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## **Meet the editor**

Dmitry Bulgin received his MD and PhD degrees from Smolensk State Medical University in 1998 and 2004, respectively. He served as an Assistant Professor at the Department of Pathology in Smolensk State Medical University from 2000 to 2004. Between 2004 and 2006, he has been a visiting researcher at the Department of Molecular Medicine of Atomic Bomb Disease Institute in

Nagasaki University Graduate School of Biomedical Sciences. Since 2006, he was appointed as a Chairman of the Department of Laboratory Medicine at Pokrovskaya Municipal Hospital in Saint Petersburg, Russia. From 2010, he served as a principal research scientist and general practitioner at the Center for Regenerative Medicine ME-DENT in Rovinj, Croatia. He is a member of professional associations such as Croatian Medical Chamber, European Society of Pathology, European Society for Artificial Organs, The Transplantation Society, European Radiation Research Society, and Tissue Engineering and Regenerative Medicine International Society. His research interests include Cancer Research, Stem Cell Biology, Molecular Medicine, Ionizing Radiation, and Tissue Regeneration.

### Contents

#### **Preface XI**


### Preface

Intensive research efforts during the past several decades have increased our understanding of carcinogenesis and have identified a cellular and molecular basis for the multi-step proc‐ ess of cancer development. Technological advances in molecular biology have proven in‐ valuable to the understanding of the pathogenesis of human cancer. The application of molecular techniques to the study of cancer has not only led to advances in tumor diagnosis and development of new treatment approaches but also provided markers for the assess‐ ment of prognosis and disease progression.

I wish to thank all the authors of this book for their excellent contributions. They share my hope that this book provides an update on recent progress in some key areas of cancer re‐ search and assists other scientists in the better understanding of the molecular pathogenesis of cancer.

I would like to express my gratitude my mentor, professor Shunichi Yamashita (Nagasaki University, Japan), and to my colleague, professor Serik Meirmanov (Ritsumeikan Asia Pa‐ cific University, Japan) for their professional support in the fields of cancer research and ra‐ diation biology.

> **Dmitry Bulgin** Center for Regenerative Medicine "ME-DENT", Rovinj Croatia

### **Mechanisms of Oncogene Activation**

Anca Botezatu, Iulia V. Iancu, Oana Popa, Adriana Plesa, Dana Manda, Irina Huica, Suzana Vladoiu, Gabriela Anton and Corin Badiu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61249

#### **Abstract**

The main modifications that characterize cancer are represented by alterations in onco‐ genes, tumor-suppressor genes, and non-coding RNA genes. Most of these alterations are somatic and the process is a multistep one. Tumors often arise from an initial trans‐ formed cell, and after subsequent genetic alterations different cytogenetically clones lead to tumor heterogeneity.

Oncogenes encode proteins that control cell processes such as proliferation and apopto‐ sis. Among these proteins are transcription factors, chromatin remodelers, growth fac‐ tors, growth factor receptors, signal transducers, and apoptosis regulators. Oncogenes activation by structural alteration (chromosomal rearrangement, gene fusion, mutation, and gene amplification) or epigenetic modification (gene promoter hypomethylation, mi‐ croRNA expression pattern) confers an increased or a deregulated expression. Therefore, cells with such alterations possess a growth advantage or an increased survival rate. Giv‐ en the fact that expression profiling of these alterations determines specific signatures as‐ sociated with tumor classification, diagnosis, staging, prognosis, and response to treatment, it highlights the importance of studying oncogenes activation mechanisms and the great potential that they hold as therapeutic tools in the near future.

**Keywords:** Oncogenes, genomic instability, epigenetic modification

#### **1. Introduction**

The main modifications that characterize cancer are represented by alterations in oncogenes, tumor-suppressor genes, and non-coding RNA genes. Most of these alterations are somatic and the process is a multistep one, although germ-line mutations can predispose a person to heritable or familial cancer.

© 2016 The Author(s). Licensee InTech. 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.

Tumors often arise from an initial transformed cell, and after subsequent genetic altera‐ tions different cytogenetically clones lead to tumor heterogeneity. Tumor heterogeneity determines different clinical phenotypes, leading to an individual response to treatment for tumors with the same diagnostic type.

Oncogenes encode proteins that control cell processes such as proliferation and apoptosis. Among these proteins are transcription factors, chromatin remodelers, growth factors, growth factor receptors, signal transducers, and apoptosis regulators. Activation of oncogenes by structural alterations (chromosomal rearrangement, gene fusion, mutation, and gene amplification) or epigenetic modification (gene promoter hypomethylation) confers an increased or a deregulated expression. Therefore, cells with such alterations possess a growth advantage or an increased survival rate. Translocations and mutations occur early on in tumor progression, whereas amplification usually occurs during late tumor stages.

A proto-oncogene is a normal gene that presents a potential to become an oncogene after a genetic alteration (mutation), leading to an increased expression. Usually, proto-onco‐ genes code for proteins that control cell growth and differentiation through signal transduc‐ tion and execution of mitogenic signals. Upon activation, a proto-oncogene (or its product onco-protein) becomes a tumor-inducing agent. Most known examples of proto-onco‐ genes include *RAS, WNT, MYC, ERK*, and *TRK*. Another oncogene is the *BCR-ABL* gene found on the Philadelphia chromosome, a piece of genetic material seen in chronic myelogenous leukemia caused by the translocation of pieces from chromosomes 9 and 22 t (9; 22).

Oncogene products can comprise a variety of molecules such as transcription factors, chro‐ matin remodelers, growth factors, growth factor receptors, signal transducers, and apoptosis regulators, each playing an important role in neoplastic transformation. For example, studies have shown that in prostate carcinomas the fusion between the *TMPR552* gene and two transcription factors *ERG*1 or *ETV1* creates a fusion protein that increases proliferation and inhibits apoptosis of cells in the prostate gland, thereby facilitating their transformation into cancer cells [1]. Another example is represented by chromatin remodeler factors such as the *MLL* gene that plays a critical role in acute lymphocytic leukemia and acute myelogenous leukemia [2]. Also, an essential role in cancer development is played by apoptosis regulators such as the *BCL2* gene, which is involved in the initiation of almost all follicular lymphomas and some diffuse large B-cell lymphomas [3].

#### **2. Mutations**

Mutations in an oncogene may lead to a change in the structure of encoded protein, enhancing its transforming activity. Oncogenes are activated by point mutations (substitutions) and may either enhance or degrade the function of a protein. Table 1 shows the occurrences of mutations in each oncogene among some tissues [4].


**Table 1.** Frequently mutated oncogenes in various type of cancers

Tumors often arise from an initial transformed cell, and after subsequent genetic altera‐ tions different cytogenetically clones lead to tumor heterogeneity. Tumor heterogeneity determines different clinical phenotypes, leading to an individual response to treatment for

Oncogenes encode proteins that control cell processes such as proliferation and apoptosis. Among these proteins are transcription factors, chromatin remodelers, growth factors, growth factor receptors, signal transducers, and apoptosis regulators. Activation of oncogenes by structural alterations (chromosomal rearrangement, gene fusion, mutation, and gene amplification) or epigenetic modification (gene promoter hypomethylation) confers an increased or a deregulated expression. Therefore, cells with such alterations possess a growth advantage or an increased survival rate. Translocations and mutations occur early on in tumor progression, whereas amplification usually occurs during late tumor

A proto-oncogene is a normal gene that presents a potential to become an oncogene after a genetic alteration (mutation), leading to an increased expression. Usually, proto-onco‐ genes code for proteins that control cell growth and differentiation through signal transduc‐ tion and execution of mitogenic signals. Upon activation, a proto-oncogene (or its product onco-protein) becomes a tumor-inducing agent. Most known examples of proto-onco‐ genes include *RAS, WNT, MYC, ERK*, and *TRK*. Another oncogene is the *BCR-ABL* gene found on the Philadelphia chromosome, a piece of genetic material seen in chronic myelogenous leukemia caused by the translocation of pieces from chromosomes 9 and 22

Oncogene products can comprise a variety of molecules such as transcription factors, chro‐ matin remodelers, growth factors, growth factor receptors, signal transducers, and apoptosis regulators, each playing an important role in neoplastic transformation. For example, studies have shown that in prostate carcinomas the fusion between the *TMPR552* gene and two transcription factors *ERG*1 or *ETV1* creates a fusion protein that increases proliferation and inhibits apoptosis of cells in the prostate gland, thereby facilitating their transformation into cancer cells [1]. Another example is represented by chromatin remodeler factors such as the *MLL* gene that plays a critical role in acute lymphocytic leukemia and acute myelogenous leukemia [2]. Also, an essential role in cancer development is played by apoptosis regulators such as the *BCL2* gene, which is involved in the initiation of almost all follicular lymphomas

Mutations in an oncogene may lead to a change in the structure of encoded protein, enhancing its transforming activity. Oncogenes are activated by point mutations (substitutions) and may either enhance or degrade the function of a protein. Table 1 shows the occurrences of mutations

tumors with the same diagnostic type.

2 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

and some diffuse large B-cell lymphomas [3].

in each oncogene among some tissues [4].

stages.

t (9; 22).

**2. Mutations**

In cancer, mutations occur in many oncogenes, most notable being *RAS* and *BRAF*. The *RAS* family represents the upstream component of the RAS/RAF/MAPK pathway and mutations in *RAS* are one of the most common activating events in most of cancers. Mutated *RAS* oncogene (*KRAS, HRAS*, and *NRAS*) encodes for a protein that remains in the active state and transduces signals for continuous cell growth. *KRAS* mutations are common in carcinomas of the lung, colon, and pancreas [5], whereas mutations of *NRAS* occur in acute myelogenous leukemia and the myelodysplastic syndrome [6].

BRAF is a protein member of the *RAF* family (RAF1, BRAF, ARAF), also regulated by RAS binding. Mutated *BRAF* oncogene encodes for a protein with a modified kinase domain, resulting in a constitutively active protein that uncontrollably stimulates the MAP kinase cascade [7].

#### **2.1. Melanoma**

*BRAF* mutations are the most common somatic mutations in cutaneous melanoma and are extremely rare in mucosal melanoma. There are found in 48% of metastatic biopsy specimens and can precede neoplastic transformation [8, 9]. Over 90% of the identified mutations in *BRAF* are in codon 600. The most common is *BRAFV600E*, resulting in substitution of glutamic acid for valine (*BRAFV600E*: nucleotide 1799 T>A; codon GTG>GAG). The second most common mutation is *BRAFV600K* (5–6%) substituting lysine for valine, (GTG>AAG), followed by *BRAFV600R* (GTG>AGG), *BRAFV600′E2′* (GTG>GAA). Less common *BRAF* mutations found in cutaneous melanoma are *BRAF V600D* (GTG>GAT) and *L597R* [10–12].

There were identified mutations in hotspot codons (12, 13, and 61) of different *RAS* genes (*HRAS, NRAS*, or *KRAS*), but the most prevalent were *HRAS* substitutions that occurred preponderent at codon 61 (*HRAS Q61L* mutation), with fewer mutations at codon 12 and codon 13 [13]. Mutations in *N-RAS* appear to be significant in melanoma even earlier than the discovery of *BRAF* mutations [14]. The base change at position 61 seems to be important in the activation of *N-RAS* genes, transforming activity being detected only when mutant codon 61 was present. *BRAFV600E* mutations are more common in younger persons and in tumors arising from intermittently sun-exposed skin, exclusive with *N-RAS* [15].

C-KIT gene encodes a receptor tyrosine kinase (KIT). All the mutations were founded in exon 11, 13, and 17. The most common is V559A mutation that results in an amino acid substitution at position 559 in KIT, from a valine (V) to an alanine (A) [16]. While BRAF and NRAS mutations are common and significant in cutaneous melanomas, C-KIT mutations were detected in acral melanomas, mucosal melanomas, conjunctival melanomas, and cutaneous melanomas [17]

#### **2.2. Colorectal cancer**

The development of colorectal cancer (CRC) is a multistep process that occurs due to the accumulation of several genetic alterations, which are associated with oncogenes and tumor suppressor genes, as well as genes involved in DNA damage recognition and repair.

Most of the *BRAF* mutations associated with CRC are located in exons 11 and 15, coding for the kinase domain. The hotspot mutation at 1796 nucleotide is the T-to-A transversion that corresponds to the *V600E* mutation (7–15%) [18].

In colorectal cancer, *RAS* gene mutations have been reported in 40–50% and the frequency of *KRAS* mutations varies between 24–50%. *KRAS* mutation occurs most commonly in codon 12 and 13 rather than in codon 61, with the most frequent mutations: *G12D, G12V, G12C, G13D, Q61H* [19]. *KRAS* mutations exist in the presence of a vast majority of wild-type *KRAS* cells, which is why they not are detected in initial disease. Thirty-eight percent of patients whose tumors were initially *KRAS* wild-type developed *KRAS* mutations that were detectable in their sera after 5–6 months of treatment [20]. *KRAS* is mutated much more frequently than *NRAS*. *KRAS* mutations were studied to determine their role in the predictability of response to chemotherapy treatment.

#### **2.3. Thyroid cancer**

BRAF is a protein member of the *RAF* family (RAF1, BRAF, ARAF), also regulated by RAS binding. Mutated *BRAF* oncogene encodes for a protein with a modified kinase domain, resulting in a constitutively active protein that uncontrollably stimulates the MAP kinase

*BRAF* mutations are the most common somatic mutations in cutaneous melanoma and are extremely rare in mucosal melanoma. There are found in 48% of metastatic biopsy specimens and can precede neoplastic transformation [8, 9]. Over 90% of the identified mutations in *BRAF* are in codon 600. The most common is *BRAFV600E*, resulting in substitution of glutamic acid for valine (*BRAFV600E*: nucleotide 1799 T>A; codon GTG>GAG). The second most common mutation is *BRAFV600K* (5–6%) substituting lysine for valine, (GTG>AAG), followed by *BRAFV600R* (GTG>AGG), *BRAFV600′E2′* (GTG>GAA). Less common *BRAF* mutations found

There were identified mutations in hotspot codons (12, 13, and 61) of different *RAS* genes (*HRAS, NRAS*, or *KRAS*), but the most prevalent were *HRAS* substitutions that occurred preponderent at codon 61 (*HRAS Q61L* mutation), with fewer mutations at codon 12 and codon 13 [13]. Mutations in *N-RAS* appear to be significant in melanoma even earlier than the discovery of *BRAF* mutations [14]. The base change at position 61 seems to be important in the activation of *N-RAS* genes, transforming activity being detected only when mutant codon 61 was present. *BRAFV600E* mutations are more common in younger persons and in tumors

C-KIT gene encodes a receptor tyrosine kinase (KIT). All the mutations were founded in exon 11, 13, and 17. The most common is V559A mutation that results in an amino acid substitution at position 559 in KIT, from a valine (V) to an alanine (A) [16]. While BRAF and NRAS mutations are common and significant in cutaneous melanomas, C-KIT mutations were detected in acral melanomas, mucosal melanomas, conjunctival melanomas, and cutaneous

The development of colorectal cancer (CRC) is a multistep process that occurs due to the accumulation of several genetic alterations, which are associated with oncogenes and tumor

Most of the *BRAF* mutations associated with CRC are located in exons 11 and 15, coding for the kinase domain. The hotspot mutation at 1796 nucleotide is the T-to-A transversion that

In colorectal cancer, *RAS* gene mutations have been reported in 40–50% and the frequency of *KRAS* mutations varies between 24–50%. *KRAS* mutation occurs most commonly in codon 12 and 13 rather than in codon 61, with the most frequent mutations: *G12D, G12V, G12C, G13D, Q61H* [19]. *KRAS* mutations exist in the presence of a vast majority of wild-type *KRAS* cells,

suppressor genes, as well as genes involved in DNA damage recognition and repair.

in cutaneous melanoma are *BRAF V600D* (GTG>GAT) and *L597R* [10–12].

4 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

arising from intermittently sun-exposed skin, exclusive with *N-RAS* [15].

cascade [7].

**2.1. Melanoma**

melanomas [17]

**2.2. Colorectal cancer**

corresponds to the *V600E* mutation (7–15%) [18].

*RAS* mutations in thyroid cancer occur in both benign tumors and thyroid cancers (both papillary thyroid carcinoma (PTC) and follicular thyroid cancer (FTC)), with variable frequen‐ cy in anaplastic thyroid cancers. PTCs with *RAS* mutations tend to display a lower rate of lymph node metastasis [21]. PTCs with *RAS* mutations often present a follicular architecture and a follicular variant of papillary thyroid carcinoma (FVPTC). There are two most common *RAS* mutations associated with thyroid cancer: mutations of *H-RAS* codon 61 and *N-RAS* codon 61 [22].

*BRAF* mutations were first detected missense mutations in thyroid cancer [23], which occurs in exon 15, due to the substitution of the amino acid valine for glutamic acid at residue position 600 [24]. This mutation is the most frequent genetic change in PTC [25], being found in 36–69% of PTC cases. *BRAF* mutation is responsible for the suppression of the sodium/iodide sym‐ porter (NIS), which is involved in iodine metabolism [26]. The *V600E* mutation comprises more than 90% of observed *BRAF* mutations, with the highest rate (77%) in the tall cell variant of papillary cancer, and the lowest percentage (12%) in the FVPTC. In PTC, *BRAF* mutation is more frequent in older patients, associated with extrathyroidal invasion [27]. By contrast, others have found that the *BRAF* mutation is not associated with age, gender, multicentricity, recurrence rate, lymphovascular invasion, or distant metastasis [28].

Activating point mutations of *RET* oncogenes are associated with hereditary cancer syndrome (multiple endocrine neoplasia type 2 -MEN 2). *RET* mutations are mostly missense and located in exons 10, 11 (extracellular domain of RET), 13, 14, 15, and 16 (in the TK domain) [29–31]. Mutation of the extracellular cysteine in codon 634 in exon 11 of RET causes ligand-independ‐ ent dimerization of receptor molecules and enhances phosphorylation of intracellular sub‐ strates and cell transformation. Mutation of the intracellular TK (codon 918) results in cellular transformation [32].

There is a high correlation between the position of the point mutation and the phenotype of the disease. Three subtypes based on clinical presentation are defined: MEN 2A, MEN 2B, and FMTC. *RET* mutations are observed in 98% of MEN2A, 95% of MEN 2B, and 88% of familial medullary thyroid carcinoma (FMTC) [33]. Activating mutations of *RET* involving exons 10, 11, 13, 14, and 15 (encoding the highly conserved cysteine-rich domain) have been proven to cause MEN2A. [34]. The mutations for MEN2A are mostly located in exon 10 (10–15%), including codons 609, 611, 618, and 620, and exon 11 (80–85%), as well as codons 630 and 634 [35]. The mutations characteristic of FMTC occur in exons 10 and 11. However, non-cysteine point mutations also have been found in exon 8 (codons 532 and 533), exon 13 (codons 768, 790, and 791), exon 14 (codons 804 and 844), exon 15 (codon 891), and exon 16 (codon 912) [35– 37]. About 95% of MEN2B patients carry a *M918T* mutation within exon 16 and 5% have an *A883F* mutation in exon 15. Mutation in codon 918 gives a more aggressive phenotype [38].

#### **2.4. Hepatocellular Carcinomas (HCC)**

In HCC, only one mutation (*KRAS* codon 13; Gly to Asp) was detected among patients and no mutations were found in codons 12 and 61 of *KRAS* or codons 12, 13, and 61 of the *NRAS* and *HRAS* genes. So, the activation of *RAS* oncogenes by point mutations does not play a major role in hepatocellular carcinogenesis [39]. Activating mutations in the *BRAF* oncogene have been found in a small fraction of hepatocellular carcinomas. *KRAS* and *BRAF* mutations are rare events in HCC and therefore not a key event in hepatocarcinogenesis [40].

#### **2.5. Pancreatic cancer**

The highest incidence of *KRAS* mutations are found in adenocarcinomas of the pancreas (90%), with activating point mutations in codon 12 of the KRAS protein, leading to a glycine (G) to aspartic acid (D) or valine (V) substitution [41]. Single amino acid substitutions at G12, 13, or Q61 lead to the formation of mutated *KRAS* that are insensitive to GAP stimulation. This leads to the accumulation of persistently GTP-bound and active KRAS, which leads to pancreatic cancer formation [42].

#### **2.6. Cervical cancer**

Cervical cancer harbors high rates of potentially targetable oncogenic mutations. *KRAS* mutations were identified in low percentage (17%) exclusively in cervical adenocarcinomas. Most mutations were missense mutations of codon G12, well-described activating mutations, which have been associated with a worse prognosis in the metastatic process [43].

*EGFR* mutations were identified in 7.5% of cervical squamous cell carcinomas; a missense mutation in exon 15 of the *EGFR* gene produces an alternate spliced transcript (isoform D). Its presence in both tumor and adjacent normal tissue suggests that EGFR S703F may be a germline mutation [44, 45].

*PIK3CA* mutations are present in both squamous cell carcinomas and adenocarcinomas (31%). The *PIK3CA* mutations were located in the exon 9 helical domain in two hotspot mutations (*E545K* and *E542K*), which result in the constitutive activation of cellular signaling [46]. *PI3KCA* mutations may impart a more aggressive and treatment-resistant phenotype and decreased survival among patients with these mutations in early stage cancers [47].

#### **3. Gene amplification and chromosomal translocations**

The interest regarding the role of genomic context in promoting amplification was intensely investigated, but is still under debate. An important interest remains to establish the tendency of some genomic region to be subject to amplification. Past researches showed that different regions of the genome were more subjected to be amplified than others, but the molecular substrate was unknown [48]. At present, several mechanisms and models have been proposed to explain gene amplification in oncogenesis.

#### **3.1. Gene amplification**

37]. About 95% of MEN2B patients carry a *M918T* mutation within exon 16 and 5% have an *A883F* mutation in exon 15. Mutation in codon 918 gives a more aggressive phenotype [38].

In HCC, only one mutation (*KRAS* codon 13; Gly to Asp) was detected among patients and no mutations were found in codons 12 and 61 of *KRAS* or codons 12, 13, and 61 of the *NRAS* and *HRAS* genes. So, the activation of *RAS* oncogenes by point mutations does not play a major role in hepatocellular carcinogenesis [39]. Activating mutations in the *BRAF* oncogene have been found in a small fraction of hepatocellular carcinomas. *KRAS* and *BRAF* mutations are

The highest incidence of *KRAS* mutations are found in adenocarcinomas of the pancreas (90%), with activating point mutations in codon 12 of the KRAS protein, leading to a glycine (G) to aspartic acid (D) or valine (V) substitution [41]. Single amino acid substitutions at G12, 13, or Q61 lead to the formation of mutated *KRAS* that are insensitive to GAP stimulation. This leads to the accumulation of persistently GTP-bound and active KRAS, which leads to pancreatic

Cervical cancer harbors high rates of potentially targetable oncogenic mutations. *KRAS* mutations were identified in low percentage (17%) exclusively in cervical adenocarcinomas. Most mutations were missense mutations of codon G12, well-described activating mutations,

*EGFR* mutations were identified in 7.5% of cervical squamous cell carcinomas; a missense mutation in exon 15 of the *EGFR* gene produces an alternate spliced transcript (isoform D). Its presence in both tumor and adjacent normal tissue suggests that EGFR S703F may be a

*PIK3CA* mutations are present in both squamous cell carcinomas and adenocarcinomas (31%). The *PIK3CA* mutations were located in the exon 9 helical domain in two hotspot mutations (*E545K* and *E542K*), which result in the constitutive activation of cellular signaling [46]. *PI3KCA* mutations may impart a more aggressive and treatment-resistant phenotype and decreased

The interest regarding the role of genomic context in promoting amplification was intensely investigated, but is still under debate. An important interest remains to establish the tendency of some genomic region to be subject to amplification. Past researches showed that different

which have been associated with a worse prognosis in the metastatic process [43].

survival among patients with these mutations in early stage cancers [47].

**3. Gene amplification and chromosomal translocations**

rare events in HCC and therefore not a key event in hepatocarcinogenesis [40].

**2.4. Hepatocellular Carcinomas (HCC)**

6 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**2.5. Pancreatic cancer**

cancer formation [42].

**2.6. Cervical cancer**

germline mutation [44, 45].

Besides point mutations resulting in amino acid substitutions, a proto-oncogene may be activated by chromosomal alterations. Among the most important cromosomal abnormalities is gene amplification, which is an increase of the copy number for a specific chromosomal region. The consequence of chromosome fragment amplification is associated with overex‐ pression of the amplified gene(s) and is a characteristic of cancer [49]. Amplified genes (hundreds of copies of normally diploid genes) may be organized as extrachromosomal elements (double minute chromosomes) as repeated units at a single locus or scattered throughout the genome.

At this moment, the relationships between the two forms of gene amplification found in tumors, the intrachromosomal homogeneously staining regions (HSRs) and the extrachromo‐ somal DNA molecules, double minutes (dmins), are not well understood [50].

Several models for initiation of amplification have been described involving defects in DNA replication or telomere dysfunction and chromosomal fragile sites. Regarding the DNA replication initial proposals, based on extra rounds of replication due to replication origins misfiring appear to be incorrect modification of models invoking replication of extrachromo‐ somal DNA [51]. Another theory involves the double-strand DNA breaks (frequent in replicating cells) generated by the collapse of replication forks that are unable to progress due to DNA structure lesions, therefore providing an opportunity to initiate the amplification process [49].

Telomeres are repetitive nucleotide sequences, with the role to prevent the loss of DNA sequences, resulted as a consequence of the incomplete DNA replication at the chromosome ends. Telomere shortening can block cell division; this mechanism appears to prevent genomic instability and development of cancer in aged cells by limiting the number of cell divisions [52]. Telomerase is responsible for telomere replication and is inactive in most somatic cells. With every cell division, the DNA telomere sequence is shortened by 40–50 bp. Telomere shortening in humans can induce replicative senescence, which blocks cell division. When telomeres are short to a critical length (replicative limit), cellular senescence is induced and normal cells cease to proliferate. This mechanism appears to prevent genomic instability and development of cancer in aged cells by limiting the number of cell divisions. In cancer, tumor cells escape replicative limit and acquire the capability to maintain telomere length through cell divisions by telomerase reactivation, or by using a recombination-based mechanism and alternate lengthening of telomeres (ALT) [53, 54]. An experimental murine model (lacking the RNA component of telomerase-TercK/K mice) for telomere dysfunction demonstrated the promo‐ tion of gene amplification. Tumor genomes arising in mutant mice contain chromosomal rearrangements, amplifications, and deletions commonly associated with human tumors [55, 56]. Despite the established correlation between telomerase reactivation and telomeres lengthening in cancer, recent literature review and analysis [52] suggest this is unlikely, because shorter telomeres and telomerase inactivation is more often associated with increased cancer rates, and the mortality from cancer occurs late in life.

In humans, shorter telomeres were associated with poorer health and aging and were also observed in preneoplastic stages, supporting a role for this mechanism in generating genomic aberrations in oncogenesis [57–60]. The model for gene amplification due to telomere abnor‐ malities and the break at fragile sites (discussed below) was first described in maize and results from the breakage/fusion/bridge (B/F/B) cycles [61]. B/F/B cycles are initiated when broken ends of chromosomes fuse, resulting in a dicentric chromosome. During anaphase, the two centromeres are pulled in opposite directions and the dicentric chromosome generates a chromosome with an inverted duplication of terminal sequences to break. The B/F/B cycle continues in the next cell cycle because this chromosome also has broken ends. The B/F/B cycles were observed like primary mechanism for gene amplification in hamster cells [62].

In human cancer, evidence of B/F/B cycles was provided by the high frequency of anaphase bridges in early passage tumor cells and tumors [63, 64]. On the other hand, it was proven that human tumor cells in culture presenting gene amplification contain DM chromosomes, and the clones with low-copy amplification contained structures related to B/F/B cycles [65, 66]. There are evidences that B/F/B cycles may generate amplicons. These results were obtained by cytogenetic analyses of HSRs in tumor cell lines and in model systems with amplifications following drug treatments [67, 68]. The model explains that loss of the DNA sequences distal to the gene under selection or their translocation to another chromosome is also possible.

HSR may arise from the integration or fusion of double minute with a chromosome [50]. Currently, the data available suggests that fusion and reintegration constitute a pathway for the evolution of extrachromosomal elements, but the site of HSR insertion has never been characterized at a nucleotide resolution [50].

#### **3.2. Fragile sites**

Fragile sites are part of normal chromosome structures existing in each individual and represent chromosome regions that are late in replicating and prone to breakage under conditions of replication stress. Fragile sites occur after partial inhibition of DNA synthesis and are constituted in regions presenting site-specific gaps and breaks on metaphase chromo‐ somes. Common fragile sites are normally stable in somatic cells, but it was observed that following treatment of cultured cells with replication inhibitors, fragile sites display gaps, breaks, rearrangements [69, 70]. Fragile sites extend over large regions of high DNA flexibility and are associated with genes.

The molecular nature and mechanisms involved in fragile site instability was unknown till recently. In many cancer cells, fragile sites and associated genes suffer frequent deletions and/ or rearrangement, demonstrating their role in genome instability during the oncogenesis process. As a group, fragile sites are heterogeneous and seem to extend over broad regions 0.3–9-Mb long. The regions comprising fragile site are particularly associated with a high frequency of recombinogenic events, including co-localization with chromosome aberrations sites related to various cancers [69].

Accordingly to several studies, there are around 127 known fragile sites in the human genome, defined as "common" or "rare" based on their frequency [71, 72]. Common fragile sites (CFSs) are a normal part of the human genome and are typically replicative stable [73]. CFSs are not the result of nucleotide repeat expansion mutations. The majority of breakages at CFSs are further distinguished depending on their sensitivity to the drugs used to induce their expres‐ sion (e.g., low doses of the antibiotic aphidocilin (APH)) [74].

because shorter telomeres and telomerase inactivation is more often associated with increased

In humans, shorter telomeres were associated with poorer health and aging and were also observed in preneoplastic stages, supporting a role for this mechanism in generating genomic aberrations in oncogenesis [57–60]. The model for gene amplification due to telomere abnor‐ malities and the break at fragile sites (discussed below) was first described in maize and results from the breakage/fusion/bridge (B/F/B) cycles [61]. B/F/B cycles are initiated when broken ends of chromosomes fuse, resulting in a dicentric chromosome. During anaphase, the two centromeres are pulled in opposite directions and the dicentric chromosome generates a chromosome with an inverted duplication of terminal sequences to break. The B/F/B cycle continues in the next cell cycle because this chromosome also has broken ends. The B/F/B cycles

were observed like primary mechanism for gene amplification in hamster cells [62].

In human cancer, evidence of B/F/B cycles was provided by the high frequency of anaphase bridges in early passage tumor cells and tumors [63, 64]. On the other hand, it was proven that human tumor cells in culture presenting gene amplification contain DM chromosomes, and the clones with low-copy amplification contained structures related to B/F/B cycles [65, 66]. There are evidences that B/F/B cycles may generate amplicons. These results were obtained by cytogenetic analyses of HSRs in tumor cell lines and in model systems with amplifications following drug treatments [67, 68]. The model explains that loss of the DNA sequences distal to the gene under selection or their translocation to another chromosome is also possible.

HSR may arise from the integration or fusion of double minute with a chromosome [50]. Currently, the data available suggests that fusion and reintegration constitute a pathway for the evolution of extrachromosomal elements, but the site of HSR insertion has never been

Fragile sites are part of normal chromosome structures existing in each individual and represent chromosome regions that are late in replicating and prone to breakage under conditions of replication stress. Fragile sites occur after partial inhibition of DNA synthesis and are constituted in regions presenting site-specific gaps and breaks on metaphase chromo‐ somes. Common fragile sites are normally stable in somatic cells, but it was observed that following treatment of cultured cells with replication inhibitors, fragile sites display gaps, breaks, rearrangements [69, 70]. Fragile sites extend over large regions of high DNA flexibility

The molecular nature and mechanisms involved in fragile site instability was unknown till recently. In many cancer cells, fragile sites and associated genes suffer frequent deletions and/ or rearrangement, demonstrating their role in genome instability during the oncogenesis process. As a group, fragile sites are heterogeneous and seem to extend over broad regions 0.3–9-Mb long. The regions comprising fragile site are particularly associated with a high frequency of recombinogenic events, including co-localization with chromosome aberrations

cancer rates, and the mortality from cancer occurs late in life.

8 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

characterized at a nucleotide resolution [50].

**3.2. Fragile sites**

and are associated with genes.

sites related to various cancers [69].

The breakage effect of APH may be reduced by using a co-treatment with low concentrations of the topoisomerase I inhibitor and camptothecin (CPT) [75]. CFS regions are highly conserved in vertebrate species, including mouse and primates [76–78]. CFSs initiate proper replication but slow to complete it, introducing breaks from unreplicated regions of DNA [79]. The mechanism proposed for CFS instability resides in this late replication. Late replication may occur due to formation of non-B DNA structures such as hairpins and toroids that block the replication fork in AT rich regions [80].

Rare fragile sites (RFSs) are classified into two sub-groups based on the compounds that induce breakage, folate-sensitive groups and nonfolate-sensitive groups, which are sensitive at bromodeoxyuridine (BrdU) or distamycin A, an antibiotic that binds to AT-pairs of the DNA sequence. The folate-sensitive group is characterized by an expansion of CGG repeats, while the nonfolate-sensitive group contains many AT-rich minisatellite repeats [81–83]. The genome instability mechanism of CGG and AT-rich repeats characteristic for RFSs can form DNA structure (hairpins and other non-B DNA) replication forks, leading to breakage [84, 85]. On the other hand, it was demonstrated that DNA polymerase stops at CTG and CGG triplet repeat sequences, which can result in continuous DNA synthesis via slippage [79].

Fragile site regions are stable in normal cells and become unstable in tumor cells. The breakage of the fragile sites may be caused by mutations leading to a blockage of replication, or by a cell cycle perturbation and gene involved in the DNA repair process deregulation [86]. Several reports developed the concepts that underlie the mechanisms leading to fragile site expression and chromosomal rearrangements at fragile sites in tumors. The analysis of DNA damage response in various tumor types, including bladder, breast, colorectal, and lung tumors, found that early stages of cancer development are associated with an active DNA damage response, including phosphorylated ATR (ataxia telangiectasia and Rad3-related protein), ATM (ataxia telangiectasia mutated), CHK1 (checkpoint kinases), CHK2 kinases, phosphorylated histone H2AX, and p53 [87-88].

These events are linked to a high frequency of LOH (loss of heterozygoty) at known fragile site regions. The explained mechanisms sustained that in precancerous lesions, the blockage or collapsed replication leads to ATR activation and with subsequent DNA double strand breaks. Tumor cells that escape apoptosis or cell cycle arrest will exhibit allelic imbalances, especially at target fragile sites because of replication sensitivity. Further, the model sustains the necessity of p53 mutation and/or other genes involved in checkpoints control, leading therefore to cancer progression. Lesions at common fragile sites are indicators of replication stress during early stages of tumorigenesis [70].

Fragile sites regions are targets for the initiation of the amplification process due to breakage. Several studies showed that boundaries of some amplicons generated through the amplifica‐ tion process mapped to common chromosomal fragile sites in hamster cells [89–90]. Evidences of the role of fragile sites in human cancer regarding gene amplification are scarce. One example of cell line model is for the *MET* amplicons in the esophageal adenocarcinoma map within the fragile site FRA7G [91].

Aphidicolin-sensitive fragile sites FRA5D, FRA5F, and FRA5C, which map distal to dihydrofolate reductase gene (*DHFR)* on 5q, are infrequently expressed and are less likely to contribute to the amplification process. In order for the gene amplification process to take effect the target gene must be in the close proximity of fragile sites, similar to the *MET* amplicon. The breakage at specific genomic sites may not contribute to the amplification process, and no evidence of recurrent amplicon boundaries was found using array CGH in a human cell culture system [92].

#### **3.3. Amplified genes in cancer**

The amplification process is important for deciphering oncogenesis molecular biology, prognosis, and targeted therapies. A good example of gene amplification is dihydrofolate reductase gene (*DHFR*), which usually occurs during progression of methotrexate-resistant acute lymphoblastic leukemia [93]. In cancer, the most amplified genes are members of four different oncogene families: *MYC*, cyclin D1 (or *CCND1*), *EGFR*, and *RAS*. The amplified DNA segment usually involves several hundred kilobases and can contain many genes.

In breast cancer, *MYC, ERBB2, CCND1, EGFR*, or *MDM2* were found to be amplified concom‐ itantly [94]. Moreover, it has been reported that there is a direct correlation between the number of amplifications and an advanced breast cancer and poor survival [95]. *MYC* oncogene is amplified in many types of cancer such as small-cell lung cancer, breast cancer, esophageal cancer, cervical cancer, ovarian cancer, and head and neck cancer [96].

Among the best-known oncogenes that are amplified in cancer cells is *N-MYC*. This gene codes for a transcription factor that plays a physiologic role in stimulating cellular proliferation and is commonly amplified in neuroblastoma where patients have poor clinical prognosis. Amplification of *N-MY*C in neuroblastoma has a valuable prognostic significance, and is correlated with an advanced tumor stage [97], along with *MYC* and *ERBB2* in breast cancer [94].

In malignant thyroid tumors, *C-MYC* gene overexpression and amplification has also been correlated with tumor aggressiveness [98]. Overexpression of cyclins is also an important element in thyroid oncogenesis, playing a crucial role in PTC pathogenesis [98]. *CCND1*, a cell cycle key regulator of G1/S transition, is a frequent target of mutagenesis in many tumors; amplification and rearrangement of its gene can lead to the over-production of this cell cycle regulatory protein. *CCND1* amplification also occurs in breast, esophageal, hepatocellular, and head and neck cancer [99].

*EGFR* (*ERBB1*) is amplified in glioblastoma and head and neck cancer. Amplification of *ERBB2* (also called *HER2/neu*) in breast cancer correlates with a poor prognosis. A monoclonal antibody against the product of this oncogene (trastuzumab) is effective in breast cancers that overexpress HER2/neu.

Fragile sites regions are targets for the initiation of the amplification process due to breakage. Several studies showed that boundaries of some amplicons generated through the amplifica‐ tion process mapped to common chromosomal fragile sites in hamster cells [89–90]. Evidences of the role of fragile sites in human cancer regarding gene amplification are scarce. One example of cell line model is for the *MET* amplicons in the esophageal adenocarcinoma map

Aphidicolin-sensitive fragile sites FRA5D, FRA5F, and FRA5C, which map distal to dihydrofolate reductase gene (*DHFR)* on 5q, are infrequently expressed and are less likely to contribute to the amplification process. In order for the gene amplification process to take effect the target gene must be in the close proximity of fragile sites, similar to the *MET* amplicon. The breakage at specific genomic sites may not contribute to the amplification process, and no evidence of recurrent amplicon boundaries was found using array CGH in

The amplification process is important for deciphering oncogenesis molecular biology, prognosis, and targeted therapies. A good example of gene amplification is dihydrofolate reductase gene (*DHFR*), which usually occurs during progression of methotrexate-resistant acute lymphoblastic leukemia [93]. In cancer, the most amplified genes are members of four different oncogene families: *MYC*, cyclin D1 (or *CCND1*), *EGFR*, and *RAS*. The amplified DNA

In breast cancer, *MYC, ERBB2, CCND1, EGFR*, or *MDM2* were found to be amplified concom‐ itantly [94]. Moreover, it has been reported that there is a direct correlation between the number of amplifications and an advanced breast cancer and poor survival [95]. *MYC* oncogene is amplified in many types of cancer such as small-cell lung cancer, breast cancer, esophageal

Among the best-known oncogenes that are amplified in cancer cells is *N-MYC*. This gene codes for a transcription factor that plays a physiologic role in stimulating cellular proliferation and is commonly amplified in neuroblastoma where patients have poor clinical prognosis. Amplification of *N-MY*C in neuroblastoma has a valuable prognostic significance, and is correlated with an advanced tumor stage [97], along with *MYC* and *ERBB2* in breast cancer [94].

In malignant thyroid tumors, *C-MYC* gene overexpression and amplification has also been correlated with tumor aggressiveness [98]. Overexpression of cyclins is also an important element in thyroid oncogenesis, playing a crucial role in PTC pathogenesis [98]. *CCND1*, a cell cycle key regulator of G1/S transition, is a frequent target of mutagenesis in many tumors; amplification and rearrangement of its gene can lead to the over-production of this cell cycle regulatory protein. *CCND1* amplification also occurs in breast, esophageal, hepatocellular, and

*EGFR* (*ERBB1*) is amplified in glioblastoma and head and neck cancer. Amplification of *ERBB2* (also called *HER2/neu*) in breast cancer correlates with a poor prognosis. A monoclonal

segment usually involves several hundred kilobases and can contain many genes.

cancer, cervical cancer, ovarian cancer, and head and neck cancer [96].

within the fragile site FRA7G [91].

10 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

a human cell culture system [92].

**3.3. Amplified genes in cancer**

head and neck cancer [99].

New data were acquired using array-CGH technique, bringing to knowledge the complex aspect of oncogene amplification in cancer. Using array-CGH analysis on identification of an 8p12 amplicon in anaplastic thyroid carcinoma (ATC) cell lines, six genes were found to be amplified, *DUSP26*,*MET*, *MYC*, *PVT1*, *YAP1* and *CIAP1* [100]*. DUSP26* effectively dephos‐ phorylates *p38* and formed a physical complex with *p38,* promoting the survival of ATC cells by inhibiting *p38-*mediated apoptosis.

The *AIB1* oncogene is located on chromosome 20q, a region frequently amplified and overex‐ pressed in breast cancer [101–102]. High levels of *AIB1* mRNA or protein predict significantly worse prognosis and overall survival in breast cancer patients [103]. *AIB1* is a transcriptional co-activator that promotes the transcriptional activity of multiple nuclear receptors such as the estrogen and progesterone receptors [104].

In cervical cancer, the array-CGH technique revealed that the 3q26.3 amplification was the most consistent chromosomal aberration in primary tissues of cervical carcinoma, and an increased copy number of *PIK3CA* gene was identified [105]. *PIK3CA* is known to be involved in the PI 3-kinase/AKT signaling pathway, which plays an important role in regulating cell growth and apoptosis.

In pancreatic cancer, chromosome 19q13 was found amplified containing *PAK4* gene [106]. *PAK* proteins are critical effectors that link *Rho GTPases* to cytoskeleton reorganization and nuclear signaling. *PAK4* interacts specifically with the GTP-bound form of *Cdc42Hs* and weakly activates the *JNK* family of *MAP* kinases. *PAK4* gene is not in a mutated oncogenic form but the activation of the *PAK4* gene promotes *KRAS2* gene mutation, a very frequent event in pancreatic cancer [106].

DNA amplification represents an important mechanism during human multistep hepatocar‐ cinogenesis. Several genes were found to be amplified within 1q21 amplicon in hepatocellular carcinoma: *CREB3L4* (cyclic AMP responsive element binding protein3-like 4); *JTB* (Jumping Translocation Breakpoint) is a transmembrane protein that suffers an unbalanced translocation in various types of cancers [107]; *INTS3* and *SNAPAP,* whose role in oncogenesis remains to be defined; *SHC1* is involved in signal transduction from receptor tyrosine kinases to down‐ stream signal to *RAS* [108–109]; *CKS1B* (CDC28 protein kinase regulatory subunit 1B), Cks1 expression was closely associated with poor differentiation and also negatively associated with p27kip1 in hepatocellular carcinoma [110]; *CHD1L* (Chromodomain Helicase/ATPase DNA Binding Protein 1-Like, also known as Amplified in Liver Cancer 1, *ALC1*) whose increased expression was associated with clinicopathological features such as microsatellite tumor formation, venous infiltration, and advanced tumor stage, overall survival time, and the disease-free survival rate [111]. Moreover, *Glyoxalase 1 (Glo1)* gene aberrations are associated with tumorigenesis and progression in numerous cancers. Hepatocarcinoma cells with genetic amplified Glo1 gene express higher levels of Glo1 and are more sensitive to cell killing effects if Glo1 expression is down-regulated [112]. The study supports the potential of Glo1 as therapeutic target in patients with hepatocellular carcinoma and genetic Glo1 amplification.

Human oncogene *JUN* gene amplification/overexpression was found in highly aggressive sarcomas and in hepatocellular carcinomas, along with amplification/overexpression of MAP3K5. *JUN* overexpression could interfere with adipocytic differentiation and promote angiogenesis [113, 114].

Amplification of the *FGFR2* gene was identified in a subset of Chinese and Caucasian patients with gastric cancer. Fibroblast growth factor receptor family members (*FGFR1–4*) belong to the *RTK* superfamily. Through interaction with FGF ligands, the receptors are involved in diverse cellular functions including regulation of development processes, mediation of cell proliferation, and differentiation, as well as angiogenesis and tissue regeneration [115, 116]. FGF ligand binding leads to kinase activation and downstream signaling to phosphoinositide 3-kinase (PI3K)-AKT and mitogen-activated protein kinase–extracellular signal–regulated kinase (MAPK-ERK) pathways [117]. Genetic modifications or overexpression of FGFRs have been associated with tumorigenesis and progression in breast, prostate, stomach, and hema‐ tologic malignancies [118, 119]. *FGFR2* amplification leads to constitutive activation of the FGFR2 signaling pathway in gastric cancer, and furthermore inhibition of this pathway using a well-tolerated, potent, and selective inhibitor can lead to rapid and durable tumor regressions in *FGFR2*-amplified gastric cancer xenograft models, representing an important treatment target [120].

#### **3.4. Chromosomal translocations**

Chromosomal translocations (CTs) are very common in human cancer, and the molecular mechanisms involved are complex and poorly understood. CTs are involved in several types of cancer, particularly in hematopoietic and lymphoid tumors [121]. This type of chromosomal abnormality seems to provide a selective growth advantage for some stem or progenitor cells, which may further initiate the development of some malignant tumors. In case of oncogenes, CTs may change the original locations of proto-oncogenes, generating effects on the gene products through two major ways [122, 123]. One is to generate oncogenic fusion proteins and the other way is that proto-oncogenes are brought into proximity with regulatory elements, causing the overexpression of proto-oncogene.

The first specific chromosomal translocation identified in human cancer was the Philadelphia chromosome [t(9;22)], which underlies chronic myeloid leukemia (CML). The fusion of chromosomes 9 and 22 leads to the joining of two unrelated genes, the *C-ABL* gene, which encodes a tyrosine kinase and is located on chromosome 9, and the gene *BCR* (for breakpoint recombination) located on chromosome 22.10 [124]. A chimeric protein (BCR-ABL) with novel transforming properties is formed from this specific chromosomal rearrangement. BCR-ABL oncoprotein has an abnormal tyrosine kinase activity and is associated with the tumorigenesis of CML and acute lymphoblastic leukemia (ALL) [124]. Duplication of the Philadelphia chromosome leads to accelerated CML blast phase, suggesting that increased copies of this aberrant gene confer a dose-dependent transforming effect [125]. Similar to t(9;22) in acute promyelocytic leukemia (APL), a chromosomal rearrangement joins a novel gene t(15;17), resulting in the formation of promyelocytic leukemia-retinoic acid receptor α (*PML-RARα*) fusion oncoprotein [126].

*PML-RAR* function is unknown, but this translocation underlies the response of this leukemia type to treatment with trans-retinoic acid. Another intergenic, CT t(12;21), leads to a novel chromosomal translocation product, *TEL-AML1*, which requires a specific treatment for pediatric acute lymphoblastic leukemia [127].

Human oncogene *JUN* gene amplification/overexpression was found in highly aggressive sarcomas and in hepatocellular carcinomas, along with amplification/overexpression of MAP3K5. *JUN* overexpression could interfere with adipocytic differentiation and promote

12 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Amplification of the *FGFR2* gene was identified in a subset of Chinese and Caucasian patients with gastric cancer. Fibroblast growth factor receptor family members (*FGFR1–4*) belong to the *RTK* superfamily. Through interaction with FGF ligands, the receptors are involved in diverse cellular functions including regulation of development processes, mediation of cell proliferation, and differentiation, as well as angiogenesis and tissue regeneration [115, 116]. FGF ligand binding leads to kinase activation and downstream signaling to phosphoinositide 3-kinase (PI3K)-AKT and mitogen-activated protein kinase–extracellular signal–regulated kinase (MAPK-ERK) pathways [117]. Genetic modifications or overexpression of FGFRs have been associated with tumorigenesis and progression in breast, prostate, stomach, and hema‐ tologic malignancies [118, 119]. *FGFR2* amplification leads to constitutive activation of the FGFR2 signaling pathway in gastric cancer, and furthermore inhibition of this pathway using a well-tolerated, potent, and selective inhibitor can lead to rapid and durable tumor regressions in *FGFR2*-amplified gastric cancer xenograft models, representing an important treatment

Chromosomal translocations (CTs) are very common in human cancer, and the molecular mechanisms involved are complex and poorly understood. CTs are involved in several types of cancer, particularly in hematopoietic and lymphoid tumors [121]. This type of chromosomal abnormality seems to provide a selective growth advantage for some stem or progenitor cells, which may further initiate the development of some malignant tumors. In case of oncogenes, CTs may change the original locations of proto-oncogenes, generating effects on the gene products through two major ways [122, 123]. One is to generate oncogenic fusion proteins and the other way is that proto-oncogenes are brought into proximity with regulatory elements,

The first specific chromosomal translocation identified in human cancer was the Philadelphia chromosome [t(9;22)], which underlies chronic myeloid leukemia (CML). The fusion of chromosomes 9 and 22 leads to the joining of two unrelated genes, the *C-ABL* gene, which encodes a tyrosine kinase and is located on chromosome 9, and the gene *BCR* (for breakpoint recombination) located on chromosome 22.10 [124]. A chimeric protein (BCR-ABL) with novel transforming properties is formed from this specific chromosomal rearrangement. BCR-ABL oncoprotein has an abnormal tyrosine kinase activity and is associated with the tumorigenesis of CML and acute lymphoblastic leukemia (ALL) [124]. Duplication of the Philadelphia chromosome leads to accelerated CML blast phase, suggesting that increased copies of this aberrant gene confer a dose-dependent transforming effect [125]. Similar to t(9;22) in acute promyelocytic leukemia (APL), a chromosomal rearrangement joins a novel gene t(15;17), resulting in the formation of promyelocytic leukemia-retinoic acid receptor α (*PML-RARα*)

angiogenesis [113, 114].

target [120].

**3.4. Chromosomal translocations**

fusion oncoprotein [126].

causing the overexpression of proto-oncogene.

A classic example is the overexpression of proto-oncogene c-MYC in Burkitt lymphoma due to t(8;14) that results in *c-MYC* gene juxtaposed to immunoglobulin heavy chain (IGH) regulatory elements [128, 129]. Further expression of the gene is directed by the strong immunoglobulin heavy-chain enhancer, which is constitutively active in B lymphocytes. Thus, c-*MYC* overexpression is a potent force driving cellular proliferation.

The t(11;14) translocation juxtaposes *CCND1* and immunoglobulin enhancer elements and is characteristic of mantle-cell lymphoma. The t(11;14) translocation juxtaposes *CCND1* and immunoglobulin enhancer elements [130].

The ability to grow leukemic cells in culture long enough to allow cytogenetic analysis has facilitated the characterization of chromosomal translocations in leukemia. However, specific chromosomal translocations have also been observed in solid tumors. Aside from interchro‐ mosomal translocations, intrachromosomal translocations are also associated with cancer. Around 60–70% of PTCs have a characteristic inv(10)(q11.2q21). The breakpoint is represented by *RET* gene locus (10q11.2), which is relegated to the opposite breakpoint of the *H4* (D10S170) or *NCOA4* (ELE1) gene (10q21) in the same chromosome [131]. The H4 protein is widely expressed in the nucleus and cytoplasm and its function is unknown [132]. In PTC, many types of rearrangement loci (11 rearranged forms) were noted and PTC1(H4, CCDC6)-RET and PTC3(NCOA4)-RET are the most common [133]. PTC2-RET is a less common type of PTC-RET [134]. These rearrangements can lead to constitutively ligand-independent RET activity involved in thyroid carcinogenesis. The hypothesis sustain that the distances between RET and H4 loci are 18 Mb, therefore chromosome folding may close the two loci to each other in thyroid cells, increasing the probability of recombination between them in the interphase nuclei. This chromosomal folding is specific for thyroid cells, and this may explain why inv(10) (q11.2q21) is frequently seen in PTC [135].

It has been shown that in prostate carcinomas, the fusion between *TMPR552* gene and two transcription factors *ERG*1 or *ETV1* creates a fusion protein that increases proliferation and inhibits apoptosis of cells in the prostate gland, thereby facilitating their transformation into cancer cells [1].

The translocations of ETS are often found in human cancer, such as Ewing sarcoma [136–137], leukemia [138–139], prostate cancer [140], and breast cancer [141]. These once disparate tumors are now defined by a chromosomal translocation fusing the EWS gene to a number of tran‐ scription factors of the ETS gene family (the most common chimeric protein is EWS-FLI1) [142]. This chimeric product presumably acts directly on target promoters to direct the expression of genes that induce cellular proliferation. Identification of EWS translocations allowed the molecular grouping of a class of tumors whose proliferation is driven by similar genetic alterations and that respond to similar chemotherapeutic regimens.

#### **4. Oncogene hypomethylation**

The first epigenetic modification observed in human cancer was the loss of DNA methylation at the 5'citosine level (m5C residues replaced by unmethylated C residues), reported in 1983 [143]. This discovery was often regarded as an unwelcome complication, and all of the attention was focused on the opposite effect hypermethylation of promoters of genes that are silenced in cancers (e.g., tumor-suppressor genes). Global hypomethylation of DNA in cancer was found associated especially with repeated DNA elements; this modification did not represent a research direction for many years [144]. However, changes in the pattern of DNA methylation have been a consistent modification in cancer cells. Both hypo- and hypermethylation were observed at various loci, but at this moment it is clear that DNA methylation plays an important role in carcinogenesis.

New deep sequencing methylome analyses have shown much more cancer-linked hypome‐ thylation of unique gene sequences and hypermethylation of repeated sequences than previously found [145–148]. Targeting DNA repetitive sequence, DNA hypomethylation may induce genomic instability and mutation events in cancer genomes [149–152] by altering the intranuclear positioning of chromatin enhancing recombination [153–155] and activating retroviral elements [156]. Promoter hypomethylation of some genes may be associated with the development of cancer by regulating the activity of genes [157].

#### **4.1. Genomic hypomethylation profiles in cancer**

DNA methylation principally occurs at 5' cytosine from dinucleotide CpG sites [158, 159]. CpG dinucleotides are found in C+G-rich regions in the genome termed CpG islands, localized frequently at promoter or gene regulatory level. However, the vast majority of CpG dinucleo‐ tides are localized within the intergenic and intronic regions of the DNA, particularly within repeat sequences and transposable elements. Unmethylated CpG islands at gene level are associated with gene transcription. In normal somatic cells, between 70% and 90% of CpG dinucleotides are methylated, which constitute approximately 0.75–1% of the total number of bases in the genome, while most CpG islands are unmethylated [160]. A part of genes promoter region are methylated as part of normal developmental processes or tissue specific (e.g., germline specific genes-MAGE genes) [161]. In X chromosomes in female dosage compensation (imprinted genes of X chromosomes in females), where only one of two copies is active, methylation of regulatory regions is involved in the repression of the expression of the silent loci [162].

Recently, high-resolution genome-wide analyses of DNA methylation changed the idea that considers oncogenesis being characterized predominantly of hypomethylated DNA repeats and hypermethylated gene regions [163–164]. The hallmark for cancer is represented by global losses of DNA methylation with local hypermethylation and hypomethylation of specific genes [165–167].

Evaluation of the majority of cancers showed that a major contributor to global DNA hypo‐ methylation is hypomethylation of tandem and interspersed DNA repeats [165, 168]. Several studies using CpG methylation-sensitive restriction endonucleases or sodium bisulfite reported that hypomethylation was often found at gene sequence level (including metastasisassociated genes) [157, 165].

#### **4.2. Hypomethylation of DNA repeats sequence in cancer**

**4. Oncogene hypomethylation**

14 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

role in carcinogenesis.

loci [162].

genes [165–167].

The first epigenetic modification observed in human cancer was the loss of DNA methylation at the 5'citosine level (m5C residues replaced by unmethylated C residues), reported in 1983 [143]. This discovery was often regarded as an unwelcome complication, and all of the attention was focused on the opposite effect hypermethylation of promoters of genes that are silenced in cancers (e.g., tumor-suppressor genes). Global hypomethylation of DNA in cancer was found associated especially with repeated DNA elements; this modification did not represent a research direction for many years [144]. However, changes in the pattern of DNA methylation have been a consistent modification in cancer cells. Both hypo- and hypermethylation were observed at various loci, but at this moment it is clear that DNA methylation plays an important

New deep sequencing methylome analyses have shown much more cancer-linked hypome‐ thylation of unique gene sequences and hypermethylation of repeated sequences than previously found [145–148]. Targeting DNA repetitive sequence, DNA hypomethylation may induce genomic instability and mutation events in cancer genomes [149–152] by altering the intranuclear positioning of chromatin enhancing recombination [153–155] and activating retroviral elements [156]. Promoter hypomethylation of some genes may be associated with

DNA methylation principally occurs at 5' cytosine from dinucleotide CpG sites [158, 159]. CpG dinucleotides are found in C+G-rich regions in the genome termed CpG islands, localized frequently at promoter or gene regulatory level. However, the vast majority of CpG dinucleo‐ tides are localized within the intergenic and intronic regions of the DNA, particularly within repeat sequences and transposable elements. Unmethylated CpG islands at gene level are associated with gene transcription. In normal somatic cells, between 70% and 90% of CpG dinucleotides are methylated, which constitute approximately 0.75–1% of the total number of bases in the genome, while most CpG islands are unmethylated [160]. A part of genes promoter region are methylated as part of normal developmental processes or tissue specific (e.g., germline specific genes-MAGE genes) [161]. In X chromosomes in female dosage compensation (imprinted genes of X chromosomes in females), where only one of two copies is active, methylation of regulatory regions is involved in the repression of the expression of the silent

Recently, high-resolution genome-wide analyses of DNA methylation changed the idea that considers oncogenesis being characterized predominantly of hypomethylated DNA repeats and hypermethylated gene regions [163–164]. The hallmark for cancer is represented by global losses of DNA methylation with local hypermethylation and hypomethylation of specific

Evaluation of the majority of cancers showed that a major contributor to global DNA hypo‐ methylation is hypomethylation of tandem and interspersed DNA repeats [165, 168]. Several

the development of cancer by regulating the activity of genes [157].

**4.1. Genomic hypomethylation profiles in cancer**

Repeat sequences are represented by transposable elements found interspersed throughout the genome, or simple repeat sequences, such as DNA satellites, found in pericentromeric or subtelomeric region of chromosomes. These are normally methylated within the healthy genome [169].

In cancer, hypomethylation of DNA repeats is a result of the demethylation process rather than the preexisting hypomethylation in a cancer stem cell [170]. The frequency of cancer-associated hypomethylation of DNA repeats is dependent with disease progression (tumor grade, stage) [171, 172]. Hypomethylation is also seen in tumor adjacent tissues and in benign tumors (breast fibroadenomas and ovarian cystadenomas), but at a lower level than cancer [145, 165, 173, 174].

Hypomethylation may affect transcription and hypomethylation of interspersed DNA repeats within promoter modifies the chromatin boundaries resulting in transcription activation of nearby genes [175, 176]. Along with the effects upon transcription, hypomethylation can affect alternative splicing and hypomethylation of a minor portion of interspersed DNA repeats may occasionally cause induction of retroviral element transcription [156]. Several studies reported numerous evidences for the causal relationships between DNA hypomethylation and in‐ creased transcription as well as hypomethylation and cancer [177–179].

Regions of cancer-associated changes in DNA methylation are found in short interspersed or clustered regions, as well as in long blocks [180–182]. Dante et al. described hypomethylation of LINE-1 (a highly repeated interspersed repeat) in mononuclear cells from patients with chronic lymphocytic leukemia [183]. Along with hypomethylation of LINE-1, Alu repeats were also subsequently observed hypomethylated in many other types of cancers [183–186]. In breast adenocarcinomas, ovarian epithelial cancers, and Wilms tumors, a hypomethylation of centromeric and juxtacentromeric satellite DNA was noted [173, 174, 187]. Moreover, another classes of tandem repeats (macrosatellite DNAs) and segmental duplications were found hypomethylated in various cancers [188–190]. The loss of DNA methylation in cancer varies according to the tumor type and subclasses of DNA repeat [191–193].

Gathering the result of the presented studies, we may conclude that in many types of cancer, hypomethylation of DNA repeats represents a highly informative prognostic marker and/or predictor of survival [194–197].

#### **4.3. Hypomethylation of DNA gene enhancer sequence in cancer**

Gene expression levels may be further modulated by DNA methylation levels at upstream enhancer sites [198], which can affect the binding of transcription factors at (CpG) islands [199]. In normal cells, DNA demethylation at enhancer's level is correlated with upregulation of expression of the associated gene. It was shown that the binding of FoxA1/FOXA1 transcrip‐ tional factors to enhancers is inhibited by DNA methylation at the respective binding site [200]. In this case, modification of DNA methylation status (demethylation) at the enhancer level may lead to an open chromatin state allowing the access of transcription factors at the active enhancer [201–202]. Following DNA demethylation, FoxD3 transcription factor binds at the enhancer level, allowing the recruitment of FoxA1 and conversion of the enhancer to a state that is set for activity. Local DNA demethylation leads also to changes in histone H3K27 or H3K9 methylation [200]. FOXA1 is an important factor for oncogenesis being involved in various types of cancer [203]. Thus, DNA hypomethylation from transcription regulatory regions may cause changes in expression [204].

#### **4.4. Genomic hypomethylation in promoters and within gene bodies**

Hypomethylation of transcription regulatory regions is less frequent than hypermethylation of CpG island promoters in cancer. Some of the gene regions (including transcription control sequences) were associated with loss of DNA methylation. Currently, there are data that sustain that promoter hypomethylation of some genes may be associated with the develop‐ ment of cancer, regulating the activity of genes [157]. For example, promoter hypomethylation of specific immunity-related genes (e.g., cytokine IL-10) may activate the specific gene expression to inhibit the immune response in breast cancer [205], and the promoter hypome‐ thylation of SPAN-Xb, an immunogenic antigen, can induce de novo B-cell response in myeloma cells [206]. However, the biological significance of promoter hypomethylation in cancer is still poorly understood [144]. Hypomethylation of gene promoters must cooperate with other key activators such as transcriptional factors to control gene expression [207, 208].

Promoters may overlap tissue-specific (T-DMR) or cancer-specific (C-DMR) differentially methylated DNA regions [209]. Most of the non-imprinted, autosomal T-DMR promoters are not the main type of vertebrate DNA promoters, and the genes presenting T-DMR promoters become activated after experimentally induced demethylation 5-deoxyazacytidine [209].

Intragenic epigenetic marks have been also involved in normal gene expression regulation and inverse relationships between imprinted gene expression and DNA methylation level was observed [210]. T-DMR regions were found not only inside many genes, but also in down‐ stream promoters, flanking certain subsets of genes [211, 212]. Moreover, besides first exon, T-DMRs are also present at exonic and intronic sequences, insulators, intragenic ncRNA genes, and 3 'terminal regions [213, 214].

The role of these regions is to connect DNA and chromatin, inducing tissue-specific chromatin epigenetic marks inside genes [215, 216]. This relationship between DNA and chromatin modification at gene level may help determine alternative promoter usage, modulate the rate of transcription initiation or elongation, and direct the choice of alternative splice sites [217, 218]. For moderately expressed genes, DNA methylation level in the middle of the gene is correlated with higher transcription rates, being related to nucleosome positioning [219]. In genes with CpG-poor promoters, methylated sequences located downstream binds Polycomb repressor complexes [212], which are being associated with repression of promoters [220].

On the other hand, certain histone modifications may direct the choice of splice junction through direct interactions with proteins that mark exon–intron junctions, altering rates of transcription and nucleosome positioning [221, 222]. As we mentioned before, DNA methyl‐ ation may also be involved in regulating alternative splicing, intron–exon junctions being enriched in sharp transitions in DNA methylation levels [223] (e.g., malignant prostate cancer cells have enrichment of DNA hypermethylation at exon–intron junctions [224]). Therefore, these findings highlight the involvement of DNA methylation levels in determining alternative splicing in tumor cells, suggesting that cancer-associated DNA hypomethylation in intronic and exonic sequences can modulate the amount and type of gene products and thereby contribute to tumor formation or progression.

tional factors to enhancers is inhibited by DNA methylation at the respective binding site [200]. In this case, modification of DNA methylation status (demethylation) at the enhancer level may lead to an open chromatin state allowing the access of transcription factors at the active enhancer [201–202]. Following DNA demethylation, FoxD3 transcription factor binds at the enhancer level, allowing the recruitment of FoxA1 and conversion of the enhancer to a state that is set for activity. Local DNA demethylation leads also to changes in histone H3K27 or H3K9 methylation [200]. FOXA1 is an important factor for oncogenesis being involved in various types of cancer [203]. Thus, DNA hypomethylation from transcription regulatory

Hypomethylation of transcription regulatory regions is less frequent than hypermethylation of CpG island promoters in cancer. Some of the gene regions (including transcription control sequences) were associated with loss of DNA methylation. Currently, there are data that sustain that promoter hypomethylation of some genes may be associated with the develop‐ ment of cancer, regulating the activity of genes [157]. For example, promoter hypomethylation of specific immunity-related genes (e.g., cytokine IL-10) may activate the specific gene expression to inhibit the immune response in breast cancer [205], and the promoter hypome‐ thylation of SPAN-Xb, an immunogenic antigen, can induce de novo B-cell response in myeloma cells [206]. However, the biological significance of promoter hypomethylation in cancer is still poorly understood [144]. Hypomethylation of gene promoters must cooperate with other key activators such as transcriptional factors to control gene expression [207, 208].

Promoters may overlap tissue-specific (T-DMR) or cancer-specific (C-DMR) differentially methylated DNA regions [209]. Most of the non-imprinted, autosomal T-DMR promoters are not the main type of vertebrate DNA promoters, and the genes presenting T-DMR promoters become activated after experimentally induced demethylation 5-deoxyazacytidine [209].

Intragenic epigenetic marks have been also involved in normal gene expression regulation and inverse relationships between imprinted gene expression and DNA methylation level was observed [210]. T-DMR regions were found not only inside many genes, but also in down‐ stream promoters, flanking certain subsets of genes [211, 212]. Moreover, besides first exon, T-DMRs are also present at exonic and intronic sequences, insulators, intragenic ncRNA genes,

The role of these regions is to connect DNA and chromatin, inducing tissue-specific chromatin epigenetic marks inside genes [215, 216]. This relationship between DNA and chromatin modification at gene level may help determine alternative promoter usage, modulate the rate of transcription initiation or elongation, and direct the choice of alternative splice sites [217, 218]. For moderately expressed genes, DNA methylation level in the middle of the gene is correlated with higher transcription rates, being related to nucleosome positioning [219]. In genes with CpG-poor promoters, methylated sequences located downstream binds Polycomb repressor complexes [212], which are being associated with repression of promoters [220].

regions may cause changes in expression [204].

16 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

and 3 'terminal regions [213, 214].

**4.4. Genomic hypomethylation in promoters and within gene bodies**

*TGFB2* gene contains an intronic Alu repeat that was found hypomethylated in some cancer cell lines. Their hypomethylation at this site might be related to the significant upregulation of *TGFB2* gene, being an example of cancer-associated hypomethylation and a target chromatin associated epigenetic changes [225]. *PRDM16* presents gene-body hypomethylation (overlap‐ ping an exon) in some of the cancer cell lines, whereas *NOTCH2* also showed gene-body hypomethylation (in a subregion of repetitive DNA).

Gene encoding the protease urokinase (*PLAU/uPA*) is overexpressed and was found hypo‐ methylated along with tumor progression in breast cancers and prostate cancers [157]. Also, other genes were observed to display hypomethylation and transcriptional activation in cancer, *S100A4*, mesothelin, claudin4, trefoil factor 2, maspin, *PGP9.5, POMC*, and the heparinase gene [144].

DNA hypomethylation is closely associated with morphological dedifferentiation in thyroid cancers. Four oncogenes (*INSL4, DPPA2, TCL1B* and *NOTCH4*) were frequently regulated by hypomethylation in anaplastic and medulary carcinoma [226].

*Hematopoietin, TNF, IL1, IL10*, and *IL17* families of cytokines had a significant tendency to be hypomethylated in five cancer types (colon, kidney, stomach, lung, and breast) [227].

Hypomethylation and increased expression in cancer has been shown for *R-RAS* [228]. A strong association of *CDH3* promoter demethylation and *P-cadherin* expression evident with histological grade and invasiveness in breast cancer was observed [229]. In Stage III and IV gastric cancer *cyclin D*2 activation is associated with promoter demethylation, activation of *synuclein γ* is associated with progression and metastatic potential in a range of solid tumors, and *maspin* expression in colorectal cancer is associated with microsatellite unstable tumors [230–232].

Hypomethylation and overexpression of some imprinted genes, including the *IGF-II*and *H19* genes, are implicated in carcinogenesis [233–235].

The putative oncogene, *ELMO3*, is overexpressed in non-small cell lung cancer in combination with hypomethylation of its promoter and these cancer-specific events are associated with the formation of metastases [236].

Aberrant hypomethylation and overexpression of *WNT5A* may be functionally important in the progression of prostate cancer. Along with *WNT5A, S100P,* and *CRIP1*, which have been previously implicated in cancer progression, are also regulated at the transcriptional level in prostate cancer by hypomethylation [237].

Evidence is accumulating for the biological significance and clinical relevance of DNA hypomethylation in cancer and for cancer-linked demethylation, and those seem to be highly dynamic processes.

#### **5. MicroRNA genes**

At present, a special consideration is given to small non-coding RNA molecules (microRNA) to their functions and involvement in human diseases. There are an extensive number of studies that link microRNA alterations to cancer pathogenesis. MicroRNA genes encode for a single RNA strand of about 21 to 23 nucleotides, which regulate gene expression by specifically targeting certain mRNAs in order to prevent them from coding for a specific protein. Some microRNA genes are mapped in chromosomal regions that undergo rearrangements, dele‐ tions, and amplifications in cancer. A growing amount of data demonstrates that microRNA genes display a different pattern of expression in various malignancies; they are found upregulated or down-regulated and therefore can function either as oncogenes activating the malignant transformation (by down-regulating tumor-suppressor genes), or as tumorsuppressor genes blocking the malignant transformation (by down-regulating oncogenes). In numerous types of cancers, many different microRNA have been shown to act as oncogenes, their expression profiling presenting specific signatures associated with malignant transfor‐ mation. Cancer-associated microRNA molecules are also called oncomir (oncomiR).

The first microRNA that has been proven to act as oncogene in human cancer was *miR-17/92* polycistronic cluster known as *OncomiR-1*, which comprises six microRNAs: *miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1* [238]. The *miR-17/92* cluster is located in the locus of intron 3 of *C13orf25* gene at 13q31.3, in a region frequently amplified in several types of lymphomas and solid tumor. It has been shown that the locus is amplified and overexpressed in human B cell lymphomas, malignant lymphoma cell lines and in lung cancers especially with small-cell lung cancer histology [239, 240]. Insertional mutagenesis studies using retroviruses indicates that *miR-17/92* acts as an oncogene in T cell lymphomas; it was shown that soon after SL3-3 murine leukemia virus infection, mice developed tumors if provirus integrates into the proximity of the gene encoding *miR-17/92* cistron [241]. Moreover, other studies uncovered that *C-MYC* and *E2F3* gene products may induce *miR-17/92* polycistronic expression through direct binding to the cluster promoter. Two microRNAs belonging to the cluster, *miR-17-5p* and *miR-20a* negatively regulate *E2F1* activity, which confirms that the *miR-17/92* can promote cell proliferation through the exchange of *E2F1* to *E2F3* pro-apoptotic proliferative [242]. Thus, *miR-17/92* represents an anti-apoptotic oncogene and *miR-20a* inhibition using antisense oligonucleotides can induce apoptosis after treatment with doxor‐ ubicin [243].

Along with the *miR-17/92* cluster from the *miR-17* family, two other paralogue miRNA gene clusters are produced, *miR-106b/25* and *miR-106a/363*, which possess oncogenic potential and are known to be involved in wide types of cancers. The *miR-106b/25* cluster is located in intron 13 of the minichromosome maintenance complex component 7 (*MCM7*) oncogene at 7q22.1 and it contains the following three miRNAs: *miR-106b*, *miR-93,* and *miR-25*. Recently, findings sustain the oncogenic potential of this cluster and reports correlate *miR-106b/25* member expression levels with processes such as tumor growth, cell survival, and angiogenesis [244, 245]. The oncogenic potential of the *miR-106b/25* cluster in malignant transformation is achieved by targeting and down-regulation of several tumor-suppressor genes such as *p21, E2F1,* and *PTEN* [246–248]. Furthermore, other recent work suggests that in breast cancer cells, *miR-106b/25* cluster overexpression leads to overcoming doxorubicin-induced senescence and cells become drug resistant through a mechanism that involves targeting *E-cadherin* transcrip‐ tional activators *EP300* [249]. The second cluster, *miR-106a/363*, is located on chromosome X (Xq26.2) and comprises of six miRNAs: *miR-106a*, *miR-18b, miR-20b, miR-19b-2, miR-92a-2, and miR-363*. A series of reports indicates an oncogenic potential for members of the cluster, for example *miR-106a* and *miR-92-2* were found overexpressed in colon and prostate cancer and also in leukemia and Ewing Sarcoma [250–252].

previously implicated in cancer progression, are also regulated at the transcriptional level in

Evidence is accumulating for the biological significance and clinical relevance of DNA hypomethylation in cancer and for cancer-linked demethylation, and those seem to be highly

At present, a special consideration is given to small non-coding RNA molecules (microRNA) to their functions and involvement in human diseases. There are an extensive number of studies that link microRNA alterations to cancer pathogenesis. MicroRNA genes encode for a single RNA strand of about 21 to 23 nucleotides, which regulate gene expression by specifically targeting certain mRNAs in order to prevent them from coding for a specific protein. Some microRNA genes are mapped in chromosomal regions that undergo rearrangements, dele‐ tions, and amplifications in cancer. A growing amount of data demonstrates that microRNA genes display a different pattern of expression in various malignancies; they are found upregulated or down-regulated and therefore can function either as oncogenes activating the malignant transformation (by down-regulating tumor-suppressor genes), or as tumorsuppressor genes blocking the malignant transformation (by down-regulating oncogenes). In numerous types of cancers, many different microRNA have been shown to act as oncogenes, their expression profiling presenting specific signatures associated with malignant transfor‐

mation. Cancer-associated microRNA molecules are also called oncomir (oncomiR).

The first microRNA that has been proven to act as oncogene in human cancer was *miR-17/92* polycistronic cluster known as *OncomiR-1*, which comprises six microRNAs: *miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1* [238]. The *miR-17/92* cluster is located in the locus of intron 3 of *C13orf25* gene at 13q31.3, in a region frequently amplified in several types of lymphomas and solid tumor. It has been shown that the locus is amplified and overexpressed in human B cell lymphomas, malignant lymphoma cell lines and in lung cancers especially with small-cell lung cancer histology [239, 240]. Insertional mutagenesis studies using retroviruses indicates that *miR-17/92* acts as an oncogene in T cell lymphomas; it was shown that soon after SL3-3 murine leukemia virus infection, mice developed tumors if provirus integrates into the proximity of the gene encoding *miR-17/92* cistron [241]. Moreover, other studies uncovered that *C-MYC* and *E2F3* gene products may induce *miR-17/92* polycistronic expression through direct binding to the cluster promoter. Two microRNAs belonging to the cluster, *miR-17-5p* and *miR-20a* negatively regulate *E2F1* activity, which confirms that the *miR-17/92* can promote cell proliferation through the exchange of *E2F1* to *E2F3* pro-apoptotic proliferative [242]. Thus, *miR-17/92* represents an anti-apoptotic oncogene and *miR-20a* inhibition using antisense oligonucleotides can induce apoptosis after treatment with doxor‐

Along with the *miR-17/92* cluster from the *miR-17* family, two other paralogue miRNA gene clusters are produced, *miR-106b/25* and *miR-106a/363*, which possess oncogenic potential and

prostate cancer by hypomethylation [237].

18 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

dynamic processes.

ubicin [243].

**5. MicroRNA genes**

Another important oncomir is represented by *miR-155* found overexpressed in several malignancies: chronic lymphocytic leukemia (CLL), B cell lymphoma, Hodgkin's lymphoma or Burkitt's type, and breast cancer. Some reports have shown that clinical isolates from B cell lymphomas, including those with large cells, contain a number of copies of *miR-155,* about 30 times higher than normal B cells [253–256]. Also, results suggest that the pancreatic ductal adenocarcinomas overexpression of *miR-155* determined decreased levels of *TP53INP1* leading to apoptosis elusion and cell growth development [257].

A promising oncomir is also *miR-21*, one of the most common miRNA associated with human cancers. *MicroRNA-21* high expression has been found in a variety of cancers including breast cancer, brain malignant tumors, glioblastomas, pancreatic, colorectal, liver, gastric, lung, skin, thyroid, ovarian, esophagus, prostate, cervical, and different lymphatic and hematopoietic cancers [250, 258–263]. Elevated *miR-21* levels have been linked to cell proliferation, apoptosis reduction, and cell migration in neoplastic transformation; it has been found that this oncomir targets and down-regulates a number of tumor-suppressor genes including *PTEN, PDCD4, BCL2, RECK, JAG1, HNRPK, BTG2, TGFBRII*, and thus sustaining cancer's invasion and metastasis [264, 265]. Moreover, experiments using transfection of MCF-7 cell lines with antimiR-21 oligonucleotide conducted to cell growth suppression in vitro and tumor growth in vivo had an increase of programmed cell death rate [266].

Altogether, these studies illustrate a major role for microRNA genes in cancer pathogenesis (Table 2); many of them have oncogenic activity and could represent valuable biomarkers very useful for cancer screening or assessment of the therapeutic effects of anti-cancer treatments.




**MicroRNA Cancer type miRNA function Potential targets Reference**

Increase cell growth Increase metastasis

Promote cell growth Promote cell migration Promote tumor growth


Promote cell invasion Promote metastasis

Increase cell Proliferation Inhibit apoptosis Increase cell migration

Promote epithelialmesenchymal transition

> Induce tumor angiogenesis Activate AKT/ERK signaling

Increase docetaxel resistance

Promote cell proliferation

Inhibit apoptosis Promote cell motility Promote cell invasion

Promote cell migration Reduce apoptosis Promote cell growth

Promote cell cycle G1/S transition Increase colony formation

Increase cell migration Increase cell invasion epithelial-mesenchymal transition


*PDCD4 PTEN BCL2*

*TPM1 PDCD4 SERPINB5*

Promote cell proliferation *PDCD4* [274]

Promote tumor growth [278]

*PTEN* [269, 270]

[272]

[265]

[276]

*ANKRD46* [271]

*CCL20* [273]

*BTG2* [275]

*PDCD4* [277]

*MARCKS* [279]

*PDCD4* [280, 281]

*CDKN1B* [282]

*TRPS1* [283]

Breast

20 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Cervix

Prostate

Blood

Prostate

Breast

*miR-221 and 222*


**Table 2.** OncomiRs in human cancer

#### **6. Concluding remarks**

Oncogene activation by structural alteration (chromosomal rearrangement, gene fusion, mutation, and gene amplification) or epigenetic modification (gene promoter hypomethyla‐ tion, microRNA expression pattern) confers an increased or a deregulated expression. Therefore, cells with such alterations possess a growth advantage or an increased survival rate. Given the fact that expression profiling of these alterations determines specific signatures associated with tumor classification, diagnosis, staging, prognosis and response to treatment, it highlights the importance of studying oncogenes activation mechanisms and the great potential that they hold as therapeutic tools in the near future.

#### **Acknowledgements**

This work was supported by Romanian Research Grant PCCA135/2012 and POSDRU/159/1.5 / S/135760

#### **Author details**

**MicroRNA Cancer type miRNA function Potential targets Reference**

Activate TGF-beta signaling Induce epithelial mesenchymal transition Induce a tumor initiating cell phenotype

Promote epithelialmesenchymal transition Increase cell migration Increase cell invasion

Promote cell proliferation Inhibit apoptosis Promote tumor growth

(Source: OncomiRDB: http://bioinfo.au.tsinghua.edu.cn/member/jgu/oncomirdb/) [310].

potential that they hold as therapeutic tools in the near future.

Gastric *E2F1* [306]

Gastric *NDST1* [309]

Oncogene activation by structural alteration (chromosomal rearrangement, gene fusion, mutation, and gene amplification) or epigenetic modification (gene promoter hypomethyla‐ tion, microRNA expression pattern) confers an increased or a deregulated expression. Therefore, cells with such alterations possess a growth advantage or an increased survival rate. Given the fact that expression profiling of these alterations determines specific signatures associated with tumor classification, diagnosis, staging, prognosis and response to treatment, it highlights the importance of studying oncogenes activation mechanisms and the great

This work was supported by Romanian Research Grant PCCA135/2012 and POSDRU/159/1.5 /

*miR-106b-25 cluster*

*miR-191*

**Table 2.** OncomiRs in human cancer

**6. Concluding remarks**

**Acknowledgements**

S/135760

Breast

22 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Liver

*HIF-1a, PTEN, BCL2L11, CDKNA and TSP-1.*

*SMAD7* [305]

*TIMP3* [307]

[303,304]

[308]

Anca Botezatu1 , Iulia V. Iancu1 , Oana Popa2 , Adriana Plesa1 , Dana Manda2 , Irina Huica1 , Suzana Vladoiu2 , Gabriela Anton1 and Corin Badiu3

1 Molecular Virology, Stefan S. Nicolau Institute of Virology, Romania

2 Research Laboratory, "CI Parhon" National Institute of Endocrinology, Bucharest, Romania

3 Department of Endocrinology, "CI Parhon" National Institute of Endocrinology, Bucharest, Romania

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### **Cell Cycle Regulation via the p53, PTEN, and BRCA1 Tumor Suppressors**

Akari Minami, Toshiyuki Murai, Atsuko Nakanishi, Yasuko Kitagishi, Mayuko Ichimura and Satoru Matsuda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61116

#### **Abstract**

Multiple cell cycle regulatory proteins play an important role in oncogenesis. Cancer cells may arise from dysregulation of various genes involved in the regulation of the cell cycle. In addition, cyclin-dependent kinase inhibitors are regarded as key regulators for cancer cell proliferation. Accordingly, permission of impaired cells by cell cycle checkpoints suppresses carcinogenesis. P53, a multifunctional protein, controls G1-S transition, which is the strongest tumor suppressor involved in the regulation of cell cycle. The p53 is stimulated by cellular stress like oxidative stress. Upon activation, p53 leads to cell cycle arrest and promotes DNA repair; otherwise, it induces apoptosis. One of the target effectors of p53 is the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). The tumor suppressor PTEN is a dualspecificity phosphatase which has protein phosphatase activity and lipid phosphatase activity that antagonizes PI3K/AKT activity. The PI3K/AKT cell survival pathway is shown as regulator of cell proliferation. The p53 cooperates with PTEN and might be an essential barrier in development of cancers. BRCA1 plays an important role in DNA repair processes related to maintenance of genomic integrity and control of cell growth. The inactivation of these tumor suppressor proteins confers a growth advantage of cancer. This chapter summarizes the function of several tumor suppres‐ sors in the cell cycle regulation.

**Keywords:** p53, PTEN, BRCA1, AKT, MDM2, p21WAF1, protein interaction, pro‐ tein expression, cell signaling, DNA repair, cell cycle regulation

© 2016 The Author(s). Licensee InTech. 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.

#### **1. Introduction**

Mechanisms of cell cycle are predominantly controlled by p53 tumor suppressor [1, 2]. The p53 transcription factor can bring G1 arrest of the cell cycle by transactivating several down‐ stream molecules [3, 4], which regulates various signaling pathways involved in the cellular response to genome stress and DNA damage. Through the stress-induced activation, p53 triggers the expression of target genes that protect the genetic reliability of cells [5]. Germinal mutations of the *p53* gene constitute an etiological base of Li-Fraumeni syndrome, which is a sporadic heterogeneous autosomal dominant inherited oncogenic disorder [6]. BRCA1, a famous breast cancer tumor suppressor, is associated with breast and ovarian cancer risk and genetic susceptibility [7]. Studies have shown that p53 as well as BRCA1 plays a key role in DNA damage responses [8]. In addition, BRCA1 could work together with phosphatase and tensin homolog deleted in chromosome 10 (*PTEN*), which is also a tumor suppressor gene that is deleted or mutated in a range of human cancers [7, 9], and may be a critical protection in development of several tumors [7, 9]. Actually, PI3K/AKT pathway is constitutively active in BRCA1 defective human cancer cells [10]. Both *PTEN* and *BRCA1* genes are documented as one of the most frequently deleted and/or mutated in various human cancers. Loss or decrease of these PTEN or BRCA1 activities, by either mutation or reduced expression, seems to have a critical role in various cancer developments. PTEN prevents the activation of PI3K/AKT pathway by dephosphorylating the membrane phospholipid PIP3 [11]. Loss of PTEN results in increased AKT recruitment to the plasma membrane and stimulates the signaling pathway. Mutations in *PTEN* genome are the cause of distinctive hamartoma syndromes (*PTEN*-related Proteus syndrome, Cowden syndrome, Proteus-like syndrome, Bannayan-Riley-Ruvalcaba syndrome) with higher risk for a development of several tumors [12]. Furthermore, *BRCA1* and *PTEN* have been shown to be involved in a complex linkage on the interaction with the p53 as presented in Figure 1. Although they are functionally distinct, mutual cooperation has been proposed. These tumor suppressors regulate diverse cellular activities including DNA damage repair, cell cycle arrest, cell differentiation, cell proliferation, cell migration, and cell apoptosis [13]. Importantly, mutations in all of these genes have been associated with increased risk of developing cancers. This review summarizes the function of the tumor suppressors in DNA repair and cell cycle regulation. We will also discuss the role of cellular signaling through protein interaction pathways for the potential implications in the fundamental DNA repair and cell cycle regulation.

#### **2. Characteristics of** *p53***,** *PTEN***, and** *BRCA1*

The *p53* gene encodes a nuclear 393-amino-acid protein which is a transcription factor (Figure 2). The p53 tumor suppressor plays an essential role in regulating cellular processes including cell cycle arrest, apoptosis, cell metabolism, and cell senescence. Inactivation of *p53* gene is related to the development of most types of cancers [14], suggesting that *p53* also plays a critical role in preventing normal cells to becoming cancer cells. In addition, importance of *p53* as an inherited cancer susceptibility gene has been revealed in the Li-Fraumeni syndrome [15].

**1. Introduction**

54 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

and cell cycle regulation.

**2. Characteristics of** *p53***,** *PTEN***, and** *BRCA1*

The *p53* gene encodes a nuclear 393-amino-acid protein which is a transcription factor (Figure 2). The p53 tumor suppressor plays an essential role in regulating cellular processes including cell cycle arrest, apoptosis, cell metabolism, and cell senescence. Inactivation of *p53* gene is related to the development of most types of cancers [14], suggesting that *p53* also plays a critical role in preventing normal cells to becoming cancer cells. In addition, importance of *p53* as an inherited cancer susceptibility gene has been revealed in the Li-Fraumeni syndrome [15].

Mechanisms of cell cycle are predominantly controlled by p53 tumor suppressor [1, 2]. The p53 transcription factor can bring G1 arrest of the cell cycle by transactivating several down‐ stream molecules [3, 4], which regulates various signaling pathways involved in the cellular response to genome stress and DNA damage. Through the stress-induced activation, p53 triggers the expression of target genes that protect the genetic reliability of cells [5]. Germinal mutations of the *p53* gene constitute an etiological base of Li-Fraumeni syndrome, which is a sporadic heterogeneous autosomal dominant inherited oncogenic disorder [6]. BRCA1, a famous breast cancer tumor suppressor, is associated with breast and ovarian cancer risk and genetic susceptibility [7]. Studies have shown that p53 as well as BRCA1 plays a key role in DNA damage responses [8]. In addition, BRCA1 could work together with phosphatase and tensin homolog deleted in chromosome 10 (*PTEN*), which is also a tumor suppressor gene that is deleted or mutated in a range of human cancers [7, 9], and may be a critical protection in development of several tumors [7, 9]. Actually, PI3K/AKT pathway is constitutively active in BRCA1 defective human cancer cells [10]. Both *PTEN* and *BRCA1* genes are documented as one of the most frequently deleted and/or mutated in various human cancers. Loss or decrease of these PTEN or BRCA1 activities, by either mutation or reduced expression, seems to have a critical role in various cancer developments. PTEN prevents the activation of PI3K/AKT pathway by dephosphorylating the membrane phospholipid PIP3 [11]. Loss of PTEN results in increased AKT recruitment to the plasma membrane and stimulates the signaling pathway. Mutations in *PTEN* genome are the cause of distinctive hamartoma syndromes (*PTEN*-related Proteus syndrome, Cowden syndrome, Proteus-like syndrome, Bannayan-Riley-Ruvalcaba syndrome) with higher risk for a development of several tumors [12]. Furthermore, *BRCA1* and *PTEN* have been shown to be involved in a complex linkage on the interaction with the p53 as presented in Figure 1. Although they are functionally distinct, mutual cooperation has been proposed. These tumor suppressors regulate diverse cellular activities including DNA damage repair, cell cycle arrest, cell differentiation, cell proliferation, cell migration, and cell apoptosis [13]. Importantly, mutations in all of these genes have been associated with increased risk of developing cancers. This review summarizes the function of the tumor suppressors in DNA repair and cell cycle regulation. We will also discuss the role of cellular signaling through protein interaction pathways for the potential implications in the fundamental DNA repair

**Figure 1.** Schematic illustration of the integrative signaling model of tumor suppressors including p53, PTEN, and BRCA1 is shown. Typical examples of molecules known to act on the cell proliferation, DNA damage response, and cell cycle regulation via the regulatory pathway are shown. Note that some critical pathways have been omitted for clarity.

Multiple mechanisms have been shown to accomplish the regulation of p53 activity, which controls the selectivity of p53 for specific transcriptional targets [16]. Discharge of p53 from normal repression by binding with molecules such as MdmX or Mdm2 may be a crucial step in the activation of p53 [17, 18]. Functional activation of p53 links with its higher DNA-binding ability, transcriptional activation, then increased expression of the target genes of p53, which are all related to cell cycle regulation and/or cellular apoptosis.

*PTEN* tumor suppressor gene is also frequently deleted and mutated in several human cancers [19]. The gene product is a 53-kDa protein with homology to tensin and protein tyrosine phosphatases. Human genomic locus of the *PTEN* consists of 9 exons on chromosome 10q23.3 encoding a 5.5-kb mRNA that postulates a 403-amino-acid open reading frame [20]. Schematic construction of the predicted PTEN protein is presented in Figure 2. The PTEN protein consists of amino-terminal phosphatase, carboxyl-terminal C2, and PDZ (PSD-95, DLG1, and ZO-1) binding domains [21]. The structure offers PTEN with its preference for acidic phospholipid substrates including phosphatidylinositol 3,4,5-triphosphate (PIP3), as the PTEN CX5R(S/T) motif resides within an active site that surrounds the catalytic core with three basic residues, which are critical for PTEN lipid phosphatase activity [22]. PTEN negatively regulates the activity of PI3K/AKT signaling through converting PIP3 into phosphatidylinositol 4,5 bisphosphate (PIP2) [23]. PIP3 is the principal second messenger of the PI3K pathway that

**Figure 2.** Schematic structures of p53, PTEN, and BRCA1 proteins are shown. The predicted consensual domain struc‐ tures for each protein are depicted. The functionally key sites including the sites of protein phosphorylation are shown. Note that the sizes of protein are modified for clarity. Activation = transactivation domain; PxxP = proline-rich region; RING = (Really Interesting New Gene) finger domain, NLS = Nuclear Localization Signal, BRCT = BRCA1 C Terminus; C2 domain = a protein structural domain involved in targeting proteins to cell membranes; PDZ = a common structural domain in signaling proteins (PSD95, Dlg, ZO-1, etc.)

mediates receptor tyrosine kinase (RTK) signaling to the cell survival kinase AKT. In general, growth factors stimulate RTKs, then activate PI3K and AKT. Upon activation, the inositol ring phosphorylated by PI3K serves to fix AKT to the plasma membrane, where it is sequentially phosphorylated and completely activated by 3-phosphoinositide-dependent kinases PDK1 and PDK2 [24]. Subsequently, activated AKT phosphorylates target proteins involved in cell survival, cell cycling, proliferation, and cell migration [25]. PTEN may act as a regulator of keeping basal levels of PIP3 under a threshold for those signaling activation. Overexpression of PTEN inhibits cell growth by supporting cell cycle arrest, which requires the lipid phos‐ phatase activity of PTEN [26], which correlates with reduced levels and nuclear localization of cyclin-D1 [27], an important molecule of cell cycle regulated by PTEN and AKT. In addition, PTEN induces the cell cycle arrest by upregulating the cell cycle inhibitor p27KIP1 [28]. However, studies have shown many tumor suppressive activities for PTEN that are working within the nucleus, where catalysis of PIP3 does not appear to characterize a main function of the enzyme. The nuclear PTEN activities may include the regulation of genomic stability and several gene expression, indeed despite that the central role of PTEN is as a negative regulator of the PI3K pathway. PTEN activity can be regulated by posttranslational regulation including phosphorylation, oxidation, and acetylation [29, 30].

*BRCA1* cDNA encodes for 1863-amino-acid protein with two nuclear localization signals (NLS) and an amino terminal conserved RING finger motif which is the shared motif present in E3 ubiquitin ligases [31] (Figure 2). The RING finger domain interacts with E2 ubiquitin ligases and applies E3 ligase activity [32]. Knock-in mice with deficient BRCA1 activity exhibit diverse genomic instability and tumor-forming phenotypes [33]. Exon 11 encodes an unstructured region of the BRCA1 protein that is phosphorylated by the ATM and Chk2 kinases in a DNAdamage-dependent manner [34, 35], and the specific function of BRCA1 may be regulated by phosphorylation. BRCA1 becomes more phosphorylated after exposure to the DNA-damaging agents [36]. The carboxyl-terminal domain of BRCA1 is involved in association with specific phosphorylated proteins [37]. Because BRCA1 plays a crucial role in maintaining genome stability, the mutation of BRCA1 is associated with increased genomic instability in cells, which consequently accelerates the mutation rate of the other critical genes. Inherited BRCA1 germline mutation is revealed as a genetic susceptibility leading to high risk of breast and ovarian cancers [38]. Although *BRCA1* gene mutations are rare in breast and/or ovarian cancers, BRCA1 protein expression is often decreased in sporadic cancer specimens. Princi‐ pally, the role of BRCA1 in cell cycle control has been understood by its ability to interact with various cyclin proteins and various cyclin-dependent kinases [39, 40].

#### **3. Relationship among PTEN, p53, BRCA1, and MDM2**

mediates receptor tyrosine kinase (RTK) signaling to the cell survival kinase AKT. In general, growth factors stimulate RTKs, then activate PI3K and AKT. Upon activation, the inositol ring phosphorylated by PI3K serves to fix AKT to the plasma membrane, where it is sequentially phosphorylated and completely activated by 3-phosphoinositide-dependent kinases PDK1 and PDK2 [24]. Subsequently, activated AKT phosphorylates target proteins involved in cell survival, cell cycling, proliferation, and cell migration [25]. PTEN may act as a regulator of keeping basal levels of PIP3 under a threshold for those signaling activation. Overexpression of PTEN inhibits cell growth by supporting cell cycle arrest, which requires the lipid phos‐ phatase activity of PTEN [26], which correlates with reduced levels and nuclear localization of cyclin-D1 [27], an important molecule of cell cycle regulated by PTEN and AKT. In addition, PTEN induces the cell cycle arrest by upregulating the cell cycle inhibitor p27KIP1 [28]. However, studies have shown many tumor suppressive activities for PTEN that are working within the nucleus, where catalysis of PIP3 does not appear to characterize a main function of the enzyme. The nuclear PTEN activities may include the regulation of genomic stability and several gene expression, indeed despite that the central role of PTEN is as a negative regulator of the PI3K pathway. PTEN activity can be regulated by posttranslational regulation including

**Figure 2.** Schematic structures of p53, PTEN, and BRCA1 proteins are shown. The predicted consensual domain struc‐ tures for each protein are depicted. The functionally key sites including the sites of protein phosphorylation are shown. Note that the sizes of protein are modified for clarity. Activation = transactivation domain; PxxP = proline-rich region; RING = (Really Interesting New Gene) finger domain, NLS = Nuclear Localization Signal, BRCT = BRCA1 C Terminus; C2 domain = a protein structural domain involved in targeting proteins to cell membranes; PDZ = a common structural

*BRCA1* cDNA encodes for 1863-amino-acid protein with two nuclear localization signals (NLS) and an amino terminal conserved RING finger motif which is the shared motif present in E3 ubiquitin ligases [31] (Figure 2). The RING finger domain interacts with E2 ubiquitin ligases and applies E3 ligase activity [32]. Knock-in mice with deficient BRCA1 activity exhibit diverse genomic instability and tumor-forming phenotypes [33]. Exon 11 encodes an unstructured region of the BRCA1 protein that is phosphorylated by the ATM and Chk2 kinases in a DNAdamage-dependent manner [34, 35], and the specific function of BRCA1 may be regulated by

phosphorylation, oxidation, and acetylation [29, 30].

domain in signaling proteins (PSD95, Dlg, ZO-1, etc.)

56 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

The PTEN and p53 complex augments the p53-DNA binding and the transcriptional action [41], which may upregulate the expression of PTEN itself and p21WAF1, which is a key molecule involved in cell cycle arrest [42]. Indeed, a superior function of p53 is to work as a transcription factor by attaching to the definite DNA consensus sequence on the p53 responsive genes. Consequently, p53 indirectly inhibits production of PIP3 by inducing the expression of this PTEN. In addition, PTEN associates with p53 and regulates the transcriptional activity of p53 by modulating its DNA binding. PTEN is also required for the maintenance of p53 acetylation [43], which is essential for target gene transcription. An adjacent function of PTEN as a tumor suppressor is accomplished through the stabilization of the p53 protein. PTEN and p53 form a complex in the nucleus under hypoxic conditions [44]. Nuclear PTEN is sufficient to reduce cancer progression in a p53-dependent manner [45]. In addition, the nuclear PTEN seems to mediate DNA damage repair through modulating the activity of DNA repair molecules. The PI3K-dependent activation of AKT indirectly leads to the inhibition of p53 functions by activating another tumor suppressor MDM2 [46]. Activation of AKT has the potential of reducing the p53-mediated cell cycle checkpoints through phosphorylation and appropriation of p21WAF1 [47]. By the way, several PI3K inhibitors favorably reduce prolif‐ eration of BRCA1-defective breast cancer cells. For example, BEZ235 inhibits not only PI3K/ mTOR but also ATM/ATR [48, 49]. It is possible that ATM pathways are involved in upregu‐ lation of the PI3K/AKT pathway in BRCA1-defective cancer cells. In contrast, BRCA1 may regulate the PI3K/AKT pathway by acting on upstream kinases of AKT. Overexpression of wild-type BRCA1 could further reduce basal phosphorylation of AKT levels in MCF7 cells [10, 50]. In addition, reduced levels of PTEN are associated with radio-resistance which can be suppressed by the ectopic PTEN expression [51, 52].

MDM2 controls carcinogenesis, whose mRNA level is also transcriptionally regulated by p53 in response to DNA damage [53]. MDM2 protein and subcellular localization are posttranslationally modulated by AKT [53]. Besides inhibiting the PI3K/AKT signaling, PTEN also promotes translocation of the MDM2 into the nucleus. Furthermore, PTEN modulates *MDM2* transcription by negatively regulating its promoter [41]. PTEN controls *MDM2* promoter activity through its lipid phosphatase activity, independent of the p53 activity [53]. In *PTEN*- null cells, *MDM2* promoter activity is upregulated, resulting in increased *MDM2* expression [53]. MDM2 also regulates the activity of p53 protein by transferring the nuclear p53 protein into the cytoplasm and by promoting the degradation of the p53 protein [54]. PTEN upregu‐ lates the p53 level as well as its activity by downregulating *MDM2* transcription [55]. However, in the absence of p53, PTEN may have a role in inhibiting MDM2-mediated carcinogenesis through regulation of *MDM2* transcription. The p53 and MDM2 complex transports from the nucleus into the cytoplasm, where MDM2 serves as an E3 ubiquitin ligase [56]. Therefore, p53 and MDM2 form a regulatory feedback loop in which p53 positively regulates MDM2 activity. Inactivation of either gene should result in lower protein levels of the other gene. The ability of PTEN to inhibit the nuclear entry of MDM2 increases the cellular content. The BRCA1 carboxyl-terminal region can also stimulate transcription of the p53-responsive promoter of MDM2 [57]. BRCA1 has been shown to affect the gene transcription, but how it does so remains elusive. Essentially, the most important molecule for the DNA damage recognition may be ATM, which is a key checkpoint kinase that phosphorylates various proteins including BRCA1 and/or p53 in response to the DNA damage [58]. BRCA1 activates the CDK inhibitor p21WAF1 and the p53 tumor suppressor protein, which regulates several genes that control the cell cycle checkpoints [59, 60]. Inhibition of this important DNA repair pathway seems to block the mechanisms that are required for normal cell survival in the presence of oncogenic mutations due to DNA damage.

### **4. Involvement of the p53-PTEN-MDM2-BRCA1 loop in cell cycle regulation**

The levels of p53 could vary and is positively related to the amount of DNA damage [61]. Low levels of p53 may induce cell cycle arrest, whereas high levels of p53 may induce apoptosis. On the other hand, growth factor-activated AKT signaling supports progression of cell cycle by acting on several factors involved in the G1/S or G2/M cell cycle transitions. Because the ability of p53 to induce cell cycle arrest and/or cell apoptosis can be provoked by cell survival signals including the AKT pathway, the cell growth signal circuitously leads to the inhibition of p53 by triggering its negative regulators [62]. The p53 protein also regulates BRCA1 transcription both in vitro and in vivo, and BRCA1 participates in p53 accumulation after irradiation through regulation of its phosphorylation and MDM2 expression [63]. MDM2 can act as a modifier of BRCA1 mutant and may accelerate breast and ovarian carcinogenesis [64]. In addition, p53 and PTEN are known to interact and to regulate each other at the transcription as well as protein level, which could be at the important control machinery for switching between survival and death. Given the ability of PTEN to stabilize p53 protein through provoking the AKT-MDM2 complex or by increasing p53 acetylation, the decreased p53 activity in PTEN-lacking tumor cells could be plausible. PTEN and BRCA1 may be regulated and interact with each other at multiple levels including transcription, protein modulation, and protein stability. Therefore, the p53-PTEN-MDM2-BRCA1 loop in cell cycle regulation now becomes dominant (Figure 3). These cross talks are frequently a combination of recipro‐ cally antagonistic pathways, which may often serve as an additional regulatory effect on the expression of key genes involved in cell cycle and carcinogenesis. Interestingly, genistein, which is a soy isoflavone, brings regulation between PTEN and p53 to support cell cycle arrest [65]. Genistein induces PTEN expression and nuclear accumulation, which elicits a sequence of PTEN-dependent nuclear p53 accumulation and recruitment of the PTEN/p53 complex to the p53 binding sites [65], then attenuates expression of cell proliferative genes [65]. In addition, genistein inhibits cell proliferation and induces cell apoptosis more proficiently in BRCA1 mutant cells than in cells expressing wild-type BRCA1 protein [66]. BRCA1-mutant breast cancer cells are highly sensitive to genistein treatment, and AKT could be genistein targets in these cells [66]. Accordingly, genetic variants in the molecules of p53, PTEN, BRCA1, and MDM2 may play roles in tumor suppressor network mediating a susceptibility to cancer. Remarkably, it has also been presented that zinc deficiency modulates the p53-PTEN-BRCA1- MDM2 signaling network in normal cells [67].

**Figure 3.** Suggestion of various regulatory loops involving the p53-PTEN-MDM2-BRCA1 network on cell cycle regula‐ tion. Interactions are shown as arrows to mean activation, while hammerheads, to mean inhibition. Expression of these tumor suppressor genes is regulated by genetic, epigenetic, and transcriptional changes, which may result in the DNA repair and cell cycle regulation in a cell. Downregulation of the function can contribute to genomic instability, which promotes malignant transformation of cells. Note that some critical pathways have been omitted for clarity.

#### **5. Perspective**

null cells, *MDM2* promoter activity is upregulated, resulting in increased *MDM2* expression [53]. MDM2 also regulates the activity of p53 protein by transferring the nuclear p53 protein into the cytoplasm and by promoting the degradation of the p53 protein [54]. PTEN upregu‐ lates the p53 level as well as its activity by downregulating *MDM2* transcription [55]. However, in the absence of p53, PTEN may have a role in inhibiting MDM2-mediated carcinogenesis through regulation of *MDM2* transcription. The p53 and MDM2 complex transports from the nucleus into the cytoplasm, where MDM2 serves as an E3 ubiquitin ligase [56]. Therefore, p53 and MDM2 form a regulatory feedback loop in which p53 positively regulates MDM2 activity. Inactivation of either gene should result in lower protein levels of the other gene. The ability of PTEN to inhibit the nuclear entry of MDM2 increases the cellular content. The BRCA1 carboxyl-terminal region can also stimulate transcription of the p53-responsive promoter of MDM2 [57]. BRCA1 has been shown to affect the gene transcription, but how it does so remains elusive. Essentially, the most important molecule for the DNA damage recognition may be ATM, which is a key checkpoint kinase that phosphorylates various proteins including BRCA1 and/or p53 in response to the DNA damage [58]. BRCA1 activates the CDK inhibitor p21WAF1 and the p53 tumor suppressor protein, which regulates several genes that control the cell cycle checkpoints [59, 60]. Inhibition of this important DNA repair pathway seems to block the mechanisms that are required for normal cell survival in the presence of oncogenic mutations

58 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**4. Involvement of the p53-PTEN-MDM2-BRCA1 loop in cell cycle**

The levels of p53 could vary and is positively related to the amount of DNA damage [61]. Low levels of p53 may induce cell cycle arrest, whereas high levels of p53 may induce apoptosis. On the other hand, growth factor-activated AKT signaling supports progression of cell cycle by acting on several factors involved in the G1/S or G2/M cell cycle transitions. Because the ability of p53 to induce cell cycle arrest and/or cell apoptosis can be provoked by cell survival signals including the AKT pathway, the cell growth signal circuitously leads to the inhibition of p53 by triggering its negative regulators [62]. The p53 protein also regulates BRCA1 transcription both in vitro and in vivo, and BRCA1 participates in p53 accumulation after irradiation through regulation of its phosphorylation and MDM2 expression [63]. MDM2 can act as a modifier of BRCA1 mutant and may accelerate breast and ovarian carcinogenesis [64]. In addition, p53 and PTEN are known to interact and to regulate each other at the transcription as well as protein level, which could be at the important control machinery for switching between survival and death. Given the ability of PTEN to stabilize p53 protein through provoking the AKT-MDM2 complex or by increasing p53 acetylation, the decreased p53 activity in PTEN-lacking tumor cells could be plausible. PTEN and BRCA1 may be regulated and interact with each other at multiple levels including transcription, protein modulation, and protein stability. Therefore, the p53-PTEN-MDM2-BRCA1 loop in cell cycle regulation now becomes dominant (Figure 3). These cross talks are frequently a combination of recipro‐ cally antagonistic pathways, which may often serve as an additional regulatory effect on the

due to DNA damage.

**regulation**

In unstressed cells, p53 may be regularly kept at low levels by its negative regulator MDM2. This feedback loop among the p53-PTEN-MDM2-BRCA1 may function for the accurate regulation of the DNA repair and cell cycle (Figure 3). When stressed, the tumor suppressor p53 predominantly induces cell cycle arrest or apoptosis in the response to DNA damage. Indeed, the regulation is crucial for the effective design of novel cancer therapeutics. Further mechanistic studies are needed in order to understand the exact molecular mechanisms for the effective treatment of cancers with the functional alterations in the cellular signaling loop. Targets within this pathway could provide strategies therapeutically valuable for several cancer treatments. It is important to investigate the linkage among the molecules, and elucidation of interaction-specific functions may provide insight into regulatory aspects of these tumor suppressors as well as opportunities for therapeutic intervention. Such molecular interactions may sustain the biological plausibility that the combination of variants of the p53- PTEN-MDM2-BRCA1 network could result in more comprehensive. Genetic analysis for germline mutations in these key genes allows for the identification of characters at increased risk of cancers. However, they may be regulated and interact with each other at multiple levels including transcription, protein modulation, and protein stability. Obviously, understanding the regulation is crucial for the effective design of novel cancer prevention and therapeutics. Further studies are needed to understand molecular mechanisms in more detail.

#### **6. Abbreviations**

ATM: ataxia telangiectasia-mutated *BRCA1*: breast cancer susceptibility gene 1 HDM2: human homolog of MDM2 LOH: Loss of heterozygosity MDM2: murine double minute 2 NEDL1: NEDD4-like ubiquitin protein ligase-1 NF-*κ*B: nuclear factor kappaB NLS: Nuclear Localization Signal mTOR: mammalian target of rapamycin PDZ: PSD-95, DLG1, and ZO-1 PEST: proline, glutamic acid, serine and threonine PTEN: phosphatase and tensin homolog deleted on chromosome 10 PIP2: phosphatidylinositol 4,5- bisphosphate PIP3: phosphatidylinositol 3,4,5-triphosphate PI3K: phosphoinositide-3 kinase RING: really interesting new gene finger domain RTK: receptor tyrosine kinase ROS: reactive oxidative species

#### **Acknowledgements**

mechanistic studies are needed in order to understand the exact molecular mechanisms for the effective treatment of cancers with the functional alterations in the cellular signaling loop. Targets within this pathway could provide strategies therapeutically valuable for several cancer treatments. It is important to investigate the linkage among the molecules, and elucidation of interaction-specific functions may provide insight into regulatory aspects of these tumor suppressors as well as opportunities for therapeutic intervention. Such molecular interactions may sustain the biological plausibility that the combination of variants of the p53- PTEN-MDM2-BRCA1 network could result in more comprehensive. Genetic analysis for germline mutations in these key genes allows for the identification of characters at increased risk of cancers. However, they may be regulated and interact with each other at multiple levels including transcription, protein modulation, and protein stability. Obviously, understanding the regulation is crucial for the effective design of novel cancer prevention and therapeutics.

60 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

Further studies are needed to understand molecular mechanisms in more detail.

**6. Abbreviations**

ATM: ataxia telangiectasia-mutated

HDM2: human homolog of MDM2

LOH: Loss of heterozygosity

NF-*κ*B: nuclear factor kappaB

NLS: Nuclear Localization Signal

PDZ: PSD-95, DLG1, and ZO-1

PI3K: phosphoinositide-3 kinase

RTK: receptor tyrosine kinase ROS: reactive oxidative species

mTOR: mammalian target of rapamycin

MDM2: murine double minute 2

*BRCA1*: breast cancer susceptibility gene 1

NEDL1: NEDD4-like ubiquitin protein ligase-1

PEST: proline, glutamic acid, serine and threonine

PIP2: phosphatidylinositol 4,5- bisphosphate PIP3: phosphatidylinositol 3,4,5-triphosphate

RING: really interesting new gene finger domain

PTEN: phosphatase and tensin homolog deleted on chromosome 10

This work was supported by JSPS KAKENHI Grant Number 25560050, 26-12035, 24240098.

#### **Author details**

Akari Minami1 , Toshiyuki Murai2 , Atsuko Nakanishi1 , Yasuko Kitagishi1 , Mayuko Ichimura1 and Satoru Matsuda1\*

\*Address all correspondence to: smatsuda@cc.nara-wu.ac.jp

1 Department of Food Science and Nutrition, Nara Women's University, Kita-Uoya Nishimachi, Nara, Japan

2 Department of Microbiology and Immunology and Department of Genome Biology, Graduate School of Medicine, Osaka University, -2 Yamada-oka, Suita, Japan

Akari Minami and Satoru Matsuda contributed equally to this work. **Competing interests statement**: The authors declare that they have no competing financial interests.

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### **Epigenetic Mechanisms in Head and Neck Cancer**

Julio Cesar Osorio and Andres Castillo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61135

#### **Abstract**

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Head and neck cancer (HNC) is one of the most prevalent human malignancies, af‐ fecting different anatomic sites of the upper aerodigestive tract (UADT) such as the oral cavity, larynx, and naso-, oro-, and hypopharynx. HNC develops through the ac‐ cumulation of multiple genetic and epigenetic alterations in a multistep process. In this issue, the aim is to describe epigenetic mechanisms behind HNC. The main mech‐ anisms evaluated are DNA methylation, posttranslational histone modification, and noncoding RNAs.

**Keywords:** Methylation, Histone modification, microRNA, Long non-coding RNAs

#### **1. Introduction**

Genetic information flow (transcription, translation, and subsequent protein modification) in a normal cell represents the machine of cell gene expression.[1] Each cell has the same information, but its expression change between different types of cells. The control of gene expression is therefore at the heart of differentiation and development.[1]

In addition to inheriting genetic information, cells inherit chemical modifications that are not encoded in the nucleotide sequence of DNA, and this modification impacts the program of gene expression. This type of modification has been termed epigenetic information.[1]

Epigenetic refers specifically to the study of mitotically and meiotically heritable changes in the control of gene expression that occur without changes in the nucleotide sequence of genome and chromatin.[2]

In both genetic and epigenetic heritage, mitosis distributes genetic information or the chemical modification through the ontogeny. Meiosis distributes genetic information or chemical modification through the formation of gametes. This means that sperm and egg carry genetic

© 2016 The Author(s). Licensee InTech. 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.

and epigenetic information. When the embryo is formed, this information is distributed through the all the cells by mitosis (Figure 1). When it comes to population level, natural selection determines which genetically and epigenetically inherited information is present.

**Figure 1.** Genetic and epigenetic information in gametes and a new embryo.

Gene regulation and genome function are intimately related to the physical organization of genomic DNA and, in particular, to the way it is packaged into chromatin.[3] There are many chemical modifications affecting DNA, RNA, and proteins that create different epigenetic process.[4] These modifications are related to DNA methylation, histone modifications, chromatin remodeling factors (associated with nucleosome positioning), and noncoding RNAs.[4] The changes will influence the chromatin states and impact gene expression patterns (Figure 2).[5] Epigenetic alterations are associated with chromosomal instability and changes in transcriptional control, which influence the overall gene expression differences seen in many human malignancies.[5]

The carcinogenesis of head and neck cancer (HNC) is a human malignancy influenced by genetic factors, age, geography, and lifestyles, among which include smoking, oral hygiene, and human papillomavirus.[6, 7] In general, different types of carcinogens attack the oral mucosa through the accumulation of multiple genetic and epigenetic alterations in a multistep process.[8] The clinical appearance of HNC preoncogenic lesion of the mucosal surfaces include leukoplakia, erythroplakia, or speckled leukoplakia reflecting the presence of white, red, or mixed white/red lesion, respectively.[9]

HNC is one of the most prevalent human malignancies, affecting different anatomic sites of the upper aerodigestive tract (UADT) such as the oral cavity, larynx, and naso-, oro-, and hypopharynx.[10] The most common histologic type among the head and neck tumors are the squamous cell carcinomas (head and neck squamous cell carcinomas [HNSCCs]).[7]

**Figure 2.** Genetic and epigenetic information.

and epigenetic information. When the embryo is formed, this information is distributed through the all the cells by mitosis (Figure 1). When it comes to population level, natural selection determines which genetically and epigenetically inherited information is present.

Gene regulation and genome function are intimately related to the physical organization of genomic DNA and, in particular, to the way it is packaged into chromatin.[3] There are many chemical modifications affecting DNA, RNA, and proteins that create different epigenetic process.[4] These modifications are related to DNA methylation, histone modifications, chromatin remodeling factors (associated with nucleosome positioning), and noncoding RNAs.[4] The changes will influence the chromatin states and impact gene expression patterns (Figure 2).[5] Epigenetic alterations are associated with chromosomal instability and changes in transcriptional control, which influence the overall gene expression differences seen in many

The carcinogenesis of head and neck cancer (HNC) is a human malignancy influenced by genetic factors, age, geography, and lifestyles, among which include smoking, oral hygiene, and human papillomavirus.[6, 7] In general, different types of carcinogens attack the oral mucosa through the accumulation of multiple genetic and epigenetic alterations in a multistep process.[8] The clinical appearance of HNC preoncogenic lesion of the mucosal surfaces include leukoplakia, erythroplakia, or speckled leukoplakia reflecting the presence of white,

HNC is one of the most prevalent human malignancies, affecting different anatomic sites of the upper aerodigestive tract (UADT) such as the oral cavity, larynx, and naso-, oro-, and hypopharynx.[10] The most common histologic type among the head and neck tumors are the

squamous cell carcinomas (head and neck squamous cell carcinomas [HNSCCs]).[7]

**Figure 1.** Genetic and epigenetic information in gametes and a new embryo.

68 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

human malignancies.[5]

red, or mixed white/red lesion, respectively.[9]

It was reported that 42,440 new cases of HNSCCs were diagnosed in the United States in 2014, and 8,390 deaths were due to this disease.[11] The mortality rate (from 2003 to 2007) was of 2.5 per 100,000 persons per year. U.S. incidence and mortality rates are about 2.5 and 2.8 in men and women, respectively.[12]

Generally, the highest HNSCC rates are found in Melanesia, South-Central Asia, and Central and Eastern Europe and the lowest in Africa, Central America, and Eastern Asia for both males and females.[13]

The incidence rates for HNSCC related to HPV infections, such as oropharynx, tonsil, and tongue base, are increasing in young adults in the United States and in some countries in Europe, which is hypothesized to be in part due to changes in oral sexual behavior.[14]

In this issue, the aim is to describe epigenetic mechanisms behind HNSCC. The main mecha‐ nisms evaluated are DNA methylation, posttranslational histone modification, and noncoding RNAs

#### **2. Epigenetic mechanisms**

#### **2.1. DNA methylation**

DNA methylation is a chemical marking system for annotating genetic information by causing gene repression through its ability to affect factor binding and chromatin structure.[14] This system has gene expression patterns that are regulated in a spatial and time-dependent manner.[5] Heritable information is carried by chemical modifications of both DNA and chromatin-associated proteins and modulates chromatin structure and DNA accessibility.[5]

DNA methylation refers to the methylation of DNA cytosine residues at the carbon 5 position. [15, 16] DNA methyltransferases (DNMTs) catalyze the transfer of a methyl group to position 5 of a cytosine, generating 5-methylcytosine (5mC).[17] This chemical modification affects gene expression when CpG-rich areas are present inside promoter regions (figure 3).[16, 18] This is accomplished by the activities of one or more DNA methyltransferases (DNMTs), which use S-adenosylmethionine (AdoMet) as a cofactor.[19]

**Figure 3.** DNA methylation.

About 60% of human gene promoters are associated with CpG islands and are usually unmethylated in normal cells, although some of them (~6%) become methylated in a tissuespecific manner during early development or in differentiated tissues.[20]

Recent findings also suggest that extensive DNA methylation changes caused by differentia‐ tion take place at CpG island "shores," regions of comparatively low CpG density close to CpG islands.[2]

A relationship between DNA methylation and cancer has been found, given that DNA methyltransferases can be genetically altered in malignancies, that is, occur with DNMT3A20 and DNMT3B.[21] Aberrant DNA methylation is an epigenetic mechanism that contributes to the development of a wide variety of human cancers, either in the form of promoter-specific hypermethylation or genome-wide hypomethylation.[22] Hypermethylation in gene promot‐ er regions is usually associated with expression suppression[23] and is the most common alterations in human cancers, leading to the abnormal expression of a broad spectrum of genes. [22] On the other hand, hypomethylation occurs in a large percentage in repetitive DNA elements.[24]

#### **2.2. Posttranslational covalent histone modifications**

Chromatin is the state in which DNA is packaged within the cell.[25] The chromatin architec‐ ture can be remodeled by a network of protein mediators called histones that play an important role in gene regulation by compacting DNA.[26] The nucleosome is the fundamental unit of chromatin, and it is composed of an octamer of histones (H2A, H2B, H3, and H4) around which 147 base pairs of DNA are wrapped.[25] Dynamic variations in the organization of chromatin structure are mediated by histone acetylation and deacetylation (figure 4).[25]

**Figure 4.** Posttranslational covalent histone modifications.

DNA methylation refers to the methylation of DNA cytosine residues at the carbon 5 position. [15, 16] DNA methyltransferases (DNMTs) catalyze the transfer of a methyl group to position 5 of a cytosine, generating 5-methylcytosine (5mC).[17] This chemical modification affects gene expression when CpG-rich areas are present inside promoter regions (figure 3).[16, 18] This is accomplished by the activities of one or more DNA methyltransferases (DNMTs), which use

About 60% of human gene promoters are associated with CpG islands and are usually unmethylated in normal cells, although some of them (~6%) become methylated in a tissue-

Recent findings also suggest that extensive DNA methylation changes caused by differentia‐ tion take place at CpG island "shores," regions of comparatively low CpG density close to CpG

A relationship between DNA methylation and cancer has been found, given that DNA methyltransferases can be genetically altered in malignancies, that is, occur with DNMT3A20 and DNMT3B.[21] Aberrant DNA methylation is an epigenetic mechanism that contributes to the development of a wide variety of human cancers, either in the form of promoter-specific hypermethylation or genome-wide hypomethylation.[22] Hypermethylation in gene promot‐ er regions is usually associated with expression suppression[23] and is the most common alterations in human cancers, leading to the abnormal expression of a broad spectrum of genes. [22] On the other hand, hypomethylation occurs in a large percentage in repetitive DNA

Chromatin is the state in which DNA is packaged within the cell.[25] The chromatin architec‐ ture can be remodeled by a network of protein mediators called histones that play an important

specific manner during early development or in differentiated tissues.[20]

**2.2. Posttranslational covalent histone modifications**

S-adenosylmethionine (AdoMet) as a cofactor.[19]

70 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**Figure 3.** DNA methylation.

islands.[2]

elements.[24]

Acetylation neutralizes the positive charge of lysine residues, weakening charge-dependent interactions between histone and nucleosomal DNA, linker DNA, or adjacent histones, thus increasing the accessibility of DNA to the transcription machinery. Histone lysine acetylation also functions in other cellular processes that require DNA access.[27]

The posttranslational modification of histones by methylation is an important and widespread type of chromatin modification that is known to influence biological processes in the context of development and cellular responses.[28] Other modifications can be performed through lysine ubiquitination, serine phosphorylation, sumoylation, and methylation of lysines and arginines.[29]

Sumoylation is a process in which proteins are functionally or structurally similar to ubiquitin and are termed ubiquitin-like proteins (UBLs). UBLs include SUMO, NEDD8, ISG15, and FAT10.[30]

Histone acetyltransferases (HAT) catalyze the transfer of an acetyl group from acetyl-CoA to ε-amino group of a histone lysine residue.[31] The action of histone deacetylases (HDAC) is different because the effect is on the lysine residues. The first process involves chromatin decondensation, and the second process involves chromatin compaction. Either one or the other affects the gene transcription due to conformation changes in the chromatin.[31]

#### **2.3. Noncoding RNAs**

The noncoding RNAs are another level of epigenetic control for their capacity to establish other epigenetic marks and control gene expression.[4] Noncoding RNA (ncRNA) is a type of RNA that does not code for protein but has enzymatic, structural, or regulatory function.[32] ncRNAs can be classed as either small or long ncRNA, based on their transcript length.[33, 34]

#### **2.4. MicroRNAs**

MicroRNAs (miRNAs) are a set of non-protein-coding RNAs that bind to partially comple‐ mentary sites in the 3'-untranslated regions of their messenger RNA targets.[35] MicroRNAs are the 21–23 nucleotide single-stranded RNA molecules found in eukaryotic cells.[36] The miRNAs interfere with messenger RNA translation or cause messenger RNA degradation, thereby repressing gene expression posttranscriptionally (Figure 5).[37] This is possible through imperfect base pairing with the 3'-untranslated region (3'-UTR) of target mRNAs of protein-coding genes, leading to the cleavage of homologous mRNA or translational inhibi‐ tion.[38]

**Figure 5.** RNA degradation by miRNA.

miRNAs are transcribed for the most part by RNA polymerase II as long primary transcripts characterized by hairpin structures (pri-miRNA) and are processed in the nucleus by RNase III Drosha into 70–100 nucleotide long precursor miRNAs (pre-miRNAs).[39]

MicroRNAs are partial complementarity between their target transcripts. A single microRNA is capable of simultaneously regulating up to hundreds of genes, giving rise to an enormous modulatory potential.[40]

MicroRNAs are involved in a variety of cellular processes, including the regulation of cellular differentiation, proliferation, and apoptosis, and an aberrant expression of miRNA is known to induce various human malignancies.[41] New evidence suggests that miRNAs can act as oncogenes or tumor suppressors, exerting a key function in tumorigenesis.[42] Recently, a new function mediating tumor metastasis in breast cancer has been assigned to miRNAs, by which this malignant step is promoted or suppressed.[42]

#### **2.5. Long noncoding RNAs**

that does not code for protein but has enzymatic, structural, or regulatory function.[32] ncRNAs can be classed as either small or long ncRNA, based on their transcript length.[33, 34]

72 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

MicroRNAs (miRNAs) are a set of non-protein-coding RNAs that bind to partially comple‐ mentary sites in the 3'-untranslated regions of their messenger RNA targets.[35] MicroRNAs are the 21–23 nucleotide single-stranded RNA molecules found in eukaryotic cells.[36] The miRNAs interfere with messenger RNA translation or cause messenger RNA degradation, thereby repressing gene expression posttranscriptionally (Figure 5).[37] This is possible through imperfect base pairing with the 3'-untranslated region (3'-UTR) of target mRNAs of protein-coding genes, leading to the cleavage of homologous mRNA or translational inhibi‐

miRNAs are transcribed for the most part by RNA polymerase II as long primary transcripts characterized by hairpin structures (pri-miRNA) and are processed in the nucleus by RNase

MicroRNAs are partial complementarity between their target transcripts. A single microRNA is capable of simultaneously regulating up to hundreds of genes, giving rise to an enormous

MicroRNAs are involved in a variety of cellular processes, including the regulation of cellular differentiation, proliferation, and apoptosis, and an aberrant expression of miRNA is known to induce various human malignancies.[41] New evidence suggests that miRNAs can act as

III Drosha into 70–100 nucleotide long precursor miRNAs (pre-miRNAs).[39]

**2.4. MicroRNAs**

tion.[38]

**Figure 5.** RNA degradation by miRNA.

modulatory potential.[40]

Long noncoding RNAs (lncRNAs) are generated by the same transcriptional machinery as are other mRNAs. These lncRNAs have a 5′ terminal methylguanosine cap and are often spliced and polyadenylated.[45] lncRNAs have length greater than 200 nucleotides without proteincoding potential but with a wide range of structural and functional roles.[34] Some of this process can include the chromatin remodeling and transcriptional control assemblies.[31] They are generally transcriptionally activated or repressed by associated transcription factors and function as molecular mediators of gene expression.[43] When the lncRNAs act as molecular scaffolds, they can guide the chromatin-modifying complexes to bind into specific genomic loci. This way, they impart a repressive or activating effect on gene expression (Figure 6).[43]

**Figure 6.** Long noncoding RNA.

According to their association with mRNA, the LncRNAs can be divided in different catego‐ ries[44]: this can overlap with coding regions of a transcript on the same strand and with coding regions of a transcript on the opposite strand. This can be intronic lncRNAs from other transcript and can be intergenic lncRNAs between two genes on the same strand.[45]

The functions of the RNA in regulation of gene expression can be summarizing at transcrip‐ tional levels, RNA processing, and translation. Furthermore, they can protect genomes from foreign nucleic acids. At chromatin level, they can modulate the genome rearrangement. Finally, the ncRNAs can operate as RNA–protein complexes, including ribosomes, snRNPs, snoRNPs, telomerase, microRNAs, and long ncRNAs.[46]

Long noncoding RNAs have also been shown to be necessary for targeting histone-modifying activities. Histone methylation is the end result of the transcription of long noncoding RNAs and the subsequent nucleation and targeting of histone modifying completes.[27]

The aberrant expression of lncRNAs has been associated with human cancers, suggesting a critical role in tumorigenesis.[47, 48] It has been demonstrated that a novel lncRNA, HOTAIR, was up-regulated and promoted cancer metastasis and predicted poor prognosis in ESCC.[34] Additionally, the association of dysregulated lncRNAs with specific developmental stages and clinical outcomes indicates their potential as strong diagnostic and prognostic predictors as well as therapeutic targets.[49]

#### **3. Epigenetic changes in head and neck cancer**

#### **3.1. DNA methylation in head and neck cancer**

The DNA methylation events in HNSCC include genes involved in cell cycle regulation, signal transduction, secreted protein, transmembrane protein, transcription factors, prosta‐ glandin metabolism, metal ion homeostasis, oxidative stress, and oncoviruses.[50] Addition‐ ally, HNSCCs have genes involved in DNA damage repair, apoptosis, Wnt signaling, signal transduction, tissue invasion/metastasis, tumor suppression, and others (Figure 7, Table 1). [13, 51]

**Figure 7.** Genes and DNA methylation in HNSCC. Modified from Magić et al.,[13] Polanska et al.,[50] and Kaabi et al. [51]


Finally, the ncRNAs can operate as RNA–protein complexes, including ribosomes, snRNPs,

Long noncoding RNAs have also been shown to be necessary for targeting histone-modifying activities. Histone methylation is the end result of the transcription of long noncoding RNAs

The aberrant expression of lncRNAs has been associated with human cancers, suggesting a critical role in tumorigenesis.[47, 48] It has been demonstrated that a novel lncRNA, HOTAIR, was up-regulated and promoted cancer metastasis and predicted poor prognosis in ESCC.[34] Additionally, the association of dysregulated lncRNAs with specific developmental stages and clinical outcomes indicates their potential as strong diagnostic and prognostic predictors as

The DNA methylation events in HNSCC include genes involved in cell cycle regulation, signal transduction, secreted protein, transmembrane protein, transcription factors, prosta‐ glandin metabolism, metal ion homeostasis, oxidative stress, and oncoviruses.[50] Addition‐ ally, HNSCCs have genes involved in DNA damage repair, apoptosis, Wnt signaling, signal transduction, tissue invasion/metastasis, tumor suppression, and others (Figure 7, Table 1).

**Figure 7.** Genes and DNA methylation in HNSCC. Modified from Magić et al.,[13] Polanska et al.,[50] and Kaabi et al.

and the subsequent nucleation and targeting of histone modifying completes.[27]

snoRNPs, telomerase, microRNAs, and long ncRNAs.[46]

74 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**3. Epigenetic changes in head and neck cancer**

**3.1. DNA methylation in head and neck cancer**

well as therapeutic targets.[49]

[13, 51]

[51]


**Table 1.** Genes and DNA methylation in HNSCC. Modified from Magić et al.,[13] Polanska et al.,[50] and Kaabi et al. [51]

The primary risk factors for the development of HNSCC include tobacco use, alcohol con‐ sumption, human papillomavirus (HPV) infections (mainly for oropharyngeal cancers), and Epstein–Barr virus (EBV) infections (for nasopharyngeal cancer).[52]

In promoter methylation, the p16 and p15 genes are commonly observed in human epithelial malignancies, including HNSCC. Histologically normal surgical margin epithelium of HNSCC patients with chronic smoking and drinking habits has a significantly higher prevalence of p15 methylation compared with nonsmokers and nondrinkers.[53]

Between genes that have been associated with hypermethylation, the p16 and the p14 genes undergo inactivation due to promoter hypermethylation.[13] Encoded by the CDKN2A gene, p16 inhibits cyclin-dependent kinases 4 and 6, thus blocking the promotion of cells from the G1 to the S phase of the cell cycle.[54] CDKN2A (p16) inactivation is common in lung cancer and occurs via homozygous deletions, methylation of promoter region, or point mutations.[55] CDKN2A (p16) disruption is reported as a frequent event in head and neck squamous cell carcinomas that confers poor prognosis.[56] Other genes as DAP-K, RASSF1A, RARß2, and MGMT have been reported as genes under hypermethylation promotor but with functions in DNA repair. These genes removed mutagenic (O6-guanine) adducts from DNA.[57]

**Major classes Member Function**

Apoptosis DAPK p53-dependent apoptosis

76 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

p14 Proapoptosis

WIF1 Secreted Wnt antagonist

EDNRB Endothelin receptor type B RUNX3 Wnt pathway antagonist

DCC Cell–cell adhesion

Signal transduction SFRP1 Antagonists of the Wnt pathway

DAPK1 Proapoptosis DCC Proapoptosis

Tumor suppression HIN1 Inhibitor Ras pathway

[51]

Wnt signaling APC Wnt signaling and adhesion

RASSF1A RAS pathway regulation and tumor suppression

E-cad Cell adhesion, forming adherens junctions

Others KIF1A Cell division and microtubule-dependent intracellular organelle

**Table 1.** Genes and DNA methylation in HNSCC. Modified from Magić et al.,[13] Polanska et al.,[50] and Kaabi et al.

The primary risk factors for the development of HNSCC include tobacco use, alcohol con‐ sumption, human papillomavirus (HPV) infections (mainly for oropharyngeal cancers), and

In promoter methylation, the p16 and p15 genes are commonly observed in human epithelial malignancies, including HNSCC. Histologically normal surgical margin epithelium of HNSCC patients with chronic smoking and drinking habits has a significantly higher prevalence of p15

Between genes that have been associated with hypermethylation, the p16 and the p14 genes undergo inactivation due to promoter hypermethylation.[13] Encoded by the CDKN2A gene, p16 inhibits cyclin-dependent kinases 4 and 6, thus blocking the promotion of cells from the G1 to the S phase of the cell cycle.[54] CDKN2A (p16) inactivation is common in lung cancer and occurs via homozygous deletions, methylation of promoter region, or point mutations.[55] CDKN2A (p16) disruption is reported as a frequent event in head and neck squamous cell

transport

Epstein–Barr virus (EBV) infections (for nasopharyngeal cancer).[52]

methylation compared with nonsmokers and nondrinkers.[53]

RUNX3 Wnt signaling inhibitor, TGF-β-induced tumor sup pression

RASSF1A/RASSF2 Negative RAS effector, proapoptotic, microtubule stabilization

RARβ Regulatory protein and apoptosis

DAPK and RASSF1A genes have shown methylation in HNSCC.[58] The methylation of p16 could be an initial process that might address abnormalities or deregulation of cell cycle controls.[59]

The ataxia-telangiectasia-mutated (*ATM*) gene produces a protein kinase that functions as a tumor suppressor by triggering appropriate cellular response to genome damage resulting from ionizing radiation or chemical carcinogen exposure.[60] It is currently unknown whether ATM is lost in HNSCCs displaying the deletion in the 11q22–23 locus.[61]

Aggressive HNSCC has been linked to expression loss of E-cadherin (ECAD) protein.[51] The protein ECAD can be inactivated by promoter hypermethylation.[62] In patients with HNSCC who are low smokers, the hypermethylation of CDH1 occurs more commonly, suggesting that an additional factor may be driving this epigenetic alteration.[62]

Cyclooxygenase-2 (Cox-2) is presumed to contribute to cancer progression through its multifaceted function, and recently its inverse relationship with E-cadherin was suggested. Increased expression of Cox-2 has been found in a variety of human malignancies, including HNSCC. [63]

The death-associated protein kinase (DAPk) family contains three closely related serine/ threonine kinases, namely, DAPk, ZIPk, and DRP-1, which display a high degree of homology in their catalytic domains.[64] The methylation profile of DAPK in HNSCC (including oral squamous cell carcinoma) is a promising biomarker for the follow-up and early detection of head and neck cancer recurrence.[65]

The Ras association domain family protein 1A (RASSF1A) is arguably one of the most frequently inactivated tumor suppressors in human cancer. RASSF1A modulates apoptosis via the Hippo and Bax pathways but also modulates the cell cycle.[66] The epigenetic inacti‐ vation of RASSF1A plays an important role in the development of cancer.[67]

TP53, once activated, leads to apoptosis and growth arrest (either cell cycle arrest or senes‐ cence). It is clear that TP53 mutation is common in HNSCC.[68] The p53 transcription factor stands out as a key tumor suppressor and a master regulator of various signaling pathways involved in this process.[69] Tobacco smoke is the best known and studied mutagen involved in lung carcinogenesis, and TP53 mutational patterns differ between smokers and nonsmokers, with an excess of G to T transversions in smoking-associated cancer.[69]

The Cancer Genome Atlas has informed about smoking-related HNSCCs and its relations with the universal loss of function of TP53 mutations and CDKN2A. This inactivation is accompanied with frequent copy number alterations, including amplification of 3q26/28 and 11q13/22.[70]

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway is antagonized by phospha‐ tase and tensin homologue (PTEN). PTEN protein is a tumor suppressor frequently disrupted in cancer, altering tumor cell growth and survival.[71] The decrease in PTEN function in HNSCC is due to several genetic and epigenetic alterations. Recently, promoter hypermethy‐ lation has been implicated in the down-regulation of PTEN in HNSCC cell lines.[72]

HNSCCs also exhibit many chromosomal abnormalities, including amplifications of the 11q13 region containing the cyclin D1 gene and the 7p11 region encoding EGFR, which lead to protooncogene activation.[73] The most critical point in regulation of the cell cycle is the G1 checkpoint. Cyclin D1, a G1 cyclin, has been implicated in the regulation of the G1 to S phase progression in many different cell types. Cyclin D1 forms active complexes that promote the phosphorylation of retinoblastoma protein (RB) and the activation of E2F-responsive gene.[74]

PIK3CA is a human gene that regulates various cellular functions, including proliferation and invasion. Because it is an oncogene, its activation by either gene amplification or mutation results in a cellular growth advantage contributing toward cancer formation and progression. [75] Mutations in PIK3CA have cases displayed of concurrent amplification. Additionally, some tumors (20%) contain focal amplification without evidence of mutation. The largest mutation proportions of PIK3CA are localized to hotspots that promote activation.[70] PIK3CA is an active mutation that is common in conjunction with infrequent copy number alteration, and it forms part of a subgroup of oral cavity tumors with favorable clinical outcomes.[70]

Bmi1 (B-cell-specific Moloney Murine Leukemia virus insertion site 1) is a transcription repressor for cell senescence, implicated in the self-renewal of stem cell. Bmi1 is highly expressed in the CD44+ cell population sorted from oral SCC tumors.[76]

E3 ubiquitin-protein ligase (CHFR) is a gene involved in a checkpoint regulating entry to mitosis.[77] Loss of CHFR leads to mitotic catastrophe and apoptosis due to mitotic spindle alteration. Aberrant methylation of the gene has been reported in several primary tumor genes. EGFR is a potential prognostic biomarker.[78]

Epidermal growth factor receptor (erb-B1) is a member of the erbB family of tyrosine kinase receptor proteins.[79] Previous studies have shown that EGFR is expressed or highly expressed in various human tumor cells.[79] The overexpression of EGFR is attributed to gene amplifi‐ cation, which is noted to be about 12 copies per cell in relation to head and neck squamous cell carcinomas. The constitutive EGFR activation caused via autocrine stimulation and through the coexpression of EGFR with its ligands, TGFα, has been observed and is indicative of its poor prognosis.[72] EGFR-targeted therapy is commonly used for the treatment of advanced HNSCC due to numerous findings that describe overexpression and/or high activity of EGFR in the majority of HNSCC.[80]

Various factors are known to regulate angiogenesis; for example, vascular endothelial cell growth factor (VEGF) has potent angiogenic effects. The presence of VEGF has been reported in approximately 40% of head and neck squamous cell carcinomas (HNSCCs), and its presence is associated with a poor prognosis.[72]

Other mechanisms included in the tumorous angiogenesis lie in the intake and utilization of locally stored fibroblast growth factors (FGFs).[50] FGF-1 (aFGF) and FGF-2 (bFGF) are found in most embryonic and adult normal and tumor tissues, where they are immobilized in the extracellular matrix (ECM).[81]

Fibroblast activation protein (FAP) is a member of the serine protease family that is selectively expressed in the stromal fibroblasts associated with epithelial cancers and is expressed at low or undetectable levels in the resting fibroblasts of normal adult tissues. FAP is expressed in more than 90% of epithelial carcinomas, which makes it a promising target.[82]

in cancer, altering tumor cell growth and survival.[71] The decrease in PTEN function in HNSCC is due to several genetic and epigenetic alterations. Recently, promoter hypermethy‐

HNSCCs also exhibit many chromosomal abnormalities, including amplifications of the 11q13 region containing the cyclin D1 gene and the 7p11 region encoding EGFR, which lead to protooncogene activation.[73] The most critical point in regulation of the cell cycle is the G1 checkpoint. Cyclin D1, a G1 cyclin, has been implicated in the regulation of the G1 to S phase progression in many different cell types. Cyclin D1 forms active complexes that promote the phosphorylation of retinoblastoma protein (RB) and the activation of E2F-responsive gene.[74] PIK3CA is a human gene that regulates various cellular functions, including proliferation and invasion. Because it is an oncogene, its activation by either gene amplification or mutation results in a cellular growth advantage contributing toward cancer formation and progression. [75] Mutations in PIK3CA have cases displayed of concurrent amplification. Additionally, some tumors (20%) contain focal amplification without evidence of mutation. The largest mutation proportions of PIK3CA are localized to hotspots that promote activation.[70] PIK3CA is an active mutation that is common in conjunction with infrequent copy number alteration, and it forms part of a subgroup of oral cavity tumors with favorable clinical outcomes.[70] Bmi1 (B-cell-specific Moloney Murine Leukemia virus insertion site 1) is a transcription repressor for cell senescence, implicated in the self-renewal of stem cell. Bmi1 is highly

lation has been implicated in the down-regulation of PTEN in HNSCC cell lines.[72]

78 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

expressed in the CD44+ cell population sorted from oral SCC tumors.[76]

EGFR is a potential prognostic biomarker.[78]

in the majority of HNSCC.[80]

extracellular matrix (ECM).[81]

is associated with a poor prognosis.[72]

E3 ubiquitin-protein ligase (CHFR) is a gene involved in a checkpoint regulating entry to mitosis.[77] Loss of CHFR leads to mitotic catastrophe and apoptosis due to mitotic spindle alteration. Aberrant methylation of the gene has been reported in several primary tumor genes.

Epidermal growth factor receptor (erb-B1) is a member of the erbB family of tyrosine kinase receptor proteins.[79] Previous studies have shown that EGFR is expressed or highly expressed in various human tumor cells.[79] The overexpression of EGFR is attributed to gene amplifi‐ cation, which is noted to be about 12 copies per cell in relation to head and neck squamous cell carcinomas. The constitutive EGFR activation caused via autocrine stimulation and through the coexpression of EGFR with its ligands, TGFα, has been observed and is indicative of its poor prognosis.[72] EGFR-targeted therapy is commonly used for the treatment of advanced HNSCC due to numerous findings that describe overexpression and/or high activity of EGFR

Various factors are known to regulate angiogenesis; for example, vascular endothelial cell growth factor (VEGF) has potent angiogenic effects. The presence of VEGF has been reported in approximately 40% of head and neck squamous cell carcinomas (HNSCCs), and its presence

Other mechanisms included in the tumorous angiogenesis lie in the intake and utilization of locally stored fibroblast growth factors (FGFs).[50] FGF-1 (aFGF) and FGF-2 (bFGF) are found in most embryonic and adult normal and tumor tissues, where they are immobilized in the Oral cancer overexpressed 1 and 2 (ORAOV1-ORAOV2) overexpression was reported in HNSCC.[50, 83] The first is required for cell growth and tumor angiogenesis,[84] and the second is required in the modulation of TMEM16A activity in various epithelial tissues.[85]

Multiple signaling pathways have been linked to tumor resistance, including activation of nuclear factor kappa B (NFκB).[86] NFκB is an epigenetic modifier that plays a major role in malignant transformation, and this pathway serves as a target for epigenetic drugs.[26] The constitutive activation of NFκB signaling is often observed in HNSCC, suggesting a common epigenetic mechanism in HNSCC biology. Indeed, the activation of NFκB signaling in HNSCC induced chromatin compaction and acquisition of resistance to chemotherapy.[26]

Metallothionein (MT) is a family of cysteine-rich, low molecular weight (500–14,000 Da) proteins. MTs have been proposed to play important roles in protecting against DNA damage, apoptosis, and oxidative stress. MT is a tumor suppressor reported to show promoter hyper‐ methylated in various cancers.[87]

Human mismatch repair genes (hMMR) have the ability to repair both mismatched bases and insertion loop errors during DNA replication.[88] Suboptimal DNA repair could result in disrupting the pattern of repeat sequences, causing chromosomal aberrations in the genome of patients suffering from instability syndromes. Significant proportions of carcinomas develop through DNA mismatch repair genes (MMR) deficiency and exhibit frequent micro‐ satellite alteration (MA).[88]

The hypermethylated adenomatosis polyposis coli (APC) tumor suppressor gene has reduced expression levels along with loss of heterozygosity (LOH), leading to the altered functioning of the APC tumor suppressor proteins, which play a role for the integrity and function of the β-catenin destruction complex.[72] The APC protein, a negative regulator of this pathway, has been strongly implicated in the development of colon cancer but still has an undetermined role in the formation of oral cancer.[89]

Loss of heterozygosity of the APC gene and epigenetic events lead to the decreased expression of APC and the Wnt antagonists, the secreted frizzled-related proteins (SFRPs), Wnt inhibitory factors (WIFs), and Dickkopf family members (DKKs), primarily by promoter hypermethyla‐ tion.[90] The persistent β-catenin signaling contributes to increased growth, metastatic potential, and resistance to chemotherapy in HNSCC and their tumor-initiating cells.[90] The methylation of WIF-1 correlated with shorter survival in oral cancer patients. The methylation of WIF1 may be considered a prognostic marker in oral cancers.[91]

Wnt pathway stimulates several intracellular signal transduction cascades (canonical and noncanonical).[92] The possible role of RUNX3 as a tumor suppressor in HNSCC has been reported. The promoter hypermethylation of antagonist's genes to Wnt (RUNX3) has been identified as a common event in cancer.[93]

Deleted in colorectal cancer (DCC) is a candidate tumor suppressor gene located at chromo‐ some 18q21.[94] DCC promoter region hypermethylation was found in 75% of primary HNSCC. There was a significant correlation between DCC promoter region hypermethylation and DCC expression.[94] DCC is a putative conditional tumor suppressor gene that is epigenetically inactivated by promoter hypermethylation in a majority of HNSCC.[94, 95]

Secreted frizzled-related protein (SFRP1) is epigenetically silenced and functions as a tumor suppressor in oral squamous cell carcinoma (OSCC). The loss of SFRP2 expression is associated with hypermethylation of its promoter.[96]

The methylation of the *KIF1A* and *EDNRB* gene promoters is a frequent event in HNSCC.[97] KIF1A (kinesin family member 1A) encodes a protein that is a microtubule-dependent molecular motor involved in important intracellular functions such as organelle transport and cell division.[98] Endothelin receptor type B (EDNRB) is a G-protein-coupled receptor that activates a phosphatidylinositol calcium second messenger system.[97, 99]

Runt domain transcription factors (RUNXs) are homologous to products encoded by the Drosophila segmentation genes runt and lozenge.[100] The *RUNX3* gene is located on human chromosome 1p36, a region that has long been suspected to harbor one or more suppres‐ sors of various tumors.[101] Inactivation of RUNX3, which is caused mainly by epigenetic alteration, is closely associated with bladder tumor development, recurrence, and progres‐ sion.[100, 101]

HIN-1 (high in normal-1) is a putative cytokine with growth inhibitory activities and is downregulated by aberrant methylation in breast cancers.[102] Evidence suggests that HIN-1 is a potential tumor suppressor gene in non-small cell lung cancer (NSCLC), silenced by promoter hypermethylation and negatively regulated by AKT signaling pathway.[103] Silencing of HIN-1 expression and methylation of its promoter occurs in multiple human cancer types, suggesting that the elimination of HIN-1 function may contribute to several forms of epithelial tumorigenesis.[104]

#### **3.2. Histone modifications in head and neck cancer**

Aberrant regulation of the demethylases controlling H3K9me3 and H4K20me3 levels could contribute to the oncogenic potential. For instance, levels of H3K4me2 and me3 are signifi‐ cantly different in oral squamous cell carcinoma in comparison with cells of the healthy tissues; the level of H3K4me2 is increased while that of H3K4me3 is decreased.[102, 105]

A similar trend was observed in tongue squamous cell carcinoma (SCC) cells where the levels of the H3K27me3 marks at chromatin near homeobox genes were inversely correlated with the transcript levels in nontumorigenic, immortalized human oral keratinocytes (OKF6- TERT1R) and tumorigenic oral SCC-9 cells.[25] This investigation found that the levels of the H3K27me3 marks at chromatin near homeobox genes were inversely correlated with the transcript levels in nontumorigenic, immortalized human oral keratinocytes (OKF6- TERT1R) and tumorigenic oral SCC-9 cells.[25]

The emerging importance of the regulation of the H3K27me3 mark as a driver of squamous differentiation suggests that SCCs may harbor defects in the epigenetic regulation of squamous differentiation.[106] The dysregulation of squamous differentiation is fundamental to the development of SCC and has been reported to occur early in premalignant lesions.[107]

On the other hand, serine phosphorylation plays an important role in assembling the DNA damage response complex by identifying DNA double-strand breaks (DSB) in the chromatin. Upon DSB, ATM induces the phosphorylation of γH2AX at serine 139, resulting in the recruitment of BRCA1, BRCA2, Rad51, Mre11, NBS1, FANCD2, and p53 repair proteins to sites of DNA damage.[108]

In addition, the phosphorylation of serine 536 involved in the phosphorylation of RELA has also been reported. RELA, also known as p65, is an REL-associated protein involved in NF-κB heterodimer formation, nuclear translocation, and activation.[109] The phosphorylation of serine 468 is also associated with RELA.[109]

Regarding sumoylation, there are also desumoylating enzymes called SENPs, which remove the SUMO residues from the sumoylated proteins. Two molecules involved in the SUMO pathway, Ubc9 and SENP5, are up-regulated in SCCs. The up-regulation of SENP5 is found in oral SCCs, and strong SENP5 expression is correlated with poor prognosis.[110]

Finally, the lysine methylation of HSP90AB1 is important for its homodimerization and its interaction with stress-induced phosphoprotein 1 (STIP1) and cell division cycle 37 (CDC37), which are co-chaperones of HSP90AB1 in human cancer cells, resulting in enhancement of cancer cell growth.[111]

#### **3.3. miRNAs in head and neck cancer**

Deleted in colorectal cancer (DCC) is a candidate tumor suppressor gene located at chromo‐ some 18q21.[94] DCC promoter region hypermethylation was found in 75% of primary HNSCC. There was a significant correlation between DCC promoter region hypermethylation and DCC expression.[94] DCC is a putative conditional tumor suppressor gene that is epigenetically inactivated by promoter hypermethylation in a majority of HNSCC.[94, 95]

Secreted frizzled-related protein (SFRP1) is epigenetically silenced and functions as a tumor suppressor in oral squamous cell carcinoma (OSCC). The loss of SFRP2 expression is associated

The methylation of the *KIF1A* and *EDNRB* gene promoters is a frequent event in HNSCC.[97] KIF1A (kinesin family member 1A) encodes a protein that is a microtubule-dependent molecular motor involved in important intracellular functions such as organelle transport and cell division.[98] Endothelin receptor type B (EDNRB) is a G-protein-coupled receptor that

Runt domain transcription factors (RUNXs) are homologous to products encoded by the Drosophila segmentation genes runt and lozenge.[100] The *RUNX3* gene is located on human chromosome 1p36, a region that has long been suspected to harbor one or more suppres‐ sors of various tumors.[101] Inactivation of RUNX3, which is caused mainly by epigenetic alteration, is closely associated with bladder tumor development, recurrence, and progres‐

HIN-1 (high in normal-1) is a putative cytokine with growth inhibitory activities and is downregulated by aberrant methylation in breast cancers.[102] Evidence suggests that HIN-1 is a potential tumor suppressor gene in non-small cell lung cancer (NSCLC), silenced by promoter hypermethylation and negatively regulated by AKT signaling pathway.[103] Silencing of HIN-1 expression and methylation of its promoter occurs in multiple human cancer types, suggesting that the elimination of HIN-1 function may contribute to several forms of epithelial

Aberrant regulation of the demethylases controlling H3K9me3 and H4K20me3 levels could contribute to the oncogenic potential. For instance, levels of H3K4me2 and me3 are signifi‐ cantly different in oral squamous cell carcinoma in comparison with cells of the healthy tissues;

A similar trend was observed in tongue squamous cell carcinoma (SCC) cells where the levels of the H3K27me3 marks at chromatin near homeobox genes were inversely correlated with the transcript levels in nontumorigenic, immortalized human oral keratinocytes (OKF6- TERT1R) and tumorigenic oral SCC-9 cells.[25] This investigation found that the levels of the H3K27me3 marks at chromatin near homeobox genes were inversely correlated with the transcript levels in nontumorigenic, immortalized human oral keratinocytes (OKF6- TERT1R)

the level of H3K4me2 is increased while that of H3K4me3 is decreased.[102, 105]

activates a phosphatidylinositol calcium second messenger system.[97, 99]

with hypermethylation of its promoter.[96]

80 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

sion.[100, 101]

tumorigenesis.[104]

**3.2. Histone modifications in head and neck cancer**

and tumorigenic oral SCC-9 cells.[25]

DNA copy number variations or deregulation of miRNA expression has been shown to contribute to carcinogenesis, including carcinogenesis of HNSCC.[112] Several studies have reported global miRNA expression changes in carcinogenesis of HNSCC, using various samples sizes, anatomical sites, and profiling methodologies.[113]

It has been demonstrated the differential expression patterns of miRNAs in numerous types of cancer. MiR-92a, miR-103/107, miR-21, miR143, miR145, miR-205 and miR-296, among others, have been confirmed to be involved in the development of esophageal squamous cell carcinoma (ESCC).[34]

It has been reported that members of the miR-17-92 cluster were deregulated in 15 patients with OSCC (tongue and floor of the mouth) and 35 patients with HNSCC. The miR-19a and the miR-19b were strongly up-regulated. The miR-17-3p/miR-17-5p and the miR-92b were moderately up-regulated. Evidence has been found that the miR-17-92 cluster is up-regulated in many cancer types.[114] Furthermore, the miR-196a and the miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation.[114]

Other studies informed that miR-21, miR-31, miR-504, and miR-10b are target tumor suppres‐ sor genes. These miRNAs are involved in HNSCC.[115] Many studies have confirmed the tumor suppressor roles of the let-7 family (the miR-99 family, miR-107, miR-133a, miR-137, miR-138, and miR-375). Other miRNAs such as miR-21, let-7, miR-107, miR-138, and miR-200c are involved in regulating stemness or the epithelial–mesenchymal transition of tumor cells. [115]

It has also been evaluated that miR-34a is significantly down-regulated in HNSCC tumors and cell lines.[116] The ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation, and migration. Tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survival as well as between miR-34a and E2F3 levels.[116]

In silico analysis identified three putative microRNA-107 (miR-107) binding sites in the 3' untranslated region (UTR) of PKCε.[117] An inverse relationship was revealed between miR-107 and PKCε in HNSCC cell lines. These data demonstrated that PKCε is directly regulated by miR-107 and, moreover, suggest that miR-107 may be a potential anticancer therapeutic for HNSCC.[117]

New computational approach strategies complementary to microRNA profiling are capable of simultaneously predicting tumor suppressor microRNAs as well as their functional targets from gene expression.[118] It provided a plausible mechanism that loss of the tumor suppres‐ sor function of miR-204 as a result of allelic imbalance at 9q21.1–q22.3 may significantly increase genetic susceptibility to HNSCC oncogenesis and progression.[118] The complete suppression of miR-204 and its host gene TRPM3 has become possible that the mRNA expression may serve as a marker the expression status in HNSCC.[118]

#### **3.4. lncRNAs in head and neck cancer**

LncRNAs have been linked to essential growth-promoting activities, and their deregulation contributes to tumor cell survival. A prominent example is the Hox transcript antisense intergenic lncRNA, HOTAIR.[119] The HOTAIR gene controls gene expression, and its expression is deregulated in a spectrum of cancers. Furthermore, HOTAIR expression correlates with patient survival.[119, 120]

HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5′ domain of HOTAIR binds Polycomb Repressive Complex 2 (PRC2), while a 3′ domain of HOTAIR binds the LSD1/CoREST/REST complex. lncRNAPCAT-1, a target gene of polycomb repressive complex 2, has been implicated in disease progression by promoting cell prolifer‐ ation.[121] The ability to link two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3 lysine 27 methylation and lysine 4 demethylation.[122]

In prostate cancer, the up-regulation of antisense noncoding RNA in the INK4 locus (ANRIL) is required for the expression of the tumor suppressors INK4a/p16 and INK4b/p15.[121]

Some examples of lncRNAs that has a role in chromatin remolding include *XIST*, which acts by recruiting the PRC2 complex to initiate X-chromosome inactivation as well as *MALAT* and *NEAT1*, both of which play a role in mRNA processing and nuclear organization.[123]

The expression of LINC00312 in nasopharyngeal carcinoma has a tumor-suppressive function. Under physiological conditions, LINC00312 inhibits proliferation in nasopharyngeal epithe‐ lium by preventing cell cycle passage from the G1 into S phase but increases cell adhesion, motility, and invasion by down-regulating the expression of estrogen receptor alpha (ERα). [124]

Other genes regulated by lncRNAs that have been implicated in cancer include NDM29, BACE1AS, and Drosophila hsr-ω gene.[125] Neuroblastoma differentiation Marker 29 (NDM29) is an RNA polymerase (pol) III-transcribed noncoding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype.[126] BACE1 plays a pivotal role in the accumulation of β-amyloid plaques and has been shown to regulate the expression of BACE1 by increasing BACE1 mRNA stability.[127] The heat shock RNA omega (hsrω) gene of *Drosophila melanogaster* is inducible by cell stress and provides structural base for sequestering diverse RNA-processing/regulatory proteins.[128]

#### **4. Clinical applications in head and neck cancer**

tumor suppressor roles of the let-7 family (the miR-99 family, miR-107, miR-133a, miR-137, miR-138, and miR-375). Other miRNAs such as miR-21, let-7, miR-107, miR-138, and miR-200c are involved in regulating stemness or the epithelial–mesenchymal transition of tumor cells.

82 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

It has also been evaluated that miR-34a is significantly down-regulated in HNSCC tumors and cell lines.[116] The ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation, and migration. Tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survival as well as between

In silico analysis identified three putative microRNA-107 (miR-107) binding sites in the 3' untranslated region (UTR) of PKCε.[117] An inverse relationship was revealed between miR-107 and PKCε in HNSCC cell lines. These data demonstrated that PKCε is directly regulated by miR-107 and, moreover, suggest that miR-107 may be a potential anticancer

New computational approach strategies complementary to microRNA profiling are capable of simultaneously predicting tumor suppressor microRNAs as well as their functional targets from gene expression.[118] It provided a plausible mechanism that loss of the tumor suppres‐ sor function of miR-204 as a result of allelic imbalance at 9q21.1–q22.3 may significantly increase genetic susceptibility to HNSCC oncogenesis and progression.[118] The complete suppression of miR-204 and its host gene TRPM3 has become possible that the mRNA

LncRNAs have been linked to essential growth-promoting activities, and their deregulation contributes to tumor cell survival. A prominent example is the Hox transcript antisense intergenic lncRNA, HOTAIR.[119] The HOTAIR gene controls gene expression, and its expression is deregulated in a spectrum of cancers. Furthermore, HOTAIR expression

HOTAIR serves as a scaffold for at least two distinct histone modification complexes. A 5′ domain of HOTAIR binds Polycomb Repressive Complex 2 (PRC2), while a 3′ domain of HOTAIR binds the LSD1/CoREST/REST complex. lncRNAPCAT-1, a target gene of polycomb repressive complex 2, has been implicated in disease progression by promoting cell prolifer‐ ation.[121] The ability to link two distinct complexes enables RNA-mediated assembly of PRC2 and LSD1 and coordinates targeting of PRC2 and LSD1 to chromatin for coupled histone H3

In prostate cancer, the up-regulation of antisense noncoding RNA in the INK4 locus (ANRIL) is required for the expression of the tumor suppressors INK4a/p16 and INK4b/p15.[121]

Some examples of lncRNAs that has a role in chromatin remolding include *XIST*, which acts by recruiting the PRC2 complex to initiate X-chromosome inactivation as well as *MALAT* and *NEAT1*, both of which play a role in mRNA processing and nuclear organization.[123]

expression may serve as a marker the expression status in HNSCC.[118]

[115]

miR-34a and E2F3 levels.[116]

therapeutic for HNSCC.[117]

**3.4. lncRNAs in head and neck cancer**

correlates with patient survival.[119, 120]

lysine 27 methylation and lysine 4 demethylation.[122]

To date, four epigenetic inhibitors have been approved by the U.S. Food and Drug Adminis‐ tration (FDA) for cancer treatment.[129] The DNMT inhibitors as 5-aza-cytidine and 5-aza-2′ deoxycytidine are widely used in in vitro in research. The cytosine analogs are converted to deoxynucleotide triphosphates inside the cell and then incorporated into the DNA during replication in the original C positions.[130]

Other inhibitors of HATs approved by FDA are p300, lysine methyltransferases (H3K79 methyltransferase DOT1L, or the polycomb complex member EZH2), and lysine demethylases (LSd1). Furthermore, small molecule inhibitors targeting the histone reader, BRD4, have also shown promise as therapeutic agent in many cancer types.[131]

The effects of an epigenetic inhibitor as lysine residues on histone tails are HDAC inhibitors that counteract the global overexpression of HDACs in cancer and reinstate a more permissive nucleosome structure for transcription.[132] Vorinostat (a pan-HDAC inhibitor) and romi‐ depsin (a class I HDAC inhibitor) have each shown >30% response rates against cutaneous Tcell lymphoma (CTCL) in phase II trials.[132]

Cetuximab is an inhibitor of the epidermal growth factor receptor (EGFR) that is used in radiation therapy. It was found to enhance HNSCC patient survival compared with radiation therapy alone.[133] The FDA has approved cetuximab to treat HNSCC; the drug has a response rate of about 10% when used as a single agent in recurrent/metastatic disease.[134] However, despite approval of cetuximab, improvement in patient survival with the use of this agent has been only modestly incremental.[133]

Advances in oncogenomics have also identified mutations in epigenetic-associated genes that encode histones and their linkers, proteins associated with the recruitment of DNA-binding proteins, HDAC I and II interacting proteins, corepressor proteins, and transcriptional activators and coactivators.[135]

The understanding of which gene mutations, DNA methylation, posttranslational histone modification, and noncoding RNAs drive the carcinogenesis may help us understand how tumor susceptibility guides the development of new combination therapies. However, it is important remember that not all cancers are equally susceptible to epigenetic therapies. The biology underpinning this observation urgently warrants our attention if epigenetic therapies are to be more widely applicable.[136]

#### **5. Conclusions**

Understanding the complexity of the epigenome, the different dynamics, and the different subunits is complex and intimidating. Gene mutations, DNA methylation, posttranslational histone modification, and noncoding RNAs are actors involved in modulating its interactions with genomic sequences, and this is fundamental for health and disease.

Only our hope and desire can continue creating new ways to interact with the epigenome, and it will be possible to build a new world where the HNSCC and other types of cancer will be able to have new therapies and new opportunities for a better life.

#### **Author details**

Julio Cesar Osorio1 and Andres Castillo2\*

\*Address all correspondence to: acastillo.doc@gmail.com

1 School of Biomedical Sciences, Universidad del Valle, Cali, Colombia

2 Biology Department, Faculty of Exact and Natural Sciences of Universidad del Valle, Cali, Colombia

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The understanding of which gene mutations, DNA methylation, posttranslational histone modification, and noncoding RNAs drive the carcinogenesis may help us understand how tumor susceptibility guides the development of new combination therapies. However, it is important remember that not all cancers are equally susceptible to epigenetic therapies. The biology underpinning this observation urgently warrants our attention if epigenetic therapies

Understanding the complexity of the epigenome, the different dynamics, and the different subunits is complex and intimidating. Gene mutations, DNA methylation, posttranslational histone modification, and noncoding RNAs are actors involved in modulating its interactions

Only our hope and desire can continue creating new ways to interact with the epigenome, and it will be possible to build a new world where the HNSCC and other types of cancer will be

2 Biology Department, Faculty of Exact and Natural Sciences of Universidad del Valle, Cali,

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1 School of Biomedical Sciences, Universidad del Valle, Cali, Colombia

\*Address all correspondence to: acastillo.doc@gmail.com

are to be more widely applicable.[136]

84 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**5. Conclusions**

**Author details**

Julio Cesar Osorio1

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### **The Role of Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma and Its Clinical Implications**

Kaveh Karimnejad, Nathan Lindquist and Reigh-Yi Lin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/61151

#### **Abstract**

Worldwide, more than 550, 000 new cases of head and neck squamous cell carcinoma (HNSCC) are estimated to occur annually, making it the sixth most common human ma‐ lignancy. Since their discovery in 2007, cancer stem cells (CSCs) in HNSCC have garnered increased interest secondary to their properties of tumorigenicity, differenti- ation, prolif‐ eration, and self-renewal. CSCs are intrinsically more resistant to tradi- tional treatments such as radiation and chemotherapy, contributing to potential metastasis and recurrence of HNSCC. This chapter focuses first on normal head and neck stem cells, providing background for the discussion of a number of topics pertaining to the study of HNSCC CSCs including molecular biomarkers and clinical implications. Continued research to elucidate the properties of CSCs will undoubt- edly expand our knowledge surrounding the pathogenesis, metastasis, and relapse of HNSCC. Ultimately, a better understanding of CSC biomarkers, signaling pathways, and mechanisms of resistance will improve therapies and patient outcomes through targeted interventions.

**Keywords:** Head and neck squamous cell carcinoma, Stem cells, Cancer stem cells, Che‐ moresistance, Metastasis

#### **1. Introduction**

In the United States, over 53, 000 new cases of head and neck squamous cell carcinoma (HNSCC) are estimated to occur each year, with roughly 11, 000 deaths annually [1]. Across the globe, HNSCC has an annual incidence of over 550, 000 cases, making it the sixth most common cancer worldwide. HNSCC accounts for 90% of the malignancies in the head and neck region, affecting the nasal vestibule, nasal cavity, paranasal sinuses, nasopharynx, lips, oral cavity, oropharynx, pharynx, hypopharynx, and larynx. The foundation of treatment for head and neck cancer has been surgery and radiation therapy, while chemotherapy may also

© 2016 The Author(s). Licensee InTech. 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.

be employed as an adjunctive treatment. Advancements in surgical technique, radiation strategies and technologies, as well as chemotherapeutic drugs have led to improvements in patient's overall quality of life. However, the prognosis for HNSCC has improved only marginally over the past thirty years, with the five-year survival rate remaining at 50% [2-4]. Even after standard therapy, patients with HNSCC exhibit relatively high rates of local recurrence, regional cervical lymph node recurrence, and to a lesser degree distant metastasis, all of which contribute to significant morbidity and mortality [5]. Often, these recurrences and metastases are more resistant to traditional treatment modalities such as chemoradiation. Unfortunately, primary site recurrence occurs in 10-30% of all patients [6].

Traditional approaches to understanding and treating HNSCC are based on the stochastic model of cancer, where all tumor cells are identical (Figure 1). More recently, the cancer stem cell (CSC) hypothesis has gained increasing traction in explaining tumorigenesis [7]. This theory proposes that cancer maintains a hierarchical order of cells, with only a small subpopulation of CSCs capable of tumor initiation, propagation, and regeneration [8]. Conventional therapies that target rapidly cycling cells are less effective in killing CSCs. CSCs also display increased intrinsic resistance to chemotherapy and radiation therapy. As a result, CSCs are likely to contribute to cancer relapse.

This review aims to provide a succinct yet thorough overview of our current basis for the CSC hypothesis as it pertains to HNSCC. We will start with a brief discussion of the normal epithelium of the head and neck region as well as our current understanding of normal endogenous stem cells of the head and neck region. Evidence for the CSC hypothesis of

**Figure 1.** In the stochastic model, all tumor cells have equal abilities to propagate, initiate tumors, and seed metastases. The heterogeneity of tumors in this model is derived from spontaneous phenotypic shifts. The emerging cancer stem cell hypothesis dictates the hierarchical model, in which asymmetric division results in specific and well-defined popu‐ lations of cancer stem cells and other cancer cells that do not initiate tumors or seed metastases. These cells may consist of populations with decreased proliferative ability (i.e., transit-amplifying cells) or post-mitotic differentiated cells with no further proliferative ability or activity.

HNSCC will include a discussion of the prospective markers for CSCs in HNSCC, as well as a closer look at the cellular regulation of these CSCs and the clinical implications of these cancer- initiating and propagating cells.

#### **2. Head and neck stem cells in normal tissues**

be employed as an adjunctive treatment. Advancements in surgical technique, radiation strategies and technologies, as well as chemotherapeutic drugs have led to improvements in patient's overall quality of life. However, the prognosis for HNSCC has improved only marginally over the past thirty years, with the five-year survival rate remaining at 50% [2-4]. Even after standard therapy, patients with HNSCC exhibit relatively high rates of local recurrence, regional cervical lymph node recurrence, and to a lesser degree distant metastasis, all of which contribute to significant morbidity and mortality [5]. Often, these recurrences and metastases are more resistant to traditional treatment modalities such as chemoradiation. Unfortunately,

Traditional approaches to understanding and treating HNSCC are based on the stochastic model of cancer, where all tumor cells are identical (Figure 1). More recently, the cancer stem cell (CSC) hypothesis has gained increasing traction in explaining tumorigenesis [7]. This theory proposes that cancer maintains a hierarchical order of cells, with only a small subpopulation of CSCs capable of tumor initiation, propagation, and regeneration [8]. Conventional therapies that target rapidly cycling cells are less effective in killing CSCs. CSCs also display increased intrinsic resistance to chemotherapy and radiation therapy. As a result, CSCs are

This review aims to provide a succinct yet thorough overview of our current basis for the CSC hypothesis as it pertains to HNSCC. We will start with a brief discussion of the normal epithelium of the head and neck region as well as our current understanding of normal endogenous stem cells of the head and neck region. Evidence for the CSC hypothesis of

**Figure 1.** In the stochastic model, all tumor cells have equal abilities to propagate, initiate tumors, and seed metastases. The heterogeneity of tumors in this model is derived from spontaneous phenotypic shifts. The emerging cancer stem cell hypothesis dictates the hierarchical model, in which asymmetric division results in specific and well-defined popu‐ lations of cancer stem cells and other cancer cells that do not initiate tumors or seed metastases. These cells may consist of populations with decreased proliferative ability (i.e., transit-amplifying cells) or post-mitotic differentiated cells

primary site recurrence occurs in 10-30% of all patients [6].

98 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

likely to contribute to cancer relapse.

with no further proliferative ability or activity.

Stem cells are unique in their ability to maintain self-renewal, differentiate into multiple lineage types in the same tissue, and display a high degree of proliferative potential [9]. The somatic stem cell microenvironment or cellular "niche" is vital in maintaining the delicate distinction between self-renewal and uncontrolled proliferation of stem cells [10]. Stem cells and the extracellular matrix secrete factors to maintain the microenvironment, while inhibitory signals from this local microenvironment provide necessary control of proliferation and differentiation to sustain this important subpopulation of cells [11, 12]. Importantly, these quiescent, undifferentiated somatic stem cells also depend on the niche for the transient stimulatory signals necessary for cell division and tissue regeneration [13]. Unlike most of the gastrointestinal tract, which contains a simple epithelial layer to allow for increased absorption, the oral cavity, pharynx, and esophagus are covered by a stratified epithelium that is more similar in structure to other tissues such as skin [14]. The stratified epithelium is composed of multiple layers of cell types oriented in order of increasing differentiation from basal to superficial.

In the normal squamous stratified epithelium, stem cells are located in the basal layer. To replenish the more superficial layers, these stem cells divide asymmetrically to self-renew and produce cells to undergo subsequent differentiation and amplification [11]. Cell division in stratified squamous epithelium results in differentiation, superficial cellular movement, stratification, and ultimately, tissue turnover. In the stratified squamous epithelium, for example, a layer of small, cuboidal, basal stem cells are responsible for cell division and regeneration [15]. Moving superficially, these committed cells further differentiate to increase keratin filament production, flatten, and decrease the size and volume of the nucleus and organelles. The most superficial (corneal or superficial) layers of the oral mucosa demonstrate cell flattening, mem- brane thickening, decreased desmosomes, and eventual sloughing of cells into the oral cavity [16]. The hierarchical normal oral epithelium is renewed approximately every 14–24 days [17]. Increasingly, endogenous oral cavity stem cells are theorized to precede CSCs, as these are the only cells with life span sufficient to accumulate the genetic mutations necessary for malignant transformation [18].

#### **3. Head and neck stem cell markers**

In contrast to other tissues or organ systems, relatively few markers have been identified or characterized for normal endogenous stem cells of the head and neck region. To date, most of our knowledge of normal head and neck stem cells is based largely on work on oral epithelial stem cells (OESCs) and corollaries from the skin and hair follicle, which also maintain a squamous stratified epithelium.

In the 1960s, the first experiments to identify potential stem cells in the oral mucosa utilized pulse-chase experiments with tritiated-thymidine (3H-TdR) to elucidate cell turnover rates in the skin and oral mucosa and identify label-retaining cells in the basal layer [19]. Of the few candidate cell surface markers for OESCs, most are also expressed in other normal oral epithelial basal cells, meaning that much of the research involving OESC markers has involved purification rather than isolation of these cells [20]. Such work is often accomplished based on sorting for cell markers and performing subsequent *in vitro* experiments to test the self-renewal and proliferative properties of these subpopulations. So far, notable candidate oral stem cell markers include keratins K5, 14, 15, and 19, β1- and α6-integrins, integrin α6β4, melanoma chondroitin sulfate proteoglycan (MCSP), p75NGFR, B-cell-specific Moloney murine leukemia virus integration site 1 (BMI1), and the p63 transcription factor (Table 1) [20-37].

Aida *et al.* performed telomere analysis of different cell types in normal lingual epithelium to calculate normalized telomere:centromere ratios (NTCRs). Overall, the basal cell group demonstrated the largest NTCR, with a smaller subgroup of these cells maintaining an exceptionally large NTCR, suggesting the presence of stem cells. In general, stem cells are thought to maintain relatively longer telomeres due to a lower telomere turnover rate as well as the potential for telomere upregulation. In addition, samples from older patients contained relatively shorter telomeres, confirming a measurable age-related progression. Finally, immunohistochemistry confirmed the presence of p27, p63, and K19 in the basal layer with relatively scant staining for Ki-67, a well-known marker of cell proliferation [22].

In one study, the magnetic separation of oral human keratinocytes yielded a fraction of α6β +CD71 cells that could regenerate a stratified oral epithelial equivalent *in vitro*. Unlike either of the α6β +CD71+ or or α6β keratinocyte groups, α6β +CD71 cells also expressed the candidate stem cell markers p63 and Keratin 19 and were negative for two recognized markers of differentiation: cytokeratin 10 or involucrin [23].

Tao *et al.* demonstrate a method of enriching a subpopulation containing both potential stem cells and transit amplifying cells through integrin-β1 adherence to collagen IV. While their subsequent study of p63 expression could not confirm specificity for stem cells in the basal layer, the ΔNp63α and ΔNp63β isoforms may be more specific markers for undifferentiated or immature cells [21].

Through cell lineage mapping, Hogan and coworkers noted a K14+K5+Trp63+Sox2+ subpopulation of long-term stem or progenitor cells located outside the taste buds that are capable of differentiating into both mature taste bud receptor cells as well as keratinocytes. The authors suggest that a similar model may apply to the taste buds of the circumvallate papillae and soft palate, and that their work may prove a model system for future study of these endogenous stem cells [24]. This same group isolated a population of undifferentiated tongue basal cells using Krt5-eGFP transgenic mice that demonstrated self-renewal and differentiation to stratified keratinized epithelial cells *in vitro*.

stem cells (OESCs) and corollaries from the skin and hair follicle, which also maintain a

In the 1960s, the first experiments to identify potential stem cells in the oral mucosa utilized pulse-chase experiments with tritiated-thymidine (3H-TdR) to elucidate cell turnover rates in the skin and oral mucosa and identify label-retaining cells in the basal layer [19]. Of the few candidate cell surface markers for OESCs, most are also expressed in other normal oral epithelial basal cells, meaning that much of the research involving OESC markers has involved purification rather than isolation of these cells [20]. Such work is often accomplished based on sorting for cell markers and performing subsequent *in vitro* experiments to test the self-renewal and proliferative properties of these subpopulations. So far, notable candidate oral stem cell markers include keratins K5, 14, 15, and 19, β1- and α6-integrins, integrin α6β4, melanoma chondroitin sulfate proteoglycan (MCSP), p75NGFR, B-cell-specific Moloney murine leukemia

Aida *et al.* performed telomere analysis of different cell types in normal lingual epithelium to calculate normalized telomere:centromere ratios (NTCRs). Overall, the basal cell group demonstrated the largest NTCR, with a smaller subgroup of these cells maintaining an exceptionally large NTCR, suggesting the presence of stem cells. In general, stem cells are thought to maintain relatively longer telomeres due to a lower telomere turnover rate as well as the potential for telomere upregulation. In addition, samples from older patients contained relatively shorter telomeres, confirming a measurable age-related progression. Finally, immunohistochemistry confirmed the presence of p27, p63, and K19 in the basal layer with relatively

In one study, the magnetic separation of oral human keratinocytes yielded a fraction of α6β +CD71 cells that could regenerate a stratified oral epithelial equivalent *in vitro*. Unlike either

stem cell markers p63 and Keratin 19 and were negative for two recognized markers of

Tao *et al.* demonstrate a method of enriching a subpopulation containing both potential stem cells and transit amplifying cells through integrin-β1 adherence to collagen IV. While their subsequent study of p63 expression could not confirm specificity for stem cells in the basal layer, the ΔNp63α and ΔNp63β isoforms may be more specific markers for undifferentiated

Through cell lineage mapping, Hogan and coworkers noted a K14+K5+Trp63+Sox2+ subpopulation of long-term stem or progenitor cells located outside the taste buds that are capable of differentiating into both mature taste bud receptor cells as well as keratinocytes. The authors suggest that a similar model may apply to the taste buds of the circumvallate papillae and soft palate, and that their work may prove a model system for future study of these endogenous stem cells [24]. This same group isolated a population of undifferentiated tongue basal cells using Krt5-eGFP transgenic mice that demonstrated self-renewal and differentiation to strati-

cells also expressed the candidate

virus integration site 1 (BMI1), and the p63 transcription factor (Table 1) [20-37].

scant staining for Ki-67, a well-known marker of cell proliferation [22].

of the α6β +CD71+ or or α6β keratinocyte groups, α6β +CD71-

differentiation: cytokeratin 10 or involucrin [23].

or immature cells [21].

fied keratinized epithelial cells *in vitro*.

squamous stratified epithelium.

100 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis


**Table 1.** The function and significance of candidate normal stem cell markers in the head and neck region.

The low-affinity neurotrophin receptor p75NGFR is a member of the tumor necrosis factor receptor superfamily and has effects in cell survival, apoptosis, and intercellular signaling [25]. It has been put forth as a possible stem cell marker for neural crest, mesenchymal, esophageal, oral mucosa [25], and most recently laryngeal epithelium [25, 26]. In addition, p75NGFR-positive basal keratinocytes are able to migrate and initiate regeneration of damaged buccal epithelium [27]. Furthermore, expression of p75NGFR has been shown to be closely related to CSCs in esophageal squamous cell carcinoma as well as laryngeal squamous cell carcinoma [26, 28].

Recent interest in lingual epithelial stem cells has provided some evidence that keratins K5 and K14 may not be specific stem cell markers in this system. Ueno and coworkers utilized immunostaining to reveal that only a small fraction of these keratins K14/K5-positive cells were actually replicating to supply epithelial cells [29]. Rather, this group identified a group of BMI1 positive stem cells that maintain the epithelial cells and can regenerate after irradia- tioninduced tissue injury. Curiously, these potential stem cells were located in the second or third epithelial layer of the interpapillary pit of the filiform papillae. Increasingly, BMI1 is viewed as a candidate marker for CSCs of the head and neck, with the potential for prognostic value based on the location of this intracellular oncoprotein [30].

MCSP, melanoma-associated chondroitin sulfate proteoglycan; NGFR, nerve growth factor receptor; TNF, tumor necrosis factor; BMI1, B-cell-specific Moloney murine leukemia virus integration site 1

#### **4. Origin of head and neck cancer stem cells**

In most instances, HNSCC is caused by the accumulation of multiple genetic mutations based on genetic predisposition, which is induced by environmental factors such as tobacco and alcohol abuse or persistent human papilloma virus infection [38]. However, the alterations of multiple molecular and cellular pathways that lead to the development and recurrence of HNSCC are still not well understood. Recently, recurrence and therapeutic resistance of HNSCC has been attributed to a subpopulation of self-sustaining, tumor-initiating CSCs. CSCs are defined by several exclusive features that allow propagation as well as tumor formation and maintenance. These features are: (1) differentiation, giving rise to heterogeneous progeny; (2) self-renewal, which maintains a pool of stem cells which can expand; and (3) homeostatic control, which accounts for tissue specificity [4].

Multiple possible origins for CSCs have been proposed wherein a population of self-renewing cells are formed, leading to tumorigenesis (Figure 2) [39]. In one such scenario, normal stem cells undergo mutations that diminish restraint on replication, thereby creating CSCs that are unresponsive to environmental or intrinsic controls on self-renewal. Another potential source of CSCs are the more differentiated progenitor cells, also known as transit-amplifying cells, which maintain a more limited role in self-renewal yet are far more numerous than stem cells. A third motif of CSC generation explains that well-differentiated, mature cells undergo mutations to dedifferentiate and obtain greater self-renewal potential [5]. There is evidence that these dedifferentiated HNSCC cells may undergo epithelial-mesenchymal transition and invasion, leading to the development of cells with CSC- or mesenchymal characteristics [40].

#### **5. Initial clues to the concept of head and neck cancer stem cells**

The first "leukemia-initiating" CSCs were identified in 1994 by Dick and co-workers through their work with acute myeloid leukemia [41]. In 2003, Al-Hajj *et al.* reported the first CSCs in a solid tumor by separating a tumorigenic subpopulation of breast cancer cells based on the surface cell markers CD44+/CD24-/low [42]. In 2007, a landmark study by Prince *et al.* described a subpopulation of CD44+ tumor-initiating cells isolated from HNSCC, although the cell surface markers CD44s and CD44v6 were subsequently described in a majority of normal head and neck tissues as well as HNSCC [43, 44]. Other subpopulations of tumor-initiating cells have since been identified in HNSCC that also fulfill the criteria for CSCs. Furthermore, several of the putative markers of these CSC subpopulations have been linked to cancer recurrence and therapeutic resistance, augmenting the evidence for the CSC hypothesis in HNSCC. Recent interest in the identification of new and improved biomarkers for HNSCC CSCs has spiked due to the prospect of using these tools to improve treatment approaches and overall mortality in this deadly disease.

**Figure 2.** Potential origins for cancer stem cells include normal stem cells, progenitor cells, or fully differentiated cells. To give rise to cancer stem cells, the progenitor and fully differentiated cells acquire mutations to reactivate genes re‐ sponsible for increased proliferative activity, cell-division, and dedifferentiation.

#### **6. Cancer stem cell assays**

basal keratinocytes are able to migrate and initiate regeneration of damaged buccal epithelium [27]. Furthermore, expression of p75NGFR has been shown to be closely related to CSCs in esophageal squamous cell carcinoma as well as laryngeal squamous cell carcinoma [26, 28].

Recent interest in lingual epithelial stem cells has provided some evidence that keratins K5 and K14 may not be specific stem cell markers in this system. Ueno and coworkers utilized immunostaining to reveal that only a small fraction of these keratins K14/K5-positive cells were actually replicating to supply epithelial cells [29]. Rather, this group identified a group of BMI1 positive stem cells that maintain the epithelial cells and can regenerate after irradia- tioninduced tissue injury. Curiously, these potential stem cells were located in the second or third epithelial layer of the interpapillary pit of the filiform papillae. Increasingly, BMI1 is viewed as a candidate marker for CSCs of the head and neck, with the potential for prognostic value

MCSP, melanoma-associated chondroitin sulfate proteoglycan; NGFR, nerve growth factor receptor; TNF, tumor necrosis factor; BMI1, B-cell-specific Moloney murine leukemia virus

In most instances, HNSCC is caused by the accumulation of multiple genetic mutations based on genetic predisposition, which is induced by environmental factors such as tobacco and alcohol abuse or persistent human papilloma virus infection [38]. However, the alterations of multiple molecular and cellular pathways that lead to the development and recurrence of HNSCC are still not well understood. Recently, recurrence and therapeutic resistance of HNSCC has been attributed to a subpopulation of self-sustaining, tumor-initiating CSCs. CSCs are defined by several exclusive features that allow propagation as well as tumor formation and maintenance. These features are: (1) differentiation, giving rise to heterogeneous progeny; (2) self-renewal, which maintains a pool of stem cells which can expand; and (3) homeostatic

Multiple possible origins for CSCs have been proposed wherein a population of self-renewing cells are formed, leading to tumorigenesis (Figure 2) [39]. In one such scenario, normal stem cells undergo mutations that diminish restraint on replication, thereby creating CSCs that are unresponsive to environmental or intrinsic controls on self-renewal. Another potential source of CSCs are the more differentiated progenitor cells, also known as transit-amplifying cells, which maintain a more limited role in self-renewal yet are far more numerous than stem cells. A third motif of CSC generation explains that well-differentiated, mature cells undergo mutations to dedifferentiate and obtain greater self-renewal potential [5]. There is evidence that these dedifferentiated HNSCC cells may undergo epithelial-mesenchymal transition and invasion, leading to the development of cells with CSC- or mesenchymal characteristics [40].

based on the location of this intracellular oncoprotein [30].

102 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

**4. Origin of head and neck cancer stem cells**

control, which accounts for tissue specificity [4].

integration site 1

The isolation and identification of CSCs is a hefty experimental challenge, as there is no established protocol to verify putative CSCs. Current experimental goals aim to satisfy the CSC criteria of both self-renewal as well as the capacity to develop heterogeneous cell lineages capable of forming tumors identical to the original [45]. Isolation strategies attempt to exploit the unique properties of CSCs that distinguish them from their differentiated progeny. Such capacities include the efflux of vital dyes by multidrug transporters, enzymatic activity, sphere-forming capacity in low attachment conditions, and the expression of cell surface antigens [46]. There are currently four main strategies for isolation of CSCs: (1) detection of side-population phenotypes by Hoechst 33342 exclusion, (2) sphere-formation assays, (3) assessment of aldehyde dehydrogenase (ALDH) activity, and (4) identification of CSC-specific cell surface markers [45]. To date, the most common modality in identifying HNSCC CSCs relies upon the expression of membrane cell surface antigens present in stem-like cells. As a result, most potential CSC populations are detected by immunohistochemistry or flow cytometry. Many of these antigens were originally put forth as potential targets as a result of their expression in normal stem cells [47, 48]. Herein, we present a review of the most promising putative HNSCC CSC markers: CD44, CD133, and ALDH. We also include a discussion of CD24 and CD10.



**Table 2.** The function, significance, and associations of putative cancer stem cell markers in HNSCC.

#### **7. Putative head and neck cancer stem cell markers**

#### **7.1. CD44**

capacities include the efflux of vital dyes by multidrug transporters, enzymatic activity, sphere-forming capacity in low attachment conditions, and the expression of cell surface antigens [46]. There are currently four main strategies for isolation of CSCs: (1) detection of side-population phenotypes by Hoechst 33342 exclusion, (2) sphere-formation assays, (3) assessment of aldehyde dehydrogenase (ALDH) activity, and (4) identification of CSC-specific cell surface markers [45]. To date, the most common modality in identifying HNSCC CSCs relies upon the expression of membrane cell surface antigens present in stem-like cells. As a result, most potential CSC populations are detected by immunohistochemistry or flow cytometry. Many of these antigens were originally put forth as potential targets as a result of their expression in normal stem cells [47, 48]. Herein, we present a review of the most promising putative HNSCC CSC markers: CD44, CD133, and ALDH. We also include a discussion

**Biological Function Significance in HNSCC Stem Cell**

**Biology**

tumor

tumorigenesis)

survival

co-

Correlated with lymph node metastases and decreased overall

Tumor cells expressing relatively high levels of ALDH have increased tumorigenicity, stem-cell-related genes, drug-resistant genes, and EMTrelated genes. Associated with high

expression of Snail protein, which is an EMT regulator and key factor in self- renewal and tumorigenicity

Showed the ability to regenerate

*in vivo* but also abundantly expressed in normal squamous epithelia of the head and neck. Associated with Snail (chemoresistance and radioresistance) as well as high coexpression of BMI1 (important for self-renewal and

**References**

[43,44,49,50,55]

[51,52,54,55]

[47,56,65]

of CD24 and CD10.

CD44 Surface glycoprotein involved in cell

CD133 (Prominin 1) Transmembrane glycoprotein localized

ALDH Intracellular enzyme most

microvilli

detoxifies

oxidation

migration and adhesion

104 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

on membrane protrusions and

commonly found in the liver. ALDH

intracellular aldehydes through

**Putative Cancer Stem Cell Markers in HNSCC Sites Studied**

> CD44 is a surface glycoprotein involved in cell migration and adhesion. Prince *et al.* demonstrated the ability of a subpopulation of CD44+ HNSCC cells to regenerate a tumor *in vivo* after transplantation of as few as 5x103 cells into an immunocompromised mouse model [44]. CD44+ cells have been shown to express a high level of BMI1, a prospective normal stem cell bio- marker that also plays a key role in self-renewal and tumorigenesis in malignancy [49, 50]. While CD44 was the first CSC marker established for HNSCC, its role has since come into question. In contrast to earlier studies involving CD44, recent studies have demonstrated the abundant expression of CD44 in most HNSCC tumor cells. In one such study, CD44 was present in 80-100% of tumor cells [43]. However, according to the *hierarchical model*, only a small subset of cells within malignant tissue that are able to generate tumors should stain positive for CSC markers. Furthermore, high percentages of CD44+ cells have been identified in normal squamous epithelia of the head and neck, with up to 60– 95% of normal head and neck epithelial cells demonstrating CD44 positivity. Although the role of CD44 in HNSCC has raised some questions, this protein may still prove a valua‐ ble role in the identification of CSCs when used in combination with other markers. In the 2009 study by Chen *et al.*, not only did knockdown of Snail expression in CD44+ALDH+ cells decrease tumor invasion and colony formation, but it also significantly increased sensitivity to chemotherapy and radiotherapy. Specifically, following seven days of Snail siRNA treatment, the CD44+ALDH+ CSCs exhibited increased sensitivity to cisplatin and etoposide, and radiotherapy. These findings illustrate the potential utility of CSCs as unique cell targets for both chemotherapy and radiation therapy.

#### **7.2. CD133**

First described in 1997, CD133 (Prominin 1) is a transmembrane glycoprotein expressed in several normal stem cell populations and malignancies [51]. CD133 is localized to cellular membrane protrusions and microvilli [52]. It is expressed in several solid malignancies including brain, colon, liver, and lung, and has been purported as a unique, specific marker for sarcomas [38]. In 2007, Zhou *et al.* demonstrated the presence of a CD133+ subpopulation in 3.5% of the native cells from a HEp-2 laryngeal cancer cell line. These CD133+ cells continued to proliferate and expand the tumor cell population in sphere formation assays. In cell culture assays, the majority of terminal cells did not express CD133, a finding consistent with the criteria for CSC self-renewal as well as the capacity to give rise to phenotypically unique tumor daughter cells [53]. More recent studies have supported these findings, suggesting the utility of CD133 as a clinically relevant prognostic marker in HNSCC. Canis *et al.* demonstrated an inversely proportional correlation between CD133 expression in primary tumors and overall survival in addition to a positive correlation between CD133 expression and the presence of lymph node metastases [54]. In addition, Yu *et al.* describe an oral cavity squamous cell carcinoma-derived side population of cells with high expression of CD133 and ALDH showed high tumorigenic capacity [55]. Cell viability assays revealed that these side populations of cells were more chemoresistant to cisplatin, fluorouracil, or doxorubicin treatment when compared to the major population of the same cell line. The researchers hypothesized that CD133 may be crucial to modulation of chemosensitivity. They subsequently performed lentiviral-mediated transduction in the side population of cells, which resulted in significant decrease in expression of CD133 mRNA and protein. The silencing of CD133 decreased the percentage of the side population in the cancer cell lines and decreased *in vivo* tumor growth. Furthermore, cisplatin treatment of the CD133 knockdown population diminished cell invasion and clonogenicity, demonstrating the enhanced sensitivity to chemotherapy by targeting CD133. These findings support the role of HNSCC CSCs as novel therapeutic targets in the development of chemotherapeutic drugs.

#### **7.3. ALDH**

ALDH is an intracellular enzyme most commonly found in the liver [47]. ALDH detoxifies intracellular aldehydes through oxidation and may play a role in the differentiation of stem cells by oxidizing retinol into retinoic acid. With regard to cancer, high ALDH activity has been linked to subsets of multiple myeloma and acute myeloid leukemia [56]. Prior to its identification with HNSCC, ALDH was labeled a putative CSC marker in both breast and colon cancer. Ginestier *et al.* successfully used high ALDH activity to identify a tumorigenic breast cancer cell fraction capable of self-renewal and generating heterogeneous tumors. In their study, expression of ALDH as detected by immunohistochemistry correlated with a poorer prognosis for breast carcinomas [57].

Expression of ALDH1 in inflammatory breast cancer has been put forth as an independent predictor of early metastasis and decreased survival [58]. In 2009, Chen *et al.* published the first study demonstrating that cells of ALDH1+ lineage have CSC properties and play a role in self- renewal in HNSCC [3]. In a study by Clay *et al.*, HNSCC cells were categorized and isolated based on either high or low ALDH activity and subsequently implanted into immunocompromised mice. Cells with relatively high levels of ALDH represented a small percentage of cells (1% to 7.8%), but gave rise to tumors from as few as 500 cells in 53% of implantations. In contrast, only 8% of similar implantations with cells expressing low levels of ALDH formed tumor. As a result, ALDH appears to be a relatively selective marker for HNSCC CSCs [56]. In a similar study, 87% of implantations with 1000 ALDH+CD44+ HNSCC cells generated tumors, compared to only 13% of ALDH-CD44 cell implantations, despite utilizing ten times more cells: 10, 000 [59]. ALDH+ cells have also been shown to exhibit higher expression levels of stem cell-related, drug-resistance-associated, and epithelial-mesenchymal-transformationrelated (EMT) genes such as Snail [3]. In fact, Snail protein overexpression transformed ALDH cells to ALDH+ cells, resulting in increased invasion and tumorigenic properties [65]. Given this association with Snail and EMT, as well as the ability to recapitulate tumors in high percentages after *in vivo* implantation in multiple studies, ALDH may be the most wellestablished HNSCC CSC marker to date.

#### **7.4. CD24 and CD10**

**7.2. CD133**

development of chemotherapeutic drugs.

**7.3. ALDH**

for breast carcinomas [57].

First described in 1997, CD133 (Prominin 1) is a transmembrane glycoprotein expressed in several normal stem cell populations and malignancies [51]. CD133 is localized to cellular membrane protrusions and microvilli [52]. It is expressed in several solid malignancies including brain, colon, liver, and lung, and has been purported as a unique, specific marker for sarcomas [38]. In 2007, Zhou *et al.* demonstrated the presence of a CD133+ subpopulation in 3.5% of the native cells from a HEp-2 laryngeal cancer cell line. These CD133+ cells continued to proliferate and expand the tumor cell population in sphere formation assays. In cell culture assays, the majority of terminal cells did not express CD133, a finding consistent with the criteria for CSC self-renewal as well as the capacity to give rise to phenotypically unique tumor daughter cells [53]. More recent studies have supported these findings, suggesting the utility of CD133 as a clinically relevant prognostic marker in HNSCC. Canis *et al.* demonstrated an inversely proportional correlation between CD133 expression in primary tumors and overall survival in addition to a positive correlation between CD133 expression and the presence of lymph node metastases [54]. In addition, Yu *et al.* describe an oral cavity squamous cell carcinoma-derived side population of cells with high expression of CD133 and ALDH showed high tumorigenic capacity [55]. Cell viability assays revealed that these side populations of cells were more chemoresistant to cisplatin, fluorouracil, or doxorubicin treatment when compared to the major population of the same cell line. The researchers hypothesized that CD133 may be crucial to modulation of chemosensitivity. They subsequently performed lentiviral-mediated transduction in the side population of cells, which resulted in significant decrease in expression of CD133 mRNA and protein. The silencing of CD133 decreased the percentage of the side population in the cancer cell lines and decreased *in vivo* tumor growth. Furthermore, cisplatin treatment of the CD133 knockdown population diminished cell invasion and clonogenicity, demonstrating the enhanced sensitivity to chemotherapy by targeting CD133. These findings support the role of HNSCC CSCs as novel therapeutic targets in the

106 New Aspects in Molecular and Cellular Mechanisms of Human Carcinogenesis

ALDH is an intracellular enzyme most commonly found in the liver [47]. ALDH detoxifies intracellular aldehydes through oxidation and may play a role in the differentiation of stem cells by oxidizing retinol into retinoic acid. With regard to cancer, high ALDH activity has been linked to subsets of multiple myeloma and acute myeloid leukemia [56]. Prior to its identification with HNSCC, ALDH was labeled a putative CSC marker in both breast and colon cancer. Ginestier *et al.* successfully used high ALDH activity to identify a tumorigenic breast cancer cell fraction capable of self-renewal and generating heterogeneous tumors. In their study, expression of ALDH as detected by immunohistochemistry correlated with a poorer prognosis

Expression of ALDH1 in inflammatory breast cancer has been put forth as an independent predictor of early metastasis and decreased survival [58]. In 2009, Chen *et al.* published the first study demonstrating that cells of ALDH1+ lineage have CSC properties and play a role in self- renewal in HNSCC [3]. In a study by Clay *et al.*, HNSCC cells were categorized and isolated

CD24 is a mucin adhesion molecule for P-selectin and L1 expressed by pre-B lymphocytes and neutrophils during cell development [47, 60]. CD24 expression has been shown to increase tumor cell proliferation and further shown to regulate multiple cell properties which contribute to tumor growth and metastasis [60]. It has been correlated with increased spread of breast cancer and has been further identified as a putative CSC marker in pancreatic, ovarian, and colorectal cancers [61, 62]. CD24 has also been associated with tumorigenesis, tumor progression, and malignant transformation of stomach and gallbladder cancers [63]. In a study by Han *et al.* CD24+CD44+ HNSCC cells were demonstrated to be more proliferative and invasive *in vitro* and more tumorigenic *in vivo.* After implantation in immunodeficient mice, CD24+CD44+ *cells* formed larger tumors than the CD24- CD44+ group. CD24+CD44+ cells were also correlated with slightly increased resistance to chemotherapeutic agents [62]. CD24 is one of the primary surface antigens involved in solid tumors and its role has been established in various human epithelial neoplasias. However, the paucity of research concerning HNSCC precludes its inclu- sion as a CSC biomarker at the present time.

CD10 is a zinc-dependent metalloendoprotease that cleaves signaling peptides and is found in a wide range of normal tissues. It has been described as a potential marker for therapeutic resistance and tumor recurrence in HNSCC [64]. As the field of CSCs remains in its infancy, further investigation regarding the roles of CD24 and CD10 will better elucidate the role of these proteins in HNSCC.

#### **8. Clinical relevance of head and neck cancer stem cells**

Today, few studies have evaluated patient HNSCC tumors or tissues and the correlation with clinical data and outcomes. One barrier to the establishment of clinically significant CSC markers in the head and neck region is secondary to the convention of amassing malignancies from various upper aerodigestive sites with distinctly diverse embryological and biological characteristics [47]. As a result, there is little definitive data with regard to clinical implications of CSCs within HNSCC, the primary exception being prognostic value. Furthermore, no single biomarker for CSC cells in HNSCC has proven absolute in distinguishing this vital subpopulation. The continued study of current prospective CSC markers in HNSCC, combined with the investigation of putative CSC biomarkers from other malignancies, will undoubtedly augment our knowledge and improve our understanding of the pathogenesis of HNSCC. In addition, further knowledge regarding the biomarkers and regulation of normal, native stem cells in the head and neck region will serve as a strong foundation for oncological research. Ultimately, CSCs may prove to be useful diagnostic and prognostic markers for HNSCC, guiding therapy and treatment through personalized approaches and interventions.

#### **Author details**

Kaveh Karimnejad# , Nathan Lindquist# , Reigh-Yi Lin\*

\*Address all correspondence to: rlin7@slu.edu

Department of Otolaryngology – Head and Neck Surgery, Saint Louis University School of Medicine, Saint Louis, Missouri, USA
