Stress Responses as Master Keys to Epigenomic Changes in Transcriptome and Metabolome for Cancer Etiology and Therapeutics

ABSTRACT

From initiation through progression, cancer cells are subjected to a magnitude of endogenous and exogenous stresses, which aid in their neoplastic transformation. Exposure to these classes of stress induces imbalance in cellular homeostasis and, in response, cancer cells employ informative adaptive mechanisms to rebalance biochemical processes that facilitate survival and maintain their existence. Different kinds of stress stimuli trigger epigenetic alterations in cancer cells, which leads to changes in their transcriptome and metabolome, ultimately resulting in suppression of growth inhibition or induction of apoptosis. Whether cancer cells show a protective response to stress or succumb to cell death depends on the type of stress and duration of exposure. A thorough understanding of epigenetic and molecular architecture of cancer cell stress response pathways can unveil a plethora of information required to develop novel anticancer therapeutics. The present view highlights current knowledge about alterations in epigenome and transcriptome of cancer cells as a consequence of exposure to different physicochemical stressful stimuli such as reactive oxygen species (ROS), hypoxia, radiation, hyperthermia, genotoxic agents, and nutrient deprivation. Currently, an anticancer treatment scenario involving the imposition of stress to target cancer cells is gaining traction to augment or even replace conventional therapeutic regimens. Therefore, a comprehensive understanding of stress response pathways is crucial for devising and implementing novel therapeutic strategies.

INTRODUCTION

One of the most critical aspects of cellular physiology is the maintenance of homeostasis, which is governed by evolutionarily conserved and interwoven molecular cascades. The extracellular milieu or microenvironment of cancer cells has a major impact on their internal molecular architecture. Throughout the course of initiation, progression, invasion, and metastasis, cancer cells constantly adapt to various forms of exogenous and endogenous stress stimuli that challenge their internal homeostatic balance. Changes in the microenvironment, such as altered oxygen and nutrient availability, largely impact the internal molecular and epigenetic landscape of the cancer cells. Likewise, internal imbalances in the molecular equilibrium caused by genomic instability, the accumulation of incorrectly folded proteins, oxidative stress, etc., lead to many alternations in cellular signaling pathways, which cumulatively determine cell fate. Whether the cancer cell will adapt to and survive the stressful stimuli or undergo apoptosis is guided by intricate signals and is largely determined by the nature and duration of the stress stimulus.

In this context, an understanding of the epigenetic alterations employed by cancer cells as an approach to adapt to stress is of crucial significance and can be utilized to devise a potential strategy to ameliorate anticancer treatment. Chemotherapy, the first-line therapeutic regimen utilized for most cancers, induces genotoxic stress to cancer cells. However, in the majority of cases the development of chemoresistance leads to tumor recurrence, which ultimately results in a poor prognosis. Several studies have indicated that cancer cells employ elaborate epigenetic cascades to adapt to chemotherapeutic and other stresses to develop therapeutic resistance (1, 2). A complete understanding of the stress-adapting epigenetic pathways of cancer cells can unveil a plethora of target molecules, which can in turn be modulated to attenuate the stress adaptations of cancer cells.

In this review, we present a comprehensive view of the current understanding on the alternations in epigenome and transcriptome levels in cancer cells when subjected to various physicochemical stress stimuli, such as oxidative stress, hypoxia, hyperthermia, alkylation, and radiation-mediated DNA damage, nutrient deficiency, and misfolded protein accumulation. This accrued information can enlighten some previously unexplored targets that can be potentially utilized to device novel therapeutic regimens against cancer.

KEY STRESS RESPONSE PATHWAYS AND THEIR EPIGENETIC AND MOLECULAR SIGNATURES

Oxidative stress and its epigenetic response.

Basal levels of reactive oxygen species (ROS) are constantly generated in each and every cell type, as a byproduct of metabolic pathways (3, 4). However, the cellular antioxidant mechanisms scavenge the ROS to diminish the ROS-mediated intracellular damage. For example, most ROS radicals react with oxygen to form superoxide, so the human cellular and mitochondrial superoxide dismutases are master regulators of ROS that use oxidation and reduction to dismutase superoxide to water and hydrogen peroxide, the latter being removed by catalase. Under various conditions, the subtle balance between ROS generation and ROS scavenging is perturbed, leading to the accumulation of elevated levels of ROS, thereby disrupting cellular homeostasis (5). As an example of how ROS can reset the cell to a higher level of oxidative stress, the cytoplasmic superoxide dismutase dimer has free cysteines, and their oxidation reduces the protein stability, thereby reducing ROS production and removal. Various conditions, such as hypoxia, nutrient imbalance, hyperthermia, and exposure to genotoxic agents, can lead to significantly augmented production of ROS within the cell, which ultimately leads to a state of oxidative stress. Nitric oxide synthesis, which is controlled by nitric oxide synthases, can also reduce oxidative stress in cancer cells by forming dinitrosyl complexes. Loss of ROS balance can alter many aspects of cell metabolism, including signaling and cell death.

(i) Altered epigenetic landscape in oxidative stress.

Heightened accumulation of intracellular ROS damages the biomolecules and disrupts the balance of cellular metabolites, thus perturbing genomic stability and compromising the intracellular epigenetic balance (Fig. 1, upper left panel). As a consequence of these alterations, senescence, cell death, and even cancer may ensue. For instance, S-adenosylmethionine (SAM) is used as a substrate by histone and DNA methyltransferases (6); ROS-mediated oxidization of GSH to GSSH attenuates SAM production by inhibiting the formation of SAM synthetase, the enzyme responsible for SAM production, thus disrupting methyltransferase activity (7, 8), leading to alterations in DNA and histone methylation. The role of ROS in mediating DNA damage and the cellular DNA damage response pathways in this context are well documented by numerous studies (9–11). It has been indicated that induction of oxidative stress recruits a DNMT1/DNMT3b/PRC4 complex to CpG islands on various gene promoters, causing hypermethylation and silencing of those genes (12).

FIG 1.

Schematic diagram representing the different epigenetic and molecular cascades employed by cancer cells to adapt in response to oxidative stress, hypoxia, hyperthermia, radiation, protein misfolding, and nutrient deprivation.

(ii) Oxidative stress and its effect on the epigenetic machinery in the context of cancer.

ROS-mediated DNA damage has been widely correlated with the development and progression of cancer (13). Intriguingly, based upon the evolutionarily conserved unstructured domain that targets the oxidized base excision repair enzyme NEIL1 to open chromatin, its damage surveillance of oxidation-susceptible sites to preserve essential gene function and to limit instability and cancer likely originated ∼500 million years ago during the build-up of free atmospheric oxygen. Indeed, the accumulation of ROS and oxidative stress is considered to be one of the prime reasons behind DNA damage and genomic instability, which ultimately leads to malignant transformation (14). ROS is reported to modulate the activities of various epigenomic modifiers, such as DNMT1, DNMT3a, histone deacetylase 1 (HDAC1) HMT1, HAT1, and MBD4, thus acting as a neoplastic trigger (15).

The role of oxidative stress in initiation of carcinogenesis has been studied by various groups. A report by Nishida et al. (16) on chronic hepatitis C indicates that 8-hydroxy-2′-deoxyguanosine, a ROS-mediated promutagenic DNA lesion, leads transformation of chromatin activation marks (H3K4me3 and H4K16ac) to a chromatin repression mark (H3K27me3) in the promoters of tumor suppressor genes. This, in turn, acts as a risk factor for the onset of hepatocellular carcinoma. A relatively older breast cancer study established the involvement of 8-hydroxy-2′-deoxyguanosine in the development of breast carcinoma (17). Another indicator of ROS-mediated DNA damage, 8-oxo-7,8-dihydro-2-deoxyguanosine, is reported to negatively correlate with the epigenetic marker 5-methylcytosine (m5C) and act as a prerequisite for development and progression of glioma (18).

The ingenious ability of cancer cells to utilize the heightened level of ROS to promote their survival is supported by various studies. For example, ROS-mediated hyperactivation of mitogen-activated protein kinase (19), NF-κB (20), peroxisome proliferator-activated receptor gamma (PPARγ) (21), and STAT3 (22) pathways stimulates cell proliferation and ensures the progression of cancer. A study by Lim et al. (23) on hepatocellular carcinoma indicated that ROS induces hypermethylation of the E-Cadherin promoter via recruitment of HDAC1 and DNMT1 by SNAIL, thus leading to activation of the epithelial-mesenchymal transition (EMT) and metastasis. A similar study on colorectal cancer indicated that ROS increases the binding of DNMT1 and HDAC1 to the tumor suppressor Runt domain transcription factor 3 (RUNX3) promoter, leading to its hypermethylation and suppression (24), thereby promoting colorectal cancer progression (24). Long interspersed nuclear element 1 (LINE-1) is the most abundant type of transposable element found in eukaryotic genomes. In normal somatic cells, LINE-1 elements are mostly methylated and suppressed. However, hypomethylated and active, LINE-1 elements lead to genomic instability and mutagenesis (25, 26). In another study in bladder cancer, ROS-mediated hypomethylation of LINE-1 was associated with heightened genomic instability and progression of cancer (27). Therefore, all these observations point toward the ingenious adaptive ability of cancer cells to oxidative stress that ultimately leads to heightened tumor progression and poorer prognostic outcome.

Epigenetic responses to hypoxia.

Oxygen is one of the most critical components supporting life of complex organisms, and in the human body it is a defining criterion of the metabolic landscape. Extremely scarce availability of oxygen in the cellular microenvironment (i.e., hypoxia) can potentially promote stress, altering the homeostasis of cells and their metabolic architecture. Hypoxia is a hallmark of the malignant phenotype and a key feature of the microenvironment in solid tumors, where the supply of oxygen is frequently irregular despite angiogenesis. The irregularity in the spatial distribution of vasculature leads to the onset of a steep oxygen gradient toward the tumor core, resulting in hypoxia and profoundly affecting the biological behavior of the hypoxic cancer cells. Hypoxia is reported to be associated with reduced sensitivity to radiotherapy or chemotherapy (28, 29) and an increased invasive and metastatic phenotype (30). Hypoxia induces both proteomic and genomic, as well as epigenomic, changes within tumor cells. In cancer cells, the response to hypoxia is governed by a heterodimeric transcriptional regulator termed hypoxia-inducible factor (HIF) (31, 32). In response to hypoxia, HIF-1α becomes stabilized by binding to a constitutively expressed β-subunit (HIF-1β), localizing to the nucleus and activating hypoxia response elements (HREs) to augment the expression of target genes (31, 33). In turn, HIF-1-activated genes regulate cellular glucose and oxygen consumption and glucose metabolic pathways. Several studies have indicated that HIF-1 induces the expression of stemness factors (34, 35) and promotes angiogenesis and metastasis in response to hypoxia (36–38).

(i) Epigenetic regulation of HIF-1α stability.

Stabilization of HIF-1α is one of the most crucial aspects in hypoxia adaptation. Under normal conditions, HIF-1α is hydroxylated by prolyl hydroxylases (PHD). Upon hydroxylation, its oxygen-dependent degradation (ODD) domain recognized by von Hippel-Lindau tumor suppressor protein (VHL), eventually leading to proteasomal degradation by the cullin2 E3 ligase complex (39–42). In contrast, under hypoxic stress, the enzymatic activity of PHDs decreases, thereby inhibiting the hydroxylation of HIF-1α while favoring its stabilization. In addition to hydroxylation, other posttranslational modifications, such as acetylation, phosphorylation, and SUMOylation, have been reported to modulate HIF-1α. Previous reports indicated that SENP1 de-SUMOylates and stabilizes HIF-1α (43); moreover, phosphorylation of HIF-1α by p38 inhibits VHL binding, leading to its stabilization (44). In contrast, HIF-1α acetylation is associated with VHL-mediated ubiquitination and degradation (45). A recent report has revealed the presence of a methylation/demethylation cycle involving methylation of HIF-1α by SET7/9 and demethylation by LSD1 as a way to regulate HIF-1α stability under hypoxic conditions (46). The epigenetic state of crucial members of HIF-1 signaling cascade also regulates HIF stability and influences its target gene expression. For instance, hypermethylation of the VHL promoter and its inactivation increases the stabilization of HIF-1α and promotes the expression of its target genes, such as carbonic anhydrase 9 (CA9) and glucose transporter type 1 (GLUT1) (47).

DNA and histone demethylation are also related to the dynamic regulation of HIF-1α. Indeed, it has been reported that in colon cancer, demethylation of CpG islands in the HIF-1α promoter promotes binding of HIF-1α to its own promoter, thus establishing a positive-feedback loop (48). Induction of euchromatic histone-lysine N-methyltransferase 2 (G9a) by hypoxic stimuli acts as a central crossroad in hypoxia-mediated signaling pathways (49, 50). G9a leads to enhancement of H3K9me2 and subsequent repression of several hypoxia-regulated genes (50, 51) (Fig. 1, upper middle panel). For instance, Reptin, a nonhistone chromatin modifier that modulates about 24.6% of hypoxia-responsive genes, is methylated and repressed by G9a (52). It binds to, deacetylates, and transcriptionally represses VEGF, BNIP3, and PGK1 via recruitment of HDAC1 (52). In contrast, G9a methylates Pontin, a chromatin-remodeling factor that regulates about 23.5% of hypoxia-responsive genes (53). Increased expression of these genes via methylated Pontin promotes hypoxia-induced tumor progression, EMT, and metastasis. Furthermore, G9a stimulates HIF-1α-mediated target gene activation by recruiting p300 to the promoters of HIF-1α target genes, including Ets1, KDM4B, and IGFBP3 (53). In addition, acetylase activity by p300/CBP and TAT-interactive protein 60 (TIP60) regulates the transcriptional expression of HIF-1α (54), while class I histone deacetylases (HDACs) regulate its stability (55). Under severe hypoxic stress, acetylation of HIF-1α at lysine 709 by p300 increases its stability (56). In contrast, SIRT1 (NAD-dependent deacetylase sirtuin 1) (Fig. 2) deacetylates p300/CBP-associated factor (PCAF)-mediated lysine acetylation of HIF-1α, thus preventing p300/CBP recruitment and hypoxia-induced target gene activation (57). SIRT3 has also been reported to inhibit HIF-1α, thereby suppressing the metabolic reprogramming of cancer cells and mitochondrial ROS production (58). Together, these reports highlight the epigenetic regulatory network of hypoxia-responsive gene expression through various histone and DNA alterations. The activation-inactivation cycle of HIF-1α is also regulated by protein acetylation: lysine 532 of HIF is acetylated by ARD1, which stimulates its interaction with VHL and subsequent degradation, under normoxic conditions (46). Under hypoxic stress, this state is reversed, supporting the activation of HIF-1α.

FIG 2.

SIRT1 is downregulated in cancer and associated with poor survival in patients with weak expression. (A) Box plot of SIRT1 mRNA levels in 15 tumor types from TCGA. Only tumors with at least 10 matched controls were included. (B) Kaplan-Meier survival curves and hazard ratios (HR) in patients with low (below mean) and high (above mean) SIRT1 mRNA levels in KIRC, LGG, and HNSC. Tumor abbreviations: BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; PRAD, prostate adenocarcinoma; STAD, stomach adenocarcinoma; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma. Analyses were conducted as described previously (229).

(ii) Noncoding RNA-mediated regulation of HIF-1α.

The stability of HIF-1α in hypoxic cancer cells was also reported to be mediated by several noncoding RNAs. In hypoxic nasopharyngeal carcinoma cells, two long noncoding RNAs (lncRNAs), PVT1 and DANCR, play a role in stabilizing both HIF-1α mRNA and the protein, promoting hypoxia-induced tumor progression (59). Both PVT1 and DANCR are reported to be associated with poor prognosis of nasopharyngeal carcinoma (59, 60). MicroRNA 181c (miRNA-181c) in breast cancer (61) and miRNA-646 and lncRNA-MTA2TR in pancreatic cancer (62) are also involved in the regulation of HIF-1α stability and its accumulation, thus playing part in managing hypoxic stress in these cancer cells.

(iii) Epigenetic regulatory mechanisms behind hypoxia-induced EMT.

It is well established that hypoxia promotes tumor progression and metastasis. Recent reports have indicated that hypoxia-induced EMT is partly driven by HDAC3 and that its knockdown significantly impairs the metastatic potential of cancer cells under hypoxic stress (63, 64) (Fig. 1, upper middle panel). In hypoxic stress, HDAC3 is recruited to epithelial gene promoters, deacetylating H3K4 and repressing them (64). However, a bivalent domain of enhanced H3K4me2/me3 and H3K27me3 was recently reported on the promoters of epithelial genes (65). These bivalent domains supposedly maintain EMT genes repressed, while keeping them poised for activation at a later stage during mesenchymal-epithelial transition (MET). These observations support the activity of epigenetic modulatory mechanisms involved in hypoxia-induced alterations in tumor cells.

Epigenetic responses to heat stress. (i) Heat shock factors and heat shock proteins.

Hyperthermia, or heat stress, is defined as a temperature above physiologically optimum range which affects physiological and molecular properties (66). Hyperthermia activates a subset of ataxia-telangiectasia mutated effectors independent of DNA strand breaks (67). When a cell is exposed to heat stress, heat shock factors (HSFs), a family of transcription factors, regulate the expression of heat shock proteins (HSPs), an evolutionary conserved pathway known as heat shock response. The function of HSFs was first observed by the Italian geneticist Ferruccio Ritossa, who showed an induction of chromosome puffs in the salivary gland of the larvae of Drosophila busckii when exposed to an elevated temperature (68). Subsequent investigations indicated that these chromosomal puffs were the regions transcribing HSPs, which function as molecular chaperones (69). HSFs are responsible for mediating cellular adaptations to proteotoxic stress conditions, such as elevated temperature, pathogens, toxins, etc. (70, 71). It has also been suggested that HSFs play a critical role during differentiation processes such as neurogenesis and gametogenesis (70, 71).

(ii) Role of heat shock factors in cancer.

HSFs and HSPs have been studied for decades in the context of cancer. HSPs have long been correlated with evasion of apoptosis, malignant transformation, and tumor progressions, and HSP inhibitors as an approach to target cancer cells are under investigation. More recent cancer research has focused on HSFs for their ability to transcriptionally regulate HSPs.

Mammalian HSFs are a family of five transcription factors and include HSF1, HSF2, HSF3, HSF4, and HSFγ. Among these, the functional regulation of HSF1, -2, and -4 has been mostly studied (72). HSF1 has been associated with tumor progression in several studies. For instance, HSF1-null mice are significantly resistant to dimethylbenzanthracene (DMBA)-induced skin cancer and mutant p53-induced malignant transformation (73). Studies have also reported resistance to HER2/ErbB2-induced breast cancer and carcinogen-induced liver cancer in humans and in HSF1-null mice (73–75). Overexpression of HSF1 is commonly observed in various carcinomas, and its higher expression is associated with poor prognosis (76). A meta-analysis of 10,287 cancer genomes from The Cancer Genome Atlas (TCGA) and cBioPortal databases revealed that the elevated expression of HSF1 in various types of cancers is due to the amplification of chromosome 8q24.3, where the HSF1 gene resides (77). In colorectal cancer, HSF1 reportedly promotes carcinogenesis by recruiting DNMT3a on MIR137HG. This leads to the suppression of miR-137, which in turn increases the expression of GLS1 and induces GLS1-mediated mTOR activation, thus promoting tumorigenesis (78). Inhibition or knockdown of HSF1 has been shown to reduce the tumor burden and to suppress growth in colorectal cancer cells (78).

HSF2 also plays a significant role in cancer. Once activated, HSF2 asserts its function by upregulating the expression of various HSPs, such as HSP27, HSP47, HSP70, and HSP90, thus promoting cell proliferation and migration (79). Along with HSF1 and HSF2, the role of HSF4 in cancer has been highlighted recently. Loss of HSF4 expression hinders Arf/p53 mediated tumorigenesis (80); however, unlike other HSFs, HSF4 also regulates cellular senescence (80) and, when inhibited, leads to cellular senescence and inhibition of cell proliferation. These roles for HSF4 depend upon the expression of p21 and p27 but are independent of p53 (80). In summary, a comprehensive understanding of the HSF family provides for selective optimization of various possibilities of new therapeutic interventions for cancer.

(iii) DNA damage and epigenetic alterations in hyperthermia.

Among the various cellular effects of hyperthermia, one of the key responses is the induction of DNA breaks and chromosomal aberrations. This response to hyperthermia occurs at temperatures higher than 41.5°C and involves focal phosphorylation of H2AX at serine 139 (γH2AX), as seen in response to double-strand breaks (81) (Fig. 1, upper right panel). Recent studies have indicated that at higher temperatures, i.e., 43 to 45.5°C, there is increased induction of γH2AX foci, a process which shows dependence on the DSB signaling factor ATM (67, 82, 83). In this context, various studies have reported a negative association between hyperthermia and the nonhomologous end-joining (NHEJ) DNA repair pathway (84, 85). A hyperthermia treatment at temperatures of 44 to 45°C inactivates DNA binding by KU70/80 and simultaneously decreases DNA-PK activity (86–89) and the levels of BRCA1 and 53BP1 (90). In general, we predict that DNA damage responses dependent upon conformational switching, and flexibility, such as the NHEJ repair complex, will be susceptible to hyperthermia treatments (91).

Apart from NHEJ, hyperthermia also inhibits repair by homologous recombination (HR) in both human and murine cells by inducing BRCA2 degradation (92). It also reduces the levels of MRE11 and inhibits the interaction with members of the Mre11-Rad51-Nbs1 (MRN) complex, which initiates HR, thereby abolishing HR-mediated DSB repair (93–96). Thus, there is a synergistic amplification of hyperthermia effects due to its impact upon depletion of key DNA damage response (DDR) proteins. That is, the structural biochemistry of DDR is connected to the cell biology of epigenetics and chromatin dynamics. For example, MRN is a primary sensor in the process of ATM activation and chromatin remodeling at DSBs and is required for establishment of epigenetic marks after repair DNA synthesis via the ATM kinase (97).

Inhibition of both of these repair pathways by hyperthermia depends on H4K16ac status. Specifically, depletion of hMOF, one of the histone acetyl transferases and a member of the largest lysine acetyltransferase (KAT) subdivision of the MYST family (MOZ, MOF, Ybf2, Sas2, and Tip60), plays a critical role (98) and leads to the attenuation of H4K16ac, which in turn causes a delay in the γH2AX focus formation (25). Therefore, regulation of H4K16ac by MOF directly impacts chromatin structure and thus affects the DDR pathway (25, 26). MOF was also reported to interact with several other proteins of the DDR pathway, regulating their functionality (25). All of these reports establish the significance of hMOF and hyperthermia in cancer therapeutics, in line with other studies reporting tumor regression following a moderate elevation in temperature (<45°C) (99).

Epigenetic alterations in radiation stress.

Ionizing radiation (IR) is the most extensively studied mutagenic radiating agent. Examples of IR include microwaves, X-rays, and gamma rays. This radiation can induce a vast array of DNA alterations, including nucleotide modifications, single- and double-strand breaks, and the formation of DNA-DNA and DNA-protein cross-links. IR is also reported to cause chromosomal alterations, such as inversions and deletions, which are observed at metaphase. The extent of chromosomal aberrations caused by IR depends on the level of linear energy transfer (LET) (100), a measure of the density of ionization along the radiation beam. For instance, high-LET radiation can induce more complex chromosomal rearrangements than does low-LET radiation (101).

(i) IR-induced changes in DNA methylation pattern.

DNA methylation is one of the most common epigenetic alterations. Accumulating evidence indicates that variations in DNA methylation patterns might facilitate the sensitivity or resistance of tumor cells to IR. Kalinich et al. (102) indicated that cancer cells, when exposed to ⁶⁰Co gamma irradiation in vitro, show a dose-dependent decrease in the level of 5-methylcytosine. Further studies confirmed that low-LET radiation induces genome-wide DNA hypomethylation.

Radiation exposure alters DNA methylation in a locus-specific manner. A study in triple-negative breast cancer by Antwih et al. (103) reported that RB1, IGF1R, and KRAS genes were differentially methylated after radiation exposure and that their levels of transcriptional expression corresponded to the level of methylation. This study also revealed altered expression of DNMT1 following X-ray radiation above 6 Gy (103). Various in vivo studies have indicated that X-rays can induce loss of global DNA methylation in target-specific and -nonspecific tissues (104–106). The alternations in methylation profile following radiation exposure have been studied in murine models also. Koturbash et al. (107) reported that acute and fractionated whole-body irradiation of mice leads to significant decreases in cytosine DNA methylation in the thymus, which in turn predisposes mice to lymphoma. In addition, irradiation-induced murine lymphomas showed an increased frequency of homozygous deletions of the Rit1/Bcl11b tumor-suppressor genes (107). The same group reported that a 1-Gy X-ray exposure for 6 h induced global DNA hypomethylation in murine spleen tissues (108).

LINE-1 hypomethylation has been observed in nearly all human cancers and is commonly associated with a poor prognosis. Several reports have indicated that exposure to IR leads to CpG hypomethylation and activation of LINE-1 retrotransposition activity in various tissues (109–112). This, in turn, acts as a driving force for carcinogenesis, highlighting the contribution of IR in initiating and promoting neoplastic transformation.

(ii) Radiation-induced modifications of histones.

IR can cause multiple types of histone modifications. A common IR-induced histone modification is phosphorylation of histone H2AX at serine 139 (γ-H2AX) (113), which is involved in diverse DNA-damage response pathways in mammalian cells through the activation of ATM and ATR (114–117). γH2AX is regarded as a measure of DSBs in DNA and as an early response to IR (118). Capetillo et al. reported that phosphorylated H2BS14 colocalizes with γH2AX loci upon DNA damage by IR (119). In addition, IR—especially UVB—has been reported to induce phosphorylation of H3S10 and H3S28 by the action of kinases, including ERK, p38, and the Src family member Fyn (120, 121). Phosphorylation of these histone moieties is frequently related with the expression of proto-oncogenes, strengthening the conclusion that radiation initiates neoplastic transformation (122).

Apart from phosphorylation, histone methylation also plays a critical role in DNA damage repair and transcriptional modulation. Histone tails can be mono-, di-, or trimethylated at specific lysine and arginine residues by histone methyltransferases, whereas demethylation is carried out by histone demethylases. For instance, laser microirradiation has been reported to load H3K9-specific methyltransferase KMT1A at DBS sites (123). Methylation at K27 of histone H3 (H3K27) is one of the most common modifications at sites of DNA damage induced by UV-laser microirradiation. Di- and trimethylation of H3K27 (H3K27me2 and H3K27me3) is carried out by the Polycomb repressive complex PRC2 via EZH1 and EZH2 (124) (Fig. 1, lower left panel). Targeting EZH2 using shRNAs or the chemical inhibitor GSK126 significantly enhances genotoxicity and promotes radiosensitivity in cancer cells (125). IR also modulates the expression of histone demethylases and histone methyltransferases, thereby impacting the efficacy of cellular responses to DNA damage-induced radiation (126).

(iii) Radiation-induced modulation of noncoding RNA expression.

Several studies have reported alternations in noncoding RNA expression profiles in response to IR (115–118) (Fig. 1, lower left panel). Both lncRNAs and miRNAs play crucial roles in cellular adaptation to radiation stress. In a recent study, Wang et al. identified 14 lncRNAs whose expression is altered in response to gamma irradiation in cervical, breast, and lung cancers (127). The participation of different lncRNAs and miRNAs in mediating radioresistance in cancer cells has been extensively studied by various groups. For instance, TUG1 lncRNA has been reported to induce radioresistance in different types of cancers by modulating miRNA 139-5p (miR-139-5p) and HMGB1 (128). TUG1 knockdown is associated with increased radiosensitivity, suggesting that it can be utilized as a potential therapeutic strategy against different types of solid tumors (129). A recent study in breast cancer reported that exposure to radiation leads to upregulation in expression of lncRNA HOTAIR (130), which in turn stimulated radioresistance by modulating the expression of miR-449b-5p and miR-218 (130, 131). In response to radiation-induced DNA damage, cancer cells rapidly employ DNA repair machinery to avoid cell cycle arrest, which in turn leads to radioresistance. lncRNA LINP1 is overexpressed in multiple cancers and confers resistance to IR and chemotherapeutic drugs by forming polyvalent interactions with Ku, by stabilizing NHEJ complex assembly, and by promoting liquid-phase condensates of break repair proteins thought to be repair foci (132). lncRNA MALAT1 acts in different cancers by inducing DNA repair and inhibiting cell cycle arrest (133, 134). A similar study by Zhang and coworkers reported that lncRNA BOKAS promotes radioresistance by upregulating Wnt1-inducible signaling pathway protein 1 (WISP1) and DNA damage repair (135). lncRNA GAS5 was also reported to suppress radiosensitivity through miR-106b and miR-205-5p (136). The radioresistance of cervical cancer and breast cancer was reported to be mediated by lncRNA NEAT1 and lncRNA LINC00963, respectively (137, 138). NEAT1 enhances cyclin D1 expression through the activity of miR-193b-3p. Apart from these lncRNAs, a number of microRNAs have been reported to be an integrated part of the response to radiation in different cancer models (132). A meta-analysis has reported at least 28 miRNAs that become differentially expressed in response to radiation exposure. Among these, seven miRNAs—miR-150, miR-29a, miR-29b, miR-30c, miR-200b, miR-320a, and miR-30a—may serve as biomarkers due to their elevated levels in serum after exposure to radiation (139). The most widely studied miRNAs in response to radiation are miR-21 and let-7. Studies have shown that let-7 affects RAS as its downstream molecule (140), whereas the downstream target of mir-21 is PTEN (141), indicating the modulation of crucial cellular signaling cascades by these microRNAs in response to radiation stress.

In fact, such RNA-protein interactions directly link chromatin and RNA responses to the outcome to alkylation and IR-induced stress. Conserved from yeast to humans and essential for heterochromatin formation, the vigilin RNA-binding protein with 14 tandemly arranged nonidentical hnRNP K-type homology (KH) domains modulates cell sensitivity to cisplatin- or IR-induced cell death and genomic instability due to defective DNA repair (142). Vigilin connects RNA and DNA responses, as it interacts with the DDR proteins RAD51 and BRCA1, and promotes their recruitment to DSB sites (142). These reports document the response of cancer cells to radiation stress through a multipronged approach that involves different epigenetic regulatory mechanisms.

Adaptation to nutrient deprivation.

Each and every cell modulates their metabolic processes depending upon the extracellular environment and nutrient availability. The intermediates of cellular metabolic processes act as cofactor or substrate of most of the enzymes associated with chromatin modification. Thus, cellular metabolism acts as a link between extracellular environment and chromatin dynamics, regulating genomic accessibility of the transcriptional machinery (143). Fluctuations between the epigenome and metabolism alter the normal physiological balance that ultimately causes complex diseases, such as type II diabetes and cancer.

(i) Epigenetic modifications in response to nutrient deprivation.

In the late 1950s, the discovery of glycolytic enzymes in the nucleus opened a new era of metabolism research. Since then, several studies have documented a fuel-sensing machinery within the cell that regulates the expression of several metabolic enzymes to maintain the metabolome at optimum levels. Nuclear localization of various metabolic enzymes during nutrient stress regulates the activity of different epigenetic modifiers, which ultimately lead to alteration of target gene expression (Fig. 1, lower right panel). Nuclear translocation of these metabolic enzymes depends upon extracellular stimuli and cellular nutrient availability. For instance, upon altered lactate metabolism, phosphorylation of tyrosine 238 of lactate dehydrogenase (LDH) promotes its nuclear import. Within the nucleus, LDH regulates epigenetic modifications by modulating the NAD⁺ level and activating the SIRT1 deacetylase system (144, 145). Similarly, under low-glucose conditions, hexokinase II (HKII), one of the principal enzymes in glycolysis, becomes phosphorylated by Akt, which promotes translocation to the nuclear compartment (146, 147). A related study in yeast cells showed that a homolog of HKII, HXKII, binds to the transcription factor Mig1 and suppresses the transcription of carbohydrate metabolic enzymes by forming a complex with cyc8 and Tup1 (148). Also, under low-glucose availability, HXKII autophosphorylates and activates Snf1, which relieves repression at the GAL4 promoter by the Mig1 complex and promotes the activity of other transcription factors, such as Adr1 (148). A low glucose level also leads to the nuclear localization of fructose-1,6-bisphosphatase 1 (FBP1), which binds to HIF-1 and HIF-2, thus suppressing transcription of glycolytic enzymes and cell proliferation-related genes (149). Nuclear import of malate dehydrogenase 1 (MDH1) during glucose deprivation promotes apoptosis and p53-mediated cell cycle arrest (150). At normal glucose levels, pyruvate kinase M2 (PKM2) interacts with HIF-1α, inducing the activation of target glycolytic enzyme genes by promoting HIF-1 and p300 binding to their promoters (151). This signaling cascade remains inhibited during glucose starvation. In yeast, Pyk1 (a homolog of PKM2), which is a part of serine-responsive SAM-containing metabolic enzyme complex (SESAME complex), regulates histone H3 phosphorylation depending upon glucose, SAM and serine amino acid levels (152).

(ii) Epigenetic regulation of metabolic enzymes under nutrient stress.

During nutrient deprivation, different metabolic enzymes translocate into the nucleus, where they regulate the expression of different target genes by modulating various epigenetic modifiers. On one hand, acetyl coenzyme A (acetyl-CoA)-producing enzymes such as the pyruvate dehydrogenase complex (PDC), acyl-coenzyme A synthetase short-chain family member 2 (ACSS2), and ATP citrate lyase (ACLY), translocate into the nucleus, increasing the acetyl-CoA pool, which is used by histone acetyltransferases (HATs) for the activation of different target genes (153–155) (Fig. 1, lower right panel). On the other hand, nuclear PDC can form a complex with pyruvate kinase isomerase M2 (PKM2) and p300. This complex then produces acetyl-CoA from phosphoenolpyruvate (PEP), resulting in local increase of acetyl-CoA and histone acetylation by p300 (156). S-Adenosylmethionine (SAM) acts as a methyl group donor that is utilized by different methyltransferases to methylate DNA and histones (157). It is also reported that an increase in SAM pool inside the nucleus inactivates specific target genes (158).

(iii) Epigenetic adaptation to changing nutrient levels.

In cancer cells, epigenetic alterations aimed at reprogramming their metabolic pathways play a crucial role in the response to altered nutrient availability in the tumor milieu. Small molecule metabolites, such as succinate, fumarate, d,l-2-hydroxyglutarate, and β-hydroxybutyrate, inhibit different metabolic enzymes and contribute to tumorigenesis and are therefore considered “oncometabolites” (159). Depending upon the metabolic status of the cell, β-hydroxybutyrate can modulate target gene expression via hyperacetylation of H3K9 and H3K14 (160) and the inhibition of HDACs, thereby regulating cellular growth and survival (161–163).

Activation of glycolysis is part of the program used by cancer cells to support their uncontrolled proliferation. For example, several groups reported a glycolytic flux in hepatocellular carcinoma and glioblastoma by hyperacetylation of the HKII promoter (164–166). Overexpression of lysine demethylase 3A (KDM3A) in bladder cancer promotes glycolysis by activating SLC2A1 (increased GLUT1 expression), phosphoglycerate kinase 1 (PGK1), HKII, lactate dehydrogenase A (LDHA), via demethylation of histone H3K9me2 (167). It was also reported that overexpression of glucose transporter GLUT1 by hypermethylation-mediated silencing of derlin-3 results in elevated glucose uptake to sustain the energy requirement of proliferating cancer cells (168). Alternatively, suppression of gluconeogenesis by hypermethylation of fructose 1,6-biphosphatase (FBP1) was described in liver and colon cancer (169). The epigenetic regulator TCF19, a histone H3K4me3-interacting protein (170), suppresses gluconeogenesis through its selective association with the NuRD complex under high-glucose conditions (171). Interestingly, in hepatocellular carcinoma, TCF19 associates with p53 to form distinct transcriptional complexes that regulate mitochondrial energy metabolism and promote cellular stress adaptation in glucose-mediated stress scenarios (172). It has also been reported that different sirtuins play extensive roles in metabolic reprogramming in cancer cells (Fig. 2). On one hand, SIRT6 causes glycolytic gene activation by augmenting H3K9 acetylation (173, 174) and, on the other hand, SIRT1 suppresses glycolysis by deacetylating HIF-1α and phosphoglycerate mutase 1 (PGAM1), thereby acting as a tumor suppressor (175). A role for SIRT1 as tumor suppressor was also supported by our analysis of TCGA, which showed that SIRT1 mRNA levels were lower in the tumor mass than in matched controls in 13 of 15 tumor types (Fig. 2A). In addition, in 3 of the 33 tumor types comprising TCGA, patients expressing SIRT1 at the lowest levels incurred greater risk of a poor outcome than those with higher mRNA levels of SIRT1 (Fig. 2B). The enzyme pyruvate kinase M2 phosphorylates histone H3 using PEP as the phosphate donor and promotes its acetylation upon stimulation by EGF (176). These interactions increase the expression of cyclin D1 and c-Myc, promoting cell proliferation and cancer progression. All of these reports highlight the altered epigenetic cascades employed by cancer cells to adapt to the steeply reduced nutrient availability in the tumor milieu to achieve cell survival.

Endoplasmic reticulum stress and cancer.

The synthesis of about one-third of all proteins of a eukaryotic cell takes place at the endoplasmic reticulum (ER). In addition to modifying the proteins to target their destination, ER regulates folding and protein quality control. The process of protein folding is highly sensitive to the concentration of calcium ions and the metabolic status of a cell. Protein folding is also influenced by various stimuli, including hypoxia and oxidative stress (177, 178). A disruption in the internal molecular balance of a cell leads to a build-up of incorrectly folded proteins within the ER lumen, altering ER homeostasis and causing ER stress. The elevated burden of misfolded proteins disrupts the balanced interplay between different transcription factors and chromatin dynamics, thereby challenging cellular homeostasis and increasing susceptibility to diabetes, neurodegenerative disorders, heart diseases, inflammatory disorders, and cancer. Misfolded proteins can furthermore impact ROS levels and cell death, as seen for superoxide dismutase mutations that increase folded stability, promote aggregation, and cause the fatal disease amyotrophic lateral sclerosis (179).

(i) Cascade of unfolded protein response.

Upon entering the ER lumen, polypeptide chains are folded into a proper structure by chaperons and modified by different oxidoreductases and glycosylating enzymes (180, 181). Approximately one-third of the polypeptides in the ER lumen fail ER quality control and are removed by the ER-associated degradation (ERAD) machinery (182). During ER stress, the excessive load of misfolded proteins overwhelms the ERAD capability to neutralize the burden and activate the unfolded protein response (UPR), an orchestra of different signaling pathways that rebalance homeostasis (183, 184) (Fig. 1, lower middle panel). Chaperone-binding immunoglobulin protein (BiP) is dissociated from the luminal side of three ER transmembrane proteins: protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1α (IRE1α). This leads to the activation of UPR (185–187). BiP dissociation causes dimerization and autophosphorylation of IRE1α and PERK (188) and coat protein II (COPII)-mediated transport of ATF6 to the Golgi apparatus (189, 190), leading either to cell survival or cell death. If ER stress is excessive, activated IRE1α degrades mRNAs through RNase-like activities, reducing the unfolded protein load by IRE1-dependent decay (RIDD) pathway (191–193), whereas at low ER stress IRE1α triggers the production of spliced X-box binding protein 1 (XBP1s) (194). Moreover, activation of JUN N-terminal kinase (JNK) by IRE1α causes apoptosis by the activation of BAX and BAK (195). XBP1s, in turn, enhances transcription of genes related to protein folding, quality control, ERAD, and phospholipid synthesis (196, 197). In contrast, activated PERK phosphorylates eukaryotic translation initiation factor 2α (eIF2α) and suppresses protein synthesis (198). PERK also induces translation of activating transcription factor 4 (ATF4), which supports transcription of the antioxidant response, autophagy, and apoptosis-related genes (199). ATF4 also stimulates the expression of C/EBP-homologous protein (CHOP), which in turn activates growth arrest and DNA damage-inducible protein 34 (GADD34) and BCL-2 homology 3 (BH3)-only proteins and suppresses BCL-2 (200).

Mahameed et al. indicated the possibility of inducing ER stress as a treatment stratagem against cancer (201). This study has demonstrated the efficacy of a combinatorial approach, including promoting selective retention in the ER (sER) and inhibiting integrated stress response (ISR) as an anticancer regimen. The dual treatment of Nelfinavir (inducer of sER) and ISRIB (inhibitor of ISR) efficiently attenuated the growth of several cell lines, including HepG2, Mel526, HCC827, Mel624, etc. In essence, combinatorial treatment of stress induction, along with chemotherapy, hinders the acquisition of chemoresistance and increases the cellular uptake of chemotherapeutic drugs, leading to an overall reduction of tumors.

(ii) Epigenetic alternations following ER stress.

UPR modulates a magnitude of transcription factors and epigenetic modifiers, ultimately regulating the expression of various target genes. For instance, NF-Y, a transcriptional activator, binds to the ERSE box of ER stress-related gene promoters and recruits ATF6, XBP1s, and other coactivators upon specific epigenetic activation marks, such as H3K4me3 (196, 202). Under normal conditions, heterochromatin protein-like 2 (HPL2) promotes transcriptional suppression of XBP1 by facilitating heterochromatin formation, whereas ER stress inactivates HPL2, thereby activating XBP1 (203). YY1 was reported to interact with ATF6, arginine methyl transferase 1 (PRMT1), and p300 under ER stress conditions (204). Under normal conditions, HDAC1 represses the transcription of UPR genes, while the SAGA-associated factor 29 (SGF29) binds to the histone H3K4me3 mark to maintain these genes in an inactive state. Under ER stress, SGF29 promotes histone H3K14 acetylation at UPR gene promoters (205). NF-Y successively recruits ATF6, YY1, and TBP, which in turn leads to the recruitment of PRMT1 and p300. PRMT1 and p300 methylate and acetylate histone H4, respectively, causing transcriptional activation of UPR-responsive genes (204).

(iii) Epigenetic regulation of ER stress and its connection to cancer.

Mutation, nutrient deprivation, and ATP deficiency within cancer cells increase the number of incorrectly folded proteins within the ER lumen, leading to ER stress. During glucose deficiency, the PERK pathway activates several proangiogenic factors, such as VEGF, FGF2, and IL-6, and suppresses expression of angiogenic inhibitor proteins, including THBS1, CXCL14, and CXCL10 (206). Activated PERK also promotes miR-211 production, which in turn represses CHOP expression by binding to its nascent mRNAs during transcription and increases histone H3K27me3 mark (207). The transcription factor CHOP is a member of the C/EBP family and is reported as a tumor suppressor (208). CHOP is reported to be activated by UPR and induces apoptosis in cancer cells (209) and has also been reported to promote brain metastasis via miR-708 expression (192).

ER stress responsive factors are correlated with tumor progression (193–195). For example, it has been reported that, during hypoxia, HIF-1α forms a complex with XBP1s to prevent apoptosis (210). IRE1α degrades transcription repressor protein period1, thereby resulting in the expression of chemokine (C-X-C motif) ligand 3 (CXCL3), leading to tumor development (211). Ubiquitination of H2A prevents RNA polymerase II from transcribing the proapoptotic gene NOXA in cancer cells. However, the upregulation of ATF3 and ATF4 activates the expression of NOXA by disrupting H2A ubiquitination (212). These and other studies collectively point toward the association of ER stress with cancer development, survival, and metastasis.

UTILIZING STRESS IN THE THERAPEUTIC REGIMEN OF ANTICANCER THERAPY

As evidenced in discussion above, the maintenance of homeostasis and its adaptive responses to stress will largely determine whether the cancer cells can survive a stressful stimulus or will undergo apoptosis. With this in mind, the harnessing and combination of stress may provide powerful therapeutic opportunities. Most current anticancer therapeutic regimens utilize the induction of stress as an approach to target cancer cells. Chemotherapeutic regimens such as alkylating agents and intercalating agents induce genotoxic stress to target tumor cells. Indeed, the first implemented anticancer drugs were alkylating agents, which are still among the most frequently used chemotherapeutic drugs; however, we are still learning their mechanisms of action and impact on RNA, which may affect how they can be best combined with other types of stress (213). Intriguingly, some of the RNA modifications produced by alkylating agents are also elicited by physiological processes. Thus, alkylation altering epigenetic marks merits more investigation (214).

As a routine first-line treatment against most aggressive carcinomas, chemotherapeutic drugs are often combined with radiotherapy to maximize genotoxic damage. For instance, cotreatment with temozolomide and radiotherapy is a standardized procedure against glioblastoma (215), increasing the median survival of patients (216). Chemotherapeutic drugs and radiation can also increase ROS levels, inducing oxidative stress and subsequent DNA damage. Cancer cells inherently display heightened ROS levels and increased tolerance to ROS (217). When levels of ROS are further elevated in cancer cells, they disrupt redox homeostasis, overriding the defensive antioxidant system of cancer cells and resulting in cell death. The induction of programmed cancer cell death by strategically elevating the levels of ROS is the principal mechanism behind many anticancer agents. Radiation therapy and most chemotherapeutic agents lead to the induction of intracellular ROS levels and stimulation of apoptosis via both extrinsic and intrinsic pathways (218).

Targeted therapies such as PDGFR inhibitor, EGFR inhibitor, BRAF inhibitor, and several monoclonal antibodies stimulate apoptosis by inducing oxidative stress (216, 219–221). In Hodgkin’s lymphoma and brain cancers, procarbazine was reported to induce irreparable oxidative DNA damage (222). In breast cancer, bladder cancer, colon cancer, and acute lymphocytic leukemia, the routinely used chemotherapeutic agents doxorubicin and 5-fluorouracil have been reported to produce ROS via p53-dependent or -independent pathways (223, 224).

In general, we are increasingly finding that epigenetics and metabolomics are interwoven with DNA replication and repair in ways that determine adaptions to stress and cancer cell fate. Hence, important new targeted therapies are coming from molecular targets in the DDR. Building upon the success of inhibitors to the PARP1 poly(ADP-ribose) (PAR) polymerase, inhibitors to the PAR glyocohydrolase (PARG) needed to hydrolyze the “cloud” of PAR into mononucleotide ADP-ribose (ADPr) are being tested (225, 226). Furthermore, excessive or unhydrolyzed PAR depletes the key metabolite NAD⁺ and triggers cell death by parthanatos by the apoptosis-inducing factor (AIF) via an allosteric mechanism that provides an interesting molecular cancer target. AIF enables mitochondrial regulation during sustained changes in NAD⁺ levels in response to diet, aging, or chronic, disease-induced PARP-1 hyperactivation, complementing NAD⁺-driven transcriptional regulation of mitochondrial biogenesis (227). Separately and in combination with alkylating and cross-linking agents that target replicative stress, inhibitors to replication stress responses such as the replication stress kinase ATR are being examined in clinical trials, making the EXO5 nuclease responsible for ATR-mediated replication restart an attractive cancer target (228, 229).

Epigenetic changes can result in what appears to be the biggest block to replication (and thus to cancer cell proliferation), which is a collision with transcription. Indeed, the nucleotide excision repair (NER) complex, which provides resistance to alkylation therapy by repairing bulky DNA lesions, may have evolved first for transcription-coupled NER from evolutionary pressure to resolve arrested transcription blocks to DNA replication (230). These considerations suggest that epigenetic transcriptional changes may be key to replication collisions that could be manipulated to promote instability or cell death in oncogenesis.

Unfortunately, acquisition of resistance toward chemotherapy, radiation therapy, and targeted therapies such as PARP inhibitors pose a major challenge to the management of advanced cancers. This has led to development of complementary strategies to target cancer cells in a multipronged therapeutic approach; one example includes oncothermia, a method of regulated hyperthermia where the temperature in the extracellular tumor liquid is increased locally by applying deep heat. A recent study in colorectal cancer reported the upregulation of the proapoptotic markers Bax and Puma and the downregulation of antiapoptotic Xiap, Bcl-2, and Bcl-xl markers by oncothermia treatment (231). Another study in the hepatocellular carcinoma cell line HepG2 indicated that oncothermia leads to apoptosis by increasing the levels of caspase-3, −8, and −9 (232). In summary, the combinatorial treatment of oncothermic stress induction, along with chemotherapy, appears to hinder the acquisition of chemoresistance and to increase the cellular uptake of chemotherapeutic drugs, leading to an overall reduction of tumor mass (204).

Since the induction of stress can serve as a potential approach to efficaciously target cancer cells, the overall understanding of the epigenetic landscape involved in stress response can unveil a plethora of new possibilities in devising anticancer regimens. In this context, CRISPR/Cas9 technology may prove to be of unparallel potential to engineer epigenome in a way so that the cancer cells can be efficiently manipulated to succumb to stress-mediated apoptosis, ultimately leading to tumor regression (233, 234). Combinatorial strategies, including the induction of stress and the modulation of epigenome to attenuate stress resistance, can act as a dual strategy which can prove to be a potential highlight in the future avenue of anticancer research.

Overall, stress offers opportunities for amplification of cancer cell killing by unbalancing DDR. This stress-DDR rebalancing can result both in increased damage and in combined transcriptional and replicational stress that can particularly target cancer cells, as they have intrinsic elements of transcription and replication stress. This is seen, for example, with ROS-causing radiation therapy, hyperthermia, metabolite NAD⁺ decreases during excess PARylation, and epigenetic RNA alterations in alkylation therapy. However, this approach will require a mechanistic understanding of the interplay between stress and DDR. For example, DNA damage in germ cells evokes elevated resistance to heat and oxidative stress by induction of inflammation and innate immune response. This can lead to activation of the ubiquitin-proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance (235). Yet, with the mechanistic knowledge of stress response networks, we can begin to better predict therapeutic effects and the prognosis of cancer patients by monitoring the related stress pathways, aiming for more effective and individualized treatment to improve the therapeutic efficacy of current therapies and their combination with targeted stress, such as oncothermia. Going forward, we expect to see more strategic applications harnessing the stress responses outlined here in order to overcome resistance toward chemotherapy, radiation therapy, and targeted molecular therapies.