Deciphering the interplay between hypoxia, angiogenesis, and endoplasmic reticulum stress in carcinogenesis: A narrative review

Abstract

Cancer cells face oxygen and nutrient shortages, driving vascular endothelial growth factor (VEGF)-mediated angiogenesis and increasing protein-folding demand, which triggers endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR) pathways. The UPR is triggered through three major sensors: IRE1, PERK, and ATF6. Simultaneously, hypoxia stabilizes hypoxia-inducible factor (HIF) genes, enabling tumors to adapt, promote angiogenesis, and enhance survival. This review aims to decode the interconnected roles of hypoxia, angiogenesis, and ER stress in carcinogenesis, with a specific focus on how HIF-regulated signaling integrates these pathways to support tumor progression and impact clinical behavior. Researchers have found that both the UPR and hypoxia pathways influence VEGF expression by increasing the transcription factors ATF-4 and XBP-1, respectively, and by enhancing the expression of HIF genes. HIF genes are known as one of the master regulators of angiogenesis. The PERK/eIF2α pathway, IRE-1, and ATF6, all three branches of the UPR response, also help cancer cells survive under hypoxic conditions. On one hand, where PERK increases the heterodimerization between α levels at the translational level, the IRE-1 branch increases its stabilization via a process known as regulated IRE-1-dependent decay, an endoribonuclease activity. Understanding this triad will support the development of targeted therapies, including HIF inhibitors, anti-angiogenic agents, and UPR modulators, as well as biomarker-based patient selection and combination treatment strategies. Integrating hypoxia, angiogenesis, and ER stress biology reveals critical insights for designing more precise and effective anticancer interventions.

Key Words: Angiogenesis; Hypoxia; Endoplasmic reticulum stress; Cancer; Vascular endothelial growth factor; Hypoxia-inducible factor

Core Tip: This review explores the interplay between hypoxia, endoplasmic reticulum stress and angiogenesis, highlighting the central regulatory role of hypoxia-inducible factors (HIFs). It highlights how HIF signaling links oxygen deprivation with vascular adaptation and protein-folding stress responses. Understanding this integrated network provides valuable insight into how tumors exploit these pathways for survival and offers potential molecular targets for therapeutic intervention. In future, this will lead to a better understanding of their synergistic relationship and how modulation of either pathway can potentially result in restraining the aggravated vasculogenesis which promotes the aberrant cell proliferation in cancer and in some case, make them more resistant to chemo and radiotherapy.

INTRODUCTION

Hypoxia refers to a state in which body tissues receive insufficient oxygen, disrupting tissue homeostasis. This condition may arise when oxygen delivery is reduced, often due to poor blood circulation, low haemoglobin levels, or decreased blood oxygen content. Hypoxia can be localized, affecting only specific tissues (tissue hypoxia) or systemic, involving the entire body (generalized hypoxia). It may occur suddenly (acute hypoxia) or develop gradually over time (chronic hypoxia). A persistent oxygen shortage triggers a range of physiological adaptations to maintain oxygen levels. These include stimulating the production of red blood cells to enhance oxygen transport, promoting the growth of new blood vessels to improve tissue oxygenation, reprogramming cellular metabolism to minimize oxygen consumption and maintain redox balance under sustained oxygen-limiting conditions[1].

Angiogenesis refers to the formation of new blood vessels from existing ones, a process essential for organ growth, embryonic development, and wound healing[2-4]. Newly formed vessels, lined exclusively with endothelial cells, deliver oxygen and nutrients to the tissues, support immune defense by facilitating the movement of blood cells, and help in the removal of waste products[3,5]. In adults, angiogenesis rarely occurs under normal conditions; it is activated only during specific physiological events such as tissue repair, skeletal development, pregnancy, or menstrual cycle[2,3,6]. Over the last two decades, researchers have gained significant insights into the molecular processes that control angiogenesis. This process is highly regulated through a balance between pro-angiogenic and anti-angiogenic signals[7]. Pro-angiogenic factors such as basic fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) activate endothelial cells to release enzymes that break down the basement membrane. This breakdown allows endothelial cells to multiply, migrate, and produce vessel sprouts that can extend by several millimetres per day[8,9]. Angiogenesis plays a role in many pathological conditions, including cancer, psoriasis, diabetic retinopathy, arthritis, asthma, autoimmune diseases, infections, and atherosclerosis[10-12]. Due to its involvement in a wide range of disorders, understanding the genes and pathways that regulate angiogenesis is crucial for developing more effective therapeutic strategies.

The endoplasmic reticulum (ER) is a flexible, tube-like organelle inside cells that participates in critical metabolic activities, such as producing sugars through gluconeogenesis and the synthesis of lipids. It is also the primary storage site for calcium within the cell and plays a crucial role in building cellular structures, such as autophagosomes and peroxisomes. Early stages of protein maturation occur in the ER, which is essential for the correct folding of proteins produced via the secretory pathway. These proteins make up nearly 30% of the total proteome in most eukaryotic cells. In specialized secretory cells, where the demand for protein production is exceptionally high, the protein-folding machinery of the ER experiences constant stress[13]. Any of the following three main responses can manage this: First, transient adaptation involves lowering protein synthesis and its translocation to the ER; the second way is to increase the transcription of unfolded protein response (UPR) target genes, including chaperones. Lastly, if the protein homeostasis is not restored, then it leads to cell death.

During carcinogenesis, the cells undergo ER stress as well as hypoxia. The triggering of stress response mechanisms, such as the UPR and the activation of hypoxia-inducible transcription factors hypoxia-inducible factor (HIF)-1 and HIF-2, helps cells balance their survival through adverse conditions. Both of these protective systems have been extensively studied at the biochemical and molecular levels. Recent findings suggest that these pathways can be activated simultaneously, not only in tumor cells but also in other tissues with poor oxygen or nutrient supply. Interestingly, they share the same transcriptional targets, such as pro-angiogenic factor, VEGF A (VEGFA). Experiments show that during the UPR, ATF4 directly binds to the VEGFA promoter and drives its transcription, whereas in hypoxia, HIF-1 performs the same function. ATF4 is a multifunctional transcription regulatory protein involved in the cellular stress response. During ER stress and hypoxia, both ATF4 and HIF-1 have a synergistic effect on the transcription level of VEGFA level.

Additionally, ER stress enhances the activity of HIF-1 and the expression of Bcl-2 interacting protein 3 (BNIP3). Under hypoxic conditions, BNIP3 promotes the growth of tumours in breast cancer in an autophagy-dependent pathway. This reveals a new connection between the two pathways, enabling cells to adapt more effectively to challenging environments[4].

HYPOXIA: CELLULAR AND MOLECULAR RESPONSE

Oxygen plays a crucial role in maintaining cellular energy balance by supporting adenosine triphosphate (ATP) production and serving as an electron acceptor in numerous biochemical processes. As a result, the body’s response to low oxygen (hypoxia) is rapid, essential, and highly conserved throughout evolution. Many oxygen-dependent reactions occur in cells, such as aerobic respiration, fatty acid desaturation, and the activity of α-ketoglutarate-dependent dioxygenases. When blood vessels are damaged or unable to supply enough oxygen due to vascular insufficiency or fluid accumulation in tissues (edema), hypoxia can develop in various diseases. In cancer, for instant, some solid tumors grow beyond the reach of blood vessels, oxygen diffuses from vessels, resulting in the formation of oxygen-poor (hypoxic) regions. For further growth, these tumors need to develop a blood supply either by creating new vessels (angiogenesis) or by using existing ones. However, tumor blood vessels often differ from normal vessels in both structure and function, leading to poor oxygen delivery to the tissues. This ability to adapt to low oxygen is a hallmark of both primary and metastatic cancers. Cellular hypoxia (oxygen levels between 0.5%-2%) may be temporary, caused by short-term imbalances between oxygen demand and supply, or long-term due to permanent vascular problems, persistent edema, or chronic inflammation. The severity of oxygen deficiency can influence the cell’s response; for example, very low oxygen levels can disrupt protein folding and other oxygen-dependent biochemical reactions. To survive, cells integrate multiple adaptive mechanisms until oxygen levels are restored, allowing them to meet their everyday metabolic needs[14].

Cells have evolved sophisticated mechanisms to cope with the fluctuations in oxygen availability. One of the primary adaptive strategies involves the activation of genes responsible for angiogenesis, energy metabolism, and cell survival. Central to this process is the HIF family, which serves as the master regulator of oxygen homeostasis[15]. Among them, HIF-1 (HIF-1α) plays a crucial role as a transcriptional mediator that adjusts cellular functions during oxygen deprivation[16]. Structurally, they belong to the bHLH period-aryl hydrocarbon receptor nuclear translocator (ARNT)-single-minded protein (bHLH-PAS) family of transcription factors. The PAS and bHLH motifs are required for heterodimerization between α (HIF-1α) and β-subunit to bind DNA at hypoxia response elements (HREs) and initiate transcription of target genes[17]. These genes regulate essential processes, including angiogenesis, glycolysis, and erythropoietin synthesis. The transcriptional activity of HIFs is further regulated by interactions with coactivators like CREB-binding protein (CBP) and p300, which associate with the C-terminal activation domains[18,19]. CBP and p300 are closely related family members and are histone acetyltransferases. They serve as transcriptional coactivators. Regulation of HIF activity is strongly oxygen-dependent. HIF-1β is constantly produced in cells, whereas HIF-1α is tightly regulated. Under normal oxygen levels (normoxia), HIF-1α is rapidly degraded after being tagged for destruction through ubiquitination and subsequently broken down by the proteasome[20,21].

In contrast, when oxygen levels are low (hypoxia), HIF-1α becomes stabilized. Specifically, during normoxia, newly synthesized cytoplasmic HIF-1α undergoes hydroxylation at proline residues 402 and 564 within its oxygen-dependent degradation (ODD) domain. This reaction is carried out by prolyl hydroxylase domain (PHD) enzymes[22,23]. Hydroxylation serves as a critical step in HIF-1α regulation, as it enables recognition by the von Hippel-Lindau (VHL) E₃ ubiquitin ligase complex. Once bound, HIF-1α is ubiquitinated, marked for proteasomal degradation, and subsequently removed from the cell[24-26]. HIF-1α activity is also modulated by hydroxylation of asparagine (Asn) at position 803 within its C-terminal transcriptional activation domain (C-TAD). Under normal oxygen levels, Asn is hydroxylated by the enzyme factor inhibiting HIF-1. This modification prevents HIF-1α from interacting with transcriptional coactivators such as CBP/p300[27-29]. During hypoxia, the availability of oxygen and essential cofactors becomes restricted; as a result, hydroxylation of HIF-1α is reduced[30,31]. HIF-1α accumulates in the cytoplasm and later translocates into the nucleus, where it associates with HIF-1β to form a functional heterodimer. The heterodimer HIF-1α/β binds to HREs located within the promoter regions of target genes, particularly at the consensus sequence 5-RCGTG-3. Through this interaction, HIF-1 regulates the transcription of numerous oxygen-responsive genes, thereby influencing their expression[18,32,33]. HIF-1 regulates a wide range of target genes that help adapt to hypoxic conditions by influencing stress responses, metabolism, and angiogenesis. Under hypoxic states, HIF-1 enhances erythropoietin (EPO) production, which promotes red blood cell synthesis and thereby improves oxygen delivery to tissues[34]. It also reprogrammes cellular metabolism, favouring glycolysis over oxidative phosphorylation. Cells under hypoxia rely mainly on anaerobic glycolysis for ATP generation. However, glycolysis yields far less energy as compared to the tricarboxylic acid cycle at normal oxygen levels[35,36]. To preserve energy homeostasis under hypoxic conditions, HIF-1 enhances the expression of glycolytic enzymes and glucose transporters, facilitating cells to boost glucose uptake and generate sufficient ATP[37-39]. Beyond metabolism, HIF-1 supports cell growth and survival by increasing the expression of genes such as insulin growth factor-2 (IGF-2) and transforming growth factor (TGF)-α[40,41]. Hypoxia and HIF-1 signaling play a central role in angiogenesis by activating VEGF-A, along with other pro-angiogenic genes and their receptors, which promote the formation of new blood vessels[42,43]. To support the recruitment of endothelial progenitor cells to hypoxic regions, HIF-1α upregulates the expression of matrix metalloproteinase-2 (MMP-2). This enzyme breaks down the components of the extracellular matrix, thereby facilitating the migration of endothelial cells[44].

HIF GENES: STRUCTURE AND FUNCTIONS

HIF-1α

HIF proteins exist in different isoforms, three oxygen-sensitive α-subunits (HIF-1α, HIF-2α, HIF-3α) and two β-subunits (ARNT/HIF-1β and ARNT2). In parallel, HIF also induces the expression of αβ integrins, which enhance endothelial cell proliferation, adhesion, and stabilization during vessel growth[45]. Among them, HIF-1α is the most extensively characterized isoform, and much of the current understanding of HIF structure and regulation is based on the studies of mammalian HIF-1α, with HIF-2α also contributing to a lesser degree. Structurally, HIF-1α is a polypeptide of approximately 120 kDa, while HIF-1β is slightly smaller, ranging from 91 kDa to 94 kDa. These two subunits dimerize to generate the functional HIF-1 transcription factor. HIF-1α and HIF-1β are members of the bHLH-PAS transcription factor family sharing sequence homology with bHLH Drosophila proteins Per and Sim. Each subunit contains a single bHLH domain and two PAS domains (PAS-A and PAS-B), which facilitate dimerization. The bHLH region is crucial for DNA binding, allowing the HIF-1α/HIF-1β complex to interact with HREs in the promoters of target genes[46,47]. Beyond these structural regions, HIF-1α possesses two transcriptional activation domain (TADs) located in its C-terminal, which are distinct from the DNA-binding and dimerization domains and are critical for hypoxia-responsive transcriptional activity[33,47]. These independent TADs are the N-terminal TAD (N-TAD, residues 531-575) and the (C-TAD, residues 786-826). Both are enriched in acidic and hydrophobic amino acids[18], but their activity is repressed under normal oxygen levels by an intervening inhibitory domain (residues 576-785)[18,33]. HIF-1α, being the oxygen-sensitive subunit, is primarily responsible for HIF-1-mediated transcription, while HIF-1β mainly acts as a dimerization partner[47,48]. The activity of HIF-1α is modulated by oxygen availability[18,49]. The recruitment of coactivators, such as CBP and p300, promotes the transcription of C-TAD[27]. The N-TAD, located in the area known as the ODD domain (residues 400-600), is recognized by pVHL in normoxia, triggering ubiquitin-proteasome degradation and thereby tightly controlling HIF-1α stability[33,50]. pVHL is a tumor suppressor that senses the cellular oxygen levels and controls cell growth. The ODD domain comprises two critical proline residues that undergo hydroxylation under normoxic conditions, thereby regulating the stability and activity of the HIF-1α subunit. Its C-terminal region functions as the protein stability domain[33,51,52]. HIF-1α contains both N-TAD and oxygen-dependent degradation domain (ODDD) regions, which are absent in HIF-1β. While HIF-1β has only the C-TAD, both HIF-1α and HIF-1β mRNAs are expressed across human tissues. Unlike HIF-1β, the expression of HIF-1α is not strongly influenced by oxygen levels in vitro. Still, it rises significantly under hypoxia in vivo, especially in organs such as the brain, heart, kidneys, lungs, and skeletal muscles[46,53-55]. Functionally, HIF-1β remains constitutively active and stable in both normoxic and hypoxic states, whereas HIF-1α is unstable in normoxia and undergoes rapid degradation via the ubiquitin-proteasome system[16,56]. The stability of HIF-1α largely determines overall HIF-1 activity[53]. HIF-1α functions as a conditionally regulated transcription factor, while HIF-1β remains active under all conditions. Unlike HIF-1α, HIF-1β can interact with additional proteins, such as the aryl hydrocarbon receptor (AhR), because it is identical to the ARNT protein, which is essential for AhR signaling[46]. Moreover, HIF-1β is capable of forming homodimers in vitro, whereas this is not the case for HIF-1α (Figure 1).

Figure 1 Schematic representation of the functional domains and potential roles of hypoxia inducible factor isoforms: Each column represents a distinct domain, with hydroxylation sites indicated above. All hypoxia inducible factor (HIF) isoforms belong to the bHLH-PAS family containing a bHLH and two PAS domains (PAS-A and PAS-B) essential for HIF-1α and HIF-1β heterodimerization. Unlike HIF-1β, HIF-1α subunits possess an oxygen-dependent degradation domain (ODDD) that mediates proline hydroxylation and lysine acetylation, leading to proteasomal degradation. The ODDD contains an N-terminal activation domain and a C-terminal transactivation domain that regulate transcriptional activity. Conserved proline residues are present in HIF-1α and HIF-2α. Several HIF-3α splice variants exist, including variant 1, which lacks the C-terminal transcriptional activation domain, and variant 2, which contains a leucine zipper domain involved in DNA binding and protein interactions. HIF: Hypoxia inducible factor; N-TAD: N-terminal activation domain; C-TAD: C-terminal transcriptional activation domain; ODDD: Oxygen-dependent degradation domain; LZIP: Leucine zipper.

HIF-2α

HIF-2α and HIF-3α are close homologues of HIF-1α. HIF-2α, also known as endothelial PAS domain protein 1, HIF-1α-like factor, HIF-1α related factor, and member of the PAS superfamily-1 (MOP-1)[57-60]. HIF-2α shares nearly half of its amino acid sequence and domain structure with HIF-1α[51,57,58]. Despite similarities in HIF-1α and HIF-2α in heterodimerizing with HIF-1β, binding to hypoxia-inducible genes, and transcriptional activation, both proteins differ in their tissue distribution and developmental roles[51,57-60]. HIF-2α is predominantly expressed in embryonic tissues, vascular endothelium, lungs, placenta, and heart, while HIF-1α is broadly present in nearly all mammalian tissues, especially heart and kidney[56,58,60,61]. They also differ in transcriptional specificity. HIF-1α primarily regulates glycolytic enzymes, such as lactate dehydrogenase and carbonic anhydrase IX (CA IX), whereas HIF-2α is more closely associated with EPO regulation and genes involved in iron metabolism. Some genes, including VEGF and glucose transporter 1, are influenced by both HIF-1α and HIF-2α[62,63] (Figure 1).

HIF-3α

HIF-3α was initially described in mice as a novel bHLH-PAS protein with about 662 amino acids[64]. It was later identified in humans with high similarity to HIF-1α and HIF-2α in their bHLH and PAS domains. Compared to HIF-1α, it shares around 57% sequence identity in the bHLH-PAS region and 61% in the ODD domain. Unlike HIF-1α, HIF-3α contains an N-TAD but lacks the C-TAD domain, although certain isoforms possess a leucine zipper domain instead[65,66]. HIF-3α is expressed in several adult tissues, including thymus, lungs, brain, heart, and kidney, and can form heterodimers with HIF-1β both in vitro and in vivo[64]. HIF-3α expression increases in mice, rats, and humans under hypoxic conditions[67,68]. HIF-2α further enhances HIF-3α transcription by stimulating its mRNA expression[69]. A shorter splice variant of HIF-3α known as inhibitory PAS domain protein (IPAS) has been identified as a negative regulator of hypoxia-inducible gene expression in mice[70]. Unlike other HIFs, IPAS lacks N-TAD and C-TAD domains, giving it a unique ability to suppress the transcriptional activity of HIF-1α and HIF-2α. This adds complexity to the regulation of hypoxia-inducible genes by the HIFs. IPAS inhibits HIF-1α by binding to its bHLH/PAS domain, leading to the formation of a defective complex with HIF-1α in the nucleus[71]. Unlike HIF-1α, IPAS is expressed in specific tissues, such as the Purkinje cells of the cerebellum and the corneal epithelium, where it plays a protective role. In the cornea, IPAS counteracts HIF-1α by suppressing VEGF signaling and neovascularization, which is crucial for maintaining transparency and vision. Its selective action on HIF-1α without influencing other bHLH/PAS transcription factors makes it a potential target for limiting tumor angiogenesis and other HIF-1α-driven pathologies. Experimental studies have shown that tumors derived from IPAS-expressing hepatoma cells grow more slowly and exhibit reduced vascularization compared to controls[70]. Another splice variant of HIF-3α, known as neonatal and embryonic PAS (NEPAS), is mainly expressed during embryonic and early postnatal development. NEPAS negatively regulates HIF-1α and HIF-2α activity by dimerizing with HIF-1β[72]. Additional HIF-3α splice forms exist in humans[66,73,74], and although all are induced by hypoxia through HIF-1α but not by HIF-2α[74]. Some evidence suggests species-specific differences, for example, in zebrafish, hypoxia increases HIF-3α expression independently of HIF-1α, with regulation occurring in a tissue-specific manner[75] (Figure 1).

MECHANISM OF HIF-1/2Α REGULATION: REGULATION OF HIF-1/2Α THROUGH THE CANONICAL MECHANISM

The activity and stability of HIF-α subunits are controlled mainly through oxygen-dependent hydroxylation. Under normal oxygen conditions, hydroxylation promotes the rapid degradation of HIF-α, whereas low oxygen levels prevent this process, leading to HIF stabilization. This regulation is mediated by the pVHL complex[76,77]. pVHL complex includes VHL, ElonginB/C, RBX1, and the scaffold protein CUL2, responsible for targeting hydroxylated HIF-α for ubiquitination and proteasomal degradation. Hydroxylation occurs at specific proline residues within HIF-1α (P402, P564) and HIF-2α (P405, P531)[25,78], catalysed by PHD1-4, particularly PHD2. These enzymes require oxygen, iron, ascorbate, and α-ketoglutarate and generate CO₂ and succinate as byproducts. In addition to prolyl hydroxylase, the enzyme factor inhibiting HIF (FIH), an asparaginyl hydroxylase, modulates HIF-1α and HIF-2α under normal oxygen conditions. It hydroxylates Asn 803 in HIF-1α and Asn 847 in HIF-2α within their C-TADs, thereby preventing their interaction with CBP/p300 transcriptional coactivators[33,79]. This reaction is mediated by FIH, an iron- and α-ketoglutarate-dependent dioxygenase enzyme that requires oxygen for activation. Under hypoxia, the functions of both PHDs and FIH are impaired, leading to the stabilization of HIF-1α and HIF-2α and an enhancement of their transcriptional activity. The suppression of these hydroxylases during low oxygen levels is partly due to the oxidation of their Fe(II) cofactor by mitochondrial reactive oxygen species (ROS) generated through complex III[80]. Additionally, ROS from NADPH oxidase, especially NOX1 and NOX4, further promote HIF signaling[81]. In renal carcinoma, this ROS-driven stabilization of HIF-2α supports tumor progression[82].

REGULATION OF HIF-1/2Α THROUGH A NON-CANONICAL MECHANISM

While the oxygen-dependent pVHL pathway is the primary regulator of HIF1/2α stability, other mechanisms also contribute by modulating their stability, synthesis, and transcriptional activity under both hypoxic and normoxic conditions. Current evidence suggests that the oxygen-independent regulation of HIF-1α is well characterized; however, the similar oxygen-independent control of HIF-2α remains less understood and requires further research.

Acetylation and deacetylation of HIF-1/2α

Post-translational acetylation and deacetylation are important for regulating the stability and transcriptional activity of HIF-1/2α, but their exact roles remain unclear due to conflict findings. Lysine acetylation at multiple sites on HIF-1α leads to distinct downstream effects. For instance, acetylation within the ODD domain promotes pVHL binding and degradation of HIF-1α. Specifically, acetylation of lysine (K532) by arrest defective-1 (ARD1) enhances pVHL recognition[83], marking the HIF-1α for proteasomal degradation. However, this acetylation is counteracted by metastasis-associated protein 1 (MTA1), which induces the deacetylation of HIF-1α via histone deacetylase 1 (HDAC1). This process enhances both the stability and transcriptional activity of HIF-1α, especially under hypoxic conditions where MTA expression is upregulated and closely associated with HIF-1α[84]. MTA1 and HIF-1α are therefore thought to promote tumor growth and metastasis. Similarly, metastasis-associated protein 2 also deacetylates HIF-1α through HDAC1, stabilizing the protein in pancreatic carcinoma[85]. The K532R substitution showed no impact on the interaction between the HIF-1α ODDD region and human ARD1, indicating that hARD1 does not acetylate HIF-1α or promote its destabilization[86]. Studies suggest that altering ARD1 expression does not significantly impact basal HIF-1α levels or hypoxic responses[87]. In contrast, acetylation at lysine residues within the C-terminal region of HIF-1α plays a critical role in its interaction with transcriptional coactivator p300 and in regulating HIF-1 activity. Specifically, acetylation of lysine 709 by p300 enhances HIF-1α stability under both normoxic and hypoxic conditions, though this interaction is counteracted by HDAC1[88]. Similarly, acetylation at lysine 674 mediated by CBP/PCAF increases HIF-1α levels and promotes p300 binding[89]. Regulation is further refined by deacetylases, such as sirtuin (SIRT1), which removes the acetyl group from lysine 674, thereby preventing p300 recruitment and suppressing HIF-1 target gene activation. For HIF-2α, CBP-driven acetylation at multiple lysine sites in the C-terminal region can also be reversed by SIRT1, which selectively enhances HIF-2 signaling[90,91].

PI3K/AKT and MAPK/ERK pathways in HIF regulation

Under non-hypoxic conditions, growth factors or activated tyrosine kinases can stimulate HIF-1α production through the PI3K/AKT or MAPK/ERK signaling cascades. Tumor-related mutations often alter these pathways, promoting uncontrolled proliferation and blocking apoptosis. PI3K signaling regulates HIF-1α translation mainly via AKT and the mTOR complexes. mTORC 1 drives phosphorylation of p70S6K and 4E-BP1 thereby releasing eIF-4E to initiate protein synthesis including HIF-1α[92]. While HIF-1α depends on both mTORC1 and mTORC2, HIF-2α expression in renal carcinoma cells is more dependent on mTORC2[93]. Similarly, the MAPK/ERK pathway enhances HIF-1α synthesis through ERK-mediated activation of MNK kinases, which directly stimulate eIF-4E, a key member of translation initiation machinery. Growth factors such as IGF-1, IGF-2, EGF, and FGF-2 also upregulate HIF-1α[40,94] and protein synthesis via both the PI3K/AKT and MAPK signaling pathways[93,95]. IGF can enhance HIF-2α transcription through the PI3K-mTORC2 pathway and promote vascularization in neuroblastoma[95]. HIF-1α plays a key role in cancer by driving IGF-2 expression, particularly in colon cancer[96]. In breast cancer, activation of the HER-2 receptor promotes HIF-1α accumulation and VEGF production through the PI3K/AKT signaling pathway, thereby enhancing angiogenesis[97,98]. Similarly, prostate carcinoma and glioblastoma often show elevated HIF-1α levels when tumor suppressors like PTEN, VHL, or p53 are lost or inactivated[99-101]. Normally, these suppressors limit PI3K activity or target HIF-1α for degradation[102]. Protein c-Jun activation domain-binding protein-1 (Jab1), also known as a subunit of constitutive photomorphogenesis nine signalosomes (CSN), is implicated in DNA checkpoint and repair, as well as apoptosis (Jab1/CSN5). Further, regulates HIF-1α stability, while oncogenic mutations enhance its transcriptional activity. For instance, MTA overexpression in pancreatic cancer and v-Src (an oncogene) activation in other malignancies both boost HIF-1α and VEGF expression, triggering tumor growth and vascularization through PI3K/AKT and HIF-dependent pathways[103-106].

HIF-1/2α phosphorylation and regulation

Beyond controlling HIF-1α synthesis, phosphorylation also shapes its stability and activity. The MAPK/ERK pathway enhances HIF-1α transcription by promoting interaction with CBP/p300 while ERK2 directly phosphorylates serine residues in HIF-1α to block its nuclear localization[107,108]. Under hypoxic conditions, HIF-1α and HIF-2α undergo specific phosphorylation events that increase their interaction with coactivators and affect transcription[109]. Various kinases such as PKA[110], ATM[111], CDK1[112], GSK3β[113], PLK3[114] and CK1δ target different residues on HIF-1/2α with outcomes ranging from enhanced stability and nuclear accumulation to accelerated degradation. For example, CDK1 activity supports HIF-1α stability, whereas the phosphorylation of GSK3β and PLK3 can drive its breakdown. Similarly, CK1δ modifies HIF-2α to regulate its nuclear presence[115] and interaction with HIF-1β[116,117]. Phosphorylation provides a critical layer of control over HIF proteins, balancing their nuclear localization and transcriptional function under different cellular conditions.

Hsp 90, hypoxia-associated factor, and Hsp70

Hsp90 stabilizes HIF-1α by protecting it from degradation and supporting its nuclear accumulation under normal oxygen conditions[118,119]. During hypoxia, Hsp90 interacts with additional partners, such as the RACK1, which destabilizes HIF-1α through proteasomal pathways[120]. Hsp90 also ensures that HIF-1α adopts the correct structure for binding DNA at hypoxia-responsive elements[121]. Short-term hypoxia increases HIF-1α, but prolonged low-oxygen exposure can reduce its levels, whereas HIF-2α remains stable. Hypoxia-associated factor directs the degradation of HIF-1α but simultaneously promotes HIF-2α activity, thereby shifting the balance of hypoxia responses and supporting tumor progression during chronic hypoxia[122,123]. Hsp70 promotes HIF-1α degradation by recruiting ubiquitin ligases[124] while Hsp90 supports its stability. Together, these chaperones play opposing roles in regulating HIF genes.

ANGIOGENESIS: MECHANISM AND REGULATION

Angiogenesis is a regulated, multi-step process influenced by both stimulatory and inhibitory signals. Disruption of this balance can lead to either excessive or insufficient vessel growth, contributing to various diseases[125,126]. In cancer, angiogenesis is essential for its progression as tumors depend on new blood vessels for the supply of oxygen, nutrients, and metastasis. Without this vascular support, tumors may regress or undergo cell death[126,127]. Neovascularization typically begins with hypoxia and basement membrane injury, followed by endothelial cell activation, migration, proliferation, and maturation into functional vessels[128-131]. Angiogenesis is controlled by a complex network of biomolecules that act through interconnected pathways. The most significant of these include growth factor signaling, extracellular matrix interactions, and integrin signaling. FGFs enhance endothelial cell migration and proliferation by stimulating VEGF production, increasing integrin expression, and activating proteolytic enzymes via FGF receptors[132,133]. VEGF binds to VEGF receptors (VEGFRs), triggering intracellular signaling cascades that promote the proliferation, migration, and survival of endothelial cells[134]. Similarly, PDGFs support the expansion of pericytes and smooth muscle cells, promote DNA replication in endothelial cells, and facilitate sprouting and vessel stabilization through binding to PDGF-β receptors[135]. TGF-β exhibits dual roles depending on its concentration: At low levels, it enhances angiogenic signals such as VEGF, whereas at higher levels, it facilitates basement membrane deposition and limits endothelial growth[132,133]. Endothelial cells involved in sprouting angiogenesis are broadly categorized into two types: Tip cells and stalk cells. Tip cells lead new sprout formation, extending filopodia to sense VEGF gradients, while stalk cells form the structural body of the sprout and proliferate to extend the vessel. Tip cells can be identified by markers like CD34 (Sialomucin)[136]. VEGFR2, which is expressed on quiescent endothelial cells and becomes activated[137] when VEGF-A binds, converting these quiescent cells into tip cells that migrate directionally along VEGF-A gradients[138,139]. The distinct role of tip and stalk cells during angiogenesis is regulated by VEGF and Notch signaling.

VEGF-A activates VEGFR2 on tip cells, which enhances the expression of the Notch ligand DII4. DII4 then interacts with Notch receptors on neighbouring stalk cells, leading to the suppression of VEGFR2 expression in these cells[137]. This Notch-mediated inhibition ensures that stalk cells remain less responsive to VEGF stimulation. Notch activity itself is influenced by mechanical cues such as shear stress[140-142]. Within tip cells, VEGF-driven induction of DII4 generates a negative feedback loop where it binds to Notch on stalk cells and reduces VEGFR2/3 signaling[137]. This regulatory mechanism prevents excessive tip cell formation, maintains a proper balance between tip and stalk cells, and shapes the branching architecture of new vessels[143]. This VEGF-DII4-Notch pathway operates in a dynamic, oscillatory manner, coordinating the continuous switching of tip and stalk cell identities during angiogenic sprouting[144].

Role of HIF-α in hypoxia-mediated regulation of angiogenesis

Hypoxia occurs when cells experience an inadequate oxygen supply, disrupting their ability to maintain normal physiological balance. This condition can result from vascular and pulmonary disorders and is a common feature of tumor growth[145]. Angiogenesis regulated by hypoxia represents a fundamental mechanism by which tissues adjust vascular oxygen levels according to metabolic requirements[146]. HIFs act as transcriptional regulators; they adjust gene expression in response to oxygen availability. In many cancers, HIF-1 is overexpressed in solid tumors, where it contributes to tumor progression and aggressiveness by stimulating vascular remodeling and modifying metabolic reprogramming[147]. As HIF-1 levels rise, it activates the transcription of several pro-angiogenic genes, which play a critical role in initiating and supporting the sprouting of new blood vessels[148]. HIF is widely recognized as the master regulator of angiogenesis due to its essential role and signaling pathways[149]. HIF-1 regulates oxygen homeostasis by inducing genes that promote vascular adaptation to improve oxygen supply, or by reducing oxygen use through a metabolic shift toward glycolysis[150]. Under physiological or pathological conditions, HIF-1 supports vessel formation through coordinated action with pro-angiogenic factors like VEGF, VEGFR, angiopoietin-1, placental growth factor, PDGF, MMP-2, MMP-9, and Tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie2)[151]. This process, termed “angiogenic switching”, enables tumors to stimulate new vessel growth, ensuring oxygen and nutrient supply for their expansion. Thus, hypoxia promotes tumor formation and metastasis, whereas angiogenesis is central to tumor advancement.

Crosstalk between hypoxia, ER stress, and angiogenesis

UPR and hypoxia-driven pathways protect cells in nutrient- or oxygen-deprived environments by enhancing VEGF expression, thereby promoting the formation of new blood vessels. It occurs both in normal physiological processes, such as placental vascular formation[152] and wound repair[153,154], and in pathological conditions, including the promotion of tumor blood vessel growth[155,156]. Researchers have demonstrated that both the UPR and hypoxia pathways influence VEGF expression; however, the interaction between them at the transcriptional level remains unclear[157-159]. To address this, the role of UPR-regulated transcription factors in VEGF control has been examined. ATF4, a stress-responsive factor, is found to significantly enhance VEGF transcription under ER stress, whereas XBP-1 (S) had a normal effect despite binding to the VEGF promoter. Reducing ATF4 levels lowered VEGF expression, confirming its importance in stress adaptation[156]. Although XBP-1(S) itself strongly regulates VEGF secretion, likely by altering ER chaperone activity required for VEGF processing[160]. Supporting this, chaperons such as glucose-regulated protein/oxygen-regulated protein 150 (GRP170/ORP150) have been shown to aid VEGF maturation and secretion[161]. Experiments conducted across different cell lines demonstrated that activating both signaling pathways simultaneously enhanced VEGFA gene transcription and secretion more strongly than activating either pathway alone. This was expected, as each pathway generates transcription factors that target distinct regions of the VEGF promoter[157,162]. Interestingly, the increased VEGFA expression was driven entirely by HIF-1α with minimal involvement from the UPR-regulated factor ATF4. Although ATF4 remains active on other genes, researchers could not confirm its binding to the VEGF promoter, and lowering its levels had no impact on VEGF expression. It is uncertain whether HIF-1 blocks ATF4 by binding more effectively to the VEGF promoter or whether hypoxia alters chromatin structure to reduce ATF4 binding ability.

Findings suggest that the increased transcription of VEGF under simultaneous activation of the UPR and hypoxia pathways is mediated by a UPR-dependent enhancement of HIF-1 activity, as indicated by the elevated expression of BNIP3, another HIF-1 target gene[163-165]. Based on prior studies, several mechanisms were considered to explain this effect. However, no changes were observed in HIF-1α mRNA or HIF-1β protein levels, nor was enhanced nuclear localization of HIF-1α detected. Phosphorylation of HIF-1α is known to stabilize this transcription factor[166] and to either positively[111,167] or negatively[113,114] regulate its activity. Studies revealed increased phosphorylation of HIF-1α when both stress pathways were combined in neuroblastoma cells. Multiple kinases have been identified that phosphorylate HIF-1α and enhance its activity, including p38MAPK[166], CK2[167], and ERK1/2[168]. Notably, p38MAPK activation through the UPR emerges as a strong candidate for enhancing HIF-1α activity under combined stress conditions. To explore the relative impact of ATF4-driven VEGF expression and HIF-1-mediated VEGF regulation, researchers mapped the activity of each pathway within tumors. They observed that BNIP3 (a HIF1 target) was expressed only in hypoxic regions distant from blood vessels[169].

In contrast, UPR targets, including CHOP, BiP, and ERdj4, were more widely distributed across metabolically active areas. This suggested that UPR signaling extended beyond zones of severe hypoxia. No regions were found where only HIF1 was active; however, some areas exhibited UPR activity in the absence of HIF1. In such regions, ATF4 likely played a significant role in VEGF induction. However, in areas where both pathways overlapped, VEGF expression appeared to be mainly driven by HIF1, with UPR, which further enhances its effect by increasing ER chaperones. These findings indicate that ATF4 plays a role in sustaining VEGF production in less hypoxic tumor regions, whereas HIF-1α predominates under severe oxygen deprivation. The study highlights a context-dependent regulation of tumor vascularization where UPR and HIF1 either act independently or synergistically[156,170,171] (Figure 2).

Figure 2 Diagram showing the interplay between hypoxia, endoplasmic reticulum stress, and angiogenesis in cancer progression: Under hypoxic conditions, cancer cells upregulate the expression of hypoxia inducible factor-1α and hypoxia inducible factor-2α, which in turn enhances vascular endothelial growth factor transcription, promoting angiogenesis. Simultaneously, hypoxia disrupts protein folding within the endoplasmic reticulum (ER), leading to the ER stress and activation of the unfolded protein response through its three primary signaling branches: PERK, IRE1, and ATF6 The PERK and IRE1 branches further contribute to vascular endothelial growth factor transcription and secretion, helping to regulate HIF-1 protein stability and thereby creating a feedback loop that supports tumor vascularization and survival under low-oxygen conditions. HIF: Hypoxia inducible factor; VEGF: Vascular endothelial growth factor; ER: Endoplasmic reticulum.

Various clinical and experimental evidences show the complex interplay and its implications. These connections between the pathways can be understood in subsections. For example, the induction of VEGF-driven angiogenesis occurs after the stabilization of HIF in hypoxic conditions. Immunohistochemistry of tumor tissues has shown that increased level of HIF-1 and VEGF expression correlates with high microvessel density and advanced tumor stage. Similarly, hypoxia-linked activation of UPR markers like PERK, IRE-1, ATF6, and GRP78 has been associated with poor prognosis and resistance to therapy. This clearly depicts the link between hypoxia leading to ER stress, which further activates the UPR. The cooperative signalling between PERK-ATF4, in other words, ER stress, and HIF genes is evident, as PERK activation induces ATF4, which cooperates with HIF to augment the expression of CA IX. This enzyme helps regulate the pH of cells. A list of such clinical and experimental evidence has been tabulated in Table 1.

Table 1 Interplay between hypoxia, angiogenesis, and endoplasmic reticulum stress in carcinogenesis.

Link (biological connection)	Experimental evidence	Clinical evidence
Hypoxia → HIF stabilization → VEGF-driven angiogenesis	When tumors become hypoxic, HIF-1 levels rise and activate several angiogenic genes, especially VEGF and its related signaling molecules. These factors help new blood vessels sprout, ensuring that the growing tumor receives enough oxygen and nutrients. As a result, hypoxia-induced HIF-1 activation directly drives VEGF-mediated angiogenesis, which supports tumor survival, progression, and metastasis[148,151]	Tumor IHC studies show high HIF-1α and VEGF expression correlating with high microvessel density, advanced tumor stage, and poor survival rate. HIF-1α showed a positive correlation with VEGF expression in colorectal cancer[172]
Hypoxia → ER stress → UPR activation (IRE1, PERK, ATF6)	Hypoxia disrupts ER protein folding and activates UPR pathways. Hypoxic cells show increased BiP, CHOP, ATF4, and XBP1s. Blocking UPR increases hypoxia-induced cell death[173]	Hypoxia-linked activation of UPR markers (GRP78, PERK, IRE1α, ATF6), which correlates with aggressive behavior, poor prognosis, and therapy resistance[174]
PERK-ATF4 ↔ HIF (cooperative signaling)	PERK activation induces ATF4, which cooperates with HIF to support metabolic adaptation (e.g., CA9 regulation). Inhibiting PERK or ATF4 reduces hypoxic survival[175]	Clinical studies summarized in[175] demonstrate that patient tumors exhibit co-expression of hypoxia (HIF-1α) and UPR markers (GRP78, PERK, ATF4, XBP1s) in aggressive, therapy-resistant regions, correlating with poor clinical outcomes
IRE1-XBP1 interaction with HIF to promote survival/angiogenesis	XBP1s interacts with HIF to enhance pro-survival and angiogenic programs. Blocking IRE1/XBP1 sensitizes cells to hypoxia[175]	Patient tumors with concurrent activation of the IRE1-XBP1 arm of the UPR and HIF-1α signaling exhibit increased angiogenesis, hypoxia tolerance, and therapy resistance, correlating with aggressive disease and poor prognosis[175]
UPR (ER stress) → increased angiogenic signaling	UPR activation upregulates VEGF, IL-8, and other angiogenic factors. Blocking UPR decreases the tumor angiogenesis in vivo[176]	Clinical observation reveals that tumors with elevated UPR markers (GRP78, PERK, XBP1) exhibit increased HIF-1α and VEGF expression, linking ER stress to enhanced angiogenic signaling and aggressive tumor behavior[174]
Hypoxia + ER stress → therapy resistance and poorer prognosis	Hypoxia and ER stress synergize to induce UPR-dependent autophagy, which promotes tumor cell survival and therapy resistance[174]	High HIF-1α and combined hypoxia/UPR signatures in tumors associate with advanced disease, metastasis, and poor survival rate[172]

VEGF: Vascular endothelial growth factor; UPR: Unfolded protein response; ER: Endoplasmic reticulum; HIF: Hypoxia inducible factor; IL-8: Interleukin-8; IHC: Immunohistochemistry.

The interconnected roles of hypoxia, angiogenesis, and ER stress, regulated largely through HIF signaling, have critical clinical relevance in cancer management. As tumors expand and rapidly outpace their oxygen supply, hypoxia stabilizes HIF-1α, which in turn drives VEGF-mediated angiogenesis and remodels the tumor vasculature. Clinically elevated HIF-1α and VEGF expression correlate with poor survival rates, increased metastatic risk, and more aggressive tumor behaviour, making them important prognostic markers in several cancers[172-176].

Simultaneously, hypoxia-induced ER stress activates the UPR, promoting tumor cells' survival in harsh microenvironmental conditions, such as nutrient deprivation, oxidative stress, and limited blood flow. Persistent UPR activation contributes to chemoresistance, radiotherapy resistance, immune evasion, and epithelial-mesenchymal transition. All of which complicates treatment and worsens patient outcomes.

Collectively, these interconnected pathways highlight multiple therapeutic opportunities. Agents targeting HIF stabilization, VEGF-driven angiogenesis, or dysregulated UPR signaling provide promising approaches for personalized and combination-based cancer therapy. This integration with existing treatments like chemotherapy, immunotherapy, and radiotherapy may help to overcome resistance mechanisms driven by hypoxia and ER stress. Thus, deciphering this triad provides a framework for improved risk stratification, prognostic assessment, and development of more effective targeted therapies in carcinogenesis.

CONCLUSION

To meet the demand of high nutrients and oxygen during carcinogenesis, cells promote angiogenesis by enhancing the expression of VEGF proteins. Due to increased protein synthesis and folding, proteostatic machinery becomes overwhelmed, and the cell encounters ER stress and activate UPR pathway. ER stress results in the activation of the PERK pathway, which leads to the stabilization of HIF genes; similarly, hypoxia also activates HIF genes. UPR and hypoxia pathways influence VEGF expression by increasing the transcription factors ATF-4 and XBP-1, and by increasing the expression of HIF genes, respectively. Thus, an interplay exists between hypoxia, ER stress, and angiogenesis, which ensures the survival of cells during carcinogenesis. A better understanding of this crosstalk will enable us to design various inhibitors that can target the pathways and help in the management of carcinogenesis.

In the future, this will lead to a better understanding of their synergistic relationship and how modulation of either pathway can potentially restrain the aggravated vasculogenesis, which promotes aberrant cell proliferation in cancer, and in some cases, make them more resistant to chemotherapy and radiotherapy.

ACKNOWLEDGEMENTS

We acknowledge Mahatma Gandhi Central University, Motihari, for providing the necessary facilities to carry out this work. Shikha Bhardwaj is a recipient of CSIR Junior Research Fellowship.