Inducción de enraizamiento de brotes de E. purpurea

Activating invasion and enabling metastasis is not only a hallmark of cancer in general but also causes most cancer-related deaths, especially in malignant melanoma [202]. Invasion and metastatic spreading are processes that alter tumor cell shape and their attachment to other cells and the extracellular matrix (ECM). Alterations in cadherin expression represent a well characterized feature of invasive cells [202]. Typically, invasive cells lose their expression of E-cadherin (CDH1), which is a key cell-to-cell adhesion molecule. In contrast, N-cadherin (CDH2) is upregulated in invasive tumor cells and gives the cells a more meschenchymal phenotype [202]. In 2003, a depiction of the connections between invasive properties of cancer cells and metastatic spreading was described by Fidler [324, 325]. There are different stages in tumor cell invasion and metastatic dissemination. First, local invasion is detectable, followed by intravasation of tumor cells into neighboring blood and lymphatic vessels. The tumor cells are transported through these circulation systems, then escape from lumina into distant tissues by extravasation and finally, they form small nodules of tumor cells, i.e. micrometastases [202, 324, 325].

The EMT has been implicated in regulating both, tumor cell invasion and metastatic spread. This developmental regulatory process allows tumor cells to gain abilities to resist apoptosis,

invade and disseminate from the primary tumor [202, 218]. A set of transcription factors, mainly SNAI1 (SNAIL), SNAI2 (SLUG), TWIST and ZEB1/2 are responsible for orchestrating the EMT process and many of these factors have been implicated in melanoma invasion and metastasis [218]. These transcription factors are responsible for several hallmark features of invasive cancer cells including: loss of adherent junctions, changes in morphology and polarity [202].

To date, many associations have been made between SOX2 expression and tumor cell invasion (reviewed in [262]). For example, SOX2 has been implicated in cellular migration and invasion in vitro by regulating MMP-2 in colorectal cancer cells [326]. Likewise, the overexpression of SOX2 in a glioma cell line resulted in significant increase in migratory and invasive properties of cells [273]. This association has also been demonstrated in melanoma. A SOX2 knockdown study in the human melanoma cell line A375 showed a 4.5-fold decrease in invasive capacity [284]. Results from this current study confirm this by demonstrating that both, migratory and invasive capacities were significantly increased in the human melanoma cell line SK Mel 30 upon SOX2 overexpression (Figure 10a & c) In agreement, shRNA-mediated knock down of SOX2 expression in the human melanoma cell line SK Mel 103 confirms a significant decrease in invasion capacity (Figure 11e). Taken together, the current study confirms that SOX2 is required for melanoma cell invasion, which is in line with results from previous studies.

SOX2 expression has also been investigated in melanoma patients. In preliminary analyses SOX2 immunopositivity correlated with dermal invasion and was found in close proximity to invasive-fronts of a melanoma [282, 284]. Here, using immunohistochemistry analysis, high expression of SOX2 in primary and metastatic melanoma patient samples compared to melanocytic nevi was confirmed (Figure 8d). Moreover, a drastically increased immunopositivity of SOX2 was found in deeper regions of primary melanoma samples (Figure 10e). In line with previous studies and the functional data, high SOX2-positive cells were observed invading into the extracellular matrix (Figure 10f), between fat cells (Figure S4b), into the epidermis (Figure S4a) and along nerve cells (Figure 10g). These results further indicate an important role for SOX2 in melanoma cell invasion, not only in vitro, but also in melanoma patients in vivo.

Tumor cell invasion is mainly regulated and orchestrated by a set of transcription factors (TFs) governing the EMT process. SOX2 has been demonstrated in many cancer types to be associated with these EMT-TFs. For example, in colorectal cancer cells, SOX2 induced EMT-TFs which led to the downregulation of E-cadherin [327]. Melanoma represents a unique case in EMT-TF expression because normal melanocytes express SNAIL2 and ZEB2

and these markers activate the MITF differentiation program resulting in tumor-suppressive signaling. However, upon melanoma initiation, either by BRAF or NRAS hyperactivation, the EMT-TF network undergoes dramatic restructuring and the expression of TWIST1 and ZEB1 are favored [203]. Moreover, after this reorganization, melanoma cells are more dedifferentiated and invasion is enhanced. Results from the current study are in agreement with this work since SOX2 leads to melanoma cell dedifferentiation and is required for melanoma cell invasion (Figure 9-10). Furthermore, upon the overexpression of SOX2 in the human melanoma cell line SK Mel 30, an upregulation of both ZEB1 and TWIST1 expression was observed (Figure 10b). Additionally, in the knockdown of SOX2 in SK Mel 103 melanoma cell lines, a slight increase in SNAI2 and ZEB2 expression was demonstrated (Figure 11f). Taken together, this suggests that SOX2 may be involved in regulating several aspects of the melanoma invasive switch, by controlling dedifferentiation which may in part be caused via repressing the MITF-M promoter, being required for melanoma invasive capacity and enhancing EMT-TFs, TWIST1 and ZEB1, identified to be involved in melanoma progression.

Linking SOX2 to these aspects further implicates SOX2 in the melanoma phenotype switch. This switch describes highly proliferative cells with low invasion capacity, which is converted into highly invasive and slow proliferating cells during melanoma progression. This switch provides melanoma cells with mechanisms in progression, maintenance and evasion of melanoma therapy [304]. After investigating SOX2 in melanoma cell invasion, the effect of SOX2 overexpression on melanoma cell metabolism was investigated. Thereby, a significant decrease in melanoma cell metabolism in the SOX2 overexpressing cells was revealed (Figure 10d) although knockdown experiments had little effect on the metabolism (Figure 11d). It is important to mention that the alamarBlue® assay is a metabolism-based method and therefore one cannot rule out that metabolic changes may lead to altered reduction of the dye without true impact on the cell cycle. Further cell cycle analysis using fluorescence- activated cell sorting (FACS) is needed to reveal whether SOX2 overexpression had a cytostatic effect or induced apoptosis resulting in cellular cytotoxicity. Furthermore, one explanation why there was not a significant increase in invasion and in turn significant decrease in cellular metabolism in the SK Mel 173-OE may be explained by the lower levels SOX2 overexpression observed in this cell line compared to the high levels of SOX2 in the SK Mel 30-OE (Figure 9b). Therefore, the effect seen is only a slight tendency but not significant. Nonetheless, these studies suggest that SOX2 overexpression induces a highly invasive, slowly proliferative phenotype and taken together with the knockdown studies, SOX2 is required for melanoma cell invasion in vitro, but not cellular metabolism.

In summary, SOX2 is a key regulator in melanoma cell plasticity. This stem cell-marker dedifferentiates human melanoma cells by repressing the MITF-M promoter, is required for melanoma cell invasion and upregulates EMT-TFs related to dedifferentiation and an invasive phenotype.