Pathways of Hypoxia-Inducible Factor (HIF) in the Orchestration of Uterine Fibroids Development

The benign nature of UF suggests that the cells maintain the main characteristics of normal myocytes. However, they are also distinguished by specific features. In tumors, metabolism always shifts to a hypoxic state due to two factors. The first one is that even under normoxic conditions, tumor cells fail to utilize pyruvate in the tricarbonic acids cycle but use it to produce lactate. This is one of the most typical phenomena called the Warburg effect [ 8 ]. The mechanisms underlying this effect have yet to be understood in detail, but it allows tumor cells to benefit by accelerating anabolic processes [ 9 ]. The second factor is that the rapid growth of the tumor cells overtakes its vascularization, causing imbalanced feeding and insufficient blood supply. As a result, metabolic products are accumulated and the cells send neighbors “hypoxic” help signaling, which increases vascularization, Fe-accumulation, glucose uptake and inflammatory response [ 10 ]. Hypoxic signaling also affects cells of normal myometrium (NM), endowing them with traits inherent in UF such as aberrant cell cycle dynamics or abnormal metabolism [ 11 ]. UF is characterized by high oxidative stress due to impaired redox metabolism [ 12 ] and increased reactive oxygen species production in response to hypoxia [ 13 ]. Moreover, when the metabolism of UF is switched to the direction of hypoxia, the tumor obviously increases its chances of malignant transformation.

Since aerobic metabolism is a universal sign of all Metazoa , the identification of molecular sensors of oxygen can be called one of the most important discoveries in the last 30 years [ 14 ]. This origin of this discovery goes back to the late 1970s with the identification of a hypoxia-responsive element of human EPO 3′ enchancer in liver and renal cells [ 15 ]. It was suggested that this specific oxygen-sensing mechanism is specific for EPO-producing cells. However, further studies revealed hypoxia-responsive element-binding activity in tissues not expressing EPO: lungs, aortic endothelium, etc. [ 16 ]. The next landmark was the identification of the main sensor of hypoxia, the HIF itself [ 17 ], as well as upstream and downstream members of its pathway: the complex of von Hippel–Lindau (VHL)–Elongin B and C-CUL2 [ 18 , 19 ]. Vascular endothelial growth factor (VEGF) [ 20 ], the prolyl-hydroxylase family [ 21 ], and factor inhibiting HIF [ 22 , 23 ]. There are three isoforms of HIF proteins, each consisting of alpha (α) and beta (β) subunits. HIF-1 and HIF-2 are the most studied members, characterized by similar protein structures. Notably, HIF-1 is a master regulator of the acute response to oxygen depletion, whereas HIF-2 is responsible for adaptation to prolonged hypoxia. Molecular biology and the particular role of HIF-3 have yet to be studied in detail; however, it regulates various pathways, including HIF-1 and HIF-2 [ 24 , 25 ]. The alpha subunit of each isoform is an inducible part normally located in the cytoplasm. The second one, beta, is a constitutively expressed part located in the nucleus. During normoxia, prolyl hydroxylases add hydroxyl residues to the α-subunit, making it a target for ubiquitin-dependent breakdown via proteasome degradation. Another regulator of HIF activity is the factor inhibiting HIF, which adds hydroxyl residues to the asparagine and thus interferes with the binding of HIF and transcriptional coactivators. Since both factors function in an oxygen-dependent manner, they are unfunctional during hypoxia. Thus, as one’s oxygen level decreases, factors inhibiting HIF and prolyl hydroxylase domain proteins are inhibited, enabling the alpha subunit to be stabilized with further translocation into the nucleus and assembly with the beta subunit. The formed HIF heterodimeric complex binds to the promoter of HIF-responsive genes in order to modulate their expression [ 26 ].

In keeping with its essential physiological role, HIF-1 has been demonstrated to contribute in a widest spectrum of human pathologies, from oncological to neurodegenerative and infectious [ 15 ]. Not surprisingly, HIF-1 is also strongly involved in various gynecological diseases—preeclampsia [ 27 ], polycystic ovary syndrome [ 28 ], cancer [ 29 ], endometriosis [ 30 ] and others [ 31 , 32 ]. Several studies have expectedly reported increased HIF1-α expression in UF [ 33 , 34 , 35 ]. In order to further elucidate this link, we carried out an analysis of available data related to expression of genes involved in the HIF-1 signaling pathway in UF based on the KEGG pathway database. It is known that the HIF pathway includes many genes with major effects on the processes typical for UF pathogenesis: apoptosis, proliferation, metabolism, regulation of the cellular availability of oxygen and nutrients [ 36 ]. In particular, such proteins as VEGF-A, ANG-1 (angiopoietin-1), VEGFR-1, EGF, SERPINE1 (PAI1), TEK, and TIMP1 regulate the division, proliferation, and motility of vascular cells as well as the disorganization of the extracellular matrix, thus favoring angiogenesis [ 37 ]. Factors like HMOX1, EDN1, iNOS, and cNOS provide control of vascular tone by balancing nutrients and oxygen supply [ 38 ]. Additionally, NO-synthases are involved in the regulation of apoptosis in UF cells by nytrosylation of caspase 3, downregulating Caspase 3 activity, and increasing the Bcl2/Bax ratio [ 39 ]. Finally, the HIF-dependent response to oxygen depletion is critically dependent on the glucose carrier protein GLUT-1 and glycolytic enzymes: HK, ALDOA, ENO1, PGK1, PFK/FBPase 3, and LDHA [ 40 ] ( Table 1 ).

GWAS remains one of the most effective approaches to the search for “susceptibility” genes to human multifactorial diseases [ 69 , 70 , 71 , 72 ]. Using the GWAS catalog, we found that variations in 174 genes are associated with UF risk in different populations around the world (for instance, rs16991615 MCM8 , rs17631680 DNMT3AP1 , rs2306022 ITGA11 , rs2456181 ZNF346 , rs3820282 WNT4 , rs4335411 ZNF692 , rs5930554 AGKP2 , rs66998222 HNRNPA1P48 , rs78378222 TP53 , rs7986407 FOXO1 , rs8105767 ZNF257 , and rs9548898 COG6 ). We found it interesting to analyze how genes that encode HIF subunits ( HIF1A , HIF1B ( ARNT ), HIF2A ( EPAS1 ), HIF3A , HIF2B , HIF3B ) interact with those GWAS-selected genes in a functional way. For this purpose, we have used the bioinformatics tool STRING ( https://string-db.org/ (accessed on 3 June 2023)). The interactome network of protein–protein interactions (PPI) obtained using the source is shown in Figure 1 . HIF2B and HIF3B were not included in the analysis of the PPI network since the STRING database does not provide data on them. It turned out that 72 GWAS genes, directly or indirectly through other functional partners, are involved in the interaction with HIF1A , HIF1B , HIF2A , and HIF3A . During analysis of the STRING network, we identified 13 GWAS genes that directly interact with genes encoding HIF subunits: ATM , CD44 , CDC42 , FOXO1 , IGF1 , RBMS1 , SIM2 , SIRT3 , TERT , THRB , TP53 , WT1 , ZEB1 (marked with red circles in Figure 1 ). Together with HIF1A , HIF1B , HIF2A , and HIF3A , these genes jointly participate in a large number of pathogenetically significant UF biological processes, characterizing hypoxia, oxidative stress apoptosis, proliferation, cell growth, adhesion and migration, myoblast differentiation, metabolic changes, morphogenesis of vessels, and others ( Table 2 ). In particular, we would like to note the participation of this set of genes in overrepresented biological processes involved in the mechanisms of cellular reply in response to hypoxia and oxidative stress; in the regulation of apoptosis directly and through the P53 signaling; in the regulation of vascular homeostasis through nitric-oxide synthase activity; participation in neoangiogenesis through the regulation of vascular associated smooth muscle cell proliferation; as well as participation in the processes that directly control the initiation and progression of the tumor: “myoblast differentiation”, “positive regulation of cell growth, “mesenchymal cell differentiation”. Taking into account the fact that these processes are reflectors of high-functional HIFs’ interaction with genes, established as a result of human genome-wide association studies (GWAS), this provides conclusive evidence of the role of the HIF pathway genes we have studied in key molecular determinants of tumor growth. Altogether, the summarized data reflecting previous research and our bioinformatic findings are interpretated in Figure 2 .

Although the presented data provide a solid ground to bridge HIF and pathogenesis of UF, there is no rationale to distinguish whether HIF is involved in growth, onset, or both. As upregulated levels of HIF and its downstream effectors were detected in UF samples, HIF is involved in progression of the disease. However, future studies can help to elucidate its role in etiology of the disease. Further investigation can also focus on identifying specific markers associated with HIF expression and downstream factors in UF. Understanding these markers could aid in predicting disease progression, treatment response, and potential complications. The recognition of HIF involvement in UF suggests that targeting HIF pathways could be a potential therapeutic strategy. Developing medications that effectively modulate HIF expression or its downstream factors may offer improved treatment options for UF patients. Further research are needed to elucidate the complete mechanisms by which HIF influences UF development. This sort of research includes studying the interactions between HIF and other molecular pathways implicated in UF pathogenesis, such as hormonal factors, epigenetic regulation and immune responses. Obtaining a comprehensive understanding of the complex biology underlying UF will contribute to improved diagnostic and therapeutic strategies. In recent decades, there has been a growing interest in the study of HIF and its targets in oncology. This attention is of highest clinical significance first of all because of the opportunity to develop new pharmacological avenues for the treatment of various pathological conditions, first of all cancer [ 73 ]. In this respect, one may observe a number of HIF-targeted medications undergoing clinical trials in cancer patients. Most HIF inhibitors are characterized by suppressive activity against cancer. For instance, PX-478 promotes apoptosis, suppresses tumor proliferation, epithelial-mesenchymal transition, and arrests the cell cycle in G2 phase, thus inhibiting tumor growth [ 74 ]. There are also several HIF-2a inhibitors undergoing clinical studies. Results of completed phase II clinical trials of HIF-2a inhibitor PT2385 in treating patients suffered from recurrent glioblastoma were posted in 2020 ( https://beta.clinicaltrials.gov/study/NCT03216499?tab=history (accessed on 10 June 2023)). A few clinical trials of HIF-2α inhibitors were started in 2021. NKT2152 is being studied in a phase I/II clinical trial in patients suffering from advanced clear-cell renal cell carcinoma. https://clinicaltrials.gov/ct2/show/study/NCT05119335?term=HIF+inhibitor&cond=cancer&draw=2&rank=4 (accessed on 10 June 2023) [ 75 ]. DFF332, Another HIF2-α inhibitor for patients with renal cell carcinoma and other tumors (such as HLRCC and VHL disease), is already undergoing phase I/Ib ( https://clinicaltrials.gov/ct2/show/NCT04895748?term=DFF332&draw=2&rank=1 (accessed on 10 June 2023)). Such a wide range of pharmacological tools gives a reason to hope for near-term applications targeting HIF in UF patients. Further studies can help to evaluate the long-term effects of HIF-suppressing therapy in order to find the most efficient and safest drugs. In this respect, one may probably expect the appearance of a rationale to use some of the mentioned medicines to treat UF. Our review gave a good reason to consider HIF-suppressing pathways to decelerate the growth rates of the UF. In this respect, potential application of HIF-inhibitors may serve a new avenue to prevent some of complications related to the disease. For instance, apart from bleeding UF can potentially cause infertility by blocking the fallopian tubes or stopping a fertilized egg from implanting in the uterus [ 76 ]. If the inhibition of HIF turns out successful therapy, women with poor fertility prognosis, miscarriages or complicated pregnancies in anamnesis would may be prescribed a new type of nonsurgical interventions to be treated. Noteworthy, that anatomical features of UF allow topical application of the medical compounds thus minimizing systemic side effects. Thus, we are assuming that the selective and local inhibition of HIF pathways may serve a novel approach to the treatment of UF.

This work was based on the literature review and did not imply following classical principles of meta-analysis such as assessing of statistical power and strict inclusion/exclusion criteria. The authors admit the limitations related to a low number of studies directly evaluating the role of HIF protein in UF. All the theoretical justifications require experimental approval.

Uterine fibroids (UF), also known as uterine myoma or uterine leiomyoma, are the widespread benign tumors of the female reproductive system, affecting approximately 70% of white women and 80% of women of African descent during their lifetime [ 1 ]. In approximately 30% of cases, UF manifest with abnormal uterine bleeding, pelvic pain, pressure, or discomfort, as well as anemia. Whereas malignant transformation of UF is a relatively rare phenomenon [ 2 ], the conditions associated with fibroids are still severe and might include infertility. Moreover, whereas UF could be the only cause of infertility in 2–3% of cases [ 3 ], these neoplasms cause complications during pregnancy and delivery [ 4 ]. Unfortunately, the management of patients with UF frequently relies on surgical approaches, potentially resulting in hysterectomy, which is one of the worst scenarios for women desiring future pregnancy. Moreover, while surgical interventions may decrease the chances of a healthy pregnancy, current pharmacological approaches demonstrate modest efficacy or serious adverse effects. For instance, currently available medications modulating different levels of the hypothalamic–pituitary–gonadal axis provide only short-term relief [ 5 ] and may cause adverse effects—bone loss [ 6 ] and a wide spectrum of other effects [ 7 ]. Additional pharmacological options such as nonsteroidal anti-inflammatory drugs and tranexamic acid help to reduce blood loss, but they do not have any proven effect on tumoral growth. Obviously, further studies of the molecular pathways underlying UF development and malignant transformation may provide novel insights into more effective and safe therapies. In this regard, herein, we review the role of crosstalk between molecular pathways driving UF and HIFs, the family of well-known proteins orchestrating cellular oxygen sensing and response to hypoxia.

Hypothesizing the role of HIF pathways in UF, we performed a literature analysis via PubMed and Google Scholar to identify relevant studies disclosing the role of HIF-proteins in benign myometrium/leiomyoma tumors. Outlining basic evidences bridging HIF and UF we also extracted studies revealing the contribution of HIF-responsive genes/proteins (the list was found in the Kegg pathway database). Combinations of the key words were as follows: uterine fibroids, leiomyoma, myoma, expression, names of chosen genes/proteins: HIF, ANGPT1 , VEGF, FLT1 , EGF , PAI-1 , SEPRINE1, TIE-2 , TEK , TIMP-1, Endothelin-1, EDN1 , iNOS, eNOS, ANP , EPO , TF , HMOX1 , TFRC , GLUT-1, SLC2A1 , PDK-1, HK, PFKL , GAPDH , ALDOA, ENO1, PGK, PFK2 , LDHA , Bcl-2, p21, cyclin-dependent kinase inhibitor 1A, and CD18 . Bibliographies were cross-referenced to identify additional studies. All relevant articles and additional articles cited in primary references are included. To search for genes associated with the risk of UF at the whole-genome level of significance, we used the GWAS catalog ( https://www.ebi.ac.uk/gwas/ (accessed on 1 June 2023)). Then, the STRING database online bioinformatics tool ( https://string-db.org/ (accessed on 3 June 2023)) was used to determine which GWAS genes are in direct interaction with the genes encoding HIF subunits. The STRING database contains information about mechanisms of protein–protein association within a common framework. The search for common biological processes (the so-called overrepresented biological processes) involving the HIF genes and the GWAS genes directly interacting with them was carried out using the Gene Ontology online tool ( http://geneontology.org/ (accessed on 4 June 2023)).

This article has demonstrated for the first time a growing understanding of molecular and genetic determinants of UF development, orchestrated by HIF. Moreover, as far as we are aware, this work is the first review emphasizing the role of HIF in benign tumors. The HIF pathway activates expression of a number of genes responsible for processes known to be imbalanced in tumors. Analysis of patterns of mRNA and protein expression of HIF-target genes, which play a key role in UF development, showed that UF cells displayed their altered status. Additionally, we revealed that genes encoding HIF subunits interact with genes associated with the development of UF in a functional way. Thirteen proteins encoded by those genes are directly interacting with HIFs: Additionally, we distinguished the main biological processes, provided by the revealed genes. These processes cover the whole spectrum of the UF-driving mechanisms: proliferation, cell growth, adhesion and migration, myoblast differentiation, metabolic changes, morphogenesis of vessels, and others. Summarizing the considerable role of hypoxia and HIF in UF pathogenesis, we expect that further studies of this disease narrowly focused on HIF-related pathways may bring new insight on more effective therapeutic options.