DUBs, Hypoxia, and Cancer

Ubiquitylation and its reversal by DUBs is key in the control of protein homeostasis, degradation, trafficking, localization, and activity. Dysfunction can lead to various diseases including cancer.HIFα transcription factors are important for the physiologic hypoxia response but also affect various processes during carcinogenesis, including ubiquitylation, through known and, as yet, unknown mechanisms.The abundance of HIF-1α and HIF-2α proteins is largely increased in various cancers with high mortality. HIFα proteins are attractive targets in cancer and, since they are regulated by ubiquitylation and proteasomal degradation, DUB inhibitors may provide a therapeutic option.However, the use of DUB inhibitors in cancer therapy is complicated by the fact that several DUBs are downregulated by hypoxia, and by the uncertainty to which extent the action of DUB inhibitors are specific/selective, as well as the co-occurrence of hypoxia-mediated downregulation of proteasomal components. Alterations in protein ubiquitylation and hypoxia are commonly associated with cancer. Ubiquitylation is carried out by three sequentially acting ubiquitylating enzymes and can be opposed by deubiquitinases (DUBs), which have emerged as promising drug targets. Apart from protein localization and activity, ubiquitylation regulates degradation of proteins, among them hypoxia-inducible factors (HIFs). Thereby, various E3 ubiquitin ligases and DUBs regulate HIF abundance. Conversely, several E3s and DUBs are regulated by hypoxia. While hypoxia is a powerful HIF regulator, less is known about hypoxia-regulated DUBs and their impact on HIFs. Here, we review current knowledge about the relationship of E3s, DUBs, and hypoxia signaling. We also discuss the reciprocal regulation of DUBs by hypoxia and use of DUB-specific drugs in cancer. Alterations in protein ubiquitylation and hypoxia are commonly associated with cancer. Ubiquitylation is carried out by three sequentially acting ubiquitylating enzymes and can be opposed by deubiquitinases (DUBs), which have emerged as promising drug targets. Apart from protein localization and activity, ubiquitylation regulates degradation of proteins, among them hypoxia-inducible factors (HIFs). Thereby, various E3 ubiquitin ligases and DUBs regulate HIF abundance. Conversely, several E3s and DUBs are regulated by hypoxia. While hypoxia is a powerful HIF regulator, less is known about hypoxia-regulated DUBs and their impact on HIFs. Here, we review current knowledge about the relationship of E3s, DUBs, and hypoxia signaling. We also discuss the reciprocal regulation of DUBs by hypoxia and use of DUB-specific drugs in cancer. Cancer growth is associated with excessive protein synthesis and degradation. Among the pathways that regulate protein homeostasis, proteasomal protein degradation mediated by ubiquitylation (see Glossary) is gaining attention in cancer therapy because it is highly selective and, as a result, proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib have been approved for mantle cell lymphoma and multiple myeloma therapy [1Moreau P. et al.Proteasome inhibitors in multiple myeloma: 10 years later.Blood. 2012; 120: 947-959Crossref PubMed Scopus (300) Google Scholar]. In addition, cancer growth is also associated with the development of limited oxygenation (commonly called tumor hypoxia). This is often exemplified by an increased amount of proteins from the hypoxia-inducible transcription factor-α family (HIFs). Commonly, in cancers of the bladder, brain, breast, colon, esophagus, head/neck, liver, lung, pancreas, skin, stomach, and uterus, as well as in acute lymphocytic and myeloid leukemias, the appearance of HIFs is associated with a poor prognosis [2Semenza G.L. Oxygen sensing, hypoxia-inducible factors, and disease pathophysiology.Annu. Rev. Pathol. 2014; 9: 47-71Crossref PubMed Scopus (404) Google Scholar]. While hypoxia appears to be a powerful regulator of the HIF ubiquitylation process [3Ivan M. Kaelin Jr., W.G. The EGLN-HIF O2-sensing system: multiple inputs and feedbacks.Mol. Cell. 2017; 66: 772-779Abstract Full Text Full Text PDF PubMed Google Scholar], much less is known about hypoxia-regulated deubiquitinases (DUBs) and the impact of DUBs on the HIF system. DUBs act as both tumor suppressors and oncogenes and have thus emerged as promising therapeutic targets in cancer. Here, we summarize the current knowledge about the reciprocal connection of DUBs with hypoxia signaling and the use of DUB-specific drugs in cancer. Ubiquitylation is a process where the small molecule ubiquitin is attached to lysine (K) residues in target proteins, either as monoubiquitin or in polyubiquitin chains. This requires three different types of enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-ligases (E3). Although ubiquitylation is mainly involved in the regulation of degradation of proteins by the 26S proteasome, it also affects many other cellular processes, such as transcription and DNA repair, cell cycle control, inflammation, and apoptosis [4Komander D. Rape M. The ubiquitin code.Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (1254) Google Scholar] (Figure 1 and Box 1).Box 1Functional Consequences of Protein UbiquitylationThe diversity of ubiquitin-modified target proteins is reflected in the number of different E3 enzymes. In humans, about 30–40 E2 and more than 600 different E3 ligases are known. Depending on their substrate specificity, E3 ligases can often act as a tumor suppressor or oncogene. E3 ligases can be divided into two main classes: (i) homologous to the E6-AP-carboxyl terminus (HECT) domain E3s, or (ii) really interesting new gene (RING) domain E3s. While HECT E3 ligases transfer ubiquitin first from the E2 to the HECT active-site cysteine and from there to a lysine in the substrate, RING E3 ligases (and the structurally related U-box E3s) act as a scaffold and transfer ubiquitin directly from the E2 to the substrate lysine. In the cell, the majority of E3s (∼92%) are RING-domain-containing E3s and the rest are HECT and other smaller families of ligases (plant homology domain, zinc finger, and U-box) [87De Bie P. Ciechanover A. Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms.Cell Death Differ. 2011; 18: 1393-1402Crossref PubMed Scopus (118) Google Scholar].Three major ubiquitylations with different functional consequences are known: (i) mono-ubiquitylation; (ii) multi-ubiquitylation (i.e., attachment of several single ubiquitin molecules to the target protein); and (iii) polyubiquitylation [i.e., attachment of polyubiquitin chains to one or several lysines (K) of the substrate].Figure IAbbreviations: E2, ubiquitin-conjugating enzyme; E3, ubiquitin-ligase; HECT, homologous to the E6-AP-carboxyl terminus; RING, really interesting new gene; Ub, ubiquitin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The diversity of ubiquitin-modified target proteins is reflected in the number of different E3 enzymes. In humans, about 30–40 E2 and more than 600 different E3 ligases are known. Depending on their substrate specificity, E3 ligases can often act as a tumor suppressor or oncogene. E3 ligases can be divided into two main classes: (i) homologous to the E6-AP-carboxyl terminus (HECT) domain E3s, or (ii) really interesting new gene (RING) domain E3s. While HECT E3 ligases transfer ubiquitin first from the E2 to the HECT active-site cysteine and from there to a lysine in the substrate, RING E3 ligases (and the structurally related U-box E3s) act as a scaffold and transfer ubiquitin directly from the E2 to the substrate lysine. In the cell, the majority of E3s (∼92%) are RING-domain-containing E3s and the rest are HECT and other smaller families of ligases (plant homology domain, zinc finger, and U-box) [87De Bie P. Ciechanover A. Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms.Cell Death Differ. 2011; 18: 1393-1402Crossref PubMed Scopus (118) Google Scholar]. Three major ubiquitylations with different functional consequences are known: (i) mono-ubiquitylation; (ii) multi-ubiquitylation (i.e., attachment of several single ubiquitin molecules to the target protein); and (iii) polyubiquitylation [i.e., attachment of polyubiquitin chains to one or several lysines (K) of the substrate]. DUBs are enzymes that can reverse ubiquitylation. The human genome encodes for up to 100 DUBs, divided into seven subgroups, depending on sequence and structure. The largest and most heterogeneous group consists of the ubiquitin-specific proteases (USPs); other groups are the ubiquitin carboxyl-terminal hydrolases (UCHs), the otubain/ovarian tumor-domain containing proteins (OTUs), the Machado-Joseph disease domain superfamily (MJDs), the JAB1/MPN/MOV34 proteases (JAMMs), the monocyte chemotactic protein-induced proteins (MCPIPs), and the novel motif interacting with ubiquitin-containing DUB family (MINDY). All DUBs are cysteine proteases [5Fraile J.M. et al.Deubiquitinases in cancer: new functions and therapeutic options.Oncogene. 2012; 31: 2373-2388Crossref PubMed Scopus (223) Google Scholar, 6Abdul Rehman S.A. et al.MINDY-1 is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes.Mol. Cell. 2016; 63: 146-155Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar], except for JAMMs, which are Zn2+ metalloproteases. The major role of DUBs is the removal of ubiquitin chains from proteins, which results in protein stabilization and protection from proteasomal degradation. In addition, DUBs are involved in: (i) processing and maturation of ubiquitin precursors, (ii) recycling of ubiquitin, and (iii) editing of ubiquitin chains [7Haq S. Ramakrishna S. Deubiquitylation of deubiquitylases.Open Biol. 2017; 7170016Crossref PubMed Scopus (4) Google Scholar]. Thus, it is not a surprise that inappropriate activity or expression of DUBs contributes to many diseases, including cancer, by directly or indirectly affecting various signaling pathways. As a result, exploration of DUBs and drugs modifying the action of DUBs has become an important research focus world-wide [8Heideker J. Wertz I.E. DUBs, the regulation of cell identity and disease.Biochem. J. 2015; 465: 1-26Crossref PubMed Scopus (0) Google Scholar]. Hypoxia is seen in many solid tumors due to rapidly increasing cell proliferation and increasing tumor mass. To overcome low oxygen, cancer cells activate several survival pathways and the formation of tumor vasculature [9Muz B. et al.The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy.Hypoxia (Auckl). 2015; 3: 83-92Crossref PubMed Google Scholar]. The HIFs are key regulators of this adaptive program. There are three known HIF α-subunits (Box 2) labile in the presence of oxygen (O2) that become stabilized under hypoxia and form heterodimers with the beta-subunit ARNT and then bind to hypoxia responsive elements (HREs) in target genes [10Suzuki N. et al.Regulation of hypoxia-inducible gene expression after HIF activation.Exp. Cell Res. 2017; 356: 182-186Crossref PubMed Scopus (9) Google Scholar]. Recent findings indicate that HIF-1α and HIF-2α containing complexes occupy distinct genomic sites, which vary with cell type. Further, none of the two HIFα subunits can compensate for the lack of the other protein [11Smythies J.A. et al.Inherent DNA-binding specificities of the HIF-1alpha and HIF-2alpha transcription factors in chromatin.EMBO Rep. 2019; e46401: 20Google Scholar]. This suggests overlapping and distinct functions for these subunits, with HIF-1α promoting an acute response to hypoxia and HIF-2α promoting a chronic response [12Murugesan T. et al.Targeting HIF-2alpha as therapy for advanced cancers.Drug Discov. Today. 2018; 23: 1444-1451Crossref PubMed Scopus (4) Google Scholar]. The function of HIF-3α and its numerous splice variants [13Heikkila M. et al.Roles of the human hypoxia-inducible factor (HIF)-3alpha variants in the hypoxia response.Cell. Mol. Life Sci. 2011; 68: 3885-3901Crossref PubMed Scopus (0) Google Scholar] is not known in detail, but some variants appear to activate gene expression [14Zhang P. et al.Molecular, functional, and gene expression analysis of zebrafish hypoxia-inducible factor-3alpha.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2012; 303: R1165-R1174Crossref PubMed Scopus (0) Google Scholar, 15Scharf J.G. et al.Oxygen-dependent modulation of insulin-like growth factor binding protein biosynthesis in primary cultures of rat hepatocytes.Endocrinology. 2005; 146: 5433-5443Crossref PubMed Scopus (0) Google Scholar], while others might be negative regulators of HIF-1α and HIF-2α [16Hara S. et al.Expression and characterization of hypoxia-inducible factor (HIF)-3alpha in human kidney: suppression of HIF-mediated gene expression by HIF-3alpha.Biochem. Biophys. Res. Commun. 2001; 287: 808-813Crossref PubMed Scopus (172) Google Scholar, 17Makino Y. et al.Inhibitory PAS domain protein (IPAS) is a hypoxia-inducible splicing variant of the hypoxia-inducible factor-3alpha locus.J. Biol. Chem. 2002; 277: 32405-32408Crossref PubMed Scopus (0) Google Scholar, 18Wang V. et al.Differential gene up-regulation by hypoxia-inducible factor-1alpha and hypoxia-inducible factor-2alpha in HEK293T cells.Cancer Res. 2005; 65: 3299-3306Crossref PubMed Scopus (60) Google Scholar].Box 2Oxygen-Dependent Degradation of HIFsAll three different HIF α-subunits contain a bHLH, two Per-ARNT-Sim (PAS-A and PAS-B) domains, a nuclear localization signal, and an oxygen-dependent degradation domain (ODD) (Figure IA). The transactivation domains (N-TAD and C-TAD) are required for the transcriptional activity of HIF-1α and HIF-2α. The ODD is located within the N-TAD. HIF-3α subunit lacks the C-TAD and instead contains a leucine-zipper (L-ZIP) domain. The protein stability of HIFα is primarily regulated by the oxygen-dependent hydroxylation of two proline residues (P402 and P564 in human HIF-1α; P405 and P531 in human HIF-2α; P492 in human HIF-3α) (Figure IB). The hydroxylations are performed by at least three proline 4-hydroxylase domain-containing enzyme family members [known as PHD1-3 or EGL Nine (Caenorhabditis elegans) Homologs (EGLN1-3)], which function as cellular O2 sensors. Apart from O2, the reaction also requires 2-oxoglutarate (2-OG), Fe2+, and ascorbate. The proline residue hydroxylation tags the HIF α-subunits for ubiquitylation by the von Hippel-Lindau (VHL)-containing E3 ligase complex and for subsequent proteasomal degradation. So far, the tumor suppressor protein VHL is the only reported E3 ligase substrate-recognition component in the oxygen-dependent HIFα degradation. The mutation of the VHL gene in patients leads to VHL syndrome, which is (also due to high HIF levels) frequently associated with angiomatosis, hemangioblastomas, pheochromocytomas, and renal clear cell carcinomas [92Kaelin Jr., W.G. The VHL tumor suppressor gene: insights into oxygen sensing and cancer.Trans. Am. Clin. Climatol. Assoc. 2017; 128: 298-307PubMed Google Scholar].HIF α-subunits are also hydroxylated by the factor inhibiting HIF (FIH) at a C-terminal asparagine residue. As a result, recruitment of the transcriptional coactivator p300 is prevented [93Markolovic S. et al.Protein hydroxylation catalyzed by 2-oxoglutarate-dependent oxygenases.J. Biol. Chem. 2015; 290: 20712-20722Crossref PubMed Scopus (59) Google Scholar]. The latter action is specific for HIF-1α and HIF-2α, since even full-length HIF-3α lacks a C-terminal transactivation domain.If oxygen becomes limited, the HIF prolyl 4-hydroxylases and FIH become inhibited and HIFs escape proteasomal degradation, which allows their accumulation, nuclear transport, heterodimerization with ARNT, recruitment of coactivators, and activation of target gene expression. Although both FIH and PHDs belong to the 2-OG-dependent dioxygenases, they are not functionally redundant as FIH has a higher O2 affinity than PHD [94Ehrismann D. et al.Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay.Biochem. J. 2007; 401: 227-234Crossref PubMed Scopus (140) Google Scholar] and HIF-1α is more susceptible to FIH modification than HIF-2α [95Bracken C.P. et al.The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses.Cell. Mol. Life Sci. 2003; 60: 1376-1393Crossref PubMed Scopus (162) Google Scholar]. Based on this, FIH maintains sufficient activity at intermediate levels of hypoxia where PHDs are inactive and enable stabilization of HIF-1α. Further, the presence of active FIH in normoxic pVHL-defective kidney cancers does not prevent from -involving a HIF program because these tumors are driven largely by HIF-2 rather than HIF-1 [92Kaelin Jr., W.G. The VHL tumor suppressor gene: insights into oxygen sensing and cancer.Trans. Am. Clin. Climatol. Assoc. 2017; 128: 298-307PubMed Google Scholar]. However, FIH seems to be physiologically important in situations with a rapid onset of hypoxia (e.g., ischemia) and in tissues largely depending on oxidative metabolism, such as heart and skeletal muscle. Indeed, recent findings from FIH-deficient mice led to a model where loss of FIH accelerates oxidative processes. In turn, this would reduce intracellular O2 levels, potentiate PHD inhibition, and lead to HIF-1α accumulation. Consequently, HIF accumulation would promote hypoxic adaptation [96Sim J. et al.The factor inhibiting HIF asparaginyl hydroxylase regulates oxidative metabolism and accelerates metabolic adaptation to hypoxia.Cell Metab. 2018; 27: 898-913Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar].Importantly, hypoxia promotes a feedback by activating EGLN1 (PHD2) and EGLN3 (PHD3) gene expression via HIFs. As a result, this leads to rehydroxylation and degradation of at least HIF-1α [97Ginouvès A. et al.PHDs overactivation during chronic hypoxia 'desensitizes' HIFa and protects cells from necrosis.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4745-4750Crossref PubMed Scopus (0) Google Scholar]. All three different HIF α-subunits contain a bHLH, two Per-ARNT-Sim (PAS-A and PAS-B) domains, a nuclear localization signal, and an oxygen-dependent degradation domain (ODD) (Figure IA). The transactivation domains (N-TAD and C-TAD) are required for the transcriptional activity of HIF-1α and HIF-2α. The ODD is located within the N-TAD. HIF-3α subunit lacks the C-TAD and instead contains a leucine-zipper (L-ZIP) domain. The protein stability of HIFα is primarily regulated by the oxygen-dependent hydroxylation of two proline residues (P402 and P564 in human HIF-1α; P405 and P531 in human HIF-2α; P492 in human HIF-3α) (Figure IB). The hydroxylations are performed by at least three proline 4-hydroxylase domain-containing enzyme family members [known as PHD1-3 or EGL Nine (Caenorhabditis elegans) Homologs (EGLN1-3)], which function as cellular O2 sensors. Apart from O2, the reaction also requires 2-oxoglutarate (2-OG), Fe2+, and ascorbate. The proline residue hydroxylation tags the HIF α-subunits for ubiquitylation by the von Hippel-Lindau (VHL)-containing E3 ligase complex and for subsequent proteasomal degradation. So far, the tumor suppressor protein VHL is the only reported E3 ligase substrate-recognition component in the oxygen-dependent HIFα degradation. The mutation of the VHL gene in patients leads to VHL syndrome, which is (also due to high HIF levels) frequently associated with angiomatosis, hemangioblastomas, pheochromocytomas, and renal clear cell carcinomas [92Kaelin Jr., W.G. The VHL tumor suppressor gene: insights into oxygen sensing and cancer.Trans. Am. Clin. Climatol. Assoc. 2017; 128: 298-307PubMed Google Scholar]. HIF α-subunits are also hydroxylated by the factor inhibiting HIF (FIH) at a C-terminal asparagine residue. As a result, recruitment of the transcriptional coactivator p300 is prevented [93Markolovic S. et al.Protein hydroxylation catalyzed by 2-oxoglutarate-dependent oxygenases.J. Biol. Chem. 2015; 290: 20712-20722Crossref PubMed Scopus (59) Google Scholar]. The latter action is specific for HIF-1α and HIF-2α, since even full-length HIF-3α lacks a C-terminal transactivation domain. If oxygen becomes limited, the HIF prolyl 4-hydroxylases and FIH become inhibited and HIFs escape proteasomal degradation, which allows their accumulation, nuclear transport, heterodimerization with ARNT, recruitment of coactivators, and activation of target gene expression. Although both FIH and PHDs belong to the 2-OG-dependent dioxygenases, they are not functionally redundant as FIH has a higher O2 affinity than PHD [94Ehrismann D. et al.Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay.Biochem. J. 2007; 401: 227-234Crossref PubMed Scopus (140) Google Scholar] and HIF-1α is more susceptible to FIH modification than HIF-2α [95Bracken C.P. et al.The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses.Cell. Mol. Life Sci. 2003; 60: 1376-1393Crossref PubMed Scopus (162) Google Scholar]. Based on this, FIH maintains sufficient activity at intermediate levels of hypoxia where PHDs are inactive and enable stabilization of HIF-1α. Further, the presence of active FIH in normoxic pVHL-defective kidney cancers does not prevent from -involving a HIF program because these tumors are driven largely by HIF-2 rather than HIF-1 [92Kaelin Jr., W.G. The VHL tumor suppressor gene: insights into oxygen sensing and cancer.Trans. Am. Clin. Climatol. Assoc. 2017; 128: 298-307PubMed Google Scholar]. However, FIH seems to be physiologically important in situations with a rapid onset of hypoxia (e.g., ischemia) and in tissues largely depending on oxidative metabolism, such as heart and skeletal muscle. Indeed, recent findings from FIH-deficient mice led to a model where loss of FIH accelerates oxidative processes. In turn, this would reduce intracellular O2 levels, potentiate PHD inhibition, and lead to HIF-1α accumulation. Consequently, HIF accumulation would promote hypoxic adaptation [96Sim J. et al.The factor inhibiting HIF asparaginyl hydroxylase regulates oxidative metabolism and accelerates metabolic adaptation to hypoxia.Cell Metab. 2018; 27: 898-913Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Importantly, hypoxia promotes a feedback by activating EGLN1 (PHD2) and EGLN3 (PHD3) gene expression via HIFs. As a result, this leads to rehydroxylation and degradation of at least HIF-1α [97Ginouvès A. et al.PHDs overactivation during chronic hypoxia 'desensitizes' HIFa and protects cells from necrosis.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4745-4750Crossref PubMed Scopus (0) Google Scholar]. The abundance of HIF α-subunits is mostly regulated on the post-translational level, although mechanisms of HIF transcription and HIF-1α mRNA translation have been described [19Kuschel A. et al.Functional regulation of HIF-1alpha under normoxia--is there more than post-translational regulation?.J. Cell. Physiol. 2012; 227: 514-524Crossref PubMed Scopus (0) Google Scholar]. In the presence of oxygen, oxygen-dependent hydroxylation of HIF α-subunits, coupled with subsequent recruitment of the von Hippel-Lindau (VHL) E3-ubiquitin ligase, leads to rapid HIF α-subunit ubiquitylation and its degradation by the proteasome. In contrast, the stability of HIF α-subunits increases in the absence of oxygen (Box 2). Although the proteasome is the primary site for HIF degradation, lysosomes also appear to contribute to HIF-1α degradation under certain circumstances, such as chaperone-mediated autophagy [20Hubbi M.E. et al.Chaperone-mediated autophagy targets hypoxia-inducible factor-1alpha (HIF-1alpha) for lysosomal degradation.J. Biol. Chem. 2013; 288: 10703-10714Crossref PubMed Scopus (96) Google Scholar]. Intriguingly, enzymes mediating hydroxylation reactions are also subject to ubiquitylation and proteasomal degradation (Box 3).Box 3Regulation of HIF Hydroxylases by UbiquitylationWhile oxygen itself regulates the activity of HIF hydroxylases, their abundance is regulated by ubiquitylation and subsequent degradation by the proteasome. The stability of the HIF prolyl 4-hydroxylases PHD1 and PHD3 is controlled by the seven in absentia homolog (SIAH1/2) E3 ligases; no E3 ligases regulating PHD2 abundance have been identified yet. Interestingly, hypoxia induced SIAH1/2 activity, as a result PHD1/3 were reduced and HIF-1α stabilized [98Nakayama K. Ronai Z. Siah: new players in the cellular response to hypoxia.Cell Cycle. 2004; 3: 1345-1347Crossref PubMed Google Scholar]. SIAH1 could also mediate FIH degradation via the proteasomal pathway under hypoxic conditions [99Fukuba H. et al.Abundance of aspargynyl-hydroxylase FIH is regulated by Siah-1 under normoxic conditions.433. 2008: 209-214Google Scholar]. Altogether, SIAH E3 ubiquitin ligases are an important part of a feedback regulation being able to affect both types of HIF hydroxylases: PHDs and FIH.In addition to SIAH1/2, a Cullin 3 and speckle-type POZ protein (SPOP) containing E3 ligase complex was reported to regulate PHD1 via polyubiquitylation and proteasomal degradation. Lack of SPOP and subsequently increased PHD1 levels could also be linked to prostate cancer progression [100Zhang L. et al.Tumor suppressor SPOP ubiquitinates and degrades EglN2 to compromise growth of prostate cancer cells.Cancer Lett. 2017; 390: 11-20Crossref PubMed Google Scholar].Conversely, HIF hydroxylases were also reported to regulate E3 ligases. In particular, the ankyrin repeat and SOCS box protein 4 (ASB4), which serves as the substrate-recognizing protein in the SCF-like Elongin-Cullin-SOCS-box E3 ubiquitin ligase complex, was found to be hydroxylated by FIH, resulting in oxygen-dependent vascular differentiation [101Ferguson III, J.E. et al.ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism.Mol. Cell. Biol. 2007; 27: 6407-6419Crossref PubMed Scopus (0) Google Scholar]. While oxygen itself regulates the activity of HIF hydroxylases, their abundance is regulated by ubiquitylation and subsequent degradation by the proteasome. The stability of the HIF prolyl 4-hydroxylases PHD1 and PHD3 is controlled by the seven in absentia homolog (SIAH1/2) E3 ligases; no E3 ligases regulating PHD2 abundance have been identified yet. Interestingly, hypoxia induced SIAH1/2 activity, as a result PHD1/3 were reduced and HIF-1α stabilized [98Nakayama K. Ronai Z. Siah: new players in the cellular response to hypoxia.Cell Cycle. 2004; 3: 1345-1347Crossref PubMed Google Scholar]. SIAH1 could also mediate FIH degradation via the proteasomal pathway under hypoxic conditions [99Fukuba H. et al.Abundance of aspargynyl-hydroxylase FIH is regulated by Siah-1 under normoxic conditions.433. 2008: 209-214Google Scholar]. Altogether, SIAH E3 ubiquitin ligases are an important part of a feedback regulation being able to affect both types of HIF hydroxylases: PHDs and FIH. In addition to SIAH1/2, a Cullin 3 and speckle-type POZ protein (SPOP) containing E3 ligase complex was reported to regulate PHD1 via polyubiquitylation and proteasomal degradation. Lack of SPOP and subsequently increased PHD1 levels could also be linked to prostate cancer progression [100Zhang L. et al.Tumor suppressor SPOP ubiquitinates and degrades EglN2 to compromise growth of prostate cancer cells.Cancer Lett. 2017; 390: 11-20Crossref PubMed Google Scholar]. Conversely, HIF hydroxylases were also reported to regulate E3 ligases. In particular, the ankyrin repeat and SOCS box protein 4 (ASB4), which serves as the substrate-recognizing protein in the SCF-like Elongin-Cullin-SOCS-box E3 ubiquitin ligase complex, was found to be hydroxylated by FIH, resulting in oxygen-dependent vascular differentiation [101Ferguson III, J.E. et al.ASB4 is a hydroxylation substrate of FIH and promotes vascular differentiation via an oxygen-dependent mechanism.Mol. Cell. Biol. 2007; 27: 6407-6419Crossref PubMed Scopus (0) Google Scholar]. In addition to oxygen-dependent degradation, several signaling pathways control the ubiquitylation and proteasomal degradation of HIF α-subunits in an oxygen (i.e., hydroxylation)-independent manner. Thereby, post-translational modifications such as phosphorylation, acetylation, and SUMOylation [21Masoud G.N. Li W. HIF-1alpha pathway: role, regulation and intervention for cancer therapy.Acta Pharm. Sin. B. 2015; 5: 378-389Crossref PubMed Scopus (197) Google Scholar] recruit E3-ubiquitin ligases to HIF α-subunits. Thus, it is not surprising that several E3 ligases and different DUBs contribute to the regulation of HIF α-subunit abundance (Tables 1 and 2).Table 1DUBs in Hypoxia Signaling and CancerDUBs(gene name)Regulation by hypoxiaInvolvement in HIF signaling regulationInvolvement in cancerRefsUSP1(USP1)↓aIn vitro.Involved in aplastic anemia and multiple myeloma progressionbIn vivo.Promotes breast cancer metastasis via KPNA2 stabilizationbIn vivo.67Oleksandr H. et al.IRE-1α regulates expression of ubiquitin specific peptidases during hypoxic response in U87 glioma cells.Endoplasm. Reticul. Stress Dis. 2016; 3: 50-62Google Scholar, 102Das D.S. et al.Blockade of deubiquitylating enzyme USP1 inhibits DNA repair and triggers apoptosis in multiple myeloma cells.Clin. Cancer Res. 2017; 23: 4280-4289Crossref PubMed Scopus (10) Google Scholar, 103Ma A. et al.USP1 inhibition destabilizes KPNA2 and suppresses breast cancer metastasis.Oncogene. 2019; 38: 2405-2419Crossref PubMed Scopus (1) Google ScholarUSP7(USP7)Deubiquitylation of HIF-1αaIn vitro.