Potential novel molecular targets for breast cancer diagnosis and treatment

Breast cancer possesses the highest incidence among cancers in women worldwide, accounting for approximately 26% of all cancers in women. Due to the discovery of specific predictive and prognostic biomarkers, breast cancer therapy has been significantly promoted in the past decades through the application of more individualized therapies to diverse subcategories with different clinical behavior. Among the established biomarkers for breast cancer, estrogen receptor alpha (ERα) and human epidermal growth factor receptor 2 (HER2) are the most potent biomarkers both in determining prognosis and predicting response to hormone therapies. However, there is a clear need to identify additional biomarkers as some subtypes of breast cancer do not express ERα and/or HER2 and additionally, there is no perfect correlation between these biomarkers and the response to targeted treatment. Therefore, the overall aim of this thesis was to evaluate potential new biomarkers for breast cancer diagnosis and to improve therapeutic strategies in breast cancer.

Estrogen signaling has been proven to play a key role in breast cell growth, differentiation and development of breast cancer. It has been well documented that ERα can directly interact with cis-regulatory elements; estrogen response elements (EREs) or indirectly with other cisregulatory elements via protein-protein interactions, such as activator protein-1 (AP-1), to regulate transcription of target genes. The AP-1 as a transcription factor is a dimeric complex that includes members of the JUN and FOS protein families. Studies have indicated a role for these proteins as potential biomarkers in breast cancer. However, a systemic analysis of the expression of all AP-1 family members as potential biomarkers in breast cancer and their interaction with ERα is still lacking.

In paper I, we examined the expression levels of seven AP-1 family members in human breast tumors and adjacent non-tumor tissues and correlated their expression with available clinicopathological parameters. We observed that the expression of all AP-1 family members except Fos-B was significantly elevated in tumor compared with adjacent non-tumor tissues. Interestingly, we observed that the Fra-1 expression level was significantly higher in the tumors classified as ERα-negative and progesterone receptor (PR) negative. Furthermore, Fra-1 expression was shown to significantly distinguish triple-negative tumors compared from luminal carcinomas.

ERα is overexpressed in the majority of breast cancers and promotes estrogen-dependent cancer progression by regulating the transcription of genes related to cell proliferation. Antiestrogens are successfully used to treat these tumors. However, in many cases resistance to this endocrine treatment develops. Therefore, insights into the molecular mechanisms that regulate ERα expression and stability are of highest importance to promote breast cancer diagnostics and therapeutics.

In paper II, we found that the atypical E3 ubiquitin ligase RNF31 can stabilize ERα and facilitate ERα-stimulated proliferation in breast cancer cell lines. This study proposes a nongenomic mechanism by which RNF31 regulates ERα expression and stability and controls the transcription of estrogen-dependent genes related to breast cancer cell proliferation.

RNF31 is one of three members in the linear ubiquitin chain assembly complex (LUBAC). In paper III, we investigated mRNA and protein expression levels of all three members of the LUBAC complex, including RBCK1, RNF31 and SHARPIN, in human breast tumors and adjacent non-tumor tissues and correlated their expression with various clinicopathological parameters. We found that all members of the LUBAC complex were significantly higher expressed in tumors compared to adjacent non-tumor tissues. We also found that the RNF31 protein expression level was significantly higher in ERα-negative tumors compared to ERα- positive tumors.

In paper IV, we identified the existence of a potential fusion transcript, called RNF31/IRF9, and a corresponding potential fusion protein. Interestingly, the potential novel fusion protein was present in the nuclei of breast tumors but not in the nuclei of normal breast tissues. In addition, the expression of the potential fusion protein was significantly higher in ERα- positive tumors compared to ERα-negative tumors.

In summary, the work presented in this thesis contribute to the understanding of estrogen signaling in breast cancer and identify and suggest a group of proteins that are candidates as potential novel biomarkers and/or drug targets to improve therapeutic strategies in breast cancer.

List of scientific papers

I. Kharman-Biz A, Gao H, Ghiasvand R, Zhao C, Zendehdel K, Dahlman-Wright K. Expression of activator protein-1 (AP-1) family members in breast cancer. BMC cancer (2013) 13: 441.
https://doi.org/10.1186/1471-2407-13-441

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

III. Kharman-Biz A, Gao H, Ghiasvand R, Haldosen LA, Zendehdel K and Dahlman-Wright K. Expression of three members ofthe linear ubiquitin assembly complex in breast cancer. [Manuscript]

IV. Kharman-Biz A, Gao H, Ghiasvand R, Kuiper R, Haldosen LA, Zendehdel K, Dahlman-Wright K. Identification of a Potential Novel Fusion Transcript, RNF31/IRF9, in Breast Cancer. [Manuscript]