Hypoxia-mediated activation of hypoxia-inducible factor-1α in head and neck squamous cell carcinoma: A review

Since the 1950s, hypoxia has been recognized as a crucial characteristic of cancer cells and their microenvironment. Indeed, hypoxia promotes the growth, survival, and metastasis of cancer cells. In the early 1990s, we found that as many phenomena in hypoxia can occur through hypoxia-inducible factor-1α (HIF1α). HIF1α is known as an angiogenesis converter in hypoxia, which promotes tumorigenesis, development, immune escape, recurrence, etc; This page goes into great detail on how HIF1α is activated during hypoxia and how the 2 signaling channels interact. It specifically emphasizes the significance of reactive oxygen species, the function of the PI3K/the serine/threonine kinase Akt/mammalian target of rapamycin cascade, and outlines the similarities between the 2 important factors (reactive oxygen species and PI3K/the serine/threonine kinase Akt/mammalian target of rapamycin cascade), nuclear factor κB, for HIF1α Important implications, in an effort to offer fresh views for the treatment of head and neck squamous cell carcinoma and HIF1α research.


Introduction
Head and neck squamous cell carcinoma (HNSCC), which encompasses a number of squamous cell carcinomas that develop along the mucous membranes of the larynx, hypopharynx, oropharynx, and oral cavity and makes up more than 95% of all head and neck malignancies. [1] With mortality rates of around 40% to 50%, HNSCC is the 6th largest prevalent malignancy worldwide. [1,2] Alcohol and tobacco use, as well as high-risk human papillomaviruses, especially type human papillomaviruses-16, are the main risk factors for HNSCC. [3] Because more than 80% of these tumors have an overexpression of the epidermal growth factor receptor (EGFR), the FDA approved Cetuximab, a monoclonal antibody against EGFR, for the treatment of HNSCC in 2006. However, It has only had modest success. [2,4] Radiotherapy with or without chemotherapeutic drugs such as cisplatin is currently the norm for treating HNSCC.
Semenza and coworkers was initially discovered hypoxia-inducible factor-1 (HIF-1) in 1991 while conducting research on the erythropoietin (EPO) gene, a gene that produces red blood cells by producing the erythropoietin hormone. [5] In this EPO gene's 30-flanking region, cis-acting DNA sequences  were discovered. Hypoxia response elements are DNA sequences that have been identified to be required for the hypoxia-induced transcriptional activation of the EPO gene (HRE). Further research discovered that the binding of a specific protein to the HRE, which is brought on by hypoxia causes numerous regulatory genes to become transcriptionally active. Later, This protein was found to be HIF-1. [6] Medicine Despite recent developments in survival rates, targeted therapy, and patient outcomes for HNSCC have not significantly increased in recent years, which, when combined with the disease's increasing occurrence, increases the need for more potent treatment options. [1,2,4] Hypoxia or scarcity of chemical elements may be markers of solid tumors, and they adapt by activating HIF-1, a transcription problem that triggers the expression of quasi-links in nursing angiogenic molecules in an anoxic atmosphere. Redox state controls the expression of angiogenic molecules, including HIF-1, as a result of its activation. The tumor microenvironment can be affected in a number of ways through alterations in the redox atmosphere. Unfortunately, tumor hypoxia reduces tumor angiogenesis and radiation efficacy, increasing therapeutic resistance and cancer recurrence, as previously mentioned. The hypoxic environment is triggered by the HIF switch. HIF has an impact on a variety of physiological processes, including apoptosis, invasion/ metastasis, immunity, cell proliferation, metabolism, and angiogenesis, through regulating over 100 genes. [5,7] Translation of the HIF1α protein and mRNA are both upregulated when the signal pathway cascade of PI3K/The serine/threonine kinase Akt (AKT)/ mammalian target of rapamycin (mTOR) is activated. [8] In actuality, hypoxia promotes growth, survival and the spread of cancerous cells. Hypoxia in the tumor is a common and significant stumbling block to effective cancer treatment.
To present, there is no FDA-approved medication to reverse tumor hypoxia, despite the fact that a variety of techniques have been used. A few inhibitors prevent the subunit dimerization of HIF-1 and/or HIF-2 mRNA or protein, or HIF interaction with co-activators; however, the majority are indirect inhibitors or have several actions. As a result, creating more targeted HIF inhibitors remains a significant problem. Unfortunately, due to safety concerns and low therapeutic efficacy, no medicines that directly inhibit the therapy of cancer patients, and HIFs have received authorization. In light of hypoxia-inducible factor-1α (HIF-1α's) significant contributions in tumor microenvironment, in solid cancers like HNSCC, targeting and correcting hypoxia may be crucial to enhancing therapeutic response. The activation of HIF, including the redox-sensitive stages in HIF1α, the primary molecular actors involved, and their interaction with the reaction to unfolded proteins, as well as the influence of the PI3K/Akt pathway on HIF, and summarizes the common points of the 2, nuclear factor κB (NF-κB), for HIF1α Important implications, is covered in this paper. It offers a fresh approach to clinical research and treatment.

The relationship between HIF-1α and HIF-1 structure
The heterodimeric transcription factor HIF-1 consists of 2 subunits: HIF-1α [or its analogs, hypoxia-inducible factor-2α (HIF-2α) and hypoxia-inducible factor-3α (HIF-3α)], and hypoxia-inducible factor-1β (HIF-1β). [9] Low oxygen levels trigger the expression of the oxygen-sensitive subunit HIF-1α. HIF-1α, on the other hand, is expressed all of the time. Aryl hydrocarbon receptor's heterodimeric partner, HIF-1β, has been discovered. Aryl hydrocarbon nuclear translocator (ARNT) is another name for HIF-1β (a heterodimeric partner of aryl hydrocarbon receptor [AhR]). HIF-1β links to AhR, allowing it to go to the nucleus more easily. [9] These 2 components share structural similarities with 2 Drosophila nuclear proteins Per-ARNT-Sim (PAS) that include the basic-helix-loop-helix (bHLH) motif, making them members of the bHLH-PAS protein family. [9] Identifiable domains set the bHLH proteins apart (b, HLH, PAS, and topologically assocaited domain) that it allows them to regulate their own transcription as well as the transcription of other family members. For HIF-1 and HIF-1 subunits to form heterodimers and bind to the HRE-DNA sequence on target genes, the bHLH-PAS motifs are necessary. Base domains' ability to bind DNA was found to be necessary for the HRE to attach to the gene, whereas dimerization with other proteins takes place at the HLH motif. It was discovered that the only protein family member with a conserved domain is PAS (among HIF-1α, ARNT, AhR, and PAS), and is not found in any other proteins. The HIF-1α subunit has NH2-terminal N-terminal transactivation domain (N-TAD) and COOH-terminal transactivation domain transactivation domains topologically assocaited domain C-terminal transactivation domain (C-TAD). HIF-1α transcriptional activity is controlled by these 2 domains. [10] Under hypoxia, CBP/p300 is 1 co-activator with which C-TAD interacts to control transcription of HIF-1α genes. HIF-1α is stabilized by N-TAD, which protects it against degradation. [11] Furthermore, Since the oxygen-dependent degradation domain (ODDD) overlaps N-TAD in all HIF-subunit structures, they all differ from HIF-1β. This ODDD domain is crucial to controll the stability of O 2 regulation. [12] Three closely similar isoforms of the HIF-1 protein include HIF-1α, HIF-2α, and HIF-3α. [10] Compared to HIF-3α, HIF-2α has a lot in common with HIF-1α in terms of the amino acid sequence (48% sequence similarity). This structural similarity may account for their ability to heterodimerize with HIF-1β and bind to HREs. HIF-1α and HIF-2α have varied tissue distribution patterns, which is interesting. While HIF-2α is only found in certain tissues, HIF-1α is expressed throughout the body. [13] Before the identification of the inhibitory PAS (IPAS), a spliced variation of HIF-3α, HIF-3α was not well known. This domain is a dominant-negative regulator of HIF-1/DNA binding ability and lacks intrinsic transactivation activity in compared to the C-TAD of HIF-1α and HIF-2α. [13] The activation of HIF1 Alpha in hypoxic conditions is the subject of the following thesis. The structure of the HIF-1α is shown in Figure 1.

The activation of HIF-1α
Due to a series of signaling processes and other signaling molecules, HIF-1α is constitutively transcribed and generated regardless. Independently from O 2 levels. In normoxic environments, HIF-1α rapidly degrades and has a brief half-life (approximately 5 minutes). [14] Through post-translational modifications like hydroxylation, acetylation, ubiquitination, and phosphorylation processes, it has been demonstrated to regulate HIF-1 stability and transcriptional activity under hypoxic environments. As discussed below the above is mostly caused by the PI3K/Akt/MTOR pathway (Fig. 2). Following that, I'll go into HIF regulation apart from the primary HIF regulatory pathways in hypoxia, including redox signaling in HIF-1α-dependent tumors. The regulatory aspects of various recent experimental studies were summarized, as well as the main regulatory role of PI3K/AKT/mTOR.

The role of redox signaling in hypoxia-induced HIF-1α activation
Reactive oxygen species (ROS) help to activate HIF-1α signaling. Late in the 1990s, It was found that the high levels of ROS produced by mitochondria during hypoxia can activate the HIF-1α signaling pathway. [15] Numerous studies proposed prolyl hydroxylase (PHD) inactivation as a mechanism of ROSmediated HIF-1α signaling activation. [16,17] which corroborated this finding. However, it is still uncertain how ROS mediates the stabilization of HIF-1α under hypoxia. [14,18] Under normoxia, however, It is undeniable that ROS can keep the HIF-1α protein stable 14.ROS has been postulated as a method for oxidizing Fe2+, a co-factor of PHDs. Reactive oxygen species may affect the transcription and translation of HIF-1α through the ERK and PI3K/AKT/mTOR signaling pathways, as well as the synthesis of microRNA-210. [14] Reactive oxygen species (ROS) are produced by mitochondria and maintain HIF stability. Complex III of the mitochondrial electron transport chain generates ROS. [16,19] Another factor that could contribute to elevated ROS levels is the Circadian locomotor output cycle protein kaput. [19] HIF-1α and NF-kB are both activated by ROS. ROS specifically impede the activity of PHD and factor inhibiting hypoxia-inducible factor in the cytoplasm. [16,19,20] Some ROS-producing enzymes have a significant impact on ROS activation. The amounts of nitric oxide (NO) are required for HIF activation. This is significant since many malignancies are associated with a rise in inducible nitric oxide synthase (iNOS) expression and NO production, both of which make the patient's prognosis worse. [21] NO S-nitrosylates Cys533 on HIF-1, resulting in improved protein stability in normoxia. NO inhibits the activity of PHD by attaching to the iron atom at the catalytic core. Contrarily, NO can prevent HIF-1 from being activated and functioning in a hypoxic environment. When coupled with ROS, NO increases the amount of calcium ions in the cytoplasm, which causes calpain, a protease that eliminates HIF-1 without the help of the 26S proteasome, to be activated. Additionally, NO restores the function of the enzymes involved in chronic hypoxia's HIF-hydroxylation through a process involving NO and ROS interaction. [20,22] When HIF-1α binds to the von Hippel Lindau protein-E3 ubiquitin ligase complex, it becomes ubiquitinated and is subsequently targeted for degradation via the ubiquitin-proteasome pathway. In hypoxic conditions, prolyl hydroxylation occurs at a slower pace (i.e., when PHDs lack access to oxygen due to scarcity), reducing HIF-1a degradation. [23] In the nucleus, stabilized HIF-1a accumulates, which induces the expression of a large number of proangiogenic factors, including vascular endothelial growth factor (VEGF), VEGF receptors (VEGFR), Angiopoietin (ANG-1 and ANG-2) receptor TIE-2, platelet derived growth factor-B, plasminogen activator mitogen 1, and certain matrix metalloproteinase family members (MMP-2 and MMP-9). [24,25] Despite the fact that they can both heterodimerize with HIF-1β and bind to genes that are induced by hypoxia and have the hypoxia response elements motif, HIF-1α and HIF-2α have different transcriptional targets. Two glycolytic enzymes, lactate dehydrogenase-A and carbonic anhydrase, are stimulated by HIF-1α. On the other hand, HIF-2α has a higher impact on the genes involved in iron metabolism and the EPO gene, but both HIF-1α and HIF-2α regulate VEGF and GLUT-1. [9] VEGFA, an endothelial mitogen, is the most notable of these HIF-1/2 targets because it is considered to be the primary controller of tumor angiogenesis. HIF-1α hydroxylation is catalyzed by prolyl hydroxylases, which introduce 1 atom of molecular oxygen into the cosubstrate a-ketoglutarate, which is then converted to succinate, and the other atom into the cosubstrate a-ketoglutarate. [23,26] PHD activity is inhibited by dimethyloxalylglycine, a competitive antagonist of a-ketoglutarate, which promotes the transcription of angiogenic factors reliant on HIF-1. Similarly, iron chelators and cobalt chloride, which increase HIF-1 activity and inhibit PHD by removing Fe (II) from the catalytic core. [23,27] Redox active chemicals that have an impact on PHDs indirectly control the activity of hypoxia-inducible factor-1. A good illustration of this kind of chemical is ascorbate. It transforms ferric iron Fe (III) complexes that are insoluble in PHDs into stable ferrous Fe (II) chelates that are soluble. This demonstrates ascorbate's function in chelating and reducing PHD-bound Fe (III) during hydroxylation, which maintains the enzyme cycle. Ascorbate functions in oncogenic activated cells as a cofactor of PHDs, reducing hypoxia-or iron chelators and cobalt chloride-induced HIF-1a activity in culture, indicating that it might increase PHDs' HIF-1hydroxylase activity. [28,29] Ascorbate supplementation decreased tumor metastasis and necrosis in ascorbate-deficient Gulo/ mice alongside the MMP-9 and VEGF production, which are HIF-1 targets, It suggests that ascorbate may slow tumor growth by preventing HIF-1. [6] Under normal circumstances, cellular antioxidant mechanisms keep ROS below spectrum of physiological conditions necessary for effective cell signaling. ROS levels can rise quickly due to increased ROS production or a lack of antioxidant defense, which can influence HIF-1 signaling (as demonstrated in a variety of tumor types).
In fact, ROS can facilitate iron oxidation, which stabilizes HIF-1α and inactivates PHD. Other redox posttranslational changes have been shown to control HIF-1α turnover in addition to hydroxylation. Numerous studies have discovered that HIF-1a is targeted by nitric oxide (NO) via S-nitrosylation at Cys533, a crucial cysteine within HIF-1a's oxygen-dependent degradation domain. Even under normoxia, HIF-1a S-nitrosylation has been shown to stabilize the protein and block its connection with VHL, simulating pseudo-hypoxic circumstances. [24] However, NO's activation of HIF-1 signaling is not always straightforward. Indeed, hypoxia has been shown to cause HIF-1a stabilization indirectly through the nitrosylation of Fe (II) situated in the catalytic region of PHDs, which inhibits PHDs hydroxylase activity. [30] HIF-1 has long been thought to be a promising therapeutic target for cancer treatment because of its well-known function in tumor angiogenesis. Both direct HIF-1 inhibitors that affect HIF-1 expression or activity and indirect HIF-1 inhibitors that act on additional molecules in associated pathways have been discovered. HIF is inhibited by the first class of inhibitors through a variety of mechanisms, including mRNA expression, protein synthesis, dimerization, DNA binding, and transcriptional activity. However, only a handful of them are being studied in clinical settings because of their pleiotropic (side) effects. The mitochondrial complex I inhibitor BAY 87 to 2243 is one of these and prevents HIF-1α stability, HIF-1 translation is inhibited by PX-478 and HIF-1α translation is inhibited by KCN-1 and its transcriptional coactivator p300 association. [31][32][33]

ROS-generating enzymes: modulators of the redox signaling
Redox signaling modulators are proteins that regulate redox signaling. Many cellular enzymes, including endoplasmic oxidore-duction1, nicotinamide adenine dinucleotide dinucleotide, oxidases, the mitochondrial electron transport chain complexes I and III, and NOS, have been shown to have an immediate impact on angiogenic mediator levels and activity. [2,21,22,[34][35][36][37][38] The regulation of some ROS-generating enzymes, their function in angiogenesis, and how their activity is related to HIF-1 will all be discussed in this section.

The important pathway regulation of HIF-1α
Oncogenic stimulation of the PI3K/AKT/MTOR pathways has a significant role in modulating HIF-1α protein levels. Under hypoxic conditions, HIF-1α protein is known to accumulate in cells as a result of growth factors, cytokines, and other signaling molecules.

Triggered of ERK through PI3K/AKT.
Signaling pathways for growth factors PI3K protein can be upregulated by activating it. [8,39,40] Through rapamycin's downstream component, mammalian target of and target protein kinase B Akt, PI3K regulates protein synthesis mTOR. By phosphorylating the eukaryotic translation initiation factor 4E binding protein, mTOR prevents cap-dependent mRNA translation eukaryotic translation initiation factor4e-binding protein1, causing these 2 components' integrity to be compromised, which increases the translation of the HIF-1 protein. [40] Furthermore, protein translation is triggered by mTOR-mediated phosphorylation of p70 S6 kinase, which increases the phosphorylation of its substrate, ribosomal protein S6. [40] Tumor suppressor protein phosphatase and tensin homolog (PTEN) prevents PI3K products from being phosphorylated. Certain growth hormones cause rat sarcoma (RAS) to become active, which encourages the RAS/ RAF/MEK/ERK kinase cascade. [39] ERK (MNK) phosphorylate eukaryotic translation initiation factor4e-binding protein1, S6 kinase, eukaryotic translation initiation factor 4E,and MAP kinase interacting kinase. [17] These signaling events increase the rate at which mRNA is translated into the HIF-1 protein as their overall effect. It's interesting to note that ERK controls both HIF-1 synthesis and transcriptional activation. The co-activator CBP/p300 is phosphorylated by ERK, which improves the formation of the HIF-1/p300 complex and subsequently increases transcriptional activity. [41] Hypoxia, both chronic and intermittent, is a cancer-causing factor.

Triggered of mitogen-activated protein kinase (MAPK)
through PI3K/AKT. Hypoxia, AP-1 Kinases, and MAPK The physiological response to low oxygen levels is greatly influenced by MAPK cascades, which are triggered during hypoxia. Reactive oxygen species or the presence of calcium ions are required for the activation of MAPK pathways in hypoxia ROS. [42] When there is hypoxia, L-type voltage-gated Ca2 + channels open, which results in a surge in calcium ions. [42] oxygen species ROS or calcium ions activate the p38 MAPK and the c-Jun N-terminal kinase (JNK) MAPK. [42,43] The levels of c-Fos and JunD MAPK cascade activity decrease as c-Jun and JunB are activated.on the other hand, does not just activate HIFs; HIF-1 could potentially influence MAPK cascade activation in the other manner. [44] Under chronic hypoxia, Increased c-Jun expression in mouse embryonic fibroblasts is dependent on HIF-1, this is comparable to the ERK MAPK cascade being activated. [25,45] C-Jun and JunB, which are part of the activating protein-1 (AP-1) complex, are crucial in the regulation of a number of genes under hypoxia. MAPK pathways can alter HIF1α activation and transcriptional activity. The ERK MAPK, p38 MAPK and JNK MAPK pathways are all activated by hypoxia. [44,46,47] HIF-1α is phosphorylated on Ser641 and Ser643 by ERK MAPK, p38 MAPK, JNK MAPK ERK. HIF-1's association with the p300 coactivator and build up in the nucleus of the cell are both influenced by this post-translational change. [46] Additionally, p38 MAPK phosphorylates HIF-1α, which improves this HIF-1 subunit's stability. [46] The 7 in absentia homolog 2 is phosphorylated by the p38 MAPK protein at the sites Thr24 and Ser29, which is another way affecting HIF-1α activation. PHD3, the enzyme that hydroxylates and degrades HIF-1α, then breaks down after this. HIF-1α is not degraded as much when p38 MAPK is activated. Dual specificity protein phosphatase (DUSP-1) and mitogen-activated protein kinase phosphatase 1 expression are upregulated in response to hypoxia. [48] generated by neuronal nitric oxide synthase, nitric oxide (NO) (nNOS) and hypoxia are required for the enhanced expression of DUSP-1 in neurons. Contrary to how NO deactivates DUSP-1, protein kinase C increases DUSP-1 expression in fibroblasts during persistent hypoxia. [49,50] Deferoxamine and cobalt chloride may also prevent histone demethylase activity, resulting in altered gene expression. [49,50] HIF-1α also reduces DUSP-2 expression in chronic hypoxia, Enzymes called DUSP-1 and DUSP-2 dephosphorylate ERK and p38 MAPK to render them inactive. [51] In chronic hypoxia, increase of DUSP-1/mitogenactivated protein kinase phosphatase 1 expression is a protective mechanism against excessive HIF1α activation by ERK MAPKs. In hypoxia, MAPK cascades improve HIF1α stability and transcriptional activity. Similar pathways are present in tumor cells under normoxia, where MAPK cascades are activated, [52] particularly in response to different growth stimuli. [53] MAPK kinases are activated by hypoxia, and NF-KB is then also activated. However, the MAPK cascade that causes this varies from model to model. In the case of HNSCC, more research is required. In the case of HNSCC, more research is required.
Although it is also regulated at the transcriptional and translational levels, HIF-1 is predominantly regulated at the protein level. When the PI3K/AKT/mTOR signaling cascade is active, HIF1α protein translation and mRNA transcription are both increased. [54] Multiple growth factors, oncogene activation, and tumor suppressor gene alterations (such as PTEN) could all stimulate HIF1α signaling via the PI3K/AKT/mTOR pathway. [54] According to reports, Glycogen Synthase Kinase-3 phosphorylates the HIF1α protein, causing VHL-independent HIF1α protein degradation. [55] We further show that lower levels of PTEN, as a result of genetic silencing and pharmacological suppression of PTEN, cause HIF-1a to accumulate in tumor cells. [54] The PI3K/AKT signal, in particular, has been shown to inactivate glycogen synthase kinase-3, resulting de HIF1α protein stabilization. [56] The RAS/RAF/MEK/ERK cascade could be activated by a variety of growth factors and oncogenic events, speeding up the production of the HIF1α protein. [57] By phosphorylating CBP/p300, a crucial co-factor for HIF1 transcriptional activity, ERK activation also increases HIF1 transcriptional activity. [45,57] 4. Important link of ROS and PI3K/AKT/mTOR pathway for HIF1α The ROS state regulates many cellular proteins. This process promotes the activity of growth factors, cytokines, and hormones by activating a number of proteins, including kinases, may activate downstream signaling pathways including phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK). [41,58] As a result, ROS directly oxidize phosphatase. Other pathways must be activated for the greatest HIF activity. Particularly during the first few hours of persistent hypoxia, NF-κB is activated. [59] Because the HIF1α gene promoter has an NF-κB binding site, HIF-1 mRNA expression is boosted. [60,61] In order to boost HIF-1a mRNA expression, P50 NF-κB and P65/RelA NF-κB were activated, but not c-REL NF-κB. [62,63] Additionally, various elements of the NF-κB activation pathway are impacted by chronic hypoxia. [64] In hypoxia, the MAPK cascade is also triggered, which causes NF-κB to become active. In addition to these mechanisms, NF-κB occurs in hypoxia through the phosphatidylinositol 3-kinase (PI3K) →AKT pathway. [65] This transcription factor is activated by Akt/ PKB kinase's phosphorylation of P65/RelA NF-κB, however this mechanism is unrelated to IκBα degradation. Moreover, TNF-α (tumor necrosis factor alpha) activates the NF-κB pathway when relevant components translocate into the nucleus, causing alterations to occur at the level of HIF1α [66,67] (Fig. 3). We can see that both ROS and PI3K/AKT/MTOR pathways have NF-κB effects in activating HIF 1α. The effect of NF-κB on HIF 1α is self-evident.

Concluding
HIF1α signaling activation can be mediated by a number of hypoxia-independent pathways, according to growing evidence. [57] Numerous important genes involved in tumor development, metastasis, and therapy resistance are controlled by HIF1α. [41,68] As already mentioned, in hypoxic conditions, a number of variables (ROS, PI3K, and NF-κB) can stabilize HIF1α and benefit cancer cells by promoting cell motility, invasion, immunological escape, and radio resistance. [18] We also described the NF-κB (the important link of ROS and PI3K/ AKT/mTOR cascade) for HIF1α implications, in an effort to offer fresh views for the treatment of head and neck squamous cell carcinoma (HNSCC) and HIF1α research. It is beneficial to provide fresh evidence for HIF inhibitor or combination inhibitor research and promote clinical treatment by examining the elements that activate HIF1α and discovering common ground among them. Thus, it offers a fresh outlook on the creation of combination inhibitors.