Gene Therapy Applications

#### **Chapter 4**

## Perspective Chapter: Topoisomerase 1 and Colo Rectal Carcinoma

*Ahmed Mohamed Nabil Helaly and Doaa Ghorab*

#### **Abstract**

Topoisomerase 1 is the main enzyme playing an important role in relaxing. The supercoiled DNA strands allow the replication fork to transcribe the DNA to RNA and finally control protein production in active and replicating cells. Blocking this essential machinery is a cornerstone mechanism in treating tumors, such as liver, breast, and metastatic colorectal carcinoma. Irinotecan is a topoisomerase inhibitor that blocks the replication ending in DNA break and tumor cell death. This chemotherapy has been successfully used in combination to overcome metastatic colorectal carcinoma. The topoisomerase-1 inhibitor makes a protein DNA complex stuck with the replicating fork creating a single DNA break, unlike topoisomerase-2, which is responsible for double DNA break. This inhibitor is exposed to drug resistance with complex machinery. Drug resistance can occur as a result of altered DNA methylation, changes in topoisomerase expression, histone recombination, or drug export pump. High expression of topoisomerase-1 is a marker of the number of tumors suggesting multiple roles of topoisomerase-1.

**Keywords:** topoisomerase-1, irinotecan, colorectal carcinoma, epigenetic, drug resistance, supercoiled DNA

#### **1. Introduction**

DNA topoisomerases are important enzymes that modify the double-stranded DNA topology. They act in both directions to relax supercoiled DNA and enforce relaxed DNA to be tenser. These 3D structure modifications share in the control of DNA transcription and finally protein translation. In humans, there are two types of isomerases: the first one is topoisomerase-1 (TOP-1); the second one is topoisomerase-2 (TOP-2). Because of the canonical functions of TOPs, these enzymes have been a target for treating overactive cells, such as cancer cells or bacteria that contain different types of isomerases [1]. TOPs covalently bind DNA and create DNA adducts aiming to release the over-twisted DNA strand. Topoisomerase inhibitors (TOP inhibitors) create TOP/DNA obstacles in front of the active replicating fork. The smash between the fork and the DNA protein complex ends in a DNA break. TOP-1 differs from TOP-2 in that the first one is responsible for a single DNA break, while the latter is involved in double DNA damage [2]. In this chapter, we are going to discuss the relationship between TOP-1 and cancer. The

chapter aims to put a spot on the role of TOP-1 in cancer colon, and TOP-1 inhibitors' role to overcome cancer colon. It is important to discuss TOP-1 resistance.

#### **1.1 Topoisomerase-1 and cancer**

Since 1989, scientists recorded the high expression of TOP-1 in aggressive tumors such as late-stage colorectal carcinoma. It seems that such tumors use TOP-1 to support their replication machine. These findings were reported in renal tumors or brain neoplasms. The excess TOP-1 activity was highest seen in leukemia besides cancer colon. Moreover, TOP-1 overactivity correlated with poor prognosis in these tumors [3, 4].

TOP-1 may exert complex relation to the process of carcinogenesis. Experimental work demonstrated that knock-out TOP-1 plays a role in oncogenic property. The knock-out cells showed less aging and more replication capacity. Interestingly, the genome was more resistant to DNA damage [5]. More recent work showed opposite results concluding that oxidative stress induces cell aging with excess TOP-1 activity [6]. It is suggested that both senescence and active replication are parts of the process of neoplasia. An immunohistochemical study of secondary pterygium showed both overexpression of TOP-1 and glutathione in the same pathology [7].

A recent study on cancer liver samples using both immunohistochemistry and microarray demonstrated upregulation of TOP-1 and TOP-2 expression in poor prognosis liver cancer candidates. Both genes were involved in the function of the guardian p53 pathway and apoptosis cascade. The study showed that it is a wise idea to consider topoisomerases as oncogenes. Furthermore, these 2 targets are potentially good potentials for cancer chemotherapy [8]. TOP-1 inhibitors include etoposide, camptothecin, and Adriamycin with wide use in clinical practice. More research on different extracts to decrease the side effects and improve the efficacy is going on [9, 10]. Studies in yeast demonstrated that active or aberrant TOP-1 induces DNA mutagenicity, and later on, unstable DNA contributes to cancer development. This mechanism is controlled in mammalians by safety SUMOylation post transcription mechanism [11]. The small ubiquitin-like modifier (SUMO) is a pathway that contributes to transcription, immunity, signaling, and stabilization of the genome. Unfortunately, defective TOP-1 and its regulatory SUMOlyation may end in tumor genesis [12].

Topoisomerase-1 gene expression is considered a marker of drug response in metastatic colorectal carcinoma. It is recommended to use irinotecan (TOP-1 inhibitor) to treat these advanced colorectal tumors where TOP-1 expression is over-expressed. It seems that these tumors over-express TOP-1 to support the tumor DNA repair making the tumor cell resistant to death [13]. TOP-1 inhibitor irinotecan has been tested on cell lines to induce acetylation of the p53 and interrupt the histone deacetylase activity, making the resistant genome of growing cancer cells suitable for DNA break. It seems that TOP-1 inhibition is challenging in the understanding of the cancer colon pathway [14]. Metabolomic analysis of colorectal carcinoma cells treated by irinotecan showed shifting to glycolysis and an increase in oxygen consumption as markers of good response to cancer chemotherapy [15].

#### **1.2 Topoisomerase inhibitors**

The TOP-1 inhibitors were first discovered from a tree growing in China named Camptotheca acuminate. The extraction product of this tree was a component of

#### *Perspective Chapter: Topoisomerase 1 and Colo Rectal Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.108988*

Chinese medicine. Later on, in the 70s and the 80s, research work managed to construct TOP-1 inhibitors in the lab and formulate them as chemotherapy [16].

Drugs inhibiting TOP-1 have been used for decades to treat malignant tumors. These chemotherapies are called TOP-1 poisons. The first-generation candidate of these compounds is camptothecin. Newer generations, include irinotecan, topotecan, and belotecan [17]. Like any chemotherapy, camptothecin derivatives have many side effects. Scientists are working with non-camptothecin generations with fewer hazards [18]. TOP-1 poisons have been prescribed for multiple tumors including colorectal cancer, ovarian tumors, small cell lung malignancy, and myeloid proliferative disorders [19]. Aggressive neoplasms over expressing TOP-1 are suggested to be good candidates for TOP-1 inhibitors as in breast or ovarian cancers besides colorectal malignancy. It is expected that TOP-1 will be radical chemotherapy for these tumors exposing their DNA to break [20–22]. The researchers were working with TOP-1 inhibitors for half a century. They managed to get the crystal structure of the enzyme. The advances in molecular docking in the last 20 years give scientists a great opportunity to design lots of derivatives to discover new drugs that are more potent with more specific functions aiming to reduce the side effects. However, the difficulty was that the mechanism of action is canonical that it is extremely difficult to avoid hazards. To overcome such obstacle, the advances in drug delivery will reduce the side effects by loading the chemotherapy dose directly onto tumor cells [23].

#### **1.3 Topoisomerase-1 inhibitors and cancer colon**

Irinotecan is considered the first drug of choice in treating metastatic colorectal carcinoma in combination with 5 fluorouracil and folinic acid. Irinotecan is a working TOP-1 inhibitor that is metabolized in the liver to a more active compound SN-38. This chemotherapy is characterized by a high volume of distribution. Fortunately, cancer cells have excess carboxylesterase enzymes that can metabolize irinotecan into its active component [24–26]. Irinotecan is a derivative of camptothecin and was approved for cancer colon in 1994. Its major side effects are neutropenia and diarrhea, which are responsible for dysbiosis. This chemotherapy was approved for children and adults. It is successful for metastatic colorectal neoplasia, as well as, solid tumors. The drug was constructed by the Japanese Yakult Honsha company. The newer generation products have been tested as new chemotherapy but failed in the late clinical phases such as rubitecan, gimatecan, lurtotecan, diflomotecan, elomotecan, silatecan, exatecan, namitecan [27, 28].

New compounds are being verified for TOP-1 inhibitors with better profiles. These compounds include belotecan and gimatecan. The first one is hydrophilic while the latter is dissolved in fat [29, 30].

Belotecan has been used to treat resistant ovarian tumors with better side effects in combination with other new modalities [31]. Other trials have been applied to treat lung carcinoma and biliary tumors [32, 33]. There is little data about the efficacy of belotecan on colorectal neoplasms. On the other hand, gimatecan showed promising results in brain tumors [34].

The advances in nanotechnology and drug delivery can overcome the side effects of traditional TOP-1. Nano liposomal irinotecan has been applied in a pancreatic neoplasm with the hope to achieve success. The results expressed better side effects, but the overall survival represented a weak response [35]. Nano liposomal irinotecan, in combination with other classic chemotherapy, showed better cancer pancreas response. Diarrhea seems to be less counted, but the patients still suffered from neutropenia in a fifth of cases treated to the new model [36].

In regard to colorectal cancer, there are promising clinical trials that liposomal TOP-1 will help in overcoming late-stage conditions. The experimental trials in mice showed better survival rates with fewer side effects [37]. Another strategy is to use a lipophilic active gradient of irinotecan SN38 to suppress advanced colorectal tumors. The results showed a successful modality and the FDA approved the drug for the treatment of late-stage cancer colon [38].

New drugs, which are non-camptothecin derivatives, have been introduced to manage different tumors. These modalities include dibenzonaphthyridines, as well as, indeno isoquinolines. They are more strong stable DNA-protein complexes. Trials of metals such as platinum, gold, copper, zinc, and others have been suggested as TOP-1 modulators [39].

More research work is concerned with indenoisoquinolines, regards the capability to inhibit TOP-1. These new groups of the drug are subjected to structural modification. They represent promising drugs with potentially fewer side effects. It is proposed that the new chemical will have a multi-mechanism of action like nuclear receptors targeting, TOP-2 interaction, modulating estrogen receptors, and manipulating VEGFR and HIF-1 alpha [40]. Studies suggested that indenoisoquinolines are weak TOP-1 inhibitors in combination with other functions. It seems that weak TOP-1 inhibitors in conjunction with other drugs, or even other pathways, are good rationales for modern cancer therapy [41].

#### *1.3.1 Indenoisoquinolines*

More than 20 years ago, a group of scientists managed to create a new anti-TOP-1 chemical 6,11-dimethyl-6,11-dihydro-5H-indeno[1,2-c]isoquinolin-5-one. This compounds the parent of quinolone family, and it exerted promising results on different human cell lines [42]. Since that time, dozens of derivatives have been introduced to be tested experimentally. These compounds have the advantage of being flat ones that strongly bind the TOP-1/DNA hybrid. The new compounds bind the TOP-1 at the arginine 364 of the enzyme TOP-1 [43]. These potential new drugs showed stronger cytotoxicity relative to their anti-TOP-1 mechanism and are expected with combined dynamic to have a less toxic profile [44]. Other pathways affected by indenoisoquinolines include induction of cell cycle arrest, stimulation of apoptotic response, and modulation of MAPK cascade. Other potential role includes phosphorylation of JUNK pathway and inhibition of the oncogene MYC pathway [45]. On the other hand, despite the strong multi-dynamic function of these drugs, they did not replace the classic TOP-1. Research is still going to tune drugs with a balance between efficacy and hazard effects.

#### **1.4 Topoisomerase-1 inhibitor resistance**

Topoisomerase inhibitors, like other chemotherapy, are subjected to resistance. Many mechanisms are involved in this process. The tumor uses efflux mechanisms to reduce the level of the drug in the tumor mass. Mutations of the TOP-1 make the drug less effective in inhibiting the enzyme. Other strategies include enhancing the DNA repair to overcome TOP-1 poison. The tumor cells stimulate p53 to support the malignant mass to survive and suppress apoptosis. Limiting the drug's bioavailability can be another way to overcome chemotherapy [46].

Recent work put the spot on cancer colon stem cell role in the development of cancer. These highly replicating cells can overexpress the ATP cassette transporters.

#### *Perspective Chapter: Topoisomerase 1 and Colo Rectal Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.108988*

The experimental work showed that the over-expressed MYC oncogene supports the tumor resistance by enriching ATP transporters [47]. The ATP cassette sub-family G isoform 2 is expected to overcome xenobiotic effects with abundance in the GIT and near blood vessels. Besides their role in cancer stem cells, they represent a defense mechanism against chemotherapy. Tyrosine kinase inhibitors, phosphodiesterase-5 inhibitors, and the fumitremorgin-type indolyl diketopiperazine have been used to support chemotherapy to overcome resistance by inhibiting the ATP transporters [48]. The resistance was marked in cases associated with the marker ATP cassette type G group member 2 [49]. To overcome the resistance obstacles, the use of an ATP cassette inhibitor has been introduced. Another method was to apply the new TOP-1 inhibitor FL118, which possesses the capacity to overcome efflux resistance. This new analog is weakly transported by ATP carriers [50]. KO143 is a potent antagonist of ATP cassette sub-family G member 2. It is stable and not easily metabolized by the cytochrome enzymes in the liver [51].

ATP-binding transporters have been involved in the resistance to TOP-1 inhibitors in cancer breast. Experimental work on cancer colon cell lines concluded that colon tumor cells expressed a similar mechanism. Furthermore, it is possible to add compounds inhibiting efflux mechanisms such as SCO-201 to the chemotherapy regimen to sensitize the tumor to respond to the chemotherapy indicating better survival rates [52]. It is important to notice that overexpression of the ATP transporters is a marker of resistance to cancer colon. It is recorded that candidates' higher expression is most likely to resist camptothecin derivatives.

#### *1.4.1 ATP efflux mechanism*

The master mechanism of resistance of colorectal tumors to chemotherapy is drug efflux mechanism. The ATP-binding cassette sub-family G member 2 (ABCG2) is responsible for decreasing anti-TOP-1 bioavailability inside the tumor microenvironment leading to failure of the therapy [53]. The promoter regulating the expression of these shuttles is epigenetically regulated by methylation. Aggressive tumors express high ATP cassettes to get rid of the xenobiotic load. Hypomethylation of the promotor-regulating ATP transporter was recorded in different tumor cell lines [54]. The ABCG2 is formed from a sequence of 655 amino acids weighing 72-kilo Dalton. The structure is homodimer with two nucleotide-binding sites to export the drugs [55, 56]. The transporters play important role in the maintenance of the tumor microenvironment. They keep the balance of osmotic pressure. They have another role in antigen processing and modulation of cell division. Cell trafficking and cholesterol metabolism are affected by the ATP transporters [57].

Recently, N6 -methyladenosine (m6 A) RNA modification has been a new modality to treat ATP transporter through epigenetic mechanism at the RNA level [58].

Another important mechanism to defeat chemotherapy is the mutation of DNA repair response. In cancer cells, positive mutations make the tumor resistant to TOP-1 effects. The tumor genes such as BRCA1, BRCA2, PLAB2, and BARD1 have been mutated. Studies recorded alterations in 86 genes related to DNA repair. Such overactivity makes the single DNA break to the TOP-1 less effective [59].

Cancer colon cells expressing both SENP-1 (sentrin specific protease) and HIF-1α (Hypoxia inducing factor alpha) are resistant to chemotherapy. Both of them overexpress proteins SUMO pathway and make the tumor resistant to hypoxia [60]. Recently, SENP1 is a target of new compounds to develop a new regimen to overcome resistance [61].

Cancer cells can develop an alternative way to resist chemotherapy, specially irinotecan by augmenting the detoxification pathway. The active metabolite SN-38 is inactivated by glucuronidation making the drug short of killing the cancer cells. The nuclear receptors pregnane X receptors and steroid/xenobiotic receptors enhance the metabolism process of SN-38. It is important to notice that they are expressed in excess in the liver and the GIT. They also induce a battery of genes that express xenobiotic transporters that make irinotecan ineffective within a short period [62].

To repel TOP-1 inhibitors, the tumor cells induce a high copy number of the TOP-1 gene to make the drug subtherapeutic to kill the neoplastic cells. Furthermore, the TOP-1 is subjected to chromosomal alterations to be transcribed from different loci. These overexpressed loci are considered biomarkers of the drug response later on [13, 63].

The cancer colon cells are smart enough to create mutated TOP-1 that is not responding to the classic inhibitors. Experimental studies on resistant cell lines demonstrated several mutations that resist camptothecin derivatives or the new generation TOP-1 drugs [64].

The antioxidant balance in the cancer metabolism plays important role in protecting the tumor cells from xenobiotic toxicity and oxidative stress. One of the common mechanisms to support the redox is to express a high amount of glutathione reductase allowing the tumor to grow in unnatural situations and protect the mass from chemotherapy [65].

Another model for TOP-1 inhibitor resistance is colorectal tumors expressing active epidermal growth factor (EGFR). It was recorded that the active metabolite SN-38 is subjected to resistance because of the over-expression of EGFR. This factor triggers a trophic response in cancer growth via a cascade of a signaling pathway [66, 67].

#### **1.5 Epigenetic therapy and cancer colon**

New work has applied epigenetic modifier agents in combination with classic chemotherapy with a promising response. The epigenetic changes improved the drug response and reduce the needed dose to improve the clinical outcome. These transformers include DNA methyl transferase, decitabine, azacytidine, and zebularine. The study also used drugs that were considered histone deacetylase inhibitors including the well-known mood stabilizer valproic acid with promising results [68]. During the process of cancer evolution, cancer cells have different strategies to survive. It was recorded that p53 is mutated and histone deacetylases (HDACs) are over-expressed. It was considered that this epigenetic mechanism corresponds to drug resistance. Experimental inhibition of HDACs by small hairpin RNAs resulted in a better response of chemotherapy against the resistant SW480 cell lines. Indeed, epigenetic modification is part of the process of carcinogenesis and drug resistance response [69].

The DNA topology is inherited by an epigenetic mechanism. However, the process is unclear. The status of DNA twisting is a unique criterion of each cell type. This information is considered a cellular memory that is transmitted from the parent cells to the daughters by mitosis. The status of DNA 3D structure controls transcription and gene expression [70]. It has been recorded that parent cells with excess supercoiling deliver daughter cells with the impaired cell cycle. DNA supercoiling is important in the process of chromatin condensation. Previously, it was thought that DNA remains quiet during the process of mitosis. However, recent evidence that a battery

of genes is still active in the mitosis process to be involved in the development of the next generation of cells [71]. The activity of the TOP-1 allows the chromatin to adapt to the DNA-positive supercoils. On the other hand, an excess of negative supercoils ends in DNA/RNA hybrid structure with resulting in DNA damage [72]. Although, DNA/RNA hybrid is considered a mechanism of regulating gene expression called an R loop [73].

#### **1.6 Topoisomerase-1 biomarkers of response**

The first line of treatment for wild-type RAS metastatic colon cancer was the anti-EGFR immunotherapy cetuximab or panitumumab [74]. Experimentation searched for the colorectal cancer biomarkers in the blood. Recently, kits to extract the tumor cDNA are available clinically to evaluate associated parameters predicting the response of the tumor to classic chemotherapy. Moreover, liquid samples assaying RAS and BRAF oncogenes have a role in chemotherapy of colorectal cancer. The assays showed that tumors with wild-type RAS and BRAF were more responsive to camptothecin chemotherapy in combination with immunotherapy as a third line of treatment for colorectal metastasis [75]. The selection of the biomarkers as a strategy of personalized medicine in colon tumors reduces the side effects for precisely predicted response to the combined TOP-1 chemotherapy-related regimen. These markers include *DYPD*, *UGT1A1, HPP1*, *HER2*, *HER3, PIK3CA*, and *PTEN* [76]. It is important to say that EGFR is modulating a battery-controlling factor or biomarker that can predict the tumor response. It influences RAS/BRAF/MEK/MAPK and PI3K/ PTEN/AKT cascades [77].

#### **1.7 Topoisomerase and other diseases**

TOP-1 is an essential enzyme in the machinery of DNA transcription by modifying the topology of the DNA helix, and it is an important factor in stabilizing the genome. Furthermore, this enzyme has another role in the process of transcription in a way not directly related to unwinding DNA. Researchers proposed TOP-1 targets as a potential treatment for autism [78]. Mitochondria depend on the imported TOP-1 to relax the DNA during the process of transcription. Mitochondrial DNA damage is involved in many, such as neurodegenerative disorders and cancer. TOP-1 with its double role is a valuable object of cancer chemotherapy [79].

Another important scope of drugs inhibiting TOP-1 is modulating the immune response on exposure to microorganisms. Experimental studies showed that loading mice with endotoxins expressed a less harmful immune reaction than candidates tested with TOP-1 inhibitors. Low-dose TOP-1 chemotherapy such as camptothecin improved the antiviral unfavorable reaction. Interestingly, the low-dose poison with mild DNA break improved the situation in fighting the viral load [80].

Recently during the COVID-19 crisis, topotecan (TOP-1) has been applied to reduce the aberrant immune response to corona infection. Experimental work on hamsters and mice showed that a topotecan dosing improved the immune response. The TOP-1 inhibitors may exert antiviral capacity [81].

#### *1.7.1 Mutated TOP-1*

Mutated TOP-1 was a part of different chronic disorders. Malfunctioning TOP-1 is associated with unstable DNA. The causes of such complex pathology are still

unknown. The hybrid TOP-1/DNA abnormal complex works as a TOP-1 inhibitor ending in excess DNA break. These phenomena were recorded in massive spinocerebellar disorders. It was reported that ATM (ataxia telangiectasia, mutated), a serine/ threonine protein kinase, plays an essential role in cell cycle activation, chromatin remodeling, DNA repair, or stimulation of apoptosis. Experimental knockout of ATM showed accumulated pathogenic TOP-1/DNA complex with excess DNA break creating neurodegenerative pathology in animal models. Furthermore, healthy ATM can mediate TOP-1 complex ubiquitination [82]. These results established the idea that healthy TOP-1 is essential for development of central nervous system.

#### **1.8 Innovations and novel strategies from a clinical perspective**

Recently, the advances in multi-omic studies provided a signature of metastatic colorectal carcinoma cases aiming to predict the response of the chemotherapy. Metastatic candidates expressed unique proteomic profiles. There is a distinct profile for colorectal tumors, associated with liver metastasis. As regarding the genomic profile, there was no significant difference between early or late-stage tumors. These findings build up personalized strategies to treat or predict poor prognosis. Metastatic colorectal patients showed overexpression of oxidative phosphorylation and Krebs cycle enzymes [58]. A new strategy to use drugs that inhibit the rate of metabolism has been introduced in clinical trials as an adjuvant in colorectal metastasis in the liver. This modality is considered as tumor micro immunity reprogramming [83].

On the other hand, the analysis of circulating tumor DNA in the blood showed a mutation signature related to the tumor outcome. The status of RAS/BRAF mutations correlated with the prognosis of patients. The RAS/BRAF mutation load correlated with overall survival. As fewer mutations were detected in the blood sample, higher rates of remission were detected [84]. Another clinical study showed a similar profile. Excessive mutation RAS/BRAF suggested poor response to the first line of treatment of colorectal carcinoma [85].

A new modality mXELIRI (capecitabine plus irinotecan) has been recently applied with or without immunotherapy. The patients tolerated therapy for colorectal tumors with acceptable efficacy and toxicity profile [86]. Other trials have been applied to combine irinotecan and vincristine in soft tissue tumors [87].

New combination chemotherapy has been introduced recently. The anti-HER3 antibody patritumab has been added to the new TOP-1 inhibitor DX-8951 derivative (DXd) to treat resistant colorectal cancer with potential success [88].

#### **2. Conclusions**

TOP-1 gene and TOP-1 target protein are important in both carcinogenesis and pharmacology of different tumors. It is concluded that TOP-1 classic inhibitors **Table 1** are the main line of treatment of metastatic colorectal carcinoma. These drugs with complex mechanism of action are associated with profound side effects and subjected to different types of machinery of drug resistance. The discovery of new drugs with multi dynamics, including mild TOP-1 in conjunction with other actions, is a promising modality to bypass the toxic effects of classic TOP-1 inhibitors as shown in **Table 2**. Too strong anti-TOP is no longer a wise strategy to apply because of the canonical TOP function in almost every cell. Epigenetic tools can be added to improve the chemotherapy outcome.


#### **Table 1.**

*In vitro studies with TOP1 inhibitors.*


#### **Table 2.**

*In vivo studies with TOP1 inhibitor.*

### **Author details**

Ahmed Mohamed Nabil Helaly1,2\* and Doaa Ghorab3,4

1 Faculty of Medicine, Forensic Medicine, and Clinical Toxicology Department, Mansoura University, Egypt

2 Faculty of Medicine, Clinical Science Department, Yarmouk University, Irbid, Jordan

3 Faculty of Medicine, Pathology Department, Mansoura University, Egypt

4 Faculty of Medicine, Basic Science, Yarmouk University, Irbid, Jordan

\*Address all correspondence to: ahmedhelaly@mans.edu.eg

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Topoisomerase 1 and Colo Rectal Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.108988*

#### **References**

[1] Bush NG, Evans-Roberts K, Maxwell A. DNA topoisomerases. EcoSal Plus. 2015;**6**:2

[2] Nitiss JL, Kiianitsa K, Sun Y, Nitiss KC, Maizels N. Topoisomerase assays. Current Protocol. 2021;**1**(10):e250

[3] Husain I, Mohler JL, Seigler HF, Besterman JM. Elevation of topoisomerase I messenger RNA, protein, and catalytic activity in human tumors: Demonstration of tumor-type specificity and implications for cancer chemotherapy. Cancer Research. 1994;**54**(2):539-546

[4] Zhao C, Yasui K, Lee CJ, Kurioka H, Hosokawa Y, Oka T, et al. Elevated expression levels of NCOA3, TOP1, and TFAP2C in breast tumors as predictors of poor prognosis. Cancer. 2003;**98**(1):18-23

[5] Humbert N, Martien S, Augert A, Da Costa M, Mauen S, Abbadie C, et al. A genetic screen identifies topoisomerase 1 as a regulator of senescence. Cancer Research. 2009;**69**(10):4101-4106

[6] Bu H, Baraldo G, Lepperdinger G, Jansen-Dürr P. mir-24 activity propagates stress-induced senescence by down regulating DNA topoisomerase 1. Experimental Gerontology. 2016;**75**:48-52

[7] Doaa G, Helaly AM. Badawi a dual pathogenesis of primary and recurrent pterygium: Immunohistochemical proof. The Open Ophthalmology. 2021;**15**:229- 235. DOI: 10.2174/1874364102115010229

[8] Liu LM, Xiong DD, Lin P, Yang H, Dang YW, Chen G. DNA topoisomerase 1 and 2A function as oncogenes in liver cancer and may be direct targets of nitidine chloride. International Journal of Oncology. 2018;**53**(5):1897-1912

[9] Kathiravan MK, Kale AN, Nilewar S. Discovery and development of topoisomerase inhibitors as anticancer agents. Mini Reviews in Medicinal Chemistry. 2016;**16**(15):1219-1229

[10] Nukuzuma S, Nakamichi K, Kameoka M, Sugiura S, Nukuzuma C, Tasaki T, et al. Suppressive effect of topoisomerase inhibitors on JC polyomavirus propagation in human neuroblastoma cells. Microbiology and Immunology. 2016;**60**(4):253-260

[11] Li M, Pokharel S, Wang JT, Xu X, Liu Y. RECQ5-dependent SUMOylation of DNA topoisomerase I prevents transcription-associated genome instability. Nature Communications. 2015;**6**:6720

[12] Han ZJ, Feng YH, Gu BH, Li YM, Chen H. The post-translational modification, SUMOylation, and cancer (Review). International Journal of Oncology. 2018;**52**(4):1081-1094

[13] Braun MS, Richman SD, Quirke P, Daly C, Adlard JW, Elliott F, et al. Predictive biomarkers of chemotherapy efficacy in colorectal cancer: Results from the UK MRC FOCUS trial. Journal of Clinical Oncology. 2008a;**26**(16):2690-2698

[14] Marx C, Sonnemann J, Beyer M, Maddocks ODK, Lilla S, Hauzenberger I, et al. Mechanistic insights into p53 regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells. Molecular in Oncology. 2021;**15**(12):3404-3429

[15] Marx C, Sonnemann J, Maddocks ODK, Marx-Blümel L, Beyer M, Hoelzer D, et al. Global metabolic alterations in colorectal cancer cells during

irinotecan-induced DNA replication stress. Cancer Metabolism. 2022;**10**(1):10

[16] Wall ME, Wani MC. Camptothecin and taxol: Discovery to clinic- thirteenth Bruce F. Cancer Research. 1995;**55**(4):753-760

[17] Martín-Encinas E, Selas A, Palacios F, Alonso C. The design and discovery of topoisomerase I inhibitors as anticancer therapies. Expert Opinion in Drug Discovery. 2022;**17**(6):581-601

[18] Talukdar A, Kundu B, Sarkar D, Goon S, Mondal MA. Topoisomerase I inhibitors: Challenges, progress and the road ahead. European Journal of Medicinal Chemistry. 2022;**236**:114304

[19] Rasheed ZA, Rubin EH. Mechanisms of resistance to topoisomerase I-targeting drugs. Oncogene. 2003;**22**(47):7296-7304

[20] O'Connor MJ. Targeting the DNA damage response in Cancer. Molecular Cell. 2015;**60**(4):547-560

[21] Pommier Y. Topoisomerase I inhibitors: Camptothecins and beyond. Nature Reviews. Cancer. 2006;**6**(10):789-802

[22] Xu Y, Her C. Inhibition of topoisomerase (DNA) I (TOP1): DNA damage repair and anticancer therapy. Biomolecules. 2015;**5**(3):1652-1670

[23] Cinelli MA. Topoisomerase 1B poisons: Over a half-century of drug leads, clinical candidates, and serendipitous discoveries. Medicinal Research Reviews. 2019;**39**(4):1294-1337

[24] Guichard S, Terret C, Hennebelle I, Lochon I, Chevreau P, Frétigny E, et al. CPT-11 converting carboxylesterase and topoisomerase activities in tumour and normal colon and liver tissues. British Journal of Cancer. 1999;**80**(3-4):364-370 [25] Mathijssen RH, van Alphen RJ, Verweij J, Loos WJ, Nooter K, Stoter G, et al. Clinical pharmacokinetics and metabolism of irinotecan (CPT-11). Clinical Cancer Research. 2001;**7**(8): 2182-2194

[26] Slatter JG, Schaaf LJ, Sams JP, Feenstra KL, Johnson MG, Bombardt PA, et al. Pharmacokinetics, metabolism, and excretion of irinotecan (CPT-11) following I.V. infusion of [(14)C]CPT-11 in cancer patients. Drug Metabolism and Disposition. 2000;**28**(4):423-433

[27] Bailly C. Irinotecan: 25 years of cancer treatment. Pharmacological Research. 2019;**148**:104398

[28] Li F, Jiang T, Li Q, Ling X. Camptothecin (CPT) and its derivatives are known to target topoisomerase I (Top1) as their mechanism of action: Did we miss something in CPT analogue molecular targets for treating human disease such as cancer? American Journal of Cancer Research. 2017;**7**(12):2350-2394

[29] Covey JM, Jaxel C, Kohn KW, Pommier Y. Protein-linked DNA strand breaks induced in mammalian cells by camptothecin, an inhibitor of topoisomerase I. Cancer Research. 1989;**49**(18):5016-5022

[30] Thomas A, Pommier Y. Targeting topoisomerase I in the era of precision medicine. Clinical Cancer Research. 2019;**25**(22):6581-6589

[31] Hur J, Ghosh M, Kim TH, Park N, Pandey K, Cho YB, et al. Synergism of AZD6738, an ATR inhibitor, in combination with belotecan, a Camptothecin analogue, in chemotherapy-resistant ovarian Cancer. International Journal of Molecular Sciences. 2021;**22**(3):1223

*Perspective Chapter: Topoisomerase 1 and Colo Rectal Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.108988*

[32] Jun E, Park Y, Lee W, Kwon J, Lee S, Kim MB, et al. The identification of candidate effective combination regimens for pancreatic cancer using the histoculture drug response assay. Scientific Reports. 2020;**10**(1):12004

[33] Kang JH, Lee KH, Kim DW, Kim SW, Kim HR, Kim JH, et al. A randomised phase 2b study comparing the efficacy and safety of belotecan vs. topotecan as monotherapy for sensitive-relapsed small-cell lung cancer. British Journal of Cancer. 2021;**124**(4):713-720

[34] Cesare DE. High efficacy of intravenous Gimatecan on human tumor xenografts. Anticancer Research. 2018;**38**(10):5783-5790

[35] Kasi A, McGinnis T, Naik G, Handa S, Williams G, Paluri R. Efficacy and tolerability of the combination of nano-liposomal irinotecan and 5-fluorouracil/leucovorin in advanced pancreatic adenocarcinoma: Postapproval clinic experience. Journal of Gastrointestinal Oncology. 2021;**12**(2):464-473

[36] Yasuoka H, Naganuma A, Kurihara E, Kobatake T, Ijima M, Tamura Y, et al. Efficacy and safety of the combination of Nano-liposomal irinotecan and 5-fluorouracil/L-leucovorin in unresectable advanced pancreatic cancer: A real-world study. Oncology. 2022;**100**(8):449-459

[37] Liu X, Jiang J, Chan R, Ji Y, Lu J, Liao YP, et al. Improved efficacy and reduced toxicity using a custom-designed irinotecan-delivering silicasome for orthotopic colon cancer. ACS Nano. 2019;**13**(1):38-53

[38] Xing J, Zhang X, Wang Z, Zhang H, Chen P, Zhou G, et al. Novel lipophilic SN38 prodrug forming stable liposomes for colorectal carcinoma therapy.

International Journal of Nanomedicine. 2019;**14**:5201-5213

[39] Gokduman K. Strategies targeting DNA topoisomerase I in Cancer chemotherapy: Camptothecins, nanocarriers for camptothecins, organic non-Camptothecin compounds and metal complexes. Current Drug Targets. 2016;**17**(16):1928-1939

[40] Cushman M. Design and synthesis of Indenoisoquinolines targeting topoisomerase I and other biological macromolecules for Cancer chemotherapy. Journal of Medicinal Chemistry. 2021;**64**(24):17572-17600

[41] Wang KB, Elsayed MSA, Wu G, Deng N, Cushman M, Yang D. Indenoisoquinoline topoisomerase inhibitors strongly bind and stabilize the *MYC* promoter G-Quadruplex and downregulate *MYC*. Journal of American Chemical Society. 2019;**141**(28):11059-11070

[42] Cho WJ, Park MJ, Imanishi T, Chung BH. A novel synthesis of benzo[c] phenanthridine skeleton and biological evaluation of isoquinoline derivatives. Chemical Pharmacy Bulletin (Tokyo). 1999;**47**(6):900-902

[43] Khadka DB, Le QM, Yang SH, Van HT, Le TN, Cho SH, et al. Design, synthesis and docking study of 5-amino substituted indeno[1,2-c]isoquinolines as novel topoisomerase I inhibitors. Bioorganic & Medicinal Chemistry. 2011;**19**(6):1924-1929

[44] Strumberg D, Pommier Y, Paull K, Jayaraman M, Nagafuji P, Cushman M. Synthesis of cytotoxic indenoisoquinoline topoisomerase I poisons. Journal of Medicinal Chemistry. 1999;**42**(3):446-457

[45] Park EJ, Kiselev E, Conda-Sheridan M, Cushman M, Pezzuto JM. Induction

of apoptosis by 3-amino-6-(3 aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline via modulation of MAPKs (p38 and c-Jun N-terminal kinase) and c-Myc in HL-60 human leukemia cells. Journal of Natural Products. 2012;**75**(3):378-384

[46] Tomicic MT, Kaina B. Topoisomerase degradation, DSB repair, p53 and IAPs in cancer cell resistance to camptothecinlike topoisomerase I inhibitors. Biochimica et Biophysica Acta. 2013;**1835**(1):11-27

[47] Zhang HL, Wang P, Lu MZ, Zhang SD, Zheng L. c-Myc maintains the self-renewal and chemoresistance properties of colon cancer stem cells. Oncology Letters. 2019;**17**(5):4487-4493

[48] Stacy AE, Jansson PJ, Richardson DR. Molecular pharmacology of ABCG2 and its role in chemoresistance. Molecular Pharmacology. 2013;**84**(5):655-669. DOI: 10.1124/mol.113.088609

[49] Tuy HD, Shiomi H, Mukaisho KI, Naka S, Shimizu T, Sonoda H, et al. ABCG2 expression in colorectal adenocarcinomas may predict resistance to irinotecan. Biochimica et Biophysica Acta. 2016;**12**(4):2752-2760

[50] Westover D, Ling X, Lam H, Welch J, Jin C, Gongora C, et al. FL118, a novel camptothecin derivative, is insensitive to ABCG2 expression and shows improved efficacy in comparison with irinotecan in colon and lung cancer models with ABCG2-induced resistance. Molecular Cancer. 2015;**14**:92

[51] Liu K, Zhu J, Huang Y, Li C, Lu J, Sachar M, et al. Metabolism of KO143, an ABCG2 inhibitor. Drug Metabolism and Pharmacokinetics. 2017;**32**(4):193-200

[52] Ambjørner SEB, Wiese M, Köhler SC, Svindt J, Lund XL, Gajhede M, et al. The

pyrazolo[3,4-d]pyrimidine derivative, SCO-201, reverses multidrug resistance mediated by ABCG2/BCRP. Cell. 2020;**9**(3):613

[53] Moon HH, Kim SH, Ku JL. Correlation between the promoter methylation status of ATP-binding cassette sub-family G member 2 and drug sensitivity in colorectal cancer cell lines. Oncology Reports. 2016;**35**(1):298-306

[54] Spitzwieser M, Pirker C, Koblmüller B, Pfeiler G, Hacker S, Berger W, et al. Promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in human cancer cell lines, multidrugresistant cell models and tumor, tumoradjacent and tumor-distant tissues from breast cancer patients. Oncotarget. 2016;**7**(45):73347-73369

[55] Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, et al. The multidrug-resistant phenotype associated with overexpression of the new ABC halftransporter, MXR (ABCG2). Journal of Cell Science. 2000;**113**(Pt 11):2011-2021

[56] Xu J, Liu Y, Yang Y, Bates S, Zhang JT. Characterization of oligomeric human half-ABC transporter ATP-binding cassette G2. The Journal of Biological Chemistry. 2004;**279**(19):19781-19789

[57] Liu X. ABC family transporters. Advances in Experimental Medicine and Biology. 2019;**1141**:13-100

[58] Li C, Sun YD, Yu GY, Cui JR, Lou Z, Zhang H, et al. Integrated omics of metastatic colorectal Cancer. Cancer Cell. 2020;**38**(5):734-747

[59] Cuella-Martin R, Hayward SB, Fan X, Chen X, Huang JW, Taglialatela A, et al. Functional interrogation of DNA damage response variants with base editing screens. Cell. 2021;**184**(4):1081-1097

*Perspective Chapter: Topoisomerase 1 and Colo Rectal Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.108988*

[60] Chen MC, Nhan DC, Hsu CH, Wang TF, Li CC, Ho TJ, et al. SENP1 participates in irinotecan resistance in human colon cancer cells. Journal of Cellular Biochemistry. 2021;**122**(10):1277-1294

[61] Wei J, Wang H, Zheng Q, Zhang J, Chen Z, Wang J, et al. Recent research and development of inhibitors targeting sentrin-specific protease 1 for the treatment of cancers. European Journal of Medicinal Chemistry. 2022;**241**:114650

[62] Ozawa S, Miura T, Terashima J, Habano W. Cellular irinotecan resistance in colorectal cancer and overcoming irinotecan refractoriness through various combination trials including DNA methyltransferase inhibitors: A review. Cancer Drug Resistant. 2021;**4**(4):946-964

[63] Rømer MU, Jensen NF, Nielsen SL, Müller S, Nielsen KV, Nielsen HJ, et al. TOP1 gene copy numbers in colorectal cancer samples and cell lines and their association to in vitro drug sensitivity. Scandinavian Journal of Gastroenterology. 2012;**47**(1):68-79

[64] Jensen NF, Agama K, Roy A, Smith DH, Pfister TD, Rømer MU, et al. Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a new pair of resistance-associated mutations. Journal of Experimental & Clinical Cancer Research. 2016;**35**:56

[65] Narayanankutty A, Job JT, Narayanankutty V. Glutathione, an antioxidant tripeptide: Dual roles in carcinogenesis and chemoprevention. Current Protein & Peptide Science. 2019;**20**(9):907-917

[66] Asano T. Drug resistance in cancer therapy and the role of epigenetics. Journal of Nippon Medical School. 2020;**87**(5):244-251

[67] Petitprez A, Larsen AK. Irinotecan resistance is accompanied by upregulation of EGFR and Src signaling in human cancer models. Current Pharmaceutical Design. 2013;**19**(5):958-964

[68] Hosokawa M, Tanaka S, Ueda K, Iwakawa S. Different scheduledependent effects of epigenetic modifiers on cytotoxicity by anticancer drugs in colorectal Cancer cells. Biological & Pharmaceutical Bulletin. 2017;**40**(12):2199-2204

[69] Alzoubi S, Brody L, Rahman S, Mahul-Mellier AL, Mercado N, Ito K, et al. Synergy between histone deacetylase inhibitors and DNAdamaging agents is mediated by histone deacetylase 2 in colorectal cancer. Oncotarget. 2016;**7**(28):44505-44521

[70] Jha RK, Levens D, Kouzine F. Mechanical determinants of chromatin topology and gene expression. Nucleus. 2022;**13**(1):94-115

[71] Palozola KC, Donahue G, Liu H, Grant GR, Becker JS, Cote A, et al. Mitotic transcription and waves of gene reactivation during mitotic exit. Science. 2017;**358**(6359):119-122

[72] Edwards DS, Maganti R, Tanksley JP, Luo J, Park JJH, Balkanska-Sinclair E, et al. BRD4 prevents R-loop formation and transcription-replication conflicts by ensuring efficient transcription elongation. Cell Reports. 2020;**32**(12):108166

[73] Yuan W, Al-Hadid Q, Wang Z, Shen L, Cho H, Wu X, et al. TDRD3 promotes DHX9 chromatin recruitment and R-loop resolution. Nucleic Acids Research. 2021;**49**(15):8573-8591

[74] Van Cutsem E, Cervantes A, Adam R, Sobrero A, Van

Krieken JH, Aderka D, et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Annals of Oncology. 2016;**27**(8):1386-1422

[75] Cremolini C, Rossini D, Dell'Aquila E, Lonardi S, Conca E, Del Re M, et al. Rechallenge for patients with RAS and BRAF wild-type metastatic colorectal cancer with acquired resistance to firstline cetuximab and irinotecan: A phase 2 single-arm clinical trial. JAMA Oncology. 2019;**5**(3):343-350

[76] Taieb J, Jung A, Sartore-Bianchi A, Peeters M, Seligmann J, Zaanan A, et al. The evolving biomarker landscape for treatment selection in metastatic colorectal Cancer. Drugs. 2019;**79**(13):1375-1394

[77] Peeters M, Price TJ, Cervantes A, Sobrero AF, Ducreux M, Hotko Y, et al. Randomized phase III study of panitumumab with fluorouracil, leucovorin, and irinotecan (FOLFIRI) compared with FOLFIRI alone as secondline treatment in patients with metastatic colorectal cancer. Journal of Clinical Oncology. 2010;**28**(31):4706-4713

[78] Li M, Liu Y. Topoisomerase I in human disease pathogenesis and treatments. Genomics Proteomics Bioinformatics. 2016;**14**(3):166-171

[79] Das BB, Ghosh A, Bhattacharjee S, Bhattacharyya A. Trapped topoisomerase-DNA covalent complexes in the mitochondria and their role in human diseases. Mitochondrion. 2021;**60**:234-244

[80] Pépin G, Nejad C, Ferrand J, Thomas BJ, Stunden HJ, Sanij E, et al. Topoisomerase 1 inhibition promotes cyclic GMP-AMP synthasedependent antiviral responses. MBio. 2017;**8**(5):e01611-e01617

[81] Ho JSY, Mok BW, Campisi L, Jordan T, Yildiz S, Parameswaran S, et al. TOP1 inhibition therapy protects against SARS-CoV-2 induced lethal inflammation. Cell. 2021;**184**(10):2618-2632

[82] Katyal S, Lee Y, Nitiss KC, Downing SM, Li Y, Shimada M, et al. Aberrant topoisomerase-1 DNA lesions are pathogenic in neurodegenerative genome instability syndromes. Nature Neuroscience. 2014;**17**(6):813-821

[83] Wu Y, Yang S, Ma J, Chen Z, Song G, Rao D, et al. Spatiotemporal immune landscape of colorectal Cancer liver metastasis at single-cell level. Cancer Discovery. 2022;**12**(1):134-153

[84] Wang F, Huang YS, Wu HX, Wang ZX, Jin Y, Yao YC, et al. Genomic temporal heterogeneity of circulating tumour DNA in unresectable metastatic colorectal cancer under first-line treatment. Gut. 2022;**71**(7):1340-1349

[85] Yao J, Zang W, Ge Y, Weygant N, Yu P, Li L, et al. RAS/BRAF circulating tumor DNA mutations as a predictor of response to first-line chemotherapy in metastatic colorectal Cancer patients. Canadian Journal of Gastroenterology & Hepatology. 2018;**2018**:4248971

[86] Xu RH, Muro K, Morita S, et al. Modified XELIRI (capecitabine plus irinotecan) versus FOLFIRI (leucovorin, fluorouracil, and irinotecan), both either with or without bevacizumab, as secondline therapy for metastatic colorectal cancer (AXEPT): A multicentre, openlabel, randomised, non-inferiority, phase 3 trial. The Lancet Oncology. 2018;**19**(5):660-671. DOI:10.1016/ S1470-2045(18)30140-2

[87] Defachelles AS, Bogart E, Casanova M, Merks JHM, Bisogno G, Calareso G, et al. Randomized phase

*Perspective Chapter: Topoisomerase 1 and Colo Rectal Carcinoma DOI: http://dx.doi.org/10.5772/intechopen.108988*

II trial of vincristine-irinotecan with or without temozolomide, in children and adults with relapsed or refractory rhabdomyosarcoma: A European paediatric soft tissue sarcoma study group and innovative therapies for children with cancer trial. Journal of Clinical Oncology. 2021;**39**(27):2979-2990

[88] Koganemaru S, Kuboki Y, Koga Y, Kojima T, Yamauchi M, Maeda N, et al. U3-1402, a novel HER3-targeting antibody-drug conjugate, for the treatment of colorectal Cancer. Molecular Cancer Therapeutics. 2019;**18**(11):2043-2050

[89] Yao Z, Zheng W, Zhang X, Xiong H, Qian Y, Fan C. Hydroxycamptothecin prevents fibrotic pathways in fibroblasts In vitro. IUBMB Life. 2019;**71**(5):653-662

[90] Laco GS, Du W, Kohlhagen G, Sayer JM, Jerina DM, Burke TG, et al. Analysis of human topoisomerase I inhibition and interaction with the cleavage site +1 deoxyguanosine, via in vitro experiments and molecular modeling studies. Bioorganic & Medicinal Chemistry. 2004;**12**(19):5225-5235

[91] Borkar MR, Martis EAF, Nandan S, Patil RH, Shelar A, Iyer KR, et al. Identification of potential antileishmanial 1,3-disubstituted-4 hydroxy-6-methylpyridin-2(1H)-ones, in vitro metabolic stability, cytotoxicity and molecular modeling studies. Chemico-Biological Interactions. 2022 Jan;**5**(351):109758

[92] Kardile RA, Sarkate AP, Borude AS, Mane RS, Lokwani DK, Tiwari SV, et al. Design and synthesis of novel conformationally constrained 7,12-dihydrodibenzo[b,h][1,6] naphthyridine and 7H-Chromeno[3,2-c] quinoline derivatives as topoisomerase I

inhibitors: In vitro screening, molecular docking and ADME predictions. Bioorganic Chemistry. 2021;**115**:105174

[93] de Souza PL, Cooper MR, Imondi AR, Myers CE. 9-Aminocamptothecin: A topoisomerase I inhibitor with preclinical activity in prostate cancer. Clinical Cancer Research. 1997;**3**(2):287-294

[94] Halder N, Dzhemileva LU, Ramazanov IR, D'yakonov VA, Dzhemilev UM, Rath H. Comparative in vitro studies of the topoisomerase I inhibition and anticancer activities of metallated N-confused porphyrins and metallated porphyrins. ChemMedChem. 2020;**15**(7):632-642

[95] Haleel A, Mahendiran D, Veena V, Sakthivel N, Rahiman AK. Antioxidant, DNA interaction, VEGFR2 kinase, topoisomerase I and in vitro cytotoxic activities of heteroleptic copper(II) complexes of tetrazolo[1,5-a]pyrimidines and diimines. Materials Science & Engineering. C, Materials for Biological Applications. 2016;**68**:366-382

[96] Parvez MM, Basit A, Jariwala PB, Gáborik Z, Kis E, Heyward S, et al. Quantitative investigation of irinotecan metabolism, transport, and gut microbiome activation. Drug Metabolism and Disposition. 2021;**49**(8):683-693

[97] Ogitani Y, Hagihara K, Oitate M, Naito H, Agatsuma T. Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity. Cancer Science. 2016;**107**(7):1039-1046

[98] Fukuda T, Nanjo Y, Fujimoto M, Yoshida K, Natsui Y, Ishibashi F, et al. Lamellarin-inspired potent topoisomerase I inhibitors with

the unprecedented benzo[g][1] benzopyrano[4,3-b]indol-6(13H) one scaffold. Bioorganic & Medicinal Chemistry. 2019;**27**(2):265-277

[99] Ki DH, Oppel F, Durbin AD, Look AT. Mechanisms underlying synergy between DNA topoisomerase I-targeted drugs and mTOR kinase inhibitors in NF1-associated malignant peripheral nerve sheath tumors. Oncogene. 2019;**38**(39):6585-6598

[100] Liu H, Lu H, Liao L, Zhang X, Gong T, Zhang Z. Lipid nanoparticles loaded with 7-ethyl-10 hydroxycamptothecin-phospholipid complex: in vitro and in vivo studies. Drug Delivery. 2015;**22**(6):701-709

[101] Lei CS, Hou YC, Pai MH, Lin MT, Yeh SL. Effects of quercetin combined with anticancer drugs on metastasisassociated factors of gastric cancer cells: in vitro and in vivo studies. The Journal of Nutritional Biochemistry. 2018;**51**:105-113

#### **Chapter 5**

### Marker Assisted Selection in Groundnut

*Diriba Beyene Goonde and Seltene Abady*

#### **Abstract**

Groundnut (*Arachis hypogaea* L.) is an important oilseed crop worldwide. Objective of this review is to highlight molecular breeding approach such as marker assisted selection on groundnut improvement with future perspectives. The review analyzed application of marker assisted selection including simple sequence repeats, random amplified polymorphism DNAs, single nucleotide polymorphism, amplified fragment length polymorphism and inter simple sequence repeats on groundnut improvement. Among the molecular markers, random amplified polymorphic DNA is a rapid method for developing genetic maps and to determine DNA fragments to characterize peanut cultivars. DArTseq is used for SNP discovery and genotyping, which enables considerable discovery of SNPs in a wide variety of non-model organisms and provides measures of genetic divergence. Polymorphism screening performed using these newly developed SSRs will greatly increase the density of SSR markers in the peanut genetic map in the future.

**Keywords:** genome, molecular markers, simple sequence repeats, groundnut, marker assisted selection

#### **1. Introduction**

Groundnut (*Arachis hypogaea* L.), also known as peanut, is a member of genus Arachis and family Leguminosae [1]. Peanuts are key oilseed and food-legume crops for both humans and livestock in tropical and subtropical regions, and globally they are the fourth largest source of edible oil and third most important source vegetable protein. Its seed contain about 50% of edible oil and the remaining 50% of the seed has high quality protein (36.4%), carbohydrate in the range (6–24.9%), minerals and vitamin [2]. It is believed to have originated in the southern Bolivia to northern Argentina region of South America. Cultivated Groundnut (A. hypogaea L., 2n = 4x = 40, AABB) is self-pollinating allotetraploid legume crop belonging to the Fabaceae family [3].

Groundnut was introduced to Ethiopia by Italian explorers in the 1920s [4]. Globally China ranks first in groundnut production with 17.39 million tonnes followed by India 6.95 million tonnes, Nigeria 2.88 million tonnes, Sudan 2.88 million tonnes and Ethiopia ranks 31th with 0.129 million tonnes with national mean yield is 1.75 tons/ha, and the total area under groundnut production is 115,291 ha [5]. The most common groundnut production constraint in Ethiopia in general and the southern region, in particular, were the lack of access to improved seeds, biotic, abiotic stress,

and the use of low-yielding local varieties [6, 7]. Therefore, the objective of this review is to highlight molecular breeding approaches such as marker assisted selection on groundnut improvement and opportunities, challenges with future perespectives of the crop.

### **2. Status production of groundnut**

Groundnut are predominantly grown in developing countries (Africa and Asia where the crop finds appropriate climate for optimum production). Although, the production is concentrated in Asia (50% global area and 68% global production) and Africa (46% global area and 24% of global production) (**Table 1**).


**Table 1.**

*The top leading country in production and productivity of groundnut in the world.*

### **3. Genomic resources**

Genomic resources such as molecular markers are powerful tools to characterize and harness the genetic variation present in the germplasm collection. Peanut has comparatively lower genomic resources (including transcriptome data) compared to other legumes like medicago, lotus and chickpea [8], robust molecular markers, specifically the genetic ones, as they provide the insight into the functional information. However, the available peanut high throughput transcriptome sequences are not complete; many have low N50 values, ranging from 500 to 750 bp [9]. Because peanut

has such a large number of genes, it is important to have a good representation of the transcriptome. On other hand, ESTs data of cultivated peanut is still remain unexplored for the development of SSR markers (**Figure 1**).

#### **4. Marker assisted selection**

Marker-assisted selection has been a plant breeding tool since it was proposed by Sax in 1923 [10]. The theory behind this method is that plant breeders could observe easy-to-score phenotypes to select difficult-to-score or low heritability traits that are linked to them. Marker assisted selection is the indirect selection of selected or desired plant phenotype depending on the closely linked DNA marker. MAS is an efficient molecular tool for breeding, in which markers linked with the desired genes are used for indirect selection for that gene in non-segregating or segregating populations. MAS is an important method for the selection of traits that are difficult, like, biotic and abiotic stress tolerance in a crop [11]. Compared with conventional phenotypic selection, MAS is not influenced by environmental conditions because it detects the structural polymorphisms at the molecular level. Further MAS is cheaper and less labour intensive, allows selection in off-season nurseries and has a potential to accelerate the breeding process [12].

Molecular markers among all genomic resources, molecular markers have direct use for germplasm characterization, trait mapping and molecular breeding. Several marker systems have been developed during the last three decades. For instance restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs) and diversity arrays technology (DArT) markers have proved their utility from time to time [13]. However, simple sequence repeats (SSRs) or microsatellites and single nucleotide polymorphism (SNP) markers are generally preferred for plant genetics and breeding


#### **Table 2.**

*Commonly used molecular markers are as below.*

applications. While SSR markers are multi-allelic, codominant and easy to use, the SNP markers are highly amenable to high-throughput genotyping approaches. Development and application of SNP markers, however, is still not routine in crop species and especially not in low-tech laboratories (**Table 2**).

#### **4.1 Markers for target traits**

The approach of identifying markers for targets traits swiftly changed with the development of linkage maps in groundnut [14]. Seed weight is controlled by a combination seed features such as Seed length, Seed width, and seed thickness. Several genes for seed-related traits have been obtained in many crops using the forward genetic strategy and reverse genetic strategies [15]. QTL analysis was used for identification of QTLs for several important traits such as drought tolerance related traits, resistance to foliar disease and nutritional quality traits (**Table 3**).

#### **4.2 Molecular markers in groundnut**

Cultivated groundnut has been analyzed by several markers systems including RFLPs, RAPDs, AFLPs and SSRs.

#### *4.2.1 Restriction fragment length polymorphism*

Restriction Fragment Length Polymorphism (RFLPs) represented the first marker system that had a large number of polymorphisms. They are used widely both to create linkage maps and to implement indirect selection strategies. In A. hypogaea, little molecular variation has been detected by using RFLP technologies have been used to analyze species in section Arachis (representing taxa that will hybridize with


*RAPD, Randomly Amplified Polymorphic DNA; RFLP, Restriction Fragment Length Polymorphism; SSR, Simple Sequence Repeat; AFLP, Amplified Fragment Length Polymorphism; AS-PCR, Allele Specific Polymerase Chain Reaction. #FAD, Fatty acid desaturase;; ELS, Early Leaf Spot; LLS, Late Leaf Spot.*

#### **Table 3.**

*Molecular markers associated with trait specific genes/QTLs in groundnut.*

*Marker Assisted Selection in Groundnut DOI: http://dx.doi.org/10.5772/intechopen.108476*

A. hypogaea) and clusters that formed using multivariate analyses [16] correspond closely with morphological groups [17]; tetraploids were clearly separated from diploids in both investigations. Stalker *et al.* [18] utilized RFLPs to examine genetic diversity among 18 accessions of A. duranensis Krapov. and We. Gregory and found a large amount of variation in the species. Individual accessions also could be uniquely identified by RFLP patterns. Kochert et al. [19] concluded that the cultivated peanut resulted from a cross between A. duranensis and A. ipaensis Krapov. and W.C. Gregory, and chloroplast analysis indicated that A. duranensis was the female progenitor of the cross. An RFLP map was developed for peanut by analyzing an F2 population from the diploid (2n = 2x = 20) interspecific cross of A. stenosperma Krapov. and W.C. Gregory (ace, HLK 410) and A. cardenasii Krapov.and W.C. Gregory (ace, GKP 10017). The linkage map covered 1063 cM with 117 markers in 11 linkage groups [20]. Fifteen unassociated markers also were reported. A second molecular map of peanut was created by Burow, Patterson, and Simpson using the tetraploid cross Florunner X 4x [A. batizocoi Krapov. and W.C. Gregory (A. cardenasii X A. diogoi Hoehne)] Burow (pers. commun.). Most of the 380 RFLP markers that have been mapped had disomic inheritance, with the exception of one linkage group which may be polysomic.

#### *4.2.2 Simple sequence repeats*

Simple sequence repeats (SSRs) are genomic fragments that consist of tandemly repeated units that are present in both coding and non-coding regions of the genome [21]. SSR markers, designed by flanking sequences, are useful for and widely applied in plant genetic analyses and marker-assisted selection breeding. Currently, g-SSR markers are common and popular for such analyses, and they have wide applications in molecular genetics and breeding, because they have multiple advantages, such as simplicity, abundance, ubiquity, variation, co-dominance, and multi-allelism [21]. Even though the peanut genome had not yet been resolved. With the recent completion of genome sequencing of peanut and two diploid progenitor species, A. duranensis and A. ipaensis, a large number of genome-wide g-SSRs were identified. SSR markers linked to resistance to early leaf spot, groundnut rosette disease, and rust and aflatoxin contamination across African cultivated groundnut varieties were identified useful to identify suitable parents for mapping populations or breeding [22].

Genotypes with similar genetic backgrounds tended to cluster in the same subgroup, indicating the effectiveness of SNP markers in assigning the tested genotypes into homogenous groups [23].

Simple sequence repeat (SSR) alleles associated with agronomic traits in at least two environments. These markers were further investigated for their potential use in genetic studies by ascertaining their genetic diversity in the natural population.

#### *4.2.3 Inter simple sequence repeats*

Inter Simple Sequence Repeats (ISSR) marker has been reported as a rapid, reproducible, and cheap fingerprinting technique based on the variation found in the regions between microsatellites. It is a fast, inexpensive genotyping technique based on variation in the regions between microsatellites (Inter Simple Sequence Repeats analyses offer breeders and geneticists with competent means to link phenotypic and genotypic variations in various fields of plant improvement) [24].

#### *4.2.4 Randomly amplified polymorphic DNA markers*

Among the molecular markers, random amplified polymorphic DNA (RAPD) is a rapid method for developing genetic maps and to determine DNA fragments to characterize peanut cultivars. PCR based Randomly Amplified Polymorphic DNA markers are good genetic markers because they give rapid results, economically convenient and use small oligonucleotide primers. With a small quantity of template, a very large number of fragments are generated from different regions of the genome and hence, multiple loci may be examined very quickly [25, 26].

#### *4.2.5 Diversity arrays technology*

Diversity Arrays Technology (DArT), which is based on genome complexity reduction and SNP detection through hybridization of PCR fragments has been used in genome-wide association studies (GWAS), construction of dense linkage maps and mapping quantitative trait loci (**Table 4**) [27].

SSR, simple sequence repeat markers, TEM, Transposable element markers, RAPD, random amplified polymorphic DNA.


#### **Table 4.**

*Some molecular marker systems developed for genetic analysis and breeding in groundnut.* Source: *[6, 7].*

#### **5. Perspectives**

Molecular markers can assist in the selection process with phenotypic selection and speed up the pace of breeding cycle, in recent times technologies such as next generation sequencing i.e. low with high throughput, Genome Selection and Genotype by sequencing can be used to achieve the desired goal in molecular breeding approaches. The genome/gene space sequence would provide the opportunities to link the phenotype with genes. The future of peanut genomics and use of molecular tools in breeding seems to be bright that will ensure the peanut improvement for different production as well as quality constraints.

#### **6. Conclusion**

Molecular markers are used to identify quantitative trait loci which enhance the efficiency of selecting complex trait in plant breeding. MAS is an efficient molecular tool for breeding, in which markers linked with the desired genes are used for indirect selection for that gene in non-segregating or segregating populations. Now a days, DArT, SSR, SNP, ISSR, etc. with high throughput technologies are very exciting markers, which enhances the crop with desired traits and induces tolerance against biotic and abiotic stresses in a short period of time.

Molecular markers can provide information that can help define the distinctiveness of species and their ranking according to the number of close relatives and their ranking according to the phylogenetic position. RAPD patterns generated from peanut cultivars could be used as genomic fingerprint to establish the identity of a given genotype. The utilization of DArT marker system may limit efficient genetic analysis of groundnut genetic resources for cultivar development. Development of highly discriminative and informative DArT markers is useful for genetic analysis and breeding in groundnut.

A desirable molecular marker should have high polymorphism, frequent occurrence, should be easy to use and should be quick, co-dominant inheritance, equally dispersed all over the genome, high transferability and reproducibility, less expensive and phenotypically neutral. However, it will still take some time before cost-effective SNP genotyping platforms are available for genotyping the tetraploid peanut germplasm collections or peanut mapping populations. Extension of SNP-based maps to the tetraploid has not been accomplished yet, and will require separation of A- and B-genome sequences, but is expected to greatly accelerate genetic mapping and marker-assisted selection.

#### **Acknowledgements**

Wallaga University and Haramaya University Fully acknowledged for giving chance to Fellow Doctor of Philosophy.

Seltene Abady (PhD) is sincerely acknowledged for guiding me the review.

#### **Author details**

Diriba Beyene Goonde1,2\* and Seltene Abady2

1 Department of Philosophy, Research and Technology, Research and Technology Park, Wallaga University, Ethiopia

2 Haramaya University, Ethiopia

\*Address all correspondence to: hordofabeyene2018@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Krapovickas A and Gregory WC. 2007. Taxonomy of the Genus Arachis (Leguminosae). Translated by DE Williams, CE Simpson. Bonplandia. 16:1-205

[2] Baraker B, Jha SK, Wani SP, Garg KK. Effect of balanced fertilizer management practices on factor of productivity on Groundnut (*Arachis hypogaea* L.) cultivation. International Journal of Chemical Studies. 2017;**5**(4):1288-1291

[3] Janila P, Nigam SN, Pandey MK, Nagesh P, Varshney RK. Groundnut improvement: Use of genetic and genomic tools. Frontiers in Plant Science. 2013;**4**:1-16

[4] Wakjira A. A review of the recent groundnut breeding activities in Ethiopia. In: Paper Presented at the First National Oilseeds Work Shop, 3-5 Dec. Addis Ababa, Ethiopia. 1991

[5] FAO. Rome, Italy: Food and Agriculture Organization of the United Nations; 2021

[6] Abady S, Shimelis H, Janila P. Farmers' perceived constraints to groundnut production, their variety choice and preferred traits in eastern Ethiopia: Implications for drought-tolerance breeding. Journal of Crop Improvement. 2019a;**33**:1-17. DOI: 10.1080/154275 28.2019. 1625836

[7] Abady S, Shimelis H, Janila P, Mashilo J. Groundnut (Arachishypogaea L.) improvement in sub-Saharan Africa: A review. Acta Agriculturae Scandinavica, Section B-Soil & Plant Science. 2019;**69**(6):528-545. DOI: 10.1080/09064710.2019.1601252

[8] Sato S, Nakamura Y, Kaneko T, Asamizu E, Kato T, Nakao M, et al. Genome structure of the legume, *Lotus japonicus*. DNA Research. 2008;**15**:227-239

[9] Guimaraes PM et al. Global transcriptome analysis of two wild relatives of peanut under drought and fungi infection. BMC Genomics. 2012;**13**:387

[10] Arus P, Moreno-Gonzalez J. Markerassisted selection. In: Hayward MD, Bosemark NO, Romagosa I, editors. Plant Breeding: Principles and Prospects. London: Champman and Hall; 1993. pp. 314-331

[11] Das G, Patra JK, Baek KH. Insight into MAS: 2017 a molecular tool for development of stress resistant and quality of rice through gene stacking. Frontiers in Plant Science. 2017;**8**:985

[12] Kumpatla SP, Buyyarapu R, Abdurakhmonov IY, Mammadov JA. Genomics-assisted plant breeding in the 21st Century. In: Abdurakhmonov I, editor. Technological Advances and Progress. In Tech; 2012. pp. 132-184

[13] Varshney RK, Hoisington DA, Tyagi AK. Advances in cereal genomics and applications in crop breeding. Trends in Biotechnology. 2006;**24**:490-499

[14] Pandey M et al. Advances in Arachis genomics for peanut improvement. Biotechnology Advances. 2012;**30**:639-651

[15] Si L, Chen J, Huang X, Gong H, Luo J, Hou Q, et al. OsSPL13 controls grain size in cultivated rice. Nature Genetics. 2016;**48**:447-456. DOI: 10.1038/ng.3518

[16] Kochert G, Halward TM, Branch WD, Simpson CE. RFLP variability in peanut cultivars and wild species. Theoretical and Applied Genetics. 1991;**81**:565-570

[17] Stalker HT. A morphological appraisal of wild species in section Arachis of peanuts. Peanut Science. 1990;**17**:117-122

[18] Stalker HT, Simpson CE. Genetic resources in Arachis. In: Pattee HE, Stalker HT, editors. Advances in Peanut Science. American Peanut Research and Education Society. Stillwater. 1995. pp. 14-53

[19] Kochert G, Stalker HT, Ginenes M, Galgaro L, Moore K. RFLP and cytogenetic evidence for the progenitor species of allotetraploid cultivated peanut, Arachis hypogaea (Leguminosae). American Journal of Botany. 1996;**83**:1282-1291

[20] Halward TM, Stalker HT, Kochert G. Development of an RFLP linkage map in diploid peanut species. Theoretical and Applied Genetics. 1993;**87**(3):379-384

[21] Haq SU, Jain R, Sharma M, Kachhwaha S, Kothari SL. Identification and characterization of microsatellites in expressed sequence tags and their cross transferability in different plants. International Journal of Genomics. 2014;**2014**:863948

[22] Kanyika BTN, Lungu D, Mweetwa AM, Kaimoyo E, Njung'e VM, Monyo ES, et al. Identification of groundnut (Arachis hypogaea) SSR markers suitable for multiple resistance traits QTL mapping in African germplasm. Electronic Journal of Biotechnology. 2015;**18**:61-67

[23] Adu BG, Badu-Apraku B, Akromah R, Garcia-Oliveira AL, Awuku FJ, Gedil M. Genetic diversity and population structure of earlymaturing tropical maize inbred lines 146 using SNP markers. PLoS One. 2019;**14**(4):e0214810. DOI: 10.1371/ journal. Pone. 0214810

[24] Tadele S, Mekbib F, Tesfaye K. Genetic diversity of coffee (Coffea arabica L.) landraces from Southern Ethiopia as revealed by inter simple sequence repeat marker. Global Advanced Research. Journal of Agricultural Science. 2014;**3**(1):024-034

[25] Edae EA, Byrne PF, Haley SD, Lopes MS, Reynolds MP. Genome-wide association mapping of yield and yield components of spring wheat under contrasting moisture regimes. Theory Applied Genetics. 2014;**127**:791-807

[26] Kumar A, Jain S, Elias EM, Ibrahim M, Sharma LK. An overview of QTL identification and marker-assisted selection for grain protein content in wheat. In: Eco-friendly Agro-biological Techniques for Enhancing Crop Productivity. Singapore: Springer; 2018. pp. 245-274

[27] Abu Zaitoun SY, Jamous RM, Shtaya MJ, Mallah OB, Eid IS, Ali-Shtayeh MS. Characterizing palestinian snake melon (Cucumis Melo Var. Flexuosus) germplasm diversity and structure using SNP and DArTseq markers. BMC Plant Biology. 2018;**18**:246

### *Edited by Ziyad S. Haidar*

*DNA Replication - Mechanisms, Epigenetics, and Gene Therapy Applications* presents the latest trends of DNA replication research by reviewing the fundamentals of molecular mechanisms of initiation and termination, functional cross-talk and/or association to the genome, DNA recombination and repair, analytical methods, recent applications, and future directions in the field. This book brings together and synthesizes contributions from active and prominent researchers in the field whose chapters address such topics as basic molecular biology, cell growth and division, DNA isolation and duplication, DNA damage response, epigenetics, DNA methylation, gene silencing, analytical and computational tools, and much more.

Published in London, UK © 2023 IntechOpen © ktsimage / iStock

DNA Replication - Epigenetic Mechanisms and Gene Therapy Applications

DNA Replication

Epigenetic Mechanisms and Gene

Therapy Applications

*Edited by Ziyad S. Haidar*