<i>TYRO3</i>-mediated regulation of MITF: a novel target in melanoma? (2009)

While the early stages of melanoma are eminently curable with adequate surgery, patients with metastatic melanoma have a dismal prognosis. Recent advances have deciphered genetic mutations that are linked to aberrant activation of oncogenic signaling pathways, thereby making melanoma a prime model for genotype- and pathway-directed targeted therapy. To date, the mitogen-activated protein (MAP) and phosphatidylinositol-3-kinase (PI3K) pathways have been in the center of targeted therapy development: e.g. about two-thirds of melanomas harbor activating somatic gene mutations of the BRAF gene, and 15–20% of cases harbor mutations in its upstream regulator NRAS (Curtin et al., 2005). Because these mutations occur in a mutually exclusive fashion, the MAP kinase pathway is constituently activated in the majority of melanomas making it an attractive target for drug development. Additionally, the MAP kinase pathway plays a central role in the interaction with other pathways, including PI3K and c-KIT and has been implicated in the control of microphthalmia-associated transcription factor (MITF), and cyclin-dependent kinase pathways. Activating mutations in BRAF are often accompanied by PTEN loss, a known cause of activation of the PI3K pathway (Curtin et al., 2005). The discovery of activating somatic gene mutations in upstream regulators of PI3K, like ERBB4 and c-KIT, and the recent observation that the two pathways seem to cooperate in melanoma progression and possibly initiation justify the development of combinatorial approaches for molecular therapy. Microphtalmia-associated transcription factor is known to be a major ‘hub’ for the activation of oncogenic survival and proliferation signaling in malignant melanoma. Microphtalmia-associated transcription factor activates multiple downstream mediators critical for normal pigment cell physiology, melanocyte survival, cell cycle regulation, and growth including proto-oncogene Bcl-2, which enhances melanocyte survival, TBX2, a suppressor of senescence, c-met and CDK2, all involved in growth-related signal transduction. Microphtalmia-associated transcription factor also plays a central role in the development and progression of melanoma, either through re-activation of early developmental pathways or dysregulation of secondary ones, because of downstream oncogenic signal activation. With MITF playing such a critical role, it is equally important to reveal what are the upstream regulators of this ‘master’ regulator. Multiple signaling pathways are known to regulate expression levels of MITF. These include Notch/Delta, Mc1R/MSH, endothelin receptor B/endothelin 3, c-Kit/SCF, MAP, and Frizzled/Wnt β-Catenin pathways. Among transcription factors implicated in the regulation of MITF are Pax3, Sox10, LEF1, and CREB and Brn2 (Mitra and Fisher, 2009). One must keep in mind that a recently proposed ‘biological rheostat’ model suggests that a tight regulation of MITF level is essential for balancing melanocyte growth and arrest (Carreira et al., 2005), thus suggesting that some of MITF’s regulators might lead to its inhibition and others to its activation. Now, a novel pathway that regulates MITF has to be added. In a recent issue of the Proceedings of the National Academy of Sciences, Zhu et al. report on the TAM receptor kinase TYRO3 as a novel upstream regulator of MITF. The authors performed a genome-wide gain-of-function screen by using the MITF-M promoter-driven luciferase reporter gene as a read out to identify genes that alter MITF transcription. Expression of cDNA expressing Mammalian Gene Collection plasmids in B16-F0 mouse melanoma cells led to the identification of 263 initial hits, of which 23 genes showed specificity for the MITF-M promoter. Of those, only the TYRO3 gene was exclusively elevated in human melanoma cells and not in other human cancer lines. TYRO3 expression levels were overexpressed (over threefold) in 50% of 40 melanoma samples. As TYRO3 expression levels correlated with increased MITF expression levels, the authors tested the possibility that TYRO3 is a major upstream oncogenic regulator of MITF in melanoma. TYRO3 regulates MITF transcription by nuclear translocation of its upstream mediator Sox10 as the authors demonstrate by Sox10 knockdown and various co-localization studies. Furthermore, Sox10 nuclear translocation and MITF upregulation were found to be dependent on the intact kinase domain of TYRO3. Using TYRO3 knockdown and Ingenuity Pathway Analysis, TYRO3 was linked to common oncogenic pathways including PI3K/Akt, Wnt/β catenin, Erk/MAPK, SAPK/Jnk, Jak/Stat, or TGFβ signaling. The authors show that TYRO3 acts as an oncogene in melanoma by demonstrating that inhibition of TYRO3 levels in the melanoma line A2058 attenuated the proliferation of these cells. TYRO3 knockdown also led to markedly reduced caspase 3 levels suggesting that TYRO3 has anti-apoptotic effects in melanoma. When TYRO3 knockdown was coupled with administration of cisplatin or docetaxol, a significant increase in caspase 3 activity with both agents was observed suggesting that TYRO3 knockdown sensitizes melanoma cells to these chemotherapeutic agents. Co-expression of TYRO3 with the BRAFV600 mutant in primary human melanocytes, which alone induces senescence but not transformation, resulted in new colony formation. These findings suggest that TYRO3 overexpression can bypass BRAFV600 induced senescence, a finding in line with the oncogenic synergism of MITF and BRAFV600 in melanoma. Conversely, TYRO3 knockdown in A2058 melanoma cells repressed tumor formation. Importantly, the authors confirmed these findings in a xenograft model: melanoma cells where TYRO3 had been knocked out showed no detectable tumor compared with A2058 cells with wild typeTYRO3 expression. Where do we go from here? As the authors point out, there are still a few ‘gaps’ to be filled in. The exact mechanisms of elevated TYRO3 expression in melanoma remain to be elucidated. It certainly would be interesting to test whether TYR03 is amplified in melanoma cells and if so, whether TYR03 amplifications are mutually exclusive with MITF amplification/somatic mutation. Also, similar to other receptor cell signaling families, one would expect that additional upstream regulators of TYRO3 exist. Consistent with this possibility is the recent discovery of cross-talk between TYRO3’s sister receptor Axl and the interleukin-15 receptor and the interaction between Src-family non-receptor tyrosine kinases and Axl1 and TYRO3 (Budagian et al., 2005). Furthermore, it remains to be determined if Sox10 is phosphorylated directly by TYRO3 or if Sox10 nuclear translocation and, most importantly, if MITF-M transcription is influenced by other pathways that may be activated by TYRO3. As shown in Figure 1, there is extensive crosstalk and synergistic signaling between the various pathways and MITF activation. For example, activated Erk is able to both directly phosphorylate MITF, which recruits a co-factor to MITF enhancing its effect on downstream targets, as well as indirectly by activation of CREB, which binds to the MITF promoter and induces its transcription. Concomitant MITF knockdown with TYRO3 overexpression could determine the exact role of MITF in TYRO3-mediated cell signaling and which effects are mediated by other pathways, like the MAP kinase or PI3K pathway (Figure 1). In this line, it also remains to be determined if the observed anti-apoptotic effects are directly mediated by TYRO3 activation, or mediated by concomitant activation of the PI3K/Akt pathway. Multiple TYRO3-activated pathways signaling pathways converge with transcription or activation of MITF [reproduced from Mitra and Fisher (2009)], an oncogenic transcription factor often dysregulated in malignant melanoma. Direct inhibition of transcription factors like MITF may be challenging although recent data have shown that histone deacetylase inhibitors (HDACi) can suppress MITF levels as well as cell proliferation (Mitra and Fisher, 2009). A small molecule inhibitor of one of the other three members of the TAM receptor tyrosine kinase family, Axl is currently under preclinical and clinical evaluation (Mahadevan et al., 2007). MP470 reduces the metabolic activity of an Axl-expressing, drug resistant gastrointestinal stromal tumor. Its effect on Mer and TYRO3 receptors is not known and autoimmune toxicity because of interruption of cell–cell signaling function mediated by TAM receptors is a concern. Nevertheless, rapid progress towards selective inhibitors of TAM receptors either in the form of small molecules, soluble receptors, or antibodies can be expected, and the results of Zhu et al. point to the possible development of therapeutic agents against TYRO3 in melanoma.