In this study, we have demonstrated that AP-1 family member JunD mediated cell survival effects in prostate cancer cells are required on GADD45α and γ family members. We demonstrate that downregulation of GADD45α and γ protein expression by JunD is an essential step in AP-1 dependent escape from programmed cell death of prostate cancer cells. JNK activation has been shown to contribute to apoptosis induction upon inhibition of NFκB in cancer cells.33
Here, we show that JNK activity contributes to apoptosis of prostate cancer cells in response to JunD inhibition as well and, similar to NFκB signaling, is dependent on GADD45α and γ, but not β, corroborating the important role of the JNK pathway in cell survival of prostate cancer.
AP-1 proteins play a role during normal cellular development and transformation, and AP-1 complexes bind to palindromic DNA sequences, resulting in the transcriptional regulation of various target genes.24
AP-1 proteins that lack potent transactivation domains, such as Fra-1, Fra-2, JunB and JunD express weak transforming activities.36
For example, mice that lack JunD are viable and do not form tumors spontaneously.37
Some AP-1 proteins that lack transforming activity can actually suppress tumorigenesis.38
The decision as to whether a particular AP-1 complex is oncogenic or anti-oncogenic may depend on the opposing activities of different AP-1 proteins, and is probably influenced by tumor type, tumor stage and the genetic background. Indeed, the role that JunD plays in the cellular environment is dependent on its partner's identity in the AP-1 heterodimer-complex such as Fra-1, on post-transcriptional and post-translational regulation and additional transcription factors that synergize with JunD protein. For example, AP-1 activity is not only regulated as a result of formation of a particular AP-1/DNA complex but also by interactions with NFκB and MAPK or PI3K signaling pathways.27
Shin et al. recently implicated AP-1 proteins and certain AP-1/DNA complexes, including JunD, in the malignant transformation of pancreatic cancer cells.
Cooperative interactions between transcription factors due to direct protein-protein interactions or via indirect effects on the DNA structure are common mechanisms of transcriptional regulation. Many transcription factors have their DNA binding sites adjacent to DNA binding sites for other transcription factors or overlapping with other binding sites. Depending on the sequence context and the particular cellular environment, binding of two distinct transcription factor complexes result in higher affinity interaction, synergistic repression or activation of specific target genes.40
Combinatorial control of transcriptional regulation has been demonstrated to affect transcription of a large number of enhancers or promoters. Combinations of binding sites for AP-1 and NFκB occur in a large number of promoter/enhancer elements, and functional cooperation between both is decisive for controlled expression of many genes, including several cytokines.
In this regard, we previously showed that combined aberrant activation of NFκB p50 and p65 and AP-1 JunD and Fra-1 in androgen-insensitive prostate cancer cells is the primary culprit for deregulated expression of IL-6, which plays a role in prostate cancer survival and progression.20
IL-6 is one of several proteins that activates the androgen receptor in hormone refractory prostate cancer, and it was recently suggested that IL-6 production promotes cell growth and escape from programmed cell death in hormone refractory prostate cancer via an autocrine and paracrine mechanism.41
Similarly to deregulated IL-6 expression in prostate cancer, here we demonstrate that JunD plays an essential role in cell survival and proliferation of prostate cancer cells that constitutively express active AP-1 and NFκB.20
We previously reported that NFκB-mediated cell-survival mechanisms in various types of cancer were dependent on GADD45α and γ family members,33
suggesting that both transcription factors, NFκB and JunD, are key players in repressing GADD45α and γ gene expression in prostate cancer cells. Interestingly, while in inflammatory cells NFκB-dependent survival mechanisms are primarily due to upregulation of GADD45β, in cancer cells downregulation of GADD45α and γ gene expression by JunD and NFκB is the major mechanisms for escape from programmed cell death. We did not observe a major contribution of GADD45β to apoptosis induction upon inhibition of JunD, but instead an essential role of GADD45α and γ proteins in apoptosis induction-mediated by JunD inhibition. Blockage of JunD activity by its dominant negative mutant inhibits prostate cancer tumor growth in an orthotopic xenograft model, demonstrating that JunD is an essential mediator of prostate cancer progression.
GADD45 proteins respond to environmental stresses mediating the activation of both p38 and JNK pathways via MTK1/MEKK4 kinase.34
The induction of JNK and p38 pathways modulates cell cycle decisions and promotes cell survival or apoptosis depending on the cell type and stimulus. JunD is phosphorylated by JNK at serines 90 and 100 and at threonine 117, which positively regulates trans-activation activity.42
JunD phosphorylation is decreased by the downregulation of JNK mediated by estrogen, which also decreases the expression of JunD transcripts, indicating an autocrine loop in the regulation of JunD mRNAs.43
We previously also demonstrated that JNK physically interacts with p53 and mediates p53 serine-15 phosphorylation in prostate cancer cells leading to p53 stabilization, increased p53 binding to its target genes, and induction of apoptosis in the context of the NFκB/IκB signaling pathway.44
Our findings demonstrate that blockage of JunD activity leads to induction of JNK and p38 activity and apoptosis. Cells expressing the dominant negative JunD and treated with JNK and p38 inhibitors show strongly reduced, but not abolished, apoptosis induction, which suggests that JNK and p38 contribute in a major form to apoptosis induction, although additional pathways cannot be excluded. These observations prompted us to investigate whether JNK and p38 activation and apoptosis induction are dependent of GADD45α and γ activity. Blockage of JunD leads to GADD45α and γ dependant induction of apoptosis that, interestingly, relies only on JNK activity as blockage of GADD45 gene expression by siRNA approaches only abrogates JNK activity while p38 activation is not altered. These data indicate that inhibition of JunD induces a pro-apoptotic pathway that is GADD45α and γ and JNK dependent and an additional pathway that involves p38 activation via a GADD45-independent mechanism.
While JunD and most likely Fra-1 elicit anti-apoptotic activities, at least in prostate cancer cells, not all members of the AP-1 family share the same activity. For example, c-Fos at least in the context of Trail-induced apoptosis of prostate cancer cells is essential, but not enough, to regulate TRAIL-induced apoptosis.45,46
Recently, Zhang et al.47
reported that NFκB inhibition and c-Fos activation significantly enhance prostate cancer cells sensitivity to TNFα or TRAIL-induced apoptosis. Thus, different members of the AP-1 family can trigger diverse cellular responses under specific stimuli or particular treatments.
In conclusion, our results establish the role of JunD in the maintenance of prostate cancer cell survival, controlling GADD45α or γ gene expression, JNK activity and apoptosis, supporting the notion of JunD as a novel therapeutic target in prostate cancer.