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. 2011 Jan 13;30(2):234-44.
doi: 10.1038/onc.2010.414. Epub 2010 Sep 27.

The role of the c-Jun N-terminal kinase 2-α-isoform in non-small cell lung carcinoma tumorigenesis

R T Nitta  1 , C A Del Vecchio, A H Chu, S S Mitra, A K Godwin, A J Wong
Affiliations

The role of the c-Jun N-terminal kinase 2-α-isoform in non-small cell lung carcinoma tumorigenesis

R T Nitta et al. Oncogene. .

Abstract

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2α, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2α isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2α mRNA expression than normal lung control tissue. To determine the importance of JNK2α in NSCLC progression, we reduced JNK2α expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2α had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2α induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2α can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2α2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2α is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2α.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of JNK2α in human non-small cell lung carcinoma (NSCLC). Expression of JNK isoforms in normal lung and in NSCLC tumors. (a) Protein samples were separated using 4–20% sodium dodecyl sulphate–poly acrylamide gel electrophoresis (SDS–PAGE). The numbers under each blot corresponds to the fold change in protein levels relative to normal lung. One representative normal lung sample of five samples is shown. (b) The JNK isoform mRNA levels were determined using quantitative reverse transcriptase PCR (RT–PCR).
Figure 2
Figure 2
Reduction of endogenous JNK2α in NSCLC cell lines. Retroviral infection of shRNAs targeted to JNK2α in HCC-827 or NCI-H2009 cells (shJNK2α). Uninfected cells and shRNAs targeted to a scrambled sequence (shScrambled) were used as controls. (a) Protein samples were separated using 4–20% SDS–PAGE. (b) Relative expression of JNK isoforms using QRT–PCR analysis for HCC-827-infected cells (top) and NCI-H2009-infected cells (bottom). (c) H2009 cells were serum starved for 24h and then stimulated with 500 mM sorbitol. The protein samples were separated using 4–20% SDS–PAGE and detected with the specified antibodies. **-represents the JNK2 isoform. The arrow indicates the JNK1 isoforms.
Figure 3
Figure 3
JNK2α promotes tumorigenesis in NSCLC in vitro and in vivo. (a) Cell growth analysis of NCI-H2009-infected cells. The cell lines were cultured in 1% fetal bovine serum and viable cells were counted daily. Results are from two separate experiments, each done in triplicate. (b) Cell growth analysis of HCC-827. (c) Anchorage-independent growth of each cell lines in soft agar. Results are from two separate experiments, each done in triplicate. (d) NOD/SCID mice were injected subcutaneously with the specified NCI-H2009-infected cell lines (n = 10 for each group). The tumors were removed after 7 weeks and were subsequently weighed.
Figure 4
Figure 4
JNK2α can form a complex with STAT3 in NSCLC cells. Co-immunoprecipitation reactions indicate that JNK2α can interact with STAT3 in cells. (a) 3xFlag-JNK2α2 was transfected in NCI-H2009 cells and the immunoprecipitates obtained with anti-Flag (left) or anti-STAT3 (right) were separated by SDS–PAGE and detected by immunoblotting with anti-STAT3, JNK2 or Flag. ***-represents a non-specific band in the immunoprecipitation due to a well-known contaminant (IgG chain). (b) Co-immunoprecipitation for endogenous STAT3 and JNK2 using H2009 shJNK2α or shScrambled (shScram) cells. An IgG control antibody was used as the control.
Figure 5
Figure 5
JNK2α is important for STAT Ser727 phosphorylation and STAT3 transcriptional activity in NSCLC cells. (a) In vitro kinase reactions with recombinant His-STAT3 and His-JNK2α2 wild-type (WT) or a kinase dead JNK2α2 mutant (K55R). (b) Left. Western analysis of H2009-infected cells. Right. QRT—PCR analysis of NCI-H2009-infected cells for c-MYC mRNA expression. (c) Left. Immunofluorescence of phosphorylated S727 on STAT3 in H2009-infected cells. Arrow indicates cell with a strong nuclear localization. Right. Percent of infected cells that possessed a nuclear localization. (d) Left. NCI-H2009 cells were transiently co-transfected with a STAT3 luciferase reporter construct (pSTAT3-TA-Luc) or the vector control (pTA-Luc) and a Renilla reporter construct (pRL-CMV), serum starved for 24 h and then stimulated with EGF. Data represent ratios of firefly-Luc activity derived from pSTAT3-TA-Luc over Renilla-Luc activity. The relative fold change of luciferase was calculated by normalizing to the serum-starved (-EGF) H2009-uninfected cells. Data shown represent the mean ± s.e. from three separate experiments performed in triplicate. Right. QRT–PCR analysis of serum-starved NCI-H2009-infected cells stimulated with EGF for c-MYC expression.
Figure 6
Figure 6
Reintroduction of JNK2α2 or STAT3 rescues the tumorigenic phenotypes in H2009 shJNK2α cells. Retroviral infection of JNK2α2 that is resistant to shJNK2α (JNK2α2res) or STAT3 in NCI-H2009 shJNK2α cells. (a) Protein samples were separated using 4–20% SDS–AGE and detected by immunoblot. ***-represents the YFP-JNK2α2res and the arrow depicts untagged JNK2. (b) Cell growth analysis of H2009 shJNK2α-infected cells. The cell lines were cultured with 1% fetal bovine serum and viable cells were counted daily. Results are from two separate experiments, each done in triplicate. (c) Anchorage-independent growth of each cell lines in soft agar. Results are from two separate experiments, each done in triplicate. Statistically significant differences compared with H2009 shJNK2α cells are indicated (*P values < 0.02).
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