*3.3. Fibrin*

Fibrin is a blood-derived fibrous protein formed by fibrinogen polymerization under the catalysis of thrombin, calcium ions, and/or factor XIII. Fibrinogen powder can dissolve in water to form a solution with low viscosity. Like gelatin, fibrin has excellent biocompatibility and biodegradability for various biomedical applications, such as hemostasia, wound healing, pharmacy, microencapsulated cell delivery, tissue engineering, and 3D bioprinting [104].

As stated above, layer-by-layer bioprinting is a logical choice for the manufacture of stratified organs, such as the liver, heart, kidney, and lung, containing multiple cell types. 3D bioprinting of stratified organ replacements depends on bioinks with appropriate rheological and cytocompatible properties. An obvious shortcoming of the fibrinogen solution for 3D organ bioprinting is that its viscosity is too low to be piled up in layers even in very high concentration (e.g., 4% w/v) [55]. After polymerization, the 3D printed constructs undergo further contraction or shrinkage for homogeneous or heterogeneous tissue formation. Structural stability is the major concern for pure fibrinogen as a bioink for 3D organ bioprinting.

The first pertinent fibrin 3D bioprinting was reported in 2007, in which fibrinogen was used as an additive of gelatin-based bioinks [105,106]. By blending fibrinogen with thermoresponsive gelatin solutions, high-resolution 3D constructs with excellent cell viability (i.e., ≈ 100%) have been obtained. During the 3D printing process, gelatin molecules provide fast gelation and immediate postprinting structural fidelity, while fibrinogen molecules ensure long-term mechanical stability upon thrombin polymerization. Physical blending and chemical crosslinking of fibrinogen/gelatin solutions can prevent breakdown and shrinkage of 3D printed constructs for a relatively longer period under physiological conditions (e.g., 37 ◦C).

More than 10 years later, Hinton and colleagues printed a Ca2+-mediated alginate/fibrinogen hydrogel into a gelatin slurry bath containing thrombin using a similar extrusion-based 3D printer and working principle [16,17]. When the alginate/fibinogen hydrogel was extruded from the 3D printer nozzle, it met the thrombin molecules in the bath and rapidly polymerized to solidify the Ca2+-mediated alginate/fibrinogen hydrogel. This is another double crosslinking or gelling path for 3D bioprinting with fibrinogen-containing composite bioinks and is termed as "bioplotting" (Figure 3). With different gelling paths, cells in the fibrinogen-containing hydrogel can be assembled in layers according to the predesigned CAD models.

Similarly, Burmeister and colleagues delivered ASCs via PEG–fibrin, i.e., PEGylated fibrin (FPEG) hydrogel, as an adjunct to meshed split-thickness skin grafts in a porcine model [107]. They showed that ASCs delivered in FPEG could enlarge the blood vessel size in a dose-dependent way, wherein FPEG acts as both a porous scaffold to prevent contraction and an ASC-delivery vehicle to accelerate angiogenesis.

For 3D organ bioprinting, the printing properties of fibrinogen solutions can be easily tailored by varying the proportion of its component in the composite bioinks [108–111]. Thermosensitive polymers, such as gelatin and agar, support extrusion-based 3D bioprinting of a wide range of natural polymers, including fibrinogen, chitosan, alginate, and hyaluronate. Succeeding (or tandem) chemical crosslinking and polymerization of fibrinogen-containing composite bioinks facilitates the printing results of the stratified cell-laden 3D constructs with increased stiffness and stabilization. For example, double gelation of gelatin/alginate/fibrinogen bioinks using both CaCl2 (for alginate ion crosslinking) and thrombin (for fibrinogen polymerization) is the most popular approach today for large-scale organ manufacturing (Figure 8) [30,36].

**Figure 8.** 3D bioprinting of ASC-laden gelatin/alginate/fibrin hydrogel for organ manufacturing in Prof. Wang's laboratory at Tsinghua University: (**a**) a pioneering double-nozzle 3D bioprinter made in this laboratory; (**b**) schematic description of the cell-laden gelatin/alginate/fibrin hydrogel and pancreatic islets being printed into a grid construct using the 3D bioprinter; (**c**) a large-scale 3D printed grid construct containing ASC-laden gelatin/alginate/fibrin hydrogel cultured in a plate; (**d**) a grid ASC-laden gelatin/alginate/fibrin construct after being cultured for one month; (**e**) a multicellular construct after three weeks of culture, containing both ASCs encapsulated in the gelatin/alginate/fibrin hydrogel before epidermal growth factor (EGF) engagement and relatively integrated pancreatic islets seeding in the predefined channels (immunostaining with anti-insulin in green); (**f**) some envelopes of the islets were broken after one month of culture; (**g**) immunostaining of the 3D construct with mAbs for CD31+ cells (i.e., mature endothelial cells from the ASC differentiation after three days of culture with EGF added in the culture medium) in green, having a fully confluent layer of endothelial cells (i.e.,

endothelium) on the surface of the predefined channels; (**h**) a vertical image of the 3D construct showing the fully confluent endothelium (formed from endothelial cells) and the predefined go-through channels; (**i**) immunostaining of the 3D construct with mAbs for CD34+ cells (i.e., endothelial cells) in green and pyrindine (PI) for cell nuclei (nucleus) in red; (**j**) immunostaining of the 3D construct with mAbs for CD34+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium; (**k**) immunostaining of the 3D construct with mAbs for CD31+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture with EGF added in the culture medium; (**l**) a control of (k), immunostaining of the 3D construct with mAbs for CD31+ endothelial cells di fferentiated from the ASCs in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium; ( **m**) immunostaining of the 3D construct with mAbs for CD31+ cells in green and Oil Red O staining for adipocytes in red, showing both the heterogeneous tissues coming from the ASC di fferentiation after a cocktail growth factor engagement (i.e., on the surface of the channels, the endothelium coming from the ASCs di fferentiation after being treated with EGF for 3 days; deep inside the gelatin/alginate/fibrin hydrogel, the adipose tissue coming from the ASC di fferentiation after being subsequently treated with insulin, dexamethasone, and isobutylmethylxanthine (IBMX) for another three days); (**n**) a control of (m), showing all the ASCs in the 3D construct di fferentiated into target adipose tissue after three days of treatment with insulin, dexamethasone, and IBMX but no EGF; (**o**) immunostaining of two-dimensional (2D) cultured ASCs with mAbs for CD31+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture with EGF added in the culture medium; (**p**) immunostaining of 2D cultured ASCs with mAbs for CD31+ endothelial cells in green and PI for cell nuclei (nucleus) in red after three days of culture without EGF added in the culture medium. Images reproduced with permission from [36].


Preface

*Molecular Mechanisms in Cancer*, created with experts in their fields from different countries, consists of twelve chapters organized into three sections: "Oncogenes," "Tumor Suppressor Genes," and "Viral Oncogenes."

Section 1 includes four chapters that discuss the effects and importance of oncogenes in various cancer types. Chapter 1 examines the roles and importance of alternative mRNA splices in cancer development, changes in intron-exon connections, oncogenic properties, and the roles of eukaryotic polycistronic gene mechanisms in the development of various cancers. Chapter 2 discusses molecular mechanisms in various types of skin cancers as well as the effects of external factors originating from the sun, especially ultraviolet light, and cancers that develop by changing molecular mechanisms. Chapter 3 addresses oncogene and tumor suppressor mechanisms in head and neck squamous cell cancer types and their clinical importance. Chapter 4 describes the potential application of targeted oncogene-dependent gene therapies in cancer treatments.

Section 2 includes three chapters that address the mechanisms of tumor suppressor genes in cancers and their effects on cancer development. Chapter 5 discusses tumor suppressor genes affecting breast cancer, especially the additives of BRCA1 and BRCA2. Chapter 6 examines the roles of the two functions of cellular stress and Nrf2 in cancer development, their contribution to chemoresistance, and their relationship with oxidative stress. Chapter 7 explains molecular changes in benign prostate cancers.

Section 3 includes five chapters that examine the epidemiology of viral oncogenes and their roles in various cancers. Chapter 8 discusses the oncogenic activities and epidemiological behavior of human papilloma viruses (HPVs) in Burkina Faso, West Africa. Chapter 9 provides an overview of the clinical features of viral oncogenic viruses in cervical cancer. Chapters 10 and 11 provide extensive information on the roles of HPV infections in urological cancers. Finally, Chapter 12 discusses the applications of HPV vaccines in different parts of the world and the effects of the ethical or moral behaviors of these societies.

With my respect and thanks to my father, Ali Budak, who provided everything for my education and passed away from cancer.

> **Metin Budak** Trakya University Faculty of Medicine, Biophysics Department, Mirko Tos Ear and Hearing Research Center, Trakya University, Edirne, Turkey

**Rajamanickam Rajkumar** Meenakshi Medical College Hospital and Research Institute MAHER, Kanchipuram, Tamil Nadu, India

Section 1 Oncogenes

## **Chapter 1** Splicing in Cancer

*Mehdi Moghanibashi and Parisa Mohamadynejad*

## **Abstract**

Defects in splicing, especially alternative splicing have been frequently found in cancers. Mutations in the splicing regulatory elements of important genes involved in cancers or the genes encoding regulatory splicing machinery could play a key role in carcinogenesis. Alterations in regulator factors in splicing have emerged as a new class of oncoproteins and tumor suppressor genes. Understanding the molecular mechanism of how defects in splicing and in particular alternative splicing are involved in carcinogenesis, could lead to new strategies to cancer therapy. Here, we review the molecular mechanism of splicing and regulatory factors involved in alternative splicing, as well as the aberrant splicing that affects cancer hallmarks. Finally, we summarize new approaches in cancer therapy based on splicing.

**Keywords:** splicing, alternative splicing, spliceosome, oncogene, tumor suppressor gene

## **1. Introduction**

Split genes in eukaryotes were discovered in 1977 for the first time. It was later indicated that the intron sequences should be removed by the spliceosome complex in the splicing process in the nucleus [1].

There are 20,687 protein-coding genes with introns (93%) and 1713 (7%) intron-less protein-coding genes in human genome [2]. It is interesting to note that only ∼4% of genes in the schizosaccharomyces cerevisiae contain introns and most of them are single intron genes [3]. The number of introns per gene varies in eukaryotic genomes so that in the human genome is 8.94, and in the fungi (0.05–3.43 introns per gene), plants (0.33–7.30 introns per gene), invertebrates (2.92–7.42 introns per gene) and vertebrates (7.35–10.09 introns per gene) [2].

The length of introns is less than 100 up to 100,000 bp in different genes of various organisms [3]. Unlike lower eukaryotes, there are relatively long introns and short exons in vertebrates [4] but in general, fungal introns are relatively short (93% of the introns in fungi are shorter than 250 nt). However, in invertebrates and plants, the average percentage of short introns (<250 nt) is 48% and 59%, respectively. In contrast, 48% of vertebrate introns are longer than 1000 nt [2].

In the present chapter, we explain the various types of splicing, the molecular mechanism of splicing, regulatory elements and factors in splicing, and finally the alternative splicing and the role of aberrant splicing in cancer are discussed.

## **2. Mechanism of cis-splicing**

The classic and ubiquitous of exons joining from the same pre-mRNA is called cis-splicing. Highly conserved splicing machinery in all eukaryotes shows that splicing is pivotal for all eukaryotic organisms [3]. Five small RNAs (snRNAs), including U1, U2, U4, U5, and U6 (because they are uracil-rich) and approximately 300 related proteins including hnRNPs and SR proteins, make up the spliceosome complexes and are involved in the splicing regulation.

The junction of introns and exons is identified by spliceosome and the "GU-AG" rule is mainly used. Four regions play a key role in splicing, including (a) the GU sequences at the beginning of the introns as 5′ splice-site (5′-SS or donor site), (b) AG dinucleotides at the end of intron referred to 3′ splice-site (3′-SS or acceptor site), (c) the sequences without high conservation in the upstream of the 3′SS called branch point sequence (BPS), and (d) polypyrimidine-rich sequences which is locating between the BPS and the 3′ SS [1].

For conventional splicing, first of all, the complex E (early) is formed by ATP-independent binding of U1 snRNP (small nuclear ribonucleoprotein) to 5′-SS via base-pairing, SF1 (splicing factor 1) to BPS, U2AF2 to polypyrimidine tract, and U2AF1 to 3′-SS. Second, SF1 is replaced by U2 snRNP (with the assistance of U2AF) through the base-pairing to BPS to form complex A. This step is ATP-dependent and reinforced by the SF3a and SF3b protein complexes, as well as U2AF2 and U2AF1. Third, complex B (inactive) is formed by rearranging complex A using recruiting U5/U4/U6 in such a way that U4 and U6 snRNPs bind strongly to each other due to complementary pairing of their RNA components, but U5 snRNP is weakly bound to them (through protein–protein interaction). Following several conformational changes, U1 snRNP leaves mRNA, U6 snRNP binds to 5′-SS, and simultaneously, U4 snRNP leaves mRNA so that U6 and U2 snRNPs pair together through their snRNA, leading to the formation of pre-catalytic (active) spliceosome complex B\* (**Figure 1**). Finally, removal of introns and joining of exons are performed using two transesterification reactions by complex C [1] (**Figures 1** and **2**).

**Figure 2.** *Molecular mechanism of exon joining [6].*

## **3. Regulation of splicing**

Cis-acting elements in RNA constitute a set of rules called "splicing code" that acts as an anchor for trans-acting factors to produce functional transcripts [7]. There is a competition between various splicing factors for the binding to the splicing regulatory elements and in turn the increasing or decreasing the recruitment of spliceosome complexes to premature mRNA.

Generally, the serine/arginine (SR)-rich protein family bind preferentially to exonic splicing enhancer (ESEs) and intronic splicing enhancer (ISEs) sites and play a positive role in the splicing (exon inclusion). However, heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to exonic splicing silencer (ESSs) and intronic splicing silencer (ISSs) elements and generally suppress exon inclusion (**Figure 3**) [1, 5].

In addition to the characteristic domain of RS (arginine/serine dipeptides) in SR proteins, there is an RNA recognition motif (RRM) which is the basis for the classification of SR proteins. In humans, there are 12 types of SR proteins, now called serine/arginine-rich splicing factor (SRSF) 1–12. RS domain of different SR proteins binds to ESE, could directly interact with each other and facilitate the recruitment of spliceosome machinery components including U1 snRNP or U2AF to mRNA, and more importantly, it has been suggested that the SR proteins bound to ESEs play a key role in exon definition in constitutive and alternative splicing.

**Figure 3.**

*Control of splicing by cis-acting elements and splicing factors [5, 7, 8].*

There are no canonical SR proteins in Saccharomyces cerevisiae and three SR-like proteins including Npl3, regulate splicing efficiency [3].

RRM is also the most common domain in the hnRNPs for the interactions with the premRNA, but hnRNPs also contain another domain called the RGG boxes including Arg-Gly-Gly tripeptides repeats. hnRNP proteins as same as SR proteins, bind directly to a specific site in the target premRNA, but inhibit splicing via blocking the binding of snRNPs to targets or looping out exons [3, 9].

## **4. Self-splicing**

Some introns are spliced without requiring to spliceosome complex (self-splicing), including group I introns (found in bacteria, bacteriophages and eukaryotes including organelle and nuclear genomes) and group II introns (found in bacteria, archaea, and eukaryotic organelles). In both, trans-esterification reactions need to excise the intron and ligate the exons.

The difference between them is that the splicing reaction is initiated by a guanosine cofactor and an internal adenosine in group I and group II introns, respectively. Also, during splicing, group II introns form a lariat like structure, whereas group I introns do not (**Figure 4**) [10].

**Figure 4.** *Comparison of a self-splicing group I, group II introns and spliceosome-catalyzed splicing [10].*

## **5. Trans-splicing**

In addition to cis-splicing, there is another type that joins exons on the separate pre-mRNAs and results in the chimeric mRNAs originating from two different genes so-called trans-splicing and commonly occurs in unicellular organisms and caenorhabditis elegans. Interestingly, acyl-CoA-cholesterol acyltransferase 1, RGS12 and CYP3A genes produce mRNA that derived from different genes in human [11].

## **6. Alternative splicing (AS)**

Different combination of exons in a special type of splicing, called alternative splicing, which lead to the formation of multiple protein isoforms from a single premRNA and promotes the transcriptome and proteome complexity. A recent analysis of the Encyclopedia of DNA Elements (ENCODE) project 1 has been revealed that the human genome consists of approximately 21,306 protein-coding genes [12, 13] and it is estimated that 92–94% of human genes harbor alternative splicing [3, 14, 15].

The most common type of alternative splicing mechanism (40% of events) is exon skipping (also called cassette exon) [4] in which one or several exons are not included in the final mRNA (**Figure 5a**) but the rarest mechanism of AS is mutually exclusive exons (**Figure 5d**) [17]. Changing in the selection of either 3′- or 5′-splice sites are other mechanisms of AS (**Figure 5b** and **c**). The fifth type of alternative splicing is intron retention (**Figure 5e**), in which an intron remains in the mature mRNA (the most common after the exon skipping). Applying alternative promoters and unusual polyadenylation are other alternative splicing mechanisms (**Figure 5f** and **g**) [16]. Various trans-acting factors, as well as specific cis-acting elements, play a key role in alternative splicing.

Expression alteration in splicing trans-acting factors and mutations (also, polymorphisms) including single-base substitutions, deletion, insertion, and translocation in cis-elements and trans-regulatory factors that encodes genes involved in splicing, would cause abnormal splicing and lead to diseases and

**Figure 5.** *Schematic representation of different types of alternative splicing [16].*

cancer. In addition, the post-modification of trans-regulatory factors and chromatin remodeling could affect splicing by altering the recognition of splicing factors and splicing sites (**Figure 6**) [5].

## **7. Role of splicing in cancers**

Gene duplication and overexpression of SRSF1 were the first evidences that alternative splicing plays an important role in tumor growth [18]. Recently, it has been reported that splicing may be correlated with the various types of cancers, and in particular, alternative splicing can affect various aspects of cancer hallmarks [8, 19] (**Figure 7**) and splicing modulators have emerged as a new class of oncoproteins and tumor suppressor genes. Importantly, RNA sequencing analysis has shown that one of the common mechanisms of tumor suppressor genes inactivation in cancers is intron retention [20].

Recently, RNA-seq studies have been reported that one of the major differences between transcriptome of normal and cancerous tissues are caused by aberrant splicing with AML and hepatocellular carcinoma show the highest and lowest

**Figure 6.** *Various mechanisms for aberrant splicing [5].*

#### **Figure 7.**

*Effects of aberrant alternative splicing on different hallmarks of cancer. Arrows up: Most contributor, arrows down: Least contributor, EMT (epithelial–mesenchymal transition) [8].*

frequency of alternative splicing alteration compared to the corresponding normal tissues, respectively. The most extensive study ever performed on the splicing profile of various types of cancers involving more than 8700 patients from The Cancer Genome Atlas (TCGA) data, shows that alternatively splicing in cancerous tissues is 30% more than normal tissues [7]. Expression dysregulation and frequent somatic

mutations in the splicing factors coding genes have been identified in more than 2% of tumors in several cohorts of patients, including TCGA data (**Figure 8**) [21].

## **7.1 Deregulation of splicing factors expression**

A wide range of factors involved in splicing and regulation of alternative splicing has been shown to be dysregulated in gene expression level. For example, serine and arginine-rich splicing factor 1 (SRSF1), a key player of alternative splicing, is involved in tumorigenesis promotion by splicing of RAC1, Tyrosine-protein kinase (SYK), MKI67 and HNRPLL genes. Specifically, in colorectal cancer, SRSF1 and other members of the SR family, including SRSF3, SRSF6, SRSF7 and SRSF10, are overexpressed and referred to as oncogenes [5, 22].

Bcl-x protein, which plays a key role in the regulation of intrinsic pathway of apoptosis, harbor alternative splicing and can obtain two different isoforms Bcl-xl (involved in apoptosis inhibition) and Bcl-xs (participated in apoptosis promotion) [23]. PTB protein 1 preferentially promote Bcl-xs isoform that leads to apoptosis, but down-regulation of PTB protein 1 could produce Bcl-xl, which inhibits apoptosis and result in tumorigenesis [24].

Some aspects of the oncogenic role of C-Myc in tumors, are changing splicing patterns. For example, it can overexpress the ITGA6A variant [the pro-proliferative isoform of integrin subunit α6 gene (ITGA6)] compared to ITGA6B isoform

#### **Figure 8.**

*Frequently alteration of splicing-factors in human cancers. MDS: Myelodysplastic syndrome; CMML: Chronic myelomonocytic leukemia; HN: Head and neck; w/o RS: With or without ringed sideroblasts; RARS/RCMD: Refractory anemia with ringed sideroblasts and refractory cytopenia with multilineage dysplasia and ringed sideroblasts; MPN, myeloproliferative neoplasm [21].*

in the colon cancer, which is mediated by epithelial splicing regulatory protein 2 (ESRP2) [22].

Tumor cells preferentially metabolize glucose via the aerobic glycolysis pathway compared to normal cells. Conversion of phosphoenolpyruvate (PEP) to pyruvate is performed by PKM (Pyruvate kinase) enzyme in the last step of glycolysis. PKM is encoded by PKLR (expressed mainly in the liver and hematopoietic cells) and PKM (expressed in most tissues) genes in which both of them harbor alternative splicing [25, 26]. Alternatively, splicing of the PKM gene results in PKM1 isoforms (mainly in normal cells) and PKM2 isoforms (mainly in tumor cells) [27, 28]. The polypyrimidine tract-binding protein (PTB), hnRNPA1 and hnRNPA2 genes are overexpressed by C-Myc and lead to upregulation of PKM2 isoform of pyruvate kinase (PKM) through splicing changing and lead to cell proliferation (**Figure 9**) [29, 30].

Vascular endothelial growth factor A (VEGF-A) is one of the key proteins in the angiogenesis process in tumor progression. The VEGF- A encoding gene produces various isoforms (angiogenic and antiangiogenic) using alternative 3′ and 5′-SSs in exons 6, 7, and 8 that can regulate angiogenesis (**Figure 10**) [31]. Interestingly, angiogenic VEGF isoforms are induced by Serine/arginine-rich protein-specific splicing factor kinase (Srpk1) and SRSF1 protein, which are activated by Wilms' tumor suppressor 1 (Wt1) protein [32].

### **7.2 Mutation in cis-acting elements and genes encode for trans-acting factors**

It has been suggested that various mutations, including silent or synonymous mutations, maybe results in intron retention (often in tumor suppressor genes and lead to premature stop codon) or exon skipping through create and loss of exonic splicing enhancers, silencers, or create novel splice sites [7]. It has been reported that 40% of patients with hereditary non-polyposis colorectal cancer carry the MLH1 mutation, and interestingly, the most common mutation in this gene is located at splicing sites [33] which was in line with bioinformatics analysis results [34]. The most somatic mutations in the splicing factors encoding genes have been observed in bladder cancer and uveal melanoma [7].

Recently, in several cancers, a recurrent mutation in the U1 snRNA encoding gene has been found, which mainly affects the third base of U1 snRNA at the splice

#### **Figure 9.**

*Regulation of metabolic shift between oxidative phosphorylation and aerobic glycolysis through alternative splicing of PKM gene. In tumors, c-Myc and possibly other factors are involved in the upregulation of the hnRNPA1, A2, and PTB genes which result in exon 9 exclusion and exon 10 inclusion and PKM2 generation. PEP to pyruvate conversion is catalyzed less efficiently by PKM2 compared to PKM1, resulting in enhancement of metabolites for anabolic metabolism [28].*

#### **Figure 10.**

*The structure of the VEGF-A gene includes eight exons (a) and producing of various isoforms by alternative splicing (b). Two mRNA isoform families, pro-angiogenic (VEGF-Axxx, b. left) and anti-angiogenic (VEGF-Axxxb, b. right) are generated by alternative splicing. (xxx shows the amino acid number of the mature protein). The transcriptional start site (TSS), translational start site (ATG) in exon 1, and alternative stop codons within exon 8 (TGA1 and TGA2) are indicated [31].*

site recognition sequence that pairs directly with 5′SS. In addition, mutated SF3B1 (**Table 1**) recognizes different BPS and results in cryptic 3′ splice sites selection. Also, the SRSF2 and U2AF1 mutations alter splice site recognition in a sequencedependent manner and results in alternative 3′ splice site selection and altered exon inclusion, respectively (**Figure 11** and **Table 1**) [7, 8]. It has been suggested that

*\* BLCA - Bladder Urothelial Carcinoma, COAD - Colon adenocarcinoma, DLBC - Lymphoid Neoplasm Diffuse Large B-cell Lymphoma, KICH - Kidney Chromophobe, LAML - Acute Myeloid Leukemia, LUAD - Lung adenocarcinoma, LUSC - Lung squamous cell carcinoma, READ - Rectum adenocarcinoma, SKCM - Skin Cutaneous Melanoma, STAD - Stomach adenocarcinoma, UCEC - Uterine Corpus Endometrial Carcinoma, UCS - Uterine Carcinosarcoma, UVM - Uveal Melanoma.*

#### **Table 1.**

*Mutation of splicing factors encoding genes in cancers [7].*

#### **Figure 11.**

*Effects of cancer-associated mutations in splicing factors on alternative splice site selection. Processing patterns favored by the mutations are shown by thicker lines. WT: Wild type [8].*

these mutations may also affect the protein-protein interaction domains and lead to alteration of splicing factors interaction with nucleosomes, which plays an important role in driving splice site definition [7].

## **8. Splicing and resistance to drugs in cancers**

Response of the tumors to therapy may be affected by splicing and resistance to treatment may be reinforced by alternative splicing [35]. Alternative splicing could induce resistance to drugs in cancer through changing factors that are involved in the metabolism of drugs including uptake, transportation and inactivation (**Figure 12**).

#### **Figure 12.**

*Aberrant alternative splicing in some oncogenes and resistance to therapy in cancers. ATG1 and ATG2: Start codon for full-length and short mRNA. TGA: Stop codon [36].*

One of the most mutated genes in melanoma cancer is BRAF that V600E mutation is the most prevalence mutation in this gene and result in a variant without exons 4–8 (give rise to lack of the RAS-binding domain of BRAF rendered cells insensitive to RAF inhibition) and lead to resistance to vemurafenib in 30% patients of melanoma. One of the strategies for prostate cancer therapy is using antiandrogen specially enzalutamide (antagonist of the interaction of androgens with AR) and abiraterone (blocker of androgen biosynthesis) to inhibit the activity of the androgen receptor (AR). HNRNPA1 could drive a splice variant of AR called ARv7 arises due to the inclusion of a cryptic exon CE3 and result in antiandrogen treatment [7, 36].

In addition to chemo and hormone therapy in cancers, radiotherapies are used for treatment and several factors involved in tumor sensitivity to ionizing radiation that some of them affected by alternative splicing. Interestingly, p73 (a transcription factor) is one of the most important proteins that could act as a marker for the sensitivity of tumor cells to radiation. There are two isoforms of the p73 including the full-length TAp73 and the N-terminally truncated ΔNp73 which truncated isoform has been indicated that strongly associated with poor survival and negative response to irradiation [16].

## **9. Cancer therapy based on splicing modulation**

Recently, new strategies have been used to correct splicing errors and overcome drug resistance. In different levels could be applying various molecules to reverse aberrant splicing. Blocking of splicing factors and kinases which phosphorylated them is one of the strategies for correct splicing (**Figure 13**) [36].

However, using splice switching oligonucleotides (SSOs) is the best-known method for therapy based on splicing [36]. For example, masking the proximal 5′ splice site by an antisense oligonucleotide (ASO) in BCL-X could shift splicing and induce apoptosis in tumor cells. The same strategy is used for reverse aberrant splicing of MDM4 and FAS mRNAs and inhibit p53 degradation and enhanced-cell death, respectively (**Figure 14**) [18]. In addition, ESEs and ESSs which are binding sites for SRSF and HNRNP proteins could be blocked by SSOs and affect splicing events [36].

#### **Figure 13.**

*Various potential strategies for therapeutic approaches to correct splicing errors [36].*

**Figure 14.** *Cancer therapies based on antisense oligonucleotide-mediated splicing modulation [18].*

## **10. Conclusion**

In conclusion, aberrant splicing, especially alternative splicing, can play an important role in carcinogenesis in all hallmarks of cancer (**Figure 15**). Recently, data from TCGA have been reported that many of the key player genes involved

#### **Figure 15.**

*The implication of alternative splicing of important genes (blue background) in the regulation of hallmarks of cancer (red background) [37].*

#### **Figure 16.**

*Tumor-associated isoforms of the important genes in cancers. ES, exon skipping; MXE, mutually exclusive exons; 5'ASS, 5' alternative splice site selection; IR, intron; retention.OE, overexpression; KD, knockdown. Other tumors include: adrenal, gallbladder, ampullary, bone, and brain; gynecological tumors include: ovarian, cervical, and uterine; head and neck tumors include: Oral, head and neck, tongue, esophageal, and thyroid.. ES, exon skipping; MXE, mutually exclusive exons; 5'ASS, 5*′ *alternative splice site selection; IR, intron retention [21].*

#### *Splicing in Cancer DOI: http://dx.doi.org/10.5772/intechopen.102707*

in cancers are affected by alternative splicing (**Figure 16**). It has recently been suggested that some misspliced RNA transcripts of non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs) and circular RNAs (circRNAs), may also contribute to tumorigenesis [38, 39]. One of the isoforms of protein phosphatase 1 regulatory subunit 10 (PPP1R10, also called PNUTS) gene generated by alternative splicing is lncRNA-PNUTS, which act as a competitive sponge for miR-205 in breast cancer [40]. In addition, PCGEM1 and BC200 lncRNAs interact with splicing factors such as hnRNPA1, hnRNPA2/B1 and U2AF65 and regulate the alternative splicing of the AR and BCL-x genes, respectively. Moreover, MALAT1 as an LncRNA, regulates alternative splicing by affecting the sub nuclear localization of SR proteins [21]. The role of alternative splicing in carcinogenesis and new strategies for cancer therapy based on reverse abnormal splicing or blocking aberrant splicing are emerging areas of cancer research.

## **Author details**

Mehdi Moghanibashi1 \* and Parisa Mohamadynejad2

1 Department of Genetics, School of Medicine, Kazerun Branch, Islamic Azad University, Kazerun, Iran

2 Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran

\*Address all correspondence to<EMAIL_ADDRESS>
© 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.

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## **Chapter 2**


Isocitrate Succinate + Glyoxylate

Hence, this enzyme has one substrate, isocitrate, and two products, succinate and glyoxylate. This enzyme belongs to the family of lyases, specifically the oxo-acid-lyases, which cleave carbon-carbon bonds. The systematic name of this enzyme class is isocitrate glyoxylate-lyase (succinate-forming). Isocitrate lyase is the first enzyme unique to the metabolic pathway known as the glyoxylate cycle which is required for the assimilation of fatty acids and acetate (Kelly et al., 2002). Recent reports have alluded to an additional role for this enzyme in *M. tuberculosis* metabolism, specifically for growth on propionate. A product of betaoxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the

Acetyl-CoA + H2O + Glyoxylate (S)-Malate + CoA

Fig. 2. Reaction scheme of glyoxylate shunt.

*Mycobacterium tuberculosis*: Dormancy, Persistence and Survival in the Light of Protein Synthesis 223

The structural intricacies have been deciphered for both the key enzymes successfully and all the information is at our disposal (Sharma et al., 2000; Smith et al., 2003; Anstrom & Remington, 2006). In the era of structure based drug design where high throughput screening, molecular modelling, *in silico* docking strategies have accelerated drug development timeline promising rescue of the hijacked host from the persistent mycobacteria. The TB Alliance has been strategically focusing to combat these molecular targets. ICL is a tetrameric protein with four subunits of 428 amino acids each. The highresolution structure of ICL from *M. tuberculosis* has been solved to 2.0 Å resolution (Sharma et al., 2000). The enzyme structure in complex with inhibitors, 3-nitropropionate with glyoxylate and 3-bromopyruvate has also been resolved. 3-bromopyruvate inhibits ICLactivity by forming a covalent adduct with the nucleophilic Cys191 (Sharma et al., 2000). The inhibitor bound ICL structures, on one hand, provide crucial information regarding the active site microenvironment, and on the other hand, produce valuable information on the type of interactions prevalent at those localized site adding momentum strength to the drug discovery process. Several ICL inhibitors are being tested, which mainly include 3 nitropropionate (McFadden & Purohit, 1977), 3-bromopyruvate (Ko & McFadden, 1990), 3 phosphoglycerate (Ko et al., 1989), mycenon (Hautzel et al., 1990) and itaconate (McFadden & Purohit, 1977). However, *in vivo* application of these inhibitors is yet a dream because of their potent toxicity and low activity. Sesterterpene sulphate, which has recently been

shown to effectively inhibit ICL in *Candida albicans* (Lee et al., 2008) is also promising.

equipped with permeability parameters that can reach the action targets.

Evolutionarily enzymes of glyoxylate shunt are highly conserved and have unique signature active site sequences which offer leverage to rational drug design approach thereby coming up with a broad spectrum more pharmacologically attractive target relevant to the treatment

The second enzyme of the glyoxylate shunt is encoded by a single gene identified in TB called *glcB* and encoding a 741 amino acid 80 kDa protein malate synthase (Smith et al., 2003). The enzyme catalyzes the Mg2+-dependent condensation of glyoxylate and acetylcoenzyme A and hydrolysis of the intermediate to yield malate and coenzyme A (Anstrom & Remington, 2006). The structure of MS from *M. tuberculosis* in complex with the substrate glyoxylate has been solved to 2.1 Å resolution structural analysis indicated that malate synthase is a much more druggable target by virtue of its deeper and more hydrophobic binding domain (Smith et al., 2003). Screening against this target will have a better chance of identifying tractable inhibitors as lead molecules (www.tballiance.org). Further refinement in understanding the mechanistic implications were brought forth by revised position of bound malate which is consistent with a reaction mechanism that does not require reorientation of the electrophilic substrate during the catalytic cycle(Anstrom & Remington, 2006) . These insights have been crucial in the inhibitor ergonomics. High throughput screening has been completed with a 1.4 million compound library and hits have been identified. The endeavour ahead is to confirm the potential hits and efficiently evaluate these, thus paving the pathway for identification of analogues and series for future optimization. It's also important to mention that high throughput screening initiatives for identifying inhibitors has not yielded very promising outcomes reason being the druggability of these potential targets. The challenge here is to design inhibitors that are

**3. The shunt and the hunt**

by-products of its metabolism via the methylcitrate cycle are vital due to their potentially toxic effects but no homolog of this enzyme has been found in the mycobacterium genome. This unique phenomenon points out on the dual role of isocitrate lyase in the glyoxylate and methylcitrate cycles in *M. tuberculosis* (Gould et al., 2006). ICL-deficient bacteria could not grow on propionate, suggesting that ICL might function as ICLs in the glyoxylate cycle and as MCLs in the methylcitrate cycle (Munoz-Elias et al., 2006).

The enzyme malate synthase (EC <IP_ADDRESS>) catalyses the condensation reaction between the carbonyl group of glyoxylate and the methyl group of acetyl-CoA to form a thio-ester which, after hydrolysis, generates L-malate and CoA (Dixon et al., 1960).

Catalysis by ICL and MS ensures the bypass of two oxidative steps of the tricarboxylic acid cycle, permitting net incorporation of carbon during growth of most microorganisms on acetate or fatty acids as the primary carbon source. Thus, the glyoxylate bypass conserves carbon and ensures an adequate supply of tricarboxylic acid cycle intermediates for biosynthetic purposes when cells convert lipids to carbohydrates (Sharma et al., 2000).


*3.3. Changes in Total Phenolics, Free Phenolic Acid Content, and Antioxidant Capacity*

The total phenol contents in tuberous roots of both groups increased after 21 d of storage (Figure 3A). Further, the total phenol content in tuberous roots of the CS group was significantly higher than that in the control group (*p* < 0.05) after 14 d of storage. Chlorogenic acid is the main phenolic compound in sweet potatoes and thus might have contributed to the increase in total phenolic. The chlorogenic acid content in CS tuberous roots was significantly higher (*p* < 0.05) than that in the control group after 14 d (Figure 3B).

**Figure 3.** (**A**) Total phenol and (**B**) chlorogenic acid contents, (**C**) 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) radical-scavenging activity, and (**D**) ferric reducing antioxidant power (FRAP) of sweet potato tuberous roots during 21 d storage control and CS groups. Data are the mean ± standard error of three replicates. Data with different characters indicate significant differences between treatments at the *p* < 0.05 level.

Moreover, the DPPH scavenging activity in tuberous roots of the CS group was significantly (*p* < 0.05) higher than that in the control group after 7 d of storage (Figure 3C). As shown in Figure 3D, the FRAP in the CS group also showed a significant upward trend during storage, with CS tuberous roots showing significantly higher FRAP levels than the control group (*p* < 0.05).

#### *3.4. Changes in Free Amino Acid Content in Sweet Potato Tuberous Roots during Storage*

The free amino acids analyzed herein were glycine, phenylalanine, tyrosine, and propanine. The initial phenylalanine content was approximately 90 mg kg−1 FW, followed by alanine and tyrosine. The glycine content was the lowest among the four amino acids tested. Almost all of these free amino acids tended to increase continuously between two and six times during storage at temperatures (Figure 4A–D).

**Figure 4.** Contents of (**A**) glycine, (**B**) alanine, (**C**) tyrosine, and (**D**) phenylalanine of sweet potato tuberous roots of control and CS group during 21 d storage. Results are expressed as the mean ± standard error of three replicates. Different characters indicate significant differences between means of treatments at the *p* < 0.05 level.


**7. Conclusions**

This paper proposes the MGRS model in terms of IFS based on soft binary relations over two universes. First of all, we defined granulation roughness based on two soft binary relations using IFSs with respect to the aftersets and foresets. In this way, we obtain two

IFSSs with respect to the aftersets and foresets. Some properties of OMGIFRS have been studied. Then, we generalized these concepts to granulation roughness of an IFS based on multi-soft relations and discussed their properties. We presented a decision-making algorithm regarding the aftersets and foresets with an example in practical decision-making problem. In IFS, the sum of membership degree, non-membership degree and hesitant degree of an element is less than or equal to 1. However, in some decision-making problems, the sum of membership degree, non-membership degree and hesitant degree of an element may be greater than 1. In this case, the Pythagorean fuzzy set which is an extension of IFS is the better set to deal with uncertainty. The Pythagorean fuzzy set extension makes better improvement in applicability and flexibility of IFS. Further work may be discussed about investigation of pessimistic MGRS of an IFS based on soft relations. Other OMGIFRS models with interval valued IFSs, uncertain linguistic FSs, basic uncertain information SSs, linguistic Z-number FSSs and Pythagorean FSs may be discussed in future.

**Author Contributions:** Conceptualization, S.B. and M.S.; methodology, M.Z.A.; software, S.B.; validation, M.G.A.; formal analysis, M.Z.A.; investigation, M.Z.A.; resources, M.G.A.; data curation, M.S.; writing—original draft preparation, M.Z.A.; writing—review and editing, S.B.; visualization, M.Z.A.; supervision, S.B.; project administration, M.Z.A.; funding acquisition, M.G.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** We did not use any data for this research work.

**Acknowledgments:** The researchers would like to thank the Deanship of Scientific Research, Qassim University for funding the publication of this project.

**Conflicts of Interest:** The authors declare that they have no conflicts of interest.