ROR2 promotes epithelial-mesenchymal transition by hyperactivating ERK in melanoma.

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a protein with important functions during embryogenesis that is dysregulated in human cancer. An intriguing feature of this receptor is that it plays opposite roles in different tumor types either promoting or inhibiting tumor progression. Understanding the complex role of this receptor requires a more profound exploration of both the altered biological and molecular mechanisms. Here, we describe that ROR2 promotes Epithelial-Mesenchymal Transition (EMT) by inducing cadherin switch and the upregulation of the transcription factors ZEB1, Twist, Slug, Snail, and HIF1A, together with a mesenchymal phenotype and increased migration. We show that ROR2 activates both p38 and ERK mitogen-activated protein kinase pathways independently of Wnt5a. Further, we demonstrated that the upregulation of EMT-related proteins depends on the hyperactivation of the ERK pathway far above the typical high constitutive activity observed in melanoma. In addition, ROR2 also promoted ERK phosphorylation, EMT, invasion, and necrosis in xenotransplanted mice. ROR2 also associates with EMT in tumor samples from melanoma patients where analysis of large cohorts revealed that increased ROR2 levels are linked to EMT signatures. This important role of ROR2 translates into melanoma patient' s prognosis since elevated ROR2 levels reduced overall survival and distant metastasis-free survival of patients with lymph node metastasis. In sum, these results demonstrate that ROR2 contributes to melanoma progression by inducing EMT and necrosis and can be an attractive therapeutic target for melanoma.

ROR2 has a protective role in melanoma by inhibiting Akt activity, cell-cycle progression, and proliferation.

BACKGROUND: Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a Wnt5a receptor aberrantly expressed in cancer that was shown to either suppress or promote carcinogenesis in different tumor types. Our goal was to study the role of ROR2 in melanoma. METHODS: Gain and loss-of-function strategies were applied to study the biological function of ROR2 in melanoma. Proliferation assays, flow cytometry, and western blotting were used to evaluate cell proliferation and changes in expression levels of cell-cycle and proliferation markers. The role of ROR2 in tumor growth was assessed in xenotransplantation experiments followed by immunohistochemistry analysis of the tumors. The role of ROR2 in melanoma patients was assessed by analysis of clinical data from the Leeds Melanoma Cohort. RESULTS: Unlike previous findings describing ROR2 as an oncogene in melanoma, we describe that ROR2 prevents tumor growth by inhibiting cell-cycle progression and the proliferation of melanoma cells. The effect of ROR2 is mediated by inhibition of Akt phosphorylation and activity which, in turn, regulates the expression, phosphorylation, and localization of major cell-cycle regulators including cyclins (A, B, D, and E), CDK1, CDK4, RB, p21, and p27. Xenotransplantation experiments demonstrated that ROR2 also reduces proliferation in vivo, resulting in inhibition of tumor growth. In agreement with these findings, a higher ROR2 level favors thin and non-ulcerated primary melanomas with reduced mitotic rate and better prognosis. CONCLUSION: We conclude that the expression of ROR2 slows down the growth of primary tumors and contributes to prolonging melanoma survival. Our results demonstrate that ROR2 has a far more complex role than originally described.

BCL2L10 Is Overexpressed in Melanoma Downstream of STAT3 and Promotes Cisplatin and ABT-737 Resistance.

The anti-apoptotic proteins from the Bcl-2 family are important therapeutic targets since they convey resistance to anticancer regimens. Despite the suspected functional redundancy among the six proteins of this subfamily, both basic studies and therapeutic approaches have focused mainly on BCL2, Bcl-xL, and MCL1. The role of BCL2L10, another member of this group, has been poorly studied in cancer and never has been in melanoma. We describe here that BCL2L10 is abundantly and frequently expressed both in melanoma cell lines and tumor samples. We established that BCL2L10 expression is driven by STAT3-mediated transcription, and by using reporter assays, site-directed mutagenesis, and ChIP analysis, we identified the functional STAT3 responsive elements in the BCL2L10 promoter. BCL2L10 is a pro-survival factor in melanoma since its expression reduced the cytotoxic effects of cisplatin, dacarbazine, and ABT-737 (a BCL2, Bcl-xL, and Bcl-w inhibitor). Meanwhile, both genetic and pharmacological inhibition of BCL2L10 sensitized melanoma cells to cisplatin and ABT-737. Finally, BCL2L10 inhibited the cell death upon combination treatments of PLX-4032, a BRAF inhibitor, with ABT-737 or cisplatin. In summary, we determined that BCL2L10 is expressed in melanoma and contributes to cell survival. Hence, targeting BCL2L10 may enhance the clinical efficacy of other therapies for malignant melanoma.

An Autocrine Wnt5a Loop Promotes NF-κB Pathway Activation and Cytokine/Chemokine Secretion in Melanoma.

Wnt5a signaling has been implicated in the progression of cancer by regulating multiple cellular processes, largely migration and invasion, epithelial-mesenchymal transition (EMT), and metastasis. Since Wnt5a signaling has also been involved in inflammatory processes in infectious and inflammatory diseases, we addressed the role of Wnt5a in regulating NF-κB, a pivotal mediator of inflammatory responses, in the context of cancer. The treatment of melanoma cells with Wnt5a induced phosphorylation of the NF-κB subunit p65 as well as IKK phosphorylation and IκB degradation. By using cDNA overexpression, RNA interference, and dominant negative mutants we determined that ROR1, Dvl2, and Akt (from the Wnt5a pathway) and TRAF2 and RIP (from the NF-κB pathway) are required for the Wnt5a/NF-κB crosstalk. Wnt5a also induced p65 nuclear translocation and increased NF-κB activity as evidenced by reporter assays and a NF-κB-specific upregulation of RelB, Bcl-2, and Cyclin D1. Further, stimulation of melanoma cells with Wnt5a increased the secretion of cytokines and chemokines, including IL-6, IL-8, IL-11, and IL-6 soluble receptor, MCP-1, and TNF soluble receptor I. The inhibition of endogenous Wnt5a demonstrated that an autocrine Wnt5a loop is a major regulator of the NF-κB pathway in melanoma. Taken together, these results indicate that Wnt5a activates the NF-κB pathway and has an immunomodulatory effect on melanoma through the secretion of cytokines and chemokines.

STAT3 enhances the constitutive activity of AGC kinases in melanoma by transactivating PDK1.

BACKGROUND: The PI3K/Akt and the STAT3 pathways are functionally associated in many tumor types. Both in vitro and in vivo studies have revealed that either biochemical or genetic manipulation of the STAT3 pathway activity induce changes in the same direction in Akt activity. However, the implicated mechanism has been poorly characterized. Our goal was to characterize the precise mechanism linking STAT3 with the activity of Akt and other AGC kinases in cancer using melanoma cells as a model. RESULTS: We show that active STAT3 is constitutively bound to the PDK1 promoter and positively regulate PDK1 transcription through two STAT3 responsive elements. Transduction of WM9 and UACC903 melanoma cells with STAT3-small hairpin RNA decreased both PDK1 mRNA and protein levels. STAT3 knockdown also induced a decrease of the phosphorylation of AGC kinases Akt, PKC, and SGK. The inhibitory effect of STAT3 silencing on Akt phosphorylation was restored by HA-PDK1. Along this line, HA-PDK1 expression significantly blocked the cell death induced by dacarbazine plus STAT3 knockdown. This effect might be mediated by Bcl2 proteins since HA-PDK1 rescued Bcl2, Bcl-XL, and Mcl1 levels that were down-regulated upon STAT3 silencing. CONCLUSIONS: We show that PDK1 is a transcriptional target of STAT3, linking STAT3 pathway with AGC kinases activity in melanoma. These data provide further rationale for the ongoing effort to therapeutically target STAT3 and PDK1 in melanoma and, possibly, other malignancies.