Investigation of novel RNAi and nanoparticle approaches for their anti-proliferative and drug-sensitizing effects in breast cancer

Abstract

Drug resistivity remains a major challenge in treating different cancer types. Among several strategies adapted to increase drug sensitivity in breast cancer cells, in the present thesis I studied an RNAi molecule targeting cholinergic receptor nicotinic alpha 5 subunit (CHRNA5) and a red-emitting oligomer nanoparticle, the two agents which I experimentally identified as negative regulators of cell proliferation. Cholinergic signaling is implicated in several different pathologies including cancer. Nicotinic acetylcholine receptors (nAChRs) are shown to be involved in regulation of cell proliferation, however they are mainly studied as mediators of nicotinic activity. CHRNA5 subunit has been shown to have roles in acetylcholine (ACh) production/stability, drug addiction and susceptibility to lung cancer. Few studies of lung and gastric cancers as well as high throughput RNAi screens show CHRNA5 as a modulator of cell proliferation. In the present study multiple CHRNA5 isoforms were cloned from MCF7 breast cancer cells (ER positive, TP53 positive) as in the case of lung cancer; moreover, a significant antimitotic effect of CHRNA5 RNAi application was demonstrated in MCF7 breast cancer cells. Similar effect of CHRNA5 silencing was only partially observed in BT-20 and MDA-MB-231 cells (ER negative, P53 mutant), yet in a seeding density-dependent manner. For the first time in literature the transcriptomic changes associated with CHRNA5 RNAi in the MCF7 cells were studied by microarrays from which differentially expressed gene lists were used to obtain the affected pathways. Additional assays confirmed the reduction in cell viability, DNA synthesis, G1 growth arrest, and changes in cytoskeleton complementing the microarray studies. Use of camptothecin (CPT) and doxorubicin (DOXO) in the absence or presence of CHRNA5 siRNA in MCF7, led to identification of CHRNA5’s role in drug sensitivity. Comparisons between CHRNA5 siRNA and public microarray datasets revealed common genes/networks between topoisomerase (TOPO)/cyclin-dependent kinase (CDK) inhibitors and CHRNA5 depletion profile in MCF7 cells. mRNA-miRNA network analysis of differentially expressed common gene sets between TOPO inhibitors and CHRNA5 RNAi treatment identified potential common regulatory miRNAs. In an independent study the anti-cancer as well as drug sensitivity associated effects of a novel CB7-capped, red-emitting conjugated oligomer nanoparticle (Red-CON) were characterized in MCF7 and MDA-MB-231 cells. Red-CON in its encapsulated form exhibited low toxicity and good efficacy as a drug delivery system. This nanoparticle formulation might serve well for future clinical and less toxic chemotherapeutic regimens.