**5.2. The localization of p53 and other DNA repair factors in the mitochondria and the regulation of their gene expression**

Recent studies have shown that the p53 protein not only acts as a "guardian of the genome", but also serves as a regulator of metabolism [145, 146]. Moreover, p53 has been reported to accumulate in the mitochondria in response to stress [147]. Besides p53, a number of widely known DNA repair factors, including ATM, BRCA1, PARP, PARG, and RB, localize in the mitochondria or regulate their functions [148–152]. The surveillance of the 5′-upstream regions of these DNA repair factor-encoding genes revealed that they commonly possess duplicated GGAA-motifs [53, 78].

GGAA-motif duplications are found in the bidirectional *APEX1*/*OSGEP* promoter region. The *APEX1* encodes apurinic/apyrimidinic endonuclease 1 (APE1) that regulates both the base excision repair (BER) and the mitochondrial DNA repair systems [75, 153]. The GGAA-duplication is contained in the regulatory region of the head–head configured *ACO2/PHF5A* genes [54]. The *ACO2* gene encodes aconitase, which plays an important role in the TCA cycle to produce citrate and isocitrate, and which also serves as a mitochondrial redox-sensor [154]. Importantly, aconitase and mitochondrial BER enzyme OGG1 (8-oxoguanine DNA glycosylase) cooperatively preserve mitochondrial DNA integrity [155]. We also confirmed that the duplicated GGAA-motifs were present in the 5′-upstream regions of the genes associated with Fanconi's anemia (FA) [53], which encode the DNA repair factors that are shown to regulate nucleotide excision repair and genome stability [156]. Interestingly, it was shown that mitochondrial dysfunction forces FA cells to produce energy by glycolysis [157], suggesting that FA proteins might be involved in the metabolic switch system in cancer cells. Additionally, Cockayne syndrome proteins CSA and CSB, which play roles in nucleotide excision repair (NER), accumulate in the mitochondria under oxidative stress [158]. In KRAS/LKB1-mutant lung cancer cells, carbamoyl phosphate synthetase-1 (CPS1), which is localized in mitochondria and which eliminates NH4 to initiate the urea cycle, also plays a role in controlling the pyrimidine/purine balance to regulate the integrity of nuclear DNAs [159]. In this circumstance, the silencing of the *CPS1* gene expression leads to an incomplete S-phase or apoptotic cell death due to increased DNA damage. As expected, the duplicated GGAA is present in the *CPS1*/*LANCL1* bidirectional promoter region. However, no such element is found near the TSSs of either the *CAD* or *ASS1* genes, which encode cytoplasmic enzymes carbamoyl phosphate syntetase-2 and argininosuccinate synthase, respectively. These observations suggest that expression of the mitochondria-localizing, DNA repair-associated protein-encoding genes could be cooperatively regulated by duplicated GGAA-motif binding TFs, supporting the hypothesis that mitochondrial dysfunction causes oncogenesis [8].

#### **5.3. The communication between telomeres and mitochondria may depend on the NAD<sup>+</sup> / NADH ratio**

The telomeres and mitochondria are thought to communicate with each other [160]. Several nuclear DNA repair factors play roles in the maintenance of mtDNAs, and damaged mtDNAs in turn exert signals to regulate nuclear transcription [74]. The system by which DNA repair/ energy production is monitored might be mediated by the balance of the NAD+ /NADH ratio, which is regulated by a number of enzymes in the nuclei, mitochondria, and cytosol [161]. In breast cancer cells, the crosstalk between BRCA1 and PARP1 maintains the stability of the DNA repair ability, which would be partly sensitive to the NAD+ concentration [162].

**6. The involvement of nicotinamide adenine dinucleotide (NAD<sup>+</sup>**

which are required for chromatin remodeling [86]. Moreover, NAD+

mouse stem cells [166]. Conversely, decreased concentrations of NAD+

aging-related diseases [75]. These observations suggest that the NAD+

Several drugs that induce an increase in the intracellular NAD+

which upregulates mitochondrial biogenesis, drives NAD+

aggressiveness or metastasis [171]. An increase in the cellular level of NAD+

metabolism and the protection of chromosomal DNAs, has been discussed in detail [66, 67]. A recent study showed that nuclear PAR can be utilized by NUDIX5 to supply ATP molecules,

amide have been reported to ameliorate metabolism or the mitochondrial functions [163–165].

 **restricts the generation and development of cancer by supporting the** 

tribute to the establishment of novel therapeutics for treating age-related diseases, including cancer [168]. Mitochondrial dysfunction has been suggested to be associated with the development of tumors or cancerous cells [169, 170]. In breast cancer cells, the knockdown of the subunit NDUFV1 leads to an aberration in complex I, which was shown to enhance

ated with the improvement of the mitochondrial integrity to suppress oncogenesis. PGC-1α,

stress resistance [172]. A recent study showed that PGC-1α suppresses the metastasis of melanoma, acting on the transcription program, namely the PGC-1α-ID2-TCF-integrin axis [173]. A loss of CSB, which can localize in the mitochondria [158], activates PARP1 to synthesize PAR [174], suggesting that the dysregulation of the mitochondrial functions to regulate DNA

/NADH molecular ratio.

is required for the DNA damage response and the DNA repair system [66, 67]. The inhibition of the PARP1 enzyme ameliorates the mitochondrial metabolism through the activation of SIRT1 [175]. Conversely, the over-activation of the PARP1 enzyme can lead to mitochondrial dysfunction [176]. The *PARP1* gene expression was found to be negatively regulated when poly(ADP-ribose) glycohydrolase (PARG) siRNAs were introduced into HeLa S3 cells [40], suggesting that the degradation of the PAR macromolecule is required for the transcription of the *PARP1* gene. Because the 5′-upstream regions of both the human *PARP1* and *PARG* genes commonly contain the duplicated GGAA-motifs [53, 54], these two genes may be regulated

contributes to the DNA repair, but also to the fine-tuning of the transcription of genes that


**-dependent transcription of DNA repair factor-encoding genes**


metabolism-associated DNA repair factors.

with mitohormesis [167], and that nutrient sensing molecules may control aging [57].

**oncogenesis and the aging process**

The biological relevance of the NAD+

The repletion of NAD+

**mitochondrial functions**

repair system may reduce the NAD+

It is worth noting again that the NAD+

**6.1. NAD<sup>+</sup>**

**6.2. NAD<sup>+</sup>**

by an NAD+

encode the NAD+

the NAD+

**) in** 

117

and its precursor nicotin-

http://dx.doi.org/10.5772/intechopen.71095

could cause aging or

level may be correlated

level are expected to con-

biosynthesis and thereby induces

molecule is the substrate for the PARP enzyme, which

may be associ-

/NADH ratio not only

molecule, especially in relation to its pivotal roles in

improves the mitochondrial functions to prolong the life span of adult

A New Insight into the Development of Novel Anti-Cancer Drugs that Improve the Expression…

The mitochondria might have conveniently deposited their function-associated genes into the nuclei, but need to take care, especially when DNA damage occurs. However, high-dose or repeated DNA damage may eventually activate the PARP enzyme, which consumes NAD+ as a substrate for the synthesis of PAR, to initiate the DNA repair system [66]. The decrease in the NAD+ level will subsequently cause incomplete TCA cycle progression and the dysregulation of respiration/OXPHOS, accompanied by the reduced expression of the mitochondrial function-associated genes. At this stage, the "Warburg effect" can be observed (**Figure 1**).

**Figure 1.** Toward the establishment of a novel cancer therapy. Cellular NAD+ molecules will decrease in accordance with aging or increased levels of various types of stress, especially DNA damage, which activates the PARP enzyme, which synthesizes PAR to consume NAD+ as a substrate. Subsequently, alterations in the transcriptional profile might occur, leading to a reduction or the mismanagement of the mitochondrial functions. In these circumstances, energy producing mitochondrial respiratory systems will decline or be dysregulated, while glycolysis will be enhanced providing ATP molecules that allow cells to proliferate in an unrestricted manner. Novel cancer therapies should be based on the concept that they will never kill cancer cells; rather, they should force the cells to regain their normal respiratory systems, including the TCA cycle and OXPHOS. The recovery of these mitochondria might also lead to the restoration of the mitonuclear communication system. In order to establish a gene therapy, it is necessary to reveal the molecular mechanisms that control the transcription of the mitochondrial function-associated genes.

#### **6. The involvement of nicotinamide adenine dinucleotide (NAD<sup>+</sup> ) in oncogenesis and the aging process**

**5.3. The communication between telomeres and mitochondria may depend on the** 

energy production is monitored might be mediated by the balance of the NAD+

DNA repair ability, which would be partly sensitive to the NAD+

**Figure 1.** Toward the establishment of a novel cancer therapy. Cellular NAD+

mechanisms that control the transcription of the mitochondrial function-associated genes.

synthesizes PAR to consume NAD+

The telomeres and mitochondria are thought to communicate with each other [160]. Several nuclear DNA repair factors play roles in the maintenance of mtDNAs, and damaged mtDNAs in turn exert signals to regulate nuclear transcription [74]. The system by which DNA repair/

which is regulated by a number of enzymes in the nuclei, mitochondria, and cytosol [161]. In breast cancer cells, the crosstalk between BRCA1 and PARP1 maintains the stability of the

The mitochondria might have conveniently deposited their function-associated genes into the nuclei, but need to take care, especially when DNA damage occurs. However, high-dose or repeated DNA damage may eventually activate the PARP enzyme, which consumes NAD+

a substrate for the synthesis of PAR, to initiate the DNA repair system [66]. The decrease in

lation of respiration/OXPHOS, accompanied by the reduced expression of the mitochondrial function-associated genes. At this stage, the "Warburg effect" can be observed (**Figure 1**).

aging or increased levels of various types of stress, especially DNA damage, which activates the PARP enzyme, which

leading to a reduction or the mismanagement of the mitochondrial functions. In these circumstances, energy producing mitochondrial respiratory systems will decline or be dysregulated, while glycolysis will be enhanced providing ATP molecules that allow cells to proliferate in an unrestricted manner. Novel cancer therapies should be based on the concept that they will never kill cancer cells; rather, they should force the cells to regain their normal respiratory systems, including the TCA cycle and OXPHOS. The recovery of these mitochondria might also lead to the restoration of the mitonuclear communication system. In order to establish a gene therapy, it is necessary to reveal the molecular

as a substrate. Subsequently, alterations in the transcriptional profile might occur,

level will subsequently cause incomplete TCA cycle progression and the dysregu-

/NADH ratio,

as

concentration [162].

molecules will decrease in accordance with

**NAD<sup>+</sup>**

116 Mitochondrial Diseases

the NAD+

**/ NADH ratio**

The biological relevance of the NAD+ molecule, especially in relation to its pivotal roles in metabolism and the protection of chromosomal DNAs, has been discussed in detail [66, 67]. A recent study showed that nuclear PAR can be utilized by NUDIX5 to supply ATP molecules, which are required for chromatin remodeling [86]. Moreover, NAD+ and its precursor nicotinamide have been reported to ameliorate metabolism or the mitochondrial functions [163–165]. The repletion of NAD+ improves the mitochondrial functions to prolong the life span of adult mouse stem cells [166]. Conversely, decreased concentrations of NAD+ could cause aging or aging-related diseases [75]. These observations suggest that the NAD+ level may be correlated with mitohormesis [167], and that nutrient sensing molecules may control aging [57].

#### **6.1. NAD<sup>+</sup> restricts the generation and development of cancer by supporting the mitochondrial functions**

Several drugs that induce an increase in the intracellular NAD+ level are expected to contribute to the establishment of novel therapeutics for treating age-related diseases, including cancer [168]. Mitochondrial dysfunction has been suggested to be associated with the development of tumors or cancerous cells [169, 170]. In breast cancer cells, the knockdown of the subunit NDUFV1 leads to an aberration in complex I, which was shown to enhance aggressiveness or metastasis [171]. An increase in the cellular level of NAD+ may be associated with the improvement of the mitochondrial integrity to suppress oncogenesis. PGC-1α, which upregulates mitochondrial biogenesis, drives NAD+ biosynthesis and thereby induces stress resistance [172]. A recent study showed that PGC-1α suppresses the metastasis of melanoma, acting on the transcription program, namely the PGC-1α-ID2-TCF-integrin axis [173].

A loss of CSB, which can localize in the mitochondria [158], activates PARP1 to synthesize PAR [174], suggesting that the dysregulation of the mitochondrial functions to regulate DNA repair system may reduce the NAD+ /NADH molecular ratio.

#### **6.2. NAD<sup>+</sup> -dependent transcription of DNA repair factor-encoding genes**

It is worth noting again that the NAD+ molecule is the substrate for the PARP enzyme, which is required for the DNA damage response and the DNA repair system [66, 67]. The inhibition of the PARP1 enzyme ameliorates the mitochondrial metabolism through the activation of SIRT1 [175]. Conversely, the over-activation of the PARP1 enzyme can lead to mitochondrial dysfunction [176]. The *PARP1* gene expression was found to be negatively regulated when poly(ADP-ribose) glycohydrolase (PARG) siRNAs were introduced into HeLa S3 cells [40], suggesting that the degradation of the PAR macromolecule is required for the transcription of the *PARP1* gene. Because the 5′-upstream regions of both the human *PARP1* and *PARG* genes commonly contain the duplicated GGAA-motifs [53, 54], these two genes may be regulated by an NAD+ -sensitive mechanism. The results support the concept that PARP1 is involved in the NAD+ -sensitive transcription system [85]. In summary, the NAD+ /NADH ratio not only contributes to the DNA repair, but also to the fine-tuning of the transcription of genes that encode the NAD+ metabolism-associated DNA repair factors.

As described previously, the promoter regions of a number of genes that encode TCA cycle enzymes and DNA repair factors contain duplicated GGAA (TTCC) motifs [53, 54]. Thus, the fine-tuning of the transcription of mitochondrial function-associated factor- and DNA repair factor-encoding genes would be required for cells to conduct mitochondria in response to DNA damage-inducing stress.

of NAD+

. As expected, multiple duplications of the GGAA-motif are present in the bidirec-

A New Insight into the Development of Novel Anti-Cancer Drugs that Improve the Expression…

metabolism

119

http://dx.doi.org/10.5772/intechopen.71095

/NADH balance.

tional promoter region between the *LACTB* (*MRPL56*) gene and the bidirectional partner *LOC107987798*. We confirmed that the duplicated GGAA-motif is present near the TSS of the human *PDSS2* gene, which encodes prenyl-diphosphatase synthase subunit 2, which is a modulator of the complex I–III and II–III [133]. The PDSS2 is required for the integrity of Coenzyme Q (CoQ) or ubiquinone, which can improve the mitochondrial functions [187]. Thus, PDSS2 would be one of the targets for novel anticancer agents [188, 189]. The introduction of the *LbNOX* gene, which encodes bacterial NADH oxidase, into HeLa cells via a lentiviral vector ameliorates the proliferative and metabolic defects caused by the impairment of the

regulator encoding genes, including *PARP*, *PARG,* and *NAMPT*, as well as the *PPARGC1A*, *LACTB*, *PDSS2*, and *LbNOX* genes, could be applied or targeted in anti-cancer gene therapy. Alternatively, TF-encoding genes can be applied to anti-cancer therapies that aid in the recovery of mitochondria. First, the transcription modulator CtBP might be artificially controlled to suppress oncogenesis or cancer progression [83, 84]. Second, because duplicated GGAAmotifs are present in the 5′-upstream regions of a number of DNA repair factor- and mitochondrial factor-encoding genes, GGAA-motif binding factors could upregulate the mitochondrial functions at the transcriptional level. Recently, it was reported that mouse Gabp, which is an ETS family protein, is required for mitochondrial biogenesis through the regulation of the *Tfb1m* gene [191], suggesting that a *GABP* expression vector might be designed and constructed for cancer treatment. The 5′-upstream regions of a number of human genes contain the GGAA-duplication, and it is a GC-box that is very frequently found near the GGAA-core motif [12]. Recently, it was reported that mutations on the ETS family protein-encoding *ERF* and *ERG* genes play roles in prostate oncogenesis [192], implying that imbalances in GGAAbinding TFs could lead to aberrant gene expression. In order to determine which TF-encoding genes should be chosen, the mechanisms through which each of these genes is differently

In this article, we focused on the transcription mechanism that regulates the mitochondrial functions and the DNA repair systems, both of which decline with aging. Although the molecular mechanisms underlying the regulation of the expression of these genes are not yet fully

The anti-cancer drugs that are currently in use, including metabolism inhibitors, telomerase inhibitors, and apoptosis inducers, were developed with the common intention of killing cancer cells. Although immune receptor target drugs have been applied in the clinical setting, they are similar in that they force cancer cells to die. The anti-cancer drugs that are currently in use damage both malignant cancer cells and normal healthy cells. Importantly, the undesired effects of these anti-cancer drugs are problematic with regard to the quality of life (QOL) of cancer patients, especially those who are too old to endure severe adverse effects that occur during the course of chemotherapy. In order to avoid lethal side effects, individual whole

understood, several lines of evidence suggest that it is dependent on the NAD+

electron transport chain (ETC) [190].These lines of evidence suggest that NAD+

regulated during tumorigenesis should be elucidated.

**8. Conclusions and future prospects**
