Discovery of IKBKE and CD24 siRNAs for the treatment of triple-negative breast cancer
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Triple-negative breast cancer (TNBC) represents approximately 10-20% of all newly diagnosed breast cancers and is classified as a subtype with the absence of ER, PR, and HER2 expression. TNBC displays high aggressiveness, a tendency to metastasize, a poor prognosis, and a low survival rate. Unlike other breast cancer subtypes, TNBC has limited treatment options due to the lack of targeted therapies. Therefore, there is a significant need to develop new therapies for TNBC. IKBKE, also known as IKKε or IKKi, is a member of the IKK (IκB kinase) family and exhibits high expression levels in various cancers. IKBKE functions as an oncogene in breast cancer and is overexpressed in approximately 30% of breast carcinomas. IKBKE is shown to be aberrantly amplified in TNBC and associated with proliferation, migration, and survival in TNBC cells. Breast cancer also expresses high levels of CD24, which is a heavily glycosylated glycosylphosphatidylinositol (GPI)-anchored surface protein and plays an important role in tumor growth, invasion, and metastasis. Moreover, overexpression of CD24 in tumors is associated with resistance to therapies. Small interfering RNA (siRNA) can specifically knockdown the expression of target genes. It represents a promising tool for cancer therapy as it can silence aberrant genes that are essential for the progression of cancer cells. However, successful siRNA cancer therapy relies on the development of safe and effective RNA delivery systems because naked siRNA is unstable and has limited cellular uptake. Numerous delivery carriers have been investigated to increase the stability and improve the cellular uptake of siRNAs. This dissertation focuses on two primary research objectives. The first objective centered on discovering siRNAs targeting IKBKE and CD24 for the treatment of TNBC. The second research objective aims to utilize a recently discovered anti-PD-L1 human domain antibody as an immune checkpoint inhibitor for cancer immunotherapy. In Chapter 1, we briefly introduced the background of the research, the statement of the problems, and research objectives. In Chapter 2, we reviewed potential treatment options for TNBC and provided a concise introduction to cancer immunotherapy, specifically focusing on PD-1/PD-L1 immune checkpoint inhibitors. In Chapter 3, we utilized a cholesterol peptide-based delivery system to condense IKBKE siRNA to form nanocomplexes for the treatment of TNBC. The stability, cellular uptake, and penetration capability of the cholesterol peptide/siRNA (CCP/siRNA) nanocomplex was significantly increased. Importantly, the CCP/siRNA nanocomplex significantly inhibited tumor growth in an orthotopic TNBC mouse model. These data suggest that IKBKE siRNA could be a promising therapeutic strategy for TNBC. In Chapter 4, we designed four CD24 siRNAs for the treatment of TNBC by targeting different regions of human CD24 mRNA. The results showed the pre-designed siRNA reduced the CD24 expression at both mRNA and protein levels in TNBC cells. CD24 siRNA efficiently inhibited the proliferation, migration, and invasion of TNBC cells. Moreover, silencing of CD24 induced tumor cell apoptosis and cell cycle arrest. Further, we evaluated the association of CD24 expression with doxorubicin resistance. We found that both CD24 mRNA and protein levels were upregulated in doxorubicin-treated MDA-MB-231 cells and CD24 siRNA sensitized MDA-MB-231 cells to doxorubicin which was reflected by a decreased IC50 value. Overall, targeting CD24 with siRNAs may be a promising therapeutic target for TNBC or other cancers with overexpressed CD24. In Chapter 5, we presented the work on the discovery of anti-PD-L1 human domain antibodies (dAbs) using the phage display technique for cancer immunotherapy. In this study, seven anti-PD-L1 domain antibodies were discovered. Among them, the CLV3 dAb showed the highest binding affinity to human and mouse PD-L1 proteins with KD values of 137.5 nM and 266.8 nM, respectively. The CLV3 dAb also exhibited potent binding affinity to PD-L1 overexpressing DU145 cells. CLV3 inhibited PBMC apoptosis in a co-culture system and showed better tumor penetration compared to antibody. The CLV3 dAb significantly inhibited tumor growth and increased the survival of CT26 tumor-bearing mice. The CLV3 dAb is therefore a promising PD-L1 inhibitor that can be used for cancer immunotherapy.
Table of Contents
Introduction -- Literature review -- Suppression of IKBKE with siRNA inhibits the growth of triple-negative breast cancer -- Development of CD24 siRNA for triple-negative breast cancer treatment -- Discovery of a novel human domain antibody targeting PD-L1 for cancer immunotherapy -- Summary and conclusions -- References
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Ph.D. (Doctor of Philosophy)