**Abstract**

The ectopic expression of cancer testis (CT) antigens and classic meiotic genes is characteristic and a hallmark of poor prognosis of melanoma disease. Here the potential mechanisms of meiotic influence on the cell and life cycle of malignant melanoma are reviewed in the genetic, epigenetic, and evolutionary aspects. The involved mutant B-RAF and N-RAS-induced senescence may be reversed by reprogramming, with stemness linked to meiotic landscape, possibly induced by DNA double-strand breaks at the mutual telomere hot spots. The induced by senescence mitotic slippage (reset of interphase from arrested metaphase) and resulting polyploidy trigger the meiotic ploidy cycle to function for effective DNA recombination repair, genome reduction, and escape of survivors, which enter the mitotic cycle again. The aberrant meiotic pathway in cancer is reviewed in the ancestral asexual variants; inverted meiosis is possible. The conundrum of cancer aneuploidy paradox, selection of fit clones, and the Muller's Ratchet of inevitable accumulation of harmful mutations is discussed. The bioinformatic study of the densely connected protein interaction network of CT antigen expressed genes revealed the melanomagenesis attractor composed of PRAME and small MAGEA group in primary tumors as compared with B-RAF-mutant nevi, restructured stemness network; invasive melanoma further displays the leading role of SPANX CT antigen group; meiotic genes are expressed in all three tissue cohorts.

**Keywords:** B-RAF-mutant melanoma, reversible senescence, reversible polyploidy, DSB hot spots, ancestral meiosis

## **1. Introduction**

Approximately 50% of melanomas carry mutations in the gene encoding *B-RAF* [1]. Ninety percent of activating *B-RAF* mutations affect the codon 600 and the most common missense change there is V600E [2]. This mutation leads to a constitutive activation of B-RAF, and consequently of the MAPK/ERK pathway, promoting survival and proliferation of melanoma cells. Other frequent mutations in melanoma include *N-RAS* gene, which is estimated to be present in 13–25% of melanomas [1], and being upstream of the same MAPK/ERK signal transduction pathway. The MAPK/ERK signal transduction pathway involves a signaling cascade initiated by the binding of growth factors or cytokines to their respective receptors, resulting in activation of RAS, which then recruits RAF proteins, a family of protein kinases including B-RAF, to the cell membrane. Phosphorylation of RAF allows the activation of MEK1 [MAP kinase/extracellular signal-regulated kinase 1(ERK1)], which positively regulates the extracellular signal-regulated kinases (ERK). ERK can then directly phosphorylate downstream transcription factors, leading to increased transcription and eventual cell growth and proliferation [3]. Following the discovery of the V600E mutation, the pathway targeting inhibitor drugs was developed [4–10]. However, while initial responses are impressive, therapeutic resistance develops in nearly every patient at a median of 11–15 months of treatment [6, 7, 9, 11, 12].

Human nevi (benign lesions of melanocytes) also frequently harbor V600E mutation in *B-RAF* [13]; however, in spite of the oncogenic nature of this mutation [14], they display classical characteristics of senescence [15] and remain benign in the large majority of cases. At the same time, nevi are supposed to give rise to a quarter of all melanomas [16]. This led to the concept that oncogene induced senescence (OIS) precedes transformation [15, 17, 18], in particular if induced by mutant RAS or *B-RAF*. The expression of mutant RAS in normal human tissues inducing cell proliferation arrest was first described in [19] and further widely used as a model of OIS in normal cells. For a long time, OIS as well as senescence induced either by chemotherapy or oxidative stress (so called accelerated senescence ACS) were assumed as a barrier in premalignant tumor for tumor progression [20]. However, later it was found that senescence has also an opposite side and can reverse, so promoting cancer and metastases development [21–24]. Moreover, the cells that have experienced and evaded cellular senescence are more resistant to therapy than their counterparts [25]. The same group showed also that two different types of histone H3 lysine 9 (H3K9) demethylases, the flavin-dependent amine oxidase LSD1 and the 2-oxoglutarate-dependent Jumonji C family member JMJD2C, epigenetically disable oncogenic RAS- or B-RAFinduced senescence by enabling the expression of E2F target genes, which permits restarting of proliferation cycles. In turn, the inhibition of the H3K9 demethylases restores senescence and controls tumor growth of melanoma [26]. These experiments show the important contribution of the chromatin remodeling in OIS and cancer.

Biochemically, B-RAF has the same kinase activity as the serine-threonine protein kinase MOS [27] that is the main meiotic kinase [28]. Interestingly, protooncogenes c-ras and c-raf also participate in gametogenesis and when overexpressed (even non-mutant) can impose the meiotic mechanisms onto somatic cells [29]. In tumors, this pathway elaborating MOS-kinase can be triggered from mitosis through DNA damage checkpoint and senescence, supposedly providing them with the survival advantage [30–33]. At the same time, the expression of many germline proteins specific for meiotic prophase has been found upregulated in cancers [34–36] and in melanoma [37] as well.

Below we review the literature data of the abovementioned meiosis-associated processes and pathways involved in cancer (in the wide sense) and melanoma, in particular.

### **2. Senescence, TP53 function, and polyploidy in melanoma**

Melanomas often derive from nevi, which already contain oncogenic B-RAF and N-RAS mutations. It was shown in several works that the melanoma genesis

**93**

metastatic tumors [59].

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

from these nevi is associated with the reverse of the OIS induced by these mutations. The mutual feature for all kinds of ACS (OIS, drug-, and oxidative stressinduced) is the introduction of DNA double-strand breaks (DSBs); a persistent DNA damage signaling was shown triggering senescence [38]. The response to the latter includes the activity of tumor suppressor transcription factor p53. Dysfunction of p53 is generally associated with malignant tumors and also with associated overcoming the polyploidy barrier [39]. In relation to melanoma, these issues will be briefly considered below. Wild-type (WT) p53 that is present at undetectable levels in normal tissues, when upregulated by DNA damage, is a potent inducer of apoptosis, cell cycle arrest, and cellular senescence, in general counteracting carcinogenesis [40], but also caring for stem cells by causing transient alternative splicing of POU5F1 in senescent embryonal carcinoma until the repair of DNA damage [41]. The tumor suppressor TP53 is mutated in its DNA binding domain in about half of somatic cancers [42]. In other cases, it is also mostly inactivated in other ways, e.g., by promoter methylation, etc. [43]. TP53 mutants, however, acquire additive functions, e.g., invasive features [44]. Melanoma is not an exclusion: with approximately only 10–19% disabling point mutations, WT p53 is found inactivated in approximately 90% of cases [45, 46]. The low frequency of p53 mutation in melanoma may be due to the overexpression of its counterpart oncoprotein MDM2, which is due to inactivation of *CDKN2A* locus encoding the dual tumor suppressors p16INK4A and p14ARF. Likewise, the most common somatic mutations associated with familial melanoma also disrupt the *CDKN2A* locus [47]. In the presence of oncogenic activation (B-RAF or N-RAS), p14ARF acts to directly inhibit MDM2, the major ubiquitin ligase that normally degrades and inactivates p53 [48]. The cooperation of B-RAF mutations with nonfunctional p53 in melanoma genesis was modeled by Patton and colleagues [49] in p53-deficient Zebrafish, where activated B-RAF induced formation of melanocyte lesions rapidly developed into invasive melanomas, resembling human melanomas and could be serially transplanted. Another tumor suppressor PTEN may also participate in melanoma genesis from B-RAF V600E nevi [50]. TP53 is a barrier to polyploidy [39], the latter is often reached by mitotic slippage (reset of interphase from arrested metaphase with a tetraploid genome). Mitotic slippage and thus polyploidization accompanies OIS or irradiation-drug-induced senescence in tumors with characteristic DNA damage response [51]; however, both senescence and polyploidy, induced by OIS or genotoxic treatments, can be reversed [52–55]. In this prolonged process occupying 7 and more days, the majority of giant cells succumb and the proportion of escape (de-polyploidized) cells may be rather low [56, 57] but they repopulate the tumor in the remote period of time. Mitotic slippage and DNA re-replication resulting in polyploidization was modeled in melanoma by Aurora A-kinase interference [58]. The DNA re-replication stress resulting in the foldincreased amount of DNA DSBs in the polyploidized cells was revealed. MDM2 antagonists relieved it by restoring the functional p53 and its downstream p21, interrupting re-replication of cells. Finally, the same was shown in melanoma: the experiments with prolonged expression of the oncogene N-RAS Q61K in pigment cells showed the induction of senescent multi-nucleated polyploid cells, however further overcoming OIS by the emergence of tumor-initiating mononucleated (de-polyploidized) stem-like cells from senescent cells. This progeny was dedifferentiated, highly proliferative, and anoikis-resistant, and induces fast-growing,

Besides inducing OIS, *N-RAS* and *B-RAF*-activating mutations can potentially impose meiotic features onto melanocytes (substituting by overexpressed B-RAF of meiotic MOS-MEK-kinase or alternatively triggering its pathway).

*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*

from these nevi is associated with the reverse of the OIS induced by these mutations. The mutual feature for all kinds of ACS (OIS, drug-, and oxidative stressinduced) is the introduction of DNA double-strand breaks (DSBs); a persistent DNA damage signaling was shown triggering senescence [38]. The response to the latter includes the activity of tumor suppressor transcription factor p53. Dysfunction of p53 is generally associated with malignant tumors and also with associated overcoming the polyploidy barrier [39]. In relation to melanoma, these issues will be briefly considered below. Wild-type (WT) p53 that is present at undetectable levels in normal tissues, when upregulated by DNA damage, is a potent inducer of apoptosis, cell cycle arrest, and cellular senescence, in general counteracting carcinogenesis [40], but also caring for stem cells by causing transient alternative splicing of POU5F1 in senescent embryonal carcinoma until the repair of DNA damage [41]. The tumor suppressor TP53 is mutated in its DNA binding domain in about half of somatic cancers [42]. In other cases, it is also mostly inactivated in other ways, e.g., by promoter methylation, etc. [43]. TP53 mutants, however, acquire additive functions, e.g., invasive features [44]. Melanoma is not an exclusion: with approximately only 10–19% disabling point mutations, WT p53 is found inactivated in approximately 90% of cases [45, 46]. The low frequency of p53 mutation in melanoma may be due to the overexpression of its counterpart oncoprotein MDM2, which is due to inactivation of *CDKN2A* locus encoding the dual tumor suppressors p16INK4A and p14ARF. Likewise, the most common somatic mutations associated with familial melanoma also disrupt the *CDKN2A* locus [47]. In the presence of oncogenic activation (B-RAF or N-RAS), p14ARF acts to directly inhibit MDM2, the major ubiquitin ligase that normally degrades and inactivates p53 [48]. The cooperation of B-RAF mutations with nonfunctional p53 in melanoma genesis was modeled by Patton and colleagues [49] in p53-deficient Zebrafish, where activated B-RAF induced formation of melanocyte lesions rapidly developed into invasive melanomas, resembling human melanomas and could be serially transplanted. Another tumor suppressor PTEN may also participate in melanoma genesis from B-RAF V600E nevi [50]. TP53 is a barrier to polyploidy [39], the latter is often reached by mitotic slippage (reset of interphase from arrested metaphase with a tetraploid genome). Mitotic slippage and thus polyploidization accompanies OIS or irradiation-drug-induced senescence in tumors with characteristic DNA damage response [51]; however, both senescence and polyploidy, induced by OIS or genotoxic treatments, can be reversed [52–55]. In this prolonged process occupying 7 and more days, the majority of giant cells succumb and the proportion of escape (de-polyploidized) cells may be rather low [56, 57] but they repopulate the tumor in the remote period of time. Mitotic slippage and DNA re-replication resulting in polyploidization was modeled in melanoma by Aurora A-kinase interference [58]. The DNA re-replication stress resulting in the foldincreased amount of DNA DSBs in the polyploidized cells was revealed. MDM2 antagonists relieved it by restoring the functional p53 and its downstream p21, interrupting re-replication of cells. Finally, the same was shown in melanoma: the experiments with prolonged expression of the oncogene N-RAS Q61K in pigment cells showed the induction of senescent multi-nucleated polyploid cells, however further overcoming OIS by the emergence of tumor-initiating mononucleated (de-polyploidized) stem-like cells from senescent cells. This progeny was dedifferentiated, highly proliferative, and anoikis-resistant, and induces fast-growing, metastatic tumors [59].

Besides inducing OIS, *N-RAS* and *B-RAF*-activating mutations can potentially impose meiotic features onto melanocytes (substituting by overexpressed B-RAF of meiotic MOS-MEK-kinase or alternatively triggering its pathway).

*Melanoma*

promoting survival and proliferation of melanoma cells. Other frequent mutations in melanoma include *N-RAS* gene, which is estimated to be present in 13–25% of melanomas [1], and being upstream of the same MAPK/ERK signal transduction pathway. The MAPK/ERK signal transduction pathway involves a signaling cascade initiated by the binding of growth factors or cytokines to their respective receptors, resulting in activation of RAS, which then recruits RAF proteins, a family of protein kinases including B-RAF, to the cell membrane. Phosphorylation of RAF allows the activation of MEK1 [MAP kinase/extracellular signal-regulated kinase 1(ERK1)], which positively regulates the extracellular signal-regulated kinases (ERK). ERK can then directly phosphorylate downstream transcription factors, leading to increased transcription and eventual cell growth and proliferation [3]. Following the discovery of the V600E mutation, the pathway targeting inhibitor drugs was developed [4–10]. However, while initial responses are impressive, therapeutic resistance develops in nearly every patient at a median of 11–15 months of treatment [6, 7, 9, 11, 12]. Human nevi (benign lesions of melanocytes) also frequently harbor V600E mutation in *B-RAF* [13]; however, in spite of the oncogenic nature of this mutation [14], they display classical characteristics of senescence [15] and remain benign in the large majority of cases. At the same time, nevi are supposed to give rise to a quarter of all melanomas [16]. This led to the concept that oncogene induced senescence (OIS) precedes transformation [15, 17, 18], in particular if induced by mutant RAS or *B-RAF*. The expression of mutant RAS in normal human tissues inducing cell proliferation arrest was first described in [19] and further widely used as a model of OIS in normal cells. For a long time, OIS as well as senescence induced either by chemotherapy or oxidative stress (so called accelerated senescence ACS) were assumed as a barrier in premalignant tumor for tumor progression [20]. However, later it was found that senescence has also an opposite side and can reverse, so promoting cancer and metastases development [21–24]. Moreover, the cells that have experienced and evaded cellular senescence are more resistant to therapy than their counterparts [25]. The same group showed also that two different types of histone H3 lysine 9 (H3K9) demethylases, the flavin-dependent amine oxidase LSD1 and the 2-oxoglutarate-dependent Jumonji C family member JMJD2C, epigenetically disable oncogenic RAS- or B-RAFinduced senescence by enabling the expression of E2F target genes, which permits restarting of proliferation cycles. In turn, the inhibition of the H3K9 demethylases restores senescence and controls tumor growth of melanoma [26]. These experiments show the important contribution of the chromatin remodeling in OIS and cancer. Biochemically, B-RAF has the same kinase activity as the serine-threonine protein kinase MOS [27] that is the main meiotic kinase [28]. Interestingly, protooncogenes c-ras and c-raf also participate in gametogenesis and when overexpressed (even non-mutant) can impose the meiotic mechanisms onto somatic cells [29]. In tumors, this pathway elaborating MOS-kinase can be triggered from mitosis through DNA damage checkpoint and senescence, supposedly providing them with the survival advantage [30–33]. At the same time, the expression of many germline proteins specific for meiotic prophase has been found upregulated in cancers

**92**

particular.

[34–36] and in melanoma [37] as well.

Below we review the literature data of the abovementioned meiosis-associated processes and pathways involved in cancer (in the wide sense) and melanoma, in

Melanomas often derive from nevi, which already contain oncogenic B-RAF and N-RAS mutations. It was shown in several works that the melanoma genesis

**2. Senescence, TP53 function, and polyploidy in 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 network can be potentially involved in melanoma genesis.
