Oxygen and metabolic reprogramming in the tumor microenvironment influences metastasis homing

ABSTRACT

Tumor metastasis is the leading cause of cancer mortality, often characterized by abnormal cell growth and invasion to distant organs. The cancer invasion due to epithelial to mesenchymal transition is affected by metabolic and oxygen availability in the tumor-associated micro-environment. A precise alteration in oxygen and metabolic signaling between healthy and metastatic cells is a substantial probe for understanding tumor progression and metastasis. Molecular heterogeneity in the tumor microenvironment help to sustain the metastatic cell growth during their survival shift from low to high metabolic-oxygen-rich sites and reinforces the metastatic events. This review highlighted the crucial role of oxygen and metabolites in metastatic progression and exemplified the role of metabolic rewiring and oxygen availability in cancer cell adaptation. Furthermore, we have also addressed potential applications of altered oxygen and metabolic networking with tumor type that could be a signature pattern to assess tumor growth and chemotherapeutics efficacy in managing cancer metastasis.

Introduction

Cancer is a group of diseases allied with reprogrammed metabolic, respiratory, and one or more gene function disorders. The abnormal growth pattern and cancer invasion are two significant hurdles for cancer management. In cancer, an invasion process often characterizes the metastasis, a direct extension, colonization, and proliferation of cancer cells into a new tissue host. Clinically the metastasis has already been classified as stage-four cancer, with a low survival rate and 90% of cancer deaths.¹ Metastasis is a reversible, evolutionary, and migratory survival process in which epithelial cells transform into mesenchymal cells, i.e., epithelial to mesenchymal transformation (EMT) or mesenchymal cells transform into epithelial cells, i.e., mesenchymal to epithelial transformation (MET). Several metastases (EMT/MET) inducing trans-differentiation factors such as chemokines, growth factors, hypoxia, nutrient deprivation, cellular signaling, cell-cell, and extracellular matrix (ECM) interaction have linked cancer prognosis to metastatic cells.

Metastatic inducers vary in different cancers, with metabolic (e.g., glucose, lactate, pyruvate, glutamine, and acetyl-CoA) and oxygen reprogramming (presence and absence of oxygen), the two fundamental hallmark processes that have a reported role in inducing metastasis. For instance, Marin-Valencia et al. reported the high glucose oxidation and glutamine content in glioblastoma (GBM) cells compared to tumor surrounding healthy cells and correlated the glioma cells infiltration to oxygen-rich brain tissues.² Notably, oxygen and metabolic availability regulate several steps (Figure 1) in the metastasis cascade, including extravasation, migration, angiogenesis, lymphomagenesis, invasion, survival, and colonization of metastatic cells.³ In general, metastatic cells based on primary cancer types tend to colonize toward a pre-defined metastatic niche of secondary organs such as the brain, lungs, liver, and bone. For instance, high nutritional and oxygen abundance in the brain, and low oxidative stress, and increased oxygen availability in the lungs, develop the metastatic niche at these organs. Similarly, a high metabolic rate in the liver makes it more susceptible as an organ to support secondary tumor growth. Furthermore, continuous mineral and nutritional availability and hypoxic conditions in the bone microenvironment add to circulatory cancer cells’ deposition.^4,5 A precise alteration in oxygen and metabolic signaling has been found between the healthy, cancer, and metastatic cells. Significantly, in the GBM tumor microenvironment (TME), metabolic heterogeneity favors the growth of two distinct types of cells, the fast-growing cells by aerobic glycolysis and the slow-growing cancer cells through oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO).⁶

Figure 1.

In the tumor microenvironment, together metabolites and cellular oxygen partial pressure (PO₂) regulates different steps of metastasis. Where sufficient metabolites concentration favors the proliferation of cancer cells, their shortage induce the migration of cancer cells to metabolite and oxygen rich organs. Under the oxygen shortage metabolite rewiring maintains the viability of cancer cells, or even presence and absence of oxygen also regulates the metabolite rewiring of tumor microenvironment. During the cancer cell migration, altered metabolism and oxygen environment regulates autophagy for the maintenance of cancer cell metabolite requirement, immune evasion and resistance for anoikis to facilitate migratory behavior of cancer cells. Similarly cellular redox homeostasis maintains oxidative stress of cancer cells and vasculogenesis regulates the metabolites and oxygen shortage and also facilitate the migratory route of circulatory cancer cells. Additionally, altered oxygen and metabolism also maintains the drug resistance and dormancy in cancer cells followed by reoccurrence of cancer

Metastasis is a nonrandom process with scarce nutrients and oxygen at the TME, inducing metabolic and oxidative stress. Thus, cancer cells migrate toward oxygen and nutrient-rich organs such as the lungs and the brain or trigger metabolic and oxygen reprogramming to substitute the deficient metabolites function to full fill the bio-energetic and bio-synthetic demand of cancer cells. Healthy cells generate a significant part of the energy from oxygen-dependent OXPHOS. In contrast, cancer cells shift their metabolism from OXPHOS to aerobic glycolysis (Warburg effect) to fulfill the high energy demand and anabolic biosynthesis for fast-growing cancer during oxygen scarcity (hypoxia). Additionally, metabolic rewiring also protects cancer cells from drug-induced toxicity and maintains cancer viability and cancer recurrence.

Apart from cancer cells, oxygen and metabolites also regulate different cell types such as fibroblast, macrophages, and neutrophils in the TME. From a therapeutic standpoint, both oxygen and metabolites play a significant role in cancer management. Furthermore, cancer cells have unique adaptability characteristics, which help them survive in adverse conditions. This review focused on the crucial role of oxygen and metabolites in metastatic progression and the role of metabolic rewiring and oxygen availability in cancer cell adaptation. We have also addressed the different metabolic alterations and associated potential applications as markers and therapeutic targets for managing cancer metastasis.

Maintenance of tumor microenvironment and metastasis niche

Cancer cells are terminally more advanced and develop their microenvironment to maintain proliferation, plasticity, and continuous metabolites and oxygen availability. Generally, metabolites and oxygen scarcity induces apoptosis in a healthy cell, but tumor cells continuously proliferate within the TME. Cancer cells induce hypoxia-inducible factor (HIF) and metabolites reprogramming to maintain an adequate oxygen and metabolites level in the TME. Furthermore, in response to hypoxia-induced apoptosis, HIF-1 activates PDK1 mediated aerobic glycolysis and suppresses the mitochondrial TCA cycle and OXPHOS.⁷ In the TME, most cellular glucose is utilized in aerobic glycolysis; however, cellular glutamine maintains the TCA intermediates.⁸ Notably, altered metabolism favors tumor growth and develops a metastasis niche; as Stephen Paget explained in his “seed and soil” hypothesis, the metastasis niche favors the proliferation, migration, colonization, survival, and plasticity of cancer cells (Figure 2). At the metastatic niche, the metabolic rewiring also maintains cancer cell plasticity and self-renewal property. It increases the expression of EMT-inducing factors SMAD, ZEB, Notch, TGF-β, and Wnt.⁹ The mitochondrial oxidation and high oxygen consumption favor the proliferative nature of epithelial cells. Moreover, the high glycolysis, lipogenesis, elevated pentose phosphate pathway (PPP), and glutamine-mediated TCA intermediate synthesis have been associated with the migratory potential of mesenchymal cells’ .⁸

Figure 2.

Tumor microenvironment facilitate the proliferation and migration of cancer cells to pre-metastatic niche. Cancer microenvironment also maintains the plasticity and dormancy (G₀/G₁) in cancer cells. Physiological stress or drug environment induces the dormancy in cancer cells and dormant cancer cells initiate the growth of secondary tumor or reoccurrence of cancer

To maintain metabolite homeostasis, cancer cells increase the expression of metabolites transporters such as glucose (e.g., GLUT), fatty acid (e.g., CD36, FABP4, and FATP), aspartate (e.g., SLC1A3), and glutamine transporter (e.g., SLC1A5). Besides metabolic transporters, cancer cells also increase the expression of ionic transporter for pH homeostasis. During aerobic glycolysis, lactate dehydrogenase (LDH) generates lactate, leading to acidification of TME. Through monocarboxylate transporters (MCT1/4), cancer cells continuously export lactate from the cells to suppress lactate-mediated cancer cell acidification.¹⁰ MCT1 mediated bidirectional lactate transport maintains ambient lactate concentration in the cancer cells.^10,11 Furthermore, cancer-associated fibroblast (CAFs), lymphocytes (e.g., neutrophils and macrophages), and the vascular system create a tumor-friendly microenvironment, favoring the metastatic cascade initiation. Stromal cells at the cancer site support developing TME and act as sources of metabolites, growth factors, and ECM for proliferating cancer cells. For instance, bone adipocytes act as a source of metabolites and growth factors for growing cancer mass,¹² and during glutamine starvation, they maintain glutamine homeostasis.¹³ Thus, to maintain adequate intracellular fatty acid concentration, cells increase the expression of fatty acid transporters.¹⁴

The adaptable behavior of cancer cells enables them to survive in a restrictive environment at the primary tumor site, during the circulation, and at the secondary cancer site.¹⁵ Though metabolic reprogramming not only favors cancer cells to survive under oxidative stress, metabolites shortage, immunological response, and presence of drugs, it also enhances the cancer cells’ ability to survive in the altered conditions that make them more aggressive toward their rapid advancement to secondary organs, colonization, and progressive metastasis. A recent study performed by Lin et al. showed that glioma cell’s adaptation to FAO maintains their proliferation, cellular respiration, and glycolysis. During initial tumor growth, cancer cells utilize a higher amount of glucose; however, they switch to FAO during colonization under shortage of glucose at the secondary site.¹⁶ For instance, in metastatic breast cancer cells, Weverwijk et al. observed glycolysis to FAO shift under glucose stress conditions.¹⁷

Increased vascular network at the TME provides oxygen and metabolites (e.g., lactate, succinate, and glutamine) to growing tumors, including transportation of neutrophils, macrophages, and platelets to the proliferating site. Succinate activates tumor-associated macrophage (TAM) that promotes lung tumorigenesis¹⁸ and angiogenesis at the TME.¹⁹ For instance, lactate activates endothelial NF-κβ/IL-8 (CXCL8) for angiogenesis,²⁰ and macrophage CCL5 activates invasive and EMT phenotypes in the breast cancer cells.²¹ The TME acidification also favors macrophage polarization, including suppressing immunological response against growing tumors and colonization of migratory melanoma cells.²² Furthermore, the lactate-mediated ERK/STAT3 pathway facilitates TAM-mediated proliferation, migration, invasion, and colonization of breast cancer cells.²³ Both altered metabolism and hypoxic conditions at the TME influence ECM remodeling and release cancer cells from the primary cancer site, while at the pre-metastatic niche, it favors invasion and colonization of migratory cancer cells.

A limited supply of glucose in the cancer cells reduces collagen synthesis, while glutamine-mediated elevated α-ketoglutarate maintains hydroxylation of proline and lysine for collagen synthesis.²⁴ Elevated glycolysis in cancer cells supports ECM development, resulting in colonization. Similarly, in migratory triple-negative breast cancer (TNBC) cells, denser collagen increases glycolysis, while at low collagen density, they shift their metabolism toward mitochondrial oxidation.²⁵ For instance, elevated glucose uptake supports breast cancer cells’ proliferation; thus, to maintain high glucose availability at the TME, breast cancer cells also hijack CAFs glucose uptake, and low glucose availability at the TME promotes cancer cell metastasis.²⁶ However, it is not clear how the variation in glucose absorption or other metabolites regulates cancer metastasis to date.

Nevertheless, Sullivan et al. showed that ECM digestion increases the GLUT1 mediated glucose uptake, favoring cancer cells’ proliferation and migration.²⁷ Additionally, Sai et al. also reported a critical role of the pre-metastatic niche in homing and colonization of migratory lung cancer cells in the bone marrow microenvironment.²⁸ During the homing of migratory cancer cells, both ECM and chemoattractant molecules on fibroblast cells’ surface support cancer cells’ interaction with host cells. Migratory mesenchymal cells interact with fibroblast cells of the post metastatic niche and activate them for colonization. At the metastasis niche, oral cancer cells and CAFs secrete the ECM remodeling enzymes such as MMP, LOX, and TIMP-1, which play a crucial role in cell transformation and migration.²⁹

Cell proliferation and migration

The high proliferation and migration of cells are critical for any cancer to become metastatic. Of different factors, oxygen and metabolites’ availability play a significant role in cancer cells proliferation and migration. Also, high reactive oxygen species (ROS) are the most common factor in all cancers, promoting tumor development and progression through the activation of survival (PI3K/Akt) and proliferative (MAPK/ERK) pathways.³⁰ For instance, the increased oxygen consumption in invasive melanoma cells upregulates cellular glycolysis and activates the mitochondrial ROS mediated focal adhesion kinase (FAK).³¹ Further, integrin-mediated FAK signaling that support the migration of invasive pancreatic cancer cells.³² In ROS response, cancer cells increase the expression of cellular antioxidants such as NADPH, nuclear factor erythroid 2–related factor2 (NRF2), superoxide dismutase (SOD), and glutathione, facilitating the proliferative and migratory mechanism of cancer cells.³⁰

The low cellular oxygen level also induces an oxidative environment, which transforms cancer cells to metastatic cells through up-regulation of proteins related to cell proliferation, migration, redox homeostasis, and metabolic reprogramming. Under a hypoxic environment, cancer cells’ either migrate toward the oxygen-rich organs or increase the expression of HIF-1/2. HIF-1 ubiquitously promotes vasculogenesis and Wnt/β-catenin mediated cell proliferation,³³ while HIF-2 shows contrasting proliferative effects in the lungs, kidneys, cardiac, pancreas, and intestinal, hepatocytes, and endothelial cells.³⁴ During the lung cancer cell migration toward oxygen-rich secondary sites, HIF upregulates cellular NADPH and suppresses E-cadherin expression.³⁵ Similarly, upregulated glutamate dehydrogenase-1 (GDH-1) and glutathione peroxidase (GPx) expression favor the adaptation of proliferating lung and breast cancer cells to oxidative stress.³⁶

The altered metabolism fulfills proliferative cells’ metabolic needs and acts as an inducer for cancer cell migration. In general, healthy brain cells oxidize glucose; however, the GBM cells and brain colonizing breast, melanoma, non-small cell lung carcinoma (NSCLC), renal, and endometrial cancer cells increase the oxidation of acetate.³⁷ Acetate adaptation generates acetyl-CoA, a substrate for the lipogenesis and TCA cycle, favoring the cancer cell’s proliferation and survival,³⁸ and maintaining the malignancy features in prostate cancer.³⁹ Significantly, LDH-A mediated aerobic glycolysis has been reported in cancer cells such as pancreatic, colorectal, breast, and NSCLC cells.⁴⁰ At the developing cancer site, LDH-A regulates MMP9, cathepsin B, PLAU,⁴¹ Tropomyosin (e.g., TM4, Tm5NM1, and Tm5NM5), and actin filament rearrangement during cancer cell multiplication, migration, and invasion.⁴² Additionally, LDH-A also favors the expression of metastasis-associated protein 1 (MTA1)⁴³ and proliferative marker (e.g., Ki-67) in migratory breast cancer cells.⁴⁴ In contrast, the inhibition of LDH-A has been reported to arrest neuroblastoma,⁴⁵ pediatric osteosarcoma,⁴⁶ and prostate cancer.⁴¹ In liver colonizing breast cancer cells, high lactate also increases glutamine oxidation and suppresses OXPHOs.⁴⁷ Furthermore, pyruvate dehydrogenase kinase 1 (PDK1) diverts tumor metabolism toward glycolysis under hypoxia, supporting glioma cell migration, autophagy, apoptosis resistance, and redox homeostasis.⁴⁸ However, PDK inhibition upregulates the TCA cycle α-ketoglutarate and arrests metastasis of renal cell carcinoma (RCC).⁴⁹

Under metabolic and oxygen scarcity, cancer cells maintain proliferation through shifting mitochondrial oxidation toward aerobic glycolysis. Therefore, to sustain the Warburg effect, colon cancer cells suppress the oxidation of mitochondrial pyruvate and upregulate the expression of embryonic pyruvate kinase (PKM2)⁵⁰ and mitochondrial ACAT-1.⁵¹ Subsequently, overexpression of glycolysis enzymes such as Phosphofructokinase-1 (PFK-1),⁵² phosphoglycerate mutase (PGAM), hexokinase, and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) helps to reduce metabolic stress in the growing TME.⁵³ However, the elevated Warburg effect results in high lactate production, inducing a shortage of fatty acid, acetyl-CoA, and TCA intermediates. Therefore, to maintain metabolites homeostasis, cancer cells divert glycolysis intermediates to the PPP, TCA, and amino acid synthesis⁵⁴ (Figure 3). Additionally, glutamine favors anabolic growth⁵⁵ and maintains acetate and fatty acids in RCC.⁵⁶ However, proliferating cancer cells increase the anaplerosis reactions to substitute limiting metabolites (e.g., TCA intermediates, NADPH, acetyl-CoA) with available metabolites (e.g., glucose, glutamine, α-ketoglutarate, fatty acid). The glucose-mediated anaplerosis reaction gives citric acid cycle intermediates such as oxaloacetate, pyruvate, and acetyl-CoA to proliferate GBM cells; however, glutamine acts under glucose scarcity as a gluconeogenic precursor.² Additionally, certain non-essential amino acids, such as glutamine and asparagine in cancer cells, favor cancer prognosis, while their shortage induces apoptosis of cancer cells.⁵⁷

Figure 3.

Healthy cells use a sequential regulated metabolism (glycolysis-TCA-oxidative phosphorylation and fatty acid oxidation) for energy generation and anabolic biosynthesis. But in cancer cells according to available nutrient and unfavorable surrounding they divert their sequential metabolism toward the altered metabolism. Under the oxygen limiting conditions cancer cells increase the cytosolic metabolism and reduce the mitochondrial oxidation

In general, cancer cells use carbon units of fatty acids for cell membrane expansion and ATP generation; however, aggressive cancer cells such as melanoma, breast, prostate, and ovarian divert them to synthesize structural and signaling lipid molecules,⁵⁸ and increases the expression of FAO acetyl-CoA acetyltransferase-1 (ACAT1).⁵¹ Also, acetyl-CoA synthetase (ACS) regulates the fatty acid synthesis and oxidation in lung cancer cells.⁵⁹ In contrast, adipocyte FAO promotes PI3K/AKT mediated ovarian and breast cancer cell proliferation and migration.^60,61 In addition, oleic and linoleic acid also activates the PI3K/AKT pathway to sustain the breast cancer cell migration,⁶² and in gastric cancer cells, oleic acid-mediated PI3K/AKT activation increase the invasion of cancer cells to neighboring organs.⁶³

Due to the high metabolism in the TME, elevated glucose, lactate, fatty acid, amino acid, and ion transporters increase metabolites transport into the cancer cells. For instance, the upregulated expression of MCT and SLC1A3 in proliferating GBM cells is essential to maintain the high influx of lactate and aspartate.⁶⁴ The overexpression of MCT1/4 in breast cancer cells maintains high aerobic glycolysis.⁶⁵ Furthermore, overexpressed MCT1/4 are also associated with disease progression and reduced survival of clear cell carcinoma patients.⁶⁶ In contrast, MCT’s inhibition has been reported to suppress proliferation of breast and lung cancer cells and shift aerobic glycolysis to OXPHOS.¹¹ Similarly, MCT1/4 inhibition makes colorectal cancer cells sensitive to 5-fluorouracil and reduces tumor growth.⁶⁷

Surprisingly, metabolite reprogramming for energy generation also helps the cancer cells adaptation to changing surrounding environment. For instance, the altered metabolism in cancer cells suppresses the inhibitory effect of anti-cancer drugs. In squamous carcinoma cells, mTOR inhibitors suppress aerobic glycolysis. However, cancer cells shift their metabolism toward glutaminolysis to maintain a high proliferation rate in lung cancer cells⁶⁸ or increase the mTOR expression to maintain glutamine synthetase (GS) expression in proliferating liver cancer cells.⁶⁹ Furthermore, in NSCLC cells mTOR mediated asparagine maintains protein synthesis during nutrient deficiency.⁷⁰ Additionally, glioma, breast, kidney, and pulmonary cancer cells excrete glutamine and uptake cysteine to synthesize glutathione to mimic mTOR inhibitory drug-induced oxidative stress.⁷¹

Epithelial to mesenchymal transformation and cancer stem-like cell maintenance

Before the commencement of Stephen Paget’s Seed and soil theory, the researchers believed that only embryonic and adult tissue-stem cells retain the plasticity (ability to transform) and self-renewal phenotype (ability to divide). Subsequently, in 1889, Stephen Paget explained the EMT feature of cancer cells linked to the stemness feature in the malignant TME. Unlike embryonic stem cells, the cancer cells do not possess the true stemness; however, their plasticity and phenotypic transformation endow them with cancer stem-like cells. In the TME, some breast cancer cells maintain tumor growth owing to their self-renewal ability and plasticity.⁷² PI3K/AKT mediated SMAD and Nanog expression regulates the self-renewal process of cells.⁷³ Of note, due to the plasticity, cancer cells show EMT during metastasis. In the TME, the EMT maintains the differential phenotype for dissemination and colonization of cancer cells, while the stem cell phenotype supports the polarity and outgrowth of cancer cells at the secondary site.^74,75 For instance, in breast cancer, an elevated asparagine favors breast cancer cells’ colonization into the lung, liver, and brain,⁷⁶ while L-Proline maintains the migratory mesenchymal phenotype of stem cells.⁷⁷ Similarly, isocitrate dehydrogenase maintains EMT in colorectal cancer cells.⁷⁸ Thus, stem-like features maintain the self-renewal and high plasticity in cancer cells, leading to EMT-mediated secondary tumor outgrowth.

In cancer cells, TGF-β is a well-known molecule responsible for cancer cell transformation and regulates the expression of mesenchymal markers such as Snail, ZEB1,⁷⁹ SMA, and vimentin.⁸⁰ During cellular transformation, it maintains the fatty acid synthase (FAS) mediated anabolic synthesis⁸⁰ and shift the cellular metabolism toward aerobic glycolysis and amino acid metabolism, suppressing the expression of epithelial markers such as occludin and zonula occludens-1 (ZO-1).⁸¹ At the time of EMT induction, increased glycolysis and mitochondrial oxidation maintain the high proliferation of epithelial cells. In contrast, upregulated amino acid synthesis and FAO maintain the mesenchymal phenotype of migratory breast cancer cells.³ Wnt signaling positively regulates the EMT process in colorectal cancer cells by expressing epithelial markers followed by suppressing mesenchymal markers such as MMP2, MMP9, ZEB1, N-cadherin, and vimentin.⁸²

Fatty acids in the regulation of cancer stem-like cells phenotype

In cancer cells, cellular fatty acid content has a significant role in cancer initiation and cellular transformation, that divert the glycolytic intermediates toward the anabolic biosynthesis of fatty acids, thereby increasing lipid accumulation that acts as a good energy source and protects cancer cells from oxidative stress-induced cell death.⁸³ In colon cancer, elevated stearoyl-CoA desaturase-1 (SCD-1) increases the monounsaturated fatty acid (MUFA) mediated Notch and Wnt signaling, favoring the stem cell phenotype⁸⁴ and also maintains the oxidative stress in Glioblastoma stem cells (GSC).⁸⁵ Giudetti et al. reported a differential fatty acid reprogramming in epithelial and mesenchymal breast cancer cells. The epithelial cells upregulate the accumulation of MUFAs and de novo lipogenesis, while mesenchymal cells suppress lipogenesis. However, the increase in the β-catenin mediated triacylglycerol synthesis and the accumulation of high cholesterol and polyunsaturated fatty acids (PUFA)⁸⁶ in breast cancer cells favor their colonization in the lungs.⁸⁷

Furthermore, inhibition of fatty acid synthesis arrests the expression of β-catenin mediated colorectal cancer cell metastasis cascade.⁸⁸ In breast cancer, lipogenesis inhibition increases oxidative stress, and acetyl-CoA mediated SMAD acetylation, including the expression of EMT markers in metastatic breast cancer cells.⁸⁹ Similarly, in colorectal cancer, a cascade of signaling begins due to the loss of acyl-CoA thioesterase-12 (ACOT-12), which induces the TWIST2 mediated EMT transformation epithelial marker E-cadherin suppression for the migration and invasion of cancer cells.⁹⁰

Glycolysis and acetyl-CoA production in cancer stem-like cells

The TNBC cells shift glycolysis toward acetyl-CoA synthesis by acetylating the H4 histone of stem cell genes SOX-2, KLF-4, OCT-4, and c-Myc to maintain stem cell behavior.⁹¹ Thus to keep an adequate amount of acetyl-CoA, cancer cells increase glycolysis to maintain stem cell and self-renewal properties in GBM, ovarian, and pancreatic cancer cells.⁹² For instance, glycolysis inhibition promotes the OXPHOS and lineage differentiation of stem cells.⁹³ The EMT induction results in increased glucose uptake and maintains the expression of mesenchymal markers such as Snail, SMAD, N-cadherin, and vimentin in pancreatic cancer cells.⁹⁴ Furthermore, Snail overexpression increases glucose and lactate uptake in transforming pancreatic cancer cells.⁹⁵ However, aerobic glycolysis mediated TME acidosis also increases the expression of stem cell markers such as Oct-4, OLIG2, and Nanog in glioma cells.⁹⁶

Metabolites and mitochondrial oxidation in the maintenance of cancer stem-like cells

The energy-generating mitochondria also regulate the epigenetic switches through acetyl-CoA during the somatic cell’s transformation.⁹⁷ The mitochondrial oxidation arrests the stem cell multiplication; however, its inhibition increases the proliferation of stem cells⁹⁸ and EMT progression⁹⁹ by suppression of E-cadherin and overexpression of fibronectin in colon and pancreatic cancer cells.¹⁰⁰ In general, cancer cells use glycolysis instead of mitochondrial oxidation; however, Curry et al. reported their occurrence in cancer cells to generate amino acids, fatty acids, and other metabolic intermediates.¹⁰¹ For instance, the head and neck cancer cells use aerobic glycolysis and mitochondrial oxidation to maintain stem cell features. Besides metabolites, oxygen partial pressure (PO₂) also regulates the stem cell phenotype in cancer cells. In the presence of oxygen, somatic cells use mitochondrial oxidation for energy generation; however, on the induction of stem cell phenotype, somatic cells shift their metabolism to aerobic glycolysis, or in other words, hypoxic condition favors the expression of stem cell markers.⁹⁷ Similarly, Curry et al. reported the proliferative marker (Ki-67) with the cellular energy demand and showed that OXPHOS maintains high proliferation in head and neck stem cells.¹⁰¹ However, dysfunction of mitochondrial oxidation shifts cellular metabolism toward aerobic glycolysis, resulting in increased expression of stemness markers, such as ALDH, CD44, Nanog, and Notch in ovarian cancer cells.¹⁰²

Another critical factor, the HIF-1α, not only maintains the sufficient energy level in stem cells but also regulates the cell quiescence by Pdk2/Pdk4¹⁰³ and activates the Wnt/β-catenin for the maintenance of EMT mediated cellular proliferation and plasticity.¹⁰⁴ HIF-1α also induces the increased expression of EMT markers such as N-cadherin, Snail, β-catenin, vimentin,¹⁰⁵ ZEB1, and Slug.¹⁰⁶ However, lower cellular oxygen also reduces ROS production, favoring the self-renewal property of stem cells. To maintain self-renewal under high ROS, stem cells activate the PI3K/AKT pathway¹⁰⁷ or increase the expression of cystine/glutamate transporter (SLC7A11) for the production of antioxidant molecules.¹⁰⁸ Additionally, high cellular ROS also induces the cellular transition to manage intracellular ROS levels and keep high glucose uptake followed by ERK-mediated NADPH synthesis.¹⁰⁹

Angiogenesis and lymphogenesis in the tumor microenvironment

Besides the role and importance of metabolites in tumor outgrowth and cancer cell migration, specific metabolites (e.g., fatty acid, glucose, and glutamine) also take part in the progression of vasculogenesis (e.g., angiogenesis and lymph-angiogenesis) and sustain the friendliness behavior with the TME. The initial investigation of increased vascularization in the TME marked them as a prognostic factor for tumor overgrowth and metastasis, supported by the constant supply of metabolites and oxygen. Also, during extravasation and invasion, it facilitates the migratory route to anticipating cancer cells. However, how cancer cells induce vasculogenesis in the tumor environment is still a matter of discussion. Cancer cells induce abnormal (leaky type) and normal (fully matured) vascularization in the TME. For instance, Wenes, et al. showed mTOR-mediated heterogenic TAMs and their association with glycolysis during vascular growth. Elevated macrophage glycolysis induces abnormal vascularization, i.e., leaky type blood vessels support cancer cells’ intravasation.¹¹⁰ However, the low glycolysis rate induces blood vessel maturation by increasing the deposition of perivascular cells and tight vascular endothelial (VE) cadherin junctions between endothelial cells. However, glycolysis inhibition downregulates VE-cadherin protein and suppresses blood vessels’ maturation.¹¹¹ Besides macrophages, several other types of cells such as platelets, neutrophils, endothelial cells, and CAFs also secrete the angiogenic factors including angiogenin, VEGF, FGF, PDGF, IL-8, CXCL-8/12, MMPs, Osteopontin, angiotensin, erythropoietin, and angiopoietin-2 for the vascular development from the existing vascular vessels.^112,113 Vasculogenic factors induce the proliferation and migration of endothelial cells, followed by colonization in the tube-like structures. For Instance, MMP-9 from the neutrophils digests the ECM, favoring the migration of endothelial cells and their colonization into the tube-like structures.¹¹⁴

During angiogenesis and lymphogenesis, the altered metabolism in cancer cells plays a vital role in expressing the vasculogenic factors. Elevated aerobic glycolysis not only generates sufficient ATP molecules for proliferating endothelial cells, but high lactate in endothelial cells also maintains their proliferative nature and they increase the expression of LDH-A and MCT-1.¹¹⁵ In the endothelial cells, lactate activates the NF-κβ/IL-8 mediated CXCL8 expression, which induces endothelial cells’ migration,²⁰ and PI3K/AKT mediated increased expression of VEGF-A for the new blood vessel development.¹¹⁶ Lactate also polarizes the M2-type macrophages and HIF-1α expression for angiogenesis. Additionally, amino acids (e.g., glutamine and asparagine) and TCA intermediate (e.g., α-ketoglutarate) also regulate endothelial cell migration and vessel development.¹¹⁷

Adipocytes from the surrounding tumor environment also support the proliferation and migration of endothelial cells.¹¹⁸ For instance, 1-butyryl-glycerol from adipocytes induces the angiogenesis cascade,¹¹⁹ and fatty acid Sphingosine-1-phosphate activates the ERK and p38 MAPK signaling for endothelial cell proliferation and their migration.¹²⁰ Besides, to facilitate the increased fatty acid transport in the endothelial cells, both VEGF-A and NOTCH increase the expression of FABP-4 mediated lipid transport. Other fatty acids (e.g., linoleic, and oleic acid) and cholesterol also induce vascular growth.¹²¹ However, the scarcity of fatty acids directs the metabolic shift toward glycolysis. Together, the shortage of glucose and fatty acid reduces the α-ketoglutarate, which activates the HIF-1α mediated VEGF-A expression to increase the vasculogenesis to maintain fatty acid availability.¹²² During vascular architecture development, glutamine metabolism regulates the proliferation and migratory behavior of endothelial cells.¹²³ Glutamine scarcity affects the endothelial cells’ proliferation, migration, causing disparity in TCA intermediates and fatty acid synthesis. Elevated glutamine (substituting α-ketoglutarate and asparagine) in the TME also induces the polarization of TAMs into M2-type macrophages. Subsequently, activated M2-type macrophages increase the vascular development and metastasis potential of cancer cells. During the vasculogenesis, macrophages from the TME sense the hypoxic condition and secrete the angiogenic factors in response to the hypoxia. Macrophages from the hypoxic cancer area secrete angiogenic factors such as VEGF,¹²⁴ PDGF, and FGF,¹²⁵ which increase the glycolysis in endothelial cells and stimulate blood vessel development.¹¹⁰

The hypoxic TME acts as a critical inducer for the progression of vasculogenesis. In response to reduced PO₂, HIF-1α increases aerobic glycolysis, favoring the VEGF-mediated angiogenesis.¹²⁶ However, lactate and succinate from TME also increase the expression of HIF-1α mediated VEGF secretion for the vascular growth around the growing tumor.¹²⁷ Under the hypoxic or oxidative environment, lactate maintains the oxidative environment and activates TAMs for angiogenesis.¹²⁸ For instance, the HIF-1α expression in NSCLC,¹²⁹ gall bladder carcinoma,¹³⁰ and breast cancer govern the VEGF expression and vascular density in the TME.¹³¹ Both the isoforms of HIF (1/2) regulate vasculogenesis and induce the expression of pro-angiogenic factors under hypoxia.¹³² The HIF-1α activates the β–catenin signaling pathway, which increases the proliferation and tumor development from endothelial cells. However, its suppression reduces the expression of angiogenic factors VEGF, FGF, and angiogenin in colorectal cancer patients.¹³³

In aggressive cancers such as melanoma, breast, hepatic, and lung cancers, the lymphogenesis process modulates the TME for cancer cell migration. The low fluidic shear, absence of pericytes, elastic discontinuous basement membrane, the weak cell-cell junction between lymph endothelial cells (LECs), and high expression of adhesion molecules in the lymphatic system favors the invasion and circulation of cancer cells through lymph vessels. Besides, high dietary fat and other available nutrients (e.g., glucose and glutamine) in the lymphatic fluid work as an excellent nutritional source for circulating cancer cells. The lymphatic system also circulates lymph fluid or exchanges lymph fluid with tissues to maintain ambient tissue fluid homeostasis and transport immune cells, dying cells, and cancer cells from tissue to lymph vessels supporting the extravasation.¹³⁴ For instance, in the marginal region of metastatic lung carcinoma, high VEGF C and D expression favor the lymph node invasion in lung cancer patients,¹³⁵ further inhibition of VEGFR2 not only suppresses the vasculogenesis but also arrest the metastasis of breast cancer.¹³⁶

Additionally, in hepatic cancer, CXCL-1 from cancer cells activates the F-actin in LECs and induces lymph vessel growth and lymph node metastasis.¹³⁷ Lymphangiogenic inducers from cancer cells, CAFs, endothelial cells, and TAMs also induce proliferation and migration of LECs. In the TME, lactate-induced macrophages secrete the VEGF-C to enhance LECs and vessel outgrowth.¹³⁸

During lymphatic vessel development, differential metabolism in the cancer environment also regulates LECs’ differentiation and migration. An altered metabolism leads to an increased PDGF-BB, VEGF-C, and D expression for lymph vessel outgrowth. Cancer cells also increase the glycolysis and FAO in the proliferating LECs. Elevated glycolysis maintains both the ATP and NADPH concentration, while FAO maintains the ATP, NADPH (through TCA cycle citrate), and acetyl-CoA in proliferating LECs. NADPH regulates the oxidative environment and acetyl-CoA used in the TCA cycle and fatty acid synthesis.^139,140 Fatty acids from the TME also regulate the epigenetic switch of lymphogenesis. Acetyl-CoA from the FAO acetylates the histone (H3K9) and improves the PROX1 mediated VEGFR3 expression and acetyl-CoA synthesis. PROX1 increases the expression of β-oxidation regulating enzyme carnitine palmitoyltransferase1A (CPT1A), while inhibition of CPT1A leads to suppression of new lymphatic vessel development.¹⁴¹ One critical study performed by Jung et al. on a high-fat diet in melanoma rat models reported the induction of lymphogenesis in cancer cells during metastasis. High fat induces pro lymphogenic markers M-CSF, VEGF-D, and VEGFR-3, followed by increased CCL19/21 in lymph nodes and CCR7 in tumors.¹⁴²

Immune evasion and resistance for anoikis

Anoikis, a form of programmed cell death, occurs in anchorage-dependent cells after detaching from the surrounding ECM. In general, ECM maintains morphological intactness; however, its breakdown activates the intrinsic and extrinsic apoptotic pathway in healthy cells while metastatic cancer cells show resistance to anoikis. Anchorage-independent growth or anoikis resistance, one of the cancer cells’ hallmarks, is acquired by the malignant cells for their migration and survival in the circulatory system.¹⁴³ Cancer cells also show resistance to the host immune system and escape from the host immune surveillance (immune evasion) during metastasis. Both immune evasion and resistance to anoikis adds to cancer cells’ survivor process and play a vital role in the detachment from the surrounding ECM. In cancer cells, anoikis resistance maintains cell viability, while immune evasion protects cancer cells from an ambush of the host immune system. During the onset of tumor prognosis, the host immune system recruits tumor-infiltrating lymphocytes (TILs), secrete cytotoxic agents (e.g., IFN-γ and granzyme) at the TME to suppress the tumor growth. However, glycolysis-mediated TME acidification neutralizes TILs’ action against growing tumors. Besides maintaining the immunosuppressive environment at the tumor site, cancer cells also recruit the regulatory T-cells (T_reg), antigen-presenting cells (APCs), and myeloid-derived suppressor cells (MDSCs) in the TME for the host immune suppression.¹⁴⁴ Altered metabolism and hypoxic condition at the TME make it possible for the cancer cell to escape from the immune surveillance and resist anoikis. Furthermore, low T-cell infiltration or reduced number of available T-cells (CD4⁺ and CD8⁺) at the developing tumor site is associated with poor survival of cancer patients.¹⁴⁵

Although excess aerobic glycolysis in sarcoma increases the secretion of CTL4 and PD-1 and creates a shortage of free glucose in the TME thereby reducing the T-cell glycolysis and suppressing the IFN-γ expression from T-cells.¹⁴⁶ Inhibition of IFN-γ expression from CD8⁺ T cells reduces immune cells’ infiltration into the TME.¹⁴⁷ Similarly, in ovarian cancer, T-cells’ restricted glycolysis promotes T-cells’ apoptosis.¹⁴⁸ Even so, the presence of arginine reprograms the exhausted T-cells and increases their anti-tumor activity against developing melanoma. Arginine diverts cellular metabolism toward mitochondrial oxidation,¹⁴⁹ and metabolic shifting from glycolysis to OXPHOS or FAO favors the survival of CD8⁺ T cells.¹⁵⁰ Additionally, glycolysis inhibition suppresses MDSCs and increases the T cell response against developing TNBC.¹⁵¹ However, Ho et al. proposed that T_reg (T_H1CD4⁺) cells play a critical role against developing tumor immune response by regulating both the activator and suppressive nature of T-cells. Reduced glycolysis in T-cells induces the suppressive nature of T_reg cells, and glucose deprivation results in reduced expression of IFN-γ and CD40L from the TH₁ cells.¹⁴⁴ Besides, glucose scarcity also induces ROS-mediated T_reg cell apoptosis, and apoptotic T_reg cells show a more suppressive effect than the viable T_reg cells. Dying T_reg cells express a high level of immunosuppressive molecules (e.g., PD-1, CD39, CD73, and CTLA-4), secrete many ATP, and indirectly induce immunosuppressive effects through adenosine signaling.¹⁵²

In response to tumor acidic environment, Foxp3 from T_reg cells’ shift glycolysis to OXPHOS and uplift their immune response against growing cancer.¹⁵³ In colon cancer cells, glutamine suppression arrests glycolysis, TCA, and PPP and increases CD8⁺ T cell immune activity. However, glutamine scarcity did not affect the CD8⁺ T cell activity; they maintain their metabolic plasticity through pyruvate carboxylase-mediated anaplerosis. Therefore, glutamine antagonists increase tumor infiltration of CD8⁺ T-cells. Additionally, glutamine targeting maintains anti-tumor memory of T-cells and increases their oxygen consumption, followed by a reduction in oxidative stress and a hypoxic environment. Increased oxygen consumption increases the acetate-mediated OXPHOS in CD8⁺ T-cells and shift glycolysis toward acetate-mediated OXPHOS.¹⁵⁴ However, increased glutamine metabolism in the TME also activates the HIF-1α mediated TAMs. Activated TAMs secrete IL-23 and increase the immunosuppressive IL-10 and TGF-β expression from T_reg cells.¹⁵⁵ Hypoxia also induces the ER stress-mediated kill signal (e.g., calreticulin) expression on the cancer cells. However, in response to increased calreticulin, breast cancer cells also increase the expression of do not kill signals (e.g., CD47) to protect cancer cells from immune killer cells.¹⁵⁶

A hypoxic environment also modulates the host immune system and increases immunosuppressive and inflammatory mediators’ expression to hijack the host immune surveillance. In gastric cancer, HIF-1α mediated TGF-β increases the T_reg deposition,¹⁵⁷ subsequently high CCL28 expression in the hypoxic TME¹⁵⁸ and CXCR4 expression on the T_reg cells helps their deposition on the TME.¹⁵⁹ Although under a hypoxic environment, pheochromocytomas and paragangliomas cells also increase the PD-L1¹⁶⁰ and CD137 expression to suppress the host T-cell activation in colon cancer.¹⁶¹ In RCC, HIF-1α induces GAL3ST1 mediated sulfonation of membrane fatty acids, protecting cancer cells from the attack of natural killer cells.¹⁶² Additionally, HIF-2α expression reduces RCC patients’ survival rate, and it also increases the IL-10 and TGF-β secretion from mast cells to suppress CD8⁺ T-cells.¹⁶³ Similarly, Mayer et al. observed that in brain metastasis, hypoxic regions have low T_C cell infiltration, and hypoxia suppression increases T-cells’ infiltration and reduces the growing tumor size. However, under hypoxia T_reg cells, adenosine signaling also helps to develop an immunosuppressive environment.¹⁶⁴

Although during the initiation of anoikis, cancer cells reduce mitochondrial oxidation and shift their metabolism toward aerobic glycolysis. Increased mitochondrial oxidation leads to ROS-mediated anoikis. However, mitochondrial oxidation suppression shifts aerobic glycolysis intermediates toward synthesizing antioxidant molecules through fatty acid¹⁶⁵ and PPP.¹⁶⁶ Elevated FAO and fatty acid synthesis in anoikis-resistant cells suppress the cellular ROS and increase osteosarcoma cells’ lung colonization.¹⁶⁷ After the ECM detachment, cancer autophagy regulates the cellular metabolism and maintains the anoikis resistance in ovarian cancer cells.¹⁶⁸ For instance, Yoshino et al. observed that anchorage-independent cells rely on increased aerobic glycolysis and shift the glycolytic intermediates for the acetyl-CoA and TCA cycle.¹⁶⁹ However, instead of glycolysis, anoikis-resistant ovarian cancer cells increase their mitochondrial oxidation and shift their glutamine toward the TCA cycle intermediates synthesis.¹⁷⁰

In contrast, cancer cells do not always use glycolysis for ATP generation, but they also shift glycolysis or intermediates of glycolysis toward TCA and OXPHOS for the anabolic synthesis to maintain the viability of circulatory cancer cells. In cancer cells, AMPK act as a sensor and regulator of cellular energy expenditure. After detachment from the ECM, breast cancer cells conserve their energy to maintain essential cellular processes. During low cellular ATP, especially after the ECM detachment, AMPK suppresses the mTORC1 mediated unnecessary protein synthesis.¹⁷¹ However, Bizjak et al. showed that low cellular glucose or glycolysis inhibition also activates the AMPK mediated anchorage-independent growth of breast cancer cells.¹⁷²

During ECM detachment, cellular integrin plays a vital role as an inducer of anoikis. After basement detachment, integrin/FAK induces apoptosis in detached cancer cells. Thus, to maintain anoikis resistance, breast cancer cells activate their survival pathways and suppress the expression of pro-apoptotic factors.¹⁷³ After the ECM detachment, elevated ROS activates the cancer cell’s survival pathway (EGFR/Src/ERK/AKT) and develops the anoikis resistance.¹⁷⁴ In epithelial and basal cell carcinoma, secreted angiopoietin-like protein 4 (ANGPTL-4) interacts with integrin and maintains the cellular antioxidant homeostasis followed by induction survival pathway (PI3K/ERK) and suppress pro-apoptotic factor (Bad) expression.¹⁷⁵ Additionally, Oleic acid also activates the ANGPTL-4 mediated anoikis resistance.¹⁷⁶ Similarly, under hypoxic conditions, ANGPTL-4 expression activates FAK-Src/PI3K/AKT/ERK survival pathway and suppresses Caspase-3, 8, 9, and PARP expression in gastric cancer cells.¹⁷⁷ However, in lung colonizing breast cancer cells, HIF-1α mediated PI3Ksignaling maintains anoikis resistance¹⁷⁸ and inhibits the expression of anoikis proteins (Bim and Bmf).¹⁷⁹

Autophagy and cancer dormancy

Physiological stresses such as oxidative, metabolic, hypoxic, and therapeutic stress restrict tumor growth and start the pro-survival autophagy, followed by a reduced metabolic and proliferative activity. Autophagy is a natural and regulated cell mechanism to degrade and recycle the cellular component. In cancer cells, to continue the survival process, autophagy degrades and recycles the macromolecules (e.g., glucose, proline, α-ketoglutarate, glutamine, pyruvate, lactate, nucleotide, and fatty acids) for the generation of essential biomolecules. However, under the stress condition, autophagy-mediated low metabolic activity maintains the dormancy nature or quiescence (G0/G1) state of lung cancer cells.¹⁸⁰

Autophagy also maintains the viability of circulatory and colonizing cancer cells and protects them from anti-cancer drugs. For instance, in response to chemotherapeutics, the ROS-mediated autophagy activates the survival pathway to protect abdominal cancer cells,¹⁸¹ while inhibiting autophagic metabolism restores drug sensitivity in cervical cancer cells.¹⁸² In GBM cells, starvation conditions induce autophagy-mediated quiescence and therapeutic resistance.¹⁸³ It reduces cancer cells’ proliferative nature and suppresses proliferative markers (e.g., Ki-67 and PCNA). However, the stem cell-like feature of cancer cells maintains the differentiation state by expressing stemness markers in the dormant head and neck cancer cells.¹⁸⁴ In addition to metabolic starvation, low PO₂ also increases the expression of dormancy markers (e.g., NR2F1, p27, and DEC2) and arrest breast cancer cells in the G0/G1-phase.¹⁸⁵ During the dormant condition, cancer cells increase the lactate, α-ketoglutarate, citrate, pyruvate, and FAO to maintain anabolic biosynthesis and suppress the AKT-mTOR/MAPK mediated metabolic activity. Additionally, dormant cancer cells also increase their antioxidant molecules to stop ROS-mediated oxidative stress.¹⁸⁶

In a limited nutrition environment, autophagy maintains energy homeostasis and anabolic biosynthesis. Therapeutic inhibition of aerobic glycolysis under low cellular glucose induces apoptosis in cancer cells, but colorectal cancer cells shift ATP generation toward autophagy to protect starved cells from apoptosis.¹⁸⁷ In the TME, glucose shortage activates the energy sensor AMPK, followed by ROS-mediated autophagosome formation. During the ROS-mediated autophagosome formation, pancreatic cancer cells suppress the NADPH and GPx-1 generation.¹⁸⁸ Cellular ROS helps in autophagy induction, but autophagy itself creates the oxidative environment. Thus, to balance ROS and autophagy, breast cancer cells also increase Nrf2 or glutathione expression for cancer cells’ sustained growth under the metabolic stress condition.¹⁸⁹ Furthermore, low cellular ATP also stimulates the AMPK mediated autophagy¹⁹⁰ and increases the membrane translocation of GLUT1 to increase the glucose uptake.¹⁹¹ However, for autophagy induction, metabolites also maintain the H⁺ ions for autolysosome formation. The TME acidification by aerobic glycolysis and lactate-mediated NADPH oxidation generates the H⁺ ions for autophagosome acidification.¹⁹² The TME acidosis even arrests cells in G0/G1-phase and maintains dormancy in cancer cells.¹⁹³

Cellular starvation directly regulates autophagosome development. For instance, suppression of cellular glycolysis makes alveolar rhabdomyosarcoma cells sensitive to autophagy inhibitors.¹⁹⁴ Thus, under the glucose starvation, AMPK activates ULK1 for autophagy initiation, while in the nutrient-rich condition, mTOR inhibits the ULK1 mediated autophagy induction.¹⁹⁵ Additionally, during amino acid starvation, autophagy also maintains the amino acid homeostasis and increases the free glutamine to synthesize non-essential amino acids (e.g., glutamate, proline, and arginine). However, sufficient free glutamine restores mTOR, directs protein synthesis, and inhibits autophagy.¹⁹⁶ For instance, to maintain the amino acid homeostasis, TNBC cells increase the localization of amino acid transporter (SLC38A2), while the shortage of glutamine results in proteasomal degradation of SLC38A2 and subsequently increases ROS-mediated autophagy.¹⁹⁷

Glutaminolysis inhibition shifts cancer metabolism toward fatty acid autophagy, while suppression of FAO and autophagy increases lung and breast cancer cell apoptosis.¹⁹⁸ Additionally, deleting the autophagy-related 7 (ATG7) gene in lung cancer cells makes them sensitive to starvation and induces mitochondrial dysfunction by increased fatty acid accumulation.¹⁹⁹ Thus, fatty acid autophagy plays a crucial role in maintaining mitochondrial function and TCA homeostasis. Amino acid shortage activates AMPK mediated lipid droplet generation and inhibits the mTOR pathway.²⁰⁰ Additionally, TME adipocytes also secrete fatty acids and stimulate AMPK mediated autophagy in starved cancer cells.²⁰¹

Autophagy also supports cancer cell proliferation during hypoxic condition. For instance, the hypoxic condition increases the expression of autophagy-related proteins such as AMPK, PERK, BNIP3, REDD1, and PML.²⁰² However, during low oxygen levels, both HIF isoforms (HIF-1α and HIF-2α) increase the survival autophagy²⁰³ by activating AKT/ERK1/2 and suppress the mTOR signaling.²⁰⁴ The HIF-1α mediated AMPK activation induces autophagy to maintain cell viability.²⁰⁵ Furthermore, the hypoxic condition increases ER stress and affects protein folding. PERK senses the protein misfolding in such stress conditions and activates the unfolded protein response (UPR) mediated autophagy to regulate the protein misfolding.²⁰⁶ Joshi et al. observed that cancer cells increase the ROS in hypoxic conditions and suppress the AKT/mTORC1 pathway to increase the autophagy survival cascade.²⁰⁷

In breast cancer cells, hypoxia increases autophagy and supports adapting in the low metabolic and oxygen environment. The hypoxic environment maintains both quiescence and stem cell state (CD44⁺) to regulate breast cancer cells’ proliferative nature.²⁰⁸ To maintain the dormancy, HIFs suppress proliferative PI3K/AKT signaling and reduce the metabolic activity²⁰⁹ by inhibiting mTOR-mediated protein expression. They also regulate the expression of EGFR/MEK/ERK-mediated stress factors and MAPK expression to maintain the dormant stage.²¹⁰ Elevated HIF-1α expression also increases protein phosphatase-2A to maintain low proliferation in cancer cells, uplift the cancer recurrence and reduce the disease-free survival of GBM patients.²¹¹ Additionally, TME ROS regulates glucose and FAO under hypoxia to establish AMPK mediated dormancy.²¹² Of note, hypoxia also maintains the dormancy condition under the presence of chemotherapeutics. In response to anti-estrogen drugs, HIF-1α increases the ERK1/2 activation and suppresses the ER-α expression, which further arrests proliferating breast cancer cells into the G0/G1-phase.²¹³

Drug resistance and cancer recurrence

Despite several therapeutic options for cancer management, cancer incidences are increasing every year. Indeed, many clinical studies reported drug resistance development followed by cancer recurrence and reduced disease-free survival of cancer patients. In the TME, both altered metabolism and PO₂ modulate the cancer environment and reduce chemotherapeutics’ presence and effectiveness. Additionally, cancer cells’ autophagy and stemness properties create a resistant environment and help in cancer recurrence. Furthermore, escape from the host immune system also plays a critical role in cancer recurrence; for instance, patients with high blood glucose are more susceptible to prostate cancer recurrence;²¹⁴ breast and colorectal cancer.²¹⁵ Similarly, Walenta et al. also correlated high lactate with cervical cancer recurrence.²¹⁶

Lactate from aerobic glycolysis maintains cancer cell viability, creates TME acidification, and induces resistance for PI3K/mTOR inhibitors in breast cancer cells.²¹⁷ For instance, the high glycolysis rate is also associated with cisplatin resistance in head and neck carcinoma, and a lower rate of glycolysis in cancer cells favor cisplatin therapy.²¹⁸ However, metabolic inhibitors are often used for effective cancer management, including anti-cancer drugs, but metabolic rewiring also protects cancer cells from metabolic inhibitors. For instance, the mTOR inhibitory drugs are used to regulate cancer metabolism, and in response to the mTOR inhibitory environment, GBM cells increase glutaminolysis followed by α-ketoglutarate mediated TCA cycle anaplerosis for cellular homeostasis.²¹⁹ Furthermore, glutamine deprivation sensitizes the cancer cells for chemotherapeutic drugs and induces apoptosis,²²⁰ but in response to glutamine shortage, liver cancer cells up-lift the aerobic glycolysis and activate serine mediated ERK to develop resistance against kinase inhibitors.²²¹ Excess glutamine oxidation also increases the drug resistance against gemcitabine in pancreatic cancer cells, and glutaminolysis inhibition increases cancer cells’ sensitivity to gemcitabine.²²² Elevated glutamine metabolism maintains the cellular glutathione to protect pancreatic cancer cells from drug effects,²²³ and in cisplatin-resistant lung cancer cells, it also supports the ATP and TCA cycle intermediates synthesis.²²⁴ Similarly, paclitaxel-mediated glycolysis and OXPHOS inhibition induce apoptosis in lung cancer cells, whereas FAO maintains resistance cancer cells’ viability²²⁵ and NADH homeostasis for the suppression of drug-induced ROS mediated apoptosis.²²⁶ Additionally, elevated fatty acid transporter (GPR120) maintains an adequate amount of fatty acid in breast cancer cells and increases ABC transporters’ expression to reduce drug accumulation in neoadjuvant chemotherapy-treated patients’ .²²⁷

Although, therapeutic resistance also increases the stem cell phenotype and self-renewal property of cancer cells and favors cancer cell regeneration. The previous section discussed the FAO’s role in maintaining cancer stem cells and NADPH mediated redox homeostasis. Thus, ROS homeostasis not only maintains the chemoresistance phenotype in hepatocellular cancer cells but also supports cancer recurrence.²²⁸ Similarly, another study showed that purine synthesis in glioma cells maintains cancer stemness and self-renewal properties.²²⁹ In leukemia cells, the high aerobic glycolysis PKM2 maintains the imatinib resistance;²³⁰ however, dexamethasone inhibits PKM2 and disrupts drug resistance. In response to PKM2 inhibition, peroxisome proliferator-activated receptor-α (PPAR-α) increases FAO and restores cellular metabolism in the drug environment.²³¹ In addition, elevated FAS in cancer cells protects cancer from DNA damaging drugs (e.g., mitoxantrone, doxorubicin, etoposide, cisplatin, and camptothecin). Nevertheless, it fails to protect from destructive microtubule drugs (e.g., paclitaxel and vinblastine). High FAS suppresses the drug-induced pro-apoptotic factors (e.g., TNF-α, ceramide, and caspase-8),²³² and FAS inhibition suppresses the PI3K/AKT signaling, followed by cancer cell apoptosis. Additionally, FAO and oxidative stress were also found in regenerated tumors and favor breast cancer recurrence.²³³

The angiogenesis inhibitors are often used for tumor angiogenesis suppression and create a hypoxic environment at the developing tumor site. In such type of drug-induced hypoxic environment, HIF-mediated metabolic reprogramming maintains the viability of cancer cells. Hypoxia-mediated aerobic glycolysis protects cancer cells from nintedanib effects²³⁴ and increases the expression of metabolic transporter (e.g., GLUT1 and MCT1/4) in sunitinib treated RCC cells.²³⁵ However, TME acidification under hypoxia reduces anti-cancer drugs’ effectiveness.²³⁶ Similarly, in colon cancer patients, HIF-1α mediated PDK3 expression induces the development of drug resistance and recurrence of colon cancer cells.²³⁷ For Instance, Li et al. reported the hypoxic environment’s involvement in the multi-drug-resistance development in the hepatoma cells against 5-FU, sorafenib, gemcitabine, cisplatin, adriamycin, and 6-thioguanine. Furthermore, the DNA damaging drug (5-FU) reduces the expression of the 5-FU target (thymidylate synthase) and protects cancer cells from DNA damage.²³⁸ Hypoxia-mediated pro-apoptotic factor (e.g., Bid/Bax) expression could be a possible way of induced drug resistance in cancer cells.²³⁹ In addition to metabolic reprogramming, regulators of metabolism are also differentially expressed in cancer cells and regulate different steps of metastasis cascade (Table 1).

Table 1.

Cancer metabolite rewiring regulator and their role in cancer metastasis

S No	Metabolism regulating enzyme	Metabolism	Role in metastasis	Cancer type
1.	Hexokinase	Aerobic glycolysis	Proliferation	Breast and hepatoma (Lincet et al., 2015)
2.	Pyruvate dehydrogenase kinase (PDK)		Proliferation, migration, autophagy, recurrence, drug and anoikis resistance	Glioma (Chae et al., 2016), nasopharyngeal carcinoma (Roh et al., 2016), breast (Dupuy et al., 2015) and colon (Lu et al., 2011)
3.	Phosphoglycerate mutase (PGAM)		proliferation	Lung (Li et al. 2006) and hepatoma (Ren et al. 2010)
4.	Lactate dehydrogenase (LDH)		Proliferation, immune evasion, autophagy, migration, drug resistance, and cancer recurrence	Pancreatic, breast, colon and NSCLC (Zhao et al., 2013), bladder (Zhao et al., 2015)
5.	Phosphofructokinase-1 (PFK-1)		Proliferation and migration	Breast, colon, ovarian (Lincet et al., 2015, Yi et al., 2012) prostate, lung, liver, colon and cervical (Yi et al., 2012)
6.	Pyruvate kinase M2 (PKM2)		Proliferation, drug resistance	Leukemia and colon (Schell et al., 2014)
7.	Pyruvate carboxylase (PC)	TCA cycle	Proliferation, migration, autophagy and drug resistance	Breast (Christen et al. 2016), colon, lymphoma and melanoma (Leone et al., 2019)
8.	Isocitrate dehydrogenase (IDH)		EMT	Glioblastoma and leukemia (Grassian et al., 2012)
9.	Glutamate dehydrogenase (GDH)	Amino acid metabolism	Proliferation, migration and drug resistance	Neuroblastoma (Quing et al. 2012), lung and breast (Jin et al., 2015)
10.	Asparagine synthetase		EMT, vasculogenesis, migration and colonization	Breast (Knott et al., 2018) and lung (Gwinn et al., 2018)
11.	Glutamate-oxaloacetate transaminase (GOT)		Proliferation, migration	Neuroblastoma (Quing et al., 2012)
12.	Glutamine synthetase (GS)		Proliferation, survival and vasculogenesis	Hepatocellular carcinoma (Adebayo Michael et al., 2019)
13.	Glutaminase (GLS)		Proliferation, migration, autophagy, angiogenesis and cancer cell survival	Colon, lymphoma, melanoma (Leone et al., 2019) and breast (Morotti et al., 2021), lung (Jiang et al., 2017)
14.	Acetyl-CoA carboxylase (ACC)	Fatty acid metabolism	Proliferation, migration and colonization	Breast (Garcia et al., 2017)
15.	ATP citrate lyase (ACLY)		Proliferation, migration, reoccurrence	Renal (Teng et al. 2018) and lung (Migita et al. 2008)
16.	Stearoyl-CoA desaturase-1 (SCD-1)		Stemness, migration, cell colonization and redox homeostasis	Colon cancer, glioblastoma (Choi et al., 2019)
17.	Acetyl-CoA acetyltransferase (ACAT)		Proliferation, migration and colonization	Prostate, lung, leukemia and breast (Fan et al., 2016)
18.	Acyl-CoA synthetase (ACS)		Proliferation, migration, dormancy	Lung (Padanad et al., 2016)
19.	Carnitine palmitoyltransferase-1 (CPT1)		Vasculogenesis, autophagy and dormancy	Breast (Park et al., 2016) and colon (Wen et al., 2017)
20.	Glutathione peroxidase (GPx)	Redox homeostasis	Proliferation, migration, and drug resistance,	Lung, breast (Jin et al., 2015) and pancreatic cancer (Meng et al., 2018)
21.	Glucose-6-phosphate dehydrogenase (G6PD)	Pentose phosphate pathway	Proliferation, migration, redox homeostasis, anoikis resistance	Breast (Sousa et al., 2020)

Conclusion

Our understanding of tumor development and metastasis has significantly improved over recent years; however, the role of oxygen and metabolic availability affecting the metastatic cascade programming is still not explicit. As a typical feature of solid tumors, oxygen and metabolic nutrients together add to metastatic cells’ survival process. For instance, cancer cell generally develops their TME to maintain the continuous metabolites and oxygen availability. The elevated metabolites at the TME promote the proliferation of cancer cells, while their shortage induces metabolite reprogramming or cancer cell metastasis toward metabolites-rich organs. However, it is not clear how the metabolites variation regulates cancer metastasis. Furthermore, both normoxia and hypoxia also facilitate the metabolites rewiring for cancer cell survivability; however, it is unclear how hypoxia drives tumor progression and warrants substantial investigation pertinent to the mechanism and implication for diagnosis and treatment. Of note, the TME oxygen and metabolites rewiring also regulate the EMT feature of cancer cells that promote cancer cell self-renewal ability and plasticity. Additionally, cancer cells’ autophagy and stemness properties create a resistant environment and facilitate cancer recurrence. Furthermore, tumor vasculogenesis and host immune evasion play a critical role in cancer development and recurrence, adding to the global disease burden. Overall, we have highlighted the importance of the oxygen-dependent/independent processes and different metabolic factors associated with major metastatic pathways responsible for tumor development, invasion, and recurrence of cancer.

Funding Statement

This work was supported by the Indian Institute of Technology Roorkee (IITR) [FIG100642].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

No ethical approval necessary for this review.