Exploring the genome-wide impact of transcription factor AP-1 in breast cancer

AP-1 plays crucial roles in a wide range of cellular processes in breast cancer. Through the dimeric basic leucine zipper (bZIP) domain, the mammalian AP-1 proteins bind to DNA and form homodimers or heterodimers from the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra1, Fra2), ATF and MAF family members. AP-1 is involved in several signal transduction pathways to control physiological and pathological processes, such as oncogenesis, metastasis and apoptosis. However, the mechanistic aspects of the modulatory effect of AP-1 in breast cancer are still not fully understood. Thus, to explore genome-wide transcriptional regulatory networks of the transcription factor AP-1 in breast cancer may help to identify novel strategies to develop new therapies. In Paper I, we established that AP-1 participates in estrogen-dependent gene expression and proliferation programs in breast cancer cells. In addition, we identified PKIB (cAMP-dependent protein kinase inhibitor-β) as a novel ERα/AP-1 target molecule, which is required for breast cancer cell growth.

In Paper II, we observed, by analyzing publically available datasets, that AP-1 is expressed at high levels in basal-like breast cancers and associated with poor clinical outcome. High level expression of AP-1 was also found in triple-negative breast cancer (TNBC) cell lines as determined by Western blot analysis and qPCR. Using cistrome and transcriptome analyses to investigate the signaling networks of AP-1 in TNBC cells, we identified that about 15% of AP-1 binding sites are located in the proximal 5’ region of the nearest gene. Gene expression profiling analysis identified differential expression of 419 and 690 genes upon knockdown of Fra-1 and c-Jun, respectively. Among these genes, 222 genes which were regulated by both Fra-1 and c-Jun were associated with cytokine-mediated signaling, type I interferon-mediated signaling, chemotaxis, cell adhesion, immune response, cell junction assembly, adherens junction organization and inflammatory response. Moreover, we found that proliferative phenotypes of TNBC cells were inhibited upon depletion of AP-1. In addition, silencing of AP-1 reduced the invasion ability both in vitro and in vivo. We further showed that AP-1 activation, downstream of the PI3K/Akt and MAPK/ERK pathways, repressed expression of E-cadherin by transcriptional upregulation of ZEB2.

In Paper III, we demonstrated that TNFα activated both the PI3K/Akt and MAPK/ERK signaling pathways to induce epithelial-mesenchymal transition (EMT) in TNBC cells via activation of AP-1 signaling and increased expression of the EMT regulator ZEB2. Based on published data on spliced transcripts, two alternatively spliced 5’UTR isoforms of the ZEB2 gene were found to be expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrated that AP-1, when activated by TNFα bound to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions.

In Paper IV, We defined that c-Jun regulated nearly a third of the TNFα-elicited transcriptome. Expression of a c-Jun-regulated pro-invasion gene set was shown to be strongly associated with clinical outcomes in TNBCs. We demonstrated that c-Jun drives TNFα-mediated TNBC malignant characteristics by transcriptional regulation of Ninj1. As exemplified by the c-Jun bound CXC chemokine genes clustered on chromosome 4, we demonstrated that NF-κB might be a pioneer factor and was required for the regulation of TNFα-inducible inflammatory genes, whereas c-Jun had little effect on TNFα-inducible inflammatory genes.

In conclusion, our studies give additional insights into the molecular mechanisms of AP-1 in relation to breast cancer cellular processes. We suggest that inhibition of AP-1 could be a new therapeutic strategy for treatment of breast cancer, especially TNBC.

List of scientific papers

I. Dahlman-Wright K, Qiao Y, Jonsson P, Gustafsson JÅ, Williams C, Zhao C. Interplay between AP-1 and estrogen receptor α in regulating gene expression and proliferation networks in breast cancer cells. Carcinogenesis. 2012 Sep;33(9):1684-91
https://doi.org/10.1093/carcin/bgs223

II. Zhao C, Qiao Y, Jonsson P, Wang J, Xu L, Rouhi P, Sinha I, Cao Y, Williams C, Dahlman-Wright K. Genome-wide Profiling of AP-1-Regulated Transcription Provides Insights into the Invasiveness of Triple-Negative Breast Cancer. Cancer Res. 2014 Jul 15;74(14):3983-94
https://doi.org/10.1158/0008-5472.CAN-13-3396

III. Qiao Y, Shiue C, Zhu J, Jonsson P, Zhuang T, Williams C, Wright A, Zhao C, and Dahlman-Wright K. AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer. Oncotarget. 2015 Apr 10;6(10):7804-14
https://doi.org/10.18632/oncotarget.3158

IV. Qiao Y, Jonsson P, Indranil S, Zhao C, and Dahlman-Wright K. AP-1 is a key regulator of TNFalpha-mediated triple-negative breast cancer progression. [Manuscript]