**4. Conventional meiosis: in brief**

The conventional meiotic progression is well described [28] and has been recently updated by Feichtinger and McFarlane [35]. Thus, only a short recitation of some of the main points is provided here.

Meiosis is a special mode of cell division that naturally occurs in mammalian only in the germ cells—in the male testis and female ovary. During meiosis, diploid germ cells undergo a single round of premeiotic DNA replication (4n), followed by two chromosome segregation events, meiosis I (reductional) and meiosis II (equational), creating haploid (1n) gametes. Meiosis I is marked by a prolonged prophase that is subdivided into five stages: leptotene, zygotene, pachytene, diplotene, and diakinesis, where during the first three stages, there occurs the formation of DSBs, homologous chromosome pairing, and synapsis and reciprocal homologous recombination (HR) between them. The initiation of meiosis is not fully understood in mammals, but it is thought that meiotic entry is initiated by upregulation of the stimulated by retinoic acid 8 (STRA8) gene expression—transcription activator that binds directly to the promoter regions of meiosis-specific genes [124–126].

During premeiotic DNA replication, a ring of specific cohesins is formed that holds newly formed sister chromatids together [127]. In meiosis I prophase, HR program is initiated by the generation of DNA DSBs along the chromosome axis in specific hotspots [128]. This is initiated by a protein complex, which consists of SPO11 and TOPOVIBL [129]. Generated DSBs serve as the substrates for the recombinase RAD51 and its meiosis-specific paralogue DMC1 acting as a heterodimer [130]. The hot spot selection in mammals mediates the zinc finger histone methyltransferase, PR domain containing 9 (PRDM9), which primes the DNA for DSB and exchange of DNA between chromosomes [131, 132]. Of note, in the case of meiosis, DNA DSBs are obligatory rather than the result of accidental damage, as in the mitotic cell cycle, and the recombination partners are homologous chromosomes in meiosis, whereas they are sister chromatids in DNA repair during mitosis. As the homologous chromosome bivalents after HR align on the metaphase I plate, the centromeres of sister chromatids form monopolar spindle associations. Loss of sister cohesion in the arm regions of chromosomes, but not the centromeric regions, occurs on entry into meiotic anaphase I permitting reductional segregation of homologous chromosomes. During meiosis II, centromeric cohesion is broken down and an equational segregation of the chromatids, like in mitosis, occurs [127].

#### **5. Melanoma and meiosis specific CT (meiCT) genes**

HR sites resulting in crossovers are initiated by the creation of DSBs in the leptotene prophase stage catalyzed by the protein Spo11 [132]. Spo11 is an homolog of the A subunit of type II DNA topoisomerase that together with TOPOVIBL, an homolog of B subunit, forms protein complex. The MREII exonuclease creates DNA nicks guiding the SPO11-TOPOVIBL complex to accurately catalyze DSBs along the genome in specific hotspots [133, 134]. Aberrant expression of SPO11 has been found in cell lines of melanoma and also lung cancer [135], see **Figure 3**, acute myeloid leukemia (AML) [136], cutaneous T-cell lymphoma (CTCL) [137] as well

**99**

*Bars = 10* μ*m.*

**Figure 3.**

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

as in patient samples of melanoma, [135, 138], cervical cancer [135], gastric cancer [138], and CTCL [139]. Although the exact mechanism of SPO11 reactivation in cancer cells remains elusive, it has been shown that in CTCL, it is regulated epigenetically and temporary expressed at the onset of the cell division in G1/S phase transition [139]. This expression before DNA replication seems unrelevant but, indeed, it appears that SPO11 expression in B-RAF- and TP-53 mutant melanoma

SPO11 expression in CTCL cell lines decreased after cell line treatment with histone deacetylase (HDAC) inhibitors, e.g., Vorinostat and Romidepsin [137]. Moreover, SPO11-introduced DNA DSBs have also been shown to increase the risk of genome rearrangements and mutations in the germline [140]—a potential source of the idiopathic male infertility, which is associated with the 20-fold increased risk of the germline cancer [141]. Spo11 appears to be present in all sequenced eukaryotic genomes, and indeed it may be the only truly universal meiotic protein. At the same time, in many organisms, the recombination defect in Spo11 mutants can rescue meiosis by production of DSBs from an exogenous source such as ionizing radiation [142, 143]. On the other side, SPO11 was also found in species and tissues undergoing asexual life-cycles [143] or DNA recombination for nonsexual function. e.g*.*, SPO11 was revealed in mouse germinal center B cells undergoing

*Meiotic genes, alternative telomere lengthening, and mitotic slippage in B-RAF V600E and TP53-mutant melanoma SkMel28 cell line: (A) the expression of the meiotic MOS-kinase (sc-28,789) and recombination endonuclease SPO11 (sc-377,161) in cell nuclei of non-treated cells; (B) co-expression of MOS and cyclin B1 (sc-245) in rare polyploid cells and some metaphases [14] of nontreated control; (C) the polyploid cell on day 7 after doxorubicine treatment (500 nM for 24 h) maintains telomeres (marked by TRF2, 05-521, millipore) by alternative lengthening of telomeres (ALT) in promyelocytic leukemia (PML) (PA5-80910, thermo fisher scientific) bodies; and (D) two giant cells resistant to B-RAF inhibitor vemurafenib (50 nM for 24 h), with signs of mitotic slippage and multinucleation on day 21 after treatment show positivity for SPO11.* 

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

may be not dependent on the cell cycle phase (**Figure 3**).

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

as in patient samples of melanoma, [135, 138], cervical cancer [135], gastric cancer [138], and CTCL [139]. Although the exact mechanism of SPO11 reactivation in cancer cells remains elusive, it has been shown that in CTCL, it is regulated epigenetically and temporary expressed at the onset of the cell division in G1/S phase transition [139]. This expression before DNA replication seems unrelevant but, indeed, it appears that SPO11 expression in B-RAF- and TP-53 mutant melanoma may be not dependent on the cell cycle phase (**Figure 3**).

SPO11 expression in CTCL cell lines decreased after cell line treatment with histone deacetylase (HDAC) inhibitors, e.g., Vorinostat and Romidepsin [137]. Moreover, SPO11-introduced DNA DSBs have also been shown to increase the risk of genome rearrangements and mutations in the germline [140]—a potential source of the idiopathic male infertility, which is associated with the 20-fold increased risk of the germline cancer [141]. Spo11 appears to be present in all sequenced eukaryotic genomes, and indeed it may be the only truly universal meiotic protein. At the same time, in many organisms, the recombination defect in Spo11 mutants can rescue meiosis by production of DSBs from an exogenous source such as ionizing radiation [142, 143]. On the other side, SPO11 was also found in species and tissues undergoing asexual life-cycles [143] or DNA recombination for nonsexual function. e.g*.*, SPO11 was revealed in mouse germinal center B cells undergoing

#### **Figure 3.**

*Melanoma*

**4. Conventional meiosis: in brief**

some of the main points is provided here.

meiosis-specific genes [124–126].

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].

The conventional meiotic progression is well described [28] and has been recently updated by Feichtinger and McFarlane [35]. Thus, only a short recitation of

only in the germ cells—in the male testis and female ovary. During meiosis, diploid germ cells undergo a single round of premeiotic DNA replication (4n), followed by two chromosome segregation events, meiosis I (reductional) and meiosis II (equational), creating haploid (1n) gametes. Meiosis I is marked by a prolonged prophase that is subdivided into five stages: leptotene, zygotene, pachytene, diplotene, and diakinesis, where during the first three stages, there occurs the formation of DSBs, homologous chromosome pairing, and synapsis and reciprocal homologous recombination (HR) between them. The initiation of meiosis is not fully understood in mammals, but it is thought that meiotic entry is initiated by upregulation of the stimulated by retinoic acid 8 (STRA8) gene expression—transcription activator that binds directly to the promoter regions of

Meiosis is a special mode of cell division that naturally occurs in mammalian

During premeiotic DNA replication, a ring of specific cohesins is formed that holds newly formed sister chromatids together [127]. In meiosis I prophase, HR program is initiated by the generation of DNA DSBs along the chromosome axis in specific hotspots [128]. This is initiated by a protein complex, which consists of SPO11 and TOPOVIBL [129]. Generated DSBs serve as the substrates for the recombinase RAD51 and its meiosis-specific paralogue DMC1 acting as a heterodimer [130]. The hot spot selection in mammals mediates the zinc finger histone methyltransferase, PR domain containing 9 (PRDM9), which primes the DNA for DSB and exchange of DNA between chromosomes [131, 132]. Of note, in the case of meiosis, DNA DSBs are obligatory rather than the result of accidental damage, as in the mitotic cell cycle, and the recombination partners are homologous chromosomes in meiosis, whereas they are sister chromatids in DNA repair during mitosis. As the homologous chromosome bivalents after HR align on the metaphase I plate, the centromeres of sister chromatids form monopolar spindle associations. Loss of sister cohesion in the arm regions of chromosomes, but not the centromeric regions, occurs on entry into meiotic anaphase I permitting reductional segregation of homologous chromosomes. During meiosis II, centromeric cohesion is broken down and an equational segregation of the chromatids, like in mitosis, occurs [127].

**5. Melanoma and meiosis specific CT (meiCT) genes**

HR sites resulting in crossovers are initiated by the creation of DSBs in the leptotene prophase stage catalyzed by the protein Spo11 [132]. Spo11 is an homolog of the A subunit of type II DNA topoisomerase that together with TOPOVIBL, an homolog of B subunit, forms protein complex. The MREII exonuclease creates DNA nicks guiding the SPO11-TOPOVIBL complex to accurately catalyze DSBs along the genome in specific hotspots [133, 134]. Aberrant expression of SPO11 has been found in cell lines of melanoma and also lung cancer [135], see **Figure 3**, acute myeloid leukemia (AML) [136], cutaneous T-cell lymphoma (CTCL) [137] as well

**98**

*Meiotic genes, alternative telomere lengthening, and mitotic slippage in B-RAF V600E and TP53-mutant melanoma SkMel28 cell line: (A) the expression of the meiotic MOS-kinase (sc-28,789) and recombination endonuclease SPO11 (sc-377,161) in cell nuclei of non-treated cells; (B) co-expression of MOS and cyclin B1 (sc-245) in rare polyploid cells and some metaphases [14] of nontreated control; (C) the polyploid cell on day 7 after doxorubicine treatment (500 nM for 24 h) maintains telomeres (marked by TRF2, 05-521, millipore) by alternative lengthening of telomeres (ALT) in promyelocytic leukemia (PML) (PA5-80910, thermo fisher scientific) bodies; and (D) two giant cells resistant to B-RAF inhibitor vemurafenib (50 nM for 24 h), with signs of mitotic slippage and multinucleation on day 21 after treatment show positivity for SPO11. Bars = 10* μ*m.*

immunoglobulin gene diversification and class switch recombination, but mice lacking Spo11 had no detectable immune system defects [144]. SPO11 introduces meiotic recombination breaks in the chromosome DSB hotspots [145]. So, it is possible that senescence-associated DDR affecting the DSB hot spots (at least, in p53-nonfunctional tumors) can upregulate and attract SPO11. Localized clustered hotspots are a feature of meiotic recombination in *S. pombe*, mouse, and humans as well, but the factors that determine whether a given DNA sequence will be a DSB hotspot are not well understood in any organism. Such hotspots may appear due to underreplication of DNA in the heterochromatin, particularly in telomeres, e.g., in the drug-induced senescence of tumor cells [146]. Depletion of the H3K9me3 chromatin repressive hallmarks seems rather decisive for attraction of SPO11 to the hot spots [147]. This data shows that execution of the very definitive molecular biochemical mechanism of SPO11 is dependent on the permissive epigenetic chromatin organization of the very general character. Therefore, it is interesting to highlight the breaking through report showing the reset of senescence and abrogation of invasive growth achieved in melanoma by inhibition of the DNA demethylases [26].

Spo11 is the catalytic center of the meiotic recombination initiation mechanism, but it is not sufficient to generate DSBs: numerous additional proteins are also required; the main of them is Mre11-Rad50-Xrs2 (MRX). These proteins form a complex with multiple roles in many different aspects of DNA metabolism, including DNA repair, telomere maintenance, and checkpoint signaling. Mutant MRX complex leaves SPO11 accumulated to telomere ends with the nonreleased terminal chiasmata [148]. Although the SPO11 catalytic gene part is conserved, the proteins involved in meiotic recombination are generally among the more rapidly evolving of all cellular proteins: major challenges for them represent the whole genome duplications (WGDs) and the difficulties of auto- and allo-polyploids in the meiotic reduction divisions [149, 150].

The meiosis-specific histone methyltransferase gene PRDM9 has also been reported to be activated in melanoma alongside with other cancers, like embryonal carcinoma, astrocytoma, leukemia, colon, prostate, breast, and ovary cancers [151].

Another meiosis-specific gene involved in SPO11-mediated recombination regulation, TEX15, has been reported to be overexpressed in melanoma and other cancers including bladder, head and neck, and lung carcinomas, neuroblastomas, prostate tumors, and sarcomas [152].

The synapsis of homologous chromosomes in conventional meiotic prophase is marked by synaptonemal complex (SC). SC is a large zipper-like protein complex that connects one pair of sister chromatids to the homologous pair, so stabilizing the tetrad and ensuring proper homolog pairing. SC formation starts with the formation of axial element that consists from SC proteins 2 and 3 (SCP2 and SCP3). Then, the axial elements (at this point referred lateral elements) are joined by the transverse filaments formed by the SC protein 1 (SCP1) [153–155]. The central elements consists of SC central element 1 and 2 (encoded by SYCE1 and SYCE2) [156]. Notably, SYCP1 and SYCP3 genes both have been implicated in cancer. Both mRNA are expressed in a variety of cancers and cancer cell lines including melanoma [30, 31, 157, 158]. Moreover, SCP3 protein expression correlated with activated AKT (pAKT) signaling [159]. Overexpression of SCP3 was shown prognostically unfavorable for lung cancer [160].

HORMA domain containing 1 (Hormad1) is another protein associated with SC axis. It has multiple roles, but in general it coordinates DSB formation with synapsis and the timely progression of DSB repair through HR [161]. Hormad1 is significantly upregulated in several cancers and noted also in melanoma [37, 162]. Although the mechanism of its reactivation remains elusive, hypomethylation of the HORMAD1 promoter region correlates with its increased expression in breast cancer and small cell lung cancer [163, 164], suggesting at least partial involvement of epigenetic pathways.

**101**

natural aging [175].

**6. Brainstorming session**

*1973 [181].*

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

Chromosome regulation in meiosis and in mitosis is dependent upon the cohesin complex. In mitotically dividing cells, this complex serves to hold sister chromatids until they settle in metaphase plate, becoming separated in anaphase while in conventional meiosis, sisters stay together through the whole meiosis I to ensure sister centromeres orientate to the same pole to drive the reductional segregation of bi-chromatid homologs. Although the structure of cohesin complexes involved in mitosis and meiosis is similar, the difference lies in subunit composition. In meiosis, specific paralogues of some of the cohesin proteins replace their mitotic counterparts [165]. One of the more prominent cohesin subunits that appears to be restricted to meiosis is REC8 (paralogous counterparts to the RAD21 mitotic cohesin) [165]. The upregulated expression of Rec8 protein was demonstrated in melanoma [37, 166] as well as in CTCL [139, 167], irradiated TP53-mutant lymphoma cell lines, HeLa, and breast and colon cancer cell lines [31, 168]. Recently it has been shown that REC8 imposed monopolarity of sister centromeres in mitotically dividing cells could result in uniparent disomy (UPD) at least in the model organism *S. pombe* (fission yeast) [169] possessing a facultative sex. REC8 in cooperation with *Mos*-kinase forms a monopolar spindle of octoploid lymphoma cells (after ionizing irradiation) which undergo recombination of DNA DSBs by meiotic recombinase DMC1 [62]. Interestingly, Rec8 does not appear to be incorporated into mitotic cohesin complex in HEK293 cells unless another meiosisspecific cohesin subunit, STAG3, is activated [170]. In melanoma, STAG3 as well as STAG2 (mitosis specific cohesin subunit) levels have been linked to the resistance of B-RAF inhibitors [171]. STAG cohesins also participate with CTCF in the topological suppression of transcription and it is the the reduced level of STAG3 that is

The cohesin-related regulators, SGO1/2 are also the meiosis-specific proteins that protect cohesin complex, in particular Rec8, from the protease separase-mediated cleavage at the centromeres of sister chromatids in meiosis I and retained Rec8 around the centromere until the start of anaphase II [172–174]. Upregulation of SGO2 expression has been demonstrated in melanoma [37] alongside with upregulation also in CTCL [139, 167] and SGO 1/2, along with REC8, in irradiated lymphoma cells [168]. However, the role of meiotic cohesins in cancer has not been extensively investigated. Another meiosis-specific cohesin subunit, which has gene expression tightly restricted to the testis in healthy humans, is RAD21L (also RAD21/REC8 paralogue) [165]. However, it is also important for the maintenance of female fertility during

While the majority of somatic cells are deficient in active telomerase, cancer cells not only can reactivate telomerase, but can also initiate a mechanism of the alternative telomere maintenance (ALT) in the absence of telomerase activity [176] or undergo transient ALT [177]. Some meiosis genes were found associated with supposed homology search in ALT [178, 179]. ALT requires a recombination-like mechanism to recognize the telomere end as DSBs and mediate the strand invasion of the end into a nonhomologous chromosome end. This strand invasion permits the initiation of a break-induced DNA replication process where the invaded non-homolog telomeric DNA serves as a replicative template for the invading telomere to elongate [180]. In summary, the review of the classic meiotic genes demonstrates their involvement in cancer, and melanoma in particular, although their function in cancer is ill defined.

*"Nothing in biology makes sense except in the light of evolution"—Dobzhansky* 

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

associated with resistance to B-RAF inhibitors.

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

Chromosome regulation in meiosis and in mitosis is dependent upon the cohesin complex. In mitotically dividing cells, this complex serves to hold sister chromatids until they settle in metaphase plate, becoming separated in anaphase while in conventional meiosis, sisters stay together through the whole meiosis I to ensure sister centromeres orientate to the same pole to drive the reductional segregation of bi-chromatid homologs. Although the structure of cohesin complexes involved in mitosis and meiosis is similar, the difference lies in subunit composition. In meiosis, specific paralogues of some of the cohesin proteins replace their mitotic counterparts [165]. One of the more prominent cohesin subunits that appears to be restricted to meiosis is REC8 (paralogous counterparts to the RAD21 mitotic cohesin) [165]. The upregulated expression of Rec8 protein was demonstrated in melanoma [37, 166] as well as in CTCL [139, 167], irradiated TP53-mutant lymphoma cell lines, HeLa, and breast and colon cancer cell lines [31, 168]. Recently it has been shown that REC8 imposed monopolarity of sister centromeres in mitotically dividing cells could result in uniparent disomy (UPD) at least in the model organism *S. pombe* (fission yeast) [169] possessing a facultative sex. REC8 in cooperation with *Mos*-kinase forms a monopolar spindle of octoploid lymphoma cells (after ionizing irradiation) which undergo recombination of DNA DSBs by meiotic recombinase DMC1 [62]. Interestingly, Rec8 does not appear to be incorporated into mitotic cohesin complex in HEK293 cells unless another meiosisspecific cohesin subunit, STAG3, is activated [170]. In melanoma, STAG3 as well as STAG2 (mitosis specific cohesin subunit) levels have been linked to the resistance of B-RAF inhibitors [171]. STAG cohesins also participate with CTCF in the topological suppression of transcription and it is the the reduced level of STAG3 that is associated with resistance to B-RAF inhibitors.

The cohesin-related regulators, SGO1/2 are also the meiosis-specific proteins that protect cohesin complex, in particular Rec8, from the protease separase-mediated cleavage at the centromeres of sister chromatids in meiosis I and retained Rec8 around the centromere until the start of anaphase II [172–174]. Upregulation of SGO2 expression has been demonstrated in melanoma [37] alongside with upregulation also in CTCL [139, 167] and SGO 1/2, along with REC8, in irradiated lymphoma cells [168]. However, the role of meiotic cohesins in cancer has not been extensively investigated.

Another meiosis-specific cohesin subunit, which has gene expression tightly restricted to the testis in healthy humans, is RAD21L (also RAD21/REC8 paralogue) [165]. However, it is also important for the maintenance of female fertility during natural aging [175].

While the majority of somatic cells are deficient in active telomerase, cancer cells not only can reactivate telomerase, but can also initiate a mechanism of the alternative telomere maintenance (ALT) in the absence of telomerase activity [176] or undergo transient ALT [177]. Some meiosis genes were found associated with supposed homology search in ALT [178, 179]. ALT requires a recombination-like mechanism to recognize the telomere end as DSBs and mediate the strand invasion of the end into a nonhomologous chromosome end. This strand invasion permits the initiation of a break-induced DNA replication process where the invaded non-homolog telomeric DNA serves as a replicative template for the invading telomere to elongate [180]. In summary, the review of the classic meiotic genes demonstrates their involvement in cancer, and melanoma in particular, although their function in cancer is ill defined.

### **6. Brainstorming session**

*"Nothing in biology makes sense except in the light of evolution"—Dobzhansky 1973 [181].*

*Melanoma*

immunoglobulin gene diversification and class switch recombination, but mice lacking Spo11 had no detectable immune system defects [144]. SPO11 introduces meiotic recombination breaks in the chromosome DSB hotspots [145]. So, it is possible that senescence-associated DDR affecting the DSB hot spots (at least, in p53-nonfunctional tumors) can upregulate and attract SPO11. Localized clustered hotspots are a feature of meiotic recombination in *S. pombe*, mouse, and humans as well, but the factors that determine whether a given DNA sequence will be a DSB hotspot are not well understood in any organism. Such hotspots may appear due to underreplication of DNA in the heterochromatin, particularly in telomeres, e.g., in the drug-induced senescence of tumor cells [146]. Depletion of the H3K9me3 chromatin repressive hallmarks seems rather decisive for attraction of SPO11 to the hot spots [147]. This data shows that execution of the very definitive molecular biochemical mechanism of SPO11 is dependent on the permissive epigenetic chromatin organization of the very general character. Therefore, it is interesting to highlight the breaking through report showing the reset of senescence and abrogation of invasive growth achieved in melanoma by inhibition of the DNA demethylases [26]. Spo11 is the catalytic center of the meiotic recombination initiation mechanism,

but it is not sufficient to generate DSBs: numerous additional proteins are also required; the main of them is Mre11-Rad50-Xrs2 (MRX). These proteins form a complex with multiple roles in many different aspects of DNA metabolism, including DNA repair, telomere maintenance, and checkpoint signaling. Mutant MRX complex leaves SPO11 accumulated to telomere ends with the nonreleased terminal chiasmata [148]. Although the SPO11 catalytic gene part is conserved, the proteins involved in meiotic recombination are generally among the more rapidly evolving of all cellular proteins: major challenges for them represent the whole genome duplications (WGDs) and the difficulties of auto- and allo-polyploids in the meiotic reduction divisions [149, 150]. The meiosis-specific histone methyltransferase gene PRDM9 has also been reported to be activated in melanoma alongside with other cancers, like embryonal carcinoma, astrocytoma, leukemia, colon, prostate, breast, and ovary cancers [151]. Another meiosis-specific gene involved in SPO11-mediated recombination regulation, TEX15, has been reported to be overexpressed in melanoma and other cancers including bladder, head and neck, and lung carcinomas, neuroblastomas,

The synapsis of homologous chromosomes in conventional meiotic prophase is marked by synaptonemal complex (SC). SC is a large zipper-like protein complex that connects one pair of sister chromatids to the homologous pair, so stabilizing the tetrad and ensuring proper homolog pairing. SC formation starts with the formation of axial element that consists from SC proteins 2 and 3 (SCP2 and SCP3). Then, the axial elements (at this point referred lateral elements) are joined by the transverse filaments formed by the SC protein 1 (SCP1) [153–155]. The central elements consists of SC central element 1 and 2 (encoded by SYCE1 and SYCE2) [156]. Notably, SYCP1 and SYCP3 genes both have been implicated in cancer. Both mRNA are expressed in a variety of cancers and cancer cell lines including melanoma [30, 31, 157, 158]. Moreover, SCP3 protein expression correlated with activated AKT (pAKT) signaling [159]. Overexpression of SCP3 was shown prognostically unfavorable for lung cancer [160]. HORMA domain containing 1 (Hormad1) is another protein associated with SC axis. It has multiple roles, but in general it coordinates DSB formation with synapsis and the timely progression of DSB repair through HR [161]. Hormad1 is significantly upregulated in several cancers and noted also in melanoma [37, 162]. Although the mechanism of its reactivation remains elusive, hypomethylation of the HORMAD1 promoter region correlates with its increased expression in breast cancer and small cell lung cancer [163, 164], suggesting at least partial involvement

prostate tumors, and sarcomas [152].

**100**

of epigenetic pathways.

B-RAF-mutant melanoma activates MEK-ERK proliferative pathway but cancer can be explained neither only by enhanced proliferation nor it can be reduced to somatic mutation theory, which has been shaken by cancer genome sequencing projects. Cancer is more complex than that [182]. B-RAF-and N-RAS-mutant nevi remaining quiescent and benign just support this notion. A very important role of OIS-induced cellular senescence for initiation of malignant tumors discovered by Serrano et al. [19] and the role of its epigenetic landscape have been revealed in recent years. Melanoma is interesting therefore as RAS, B-RAF mutations just produce this senescent background, which can undergo reverse by reprogramming resulting in drug resistance [25], but senescence can be again restored in invasive B-RAF-mutant melanoma by structurally unrelated silencing with H3K9 demethylases [26]. Thus, OIS senescence in cancer has a dynamic nature with the epigenetic component of the general character [183]. But melanoma is also interesting for the high overexpression of meiosis-related CT genes. Overexpression of CT antigens is prognostic for poor outcome of invasive melanoma; in addition, classic meiotic genes are known to be expressed in cancers [30, 31, 168] and also in melanoma [37]. Some authors reason that overlaying of meiotic protein aberrant activities over the normal mitotic cycle (termed "meiomitosis"), first of all of the stable cohesion of sister chromatids needed for meiosis I, is interfering with normal mitotic separation of chromatids, leading to aneuploidy, genome instability, and tumor progression [36, 37, 184, 185]. The questions arise: (1) whether the mitotic cycle in tumors is normal? (2) If the meiotic features found in tumors belong to conventional gametic meiosis? (3) If an aneuploidy can perpetuate the tumor growth? Let us begin with the latter. This problem is well known as "Aneuploidy Paradox" [186], which means that incorrect segregations of genetic material should hinder and prevent cell division; however, aneuploidy paradoxically is well known as correlating with tumor growth and aggression, which may be due to selection of the fittest aneuploid clones. This conundrum cannot be explained satisfactorily with clonal selection of rare positive mutations because the "Muller's Ratchet" [187] will inevitably accumulate deleterious mutation leading ultimately to extinction of the asexual cell line. The problem, of the "Muller Rachet", however is still explored by population evolutionists [188]. Aneuploidy in cancer arises from the inherent chromosome instability of polyploidy cells. So, we arrive here to the polyploidy which in different proportions is a very characteristic feature of all malignant tumors (comparing with their normal tissue origins), progresses with cancer aggression, and which up to now is often ignored by cancer researchers [189]. However, it is just a reversible polyploidy, which provides the extraordinary resistance of cancers to therapy [56, 190–192] and likely a cancer line immortality as such. Moreover, our studies brought us to the notion of a cancer life cycle, composed of a cell cycle (lasting 17-23 h) and ploidy cycle (reversible polyploidization which takes 1–2 weeks or more), both cycles are reciprocally linked [32, 193]. This reciprocal cancer life cycle is an analogue of the "neosis" of cancer cells, related to polyploidy and senescence with rejuvenation of reduced offsprings described by Rajaraman [194, 195] and was confirmed in tumors by multiple authors [190–192], also in melanoma [59]. Thus, the answer to the first question is that the cell cycle in cancers including melanoma is not conventional and at least, in the tumor subpopulation, it is composed of two reciprocally joined different cycles, conventional mitotic and a ploidy cycle, one being quick and another being slow. The latter is often overlooked [189] as being hidden due to the low proportion in relation to the mitotic cycles. The ploidy cycle of giant cells associated with senescence reprogramming becomes clearly manifested in resistant tumors after high dosage DNA damage with anticancer drugs and ionizing irradiation [177, 196–198]. Therefore, cancer research needs prolong follow up of

**103**

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

genetic program of meiotic regulation could evolve independently [211].

Considering the expression of CT genes not only in testis but also in ovaria, early embryo and placenta, Loyd Old [212] associated their expression with the female gametogenesis-like program in tumor cells by formulating the title of his article "Cancer is a somatic cell pregnancy." Some researchers consider a possible parthenogenetic variant of the embryological in essence theory of cancer which is known from the nineteenth century [29, 213] while ontogenetic variant of this theory for the origin of tumors termed "a life-code" has been recently suggested by Jinsong Liu [214]. An interesting asexual parthenogenetic variant for triploid tumors, which are typical for resistant cancers may be achieved by digyny (69, XXY, in case of male cancers) [215]. Some observations suggest that triploidy may exchange with diploid subline on the basis of multinucleated giant cells in the same tumor [216]. The cycle of cancer stem cells likely can start with the relic uniparental disomy. The latter is described in facultative sex of the fission yeast [169], in plants, stressed and spontaneously [217] and in senescing human cells [218]. All these parasexual mechanisms may include aberrant meiotic elements and genes activity [62] and may exist in parallel or as a complex chain of one process of the survival support and escape of resistant tumors. In fact, their studies are only started. So, the answer to the second question if we should reckon exclusively with the mechanisms of conventional gametic meiosis in somatic tumors is also negative. SC in tumors was never found although the relevant genes and proteins ectopically expressed [62, 160], including melanoma [37]. We should rather reckon with evolutionary forms of meiosis in asexual life cycles. This turn of reasoning is becoming particularly context-updated if we also consider the recent gene expression phylostratigraphic analysis showing that ancestral regulatory networks drive cancer [219]. The latter in turn is associated with polyploidy [220]. Moreover, in recent time, the ancient inverted meiosis (IM) appeared on the stage [221]. IM does not require the cohesion of sister chromatids (thus, SC is not needed): the homologs are joined by their ends, recombine by sub-telomeric sequences, segregate sisters in the first meiosis and homologs in the second. Thus, IM can repair the damaged telomeres, provide some degree of genetic diversity,

individual cells and ploidy measurements [177, 191, 199, 200]. Tumor cells enter this ploidy cycle when they senesce by OIS or get the DNA damage in any other way (e.g., by ionizing irradiation or oxidative stress). If the treatment is harsh, the majority of induced giant cells will die in the time course, during mitotic catastrophe or in unsuccessful attempts of multipolar or aberrant bipolar bridged mitoses, but a minor minority of resistant cancer cells repair the DNA damage and repopulate tumors through depolyploidization by budding or other type of ploidy reduction [33, 56, 189, 191, 201, 202]. So, in our brainstorming session, we arrived to ploidy cycles and DNA damage. Here is a right link to the origin of meiosis and sex. The whole genome duplications (WGD) is a well-known driver of gene and species evolution [203] and appeared already in prokaryotes as the first evolutionary steps toward eukaryotic sex [204]. The most immediate reasons of the meiosis origin were the necessity to repair DNA damage [205]. Another reason, coupled to the first, was the relief of mutational load of aneuploidy resulting from polyploidy when it was advantageous to have more than one copy of the genome per cell [206]. Thus, the aneuploidy paradox in cancer might be resolved by asexual (somatic) meiosis (including recombination and reduction) and this meiosis is very likely ancestral. Briefly, the evolution of meiosis in eukaryotes could start from polyploid endomitosis (insect-type, without actual karyotomy), (enriched in MOS-kinase as found in tumor cells) [207], followed by zygotic meiosis, and ending in gametic meiosis in most extant vertebrates [149, 208–210]. Meiosis originated in evolution several times; there is also a view that individual blocks of

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

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

individual cells and ploidy measurements [177, 191, 199, 200]. Tumor cells enter this ploidy cycle when they senesce by OIS or get the DNA damage in any other way (e.g., by ionizing irradiation or oxidative stress). If the treatment is harsh, the majority of induced giant cells will die in the time course, during mitotic catastrophe or in unsuccessful attempts of multipolar or aberrant bipolar bridged mitoses, but a minor minority of resistant cancer cells repair the DNA damage and repopulate tumors through depolyploidization by budding or other type of ploidy reduction [33, 56, 189, 191, 201, 202]. So, in our brainstorming session, we arrived to ploidy cycles and DNA damage. Here is a right link to the origin of meiosis and sex. The whole genome duplications (WGD) is a well-known driver of gene and species evolution [203] and appeared already in prokaryotes as the first evolutionary steps toward eukaryotic sex [204]. The most immediate reasons of the meiosis origin were the necessity to repair DNA damage [205]. Another reason, coupled to the first, was the relief of mutational load of aneuploidy resulting from polyploidy when it was advantageous to have more than one copy of the genome per cell [206]. Thus, the aneuploidy paradox in cancer might be resolved by asexual (somatic) meiosis (including recombination and reduction) and this meiosis is very likely ancestral. Briefly, the evolution of meiosis in eukaryotes could start from polyploid endomitosis (insect-type, without actual karyotomy), (enriched in MOS-kinase as found in tumor cells) [207], followed by zygotic meiosis, and ending in gametic meiosis in most extant vertebrates [149, 208–210]. Meiosis originated in evolution several times; there is also a view that individual blocks of genetic program of meiotic regulation could evolve independently [211]. Considering the expression of CT genes not only in testis but also in ovaria, early embryo and placenta, Loyd Old [212] associated their expression with the female gametogenesis-like program in tumor cells by formulating the title of his article "Cancer is a somatic cell pregnancy." Some researchers consider a possible parthenogenetic variant of the embryological in essence theory of cancer which is known from the nineteenth century [29, 213] while ontogenetic variant of this theory for the origin of tumors termed "a life-code" has been recently suggested by Jinsong Liu [214]. An interesting asexual parthenogenetic variant for triploid tumors, which are typical for resistant cancers may be achieved by digyny (69, XXY, in case of male cancers) [215]. Some observations suggest that triploidy may exchange with diploid subline on the basis of multinucleated giant cells in the same tumor [216]. The cycle of cancer stem cells likely can start with the relic uniparental disomy. The latter is described in facultative sex of the fission yeast [169], in plants, stressed and spontaneously [217] and in senescing human cells [218]. All these parasexual mechanisms may include aberrant meiotic elements and genes activity [62] and may exist in parallel or as a complex chain of one process of the survival support and escape of resistant tumors. In fact, their studies are only started. So, the answer to the second question if we should reckon exclusively with the mechanisms of conventional gametic meiosis in somatic tumors is also negative. SC in tumors was never found although the relevant genes and proteins ectopically expressed [62, 160], including melanoma [37]. We should rather reckon with evolutionary forms of meiosis in asexual life cycles. This turn of reasoning is becoming particularly context-updated if we also consider the recent gene expression phylostratigraphic analysis showing that ancestral regulatory networks drive cancer [219]. The latter in turn is associated with polyploidy [220]. Moreover, in recent time, the ancient inverted meiosis (IM) appeared on the stage [221]. IM does not require the cohesion of sister chromatids (thus, SC is not needed): the homologs are joined by their ends, recombine by sub-telomeric sequences, segregate sisters in the first meiosis and homologs in the second. Thus, IM can repair the damaged telomeres, provide some degree of genetic diversity,

*Melanoma*

B-RAF-mutant melanoma activates MEK-ERK proliferative pathway but cancer can be explained neither only by enhanced proliferation nor it can be reduced to somatic mutation theory, which has been shaken by cancer genome sequencing projects. Cancer is more complex than that [182]. B-RAF-and N-RAS-mutant nevi remaining quiescent and benign just support this notion. A very important role of OIS-induced cellular senescence for initiation of malignant tumors discovered by Serrano et al. [19] and the role of its epigenetic landscape have been revealed in recent years. Melanoma is interesting therefore as RAS, B-RAF mutations just produce this senescent background, which can undergo reverse by reprogramming resulting in drug resistance [25], but senescence can be again restored in invasive B-RAF-mutant melanoma by structurally unrelated silencing with H3K9 demethylases [26]. Thus, OIS senescence in cancer has a dynamic nature with the epigenetic component of the general character [183]. But melanoma is also interesting for the high overexpression of meiosis-related CT genes. Overexpression of CT antigens is prognostic for poor outcome of invasive melanoma; in addition, classic meiotic genes are known to be expressed in cancers [30, 31, 168] and also in melanoma [37]. Some authors reason that overlaying of meiotic protein aberrant activities over the normal mitotic cycle (termed "meiomitosis"), first of all of the stable cohesion of sister chromatids needed for meiosis I, is interfering with normal mitotic separation of chromatids, leading to aneuploidy, genome instability, and tumor progression [36, 37, 184, 185]. The questions arise: (1) whether the mitotic cycle in tumors is normal? (2) If the meiotic features found in tumors belong to conventional gametic meiosis? (3) If an aneuploidy can perpetuate the tumor growth? Let us begin with the latter. This problem is well known as "Aneuploidy Paradox" [186], which means that incorrect segregations of genetic material should hinder and prevent cell division; however, aneuploidy paradoxically is well known as correlating with tumor growth and aggression, which may be due to selection of the fittest aneuploid clones. This conundrum cannot be explained satisfactorily with clonal selection of rare positive mutations because the "Muller's Ratchet" [187] will inevitably accumulate deleterious mutation leading ultimately to extinction of the asexual cell line. The problem, of the "Muller Rachet", however is still explored by population evolutionists [188]. Aneuploidy in cancer arises from the inherent chromosome instability of polyploidy cells. So, we arrive here to the polyploidy which in different proportions is a very characteristic feature of all malignant tumors (comparing with their normal tissue origins), progresses with cancer aggression, and which up to now is often ignored by cancer researchers [189]. However, it is just a reversible polyploidy, which provides the extraordinary resistance of cancers to therapy [56, 190–192] and likely a cancer line immortality as such. Moreover, our studies brought us to the notion of a cancer life cycle, composed of a cell cycle (lasting 17-23 h) and ploidy cycle (reversible polyploidization which takes 1–2 weeks or more), both cycles are reciprocally linked [32, 193]. This reciprocal cancer life cycle is an analogue of the "neosis" of cancer cells, related to polyploidy and senescence with rejuvenation of reduced offsprings described by Rajaraman [194, 195] and was confirmed in tumors by multiple authors [190–192], also in melanoma [59]. Thus, the answer to the first question is that the cell cycle in cancers including melanoma is not conventional and at least, in the tumor subpopulation, it is composed of two reciprocally joined different cycles, conventional mitotic and a ploidy cycle, one being quick and another being slow. The latter is often overlooked [189] as being hidden due to the low proportion in relation to the mitotic cycles. The ploidy cycle of giant cells associated with senescence reprogramming becomes clearly manifested in resistant tumors after high dosage DNA damage with anticancer drugs and ionizing irradia-

tion [177, 196–198]. Therefore, cancer research needs prolong follow up of

**102**

and not the least, it can count homologous chromosome pairs, to get rid of aneuploidy. Strikingly, IM was revealed in the proportion of normal human oocytes sorting out the aneuploid embryos in a polar body [222]. Although SC is not needed, however the telomere clustering at the spindle pole body for the chromosome homology search by spinning the chromosomes, for DNA recombination between homologs, is needed. Although currently the study of IM in human cancer is in infancy [62], the IM related to telomere DSBs well fits several peculiarities found in tumors: cellular senescence linked to telomere attrition, polyploidy associated with cellular senescence, mitotic slippage, reprogramming, and alternative telomere lengthening characteristic for some cancers [62]. We proposed a hypothesis that ALT-associated PML bodies in mitotic slippage of tumor cells may serve as a site for IM recombination repair [177]. Interestingly, the meiotic genes involved in the homology search and recombination RAD21L (Rec8 paralog) and Hop2-Mnd1 heterodimer (RAD51-dependent) were found associated with ALT [178, 179]. The expression of the proteins, which may be involved in IM-related ALT (SPO11, MOS, TRF2-colocalised with PML-bodies), and mitotic slippage were also observed in polyploidy cells of B-RAF V600E mutant melanoma SkMel28 cell line treated with doxorubicin and vemurafenib (mutated B-RAF-inhibitor) (**Figure 3**). The question how much the meiotic features in tumors are stochastic and how much program-directed is central for addressing the problem. The most prominent feature of cancer is adaptation to extinction by the mechanisms acquired in the evolution of life on earth. The naturally occurring tumors are found already in *Hydra* [223]. When the organisms were challenged by extinction, they have adapted to it by transient polyploidy, epigenetic plasticity, including pluripotent stemness with its bivalency of genes, intrinsically disordered proteins, and rearrangement of the nuclear architecture domains by phase transitions these epigenetic adaptations are by two orders faster than the gene mutationselection-based process would allow [224]. In accord, the expression of stemness genes, early stress response genes, epigenetic master activator CTCFL/BORIS and in particular, CT antigens genes as universal adaptors for reconstruction of the genome functional network—all these epigenetic evolutionary adaptations are found in melanoma, which are highly mortal-risky and treatment resistant in patients. At the same time, the tumor pathways are rare evolutionary attractors of the genome multi-dimensional network [225], entrapping cancer cells by the therapy resistance—only a small number of cells, but inevitably survive and repopulate the tumors [56, 177]. These rare genome space states can be only chosen by the mechanisms of nonequilibrium thermodynamics, which is by coherating fluctuations, through the method of trial and error [224, 226]. Those are inevitably accompanied by a lot of cell death and a lot of aberrant phenotypes, which may persist as transient or axillary to reproductive cancer cell line. The fidelity of the genome achieved through the evolutionary meiosis and ploidy life cycles can counteract the aneuploidy; otherwise, tumor cells may balance between both options. The snap-shot studies, not considering this factor (e.g., the productive expression of meiotic genes in only sub-population of tumor cells) can thus bring to misleading interpretations [227]. Moreover, both forward and reverse mutations occurring by gene conversion were recently found in the oldest (from 1951) human cancer cell line cervical carcinoma HeLa [228], which is also known serving a positive control for the meiotic proteins antibodies and expresses them in reversible polyploidy cycles [31]. As suggested by Maciver in 2016 [229], gene conversion in asexual polyploid species can compensate the "Muller's Ratchet." Gene conversion is the process by which one DNA sequence replaces a homologous sequence such that the sequences become identical after the conversion event. In this case, the nonreciprocal "copy-paste" recombination is occurring which is stimulated by

**105**

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

DNA strand breaks in hot spots [230]. This type of the genetic reconstruction seems also to be compatible with tumor cell senescence, mitotic slippage, and ALT.

The CT antigens and meiotic genes enhanced expression in tumors, including B-RAF-mutant melanoma, is associated with poor prognosis for the patient survival and treatment outcomes. The review shows that the functions of CTA and meiotic genes in cancer are multilayered: they involve genetic, whole-genomic, cytogenetic, epigenomic, and posttranslational levels of regulation, which are evolutionarily evolved. That means that the expression of CT antigens and meiotic genes is in general adaptive, explaining the correlation of this expression with poor melanoma prognosis. The matter concerns some recently acknowledged biological processes, whose mechanisms and thermodynamics are not fully understood. These are reversible polyploidy and reversible senescence, transient ALT, gene conversion, and likely also several forms of evolutionary, nonconventional, asexual meiosis and parthenogenesis. The fidelity of the genome aimed through the evolutionary meiosis and ploidy life cycles can potentially compensate the aneuploidy, or the tumor cells may balance between the advantages and disadvantages of both options [150]. All these questions still remain open for future studies.

The authors thank Olga Anatskaya (St. Petersburg) for advice in bioinformatic methodology and Madara Kreismane for technical support for manuscript preparation. This work was supported by a grant from the European Regional Development Fund (ERDF) projects No. 1.1.1.1/18/A/099, 1.1.1.2/VIAA/3/19/463 for K.S. and

**Gene Symbol Log2FC** Melanoma-associated antigen 3 MAGEA3 7.645235 Melanoma-associated antigen 12 MAGEA12 7.348702 Cancer/testis antigen 2 CTAG2 7.111641 MAGE family member C2 MAGEC2 6.874003 Melanoma-associated antigen 6 MAGEA6 6.828297 Chondrosarcoma-associated Gene 1 CSAG1 6.204377 Preferentially expressed antigen of melanoma PRAME 5.821726 Melanoma-associated antigen 1 MAGEA1 5.752684 MAGE family member A2B MAGEA2B 5.738676

Student Scholarship from the University of Latvia Foundation to N.M.V.

The authors declare no conflict of interest.

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

**7. Conclusion**

**Acknowledgements**

**Conflict of interest**

**Appendix A**

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

DNA strand breaks in hot spots [230]. This type of the genetic reconstruction seems also to be compatible with tumor cell senescence, mitotic slippage, and ALT.
