Peripheral myelin protein 2 (PMP2)

attributed to known SOX10 target genes, such as MITF or MC1R [235]. Both have been shown to independently regulate melanoma cell migration but the direct correlation to SOX10 is missing and MITF has not been found upregulated in cell lines where SOX10 overexpression enhanced the invasive capacity in this study (section 4.6).

Notwithstanding, as MIA overexpression in SOX10-inhibited melanoma cells did not completely restore their invasion capacity (section 4.4.4, Figure 32) the involvement of further SOX10 target genes cannot be excluded.

the peripheral autoimmune neuropathy Guillain Barré syndrome [170]. Its structure forms a compact 10-stranded up-and-down β-barrel that can encapsulate a fatty acid molecule [123]. A predicted structure of PMP2 is shown in Figure 57. The helical lid segment of PMP2 is partially embedded in the membrane and another membrane-binding site was suggested to be on the opposite site of the protein.

The myelin sheath is a unique biological membrane that is essential for the development and function of the vertebrate nervous system. It contributes to a 100-fold amplification of the nerve impulse along the axon by acting as an isolator and by directing the localization of neuronal ion channels to the nodes of Ranvier. The myelination process includes a myelinating glial cell that wraps its differentiated plasma membrane dozens of times tightly around selected axons. This process requires coordinated membrane synthesis, compaction, and maintenance. Myelin-specific proteins play a central role in all these processes and are highly abundant in the myelin sheath [96]. Many of them are cell adhesion molecules of the immunoglobulin family type I transmembrane proteins, such as MPZ. These proteins can mediate extracellular interactions between the successive layers of myelin and the neuronal plasma membrane. MBP and PMP2 are the only two non-transmembrane proteins that are present in high abundance in the region of compact myelin. They are both positively charged and this biochemical property facilitates their binding to the negatively charged inner leaflet of the myelin membrane. Both are localized at the cytoplasmic position of two consecutive layers of the myelin membrane and were shown to act synergistically in the compaction and maintenance of the multilayered myelin membrane [262]. It was shown that PMP2 can stack membrane bilayers and affect lipid bilayer dynamics [132], [133], [168], [262]. In addition to its function on the integrity of the myelin multilayer, it has also been suggested to be important for lipid transport to and from the membrane.

PMP2 mRNA was also found highly expressed in the cerebral cortex according to the Human Protein Atlas (section 3.1.12). Interestingly, PMP2 and SOX10 were found upregulated in dermal Schwannomas compared to other cutaneous neoplasms of peripheral nerve sheath origin [241]. Strikingly, SOX10 is present in both myelinating glial cell populations, which are Schwann cells and oligodendrocytes, throughout development and after terminal differentiation [139]. SOX10 is required for myelination in both cell types [31], [256]. In Schwann cells, SOX10 directly activates the transcription factor EGR2 [82]. Together they activate myelin gene enhancers as MPZ, PMP-22, MBP, and myelin-associated glycoprotein (MAG) [148], [251].

In this study, PMP2 was only found specifically expressed on mRNA and protein level in two melanoma cell lines (WM278 and WM239A) out of 13 (sections 4.9.1 and 4.9.2.2).

The mRNA expression of PMP2 varied highly in melanoma cells and also moderate mRNA expression was found in melanocytes.

PMP2 mRNA expression was strongly reduced upon SOX10 inhibition in all investigated melanoma cell lines but reduction on protein level was only found in cell lines WM278 and WM239A (section 4.8.1, Figure 41 and section 4.9.1, Figure 46). Although SOX10 overexpression was sufficient to increase PMP2 protein expression in WM278 cells and although its mRNA levels were considerably elevated upon SOX10 overexpression, no induced or increased PMP2 protein expression was found in cell lines WM3211, WM1366, and 1205Lu (section 4.8.1, Figure 42). This discrepancy might be explained due to a substantial role for regulatory processes occurring after mRNA transcription [278]. Several possible events can restrict PMP2 expression on protein level. In mammalian cells, mRNA is produced at a much lower rate than proteins, which means two copies of a specific mRNA per hour correlates with dozens of copies of the according protein of mRNA per hour. The stability of mRNA is less than that of proteins with 2.6-7 hours versus about 46 hours, respectively. Therefore it is possible that the PMP2 mRNA is very unstable and a very high amount of mRNA production (as it is the case for cell lines WM278 and WM239A) would be necessary for PMP2 protein expression. Strikingly, PMP2 ectopic overexpression in the PMP2-negative cell line WM3211, which generated high amounts of mRNA, led to strong PMP2 expression on protein level (section 4.9.2.4, Figure 54). Regarding the fact that PMP2 mRNA was measured in several melanoma cell lines and SOX10 regulation of PMP2 expression was also found in these cells (4.8.1), it is possible that cell lines expressing PMP2 mRNA but not PMP2 protein might miss several specific regulators for PMP2 translation or modifications that affect protein stability. Differential regulation of post-transcription (RNA processing, alternative splicing or differential splicing, regulatory elements in 5’

UTR, or depletion of ternary complexes), translation, and protein degradation together with protein modifications could contribute to absent PMP2 protein. Furthermore, the presence of micro RNAs - which is certainly differing between the different cell lines - could alter protein translation.

Moreover, PMP2 expression might be regulated epigenetically. The PMP2 promoter region as well as 1023 bp upstream of the promoter and 1049 bp downstream were analyzed by two different softwares for identification of CpG islands (EMBOSS Cpgplot

and CpG Island searcher, section 3.1.12). An island was defined with the following parameters: observed GpC/expected GpC > 0.65, percentage GC > 55%, length > 200, and distance to next island > 100 bp. As no CpG islands were found DNA methylation can be excluded as a potential epigenetic regulatory mechanism for PMP2 expression.

Not only PMP2 expression but also expression of other SOX10-regulated myelin proteins, i.e., MPZ and PLP1, was found in melanocytes and melanoma cell lines (section 4.9.1, Figure 47). Strikingly, MPZ and PLP1 mRNA levels were found substantially elevated in WM278 cells compared to other melanoma cells. PLP1 mRNA levels were also found elevated in WM239A cells. Thus, the restricted expression of myelin proteins in some melanoma cell lines seems to be a cell line-specific feature.

The VGP cell lines WM278 derived from a nodular melanoma stage IV (http://www.wistar.org/lab/meenhard-herlyn-dvm-dsc/page/melanoma-cell-lines-vgp) while WM239A were established from a lymph node metastasis [293], which does not indicate that the specific expression of myelin proteins in these cell lines is related to their original tissue microenvironment. Nevertheless, the expression of myelin proteins in melanoma cells could be a forecast where the cells might preferentially metastasize according to the seed and soil hypothesis of cancer pathogenesis [68], [193]. Hereby, the term “seed” is referred to certain tumor cells that display affinity for the milieu of certain organs, denominated as “soil”.

Reed et al. [210] demonstrated that Schwann cell-resembling melanocytic nevus cells express proteins that define the earliest stages of Schwann cell development, i.e., p75, neural cell adhesion molecule (N-CAM), and growth-associated phosphoprotein-43 (GAP-43). Notwithstanding, these proteins were found limited in primary melanoma and absent in melanoma in situ. Another research group found histological and molecular characteristics of malignant peripheral nerve sheath tumors in amelanotic melanomas of their BRAF^V600E-Cdk4^R24C mouse model that seems to be also present in a subset of human melanomas (oral presentation of Jennifer Landsberg, [307], FV28). Therefore, expression of myelin proteins might be a specific feature in a subset of melanomas contributing to melanoma heterogeneity.

Due to an early downregulation of PMP2 protein by SOX10 inhibition in WM278 cells, a direct transactivation of PMP2 by SOX10 was suggested. In silico analyses identified three potential SOX10 binding sites in the PMP2 promoter region and ChIP as well as EMSA experiments indicated binding of SOX10 to the proximal PMP2 promoter region (section 4.8.2).

In order to investigate the role of PMP2 in melanoma cells, gain- and loss-of-function experiments were performed (section 4.9.2). PMP2 inhibition via RNA interference with two different siRNAs reduced cell number and viability in PMP2-positive but not in PMP2-negative cells (section 4.9.2.2, Figure 51). Since caspase 3 was activated by siPMP2b but not by siPMP2a, while p21 was induced by both siRNAs, it was suggested that PMP2 inhibition might be able to prevent cell cycle progression in PMP2-positive melanoma cells.

Matrigel analysis showed decreased invasion after PMP2 inhibition in WM278 cells, which was only significant in case of the caspase 3-activating siPMP2b (section 4.9.2.3, Figure 52). Since myelination of neurons by Schwann cells is a highly migratory process, the influence of PMP2 on melanoma cell migration was further investigated by PMP2 overexpression. Stable PMP2 overexpression significantly increased invasion of WM278 cells but it could not rescue the SOX10 knockdown phenotype in these cells (section 4.9.2.3, Figure 53). Cell viability was not influenced by PMP2 overexpression.

Moreover, PMP2 wild type and mutant variants were stably expressed in PMP2-negative WM3211 cells (section 4.9.2.4). The L27D mutant has been described recently by Ruskamo et al. [219] to reduce PMP2 binding to the membrane. The Mut3 mutant represents a mutated form of PMP2 where all three amino acids that allow fatty acid binding according to Majava et al. [168] were mutated. On mRNA level, expression of PMP2 wild type and mutants was highly upregulated compared to control cells while only wild type PMP2 and the L27D mutant were also found expressed on protein level.

It is possible that mutations of the three amino acids in Mut3 affect protein stability and thereby cause protein degradation. Notwithstanding, WM3211 stably transfected with PMP2 wild type, both PMP2 mutant forms, and control vector were subjected to a three-dimensional spheroid assay (section 4.9.2.4). Quantification of this assay revealed that PMP2 wild type-overexpressing cells substantially increased invasiveness compared to the mutant and control cells. Overexpression of the L27D mutant also considerably increased the invasive capacity compared to the control cells but to a lesser extent than PMP2 wild type-expressing cells. Two explanations are possible for this observation:

firstly, it is possible that PMP2 has another membrane binding domain that is not affected by the L27D mutation. Secondly, the membrane binding function might not be required for stimulating the invasive potential in melanoma cells.

Taken these results together, SOX10 directly transactivates PMP2 and PMP2 plays a promigratory role in melanoma cells. PMP2 overexpression increased melanoma cell

invasion but the entire functions of PMP2 in melanoma cells remain to be elucidated.

Due to its roles in the myelinating process of Schwann cells it is possible that PMP2 can modify membrane dynamics during migration processes or it might be involved in lipid signaling in the migrating cell.

5.6 SOX10 regulated genes – parallels between Schwann cell, Schwannian