Proteins that are structurally related to HIF-1α (HIF-2α, HIF-3α) and HIF-1β (ARNT2, ARNT3) have also been identified. HIF-1α and ARNT (HIF-1β) mRNA are expressed in most, if not all, human and rodent tissues. However, HIF-2α, HIF-3α, ARNT2, and ARNT3 show a more restricted pattern of expression. Of the three α-subunits, HIF-1α and HIF-2α, which have been well studied, have 48% amino acid sequence identity and similar protein structures, but are nonredundant and have distinct target genes and mechanisms of regulation. HIF-3α, whose role is not yet fully understood, has high similarity to HIF-1α and HIF-2α in the basic helix-loop-helix (bHLH) and Per-Arnt-SIM (PAS) domains but lacks the C-terminal transactivation domain (TAD-C).
In many organs, the physiological oxygen tensions are significantly lower than ambient oxygen tensions: brain (4.4%), liver (5.4%), muscle (3.8%), bone marrow (6.4%), etc. Additionally, a lot of solid tumors contain hypoxic microenvironment because of the limited diffusion of O2 from nearby vessels. As a result, cells under this physiological or pathological hypoxia developed the HIF system as a key systemic response to transactivate a large number of target genes, including those promoting angiogenesis, anaerobic metabolism, and resistance to apoptosis. Hence, in certain contexts, HIF-1 drives the initial response to hypoxia and plays essential roles in mammalian development, physiology, and disease pathogenesis.
Regulation of HIF-1 Activity
The synthesis of HIF-1α is also regulated by O2-independent mechanisms, involving many oncogenes and tumor suppressors (Majmundar et al. 2010). HIF-1α protein expression is regulated by the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. Firstly, growth factors activate the PI3K through binding to a receptor tyrosine kinase. Next, PI3K activates the downstream serine/threonine kinases AKT and mammalian target of rapamycin (mTOR). Meanwhile, in the MAPK pathway, MAP/ERK kinase (MEK) activates the extracellular-signal-regulated kinase (ERK). ERK, in turn, activates MNK. MNK and mTOR simultaneously promote HIF-1α translation through phosphorylating p70 S6 kinase (S6K). The posttranslational modifications of HIF-1α are also regulated by PHD/VHL-independent mechanism. It has been identified that the receptor of activated protein kinase C (RACK1) competes with heat shock protein (HSP90) for binding to HIF-1α, promoting PHD/VHL-independent proteasomal degradation of HIF-1α (Liu et al. 2007). HIF-1 expression is also regulated by noncoding RNAs. For example, miR-107, a microRNA expressed in human colon cancer and upregulated by p53, suppresses the expression of HIF-1β.
Being a master regulator of cellular response to hypoxic stress, HIF1 is extensively involved in many physiological and pathological processes, such as cerebrovascular disease, inflammation, Parkinson’s disease, and angiogenesis. Nevertheless, here, we focus on its function in cancer development.
HIF-1 and Cancer Metabolism
In order to adapt to the oxygen consumption under hypoxia, cells have to adjust their mitochondria activity, because the deficiency of oxygen will increase the level of mitochondrial ROS, leading to apoptotic cell death (Simon 2006). HIF-1 regulates the mitochondrial activity through different mechanisms. First, it was showed that PDK1 is a direct transcriptional target of HIF-1 (Kim et al. 2006). PDK1 can phosphorylate and inactivate PDH, which converts pyruvate to acetyl-CoA, thereby decreasing acetyl-CoA entry into the TCA cycle. Moreover, HIF-1 downregulates mitochondria activity by activating the expression of BNIP3 and BNIP3L, which triggers selective mitochondrial autophagy, resulting in a decrease in mitochondrial mass and O2 consumption. On the other hand, HIF-1 reduces mitochondrial biogenesis through upregulating MXI-1, which attenuates PGC-1β-regulated mitochondrial biogenesis by inhibiting c-Myc oncogene (Zhang et al. 2007). More importantly, HIF-1 fine-tunes mitochondrial metabolism under hypoxia by regulating the subunits conversion of cytochrome c oxidase 4 (COX 4). Under hypoxia, HIF-1 regulates COX4 subunit expression by activating transcription of the genes encoding COX4-2 and LON, a mitochondrial protease that is required for COX4-1 degradation. HIF1 can optimize the efficiency of respiration by regulating COX4 subunit switch. Furthermore, mitochondrial complexes on inner membrane play important roles in regulating respiration. Therefore, iron-sulfur clusters, as an important part of complexes, are critical for respiration. The hypoxia-induced miR-210 directly represses the expression of ISCU1/2 (iron-sulfur cluster assembly proteins), which downregulates iron-sulfur cluster biogenesis. By repressing ISCU1/2 during hypoxia, miR-210 decreases the activity of Complex I and disrupts mitochondrial respiration. Overall, HIF-1 mediates adaptive responses to hypoxia by reducing ROS levels through the regulation of mitochondrial oxidative metabolism.
Besides the established function in glucose metabolism, HIF-1 also plays important roles in lipid metabolism. It has been reported that HIF-1 can regulate the expression of fatty acid synthase (FASN) and lipin1 (LPIN1). FASN gene, which is the major enzyme for lipogenesis and catalyzes the combination of acetyl-CoA and malonyl-CoA to produce palmitic acid, is upregulated by hypoxia through induction of the SREBP-1 gene by HIF-1. Meanwhile, HIF-1 can promote transcription of lipin1, which catalyzes the penultimate step in triglyceride biosynthesis, leading to accumulation of triglycerides and lipid droplets. In cardiac myocytes, hypoxia was reported to diminish FAO (fatty acids oxidation), and in cancer cells, it was found that HIF-1 suppresses FAO by inhibiting MCAD and LCAD (the medium- and long-chain acyl-CoA dehydrogenases) expression, two enzymes belonging to the acyl-CoA dehydrogenase family that are rate-limiting enzymes in fatty acid β-oxidation, and HIF-1-mediated FAO suppression promotes cancer proliferation partly by reducing ROS as well as by regulating cell proliferation signaling PTEN pathway (Huang et al. 2014).
HIF-1 and Cancer Metastasis
The extravasation of cancer cells is a rate limit step in the metastatic process. During extravasation, cancer cells must first adhere to vascular endothelial cells (ECs), disrupting the interaction between vascular ECs. It has been found that hypoxia can increase the extravasation of cancer cells, and this effect was abrogated in HIF-knockdown cells. Two important genes, L1CAM and ANGPTL4, are involved in HIF1-regulated extravasation of cancer cells under hypoxic condition. Both L1CAM and ANGPTL4 are regulated by HIF1. L1CAM is a protein involved in cell-cell adhesion via hemophilic interaction or by heterophilic interaction with neuropilin 1(NRP1), integrin, or CD24. Overexpression of L1CAM in cancer cells promotes the adhesion of cancer cells to EC monolayers and increases the number of extravasated cancer cells in lung tissue (Zhang et al. 2012). Angiopoietin-like 4 (ANGPTL4), which is highly expressed in the primary breast cancers of women with lung metastases, inhibits EC-EC interactions. Moreover, both HIF-1 and HIF-2 contribute to ANGPTL4 expression, thereby facilitating extravasation and lung metastasis. Furthermore, HIF-1-regulated SDF-1/CXCR4 signaling may also promote the adhesion of breast cancer cells to ECs in the circulatory system.
HIF-1 also contributes to the production of secretory factors involved in premetastatic niche formation. In breast cancer cells, HIF-1 induces the expression of multiple members of the lysyl oxidase (LOX) family, including LOX, LOXL2, and LOXL4 (Wong et al. 2011). These proteins modify the collagen matrix in the lung at hydroxylated lysine residues and recruit bone-marrow-derived cells (BMDCs) to promote breast cancer premetastasic niche formation. The BMDCs promote metastasis by producing chemokines that recruit tumor cells to the lung. Interestingly, LOX, LOXL2, and LOXL4 are highly expressed in different primary human breast cancers (Wong et al. 2011). Therefore, a monoclonal antibody or small molecule inhibitor directed against LOX might be useful for breast cancer therapy. Another important mechanism is about tumor-lymphatic vessel cross-talk, leading to the formation of premetastatic niche. Lymphatic endothelial cells (LECs) are a specialized endothelium and promote lymph metastases by recruiting tumor cells to the lymphatic vessels. HIF-1 can promote VEGF expression in LECs. Furthermore, a hot topic is about whether HIF-1 signaling can promote formation of the premetastatic niche by regulation of exosomes. Studies have demonstrated that hypoxia influences exosome cargo content and promotes angiogenesis. There are also other mechanisms involved in tumor metastasis; for example, hypoxia can also promote microvesicle shedding by increasing the small guanosine triphosphatase RAB22A.
HIF-1 and Immunity
Recent studies have highlighted the importance of HIF-1 signaling in regulating immune responses, including both innate and adaptive immunity. In innate immunity, HIF-1 can influence inflammation, infection, and metabolism. (1) In regulation of inflammation, mice with HIF-1α myeloid-specific deletion showed impaired inflammatory responses. HIF-1α not only increases macrophage aggregation, invasion, and motility but also induces neutrophil survival through regulating NF-κB. HIF-1α also drives the expression of proinflammatory cytokines (TNF-α, IL-6). As a result, HIF-1 inhibition could be a potential strategy for relieving excessive inflammatory responses. (2) Since bacterial infection often leads to a hypoxic microenvironment, it is interesting to study the relationships of infection and hypoxic response. It has been reported that HIF-1α promotes granule protease production, releases nitric oxide (NO) and TNF-α, thus enhancing antimicrobial activity. (3). Myeloid cell function is broadly regulated by metabolic changes (Haschemi et al. 2012). To fight against pathogens or cancer, HIF-1α increases glycolysis and limits oxidative phosphorylation through regulating key glycolytic enzymes and PDK1 in myeloid cells.
In adaptive immunity, hypoxia and HIF-1 also play important roles, especially in T cell differentiation and function. With respect to T cell differentiation, HIF-1 regulates the balance between regulatory T cell (Treg) and Th17 differentiation. HIF-1α activates RORγt transcription and forms complexes with RORγt and P300 to activate the IL-17 gene expression during Th17 development. On the other hand, HIF-1 inhibits Treg differentiation by associating with Foxp3, leading to its proteasomal degradation (Dang et al. 2011). Lack of HIF-1α in T cells reduces the expression of glycolytic molecules and the differentiation of Th17 cells and Treg cells, which demonstrates that HIF-1 controls T cell lineage by regulating cellular metabolism. HIF-1α also regulates many molecules associated with CD8+ T cells function. For example, HIF-1α promotes the expression of important cytolytic molecules (granzyme B, perforin) and alters migration and chemokine receptor expression (Doedens et al. 2013). Therefore, HIF-1 activity in CD8+ T cells may promote pathogen clearance or diminish tumor burden.
In regard to adaptive tumor immunity, on one hand, CD4+ Th1 cells and CD8+ cytotoxic lymphocytes have been linked to a better prognosis in cancer patients. On the other hand, Treg cells can protect the host from autoimmunity by inhibiting self-reactive T cells. HIF-1 signaling contributes to tumoral immune escape from cytotoxic T lymphocytes (CTL) (Noman et al. 2009). Programmed cell death 1 (PD-1) and CTLA-4 act as coinhibitory receptors on activated T cells. PD-L1, as the ligand of PD-1, presents in tumor cell surface. HIF-1α not only promotes CTLA-4 expression in CD8+ T cells but also increases PD-L1 expression to promote immune escape (Doedens et al. 2013; Barsoum et al. 2014). Therefore, blocking CTLA-4 and PD-L1 expression is a promising strategy for cancer immunotherapy in hypoxic-tumor cells. Meanwhile, HIF-1 promotes the recruitment of regulatory T (Treg) cells by inducing expression of the chemokine CC-chemokine ligand 28 (CCL28), which promotes tumor tolerance and angiogenesis.
Perspectives and Summary
Soon after HIF-1 was discovered, it has been demonstrated that this protein participates in many human disease pathophysiology, such as ischemic cardiovascular disorders, pulmonary hypertension, and pregnancy disorders, especially in cancer which is the most intensively investigated. Interest in the role of hypoxia-inducible factor 1 (HIF-1) in cancer biology has grown exponentially in the past three decades. HIF-1 plays key roles in many crucial aspects of cancer cell biology including angiogenesis, metastasis, metabolism, and immunity. Traditionally, HIF-1 functions in cells via genetically changing the expression of target genes. However, more comprehensive studies have shed light on mechanisms underlying epigenetics (such as functional noncoding RNAs, histone protein modifications, and DNA methylation).
Clinical data indicates that HIF-1 high expression is associated with increased risk of cancer patient mortality. Thus, while intensive mechanistic studies are under way, HIF-1 targeted anticancer therapy has emerged as a new therapeutic approach for clinical application. Remarkably, several inhibitors (Trastuzumab, Imatinib, Camptothecin) of HIF-1 have been approved for human diseases (Semenza 2003), and screens are underway to identify novel small-molecule inhibitors of HIF-1 and test their efficacy as anticancer agents. Nowadays, with cancer immunotherapy emerging as a promising strategy for cancer patient treatment, novel strategies targeting HIF-1 in combination with current immunotherapy would probably provide more universal and effective therapeutic strategies, especially in the case of solid tumors.
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