Hypoxia-induced Factor-1α and its Role in Endometrial Cancer

Endometrial Cancer Histopathology and Clinic

Precancerous conditions include simple endometrial hyperplasia, glandular endometrial hyperplasia, cystic, adenomatous without atypia, atypical, low, medium and high grade. Histologically, endometrial cancer is divided into two types (10-12): Type 1 and Type 2 (Table I). The stage of clinical advancement of endometrial cancer, the so-called staging, is determined by the classification developed by the International Federation of Gynecology Obstetricians (FIGO) (Table II). The five-year survival rate for each FIGO grade is as follows: Grade I – 83%; Grade II – 73%; Grade III – 52%; Grade IV – 27%. Similar to FIGO is the tumor (primary tumor, size), node (lymph node), and metastases (distant metastases) (TNM) classification. This classification was developed by the UICC (Union for International Cancer Control) (13). The second parameter is the so-called grading, i.e., the degree of histopathological differentiation of the tumor where: G1 indicates highly differentiated neoplasm, G2 moderately differentiated, and G3 poorly differentiated (13). The five-year survival rate for each stage of histological malignancy of endometrial cancer is as follows: G1, 80-85%; G2, 74-78%; G3, 50-64%.

Clinical manifestations of endometrial cancer are: 1) abnormal uterine bleeding: (a) after menopause, and b) acyclic or abundant, hemorrhagic with premenopausal clots; 2) bloody, purulent, watery vaginal discharge; 3) rarely abdominal pain, and 4) gynecological examination per rectum – enlargement of the uterus, uterine infiltration, abdominal and metastatic tumors.

Treatments for endometrial cancer include the following: 1) Self-treatment of surgery; 2) Radiotherapy: Tele+Brachytherapy; 3) Surgery + Radiotherapy, 4) Surgery and/or radiotherapy with adjuvant chemotherapy or hormone therapy, and 5) Primary radiotherapy followed by surgery.

The prognosis for endometrial carcinoma depends on the histological type and degree of histological differentiation, the clinical stage, the depth of uterine muscle infiltration, lymph node metastases, peritoneal cytology, the age of the patient, genetic factors (DNA ploidy, nuclear atypia, expression of specific genes) and sex hormone receptors. Prognostic factors of endometrial cancer include:1) Histological type – types 1 and 2; 2) Degree of histological differentiation G 1,2,3; 3) FIGO Clinical Staging; 4) Muscle membrane infiltration; 5) Vascular infiltration; 6) Cervical infiltration; 7) Lymph node metastases; 8) Receptor status – estrogen (ER), progesterone (PR); 9) Ploidy DNA, and 10) Gene expression disorders (p53, bcl2, MSI, PTEN) (12-20).

Risk Factors for Endometrial Cancer

Endometrial cancer is a common malignant tumor of the female reproductive organs, developing in the mucous membrane lining the inside of the uterus. Hyperplasia occurs as a result of long-term stimulation of the endometrium by estrogens of both endo- and exogenous origin. Endogenous hyperestrogenism characterizes anovulatory cycles, especially in the presence of polycystic ovary syndrome (PCO), since it is associated with the absence of the formation of the corpus luteum. Estrogen-producing tumors (granuloma, capsule) cause pathological hyperplasia in 46% of patients and endometrial cancer in 10% of patients (21).

Somatic mutations in endometrial cells are very common. They occur much earlier than any lesions that can be detected histopathologically. Hence, the preclinical phase of endometrial cancer is only recognized by genetic changes in mutated cells. Genetic changes can persist for many years, being “covered” by changes related to the menstrual cycle. During this time, hormonal factors can cause positive or negative selection of mutant cells. Examples of such early changes in the endometrium include inactivation of the PTEN gene as a result of deletion or substitution of bases and the acquisition of microsatellite instability.

The mutant cells in such cases participate in the monthly regeneration of the endometrium and can be spread throughout the organ, forming microfoci of mutant clones. This stage may be referred to as the “hidden phase of carcinogenesis” and may have potential implications for the diagnosis and treatment of endometrial cancer. Clinically recognizable precancerous lesions, which can be diagnosed by a pathologist, arise as localized clonal growths of the endometrial glands, which gradually acquire further somatic mutations, leading to cancer.

The changes described above, such as PTEN inactivation and the acquisition of the microsatellite instability phenotype, often referred to as early, may occur in the latent premalignant phase or directly in the development of the malignant phenotype. Thus, mutations in the normal endometrium that do not result in a malignant phenotype until exposure to estrogens should be considered. The existence of such mutant cells may serve as a diagnostic, therapeutic, and prognostic determinant. The following diagram illustrates the transformation of normal endometrium into endometrial cancer (Figure 1).

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Figure 1.

Development of endometrial cancer.

The phenomenon of transformation from simple hyperplasia, through atypical hyperplasia, to cancer usually takes many years. Therefore, it is possible to detect it at an early enough stage and to undertake effective treatment. It is assumed that mucosal hyperplasia with mild and moderate grade atypia develop into cancer after 1-10 years. If we are dealing with a large atypia, this time is shortened to 1-3 years. Therefore, at the current stage of knowledge, the main preventive measure is strict control of women in the high-risk group (postmenopausal women, treated with tamoxifen, women with a family history of Lynch syndrome). Systematic ultrasound examinations of the genitals in these women may allow identification of an abnormality in the endometrium: increased thickness of more than 4 mm, presence of fluid in the uterine cavity, lack of middle echo and heterogeneous structure. This is an indication for in-depth diagnostics, i.e., histopathological evaluation of the endometrium.

The risk of cancer in postmenopausal women with an endometrial echo not exceeding 4 mm is 0.1-1%. Abnormal bleeding from the genital tract is one of the most common symptoms of endometrium pathology. Any woman who observes bleeding or spotting from the genital tract, especially more than 6 months after the last menstrual period, during hormone therapy, should undergo diagnostic curettage of the uterine cavity. As a result, about 70% of cancers can be diagnosed at stage I. This is associated with a high five-year survival rate (6).

Another risk factor for endometrial cancer is obesity. A Body Mass Index (BMI) above 25 kg/m² doubles the risk of developing the disease. A BMI above 30 kg/m² increases the risk up to three times (22).

Adipose tissue is the site for the conversion of androstenedione to estrone, which increases the risk of endometrial cancer by approximately 25% (23). It is estimated that being overweight is associated with the development of almost half of endometrial cancer cases in Europe and the USA (24). Elevated triglycerides, often found in obese people, may increase the risk of endometrial cancer (25). Risk factors for endometrial cancer include diabetes, hypertension and liver disease, which impair estrogen degradation and cause an increase in serum estrogen levels (26). A diet rich in animal fats predisposes to endometrial cancer. It is associated not only with an increase in estrogen levels, but also with a decrease in sex hormone-binding globulin (SHGB). The occurrence of menopause at a later time (after the age of 52) is also important for the risk of endometrial cancer, as it prolongs the total time of estrogen exposure to the endometrium (27).

Endocrine imbalances can also be caused by improperly administered hormone replacement therapy (HRT). It refers to the incorrect selection of the dose of estrogens and progestogens with a predominance of the concentration of the former. It is believed that the incidence of endometrial cancer in this case increases from 2-12 times after a period of 2-3 years of taking hormones. It reaches its highest risk after 10-15 years (28, 29). Greater availability of hormonal treatment and a more caloric diet leading to the so-called civilization diseases (obesity, diabetes, hypertension) explain the relationship between the higher incidence of endometrial cancer and the wealth of the society.

Another risk factor for endometrial cancer may be the use of high-dose tamoxifen. Tamoxifen, which belongs to the group of selective estrogen receptor modulators (SERMs) and is used in the treatment of breast cancer due to its anti-estrogenic effect in the breast, exhibits estrogenic activity in the endometrium, often leading to endometrial hypertrophy (30). Tamoxifen use for more than five years increases the risk of cancer by 2-3 times. Data indicate that estrogens may lead to the initiation of carcinogenesis in estrogen-dependent tissues (endometrium, breast) by influencing the cell cycle and secondarily on cell proliferation (31). Genetic disorders such as hereditary non-polyposis colorectal cancer (HNPCC, Lynch syndrome) are also risk factors for endometrial cancer. In this case, endometrial cancer develops about 15 years earlier than the “sporadic” form, and the probability of its occurrence throughout life is 25-50% (22, 32-34).

Some epidemiological evidence suggests that physical activity may reduce the risk of endometrial cancer. It is hypothesized that physical activity may affect insulin-dependent pathways, endogenous sex hormone levels and energy balance. Closer analysis reveals effects on inflammation, immune function, estrogen metabolism and signaling pathways in the cell (24).

It is interesting to note that smoking cigarettes may reduce the risk of endometrial cancer due to its negative effects on estrogen production and metabolism. Multiple births may also be a protective factor, as pregnancy is a period of intensive progesterone production. Oral contraceptives (combined preparations) work in a similar way by regulating the body’s hormonal balance (35, 36). Factors that reduce and increase the risk of endometrial cancer are grouped in Table III. The above-mentioned factors relate to the risk of developing type I cancer. Analogous data for non-estrogen-dependent cancers are not known (32).

Tumor Microenvironment – Hypoxia, Angiogenesis, and Growth

The microenvironment of solid tumors is characterized by hypoxia, low pH (acidosis) and a limited supply of nutrients. These conditions are a consequence of relatively rapid proliferation of cancer cells and insufficient and dysfunctional tumor vascularization (37). In these cells, the dependence of energy production on oxygen requires effective adaptation of cancer cells to hypoxic conditions. The most important aspect of the cellular response to hypoxia is the stimulation of angiogenesis and erythropoiesis and the retuning of metabolism to anaerobic (38). A change in cancer cell metabolism is a key adaptive response to hypoxia, which is characterized by an increase in glucose transport to cells and an increase in the glycolysis process, resulting in increased lactate production. The glycolysis process is the main source of energy (ATP) under hypoxia as a result of inhibition of oxidative phosphorylation and reduction of mitochondria in cells (39). In addition, hypoxia alters lipid metabolism, causing an increase in de novo fatty acid synthesis as a result of increased expression of lipogenesis enzymes (Figure 2). The intensification of the glycolysis process in cancer cells allows for an increase in the number of metabolic precursors necessary for the synthesis of nucleic acids, proteins and phospholipids, which enables cell proliferation (40). One of the reasons why it is difficult to develop new cures for cancer is that it is heterogeneous. Within the same tumor, we will find a whole range of different cells, with different mutations, and differential response to a given drug. Not only are cancer cells different, but so are the cells that surround the tumor. We collectively call them tumor microenvironment (TME). The microenvironment plays an important role in tumor formation, development, and possible metastasis (41). The cells that make up the microenvironment are the cells of the immune system, as well as the cells that make up the blood vessel or healthy tissue from which the tumor originates (42). Some of the immune cells present in the microenvironment, such as cytotoxic T cells or Natural Killer cells (NK) cells, have the potential to kill cancer cells (43). Other types of cells are also present, such as myeloid-derived suppressor cells (MDSCs), and regulatory lymphocytes (Tregs), which promote cancer growth (44). Knowledge of the microenvironment fosters new ideas – for example, microenvironmental cells can be used as targets of new drugs, or as biomarkers to select the right patients for appropriate treatments. The tumor microenvironment can inform about response to a given form of treatment and whether a drug can be administered to a given patient – because of more side effects compared to the potential benefits (45). It is believed that a thorough understanding of the molecular processes of carcinogenesis, as well as angiogenesis in tumors, would allow for the design of effective anti-cancer therapy and thus improve patient quality of life. Current cancer therapies are increasingly using treatment with antiangiogenic factors, which is due to the higher success rate in clinical trials. However, both the advantages and limitations as well as the disadvantages of this type of therapy should be taken into account. The main stimulator of angiogenesis is hypoxia of cancer cells. As the tumor structure grows, the inner regions of the tumor become necrotic. This is related to the lack of oxygen and nutrients in the developing mass. Hypoxia regulates the production of hypoxia-induced factor – HIF-1α, responsible for the production of pro-angiogenic factors, such as VEGF, PIGF, HGF, and Ang-1 (46). Cancerous tumors are therefore not structures that could be combated by regulating the supply of oxygen to their structures, since it has been proven that they remain indifferent to the lack of oxygen and, have mechanisms that allow them to survive in such conditions. Hypoxia significantly increases the density of blood vessels in tumors that is directly related to tumor expansion and affects the formation of metastases. In the initial stage of cancer development, the tumor is built by clusters of cells and its size does not exceed 1-2 mm² (47). This stage can last months or years, and its maintenance depends on the balance between cell proliferation and death (48). These are the so-called in situ cancers that do not have blood vessels in their structure.

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Figure 2.

Adaptive response of cancer cells to hypoxia. HIF-1α: α subunit of HIF-1; HIF-1β: β subunit of HIF-1; HRE: hypoxia response element.

All substrates needed for development, such as oxygen and nutrients, are supplied by diffusion from the vessels surrounding the tumor structure (49). The transition of the tumor to the next phase is directly related to the appearance of hypoxia conditions because of necrosis in the central parts of the tumor. Without the creation of new connections and the delivery of more blood to hypoxic cancer sites, it would not be possible for the cancer to grow further. The acquisition of an angiogenic phenotype is a state of permanent, irreversible, genetic modification that leads to the production of pro-angiogenic factors. This is also called angiogenic transition, and at this stage, the risk of metastasis increases due to the rapid growth of the cancer and tissue infiltration (50). The process of angiogenesis begins with an imbalance between pro- and antiangiogenic factors. Therefore, the first, general stage of angiogenesis can be considered to be an increase in the concentration of proangiogenic factors, e.g., VEGF or basic fibroblast growth factor (bFGF) (51). The second stage of angiogenesis is the increased activity of enzymes–matrix metalloproteinases (MMPs), which are found in the form of proenzymes before activation (52). They are located in the extracellular matrix and around the basement membrane. The tasks of metalloproteinases, such as stromelysin collagenases or gelatinases, include the loosening and degradation of the structures of the basement membrane and extracellular matrix. After the action of these enzymes, the structure of the blood vessels changes, and they can form a new branch. At the same time, endothelial cells are activated, and the extracellular matrix structure is relaxed. The vessel lumen expands, and migration of endothelial cells can increase. The third characteristic stage of angiogenesis is the proliferation and differentiation of endothelial cells of degraded blood vessels formed as a result of previous processes. Vascular endothelial cells are also affected through the adherence and communication with other cells. The migration of endothelial cells (ECs) to these sites allows the capillary to grow and move on to the last stage of angiogenesis – the maturation and stabilization of newly formed blood vessels (53). The mechanism of angiogenesis in cancerous tumors differs significantly from the process of natural vasculogenesis. First of all, it includes changes in the concentration of angiogenic factors, but also in the environment in which the vessels are formed. Angiogenesis in cancer is thought to be triggered by chronic inflammation and severe hypoxia (54). Endothelial sprouting is one of the best-known mechanisms of angiogenesis. It is characterized by the growth and branching of already existing blood vessels towards the avascular zone (55, 56). When discussing the complicated process of sprouting, it is impossible to ignore how the initiating factors affect it. Endothelial cells contain specific receptors that, receiving the signal, start the local reconstruction of the extracellular matrix (ECM). Factors that bind to receptors can be endo-, para- and autocrine substances. Endocrine factors activating and initiating vascular germination come from the circulatory system, paracrine factors are produced by cancer cells, stroma, and macrophages present in the environment or ECM. Autocrine initiators are those secreted by the endothelial cells themselves. Factors that affect the initiation of the endothelial cell germination process include hypoxia-induced factor – HIF-1α (57). Its over-expression in the body occurs during increasing metabolic stress caused by the development of cancer – during hypoxia – i.e., a state of imbalance between the demand for oxygen and its amount supplied to the cancerous tumor (58). Acidosis is also among the factors causing over-expression of HIF-1α. HIF triggers a cascade of expression of pro-angiogenic factors. These include: platelet-derived growth factor B (type B), hepatocyte growth factor (HGF), angiopoietin 2, placental growth factor (PIGF), epidermal growth factor (EGF) and the vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), which is the main initiator and stimulator of angiogenesis. HIF-1α also stimulates the expression of endothelial nitric oxide synthase (eNOS). By breaking down arginine, this enzyme releases nitric oxide molecules, which clearly dilate blood vessels, thus increasing the efficiency of the angiogenesis process (59). Instead of stimulating the formation of new blood vessels, tumors often use the host capillary system to supply themselves with nutrients. This type of angiogenesis, called vessel co-option, is described as one of the three deadliest types of new capillary formation in cancerous tumors (60). Together with vascular mimicry and glomerular angiogenesis, this type of angiogenesis usually indicates a high risk of metastasis. Vessel co-option has been observed in both primary and secondary cancers. A model of incorporating host capillaries into tumor tissue was first observed in brain cancer, which has the most extensive blood vessel networks in the human body. Incorporation of host blood vessels into the tumor begins with the secretion of angiopoietin-2 by primary blood vessels. The substance destabilizes the vessels present and causes their atrophy (61). Hypoxia, which occurs as a result of the degradation of blood vessels, stimulates cancer cells to produce VEGF. The signal initiates the process of endothelial cell germination and thus the production of more blood vessels in the tumor tissue. Studies on VEGF have shown an undeniable correlation between VEGF expression and hypoxic conditions. During cell hypoxia, in both physiological and pathological conditions, cells produce a compound called hypoxia-induced factor. HIF is one of the most important pro-angiogenic factors, acting on both endothelial cells and the cells that produce it, including cancer cells. It is a significant proangiogenic factor because its appearance stimulates the over-expression of other proangiogenic factors. These include VEGF and its placental homologue growth factor (PIGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor B (PDGF-B) and angiopoietin-1 (Figure 3) (62-64).

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Figure 3.

The HIF-1α subunit is synthesized under aerobic conditions. Hydroxylase, which is responsible for the attachment of OH- and thus the von Hippel-Lindau factor, redirects the α subunit to degradation through ubiquitinylation. Under hypoxia conditions, the α subunit accumulates and translocates to the cell nucleus. The combination of HIF-1α and HIF-1β leads to the formation of a full-fledged heterodimer of the hypoxia-induced factor HIF-1. It regulates approximately 40 genes responsible, among others, for the production of VEGF, PDGF, TGF-α.

Hypoxia as a Cause of Endometrial Cancer Development

As described earlier, under hypoxic conditions, the expression of oxygen-sensitive transcription factors (HIF) increases. An increase in endometrial HIF1α expression is observed as the tissue undergoes changes from normal to precancerous and then adenocarcinoma. This process is associated with increased angiogenesis in the endometrium. Therefore, HIF1α, and thus tissue hypoxia, may be a key regulator of endometrial carcinogenesis (65). A worse prognosis has been shown in patients with endometrial cancer with elevated HIF-1α (66).

The association of HIF-1α with tumor aggressiveness in other locations has also been documented (67). The hypoxic state correlates with numerous gene mutations in endometrial cancer (68). In the case of other cancers with TP53, MYC, and PTEN mutations, hypoxia plays a significant role (69). Hypoxia also impacts the mutational load (70). Based on these data, it can be assumed that imaging of the tumor hypoxia state in EC may have a prognostic value and facilitate the personalization of treatment strategies (71-73).

HIF-α

HIF was discovered in 1992 as a result of research on the promoter region of the erythropoietin (EPO) gene (74), which is responsible for the induction of EPO expression. Subsequent studies have confirmed its key role in the primary transcriptional response to hypoxia-induced stress (38).

So far, three isoforms have been detected for HIF-α and HIF-β (49). It is known that all HIF-β isoforms have the same function. The most abundant HIF-1β can dimerize with any isoform of HIF-α protein: HIF-1α, HIF-2α, HIF-3α (21). HIF-1α and HIF-2α have the highest transcriptional activity. The function of HIF-3α is not yet well understood. It is known that one of its isoforms is attributed the role of an inhibitor of HIF-1α and HIF-2α (75).

The HIF transcription factor binds to DNA in the form of a heterodimer, which is formed by HIF-α subunits and HIF-1β. HIF-α is subject to post-translational regulation in contrast to the constitutively expressive HIF-1β subunit (76), therefore the activity of the HIF factor depends on the functioning of HIF-α (77).

HIF-1 is a major factor regulating metabolism and adaptive changes (65, 72). Under conditions of oxygen deprivation, this protein induces the expression of genes encoding proteins responsible for cell adaptation to hypoxia and involved in various aspects of cancer biology (65, 68). So far, about 100 HIF-dependent genes have been identified. They can be divided into: genes regulating cellular metabolism, angiogenesis, proliferation, erythropoiesis, apoptosis, oxygen supply, anaerobic metabolism, glucose and iron transport, hematopoiesis, cell immortality, genetic instability, invasiveness and metastasis, signal transduction for growth factors and resistance to chemo- and radiotherapy (66-68, 70-72). In addition, protein-producing genes that are involved in the formation of new blood and lymphatic vessels are the first whose expression is directly regulated by the concentration of HIF-1 in a cancerous tumor (68).

HIF-1 belongs to a family of protein transcription factors containing a characteristic DNA-binding structure of the basic helix-loop-helix (hHLH) type (68). It functions as a heterodimer consisting of the β subunit and one of the three subunits HIF-1α, HIF-2α, and HIF-3α (67, 77).

HIF-α is a subunit sensitive to oxygen concentration and subject to constitutive expression, HIF-β, also known as ARNT (aryl hydrocarbon receptor nuclear translocator) (38). Both subunits belong to the Per-Arnt-Sim (PAS) family of proteins. They have a helix-loop-helix base domain hHLH and a PAS domain (PAS-A and PAS-B) in the N-terminal region. They are responsible for heterodimerization and binding to DNA (78).

The transactivation domains N-TAD and C-TAD are located near the end of the carboxy-terminal region of HIF protein (78). The HIF-β protein has only one TAD domain, which is responsible for the participation of CBP/p300 coactivators to the HIF-α/HIF-β heterodimer in the cell nucleus (79). As a result of this process, other transcription factors can attach to the HIF complex (78).

The N-TAD domain is directly adjacent to the oxygen-dependent oxygen dependent degradation domain (ODDD). ODDD is a domain that plays an important role in regulating the stability of the HIF-α protein (72, 78, 79). Reduced oxygen pressure exerts a stabilizing effect on the HIF-α protein. Normal oxygen pressure has the opposite effect, i.e., the breakdown of the HIF-α protein (80).

The half-life of HIF-α protein under normoxia conditions is about 5 min (76). One of the first steps in the degradation of HIF-α is the hydroxylation of amino acids within the N- or C- terminal transactivation domain. It involves three proline hydroxylases (PHD) and aspartate hydroxylase, which is called HIF inhibitory factor (FIH) (80). In oxygenated cells, HIF-1 is inactive because its α subunit is degraded in the cell via the proteasome (66, 68, 72).

Then, the hydroxylated subunit of α binds to the von Hippel-Lindau tumor suppressor protein (VHL) and ubiquitin via ubiquitin ligase E3, which results in the degradation of the complex in the proteosome (66, 67, 72). The family of proline hydroxylases regulating the stability of HIF protein includes: PHD1, PHD2, PHD3, which belong to the group of non-heme dioxygenases (81). They require oxygen, divalent iron ions and α-ketoglutarate for their activity, which is why at reduced oxygen content, the activity of PHD decreases, causing HIF activity. PHD2 is considered the most important hydroxylase. Cancers with a mutation in the VHL gene are characterized by high levels of the HIF-1 protein, even under conditions of good oxygenation (65, 67). Thus, HIF-1-induced high gene expression and a more malignant phenotype.

In addition to proline residues, asparagine 803 is also hydroxylated by factor inhibiting HIF-1 (FIH) (67, 81). Under hypoxia conditions, α hydroxylation is inhibited by the inactivation of PHD and FIH enzymes, whose activity depends on oxygen concentration, which leads to stabilization and accumulation of the α subunit, which is translocated to the cell nucleus (67, 72, 80). FIHs have been proven to function under moderate hypoxia conditions when PHDs no longer exhibit catalytic activity (82). In the nucleus, the α subunit fuses with the β to form a heterodimer, which binds to the hypoxia response element (HRE) leading to the stimulation of gene expression in response to the hypoxic state (66, 67). To date, a number of genes carrying the HRE sequence have been identified. Genes that are involved in many important processes have been identified in the promoter region. These genes are involved in a number of important processes including neovascularization (VEGF genes, angiopoietin 1, PDGF), apoptosis (NPM gene), glucose metabolism (GLUT1, GLUT3 gene), pH regulation (IX carbonic anhydrase gene) or adaptation of metabolism to anaerobic conditions (MMP2, UPAR genes) (83-89). Binding of the HIF-1 transcription factor leads to a number of subsequent processes and, consequently, to carcinogenesis. The key coactivator of HIF-1 is the p300 protein, which, by binding to HIF-1α in the nucleus, increases its transcriptional activity (66). In the normal oxygen state, HIF-1α translation is regulated by cytokines and growth factors (73). There are many mechanisms that regulate the level of HIF-1α protein regardless of the oxygen concentration in the cell. These include, a pathway involving the tumor suppressor protein p53, regulation by heat-shock protein (HSP 90) and the receptor of activated protein kinase C (RACK) (90). Activation of HIF-1 in cancer cells may also be independent of hypoxia as a result of activation of oncogenes (Ras, Src, PI3K) or loss of function of tumor suppressor genes (vHL, PTEN) (67). It should also be mentioned that HIF-1α participates in the repair of the endometrium during the menstrual cycle. Increased HIF-1α expression is mainly associated with the secretory phase, with a documented peak in expression in the late stage (91).

Recent studies have confirmed that the action of HIF-1α depends on decreasing progesterone levels and hypoxia, stimulating the endometrium to secrete the angiogenic factors interleukin 8 (IL-8) and VEGF (92). In addition, HIF-1α has been shown to be a coactivator of estrogen-dependent VEGF synthesis (92). HIF-1α modulates angiogenesis not only in the normal endometrium, but also in the endometrium with neoplastic changes (93, 94). However, the most important regulator of HIF-1 activity is the von Hippel-Lindau (VHL) protein (70). At present, it is not known how the gene responsible for the formation of HIF-1α is expressed under hypoxic conditions. An intracellular oxygen pressure receptor has not been identified in cells.

However, there are experimental data suggesting that the factor responsible for the expression of this gene is not a direct decrease in pO2, but a disturbance of the cell’s energy potential (65-68). In many human tumors, the α subunit of HIF-1 has been shown to be over-expressed.

Accumulation of HIF-1 α was detected in breast (56-76%), prostate (82%), rectal (70-100%), ovarian (69%), cervical (72-81%) and head and neck (58-94%) (67, 73) cancers; and also in cancerous tumors of the kidneys, pancreas, brain, endometrium and intestines (66, 72). Changes in HIF-1α expression levels appear to be a good marker for assessing disease progression and tumor aggressiveness. Poor prognosis and lack of response to treatment in cells over-expressing HIF-1α have been confirmed in head and neck cancers, gliomas, rectal cancer, nasopharyngeal cancer, pancreatic cancer, breast cancer, endometrial cancer, cervical cancer, ovarian cancer, bladder cancer, gastric cancer, and osteosarcoma (70, 72, 94).

The Role of Single Nucleotide Polymorphisms (SNPs) of the HIF-1α Gene in Tumorigenesis

In recent years, research on cancer development has considered the influence of individual factors of genetic susceptibility. There is a growing number of studies assessing the relationship between the presence of single nucleotide polymorphisms (SNPs) and susceptibility to cancer (95-97). Genetic polymorphisms, including polymorphisms within the HIF-1α gene, associated with inter-individual diversity and variability, have been identified as the most important genetic factors involved in the development of many diseases, including malignant neoplasms (98). Elucidation and understanding of the phenotypic effects of SNPs, which are based on direct effects of gene-gene and gene-environment interactions, is important for understanding cancer pathogenesis and implementing screening (79). Determination of polymorphisms is a new way to investigate the etiology of polygenic disorders linked to cancer (78, 98). The human HIF-1α gene is located on chromosome 14q21-24. It is composed of 15 exons and encodes 3913 cDNA base pairs (37, 99). A number of polymorphisms and mutations within this gene have been identified. Single-nucleotide polymorphisms in the coding regions of the HIF-1α gene can affect its structure and biological activities (100). In addition, the presence of SNPs is associated with greater stability of the HIF-1α protein and its constant activation (78, 101).

To date, the best-studied polymorphisms of the HIF-1α gene are: 1772C/T (rs11549465) and 1790G/A (rs11549467), which result in the replacement of proline with serine at codon 582 in the first case and alanine for threonine at codon 588 in the second case (95, 99, 101). Studies have shown that the 1772C/T and 1790G/T polymorphisms of the HIF-1α gene are associated with the development of various types of cancer, including endometrial cancer (101, 102).

Studies have shown that the presence of the 1772C/T polymorphism is associated with high transcription capacity and protein synthesis. It also causes structural changes; it increases the stability of the HIF-1α protein and affects the expression of downstream target genes under both normoxia and hypoxia conditions. The 1772C/T polymorphism is associated with increased microvascular density in the tumor, which contributes to tumor development and progression (97, 99, 101). It has been shown that the 1772 C/T polymorphism affects the risk of developing many cancers. In addition, this polymorphism can contribute to the spread of metastasis, but to a different extent for each tumor type (95, 98, 99, 103, 104).

Literature data indicate that the 1772C/T polymorphism of the HIF-1α gene may be a risk factor for head and neck, lung, esophagus, stomach, pancreas, colon, kidney, cervical, breast, prostate and endometrial cancers (99, 101, 103). Studies have shown that HIF-1α expression increases from the minimum values observed in normal endometrium to medium and high levels found in hyperplasia and cancer (100). It is known that the change in protein synthesis is usually caused by a change in the transcription of the encoding gene and the level of mRNA. Therefore, gene sequence variability can contribute to protein synthesis. The HIF-1α gene is highly polymorphic and in view of the significant importance of the HIF-1α protein in the development of cancer, it is worth examining how these polymorphisms affect the risk of endometrial cancer.

There is little literature data in the PubMed database on the importance of HIF-1α gene polymorphisms for the development of endometrial cancer. Among the polymorphisms known so far, the most frequently studied was the single nucleotide polymorphism - 1772C/T. In a study by Kafshdooz et al. on the 1772C/T polymorphism of the HIF-1α gene in women with endometrial cancer, a significant difference was found in the distribution of genotypes and allele frequencies between the patients and the control group (p<0.001). A statistically significant difference was found for CT, TT, CT + TT genotypes (p<005). TT homozygous was only found among patients with endometrial cancer. However, the researchers found no evidence of a relationship between the 1772C/T polymorphism and clinical-pathological features in patients with endometrial cancer (43). The above results have been confirmed by other researchers (50). A significant difference was found in the distribution of genotypes and allele frequencies between patients with cervical and endometrial cancer and the control group (p<0.001). Statistically significant differences between patients with cervical and endometrial cancer and controls were found for CT, TT CT + TT genotypes and patients with CC genotype (p<0.05) (23). Carriers of the 1772T allele (CT+TT genotype) were more common among patients with cervical cancer (68.8%) and endometrial cancer (81%) than in the control group (36.5%). In this study, there was no significant difference between patients with ovarian cancer and the control group in terms of the distribution of genotypes and alleles of the 1772 C/T polymorphism (p>0.05) and evidence of an association between clinical-pathological features and the presence of the 1772C/T polymorphism in patients with ovarian, cervical, and endometrial cancer (78). The results obtained in these studies provide evidence that the polymorphism of the HIF-1α gene may increase the risk of endometrial cancer (77, 78). However, there are reports indicating that there is no association between HIF-1α gene polymorphism and endometrial cancer; these include such as a meta-analysis of 39 studies that included 10,841 cancer patients and 14,682 healthy controls (44). The polymorphisms C1772T, G1790A, C111A and rs2057482 were analyzed. Only the C1772T and G1790A polymorphisms were associated with cancer risk. When polymorphic variants were analyzed for tumor types, the C1772T polymorphism was associated with cervical (T/T, C/T+C/C genotypes) and prostate (T vs. C) cancers. This polymorphism has not been shown to be associated with throat, breast, bowel, lung, bladder and endometrial cancer.

A meta-analysis was also carried out by the team of Yang et al. (101). The analysis included 34 studies involving 7,522 patients and 9,847 healthy controls for whom the 1772C/T polymorphism was tested, and 24 studies involving 4,884 patients and 8,154 controls that analyzed the 1790G/A polymorphism. Only one study has shown an association between 1772C/T and endometrial cancer (78). Researchers also showed racial differences (between the Caucasian and Asian populations) in the prevalence of genotypes of the 1772C/T polymorphism of the HIF-1α gene (77).

Due to the small number of reports on the association between the 1772C/T polymorphism of the HIF-1α gene and the risk of cancer, further research is necessary. In addition, a large case-control sample is required for this type of analysis. Studies should also take into account variables, such as ethnicity, environmental and risk exposure, and lifestyle.

Therapies “Targeted” at Hypoxia and Changes in Metabolism

Tumor hypoxia is a major therapeutic problem as it contributes to resistance to standard treatment regimens with chemotherapy and radiotherapy. Understanding the biological and molecular differences between normal and cancer cells is important for the development of anti-cancer drugs with selective activity (105). Hypoxia of tumors and changes in the metabolism of cancer cells are convenient targets for selective anticancer therapy, which can include bioreductive drugs, inhibition of the activity of the transcription factor HIF-1 and gene therapy (106). The trend in anti-cancer therapy is the administration of bio-reductive drugs, which are specifically activated by reductive enzymes in hypoxic cells. These prodrugs, when activated, generate cytotoxic radicals that react with DNA and cause selective apoptosis of cancer cells (107). A promising therapeutic target is the transcription factor HIF-1, the activation of which not only reprograms metabolism, but also affects many other important aspects of cancer biology, such as the stimulation of angiogenesis (108). HIF-1 is an attractive target for cancer therapy because its activity in normal tissues is negligible, and therefore the side effects on normal cells should be minimal (109). Various small molecule inhibitors of HIF-1, echinomycin and synthetic polyamides and chetomin, are being tested (110). The first two inhibitors block the binding of HIF-1 to DNA, while the chetomin inhibits the binding of the p300/cyclic-AMP-response-element binding protein (p300). This protein is a key coactivator of HIF-1, by binding to HIF-1; it increases its transcriptional activity (111). A characteristic feature of cancer cells is increased glycolysis, which enables cell survival and proliferation under hypoxia conditions. Pharmacological inhibition of this process is also a convenient target for selective therapy in oncology (112). Glycolytic inhibitors include the following compounds: 2-deoxyglucose, 3-bromopyruvate, lonidamine and lactate dehydrogenase inhibitors. 2-deoxyglucose is an unmetabolized analogue of glucose, which accumulates in the cell, leading to the blockage of glycolysis. In contrast, 3-bromopyruvate and lonidamine inhibit hexokinase activity (113). The increase in glucose transport to cancer cells is used clinically to detect tumors and their metastasis with positron emission tomography using a glucose analogue, ¹⁸F-fluorodeoxyglucose (FDG-PET) (114). Increased expression in cancer cells under the influence of hypoxia and HIF-1 activation is also used as tumor marker to detect and monitor tumor progression (115, 116). CAIX is a common prognostic marker in various human cancers, such as lung, breast, cervical, and squamous cell carcinoma of the head and neck (117). Patients with endometrial cancer are treated primarily surgically. Prognostic factors are used to determine adjuvant therapy for patient groups based on the risk of recurrence (histological type and grade, age, tumor size and lymphatic space involvement) (118).

External beam radiotherapy (EBRT) after surgery reduces the risk of vaginal and pelvic recurrence. However, it does not affect overall survival (119-122). Studies have shown that molecular classification has a high prognostic value in the case of high-risk endometrial cancer (123). For TP53 mutated tumors regardless of histological type, adjuvant chemoradiotherapy results in improved disease-free survival compared to radiotherapy alone (123). A potential improvement in endometrial cancer treatment strategies is hypoxia imaging and its association with TP53 status.

Preoperative observation of the hypoxia state could help identify patients who would benefit most from adjuvant radiation therapy. This would allow for the selection of patients who could benefit from EBRT instead of adjuvant brachytherapy alone. Additionally, in the case of hypoxic postoperative tumors with residual disease, it is possible to increase the dose with brachytherapy or external beam radiotherapy. If the tumor is hypoxic, a radio-sensitizing agent should be used, or the dose should be omitted or reduced.

Hypoxia imaging can be used in the case of locally advanced endometrial cancer (stage III) treated mainly with chemoradiotherapy. In this way, patients who would benefit from hypoxia modification with a radiosensitizer can be selected (124). When neoadjuvant chemotherapy (NAC) is used in patients with endometrial cancer, it may be possible to further refine its use if the state of tumor hypoxia, stage and tumor volume, are taken into account to select patients who are likely to respond (125). Monitoring hypoxia and its correlation with genetic instability and DNA damage repair efficiency (confirmed by the prevalence of HRD in p53-mutated endometrial cancer) may be an important predictive factor for women who benefit from the use of polyadenosineriphosphate polymerase inhibitors (PARP) (126). New research achievements in understanding differences in cancer cell metabolism provide the basis for the development of a new generation of drugs and the testing of new therapeutic strategies to more effectively and selectively destroy cancer cells and prevent drug resistance associated with hypoxia (127).