & Dvorak, 1999). HIF-1α-dependent

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ACCEPTED MANUSCRIPT expression of angiopoietin-like 4 and the L1 cell adhesion molecule further modulate intraand extravasation (H. Zhang, et al., 2012).

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Tumor cell homing to distant sites involves the HIF-regulated chemokine receptor CXCR4 (Staller, et al., 2003), which binds to stromal-derived factor-1 (SDF-1α) in secondary organs

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and drives breast cancer metastasis (Muller, et al., 2001). Primary tumor hypoxia can also

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promote metastatic spread by establishing a pre-metastatic niche in distant organs. Specifically, hypoxia-induced lysyl oxidase contributes to the pre-metastatic niche in a

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process that includes collagen crosslinking and recruitment of CD11b+ myeloid cells (Erler, et

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al., 2009). Finally, HIF expression in tumor stromal cells can also affect metastasis: HIF-2 expression in tumor endothelial cells has been shown to increase tumor cell migration and

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metastasis, while endothelial HIF-1 reduces cell migration and metastasis (Branco-Price, et

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al., 2012).

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We refer readers to (Rundqvist & Johnson, 2010) for a detailed review of HIFs in breast cancer metastasis and to (Johnson, Schipani, & Giaccia, 2015) for a review of the role of hypoxia and HIFs in bone metastasis.

3.10 Treatment resistance

Intrinsic and acquired resistance to radiotherapy (ionizing radiation; IR) and chemotherapy are major reasons for treatment failure in cancer patients. It has been known since the 1950s that hypoxia is a crucial mediator of chemo- and radioresistance (Gray, Conger, Ebert, Hornsey, & Scott, 1953; Roizin-Towle & Hall, 1978). HIF overexpression in clinical samples is associated with therapeutic resistance or decreased survival following IR or chemotherapy (Aebersold, et al., 2001; Birner, Schindl, et al., 2001; Burri, et al., 2003; Generali, et al., 2006; Koukourakis, et al., 2002; Vergis, et al., 2008). 20

ACCEPTED MANUSCRIPT Under normoxic conditions, IR generates ROS that cause irreparable DNA damage and cell death. Intratumoral hypoxia prevents ROS formation, resulting in inefficient DNA strand

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breaks and IR resistance (J. M. Brown & Wilson, 2004). HIF-1α expression is temporarily and acutely decreased after IR due to re-oxygenation of surviving hypoxic tumor cells

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(Harada, et al., 2009). ROS production later stabilizes the HIF-1α protein and increases

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expression of HIF-1α and its target genes (Dewhirst, Cao, & Moeller, 2008; Harada, et al., 2009; Moeller, Cao, Li, & Dewhirst, 2004). HIF-1α appears to exert both radiosensitizing

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(e.g., by promoting proliferation and p53-induced apoptosis) and radioresistant effects (via

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vascular radioprotection) (Moeller, et al., 2005). HIF-1α-mediated alterations in tumor glucose metabolism may also affect cellular responses to IR (Meijer, Kaanders, Span, & Bussink, 2012). Importantly, silencing or pharmacological inhibition of HIF-1α can increase

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the anti-tumor effects of IR (Harada, et al., 2012; Harada, et al., 2009; Moeller, et al., 2005; X. Zhang, et al., 2004). HIF-2α contributes to tumor cell survival after IR, and HIF-2α

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inhibition can increase the IR effect by activating the p53 pathway and increasing apoptosis (Bertout, et al., 2009). Thus, HIF inhibitors can be used in combination with radiotherapy to target radiotherapy-resistant tumor cells. Furthermore, radiosensitizers such as misonidazole mimic the effect of oxygen and can be used to enhance IR efficiency in hypoxic tumors, particularly in head and neck cancers (J. M. Brown & Wilson, 2004). A number of studies suggest that HIFs mediate chemoresistance. One main mechanism by which HIF-1 mediates chemoresistance is by activating the multidrug resistance 1 (MDR1) gene (Comerford, et al., 2002), which encodes a glycoprotein belonging to the ATP-binding cassette (ABC) transporter family and functions as a drug efflux pump. In this way, HIF-1α and MDR1 can decrease the intracellular concentration of various chemotherapeutic drugs and contribute to hypoxia-induced drug resistance (Comerford, et al., 2002). Similarly, HIF-2 can activate transcription of another ABC transporter ABCG2, suggesting a potential 21

ACCEPTED MANUSCRIPT mechanism of HIF2-induced chemoresistance (Martin, et al., 2008). Hypoxia and/or HIFs can mediate chemotherapy resistance through additional mechanisms including: a) hypoxia-

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induced decreases in tumor cell proliferation (Tannock, 1968); b) metabolic reprogramming; c) hypoxia-driven selection of p53-mutated tumor cells with less apoptotic capacity (Graeber,

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et al., 1996); and d) inhibition of DNA damage (Sullivan & Graham, 2009). Taken together,

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these data on the role of HIFs in therapeutic resistance suggest that HIF1/2 inhibitors might be effective in combination with conventional chemotherapy (L. M. Brown, et al., 2006; Erler, et

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al., 2004; He, et al., 2012; Nardinocchi, Puca, Sacchi, & D'Orazi, 2009; Roberts, et al., 2009;

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Unruh, et al., 2003; Zhao, et al., 2014). For a comprehensive review of the mechanisms underlying HIF-induced chemotherapeutic resistance, we refer the reader to (Rohwer &

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Cramer, 2011).

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4. Targeting hypoxia and HIFs in cancer Since hypoxia is a hallmark of solid tumors and mediates aggressive, metastatic, and resistant disease, it is arguably one of the most attractive therapeutic targets in cancer. Several approaches for targeting hypoxic tumor cells have been proposed including hypoxia-activated prodrugs, gene therapy, recombinant anaerobic bacteria, specific targeting of HIFs, or targeting pathways important in hypoxic cells such as the mTOR and UPR pathways (Melillo, 2007; Semenza, 2012; Wilson & Hay, 2011). We briefly discuss the mechanism of action of hypoxia-activated prodrugs before describing in detail the drugs that directly or indirectly modulate HIFs (see Figure 2 and Table 1).

4.1 Hypoxia-activated prodrugs

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ACCEPTED MANUSCRIPT A prodrug is an inactive compound that can, either spontaneously or via specific metabolic pathways, be converted to its pharmacologically active species. One way of exploiting tumor

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hypoxia has been to design hypoxic prodrugs that are activated in hypoxic tissue and thus selectively kill hypoxic tumor cells (Denny, 2000). Hypoxic prodrugs are activated by

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reduction of the prodrug by cellular reductases. One-electron reductases generate a prodrug

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radical that is either re-oxidized to the initial prodrug in oxic cells or is converted into its cytotoxic species in hypoxic cells (Figure 4). Alternatively, two-electron reductases bypass

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the prodrug radical formation step and directly convert the prodrug into the active drug. Since

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no oxygen-sensitive prodrug radical is formed in the two-electron reduction pathway, this reaction is typically (but not always) oxygen insensitive and more likely to lead to off-target effects. The cytotoxic effect of the active metabolite is mainly mediated by DNA interactions

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and by interfering with replication. Furthermore, surrounding non-hypoxic tumor cells can be killed through the so-called “bystander” effect, which can be enhanced by prolonging the

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half-life of the cytotoxic metabolite. Examples of hypoxia-activated prodrugs are provided in Table 2. For further reading on hypoxic prodrugs, we refer the reader to Phillips (Phillips, 2016).

The hypoxic prodrug tirapazamine has been extensively tested in clinical trials, but results have been disappointing (DiSilvestro, et al., 2014; Rischin, et al., 2010). A second-generation tirapazamine analog SN30000 has improved pharmocodynamic and pharmacokinetic properties and superior anti-tumor effects in xenograft models (Hicks, et al., 2010). Some of the disappointment in clinical trials may have been due to a failure to adopt a “personalized” or precision approach: to optimize the potential therapeutic effect, it is crucial that patients are stratified based on tumor-specific predictive biomarker expression that indicates sensitivity to the drug being tested. However, predictive biomarkers for hypoxic prodrugs are poorly defined. Interestingly, P450 oxidoreductase was recently identified as a biomarker of 23

ACCEPTED MANUSCRIPT sensitivity to the hypoxia prodrugs SN30000 and TH-302 (Hunter, et al., 2015). Results from recently published phase II clinical trials for TH-302 in combination with gemcitabine in

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pancreatic cancer (Borad, et al., 2015) or in combination with doxorubicin in soft tissue sarcoma (Chawla, et al., 2014) are encouraging, and phase III studies have been initiated. The

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prodrug apaziquone (EO9), a mitomycin C derivative, showed efficacy in preclinical studies,

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but clinical trials were negative. An evaluation of why apaziquone failed showed that the drug had poor pharmacokinetics: the drug could not penetrate the tumor and was rapidly degraded

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on systemic delivery (Phillips, Hendriks, Peters, Pharmacology, & Molecular Mechanism,

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2013). Therefore, loco-regional administration of apaziquone was proposed as a superior option to avoid problems with drug delivery and clearance. Loco-regional administration of apaziquone in superficial bladder cancer, an excellent model due to the capacity for direct

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prodrug delivery to the cancer via a catheter, has shown efficacy. A phase III trial of apaziquone (EOquin®) as adjuvant therapy in patients with bladder cancer treated by surgery

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was recently completed (NCT00598806).

4.2 Drugs targeting hypoxic signaling Strategies to target hypoxia include direct and indirect HIF targeting as well as targeting of downstream HIF signaling pathways. A classic example of the latter is anti-VEGF-therapy: monoclonal antibodies targeting VEGF (bevacizumab) or small molecule inhibitors targeting the VEGF receptor have shown clinical benefit in advanced cancer (Ellis & Hicklin, 2008). There are considerably fewer selective and specific HIF inhibitors compared to non-selective inhibitors that target multiple pathways including the HIF pathway. Nonetheless, the nonselective inhibition of HIFs might be an important anti-tumor mechanism in practice. In the following section (and Figure 2), we summarize the drugs that have been shown to directly or indirectly target HIFs or their pathways. 24

ACCEPTED MANUSCRIPT

4.2.1 Inhibitors of HIF mRNA or protein expression

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Aminoflavone, an aryl hydrocarbon receptor ligand, has been shown to partially inhibit HIF-

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1α mRNA expression in breast cancer cells and almost completely inhibit HIF-1α protein

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accumulation and transcription of downstream target genes in an aryl hydrocarbon receptorindependent fashion (Terzuoli, et al., 2010). However, its clinical utility has not been

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demonstrated, and several clinical trials using the prodrug AFP-464 have either been terminated or withdrawn prior to enrollment due to lack of efficacy.

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PI3K/Akt/mTOR inhibitors. Apart from the well-defined oxygen-mediated regulation of HIF-α subunits, translation of HIF-α mRNA is controlled by growth factor signaling pathways such

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as the PI3K/AKT/mTOR pathway. Thus, molecules that target this pathway presumably also

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modulate HIF activity. Consistent with this, several studies have shown that mTOR inhibition

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decreases HIF-1α and HIF-2α levels under both normoxic and hypoxic conditions (Hudson, et al., 2002; Majumder, et al., 2004; Mohlin, et al., 2015; Thomas, et al., 2006). We recently showed that hypoxia-induced HIF2A transcription in neuroblastoma cells is highly dependent on PI3K-mTORC2 but not PI3K-mTORC1, further supporting the hypothesis that targeting this pathway abrogates HIF activity (Mohlin, et al., 2015). A novel group of HIF-1 inhibitors called glyceollins (isolated from soybeans) can also block HIF-1α translation by inhibiting the PI3K/AKT/mTOR pathway under hypoxic conditions (S. H. Lee, Jee, Bae, Liu, & Lee, 2015). Topoisomerase 1 inhibitors. Irinotecan and topotecan are FDA-approved topoisomerase I inhibitors and analogues of the naturally occurring cytotoxic alkaloid camptothecin. Topotecan was identified as an inhibitor of HIF-1 activity (Rapisarda, et al., 2002) and was later shown to inhibit HIF-1α translation (Rapisarda, et al., 2004). More recently, the camptothecin-induced effects on HIF-1α levels were shown to occur as a result of changes in

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ACCEPTED MANUSCRIPT miRNA expression (Bertozzi, et al., 2014). In a small pilot trial, topotecan decreased HIF-1α protein expression in 4 out of 7 paired tumor biopsies taken from patients before and after

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treatment (Kummar, et al., 2011). In an ongoing phase II study of bevacizumab and irinotecan in young patients with brain tumors, one of the secondary outcomes is measurement of HIF-

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2α expression at baseline and association of HIF-2α expression with progression-free

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survival. SN-38 is the active metabolite of irinotecan, and attachment of polyethylene glycol (PEG) to SN-38 improves drug solubility: PEG-SN38 (or EZN-2208) has been shown to

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downregulate both HIF-1α and HIF-2α in a neuroblastoma model (Pastorino, et al., 2010), and

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a phase I study of PEG-SN38 in patients with neuroblastoma and other solid tumors showed low toxicity and supported further study of this drug in neuroblastoma (Norris, et al., 2014). Furthermore, PEG-SN38 in combination with all-trans retinoic acid was synergistic in a

Bernardi, 2015).

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mouse model of acute promyelocytic leukemia (Coltella, Valsecchi, Ponente, Ponzoni, &

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EZN-2968 is a synthetic antisense oligonucleotide comprised of 16 nucleotide residues 100% complementary to residues in the mRNA coding sequence of human HIF-1α. EZN-2968 binding leads to HIF-1α mRNA downregulation in a dose-dependent manner and results in near complete inhibition at 5 nM concentrations (Greenberger, et al., 2008). EZN-2968 has a three base pair mismatch with the HIF-2α sequence and, as a consequence, only downregulates HIF-2α mRNA by 20% at 25 nM. EZN-2968 treatment of tumor cells in vitro downregulated HIF-1α protein levels during hypoxia and reduced the growth rate. The in vivo effect of EZN-2968 has also been tested in a subcutaneous prostate cancer cell line xenograft model and, although treatment did not result in a significant reduction in tumor burden, pretreatment of cells with EZN-2968 prior to injection resulted in a significant reduction in tumor size. EZN-2968 has also been evaluated in two phase I clinical trials: results from one trial showed reduced HIF-1α mRNA in post-treatment biopsies from 4 out of 6 patients with 26

ACCEPTED MANUSCRIPT paired tumor biopsies, and HIF-1α protein and mRNA levels of target genes were reduced in biopsies from two patients. Although the trial provided preliminary proof of concept for

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modulation of HIF-1α mRNA and protein expression in response to EZN-2968, the trial closed prematurely due to suspended development of the compound (Jeong, et al., 2014). A

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third phase I trial has recently been initiated in patients with hepatocellular carcinoma to

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establish proof-of-mechanism for EZN-2968 (RO7070179).

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2-methoxyestradiol (2ME2) and analogues. 2ME2 is a microtubule-targeting estrogen metabolite that causes mitotic arrest. 2ME2 inhibits tumor growth and angiogenesis by

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downregulating HIF-1α protein expression, most likely through translational inhibition (Mabjeesh, et al., 2003). A recent report has suggested that 2ME2 also downregulates HIF-2α

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to overcome sorafenib resistance in hepatocellular carcinoma cells (Ma, et al., 2014). The

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safety and efficacy of 2ME2 (Panzem™) have been evaluated in numerous phase I and phase II studies, and there have been efforts to generate more effective 2ME2 analogues. To this

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end, ENMD-1198 has been selected as a lead analogue (LaVallee, et al., 2008) and has been tested in a phase I dose-escalation study in advanced cancer patients (Q. Zhou, et al., 2011). Heat shock protein inhibitors. Heat shock proteins (Hsp) are cellular chaperones that ensure that target proteins are correctly folded and localized. Geldanamycin (GA) is an anti-cancer antibiotic that promotes protein degradation by binding and inhibiting Hsp90. GA can induce proteasomal degradation of HIF-1α in a VHL-independent manner both under normoxic and hypoxic conditions (Isaacs, et al., 2002). The GA analogues 17-AAG (tanespimycin) and 17DMAG (alvespimycin) have been evaluated in several phase I and phase II trials either alone or in combination with other chemotherapies. In a comparative analysis, a next-generation small molecule Hsp90 inhibitor EC154 more effectively inhibited HIF-1α and HIF-2α than 17-AAG (Bohonowych, et al., 2011). This study highlights the discordance between HIF activity and HIF protein expression, emphasizing that pharmaceutical inhibition of HIF 27

ACCEPTED MANUSCRIPT activity is not necessarily equivalent to HIF tissue expression and, therefore, HIF biomarker analysis is not always a robust surrogate of HIF suppression.

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Triptolide, isolated from a Chinese herb, mediates its anti-tumor effects by inhibiting Hsp70. Somewhat unexpectedly, triptolide increased HIF-1α levels under both normoxic and hypoxic

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conditions, although target gene transcription was reduced suggesting that triptolide inhibits

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HIF-1 activity (Z. L. Zhou, et al., 2010). The triptolide prodrug Minnelid™ is currently being

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tested in a phase I trial of patients with advanced gastrointestinal tumors (NCT01927965). Histone deacetylase (HDAC) inhibitors. Histone deacetylases (HDACs) remove acetyl groups

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from histones or non-histone proteins such as transcription factors. The HDAC inhibitors vorinostat (SAHA, Zolinza®), romidepsin (Istodax®, previously known as FK228),

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panobinostat (Farydak®), and belinostat (Beleodaq®, also known as PXD101) are FDA

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approved for the treatment of various cancers. HDAC inhibitors have been shown to promote

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HIF-1α degradation and, although the exact mechanism is not completely understood, translational inhibition of HIF-1α has been proposed as vorinostat’s mechanism of action (Hutt, Roth, Vignaud, Cullin, & Bouchecareilh, 2014). Another mechanism of HDAC inhibitor action might be via Hsp90 inhibition, as described above. Both HIF-1α and HIF-2α are themselves regulated by acetylation and deacetylation events but in opposing manners (Keith, et al., 2012). Deacetylation of HIF-2α by SIRT1, a deacetylase, increases HIF-2α’s transcriptional activity, while deacetylation of HIF-1α results in transcriptional repression. This suggests that HDAC inhibitors can have direct but contrasting effects on HIF-α subunits. YC-1 was originally discovered as an activator of soluble guanylate cyclase, an enzyme that increases cGMP levels and, as a result, inhibits platelet aggregation and vasoconstriction. Additionally, YC-1 has anti-tumor effects by inhibiting HIF activity via an unknown non-

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ACCEPTED MANUSCRIPT guanylate cyclase-mediated mechanism. Treatment of hepatocellular carcinoma Hep3B cells with YC-1 decreased hypoxic induction of erythropoietin (EPO) and VEGF, decreased HIF-

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1α protein accumulation, and reduced HIF-1/DNA binding (Chun, et al., 2001). Bearing in mind that EPO is a specific HIF-2 target (Rankin, et al., 2007), the observed downregulation

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of EPO in Hep3b cells by YC-1 suggests that YC-1 also inhibits HIF-2 activity. YC-1 is

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thought to inhibit HIF-1 activity by preventing its interaction with the p300 co-activator (S. H. Li, et al., 2008), i.e., the same mechanism used by the endogenous FIH enzyme. However,

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empirically this mechanism was cell line dependent and was not observed in Hep3b cells.

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This suggests that EPO downregulation in Hep3b cells upon YC-1 treatment is not mediated by preventing HIF interaction with p300 but rather via another unknown mechanism. This is consistent with the observation that HIF-2 is not regulated to the same extent as HIF-1 by

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FIH. YC-1 also has anti-tumor and anti-angiogenic effects in several xenograft models (Yeo, et al., 2003) and has also been shown to inhibit tumor cell invasion and metastasis (Shin, et

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al., 2007), but there have been no YC-1 clinical trials to date. Based on YC-1’s structure, new analogues have been synthesized and screened for their HIF inhibitory effects (Masoud, Wang, Chen, Miller, & Li, 2015). PX-478 is derived from the alkylating agent melphalan, a cytotoxic agent used in the treatment of multiple myeloma and some other cancers including ovarian cancer. PX-478 inhibits HIF-1α at multiple levels, primarily by translational inhibition and, to a lesser extent, by promoting protein degradation in an oxygen-, pVHL-, and p53-independent manner (Koh, et al., 2008). PX-478 has shown anti-tumor activity in several xenograft models (S. Welsh, Williams, Kirkpatrick, Paine-Murrieta, & Powis, 2004), and a phase 1 dose-escalation study of oral PX-478 in patients with advanced solid tumors or lymphoma showed, at best, stable disease in 14 out of 36 evaluable patients (Ban, Uto, & Nakamura, 2011). Thioredoxin inhibitors. PX-12 is an inhibitor of the redox protein thioredoxin (Trx-1). Trx-1 29

ACCEPTED MANUSCRIPT is found at high levels in many human cancers and is also associated with increased expression of HIF-1α and HIF-regulated genes (S. J. Welsh, Bellamy, Briehl, & Powis, 2002).

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Two Trx-1 inhibitors, PX-12 and pleurotin, were initially found to decrease HIF-1α and VEGF levels in vitro and in vivo (S. J. Welsh, et al., 2003). However, a phase II trial of PX-12

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in patients with advanced pancreatic cancer was terminated early due to lack of significant

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efficacy (Ramanathan, et al., 2011). Two novel Trx-1 inhibitors, AJM290 and AW464, also inhibited HIF transcriptional activity and DNA binding but, unexpectedly and in contrast to

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other Trx-1 inhibitors, stabilized both HIF-1α and HIF-2α (Jones, Pugh, Wigfield, Stevens, &

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Harris, 2006).

Cardiac glycosides were identified as HIF inhibitors in a cell-based screen (H. Zhang, Qian,

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et al., 2008). Both HIF-1α and HIF-2α protein expression were reduced during treatment with

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the cardiac glycosides ouabain, proscillaridin A, and digoxin. In contrast, HIF-1α mRNA levels increased during treatment. Of note, the in vitro doses used were much higher than

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circulating therapeutic doses of digoxin and would, therefore, be expected to induce severe toxicity (Lopez-Lazaro, 2009). Also, species-related differences in cardiac glycoside sensitivity might contribute to the anti-tumor effects of digoxin observed in mice harboring human malignant cells. A phase II breast cancer trial recently commenced in which changes in HIF-1α protein expression in tumor tissue will be evaluated in patients taking daily oral digoxin for two weeks prior to surgery compared to no drug prior to surgery (NCT01763931). FM19G11 was identified as a novel, low cytotoxicity inhibitor of HIF transcriptional activity in a cell-based screen (Moreno-Manzano, et al., 2010). Both HIF-1α and HIF-2α protein accumulation was inhibited during hypoxia independent of proteasome activity, and FM19G11 also promoted differentiation of hypoxic neural stem cells, possibly through downregulation of Sox2 and Oct4. HIF-2α translational inhibitors. HIF-2α translation is regulated by an iron-dependent 30

ACCEPTED MANUSCRIPT mechanism involving an iron-responsive element (IRE) in the 5’-UTR of the HIF-2α mRNA (Sanchez, Galy, Muckenthaler, & Hentze, 2007). The IREs can bind iron-regulatory proteins

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(IRPs), which then repress mRNA translation. Binding of IRP1 to the IRE in the 5’-UTR of HIF-2α mRNA is impaired during hypoxia, leading to de-repression of HIF-2α mRNA

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translation. However, when intracellular iron levels are low, IRP1 still binds to the IRE and

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represses HIF-2α mRNA translation. Zimmer et al. identified compounds that could repress HIF-2α activity by promoting IRP1 binding to the IRE in the HIF-2α 5’-UTR (Zimmer, et al.,

4.2.2 Inhibitors of HIF dimerization

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2008).

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The PAS domains in the HIF-α and HIF-1β/ARNT subunits mediate HIF complex assembly,

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and small molecules that target these domains have been screened to discover inhibitors of

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this protein-protein interaction.

Acriflavine is a potent inhibitor of HIF-α-HIF-1β dimerization by directly binding to the PASB domain of both HIF-1α and HIF-2α (K. Lee, et al., 2009). Treatment with acriflavine reduced tumor growth and vascularization in prostate and hepatocellular xenograft models (K. Lee, et al., 2009).

PT2385. Based on the crystal structures of the PAS-HIF-2α and PAS-HIF-1β heterodimers, the HIF-2α PAS-B domain was found to harbor a large internal cavity (Scheuermann, et al., 2009). Interestingly, an allosteric ligand that binds in this cavity and inhibits HIF-2 transcriptional activity has recently been described (Scheuermann, et al., 2013). The ligand did not affect HIF-2α mRNA and protein levels; thus, this inhibitor is unlikely to affect HIF1β-independent HIF-2α mechanisms such as translational activation. Based on this ligand, a novel compound PT2385 has now entered a phase 1 dose-escalation trial in patients with 31

ACCEPTED MANUSCRIPT advanced ccRCC (NCT02293980). Since HIF-2α rather than HIF-1α is the major driver of VHL-mutated ccRCC (Carroll & Ashcroft, 2006; Kondo, Kim, Lechpammer, & Kaelin,

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2003), specific targeting of HIF-2 in this tumor is likely to be more beneficial.

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4.2.3 Inhibitors of HIF-DNA binding

Transcriptional activation of target genes by HIFs requires DNA binding. Inhibiting this step

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is another potential mechanism to inhibit HIF activity.

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Echinomycin is a small antibiotic molecule isolated from Streptomyces echinatus that binds DNA in a sequence-specific manner. In cell-based screening, echinomycin inhibited binding

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of HIF-1 to HREs and thus prevented induction of hypoxia-induced VEGF expression (Kong,

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et al., 2005). In a mouse model of relapsed acute myeloid leukemia, echinomycin cured mice without affecting normal hematopoietic stem cells (Y. Wang, et al., 2014). Nevertheless,

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clinical trials of echinomycin have been disappointing, and thus its therapeutic relevance remains to be established (Melillo, 2007). Polyamides. Synthetic oligomers containing N-methylpyrrole and N-methylimidazole amino acids can be modified to target specific DNA sequences to modulate gene expression. A polyamide designed to target HREs inhibited binding of HIF-1 to the VEGF HRE and blocked VEGF synthesis (Olenyuk, et al., 2004). A recent study compared polyamide uptake in different tumor xenografts and showed that there was significant variability in compound uptake in xenografts of different origin (Raskatov, Szablowski, & Dervan, 2014). Polyamides are currently not in clinical use.

4.2.4 Inhibitors of HIF transcriptional activity

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ACCEPTED MANUSCRIPT N-TAD and C-TAD, the two functional HIF domains, are responsible for mediating their transcriptional activity by interacting with co-activators such as p300/CBP. Therefore,

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transcriptional inhibition has been pursued as a therapeutic strategy by interfering with this HIF-p300 interaction.

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Chetomin, a Chaetomium fungus metabolite, was identified as a potent disrupter of the HIF-

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p300 interaction in a high-throughput screening study of >600,000 substances (Kung, et al.,

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2004). Chetomin inhibited binding of both HIF-1α and HIF-2α to p300 and in vivo anti-tumor effects were also demonstrated. In a later study, chetomin was shown to disrupt zinc-binding

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sites in the p300 CH1 domain, which hindered the HIF-p300 interaction (Cook, et al., 2009). Gliotoxin and chaetocin are other naturally occurring metabolites belonging to the same

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family as chetomin, and they have also been shown to block the HIF-p300 interaction (Reece,

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et al., 2014). However, due to chetomin’s toxicity, this drug has not been pursued clinically

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(Onnis, Rapisarda, & Melillo, 2009).

Proteasome inhibitors. Bortezomib (Velcade™) is an FDA-approved proteasome inhibitor for the treatment of multiple myeloma and mantle cell lymphoma. Bortezomib blocked hypoxiainduced CAIX, EPO, and VEGF accumulation in cell lines by inhibiting HIF-1α C-TAD’s transcriptional activity (Kaluz, Kaluzova, & Stanbridge, 2006; Shin, Chun, Lee, Huang, & Park, 2008). However, data were conflicting as to whether this was due to repression of the HIF-1-p300 interaction. Since bortezomib blocked hypoxic EPO production in Hep3B cells (Shin, et al., 2008), HIF-2 activity was also presumably compromised, but this was not investigated. As a single agent in a phase II trial, bortezomib was inactive in patients with metastatic colorectal cancer even though tumor biopsies collected pre- and post-treatment showed HIF-1α accumulation and reduced CAIX levels, indicating that hypoxic signaling pathways were affected (Mackay, et al., 2005). Falchook et al. hypothesized that bortezomib combined with bevacizumab would overcome anti-angiogenic therapy resistance by blocking 33

ACCEPTED MANUSCRIPT HIF-1α upregulation, although the published study only recruited a small number of patients and a lack of paired biopsies precluded any robust analysis of changes in HIF-1α and

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downstream targets (Falchook, et al., 2014). Amphotericin B (AmB) is used to treat systemic fungal infections, but long-term treatment is

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associated with anemia, supposedly by EPO suppression. Yeo et al. showed that AmB

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treatment downregulated EPO mRNA and protein levels in rat kidneys and Hep3B cells (Yeo,

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et al., 2006). Only HIF-1α protein levels were investigated in this study, and AmB affected neither protein levels nor HIF-1α localization, suggesting that reduced EPO was due to

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repressed HIF-1α C-TAD activity via enhanced interaction between HIF-1α and FIH-1.

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However, the downregulation of EPO implies that AmB could also inhibit HIF-2 activity.

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pseudohypoxic phenotype

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5. Neural crest-derived tumors as model systems for targeting HIF-2 and the

The neural crest-derived sympathoadrenal stem cell gives rise to a sympathetic nervous system (SNS) composed of cells from three differentiation lineages, namely the ganglionic/neuronal, chromaffin, and SIF (small intensely fluorescent) lineages (Landis & Patterson, 1981). Several observations suggest that HIF-2 signaling has specific roles during SNS development. HIF-2α is transiently and specifically expressed in human embryonal and fetal SNS cells and during rodent SNS development (Mohlin, Hamidian, & Pahlman, 2013; Nilsson, et al., 2005; Tian, Hammer, Matsumoto, Russell, & McKnight, 1998). Furthermore, hypoxia and HIFs are important regulators of the synthesis (Schnell, et al., 2003) and secretion (Kumar, et al., 1998) of catecholamines, which are key SNS neurotransmitters. Consistent with these observations, mouse embryos lacking HIF-2α have severely reduced norepinephrine levels compared to wild type mice (Tian, et al., 1998).

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ACCEPTED MANUSCRIPT Human tumors develop from at least the ganglionic and the chromaffin SNS lineages. Pheochromocytomas and paragangliomas arise from chromaffin cells in the adrenal medulla

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and extra-adrenal sympathetic paraganglia, respectively, and affect adults and, rarely, children. Neuroblastomas arise from sympathetic neural precursor cells in the adrenal medulla

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or sympathetic ganglia and are bona fide childhood tumors. The vast majority of

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pheochromocytomas and paragangliomas are benign, in contrast to neuroblastomas that present with metastases at the time of diagnosis in about a half of patients. Interestingly, the

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tumorigenesis of these closely related tumors seems to involve aberrant HIF-2 signaling,

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resulting in pseudohypoxic phenotypes linked to disseminated disease (Eisenhofer, et al., 2004; Favier, et al., 2009; Pietras, et al., 2008). Somatic gain-of-function mutations in HIF2A have been discovered in pheochromocytomas and paragangliomas (Comino-Mendez, et al.,

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2013; Lorenzo, et al., 2013; Taieb, et al., 2013; Toledo, et al., 2013; C. Yang, et al., 2013; Zhuang, et al., 2012), while high HIF-2α expression is associated with poor survival in

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patients with neuroblastoma (Holmquist-Mengelbier, et al., 2006). Interestingly, HIF-2αpositive neuroblastoma cells have been identified in perivascular niches (HolmquistMengelbier, et al., 2006; Pietras, et al., 2008), indicating that HIF-2α protein is stabilized in these cells despite sufficient oxygen and facilitating the pseudohypoxic phenotype in tumor cells. Thus, these SNS-derived tumors provide models to study HIF-2’s role in tumorigenesis and metastasis and to test novel treatment protocols that target HIF-2 directly or indirectly by interfering with upstream or downstream pathways (Mohlin, et al., 2015; Richter, Qin, Pacak, & Eisenhofer, 2013). Pheochromocytomas and paragangliomas have the largest hereditary component of all human tumors, and many of the associated germline mutations (such as those in VHL and succinate dehydrogenase (SDH)) result in pseudohypoxic phenotypes (Dahia, 2014). As noted above, VHL is a crucial mediator of HIF-α degradation at normoxia; thus, VHL loss stabilizes HIF-α 35

ACCEPTED MANUSCRIPT and results in pseudohypoxia. Mutations in SDHx (SDHA, SDHB, SDHC or SDHD) lead to succinate accumulation, which acts as a competitive inhibitor of the 2-oxoglutarate-dependent

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dioxygenases such as the PHDs, again stabilizing HIF-α and causing pseudohypoxia. Unsupervised hierarchical classification of gene expression profiles of pheochromocytomas

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and paragangliomas distinguishes two main clusters. Cluster 1 includes HIF2A-, VHL-, and

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SDHx-related tumors and is characterized by expression of genes involved in hypoxic

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signaling and high expression of HIF-2α itself (Eisenhofer, et al., 2004; Lopez-Jimenez, et al., 2010). Tumors with SDHB mutations seem to be more aggressive and metastatic (Amar, et

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al., 2007; King, et al., 2011), and although the molecular explanation for this phenomenon has not been fully established, a potential role for active HIF-2 signaling has been proposed

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(Guzy, Sharma, Bell, Chandel, & Schumacker, 2008). HIF-2α, therefore, appears to be a

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highly relevant target in most, if not all, cluster 1 pheochromocytomas and paragangliomas. Moreover, HIF-2α silencing in PC-12 rat pheochromocytoma cells led to increased expression

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of the catecholamine-synthesizing enzyme PNMT, while HIF-1α silencing had the opposite effect (Qin, et al., 2014), again suggesting opposing roles for these two HIFs in SNS-derived tumor cells.

Clinically relevant in vitro and in vivo models are required to effectively evaluate drugs that target HIF-2 in neural crest-derived tumors. A number of xenograft models and genetically engineered mouse models (GEMMs) have been developed for neuroblastoma and pheochromocytoma (Table 3 and further reviewed in (Korpershoek, Pacak, & Martiniova, 2012)). GEMMs have contributed to our understanding of tumor initiation and growth; however, GEMM tumors are by definition of murine origin and the models may not necessarily accurately recapitulate the biological and clinical features of human disease. GEMMs carrying SDHx mutations have been created, but neither SdhD+/- nor SdhB+/- mice develop pheochromocytomas or paragangliomas (Bayley, et al., 2009; Piruat, Pintado, Ortega36

ACCEPTED MANUSCRIPT Saenz, Roche, & Lopez-Barneo, 2004). This subject is further reviewed in (Louis J. Maher III, 2011).

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Many conventional human neuroblastoma cell lines exist that have contributed to progress in understanding this disease. Similarly, the rat PC-12 pheochromocytoma cell line (Greene &

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Tischler, 1976) and the mouse MPC pheochromocytoma cell line (Powers, et al., 2000) have

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been widely used. However, the degree to which conventional cancer cell lines resemble

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patient disease has been questioned due to the tumor cell differentiation and aberrant genetic deviations that arise following long-term culture in serum-containing media (J. Lee, et al.,

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2006). There is thus a need to develop relevant mouse models with disease-specific genotypes to target HIF-2 and the pseudohypoxic phenotype.

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Recently established neuroblastoma patient-derived orthotopic xenografts (PDXs), in which

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surgical resection specimens from patients are implanted to the para-adrenal space of

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immunodeficient mice, retain the geno- and phenotypes of patient tumors (Braekeveldt, et al., 2015; Braekeveldt, et al., 2016). Neuroblastoma PDXs also exhibit infiltrative tumor growth and spontaneous metastatic spread to clinically relevant locations including the bone marrow. Furthermore, PDX-derived human neuroblastoma cells cultured under serum-free stem cellpromoting conditions also retain the genotype and tumorigenic and metastatic capacity in vivo (Braekeveldt, et al., 2015). These cells can be used for drug testing (Mohlin, et al., 2015). A limitation of this model system is that the mice lack a fully functional immune system and efforts are therefore being made to reconstitute the human immune system in immunodeficient mice (Shultz, et al., 2014). Nevertheless, neuroblastoma PDXs in vivo and in vitro will facilitate the identification of drugs targeting hypoxic tumor cells and the pseudohypoxic phenotype in aggressive neuroblastoma. Although these drugs are likely to also be effective in Cluster 1 pheochromocytomas and paragangliomas, establishing PDXs and human cell lines from metastatic pheochromocytoma/paraganglioma would nevertheless 37

ACCEPTED MANUSCRIPT be highly desirable.

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6. Summary and future perspectives

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Tumor hypoxia and its main mediators the HIFs regulate many important biological hallmarks

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of cancer ranging from genetic instability and tumor cell differentiation to metabolic reprogramming and tumor vascularization. Experimental and clinical data from various tumor

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types suggest that HIFs also regulate metastasis and treatment resistance, which account for the majority of cancer-related deaths. Thus, HIF inhibitors are likely to target multiple

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important carcinogenetic processes. Intriguingly, HIF-1 and HIF-2 often have opposing effects on tumor growth, for instance on crucial cell cycle and apoptotic regulators like c-

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MYC and p53. Consequently, specific HIF-1 or HIF-2 inhibition, depending on the tumor

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type, is likely to be required for successful clinical exploitation of HIF action.

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Precision-based targeting of specific growth factors/receptors and/or pathways is currently being tested in numerous preclinical and clinical trials and some personalized therapies, such as trastuzumab in breast cancer, are now standard care. Although the targeted approach has been effective in some tumor types, most if not all of the cancers treated with targeted drugs eventually become resistant. In addition, intratumoral heterogeneity and clonal evolution, which are common in solid tumors, are also likely to contribute to resistance to some targeted therapies. In contrast, targeting the hypoxic phenotype represents a more general approach to eradicate malignant cells. Importantly, dysregulated HIF-2 is common in various tumor types regardless of their genetic and molecular diversity, and HIF-2 inhibition could potentially overcome the resistance associated with growth factor receptor inhibition (Franovic, Holterman, Payette, & Lee, 2009). In particular, glioblastoma, ccRCC, and neuroblastoma represent tumor types that might be particularly vulnerable to HIF-2 inhibition.

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ACCEPTED MANUSCRIPT Pheochromocytomas and paragangliomas are also interesting tumor types from the perspective of HIF-2 inhibition due to their HIF2A mutations and mutations in VHL and SDH

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causing a pseudohypoxic phenotype. To optimize their effects, HIF inhibitors should be combined with conventional chemotherapy and/or IR. Recent progress in clinical

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immunotherapy and discoveries into how HIFs regulate the tumor immune response suggest

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that combined immunotherapy and HIF inhibition is likely to be a powerful therapeutic

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approach.

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Figure Legends

Figure 1. Hallmarks of cancer regulated by hypoxia and HIFs.

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Hypoxia and HIFs regulate multiple cancer phenotypes. Targeting hypoxia/HIFs will thus

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inhibit several traits of tumor progression, metastasis, and treatment resistance. Abbreviations:

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VEGF, vascular endothelial growth factor; PDGF-β, platelet-derived growth factor-β; ANGPT2, angiopoietin 2; oxphos, oxidative phosphorylation; SSB, single-stranded break; TAM, tumor-associated macrophage; CTL, cytotoxic T-lymphocyte; Treg, regulatory T-cell; MDSC, myeloid-derived suppressor cell; DC, dendritic cell; MDR, multi-drug resistance; ROS, reactive oxygen species; IR, ionizing radiation Figure 2. Regulation of HIF-α under normoxic and hypoxic conditions. Under normoxic conditions, HIF-α undergoes hydroxylation at conserved residues, a process mediated by prolyl-4-hydroxylases (PHDs) and factor inhibiting HIF-1 (FIH-1) enzymes. Hydroxylation by PHDs promotes destabilization of the HIF-α protein, while hydroxylation by FIH-1 inhibits transcriptional activity by preventing interaction with CBP/p300. Degradation of HIF-α is mediated via an ubiquitin-dependent process executed by the VHLcomplex. Inactivation of PHDs and FIH-1 under hypoxic conditions leads to HIF-α 39

ACCEPTED MANUSCRIPT stabilization followed by translocation into the nucleus and dimerization with HIF-1β/ARNT to form the HIF transcription factor. Together with the co-activator CBP/p300, HIFs induce

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transcription of a plethora of target genes during hypoxia. The small molecules that can

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modulate HIF activity at different levels are also shown.

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Figure 3. Domain structures of HIF-α isoforms.

The HIF-α subunits contain a basic HLH (helix-loop-helix) domain for DNA-specific binding

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and two sequential PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) that facilitate heterodimerization with HIF-1β/ARNT. There are two transcription-activation domains

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(TADs), one N-terminal domain (N-TAD), and one C-terminal domain (C-TAD). The NTAD is located in the oxygen-dependent degradation (ODD) domain. Conserved residues

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localization signal.

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where PHDs and FIH-1 hydroxylate HIF-α are specified. Abbreviation: NLS, nuclear

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Figure 4. Activation mechanism of hypoxic prodrugs. The prodrug can be converted into its active compound either by one-electron or two-electron reduction. One-electron reduction produces a prodrug radical that can be further reduced into the active toxic compound in hypoxic cells or be re-oxidized back to the prodrug in normoxic cells. Alternatively, some prodrugs are reduced by two-electron reductases, thus bypassing the formation of a prodrug radical. Table 1. Inhibitors of HIF activity by different mechanisms and targets. Abbreviations: PI3K, phosphoinositide 3-kinase; mTOR, mechanistic/mammalian target of rapamycin; Hsp90, heat shock protein 90; HDAC, histone deacetylase; IRP1, iron-responsive element-binding protein 1; IRE, iron response element; HRE, hypoxia response element; FIH, factor inhibiting HIF; Hsp70, heat shock protein 70. Table 2. Hypoxia prodrugs in clinical trials. 40

ACCEPTED MANUSCRIPT Table 3. Animal models of the neural crest-derived tumors neuroblastoma and pheochromocytoma/paraganglioma.

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Abbreviations: BM, bone marrow; GEMMS, genetically engineered mouse models; NB,

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neuroblastoma; PGL, paraganglioma; PDX, patient-derived xenograft; Pheo, pheochromocytoma

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Acknowledgments

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This work was supported by grants from the Berta Kamprad Foundation, the Swedish Cancer Society, the Swedish Childhood Cancer Foundation, the Swedish Research Council,

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VINNOVA, the SSF Strategic Center for Translational Cancer Research-CREATE Health, the Strategic Cancer Research Program BioCARE, Crafoord Foundation, The Royal

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Physiographic Society in Lund, Gunnar Nilsson’s Cancer Foundation, Hans von Kantzows’

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Foundation, Jeanssons Stiftelser, The Krapperup Foundation, Region Skåne and Skåne

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University Hospital research funds.

Conflict of interest:

The authors declare that there are no conflicts of interest.

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inducible factor-1alpha with reduced transcriptional activity mediates the antitumor effect of triptolide. Mol Cancer, 9, 268. Zhuang, Z., Yang, C., Lorenzo, F., Merino, M., Fojo, T., Kebebew, E., Popovic, V., Stratakis, C. A., Prchal, J. T., & Pacak, K. (2012). Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. N Engl J Med, 367, 922-930. Zimmer, M., Ebert, B. L., Neil, C., Brenner, K., Papaioannou, I., Melas, A., Tolliday, N., Lamb, J., Pantopoulos, K., Golub, T., & Iliopoulos, O. (2008). Small-molecule inhibitors of HIF-2a translation link its 5'UTR iron-responsive element to oxygen sensing. Mol Cell, 32, 838-848.

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References

Wortmannin LY94002 GDC-0941 PI-103 Rapamycin PP242 Aminoflavone Glyceollins

PI3K PI3K PI3K PI3K mTOR mTOR unknown AKT/mTOR, Hsp90

Topotecan, PEG-SN38 EZN-2968

Topoisomerase 1 HIF-1α mRNA

2ME2, ENMD-1198 Geldanamycin and analogues Vorinostat YC-1 PX-478 PX-12, pleurotin Cardiac glycosides FM19G11 HIF-2α translational inhibitors

Microtubules

Jiang et al. 2001 Jiang et al. 2001, Mohlin et al. 2015 Mohlin et al. 2015 Mohlin et al. 2015 Hudson et al. 2002 Mohlin et al. 2015 Terzuoli et al. 2010 Lee et al. 2015 Rapisarda et al. 2004, Pastorino et al. 2010 Greenberger et al. 2008 Mabjeesh et al. 2003, LaVallee et al. 2008

DNA binding

Transcriptional activity

RI

SC

NU

Isaacs et al. 2002 Hutt et al. 2014 Chun et al. 2001, Li et al. 2008 Koh et al. 2008 Welsh et al. 2003 Zhang et al. 2008 Moreno-Manzano et al. 2010

IRP1/IRE interaction

Zimmer et al. 2008

HIF-1α/2α PAS-B domain HIF-2α PAS-B domain

Lee et al. 2009 Scheuermann et al. 2013

Echinomycin Polyamides

HRE HRE

Kong et al. 2005 Olenyuk et al. 2004

Chetomin

p300 recruitment

Bortezomib

p300 recruitment FIH-1 interaction and p300 recruitment Hsp70 Thioredoxin-1

Kung et al. 2004 Kaluz et al. 2006, Shin et al. 2008

MA

Hsp90 HDAC unknown unknown Thioredoxin-1 unknown unknown

TE

Acriflavine PT2385

AC CE P

HIF-α / HIF-1β dimerization

PT

Drug

D

Inhibitory mechanism mRNA/protein expression

Amphotericin B Triptolide AJM290, AW464

Yeo et al. 2006 Zhou et al. 2010 Jones et al. 2006

PI3K, phosphoinositide 3-kinase; mTOR, mechanistic/mammalian target of rapamycin; Hsp90, heat shock protein 90; HDAC, histone deacetylase; IRP1, iron-responsive element-binding protein 1; IRE, iron response element; HRE, hypoxia response element; FIH, factor inhibiting HIF; Hsp70, heat shock protein 70.

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ACCEPTED MANUSCRIPT Table 2. Examples of hypoxia prodrugs in preclinical and clinical trials.

Clinical trial status Phase III Phase III (active) Phase III (completed)

Cancer Soft tissue sarcoma Pancreatic cancer Bladder cancer

PT

Prodrug TH-302 (Evofosfamide) EO9 (Apaziquone)

SC

Multiple

NU

PR-104 Tirapazamine (TPZ)

Phase I/II Phase I /II Phase I Phase I/II (completed) Phase III (completed)

Acute leukemia Cervical cancer Head and neck cancer

MA

AQ4N

RI

Phase III (active)

Preclinical

TE

Phase II (active)

Non small cell lung cancer Squamous cell carcinoma

NCT02454842 NCT02449681

AC CE P

SN30000 (CEN-209) TH-4000

D

Lung cancer

NCT NCT01440088 NCT01746979 NCT00598806 NCT00461591 NCT01410565 NCT02563561 NCT01469221 NCT00394628 NCT00109356 NCT00090727 NCT01037556 NCT00262821 NCT00174837 NCT00094081 NCT00017459 NCT00006484

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ACCEPTED MANUSCRIPT Table 3. Animal models of neuroblastoma and pheochromocytoma/paraganglioma.

Model

Tumor

Metastasis

Reference(s)

NB NB

Rare Multiple incl. BM

NB

No

NB

No

TH-MYCN/Caspase-8-deleted

NB

BM

Lin28b overexpression

NB

Not reported

NF1+/-

Pheo

Ink4a Arf-/-

Pheo

PSA-Cre;Pten-loxP/loxP

Pheo

Met918Thr

Pheo

c-mos

Pheo

Conventional cell-line derived Orthotopic PDX

TE

Pheo/PGL

Berry, Cancer Cell, 2012 Teitz, Cancer Res, 2013

Molenaar, Nat Genetics, 2012

Not reported

Jacks, Nat Genetics, 1994

Lungs

You, PNAS, 2002

Lungs

Korpershoek, J Pathol, 2009

No

Smith-Hicks, EMBO J, 2000

Not reported

Schulz, Cancer Res, 1992

Fritz, Cancer Res, 2002 Not reported

Pellegata, PNAS, 2006

AC CE P

Cdkn1b -/-

D

Germline

SC

/MYCN

Weiss, EMBO J,1997

NU

ALK

F1174L

MA

TH-MYCN

Khanna, In vivo, 202 Braekeveldt, Int J Cancer, 2015

RI

GEMMs

PT

Xenografts

69