**3. Cancer testis (CT) genes**

CT genes were first defined as a group of tumor antigens that elicit a cytosolic T cell response and are expressed in male germ cells in the testis and various malignancies [63–65]. The first CT antigen identified was melanoma antigen 1 (MAGEA-1) [66]. Using the melanoma cell line MZ2-MEL and autologous cytotoxic T-lymphocyte (CTL) clones cytolytic to this line, MAGE-1 (subsequently re-named as MAGEA1, melanoma antigen A1) was identified as the target antigen for one of the CTL clones. This represented the first immunogenic tumor antigen shown to have elicited autologous cytotoxic T-lymphocyte responses in a cancer patient. Pursuing the same strategy, a range of other tumor-antigen genes, including MAGE-A3, another member of the MAGE-A family, as well as two additional families of antigens, namely the BAGE and GAGE gene families, were identified [64, 67–69]. The next huge step toward the identification of tumor antigens came from the screening of cDNA expression libraries with antibodies, the technology called SEREX (serological analysis of cDNA expression libraries) [70]. Very soon SEREX led to the identification of several categories of tumor antigens. To date, more than 80 families of CT genes are recognized and defined as germline restricted genes with evaluated expression in cancer [71]. As per today's definition, CT gene should simply exhibit a biased expression in the testis, ovaries [72], or the placenta [73], and in cancer.

CT genes can be divided between those that are encoded in the X chromosome (CT-X genes) and those that are distributed throughout the genome (non-X CT genes). CT-X genes are mostly members of gene families organized into complex direct and inverted repeats, and are expressed in testes primarily during the spermatogonial stage of spermatogenesis [74]. Annotation of the sequence of the human X chromosome has revealed that as many as 10% of all genes present on the chromosome are members of known CT families [75]. Further analysis of the expression patterns of genes of unknown function located in these repeated regions could even increase this estimate [76]. Melanoma has been found to have one of the highest CT antigens frequency expressions among other cancers. Moreover, higher frequency of CT antigens expression in melanoma is also correlated with worse disease outcome [77–80].

**95**

**Figure 1.**

*CT antigens constructed using String server [82].*

*The Role of the Meiotic Component in Reproduction of B-RAF-Mutated Melanoma…*

Our analyses of the NCBI's Gene Expression Omnibus [81] GSE98394 dataset including a cohort of 27 B-RAF-mutant nevi and 51 melanoma, described in details in [81] revealed the stark upregulation of many CT antigens in primary melanoma compared to nevi (Appendix Table 1). The densely connected component of protein-protein interactions (PPI) network of the upregulated melanoma CT antigens genes constructed using String Server [82] revealed the melanoma network module composed of 25 nodes, with a carcass of MAGEA-group hubs connected with the cohesin subunit SA-2 (STAG2) and the inhibitor of the differentiation-inducing retinoid acid receptor (PRAME) [83] hubs indicating to the acquired stemness (**Figure 1**). The high average node connectivity degree (5.84, PPI enrichment p-value <1.0e-16) characterizes this module as a CT antigen attractor of melanoma

Similar upregulation of many CTA, however, different from those, occurs when the primary melanoma progresses and metastasis are formed as revealed in the TCGA-SKCM dataset that includes 103 primary melanoma and 368 melanoma

The biological role of CT genes, particularly CT-X genes (a majority of them are CT antigens), in both germline tissues and tumors remains not well understood. However, studies have provided some evidence that MAGE gene expression may protect cells from programmed cell death and contribute to the development of malignancies by promoting survival [84]. It has also been shown that MAGE A2 is a strong inhibitor of the p53 tumor suppressor through histone deacetylase (HDAC)3 recruitment. In human primary melanoma cells, Mage A2 expression confers resistance to chemotherapeutic drugs by interfering with p53 acetylation [85]. Mage A2 interferes with p53 acetylation at promyelocytic leukemia (PML)-nuclear bodies (NBs) and with PMLIV-dependent activation of p53 through an HDAC-dependent mechanism, so downregulating it [86]. Usually, p53 is recruited to PML-NBs where it becomes acetylated and activated, and participates in the triggering of cellular senescence [87], a critical barrier against cell transformation (discussed above).

The mechanisms involved in the regulation of CT antigens expression appears to be promoted by DNA demethylation. Methylation of CpG islands within gene promoters is responsible for gene silencing due to both its effect on chromatin structure

*The densely connected component of protein-protein interactions (PPI) network of the upregulated melanoma* 

metastases (https://www.cancer.gov/tcga) (Appendix Table 2).

*DOI: http://dx.doi.org/10.5772/intechopen.93641*

genesis from B-RAF-mutant nevi.

#### *The Role of the Meiotic Component in Reproduction of B-RAF-Mutated Melanoma… DOI: http://dx.doi.org/10.5772/intechopen.93641*

Our analyses of the NCBI's Gene Expression Omnibus [81] GSE98394 dataset including a cohort of 27 B-RAF-mutant nevi and 51 melanoma, described in details in [81] revealed the stark upregulation of many CT antigens in primary melanoma compared to nevi (Appendix Table 1). The densely connected component of protein-protein interactions (PPI) network of the upregulated melanoma CT antigens genes constructed using String Server [82] revealed the melanoma network module composed of 25 nodes, with a carcass of MAGEA-group hubs connected with the cohesin subunit SA-2 (STAG2) and the inhibitor of the differentiation-inducing retinoid acid receptor (PRAME) [83] hubs indicating to the acquired stemness (**Figure 1**). The high average node connectivity degree (5.84, PPI enrichment p-value <1.0e-16) characterizes this module as a CT antigen attractor of melanoma genesis from B-RAF-mutant nevi.

Similar upregulation of many CTA, however, different from those, occurs when the primary melanoma progresses and metastasis are formed as revealed in the TCGA-SKCM dataset that includes 103 primary melanoma and 368 melanoma metastases (https://www.cancer.gov/tcga) (Appendix Table 2).

The biological role of CT genes, particularly CT-X genes (a majority of them are CT antigens), in both germline tissues and tumors remains not well understood. However, studies have provided some evidence that MAGE gene expression may protect cells from programmed cell death and contribute to the development of malignancies by promoting survival [84]. It has also been shown that MAGE A2 is a strong inhibitor of the p53 tumor suppressor through histone deacetylase (HDAC)3 recruitment. In human primary melanoma cells, Mage A2 expression confers resistance to chemotherapeutic drugs by interfering with p53 acetylation [85]. Mage A2 interferes with p53 acetylation at promyelocytic leukemia (PML)-nuclear bodies (NBs) and with PMLIV-dependent activation of p53 through an HDAC-dependent mechanism, so downregulating it [86]. Usually, p53 is recruited to PML-NBs where it becomes acetylated and activated, and participates in the triggering of cellular senescence [87], a critical barrier against cell transformation (discussed above).

The mechanisms involved in the regulation of CT antigens expression appears to be promoted by DNA demethylation. Methylation of CpG islands within gene promoters is responsible for gene silencing due to both its effect on chromatin structure

#### **Figure 1.**

*The densely connected component of protein-protein interactions (PPI) network of the upregulated melanoma CT antigens constructed using String server [82].*

*Melanoma*

The possibility of imposing the meiotic (oocyte maturation) program by overexpressed RAS and RAF onto somatic cells was reported in literature [29, 60, 61]. Such trigger can supposedly favor the reduction division of polyploidized tumor cells [31–33] and likely also, in collaboration with REC8, the monopolar spindle of meiotic prophase [62]. In irradiated lymphoma cell lines, MOS was activated through polyploidy only in TP53-mutants, not their WT TP53 counterparts [30], where neither polyploidy nor MOS was induced. MOS protein was shown expressed in 20 types of cancer, including melanoma (https://www.proteinatlas. org/ENSG00000172680-MOS/pathology). As shown by more recent data on OIS in melanoma [58], the persistence of DNA damage in the absence of p53 function may be a bridge to invasive melanoma. And the persistent DNA DSBs in senescing polyploid cells, in turn, may be also a bridge from the G2M DNA damage checkpoint and/or mitotic slippage to the meiotic-type recombinative prophase possessing the same molecular background [33] (see also below in the section about SPO11 nuclease). So, B-RAF and N-RAS mutation, senescence with DDR signaling, deficiency of p53 function (upregulation of MDM2), induced and reversible polyploidy, and trigger to meiotic prophase are all molecularly related and this

CT genes were first defined as a group of tumor antigens that elicit a cytosolic

antigen for one of the CTL clones. This represented the first immunogenic tumor antigen shown to have elicited autologous cytotoxic T-lymphocyte responses in a cancer patient. Pursuing the same strategy, a range of other tumor-antigen genes, including MAGE-A3, another member of the MAGE-A family, as well as two additional families of antigens, namely the BAGE and GAGE gene families, were identified [64, 67–69]. The next huge step toward the identification of tumor antigens came from the screening of cDNA expression libraries with antibodies, the technology called SEREX (serological analysis of cDNA expression libraries) [70]. Very soon SEREX led to the identification of several categories of tumor antigens. To date, more than 80 families of CT genes are recognized and defined as germline restricted genes with evaluated expression in cancer [71]. As per today's definition, CT gene should simply exhibit a biased expression in the

CT genes can be divided between those that are encoded in the X chromosome (CT-X genes) and those that are distributed throughout the genome (non-X CT genes). CT-X genes are mostly members of gene families organized into complex direct and inverted repeats, and are expressed in testes primarily during the spermatogonial stage of spermatogenesis [74]. Annotation of the sequence of the human X chromosome has revealed that as many as 10% of all genes present on the chromosome are members of known CT families [75]. Further analysis of the expression patterns of genes of unknown function located in these repeated regions could even increase this estimate [76]. Melanoma has been found to have one of the highest CT antigens frequency expressions among other cancers. Moreover, higher frequency of CT antigens expression in melanoma is also correlated with worse

T cell response and are expressed in male germ cells in the testis and various malignancies [63–65]. The first CT antigen identified was melanoma antigen 1 (MAGEA-1) [66]. Using the melanoma cell line MZ2-MEL and autologous cytotoxic T-lymphocyte (CTL) clones cytolytic to this line, MAGE-1 (subsequently re-named as MAGEA1, melanoma antigen A1) was identified as the target

network can be potentially involved in melanoma genesis.

testis, ovaries [72], or the placenta [73], and in cancer.

**3. Cancer testis (CT) genes**

**94**

disease outcome [77–80].

and binding of transcription factors [88]. "Epigenetic reprogramming," consisting of concerted DNA pan-demethylation and corresponding chromatin remodeling, occurs twice in the human life cycle: during early embryogenesis and gametogenesis of primordial germ cells (PGC) [89]. So far, all CT antigens studied have methylated CpG islands in normal somatic tissues and are activated by demethylation during spermatogenesis [90]. Experimental demethylation of CT antigens promoters induces antigen expression in cells that do not normally produce them [91]. It has been proposed that the activation of CT antigens in cancer is a consequence of the ectopic induction of gametogenic program [74, 92, 93], which thus includes the meiotic component.

As recently found, all MAGEs contain a conservative E-ring domain and assemble with E3 RING ubiquitin ligases to form MAGE-RING ligases (MRLs) that act as regulators of ubiquitination by modulating E-ring-ligase activity [94]. The latter are acting at the cross-roads between tumor suppression and oncogenesis [95]. In addition, a majority of the CT antigens [96, 97] are intrinsically disordered proteins (IDPs). IDPs lack rigid 3D structures either along their entire length or in localized regions. Despite the lack of structure, most IDPs can transit from disorder to order upon binding to various biological targets [98]. Protein intrinsic disorder can serve as the structural basis for hub protein promiscuity; thus, CT antigens proteins can provide flexible linkers between functional domains [99]. Many normal cellular processes are associated with the presence of the right amount of precisely activated IDPs at right places and at the right time, those may be altered in disease, including cancer [100, 101]. The IDPs—features of the X-linked CT antigen-encoded genes, which can change their targets, as well as the relation of the MAGE group to ubiquitin-ligases suggest their highly adaptive post-translation functions for the cancer genome and proteome networks. This property is consistent with their activation by CTCF inhibitor and pan-genome activator, the CT gene Brother of Regulator of Imprinted Sites (BORIS) located at the chromosome region 20q13.2. This region is commonly amplified in human cancers [102, 103]. BORIS expression is normally restricted to testis and becomes aberrantly expressed in different types of cancer [104]. In melanoma, BORIS expression was observed in 59% of melanoma cell lines, in 16% of primary melanomas and in 34% of melanoma metastases [105].

Normally, BORIS plays a major role in regulating de-repressing, de-methylation processes during spermatogenesis—it removes imprinting from genes during the last mitotic division of type B spermatogonia producing the first spermatocyte [106]. In particular, in melanoma, BORIS binds near the promoter of transforming growth factor-beta 1 (TFGB1), a well-recognized factor involved in the transition toward an invasive state, activating it through transcriptional reprogramming [107]. BORIS is a paralog and antagonist of CTCF. A primary role for CTCF in the global organization of chromatin architecture was shown, which suggests that CTCF may be a heritable topological repressive component of an epigenetic system regulating the interplay between DNA methylation, higher-order chromatin structure, and lineage-specific gene expression [108, 109]. Nowadays, multiple studies have indicated an oncogenic role for BORIS [110–112]. Notably, emerging evidence has shown that BORIS functions as an epigenetic modifier in modulating the whole genome gene expression [113–115], including expression of other CT genes [116, 117]. BORIS was also found to be expressed in embryonal carcinoma, ovarian cancer [118] as well as cancer stem cell (CSC)-enriched populations isolated from epithelial cancer cells [119, 120]. The mRNA isoforms of BORIS genes are expressed in normal ovary and in the altered pattern, in epithelial ovarian cancer [121]. An association of BORIS expression with CSC-like properties was also observed [119, 120]. Moreover, it has been shown that BORIS association with the CSC-like traits occurs through the epigenetic regulation of *POU5F1/OCT4* [112]. OCT4 is

**97**

**Figure 2.**

*used to construct the heat map.*

*The Role of the Meiotic Component in Reproduction of B-RAF-Mutated Melanoma…*

considered a master regulator in the maintenance of stem cell pluripotency. Many studies have demonstrated a correlation between OCT4 and CSCs in many cancers,

In relation to metastatic melanoma, using the TCGA database (https://www. cancer.gov/tcga), we assessed the expression of a number of genes selected from the POU, SOX, SALL, and NANOG gene families with relation to stemness in normal and cancer stem cells [125] and noted an increase in stemness during transition from primary melanoma to metastases. Moreover, the heat map shows the reconstruction of the landscape in the expression of stemness-associated genes indicating

Melanocytes originate from the neural crest developing in embryo very early (as the fourth germ layer) and is associated with intensive cell migration. Melanomas in patients or cell constructs upregulating the Wnt pathway, associated with neural crest development, display epithelial-to-mesenchyme-transition (EMT) phenotype, worse prognoses in patients, and resistance to drugs in vitro [129]. The role of the neural crest development factors in ectopic regulation of melanoma was also investigated in [130]. Likely, because of the origin, nearly the root of the ontogenetic

*Gene expression (in log2CPM values) of stemness genes in the cohort of 368 melanoma metastases compared to 103 primary melanoma from the TCGA-SKCM dataset (https://www.cancer.gov/tcga). The data was extracted from the TCGA database using the TCGA Biolinks Bioconductor package [126]. EdgeR [127] was used to perform differential expression analysis through the generalized linear model approach. The differentially expressed genes (DEGs) which were upregulated in metastatic melanoma (log2FC > 0, p < 0.01) were filtered for genes from the POU, SOX, SALL, and NANOG gene families with relation to stemness. Seaborn [128] was* 

*DOI: http://dx.doi.org/10.5772/intechopen.93641*

to the whole genome rearrangement (**Figure 2**).

tree, melanoma is so invasive and malignant.

including melanoma [122–124].

*The Role of the Meiotic Component in Reproduction of B-RAF-Mutated Melanoma… DOI: http://dx.doi.org/10.5772/intechopen.93641*

considered a master regulator in the maintenance of stem cell pluripotency. Many studies have demonstrated a correlation between OCT4 and CSCs in many cancers, including melanoma [122–124].

In relation to metastatic melanoma, using the TCGA database (https://www. cancer.gov/tcga), we assessed the expression of a number of genes selected from the POU, SOX, SALL, and NANOG gene families with relation to stemness in normal and cancer stem cells [125] and noted an increase in stemness during transition from primary melanoma to metastases. Moreover, the heat map shows the reconstruction of the landscape in the expression of stemness-associated genes indicating to the whole genome rearrangement (**Figure 2**).

Melanocytes originate from the neural crest developing in embryo very early (as the fourth germ layer) and is associated with intensive cell migration. Melanomas in patients or cell constructs upregulating the Wnt pathway, associated with neural crest development, display epithelial-to-mesenchyme-transition (EMT) phenotype, worse prognoses in patients, and resistance to drugs in vitro [129]. The role of the neural crest development factors in ectopic regulation of melanoma was also investigated in [130]. Likely, because of the origin, nearly the root of the ontogenetic tree, melanoma is so invasive and malignant.

#### **Figure 2.**

*Melanoma*

meiotic component.

and binding of transcription factors [88]. "Epigenetic reprogramming," consisting of concerted DNA pan-demethylation and corresponding chromatin remodeling, occurs twice in the human life cycle: during early embryogenesis and gametogenesis of primordial germ cells (PGC) [89]. So far, all CT antigens studied have methylated CpG islands in normal somatic tissues and are activated by demethylation during spermatogenesis [90]. Experimental demethylation of CT antigens promoters induces antigen expression in cells that do not normally produce them [91]. It has been proposed that the activation of CT antigens in cancer is a consequence of the ectopic induction of gametogenic program [74, 92, 93], which thus includes the

As recently found, all MAGEs contain a conservative E-ring domain and assemble with E3 RING ubiquitin ligases to form MAGE-RING ligases (MRLs) that act as regulators of ubiquitination by modulating E-ring-ligase activity [94]. The latter are acting at the cross-roads between tumor suppression and oncogenesis [95]. In addition, a majority of the CT antigens [96, 97] are intrinsically disordered proteins (IDPs). IDPs lack rigid 3D structures either along their entire length or in localized regions. Despite the lack of structure, most IDPs can transit from disorder to order upon binding to various biological targets [98]. Protein intrinsic disorder can serve as the structural basis for hub protein promiscuity; thus, CT antigens proteins can provide flexible linkers between functional domains [99]. Many normal cellular processes are associated with the presence of the right amount of precisely activated IDPs at right places and at the right time, those may be altered in disease, including cancer [100, 101]. The IDPs—features of the X-linked CT antigen-encoded genes, which can change their targets, as well as the relation of the MAGE group to ubiquitin-ligases suggest their highly adaptive post-translation functions for the cancer genome and proteome networks. This property is consistent with their activation by CTCF inhibitor and pan-genome activator, the CT gene Brother of Regulator of Imprinted Sites (BORIS) located at the chromosome region 20q13.2. This region is commonly amplified in human cancers [102, 103]. BORIS expression is normally restricted to testis and becomes aberrantly expressed in different types of cancer [104]. In melanoma, BORIS expression was observed in 59% of melanoma cell lines,

in 16% of primary melanomas and in 34% of melanoma metastases [105].

Normally, BORIS plays a major role in regulating de-repressing, de-methylation processes during spermatogenesis—it removes imprinting from genes during the last mitotic division of type B spermatogonia producing the first spermatocyte [106]. In particular, in melanoma, BORIS binds near the promoter of transforming growth factor-beta 1 (TFGB1), a well-recognized factor involved in the transition toward an invasive state, activating it through transcriptional reprogramming [107]. BORIS is a paralog and antagonist of CTCF. A primary role for CTCF in the global organization of chromatin architecture was shown, which suggests that CTCF may be a heritable topological repressive component of an epigenetic system regulating the interplay between DNA methylation, higher-order chromatin structure, and lineage-specific gene expression [108, 109]. Nowadays, multiple studies have indicated an oncogenic role for BORIS [110–112]. Notably, emerging evidence has shown that BORIS functions as an epigenetic modifier in modulating the whole genome gene expression [113–115], including expression of other CT genes [116, 117]. BORIS was also found to be expressed in embryonal carcinoma, ovarian cancer [118] as well as cancer stem cell (CSC)-enriched populations isolated from epithelial cancer cells [119, 120]. The mRNA isoforms of BORIS genes are expressed in normal ovary and in the altered pattern, in epithelial ovarian cancer [121]. An association of BORIS expression with CSC-like properties was also observed [119, 120]. Moreover, it has been shown that BORIS association with the CSC-like traits occurs through the epigenetic regulation of *POU5F1/OCT4* [112]. OCT4 is

**96**

*Gene expression (in log2CPM values) of stemness genes in the cohort of 368 melanoma metastases compared to 103 primary melanoma from the TCGA-SKCM dataset (https://www.cancer.gov/tcga). The data was extracted from the TCGA database using the TCGA Biolinks Bioconductor package [126]. EdgeR [127] was used to perform differential expression analysis through the generalized linear model approach. The differentially expressed genes (DEGs) which were upregulated in metastatic melanoma (log2FC > 0, p < 0.01) were filtered for genes from the POU, SOX, SALL, and NANOG gene families with relation to stemness. Seaborn [128] was used to construct the heat map.*

The particular interest for carcinogenesis represents the non-X CT genes or germline restricted genes that normally mediate meiotic program [30, 34–37, 131] and therefore are denoted by some authors, the meiosis-specific CT (meiCT) genes [36].
