MicroRNAs in HER2-Amplified Breast Cancer

University dissertation from Department of Clinical Sciences, Lund University

Abstract: Popular Abstract in English Breast cancer is the most common cancer in women worldwide and the leading cause of cancer-related deaths. Although survival rates are low compared to other cancers, more than 500,000 women worldwide die of breast cancer every year. It is however not a single disease but rather a collection of different diseases with similar appearance but distinct molecular mechanisms. Thus, in order to have higher chances of therapy success, it is crucial that the tumor is not only detected as early as possible but that treatment is adjusted to the subtype of disease. One of the main breast tumor classifications relies on the presence or absence of three main cellular indicators of biological state, so called biomarkers: estrogen receptor alpha (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). These subdivisions do not only have prognostic value, but also guide treatment choices. Targeted therapies against the hormone receptors (ER and PR) and HER2 have improved clinical outcomes considerably. However, resistance to targeted drugs is frequent and it has become apparent that further, more detailed classifications are required to identify patients that will respond to known therapies as well as to develop new drugs for those that do not. The biomarker HER2 is an oncogene, i.e. it promotes tumor growth when produced at higher levels. Almost a fifth of breast tumors show an increase of the HER2 gene, which contains the hereditary information for the growth factor receptor. Elevated production of HER2 is usually accompanied by lower levels of ER and resistance against ER-related drugs. Our group reported earlier that a rise in production of HER2 might also lead to the increase of a small non-coding RNA, whose gene is located within the HER2 gene. RNAs are transcribed short ‘copies’ of our hereditary information and have important roles in coding, regulation and synthesis of proteins. Non-coding RNAs, unlike coding RNAs, are not translated into proteins but rather exert their full functions on RNA level through sequence-specific or structural interactions with other cellular components. The small non-coding RNA within the HER2 gene is a so called microRNA (miRNA), an emerging class of molecules that regulate the expression of the majority of cellular proteins. The aim of this study was to investigate the levels and biological functions of this miRNA named mir-4728, and to explore its suitability as a biomarker in breast cancer. The results from the first study show that mir-4728 directly regulates ER, which establishes a direct connection between HER2 and ER, two of the main breast cancer biomarkers. The miRNA thus contributes to the observed effect that ER is present at lower levels in HER2 positive breast cancer. This finding can aid in unravelling the complex cellular interactions in breast cancer and help to understand drug resistance mechanisms. The second study focused on the potential threats of miRNA-based drugs. Some miRNAs have very important functions but are lost in diseases like cancer. One therapeutic approach is to replace the cell’s missing miRNAs to restore their function, and the first clinical studies are ongoing. We observed that if quality control is not performed properly by the manufacturers of these replacement miRNAs, there can be large differences between production batches, potentially leading to severe adverse effects with unexpected consequences. This observation is clinically relevant to prevent undesired side-effects in miRNA replacement therapy. The third study investigated miRNA isoforms, variations of the same miRNA, which differ only minimally in sequence but can have very distinct cellular functions. A miRNA that is often elevated in breast and other cancers is miR-21. Our results show that this miRNA is regulated by a degradation pathway, which appears to be low or missing in many tumors. The fourth study then continues to show that the HER2 miRNA mir-4728 actually represses this degradation pathway and thus leads to higher levels of tumor-promoting miR-21. Elevated miR-21 then downregulates the tumor suppressor programmed cell death 4 or PDCD4, which we show to be connected to worse prognoses in HER2 positive breast cancer. Together, these results open up new possibilities for therapeutic strategies and help understand molecular mechanisms in cancer. The fifth study evaluated more global effects of mir-4728. We observed that blocking the function of this miRNA led to a strong decrease of metabolism-related genes in the cell. This indicates that the active miRNA is somehow involved in upregulation of tumor metabolism, thus contributing to cancer progression. In summary, we have found that the microRNA mir-4728 can contribute to tumor development through several mechanisms. Since it is located in the gene of the important breast cancer biomarker HER2 and they are produced together, it might not contribute additional information as a biomarker. However, the molecular mechanisms this microRNA uses to contribute to cancer could be exploited for therapeutic use in the future.