Study on the Roles of Unfolded Protein Response and Ubiquitin-Proteasome System in Radiotherapy Using Nanoparticle Drug Delivery in Triple-Negative Breast Cancer

Nanometer-sized particles have novel structural and physical properties that are attracting great interests from pharmaceuticals for the targeted delivery of anticancer drugs and imaging contrast agents. Histone deacetylase inhibitors (HDACi) block the deacetylation function of HDACs, causing cell cycle arrest, endoplasmic reticulum (ER) stress, differentiation, inhibition of angiogenesis, apoptosis and autophagy in many tumors. Normal cells are relatively resistant to HDACi-induced cell death. It is worth mentioning that HDAC6 plays an important role in the different protein quality control systems. Proteins can be degraded via the ubiquitin-proteasome system (UPS) or via autophagic pathways both of which act as key protein quality control mechanisms. When the accumulation of unfolded or misfolded proteins can induce the aggresomal protein degradation, HDAC6 has an essential role because it can bind both polyubiquitinated proteins and dynein, thereby acting to recruit protein cargo to dynein for transport to aggresomes. Whereas the inhibition of unfolded or misfolded proteins degradation leads to ER stress and stimulates the activation of several ER proteins. The unfolded protein response (UPR) interacts in a coordinated manner with the UPS to alleviate protein misfolding and its cellular consequences. However, if the cells are exposed to prolonged or robust ER stress, they die. Many chemotherapeutic agents and ionizing radiation (IR)-induced ER stress can lead to apoptosis or autophagy in cancer cells. Our previous study found that BNIP3 (Bcl-2/adenovirus E1B 19 kD a protein-interacting protein 3, also known as NIP3), which is a member of Bcl-2 subfamily, is very important role in the IR combination with HDACi treated with triple receptor-negative breast cancer (TNBC). The combination therapy resulted in a significant increase in autophagy and decreased tumor tissue expression of BNIP3. In this project, we will investigate the BNIP3 siRNA or HDAC6 inhibitors (tubacin) using NP drug delivery enhances radiosensitivity in TNBC. To precisely guide and internalize the therapeutic drugs (BNIP3 siRNA or tubacin) to TNBC cells, we used epidermal growth factor receptor (EGFR) targeting RNA aptamers. EGFR is highly amplified (>97%) in TNBC cells. We will utilize iron oxide (Fe3O4) for the synthesis of drugs of NPs. The NPs have excellent biocompatibility and biodegradability due to which it has been approved by the Food and Drug Administration (FDA) for use in a number of therapeutic applications. Here, we outline research goals that hope to achieve within three year. First year: We will synthesize the nanocarriers conjugated with EGFR RNA aptamer. We also analyze the detailed physicochemical characteristics of these NPs. Furthermore, we will examine cytotoxicity and pharmacokinetics of these NPs in vivo. We will modify stable nanocarriers that provide cell targeting, drug storage and controlled drug release. Second year: To evaluate the cytotoxic effects of tubacin and IR used alone or in combination on two TNBC cell lines. Analyzing whether combined treatment can induce TNBC cells to affect UPS and ER stress and further understanding of the different ways cells death is induced by TNBC cells, including the molecular mechanisms of apoptosis and autophagy. We will determine the molecular mechanisms of the interaction of HDAC6 with dynein and induce marked accumulation of ubiquitinated proteins by tubacin. Third year: To establish the orthotopic breast cancer model of luciferase-expressing 4T1 or MDA-MB-231 cell lines. We will use in vivo molecular imaging system (IVIS) to accurately measure internally implanted orthotopic tumors. We will evaluate the anticancer effects of combined treatment with BNIP3 siRNA or tubacin NPs with EGFR RNA aptamer and IR in orthotopic TNBC tumor bearing mice.