Nucleotide Metabolism and DNA Repair: Implications in Cancer Treatments

Abstract

Cancer cells have many derailed processes due to which they have a higher proliferative capacity. The rewiring is continuously taking place to meet their metabolic demands. The demands depend on the stage of cancer, and these differences create challenges in curing them. Nucleotide metabolism plays a pivotal role in shaping cancer fate. DNA repair and other damage pathways also play a key role in cancer progression, genomic instability, errors in genetic material etc. These are discussed in this mini review so that researchers can take the lead to make an effort to combat cancer and design new therapeutics.

1. Introduction

The cancer cells have enormous proliferative capacity. They undergo a variety of rewiring and reprogram themselves to adapt to changes in the microenvironment around them to meet their metabolic needs [1,2,3]. The cancer hallmarks are characterized by dysregulated glucose and amino acid uptake, acquisition of metabolites and nutrients using multiple ways, exploitation of metabolic intermediates, nicotinamide adduct synthesis, upregulation of anti-apoptotic genes, downregulation of pro-apoptotic genes and extensive interaction with their tumor micro-environment [4,5]. The various types of cancer have multiple differences on the basis of metabolic demands; this difference depends on cancer stage and involves dynamic shifts in the metabolic pathways. Because of these differences, the targeted anti-cancer therapy is becoming challenging [6]. Nucleotide metabolism is also a role player in shaping cancer fate since de novo and salvage pathways behave differently in cancer [7]. Nucleotide metabolism is not only essential for DNA replication but also critically regulates DNA repair capacity, pathway choice, and genome stability. Cancer cells frequently reprogram nucleotide biosynthesis to sustain proliferation and cope with DNA damage, making this excess therapeutically important. Nucleotide metabolism enzymes physically interact with the repair protein [8]. Apart from the evident role of cell metabolic pathways in cancer, DNA-damage and repair pathways also play a key role in deciding cancer fate because changes in these pathways can lead to genomic instability, enormous DNA errors, mutations and intra-tumor heterogeneity [9,10,11,12].

2. Dysregulation of Metabolic Pathways in Cancer Cells

DNA damage or repair is regulated by a pool of nucleotides which are building blocks for DNA replication and repair [13,14]. Various metabolic pathways are involved in de novo nucleotide synthesis, and they impact the intracellular nucleotide pool [13,15,16]. The ribose backbone for nucleotides comes from ribose- 5-phosphate (R-5-P), a pentose-phosphate pathway (PPP) intermediate [13]. The PPP intermediates are generated from glucose-6-phosphate (G6P), an intermediate of glycolysis. The PPP intermediates are important for Nicotinamide adenine dinucleotide phosphate (NADPH), nucleotide and protein synthesis. The importance of PPP is huge in cancer metabolism [17,18]. The cancer cells show the Warburg effect, which is observed to adapt their metabolism to meet high energy demand, biosynthetic products for cell growth and division, and ultimately increased glucose consumption regardless of oxygen availability. This increased glucose consumption is also reported to fuel PPPs in multiple cancers, leading to the production of NADPH and nucleotide precursors [14]. A few other metabolic pathways are involved in purine and pyrimidine ring synthesis. Glutamine’s amide group is essential for inosine monophosphate synthesis required for de novo synthesis and required in uridine monophosphate synthesis in de novo pyrimidine synthesis [19,20]. In normal cells the alpha-ketoglutarate is converted into glutamate and finally to glutamine. Excess glutamate (a neurotransmitter) is released by neurons and gets converted into non-toxic glutamine using the enzyme Glutamine Synthetase (GS). While in glioblastoma, a brain cancer subtypes the reversal occurs where glutamine is converted into glutamate and finally to alpha-ketoglutarate to feed the TCA cycle (energy production) and provide nitrogen for nucleotide synthesis. Glutamates cause excitotoxicity which kills normal cells and makes space for tumors to grow [21,22]. Figure 1 explains the differentiation between normal and cancer cells. Likewise, aspartate is vital for the synthesis of pyrimidines, and glycine is important for purine synthesis. The aspartate is used to build pyrimidines, the “building blocks” of DNA/RNA like cytosine and thymine by the CAD enzyme complex in tumor cells [20]. In normal cells argininosuccinate synthase (ASS1) is highly expressed. Aspartate is used to produce argininosuccinate, fueling the urea cycle and maintaining arginine levels. This prevents a large buildup of aspartate in the cell. While in cancer cells argininosuccinate synthase is silenced or mutated which leads to lower arginine synthesis and aspartate buildup [15]. It is worth mentioning that the availability of amino acids and metabolic substrates plays a key role in influencing the amount and nucleotide production ratio in a cancer cell and may provide a hint at the mechanism for DNA repair.

Normal cells are attributed to metabolizing glucose to pyruvate, then oxidation to carbon dioxide via the TCA cycle, and finally the oxidative phosphorylation process in the mitochondria in the presence of oxygen. In the absence of oxygen, pyruvate is converted to lactate. On the other hand, cancer cells convert most of the glucose to lactate despite oxygen availability (Warburg effect). The lactate excess favors tumor progression (Figure 1). Glutamate from glutaminolysis is a major substrate for TCA refueling. Citrate-derived acetyl-CoA is used for lipid production. This synchronous lipid and nucleic acid synthesis favors cancer cell proliferation. Metabolism of nucleotides has a key role in carcinogenesis and cancer advancement. Researchers have targeted nucleotide metabolism for treating cancer [23,24,25]. The nucleoside analogs like 5-FU and gemcitabine can hinder nucleotide metabolism and play a key role in chemotherapy for the last many decades [26]. Plenty of cancer drugs are nucleotide analogs. These mimic natural nucleotides to inhibit DNA/RNA synthesis or cause “thymineless death” by inducing massive DNA damage.

3. DNA Repair Pathways and Their Relationship to Tumor Therapies

The cancer cells show a remarkable shift in metabolic programming in which the DNA damage response pathway’s activation is key to mention. This ultimately leads to nucleotide synthesis and glucose metabolism directed towards anabolic metabolite production [27]. The hyperactive DNA damage response pathways in cancer cells promote rapid growth and survival. The DNA damage response consists of multiple DNA repair pathways, each unique to a specific type of DNA damage [4]. During the repair pathway initiation and progression, there is enzymatic protein movement towards DNA damage sites and later chromatin remodeling [28,29]. DNA damage responses occur in response to double-stranded DNA breaks and ROS (reactive oxygen species)-mediated oxidative stress arising from exogenous sources and cellular metabolism, respectively [30]. The exogenous sources are UV light exposure, oxidative damage, and ionizing radiation [31,32,33]. Genome integrity maintenance is under tight regulation by DNA repair processes, including Base Excision Repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end-joining (NHEJ), and homology-directed repair (HDR) [34,35,36]. The disruption of the DNA damage response process fuels the cancer cell progression [5]. DNA repair proteins are essential for the survival and genomic stability of normal cells. However, the dysfunction or deficiency of these proteins prevents the accurate repair of DNA strand damage, allowing for the accumulation of mutations that ultimately drive the transformation of normal cells into cancer cells [6]. The layout of different DNA damage and repair system is given in Figure 2.

The disruption of homology-directed repair (HDR) induces a cascade of complex molecular changes that drive genomic instability and tumor progression. By bypassing the traditional DNA damage “roadblock” that triggers apoptosis in normal cells, cancer cells survive and adapt to persistent mutations. This metabolic and structural rewiring creates significant heterogeneity, ultimately dictating the divergent clinical responses seen in radiotherapy and chemotherapy. However, DNA damage is also induced by chemo- and radiotherapy in cancer cells. Use of multiple therapies like radiotherapy, topoisomerase inhibitors, for example, doxorubicin, and Poly(ADP-ribose) polymerases (PARP) inhibitors all induce DNA double breaks (DSBs) [37]. Any upheaval in DSB repair pathways can influence tumor response towards the above-mentioned therapies. It was reported that decreased expression or mutation in BRCA1 and 2 proteins may lead to HDR defects of DNA DSBs and sensitize tumor cells to PARP inhibitors and radiotherapy, inducing lesions [38]. On the other hand, upregulation of proteins involved in the non-homologous end joining pathway (NHEJ) can lead to resistance to therapies that induce double-strand breaks (DSB). This is because tumor cells can repair DNA damage and thereby avoid cell death [38]. The base excision removal system has the capacity to remove damaged bases from DNA. Anti-tumor agents such as temozolomide and floxuridine induce DNA lesions of N7mG, all of which can be repaired through the BER pathway [38]. Upregulation of BER pathway proteins can make cancer cells resistant to treatment, whereas downregulation can make cancer cells sensitive. Many inhibitors for the BER pathway are under development [34]. Mismatched DNA bases are recognized by MMR proteins, which recognize, excise and replace the mismatched bases with correct base pairing. Mlh1 and Mlh2 genes from the MMR gene family if mutated lead to the human colon cancer-prone syndrome, Lynch Syndrome. In earlier decades these mutation-based colon cancers were targeted by methotrexate, leading to oxidative damage. However, increased somatic mutations in MMR-deficient tumors can stimulate the immune system. In such cases, immune therapy has become a promising strategy for targeting MMR-deficient tumors [35]. Damaged nucleotides like pyrimidine dimers, adducts, and intrastrand cross-links are recognized by proteins of the nucleotide excision repair pathway. Alkylating agents like cisplatin can treat cancer by inducing intrastrand cross-links within the DNA, activating the NER pathway [36]. Exogenous or endogenous insults cause strand breaks, base modification, and mutation and activate specific repair pathways corresponding to the types of DNA damage [39]. Faults in the repair pathway can lead to cancer progression.

Hence, it is a known fact that changes in DNA repair pathways contribute to the cancer phenotype of cells and cause resistance to tumor therapy. On the other hand, there is huge potential for next-generation targets also to treat cancer based on targeting DNA damage repair pathways. It can turn out to be advantageous.

4. Inhibitor Development for the DNA Repair Pathway in Cancer Therapeutics

Cancer cells devise multiple metabolic reprogramming to work against cancer treatments. The role of synthetic lethality can improve cancer treatment. DDR inhibitors are one of the important synthetic lethal agents that can help in cancer cell death by inducing specific mechanisms blocking DNA repair pathways by targeting DNA repair pathway genes (Figure 3). DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a serine/threonine kinase that aids in non-homologous end joining (NHEJ) [40]. It gets self-auto-phosphorylated, which leads to a conformational shift allowing end processing enzymes to reach double-strand break ends [41]. DNA-PKcs works in combination with ATR (ataxia telangiectasia mutated and Rad3-related) and ATM (ataxia-telangiectasia mutated) to activate phosphorylation of proteins involved in cellular DNA damage response. Compound targeting the AP-binding site of these kinase domains can become the most influential candidates in inhibiting DNA-PK proteins [42]. This inhibition reduces phosphorylation of cGAS and impedes the kinase activity of DNA-Pkcs. The use of inhibitors can also sensitize cancer cells to damaging agents [43,44].

Another important class of DNA damage repair pathways’ target protein is PARP (poly ADP-ribose polymerase) (Figure 4). PARP has a role in DNA repair, maintains genomic integrity, and regulates cell death [45]. The Base Excision Repair (BER) pathway and single-strand break (SSB) repair also depend on PARP [46]. Two types of PARP, namely PARP1 and 2, are essential for cell survival. NAD+ is used as a substrate by them for PARPylation of proteins and releasing nicotinamide. These events help in the maintenance of the target protein’s activity, stability, and conformation. PARP-1 suppression leads to more fatal cancer as an important protein of homologous recombination (HR); BRCA1 and 2 are broken. DDX21 aids in ribosome production, and PARP1 is essential for DDX21 functionality [47]. So, a PARP inhibitor can suppress the progression of cancer. Various inhibitors like Olaparib, veliparib, rucaparib, niraparib, and talazoparib are being used in the medical oncology field for cancer treatment [48,49,50]. Multidimensional approaches like immunotherapy and PARP inhibition can target the immune system to treat cancers like ovarian, lung, gastrointestinal, and prostate cancers [51,52]. Overall response rate (ORR) in ovarian cancer patients by using PARP inhibitors increased [44].

Another important class of proteins is CHK1 (Checkpoint Kinase 1) which is a serine/threonine-specific protein kinase. It has an important role in DNA damage response, DNA damage repair and cell cycle regulation (Figure 5). CHK1 is phosphorylated in an ATR-mediated fashion; this event delays cell cycle progression and pushes the cell into the repair process. Hence, CHK1 is a cell cycle checkpoint that improves survival rate and increases cancer cell resistance [53]. Regulation of CHK1 can be exploited as an anticancer target in cancer treatment. CHK1 inhibitors prevent the restart of stalled replication forks and lead to cancer cell death. This inhibition also sensitizes cancer cells to drugs and induces replication stress in them. Multiple reports have cited the use of single-agent use of CHK1 inhibitors with other drugs also to inhibit tumor growth [53]. Two generations of CHK1 inhibitors are there but the second is improved over the first one which has high toxicity. LY2880070 and SRA737 inhibitors are under study. These are being tried for use in combination with antimetabolites, damaging agents [5]. Chk1 inhibition may be effective against p53-deficient cancer cells compared to p53-proficient cancer cells [54,55].

5. Challenges and Perspective

There is potential in targeting the DNA repair pathway protein against cancer. There is a long way to go to reach that stage since most of the inhibitors cannot get into a clinical setup to be prescribed to patients with a lower amount of toxicity and high selectivity against cancers. Many of the regulatory proteins in DDR are scaffold proteins having a key role in transducing signals and no enzymatic activity. This is a complicated situation where inhibitors against enzymatic activity outweigh this. There is a dire need for alternatives for the unmarketable scaffold protein of DDR pathways. PROTACs (PROteolysis-TArgeting Chimeras) are compounds that induce selective degradation of target proteins via the ubiquitin-proteasome system, making them particularly effective for targeting non-enzymatic or “undruggable” scaffold proteins. C-MYC, BET, androgen receptors, and BRD7 have been targeted by PROTACs to kill cancer cells [55,56]. CRISPR/Cas9-based selecting scissors which can cut/edit pre-cancerous genes are another potential way to deal with cancer [57]. Chimeric Antigen Receptor T-cells, or CAR-T cells have recently been in the limelight where the CRISPR technique is used to engineer T cells from patients and injected back into them where they can better kill cancer cells. There are many FDA-approved CAR-T therapies being used like ABECMA^® (idecabtagene vicleucel), BREYANZI^® (lisocabtagene maraleucel), CARVYKTITM (ciltacabtagene autoleucel), KYMRIAHTM (tisagenlecleucel), TECARTUSTM (brexucabtagene autoleucel), and YESCARTATM (axicabtagene ciloleucel). These are being registered with the UPMC Hillman Cancer Center. However, these are in infancy and need more solid outcomes with fewer toxic effects on patients suffering from aggressive cancer. The new technologies are costly for the common masses, and their clinical evaluation is under scrutiny.

6. Conclusions

Cancer has been described as a genetic disease driven by multiple mutations in the genome of cells leading to cancer cell progression [58]. In recent decades it has been attributed that metabolic reprogramming is a hallmark of cancer and often it has been regarded as a metabolic disease too [59,60], where tumor and microenvironment interaction plays an important role in defining the progression of cancer. It is important to study whether mutation overload is from selective pressures by extrinsic metabolic factors in the tumor microenvironment. There are cases where extrinsic factors like the availability of essential nutrients may resist or progress cancer. For example, low glucose, hypoxia, and acidic pH in the microenvironment modulate plasticity in cancer cells and survival [61]. It is also possible that mutations in DNA repair genes may arise due to extrinsic environmental stress conditions where genomic instability goes in favor of cancer cells. It is evident that heterogeneity in tumors is directly proportional to anti-cancer therapy resistance. Such tumors behave more aggressively than tumors arising from single clonal populations [62]. There is an importance of metabolic factors including nucleotides where they are responsible for the dynamic interplay between them and the microenvironment. These factors influence the treatment of aggressive cancer. The changes in genetic material, i.e., chromatin, DNA repair genes’ translational products and mutations allow cancer cells to diversify and have genetic heterogeneity which promotes cancer evolution and metastasis. As discussed above, the metabolic changes turn in favor of cancer cells leading to their pronounced growth rate and less reliance on oxygen for metabolic pathways from glycolysis to oxidative phosphorylation for complete oxidation of glucose. DNA damage and metabolic changes go hand in hand, and both regulate each other too. This creates a regulatory loop that helps in compensating for one pathway to another in odd conditions. The role of metabolic processes and DNA damage repair in aging declines in normal cells and studying these aspects can help us to understand what goes opposite in cancer cells and further targets for anti-cancer campaigns can be brought to the limelight. The multiple anti-cancer approaches will yield productive results in curing cancer.