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The Jun kinase 2 isoform is preferentially required for epidermal growth factor-induced transformation of human A549
Abstract:
We have previously found that epidermal growth factor (EGF) mediates growth through the Jun N-terminal kinase/stress-activated kinase (JNK/SAPK) pathway in A549 human lung carcinoma cells. As observed here, EGF treatment also greatly enhances the tumorigenicity of A549 cells, suggesting an important role for JNK in cancer cell growth (F. Bost, R. McKay, N. Dean, and D. Mercola, J. Biol. Chem. 272:33422-33429, 1997). Several isoforms families of JNK, JNK1, JNK2, and JNK3, have been isolated; they arise from alternative splicing of three different genes and have distinct substrate binding properties. Here we have used specific phosphorothioate oligonucleotides targeted against the two major isoforms, JNK1 and JNK2, to discriminate their roles in EGF-induced transformation. Multiple antisense sequences have been screened, and two high-affinity and specific candidates have been identified. Antisense JNK1 eliminated steady-state mRNA and JNK1 protein expression with a 50% effective concentration (EC50) of <0.1 microM but did not alter JNK2 mRNA or protein levels. Conversely, antisense JNK2 specifically eliminated JNK2 steady-state mRNA and protein expression with an EC50 of 0.1 microM. Antisense JNK1 and antisense JNK2 inhibited by 40 and 70%, respectively, EGF-induced total JNK activity, whereas sense and scrambled-sequence control oligonucleotides had no effect. The elimination of mRNA, protein, and JNK activities lasted 48 and 72 h following a single Lipofectin treatment with antisense JNK1 and JNK2, respectively, indicating sufficient duration for examining the impact of specific elimination on the phenotype. Direct proliferation assays demonstrated that antisense JNK2 inhibited EGF-induced doubling of growth as well as the combination of active antisense oligonucleotides did. EGF treatment also induced colony formation in soft agar. This effect was completely inhibited by antisense JNK2 and combined-antisense treatment but not altered by antisense JNK1 alone. These results show that EGF doubles the proliferation (growth in soft agar as well as tumorigenicity in athymic mice) of A549 lung carcinoma cells and that the JNK2 isoform but not JNK1 is utilized for mediating the effects of EGF. This study represents the first demonstration of a cellular phenotype regulated by a JNK isoform family, JNK2.
Insights
Epidermal growth factor (EGF) promotes lung cancer cell growth and tumorigenicity. The JNK2 (Jun N-terminal kinase 2) isoform, but not JNK1, is crucial for mediating these EGF-induced effects, highlighting JNK2 as a therapeutic target.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Epidermal growth factor (EGF) signaling is implicated in cancer cell growth.
- The Jun N-terminal kinase (JNK) pathway, particularly JNK1 and JNK2 isoforms, plays a role in EGF-mediated processes.
- Understanding the specific roles of JNK isoforms in cancer is essential for targeted therapies.
Purpose of the Study:
- To investigate the distinct roles of JNK1 and JNK2 isoforms in EGF-induced proliferation and tumorigenicity of A549 human lung carcinoma cells.
- To determine which JNK isoform mediates EGF-induced cellular transformation.
- To establish the duration of specific JNK isoform inhibition using antisense oligonucleotides.
Main Methods:
- Utilized specific phosphorothioate antisense oligonucleotides targeting JNK1 and JNK2 mRNA and protein.
- Assessed the efficacy and specificity of antisense oligonucleotides in inhibiting JNK1 and JNK2 expression and activity.
- Quantified EGF-induced proliferation, colony formation in soft agar, and tumorigenicity in athymic mice following JNK isoform inhibition.
Main Results:
- Antisense JNK1 specifically reduced JNK1 expression and activity by 40%, while antisense JNK2 reduced JNK2 expression and activity by 70%.
- Antisense JNK2, but not antisense JNK1, significantly inhibited EGF-induced proliferation and colony formation in soft agar.
- Inhibition of JNK2 by antisense oligonucleotides effectively blocked EGF-mediated tumorigenicity in A549 cells.
Conclusions:
- The JNK2 isoform is specifically required for mediating EGF-induced proliferation and tumorigenicity in A549 lung carcinoma cells.
- This study provides the first evidence of a cellular phenotype regulated by the JNK2 isoform.
- Targeting JNK2 may represent a viable strategy for inhibiting lung cancer progression driven by EGF signaling.