Receptor Targeted Theranostic Nanoparticles for Treatment of Drug-Resistant Triple-negative breast cancer Open Access

Miller-Kleinhenz, Jasmine (Fall 2017)

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Breast cancer is the most common cancer in women world-wide. About 20% of women with breast cancer are negative for estrogen receptor, progesterone receptor, and HER-2/neu receptor and are diagnosed with triple negative breast cancer (TNBC). TNBC occurs more frequently in young women and African-Americans and presents as an aggressive disease with a poor prognosis. Since those with TNBC do not express the receptors and pathways for which targeted therapies have been developed, the only therapeutic options are chemotherapy and surgery. Unfortunately, the majority of women with TNBC are resistant to chemotherapeutic treatment. Given the poor survival and lack of effective therapeutics for TNBC, the development of new therapeutic approaches for patients with drug-resistant TNBC is essential.

Drug resistance in TNBC has been associated with the presence of a high level of cancer stem-like cells (CSCs). This dissertation, through investigation of the heterogeneity of response to chemotherapy drug, Doxorubicin (Dox) or uPAR-targeted nanoparticle carrying Dox in a human patient derived xenograft (PDX) models of TNBC, identified the Wnt/β-catenin CSC pathway as being overexpressed in drug-resistant tumors compared to tumors that were sensitive to treatment. Based on the overexpression of Wnt co-receptor LRP5/6, we developed a novel Wnt-targeted therapy to effectively treat drug resistant TNBC.

Our results demonstrate that dual targeting of the Wnt and uPAR leads to decreasing of Wnt/β-catenin signaling, inhibition of the CSC phenotype, and epithelial to mesenchymal transition (EMT) in vitro. Systemic delivery of our dual targeted nanoparticles led to nanoparticle-drug delivery into PDX tumors, resulting in stronger tumor growth inhibition compared to non-targeted or single-targeted nanoparticle carrying Dox in a human breast cancer PDX model in vivo.

Altogether, this work confirms the relationship of the Wnt/β-catenin pathway to drug-resistance in TNBC. Our results provide a foundation for the long-term goal of developing targeted therapies for drug-resistant TNBC. Enhanced delivery of chemotherapeutics into breast cancer stem-like cells using dual Wnt receptor and uPAR-targeted theranostic nanoparticles will significantly impact development of novel therapy for drug-resistant TNBC tumors and thereby improve prognoses for TNBC patients.  

Table of Contents

Chapter 1: Introduction                                                                               p. 1     

           1.1 Introduction                                                                                            p. 2

           1.2 Current Treatment for TNBC                                                                  p. 3

           1.3 TNBC Subtypes and Biomarkers for Characterization                          p.5

1.4 Potential cellular targets for the development of TNBC targeted theranostic nanoparticles       p. 9

                       1.4.1 Urokinase plasminogen activator receptor (uPAR)                 p.12

                       1.4.2 Epidermal growth factor receptor (EGFR)                               p.13

                       1.4.3 Insulin growth factor 1 receptor (IGF-1R)                              p.14

                       1.4.4 Mucin 1 (MUC1)                                                                     p.14

                      1.4.5 Folate receptor                                                                          p.15

                       1.4.6 CXCR4                                                                                    p.15

                       1.4.7 Wnt/β-catenin pathway                                                            p.16

1.5 Targeting Cancer Stem Cells in TNBC                                                  p.19

1.6 Current Advances in Cancer Nanotechnology for TNBC                      p.21

                       1.6.1 Liposome Nanoparticles                                                          p.22

                      1.6.2 Polymeric Nanoparticles                                                         p.25

                       1.6.3 Carbon Nanotubes                                                                    p.28

                       1.6.4 Metallic Nanoparticles                                                              p.29

           1.7 Conclusions                                                                                              p.33

           1.8 Scope of this Dissertation                                                                       p.35

Chapter 2: Characterization of Differential Drug Response TNBC PDX tumors after treatment with uPAR targeted theranostic nanoparticles  p.36

           2.1 Introduction                                                                                             p.37

           2.2 Materials and Methods                                                                            p.41

           2.3 Results                                                                                                     p.44

                       2.3.1 Generation of Primary Orthotopic Drug-Resistant

TNBC Patient Derived Xenograft                                                    p.44

2.3.2 ATF-IONP-Dox inhibits tumor growth in

PDX of Drug-Resistant TNBC                                                          p.48

2.3.3 Cytotoxicity in PDX of Drug-Resistant TNBC                       p.51

2.3.4 Heterogenous Response in Drug-Resistant

TNBC after treatment with Dox or ATF-IONP-Dox                       p.54

2.3.5 Characterization of Heterogenous Phenotype in Drug-Resistant TNBC after treatment withDox or ATF-IONP-Dox         p.56

2.3.6 Heterogeneity of targeted delivery of ATF-IONP-Dox in TNBC PDX Tumors      p.58

2.3.7 Intra- and Inter- Phenotypic Characteristics of Drug-Resistant TNBC after treatment with Dox or ATF-IONP-Dox      p.60    

2.4 Discussion                                                                                                p.62    

Chapter 3: Dual-targeting Wnt and uPA Receptors inhibits Cancer Stem-Cell Phenotype in Chemo-Resistant Breast Cancer    p.64

           3.1 Abstract                                                                                                  p.65

           3.2 Introduction                                                                                             p.66

           3.3 Materials and Methods                                                                            p.70

           3.4 Results:                                                                                                    p.78

                       3.4.1 Chemo-resistant breast cancer cells have upregulated levels of biomarkers associated with cancer stem cells.      p.78

3.4.2 Development and characterization of Wnt/LRP and uPAR single targeted and dual targeted IONPs         p.81

3.4.3 Characterization of iWnt and iWnt-ATF24 IONPs in vitro                   p.84

3.4.4 iWnt-ATF24 IONPs decreased the activation of Wnt/ β-catenin pathway and cell invasion in human breast cancer cells in vitro.        p.87

3.4.5 Targeted IONP treatment reduced the levels of cancer stem cell associated biomarkers without affecting cell proliferation and cell cycle status.        p.90

3.4.6 Inhibition of Wnt/LRP signaling and uPAR using single or dual receptor targeted IONP carrying Dox had a similar effect on tumor cell viability as the conventional Dox in vitro.                                            p.94

3.4.7 Targeted delivery of iWnt-IONPs, ATF24-IONPs and iWnt-ATF24-IONPs into orthotopic breast PDX tumors in nude mice following systemic administration.          p.97

3.4.8 Targeted delivery of iWnt-ATF24-IONP-Dox downregulated CD44, uPAR, and Wnt signaling, leading to decreased cell proliferation and tumor growth inhibition in an orthotopic chemo-resistant breast cancer PDX model                     p.99

3.5 Discussion                                                                                               p.103

3.6 Supporting Information                                                                           p.111

Chapter 4: Discussion                                                                           p.117

           4.1 Summary                                                                                                  p.118

           4.2 Limitations of Study                                                                                p.124

           4.3 Future Directions                                                                                   p.125

           4.4 Supporting Information                                                                           p.129

References                                                                                                                p.130

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