THERAPEUTIC TARGETING OF HYPOXIA-INDUCIBLE FACTOR SIGNALING PATHWAYS- A PROMISING APPROACH IN CANCER TREATMENT

1. Department of Biotechnology, KoneruLakshmaiah Education Foundation, Andhra Pradesh, India. 2. Department of Humanities and Basic Sciences, Aditya Engineering College, Surampalem, Andhra Pradesh, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 25 July 2020 Final Accepted: 28 August 2020 Published: September 2020

Oxygen and nutrients are delivered to the cells with the help of the vascular networking system, which makes availability of oxygen as primary regulator for many processes. Low oxygen availability condition activates the Hypoxia Inducible Factors (HIF), which are transcription regulators helping in the expression of genes related to cell cycle regulation and angiogenesis. HIF is hence regarded as the master regulator of angiogenesis. The oxygen deprival is due to the increased consumption of oxygen in the tumor microenvironment and in turn leads to hypoxia. A thorough understanding of how hypoxia influences angiogenesis mediated by several pathways has become essential for identifying novel strategies targeting HIF thereby blocking angiogenesis. In this review we would discuss about the HIF signaling pathways and altered functions of immune cells due to hypoxia by considering that reducing or targeting hypoxia may in turn prevent the suppression of anti-tumor immune response.

Introduction:-
Tumour contains certain regions which are deficient of oxygen supply, necrosis areas. Cells have well exposure to oxygen if they are nearer to blood vessels. An important mechanism as adaptive response to reduced oxygen availability is regulated by HIF pathways [1]. HIF can show their effects on hypoxia responsive genes thereby promoting cancer cell progression. Most recently many potential drugs were identified that inhibit HIF and were also validated as anti-cancer drugs [2]. HIF-1 is the key regulator that helps the tumor cells to adapt in hypoxic microenvironment. Later HIF mediated pathways were discovered which helped in tracing potential anti-cancer drugs [3]. In addition, the noteworthiness of focused HIF treatment is as not completely known in a clinical setting, and huge numbers of the preclinical examinations have discovered undesirable outcomes, demonstrating that the HIF and its pathways are yet not totally understood. It is appropriate that future examination is required around the impacts of focusing on hypoxia when combined with various types of immunotherapy [4]. While there is potential for diminishing hypoxia-interceded protection from immunotherapy, a few treatments might be hampered in oxygenated tumors, bringing about decreased helpful adequacy [5].

Metabolic Changes in TME due to Hypoxia induced pathways:
Hypoxic tumor microenvironment is well characterized by the elevated levels of lactic acid. It can be well explained by Warburg effect which is a metabolic move happening in profoundly multiplying cells that can convert glucose to lactate indeed the presence of oxygen [13]. In this process mainly cancer cells obtain their requirement by depending upon glycolysis rather than TCA cycle [14]. It is well known fact that in glycolysis phenomenon 2ATP of energy is produced per glucose molecule but in TCA cycle for the same molecule 36 ATP are generated. Anyhow studies state that glycolysis has faster kinetics compared to TCA cycle which significantly makes cancerous cell choose glycolysis over TCA cycle [15]. This is a potential hallmark of tumor cells. Many evidences showed that cMyc and Ras and p53 are also involved in regulation of the Warburg effect. To meet the demands of the tumor, HIF-1 regulates aerobic glycolysis and HIF-1α activate glucose transporter gene expressions (Glut1 and 3) under hypoxia environment which helps in glucose uptake by cancer cells [16]. The influx of glucose to meet the requirement of glycolysis is ensured by the activated expression of hexokinases and phosphoglycerate kinase-1. These mechanisms are also regulated by HIF-1α. In few experiments it was demonstrated that PKM2 pyruvate kinase M2 is one of the target gene for HIF-1α and thus its expression helps in uptake of glucose in cancer cells [17]. The lactate production finally leads to acidosis of tumourmicroenvironment which in turn increases the cancer cell invasion [18]. MtoR is mammalian target of rapamycin (protein kinase) has role in regulating cellular processes, but hypoxia mediated mechanisms do not allow expression of this protein kinase resulting in activity of tumor environment [19]. Hence, hypoxia mediated mTOR signaling mechanism may regulate cell behavior and gene expression [20].

Immune cell functions in hypoxic tumourmicroenvironment:
Hypoxia is known to have great impact on immune cell functions. Function and differentiation of dendritic cells is altered under hypoxic conditions. Dendritic cell which are known for their antigen presenting function loose the capacity to present antigens in oxygen deprived conditions [21]. Hypoxic mature dendritic cells have profound change in their chemokine expression. Hypoxia plays a major role in regulation of tumour-associated macrophages (TAMS's). TAM's are attracted to hypoxic regions in TME [22].
Myeloid cells comprise granulocytes and monocytes collectively. They arise due to differentiation of progenitors of hematopoietic stem cells in the bone marrow [23]. Hypoxia-inducible factor 1α, induced by hypoxia in the tumour microenvironment is shown to regulate the function and differentiation of Myeloid-derived suppressor cells (MDSC) in the tumour microenvironment [24]. MDSC are characterized by common myeloid origin. Immature myeloid cells comprising the same phenotype as that of the MDSC are frequently generated in the bone marrow of healthy individuals whereas, in cancer cells, a regular myeloid cell instead of differentiating into a macrophage, a Dendritic cell or polymorphonuclear neutrophils (PMN), differentiates to a pathological MDSC, an immunosuppressive Tumour-associated macrophage (TAM) [25]. For this reason, MDSC is critical in regulating the immune responses in cancer. Hypoxia-Inducible Factors HIF is known to inhibit functions of tumours-infiltrating lymphocytes.
Research has shown that in vitro cultures of hypoxic T cells lead to a suppressed proliferation, essentially because of an increase in iNOS and CD69, which impacts T-cell responses detrimentally [26]. The activation of HIF 1α downregulates T cell reception signal transduction, whereas the absence of HIF 1α leads to an increase in proinflammatory cytokines, due to an increase in NF-κB activation [27].

Inhibition of HIF-A promising target for Cancer therapy:
Exploring HIF pathways and selecting it as an inhibitory target for cancer was well established [28]. As of now there are few inhibitors against HIF molecular components at phase 1 and 2 clinical trials. Studies have demonstrated that hypoxia environment showed resistance to the immunotherapy and helps in the activation of few immunosuppressive cells like MDSCs (Myeloid derived suppressor cells) [29]. There by targeting hypoxia and its pathways can prevent the suppression of immune responses against tumor. Most of the therapies related to the HIF pathways help in inhibiting angiogenesis and affecting cancer cell metabolism. The potential outcome may be observed when combination of immunotherapy and HIF inhibition therapies are used [30].
One such study has been explored in murine prostate cancer where TH-302 reduces the hypoxia. In another study, ENTPD2 ectonucleoside triphosphate diphosphohydrolase 2 inhibitors and immune checkpoint inhibitors which increased the infiltrating capability of T-cells into cancer environment [31]. Increased anti-tumor function of T-cell and tumor clearance was observed in B16 and MC38 murine tumor models when Metformin, a type-2 diabetes drug was used along with PD-1 blockade [32]. By targeting Mtor or AKT pathways inhibition of HIF can be achieved. Tuberous sclerosis complex 2 (TSC2) is regulator of mTOR and TSC2 knockouts have shown accumulation of HIF-1α and further activation of genes related to expression of VEGF [33]. Another potential drug named Rapamycin, has significantly reduced the levels of HIF-1α in TSC2 knockouts. PD-L1 is an important regulator for immune escape of cancer cells. The blockade of such regulator can enhance the immune activity [34]. It has been explored that AKT and mTOR pathways regulate PD-L1 in lung cancer, colorectal cancer and pancreatic cancer. Targeting hypoxia related pathway with immunotherapy together is a considerable approach. But sometimes immunotherapies within the tumor microenvironment become beneficial on exposure to hypoxic environment. Oncolytic HSV-1 are already used for melanomas as first line treatment [35]. Recent studies also showed that these viruses replicate more efficiently in hypoxic conditions finally which resulted in lower progression rates of tumor. But it depends on the virus because Oncolytic adeno virus replication gets hampered under hypoxic conditions.
To date, several HIF inhibitors have been synthesized and identified, to inhibit the expression and function of HIF subunits through direct or indirect mechanisms [36]. Although the classification of HIF inhibitors may not be accurate, they are tentatively classified based on the reputed mechanisms of action. These are protein synthesis, agents modulating the expression, protein accumulation and degradation, dimerization, DNA binding, and transcriptional activity of HIFs [37]. While no potential agents have been developed to selectively inhibit HIFα protein synthesis, few natural compounds such as Chrysin inhibits synthesis of HIF-1α protein by AKT signaling [38]. Whereas glyceollin, isolated from soybeans, by restricting the PI3K / AKT / mTOR pathway, significantly reduced HIF-1α synthesis. In addition, 2-methoxyestradiol (2ME2) which is naturally produced by catechol-Omethyltransferase-mediated O-methylation of 2-hydroxyestradiol has been found to inhibit the synthesis of HIF-1α 1335 and HIF-2α proteins, and suppress their nuclear translocation and transcriptional activity. KC7F2, which inhibits HIF-1α protein synthesis, is also a lead compound [39].
A compound Indenopyrazole 21 was found to strongly inhibit HIF-1 alpha transcriptional activity without affecting the accumulation of HIF-1α protein and HIF-1 heterodimerization [40]. While, YC-1, an anti-platelet aggregation agent, oppresses HIF transcriptional activity and may also affect HIF-1α protein accumulation. Chetomin, a metabolite, was found to inhibit HIF-1 alpha association with p300 by disrupting the p300 protein's tertiary structure of the CH1 domain [41]. FM19G11, a novel chemical entity, effectively represses p300, a histone acetyltransferase necessary by inhibiting histone acetylation which is a co-factor for HIF-transcription activation [42].

Conclusion:-
Studies have proven that hypoxia/HIF mechanisms are master regulators in promoting angiogenesis in TME. This review represents that HIF related mechanisms are potential targets incapability to regulate the process of angiogenesis by manipulation of HIF pathways for treating cancers. Cancer cells can evade single anti-angiogenic procedures by promoting other pro-angiogenic pathways. As HIF regulate pro-angiogenic pathways there may be increased therapeutic outcome on targeting HIF mechanisms.