Cancer metabolism challenges genomic instability and clonal evolution as therapeutic targets

Abstract Although cancer precision medicine has improved diagnosis and therapy, refractory cancers such as pancreatic cancer remain to be challenging targets. Clinical sequencing has identified the significant alterations in driver genes and traced their clonal evolutions. Recent studies indicated that the tumor microenvironment elicits alterations in cancer metabolism, although its involvement in the cause and development of genomic alterations has not been established. Genomic abnormalities can contribute to the survival of selected subpopulations, recently recognized as clonal evolution, and dysfunction can lead to DNA mutations. Here, we present the most recent studies on the mechanisms of cancer metabolism involved in the maintenance of genomic stability to update current understanding of such processes. Sirtuins, which are NAD+‐dependent protein deacetylases, appear to be involved in the control of genomic stability. Alterations of deleterious subpopulations would be exposed to selective pressure for cell survival. Recent studies indicated that a new type of cell death, ferroptosis, determines the survival of clones and exert cancer‐restricting or ‐promoting effects to surrounding cells in the tumor microenvironment. Suppressing genomic instability and eliminating deleterious clones by cell death will contribute to the improvement of cancer medicine. Furthermore, the elucidation of the mechanisms involved is seen as a bridgehead to the pharmacologic suppression of such refractory cancers as pancreatic cancer.

tions in cancer metabolism, although its involvement in the cause and development of genomic alterations has not been established. Genomic abnormalities can contribute to the survival of selected subpopulations, recently recognized as clonal evolution, and dysfunction can lead to DNA mutations. Here, we present the most recent studies on the mechanisms of cancer metabolism involved in the maintenance of genomic stability to update current understanding of such processes. Sirtuins, which are NAD+-dependent protein deacetylases, appear to be involved in the control of genomic stability. Alterations of deleterious subpopulations would be exposed to selective pressure for cell survival. Recent studies indicated that a new type of cell death, ferroptosis, determines the survival of clones and exert cancer-restricting or -promoting effects to surrounding cells in the tumor microenvironment. Suppressing genomic instability and eliminating deleterious clones by cell death will contribute to the improvement of cancer medicine. Furthermore, the elucidation of the mechanisms involved is seen as a bridgehead to the pharmacologic suppression of such refractory cancers as pancreatic cancer.

K E Y W O R D S
ferroptosis, metabolism, oxidative stress, pancreatic cancer, sirtuins

| INTRODUC TI ON
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies with a poor 5-year survival rate of 5% due to a poor early diagnosis rate and limited response to treatments, despite the findings of recent extensive studies that stratified patients according to treatment to discover subtype-specific therapies. 1 Clinical sequencing has elucidated three subtypes of PDAC, namely classical, quasi-mesenchymal, and exocrine-like ones, which correspond to their varying responses to therapy. 2 Clinical sequencing has shown that mutations of the oncogene KRAS occur in more than 90% of PDACs. Other major mutations observed include the SMAD family in the TGFb pathway and tumor suppressor genes, including TP53 and P16INK4A. 3 Recently, a study of metabolism in PDACs demonstrated that PDAC cells rely on the distinctive pathway in which glutamine supports PDAC growth through a KRAS-regulated metabolic pathway. 4 Through this pathway, glutamine is converted to oxaloacetate by aspartate transaminase (GOT1), and oxaloacetate is converted further into malate and then pyruvate. The metabolic pathway is associated with an increased NADPH/NADP+ ratio that results in the maintenance of the cellular redox state. The KRAS pathway in PDAC is considered essential and indispensable, and the targeting oncogenes may provide novel therapeutic approaches to the treatment of PDACs. 4 However, few studies have elucidated the mechanisms of cancer metabolism alterations involved in genetic alterations, that is, whether they are a result of genomic instability, as is characteristic of malignant tumors.
Dysfunction in the maintenance of genomic stability will elicit the clonal evolution of cancers. The control of cancer metabolism, genomic instability, and cell death is expected to be useful in creating a new era of cancer medicines against refractory cancer cell clones, such as those of PDACs ( Figure 1).

| S IRTU IN S CONTROL G ENOMI C S TAB ILIT Y IN PDAC S
Although metabolism and DNA repair are considered different processes, and the point of contact of their mechanisms has not been clarified, recent studies have reported that several NAD +dependent signaling pathways regulate cell cycle progression and transcriptional regulation, as well as DNA repair, in response to genotoxic insult damages. NAD+-dependent protein deacetylases, such as sirtuins, are involved in DNA damage responses, such as repair and recombination, during replication. 5 The sirtuin 1 (SIRT1) complex is involved in homologous recombination (HR) repair by reducing nucleosome density at DNA damage sites. 6 The ATPase domain of brahma-related gene 1 (BRG1) and the zinc finger domain of SIRT1 interact with poly-ADP ribose in response to DNA damage and are responsible for repairing broken F I G U R E 1 Genomic and metabolic alterations in pancreatic ductal adenocarcinoma (PDAC) development. The schema represents the development of PDAC from pancreatic intraepithelial neoplasia (PanIN) 1, 2, and 3 in the precancer stages. Clinical sequencing results indicate the mutation frequencies of KRAS (99%), TP53 (85%), and SMAD4 (55%) and telomere shortening (91%), although numerous additional mutations occur during the metastatic process. 59 Tissue abnormality has been associated with hypoxia and the Warburg effect at early and advanced stages of pancreatic carcinogenesis, 60,61 but recent studies have shown that cancer metabolism alterations such as phosphatidylinositol-3 kinase (PI3K), AKT, mammalian target of rapamycin (mTOR), and sirtuins are supposed to be involved at least from PanIN 2 stages and can elicit oxidative stress and damage responses associated with genomic instability. 62,63 Such alterations may induce further mutations in advanced stages of PDAC. Antitumorigenic adaptive immune response may be involved in early stages of pancreatic carcinogenesis, whereas protumorigenic innate immune response may be involved in advanced stages of PDAC. 64 Oxidative stress controls the function of inflammatory cell types and mechanisms underlying genomic instability in PDAC. 65 As the effect of genomic instability, numerous neoantigens can be targets of immune cells 64,65 DNA ends. At the damage sites, SIRT1 deacetylates BRG1 at lysine residues 1029 and 1033, which stimulates ATPase activity to promote HR. 6 SIRT2 and SIRT3 are reportedly involved in HR by utilizing I-SceI endonuclease-based green fluorescent protein reporter assays, which makes the precise introduction of DNA double-strand breaks (DSBs) possible. This study indicates that SIRT2 or SIRT3 is involved in the recruitment of RAD51 to DSB sites, an essential step for RAD51-dependent HR repair, as well as in the colocalization of the H2A.X variant histone (γH2AX) foci with replication protein A1 (RPA1). 7 The deacetylase SIRT6 is reportedly involved in chromatin remodeling and DNA damage response. SIRT6 was found recruited to sites of UV-induced DNA damage and stimulated the repair of such damage by targeting damage-specific DNA-binding protein 2 (DDB2). 8 Moreover, SIRT6 is responsible for more efficient DNA DSB repair. A panel study on rodent species with diverse lifespans indicated that DSB repair and SIRT6 were optimized during the evolution of longevity, which provides new targets for antiaging interventions. 9 Furthermore, SIRT6 can coordinate with the chromatin remodeler chromodomain helicase DNA-binding protein 4 (CHD4) to promote chromatin relaxation in response to DNA damage. 10 DNA damage can induce the activation of ataxia-telangiectasia mutated (ATM) protein by TIP60 acetylation and autophosphorylation, which trigger the cascade of DNA damage response and repair, whereas SIRT7 is essential to the dephosphorylation and deactivation of ATM, suggesting that SIRT7 regulates ATM activity and DNA damage repair. 11 Given that DNA damage response defects are closely associated with genomic instability, an underlying hallmark of cancer, the appropriate functions of sirtuins are essential to the maintenance of DNA fidelity during replication and resultant genomic integrity.
In pancreatic cancer, SIRT1 regulates acinar-to-ductal metaplasia differentiation by deacetylating pancreatic transcription factor-1a and β-catenin, depending on its subcellular localization, and supports cancer cell viability, which can result in the sensitization of tumor cells to the SIRT1/2 inhibitor tenovin-6. Thus, SIRT1 may be an important regulator and potential therapeutic target in pancreatic carcinogenesis. 12 Moreover, SIRT3 is involved in the hypoxia-inducible factor 1 subunit alpha (HIF1α) pathway, which is regulated by the tumor suppressor profilin 1. The mechanism to the HIF1⍺ pathway is independent of its cytoskeleton-related activity, although profilin1 was originally identified as an actin-associated protein. 13 Furthermore, a study on SIRT3 and SIRT7 as biomarkers for determining patient outcome indicated tumor-suppressing properties in the context of pancreatic cancer. 14 In addition, a previous study demonstrated that the loss of SIRT6 resulted in histone hyperacetylation at the Lin-28 homolog B (LIN28B) promoter, MYC proto-oncogene recruitment, and pronounced induction of LIN28B and downstream let-7 target genes, high-mobility group AT-hook 2 (HMGA2), insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), and IGF2BP3. 15 The overall significance of SIRT1, SIRT3, SIRT6, and SIRT7 in cancers has indicated their use as therapeutic targets against pancreatic cancer 5 Nevertheless, the involvement of sirtuins in the control of genomic stability at exact molecular levels needs further elucidation, which would be important in the development of novel drugs ( Figure 2).

| LIPID -RE AC TIVE OX YG EN S PECIE S C AN INDUCE FERROP TOS IS IN PDAC CELL S
Recent studies have indicated that the inevitable production of reactive oxygen species (ROS) during unlimited proliferation, a characteristic of tumors, is involved in the biological process of cells such as insults to DNA, repair of induced damage, and induction of cell death. 16 Given that a hallmark of cancer is unregulated proliferation, 17 16 Previous studies demonstrated that unique features of ferroptosis were frequently attributed to other cell death pathways, such as necrosis, apoptosis, autophagy, and programmed cell death, but ferroptosis appears to play a central role in multiple pathologies. 16 These significant studies indicated that the following three factors can stimulate the ferroptotic pathway: (1) long-chain polyunsaturated fatty acids contained in phospholipid membranes; (2) redox-activated iron; and (3) defects in the lipid peroxide repair system. Certain molecular substances can also target important proteins and related metabolic pathways to trigger these processes, which are associated with cancer-acquired drug resistance and immune evasion. 20 Among those factors, the redox activation system is well characterized. 20 The catalytic subunit of system X C − has a critical role in dependent antioxidant function. A recent study indicated that the

| S IRTU IN S REG UL ATE FERROP TOS IS IN DAMAG ED CELL S
A recent study has described the significant role of SIRT1 in the induction of ferroptosis in liver damage in mice. 29

| FERROP TOS IS DE TERMINE S CLONE SURVIVAL IN TUMOR MICROENVIRONMENT
As mentioned previously, the oxidative stress-dependent apoptotic The surviving cells may expand, and dead cells will be eliminated from PDAC tissues, which will give rise to clonal evolution, as demonstrated by clinical sequencing analysis 44,45 suggesting the usefulness of a combination of immune checkpoint therapy. 54 In contrast, nicotinamide inhibits SIRT2 and SIRT6 55,56 and is considered beneficial in the prevention of breast cancer recurrence. 57 In the sirtuin-targeting strategy, family-specific reagents will be necessary to be innovated, given that the standard therapeutic approaches are still not effective enough. For resectable PDAC cases, surgery followed by adjuvant chemotherapy with gemcitabine plus capecitabine is the standard of care. For borderline resectable and locally advanced unresectable PDACs, neoadjuvant protocols are utilized, whereas for metastatic PDACs, FOLFIRINOX (fluorouracil, irinotecan, and oxaliplatin) and nabpaclitaxel-gemcitabine are standard treatment options in patients with good performance status. 58 We therefore emphasize that understanding the underlying mechanisms that allow sirtuins to show apparently two opposite anticancer roles should be one of our main challenges in developing effective PDAC treatment ( Figure 4).
Improving the treatment results of pancreatic cancer has been a longstanding goal in the oncological field. The genetic changes in pancreatic cancer are relatively simple, centered on KRAS drivers; however, their biological malignancy is extremely high. As analyzed in this paper, the metabolic characteristics of pancreatic cancer are closely related to genomic changes. Clarifying the mechanism for the selection and creation of dangerous clones is a major challenge in the medical treatment of pancreatic cancer. If the dangerous clones resulting from the oxidative stress response can be eradicated, current standard treatments can be combined to improve the outcome of pancreatic cancer.

ACK N OWLED G EM ENT
We thank Professor Koshi Mimori, Kyushu University Beppu Hospital, Japan, for critically reviewing this manuscript. The authors are thankful to all lab members.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable in this review article.