Post-translational modification of estrogen receptor alpha and p53 in breast cancer cells

Estrogen receptor α (ERα/ESR1) and tumor protein p53 (p53/TP53) signaling are aberrant and play important roles in breast cancer pathogenesis and evolution. ERα is highly expressed in the majority of breast cancers and is an important contributor to the development of these tumors. p53 aberrances occur as mutations resulting in defective protein or as decreased expression due to genomic mutations or deletions and genetic hyper-methylation. Besides regulation at the genome and transcriptome levels, accumulated evidence shows that post-translational modifications of ERα and p53 also play critical roles in cancer cell proliferation and therapeutic resistance. The overall aim of this thesis was to characterize the protein modification properties of the E3 ubiquitin ligase RNF31 on ERα and p53 signaling and the effect of p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) phosphorylation on ERα signaling.

In the first study, we find that the E3 ubiquitin ligase ring finger protein 31 (RNF31) is highly expressed in breast cancers, and increases estrogen-stimulated cell proliferation by facilitating estrogen signaling. Furthermore, we show that RNF31 interacts with ERα via its RBR (Ring between ring fingers) domain and induces ERα mono-ubiquitination resulting in increased ERα levels. This modification occurs in the cytoplasm and depends on the ligase activity of RNF31.

In the second study, we investigate the epithelial-mesenchymal transition (EMT) phenomenon in triple-negative breast cancer (TNBC) cells. We provide evidence that AP-1 signaling contributes to EMT in TNBC cells via activation of its target gene ZEB2. We demonstrate that AP-1 binds to two distinct cis-regulatory regions of ZEB2 and regulates its expression by mediating long-range chromatin interactions.

In the third study, we identify that RNF31 is involved in the p53 pathway based on an unbiased approach exploring global gene expression profiling data. We show that RNF31 inhibits p53-dependent cell cycle arrest and cisplatin-induced apoptosis in wild type p53 breast cancer cells. Depletion of RNF31 increases p53 protein levels and its target genes. We demonstrate that RNF31 interacts with the p53/MDM2 complex and facilitates p53 poly-ubiquitination and degradation, possibly by modifying MDM2 stability.

In the fourth study, we investigate the effect of PAK4 phosphorylation on ERα and its association with tamoxifen sensitivity. PAK4 expression is found to correlate with poor tamoxifen response in data from multiple clinical databases. We show a feed-forward regulation between PAK4 and ERα signaling. PAK4, which has been demonstrated to be a direct target gene of ERα, increases ERα stability and phosphorylates ERα on the S305 site. This phosphorylation facilitates activation of ERα signaling.

In conclusion, our data identify the E3 ubiquitin ligase RNF31 as a modulator of both ERα and p53 protein levels, thus facilitating breast cancer cell proliferation in a dual manner. We also identify that PAK4 plays a role in ERα signaling and tamoxifen resistance and that AP-1 regulates ZEB2 and contributes to EMT phenomenon. We suggest that RNF31 and PAK4 might be useful therapeutic targets in ERα-positive breast cancer and add new knowledge about the role of ZEB2 in TNBCs.

List of scientific papers

I. J Zhu, C Zhao, A Kharman-Biz, T Zhuang, P Jonsson, N Liang, C Williams, C-Y Lin, Y Qiao, K Zendehdel, S Strömblad, E Treuter, K Dahlman-Wright: The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation. Oncogene 01/2014; 33(4340-4351).
https://doi.org/10.1038/onc.2013.573

II. Yichun Qiao, Chiou-Nan Shiue, Jian Zhu, Ting Zhuang, Philip Jonsson, Anthony P H Wright, Chunyan Zhao, Karin Dahlman-Wright: AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFalpha-induced epithelial-mesenchymal transition in triple-negative breast cancer. Oncotarget, 2015. 6(10): p. 7804-14.
https://pubmed.ncbi.nlm.nih.gov/25762639

III. Jian Zhu, Chunyan Zhao, Ting Zhuang, Philip Jonsson, Cecilia Williams, Staffan Strömblad, Karin Dahlman-Wright. RING finger protein 31 (RNF31) promotes p53 degradation in breast cancer cells. Oncogene 07/2015.
https://doi.org/10.1038/onc.2015.260

IV. Ting Zhuang, Jian Zhu, Zhilun Li, Julie Lorent, Karin Dahlman-Wright, Staffan Strömblad. Pharmacological targeting of p21-activated kinase-4 inhibits estrogen receptor alpha signaling and restores tamoxifen-sensitivity in breast cancer cells. [Manuscript]