JNK2 Is Required for the Tumorigenic Properties of Melanoma Cells

Abstract

Overexpression and activation of c-Jun N-terminal kinases (JNKs) have been observed in multiple cancer cell lines and tumor samples. Various JNK isoforms have been reported to promote lung and liver cancer, as well as keratinocyte transformation, suggesting an important role of JNK signaling in promoting tumor development. However, there are three JNK isoforms, and it is unclear how each individual isoform, especially the ubiquitously expressed JNK1 and JNK2, functions in melanoma. Our previous study found that C116S mutations in both JNK1 and JNK2 rendered them insensitive to the covalent pan-JNK inhibitor JNK-IN-8 while retaining kinase activity. To delineate the specific roles of JNK1 and JNK2 in melanoma cell proliferation and invasiveness, we expressed the wild type (WT) and C116S mutants in melanoma cell lines and used JNK-IN-8 to enable chemical-genetic dissection of JNK1 and JNK2 activity. We found that the JNK2^C116S allele consistently enhanced colony proliferation and cell invasiveness in the presence of JNK-IN-8. When cells individually expressing WT or C116S JNK1/2 were subcutaneously implanted into immunodeficient mice, we again found that bypass of JNK-IN-8-mediated inhibition of JNK signaling by expression of JNK2^C116S specifically resulted in enhanced tumor growth in vivo. In addition, we observed a high level of JNK pathway activation in some human BRAF inhibitor (BRAFi) resistant melanoma cell lines relative to their BRAFi sensitive isogenic counterparts. JNK-IN-8 significantly enhanced the response to dabrafenib in resistant cells overexpressing JNK1^WT, JNK2^WT, and JNK1^C116S but had no effect on cells expressing JNK2^C116S, suggesting that JNK2 signaling is also crucial for BRAFi resistance in a subset of melanomas. Collectively, our data show that JNK2 activity is specifically required for melanoma cell proliferation, invasiveness, and BRAFi resistance and that this activity is most important in the context of JNK1 suppression, thus providing a compelling rationale for the development of JNK2 selective inhibitors as a potential therapy for the treatment of melanoma.

Figure 1.

cBioportal data analysis of MAPK8 and MAPK9 genes. (A) Aggregate RNA expression levels of MAPK8 and MAPK9 genes in multiple cutaneous melanoma clinical cohorts show that MAPK9 is significantly more highly expressed (4.5-fold; p < 2.2 × 10⁻¹⁶). (B) Somatic variants in MAPK8 and MAPK9 are rare, with MAPK8 exhibiting deep deletion more frequently and MAPK9 exhibiting amplification more frequently.

Figure 2.

JNK2 activity promotes long-term cell proliferation. (A) Schematic of chemical genetic methods for delineating the specific roles of JNK1 and JNK2 signaling. GFP, JNK1^WT, JNK1^C116S, JNK2^WT, and JNK2^C116S were overexpressed in 501MEL, WM239A, and HMEL-T1 cell lines. Administration of JNK-IN-8 can distinguish the signaling of JNK1 and JNK2. (B) Representative Western blots of 501MEL cell lines indicated overexpression of JNK1^WT, JNK1^C116S, JNK2^WT, and JNK2^C116S. (C) Representative colony formation assay after continual treatment with JNK-IN-8 in a 501MEL cell line for 14 days, quantified by total colony area (ImageJ), showing a strong growth advantage for the cells overexpressing JNK2^C116S vs cells overexpressing JNK1^WT, JNK1^C116S, and JNK2^WT. The scale bar on the representative images is 5 cm. (D–F) Comparison of the colony area treated with JNK-IN-8 normalized to control among 501MEL, WM239A, and HMEL-T1 cell lines, respectively, overexpressing GFP, JNK1^WT, JNK1^C116S, JNK2^WT, and JNK2^C116S (*p < 0.05; **p < 0.001; ***p < 0.0001; n = 7).

Figure 3.

JNK2 significantly promotes melanoma cell invasion in vitro. Dramatic increase in the level of cell invasion in (A) 501MEL and (B) HMEL-T1 cell lines overexpressing JNK2^C116S compared to cells overexpressing GFP, JNK1^WT, JNK1^C116S, JNK2^WT, and JNK2^C116S (*p < 0.05; ****p < 0.0001; n = 10).

Figure 4.

Activation of JNK2 significantly promotes tumorigenesis in vivo. (A) Experimental design of a xenograft mouse model. (B and C) Role of JNK1 signaling in tumor formation (n = 8 for each group). (D and E) Role of JNK2 signaling in tumor formation (n = 8 for each group). (F) RPPA heat map of significant changes of potential downstream target genes of activation of the JNK2 signaling pathway. Red indicates upregulated genes. Green indicates downregulated genes. Yellow indicates no significant changes.

Figure 5.

Roles of JNK1 and JNK2 in adaptive BRAFi resistance in a subset of melanoma cell lines. (A and B) Comparison of activation of JNKs and c-Jun between melanoma paired resistant and sensitive cells. (H and N) Western blots of m229R and SK-MEL28R resistant cells, respectively, indicated overexpression of JNK1^WT, JNK1^C116S, JNK2^WT, and JNK2^C116S. (C–G and I–M) Sensitivity to BRAFi was measured by the dose–response curve of cell survival to dabrafenib. JNK-IN-8 significantly increased the inhibition effect of dabrafenib in cells overexpressing GFP, JNK1^WT, JNK1^C116S, and JNK2^WT but had no effect on cells overexpressing JNK2^C116S.

Figure 5.

Roles of JNK1 and JNK2 in adaptive BRAFi resistance in a subset of melanoma cell lines. (A and B) Comparison of activation of JNKs and c-Jun between melanoma paired resistant and sensitive cells. (H and N) Western blots of m229R and SK-MEL28R resistant cells, respectively, indicated overexpression of JNK1^WT, JNK1^C116S, JNK2^WT, and JNK2^C116S. (C–G and I–M) Sensitivity to BRAFi was measured by the dose–response curve of cell survival to dabrafenib. JNK-IN-8 significantly increased the inhibition effect of dabrafenib in cells overexpressing GFP, JNK1^WT, JNK1^C116S, and JNK2^WT but had no effect on cells overexpressing JNK2^C116S.