Polymeric Delivery of siRNA for Breast Cancer Therapy

• Author / Creator

  Parmar, Manoj B

• Breast cancer is the second most common cancer in women, and the leading cause of cancer-related deaths among women. Conventional breast cancer therapeutic strategies such as surgical removal of tumor, radiation therapy and chemotherapy have several limitations and non-specific effects on non-malignant cells, which warrant a search for alternative and specific therapeutic strategies. RNA interference (RNAi) holds a great promise as more specific and targeted therapy for breast cancer. RNAi using short-interfering RNA (siRNA) could silence a specific gene critical for the uncontrolled growth of cancer cells. Since the delivery of siRNA is a main barrier for implementation of RNAi therapy, we explored the potential of a non-viral delivery system using low-molecular weight polyethylenimines (PEIs) substituted with lipidic moieties. Among the library of modified PEIs, linoleic acid substituted PEI (PEI-LA) delivered siRNAs successfully and higher uptake of siRNA/polymer complexes was observed compared to native PEI. Using PEI-LA delivery system, we first identified potential cell cycle protein targets from the library of 169 siRNAs, and cell division cycle protein 20 (CDC20), a recombinase RAD51, and serine/threonine protein kinase CHEK1 have emerged as promising targets in breast cancer cells. After validating all identified cell cycle proteins in vitro, CDC20 siRNA was delivered in vivo. The tumor growth was successfully decreased with CDC20 siRNA delivery. We then explored the potential of combinational siRNA delivery against cell cycle and anti-apoptotic proteins as these proteins are essential for cancer cell growth and survival, and targeting both proteins simultaneously may result into synergism of therapy. After delivering two cell cycle proteins (TTK protein kinase and CDC20) and an anti-apoptotic protein (survivin), we observed synergistic effects of combinational siRNA therapy in breast cancer cells for selected siRNAs. We also determined the potential non-specific effects of combinational siRNA delivery in non-malignant cells in vitro. Non-specific effects of siRNA could be minimized with specific formulation of siRNA/polymer complexes. We then focused on to tackle metastasis of breast cancer. Several reports confirmed overexpression of protein phosphatases in metastatic breast cancer, which laid the foundation of our hypothesis to target cell cycle and phosphatase proteins simultaneously to decrease not only breast cancer cell growth, but also metastasis. We performed a library screen consisting of 267 phosphatase siRNAs, and identified PPP1R7, PTPN1, PTPN22, LHPP, PPP1R12A and DUPD1 as potential phosphatase targets to decrease migration of breast cancer cells. Down-regulation of CDC20 and identified phosphatase by siRNA inhibited breast cancer cell growth as well as migration. Here we used hyaluronic acid modified siRNA/PEI-LA polyplexes that showed higher siRNA efficacy compared to non-modified complexes. The higher efficacy of siRNA was due to improved physicochemical characteristics of polyplexes, such as better dissociation of siRNA from its carrier and better availability of siRNA in the cytoplasm. These polyplexes were also used to deliver CDC20 and survivin siRNAs, which inhibited breast cancer cell growth significantly regardless of its phenotype. However, these siRNAs inhibited non-malignant cell growth as well. A careful formulation of siRNA polyplexes is needed to minimize side-effects of siRNA delivery to normal cells. Overall, we established an importance of targeting cell cycle proteins in breast cancer as well as formulation of siRNA polyplexes with a functional and non-toxic siRNA delivery carrier (PEI-LA). The effects of siRNA therapy seem to be independent of breast cancer phenotypes, so that this therapy could be functional for a range of breast cancers and additionally in other types of cancers.

• Subjects / Keywords

  • Breast cancer therapy
  • siRNA delivery
  • Cell cycle proteins
  • Anti-apoptotic proteins
  • Protein phosphatase

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-fabb-w982

• License

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