We have applied our understanding of the embryonic neural crest and the power of our chick embryo transplant model to better understand the molecular mechanisms of melanoma metastasis, including cell dissemination and invasion. Previous work from our laboratory has shown that aggressive melanoma cells transplanted into the chick embryo respond to embryonic environmental cues and migrate along stereotypical neural crest emigration routes (Kulesa et al., 2006
). Furthermore, a subset of migrating melanoma cells re-expressed the melanocyte differentiation marker Melan-A. Together, these findings suggested that aggressive melanoma cells display a highly plastic neural crest-like phenotype and may respond to potent embryonic signals that promote migration or differentiation. Here we report results that build on these initial findings and support the hypothesis that melanoma cells are predisposed to being metastatic based on their intrinsic relationship to the neural crest and suggest a model in which melanoma cells exploit portions of the neural crest developmental program to facilitate metastasis.
Our experimental design combined laser capture microdissection with highly sensitive RT-qPCR to directly test to what extent c8161 aggressive melanoma cells utilize neural crest-related signaling pathways for invasion (see Table S2
for a summary of the results). We found that c8161 cells have a much higher propensity to respond to the embryonic neural crest microenvironment, as manifest by the induction of genes associated with the neural crest developmental program. Poorly aggressive c81-61 cells were less responsive and primary melanocytes did not significantly induce any of the analyzed genes (). Responsive genes primarily included those involved in the early stages of the chick neural crest developmental program: induction, specification, EMT and survival. Importantly, this responsiveness did not appear to be driven by the up-regulation of common tumor-promoting genes. Rather, the majority of genes assayed were expressed at lower levels in c8161 cells compared to primary melanocytes. This included several genes commonly associated with cancer pathogenesis such as the EGF receptor family members ERBB2, ERBB3,
, the purported melanoma oncogenes MITF
, and several markers of pluripotency, including NES, SOX2,
(). Yet many of these genes also exhibited dynamic regulatory properties in response to the chick embryonic neural crest microenvironment (,). The ability of c8161 cells to respond to chick embryonic neural crest microenvironmental signals suggests that receptors associated with the neural crest developmental program are functional and active. Interestingly, none of the receptors that were expressed by both c8161 cells and primary melanocytes showed increased expression in the melanoma cells. Rather, key receptors associated with the neural crest developmental program (BMPR1B, BMPR2, FGFR1) showed decreased expression in c8161 cells relative to primary melanocytes (). It is possible that pathway components downstream of these receptors may enhance receptor activity and thereby promote melanoma cell responsiveness to the chick embryonic microenvironment. Recent evidence suggests a predominant role for BMP4 in melanoma metastasis, in part through regulation of SNAI2
(Gupta et al., 2005
; Rothhammer et al., 2005
). Although we did not observe significant changes in SNAI2
gene expression, our
data support a role for BMP4 in the invasion process. BMP4,
along with the Wnt target gene AXIN2
, displayed the highest induction in c8161 cells following transplantation into the chick embryonic neural crest microenvironment (106- and 157-relative fold increases respectively, ). Wnt and Bmp are important signals in neural crest development, and are currently thought to be antagonistic, with dominant Wnt signaling driving melanocyte specification while BMP4 promotes neural and glial differentiation at the expense of pigment cells (Jin et al., 2001
). The observed up-regulation of BMP4 suggests that Bmp signaling is dominant in c8161 melanoma cells. The activation of neuronal-fate-promoting Bmp signaling supports previous findings that melanomas frequently acquire neuronal-like characteristics and may explain the high proficiency with which melanomas metastasize to the brain (Iyengar and Singh, 2010
; Madajewicz et al., 1984
). Additionally, because Axin2 is a component of the beta-catenin destruction complex, the high AXIN2 levels may result in reduced Wnt/beta-catenin signaling. The significant negative correlation observed between AXIN2
, a known target of Wnt/beta-catenin signaling, supports this possibility. Thus, overactive Bmp signaling combined with inhibition of differentiation-promoting Wnt signals may correlate with the heightened plasticity observed in c8161 cells.
Three Eph receptors also showed high correlation with BMP4 and AXIN2 (). Significantly, two of them (EPHA2 and EPHA4) were also genes expressed by c8161 cells but not detected in primary melanocytes. When combined with the notable up-regulation observed for BMP4, it is intriguing to surmise that BMP4 may directly facilitate the up-regulation of some Eph family members, although a direct link between BMP4 and Ephs has not been reported. Yet, our data clearly highlight the dynamic regulation of the Eph/ephrin families following exposure to the chick embryonic neural crest microenvironment. We demonstrated that 10 out of 13 Eph/ephrins expressed in c8161 cells diplayed inductive responses resulting from exposure to the chick neural crest embryonic microenvironment (). Importantly, this contrasts with primary melanocytes, which appear to actively repress gene expression of EPHA2 and EPHA4 following transplantation.
A role for Eph/ephrin signaling has been reported for many types of cancers, including melanoma (Pasquale, 2010
; Vogt et al., 1998
). Likewise, Eph/ephrin interactions have proven important for migration and sorting of embryonic neural crest cells (Kasemeier-Kulesa et al., 2006
; Krull et al., 1997
; Smith et al., 1997
), including melanoblasts, the melanocytic precursor (Harris et al., 2008
; Santiago and Erickson, 2002
). Many of the Eph/ephrins known to be involved in neural crest cell migration and pathfinding are expressed by c8161 cells. However, hierarchical clustering of the Eph/ephrins revealed seven different Eph/ephrins undetected in c8161 cells compared to c81-61 cells and primary melanocytes (). Although the effects of the loss of these Eph/ephrins in c8161 cells remains under investigation, it is tempting to speculate that aberrant Eph/ephrin expression could result in deregulated signaling and facilitate metastasis by allowing tumor cells to overcome repulsive cues found in the microenvironment. As an example, EFNA5
has been shown to regulate trunk neural crest cell migration (McLennan and Krull, 2002
). Our work demonstrated that knockdown of ephrin-A5 in c8161 cells allowed for their ectopic invasion into the region adjacent to rhombomere 5, an area of the head that typically excludes neural crest cell migration (Sechrist et al., 1993
exhibited a nearly ten-fold increase in the migrating c8161 population (), suggesting that ephrin-A5 may be a regulator of melanoma metastasis.
Lastly, our data revealed the re-expression of twelve neural crest-related genes in c8161 cells that were undetected in melanocytes (). The majority of these genes, including three members of the Eph receptor tyrosine kinase family, are involved in microenvironmental sensing and migratory pathfinding (EPHA2, EPHA4, EPHB2, PLXNA4), and EMT (SNAI1, TWIST1, ZEB1, MYB). Thus, the aberrant reacquisition of neural crest-related genes typically silenced during melanocyte differentiation may contribute to the enhanced environmental sensing and plasticity displayed by c8161 cells. Interestingly, we observed that several of these genes exhibited intermediate levels of expression in the poorly aggressive c81-61 melanoma cells (). Intermediate expression was also observed in c81-61 cells for several genes expressed by primary melanocytes but undetected in c8161 cells. Together, this suggested that moderate expression of these genes may highlight a transitory phase during transformation from melanocyte to malignant melanoma. We conclude that melanoma cells reacquire some neural crest-related genes associated with EMT and migration while silencing differentiation genes.
In summary, this study has revealed important insights into the molecular pathogenesis of metastatic melanoma. We postulate that malignant melanoma cells reacquire aspects of the embryonic neural crest program during neoplastic transformation, which promotes the malignant phenotype. Our data revealed the re-expression of multiple neural crest-related genes in c8161 cells that were undetected in melanocytes. The majority of these genes are involved in microenvironmental sensing, migration, and EMT. Further work will be required to decipher which of the observed transcriptional changes will be relevant to human disease. Because of the similarities between the embryonic neural crest developmental program and tumor metastasis, we postulate that many of the transcriptional changes identified by our model system may reflect novel aspects of the complex metastatic process. Our data suggest that c8161 melanoma cells have a much higher propensity to respond to the embryonic neural crest microenvironment than poorly aggressive c81-61 melanoma cells or primary melanocytes. Although we cannot rule out the possibility that pre-existing heterogeneities within the c8161 tumor cell population conferred the differing migratory abilities observed, the molecular and behavioral reproducibility of c8161 migration in our chick embryo transplant model support the premise that powerful embryonic signaling cues promote the dynamic gene expression patterns observed in the highly plastic c8161 cells. Furthermore, the fact that so few changes were observed in the poorly aggressive melanoma cells or the primary melanocytes suggests that the ability to regulate transcription may be a readout of cellular plasticity and thus may reflect enhanced metastatic potential. Together, these data suggest that a profile of metastasis, based on the expression patterns of neural crest-related genes, may be established for melanoma cells and that the relative expression of some neural crest-related genes may assist in monitoring melanotic lesions and facilitating accurate diagnosis and prognosis, while also providing novel therapeutic targets.