Development of Novel Therapeutic Strategies for Targeting Metastatic Cancer Cells and Overcoming Cancer Stem Cell Chemoresistance

摘要

90% of all cancer related patient mortalities are due to metastasis, the process by which cancer cells spread and seed secondary tumors at distant sites in the body. Currently, there are no effective therapies aimed at targeting metastasis due to the complex nature and multitude of pathways involved in this biological process. To address this important aspect of cancer biology, we sought to investigate key modulators of the extracellular matrix (ECM), as this is a critical determinant for a tumor cells capacity to escape and invade adjacent tissue structures. Matrix Metalloproteinases (MMPs) represent the most prominent family of proteinases associated with tumorigenesis yet drugs designed to inhibit their proteolytic activities largely failed in clinical trials due to issues with selectivity for individual MMPs. To combat this issue, we utilized a mutagenesis approach to investigate whether a unique and druggable region of the protein outside the catalytic domain existed for potential use as an anti-metastatic drug. To this end, we identified the hemopexin-like (PEX) domain as a potential candidate and have shown that the PEX domain of latent MMP-9 (proMMP-9) enhances cancer cell migration through self-interaction and hetero-interactions with cell surface proteins such as CD44 and alpha4beta1 integrin. In addition to this, we have also shown that these interactions can be disrupted through the use of a small molecule inhibitor 1 (N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide) which binds within the central cavity of the PEX domain of proMMP-9. Through rational drug optimization, we have since improved the potency of this lead compound without losing specificity for a single MMP. We have also demonstrated that treatment with either parental compound 1 or lead derivative compound 3c results in attenuated Src kinase activation in fibrosarcoma cells resulting in a loss of focal adhesion complex formation and an overall decrease in cancer cell migration.;Another approach outside of inhibiting the migratory and invasive potential of cancer cells was to develop a strategy to target cells that have already metastasized and seeded a secondary tumor. Metastasis is a primary driver of malignant progression by imposing a powerful selective pressure, resulting in a more aggressive population of cancer cells that can resist death and escape the harsh environment present within the circulatory system. Upon extravasation and colonization of a secondary malignancy, these tumors are often enriched for cancer stem cells (CSCs) and are notoriously hard to eradicate in the clinic. Standard oncology treatments, such as chemotherapy and radiotherapy, often shrink the bulk tumor; however, the tumor tends to relapse due to the presence of these CSCs, which are insensitive to both chemotherapy and radiotherapy. Using a patented high-throughput (HT) three-dimensional (3D) invasion and an OCT-4/NANOG reporter gene assay, we identified nortriptyline (NTP), a tricyclic antidepressant, as a potential anti-CSC drug candidate. Treatment with NTP resulted in decreased invasion and expression of stem cell-determining transcription factors, such as octamer-binding transcription factor 4 (OCT-4), SRY (Sex determining region Y)-box 2 (SOX2), and homeobox transcription factor NANOG with minimal cytotoxicity observed. Overall, we have identified a novel small molecule that targets the PEX domain of proMMP-9 inhibiting cancer cell migration and invasion in addition to a drug that has potential use for CSC differentiation therapy via target- and phenotype-based screening methods.