Aquaporins - from ion channels to human cancers

Abstract

Despite advances in diagnostic techniques and cancer care management, cancer continues to be one of the leading causes of death worldwide. This thesis used two different approaches to advance cancer treatments: i) In the first approach, we used natural compounds as promising sources of new agents for controlling cancer proliferation and metastasis, and ii) second, we developed a mass-throughput technique for exploring the role and significance of aquaporins in cancer development. In phase 1A (chapter 2), using computational and experimental biology approaches, we identified candidate mechanisms of action of a traditional Chinese medicine, Compound Kushen Injection (CKI), in a breast cancer cell line. CKI disrupts the cell cycle and induces apoptosis in breast cancer cells; however, the exact mechanism of its single compounds and their effects on cancer proliferation, migration and invasion remained unknown. High-performance liquid chromatography (HPLC) fractionation and molecular biology techniques were used to define chemical fractions required for CKI to induce apoptosis. Bioinformatic analysis of RNA-seq data revealed correlations between different compounds and gene expression and phenotype. In phase 1B (chapter 3), CKI, fractionated mixtures, and isolated components were tested on migration assays with colon (HT-29, SW-480, DLD-1), brain (U87-MG, U251- MG), and breast (MDA-MB-231) cancer cell lines. Human embryonic kidney (HEK-293) and human foreskin fibroblast (HFF) served as non-cancerous controls. Wound closure, transwell invasion, and live cell imaging showed CKI reduced motility in all eight lines. Fractionation and reconstitution of CKI study on cancer cell lines demonstrated that combinations of compounds were required for activity. Live cell imaging confirmed that whole CKI strongly reduced migration of HT-29 and MDA-MB-231 cells, moderately slowed brain cancer cells, and had no effect on HEK-293. CKI uniformly blocked invasiveness through extracellular matrix. Apoptosis was increased by CKI in breast cancer but not in non-cancerous lines. Cell viability was not affected by CKI in all cell lines. Transcriptomic analyses of MDA-MB-231 indicated down-regulation of actin cytoskeletal and focal adhesion genes with CKI, consistent with the observed impairment of cell migration. As a result, we found the pharmacological complexity of CKI is important for effective blockade of cancer proliferation, cell migration and invasion. In phase 2 (chapter 4-5), our aim was to investigate the primary dogma that aquaporins (AQP) are only permeable to water and glycerol. Aquaporins are of interest internationally as therapeutic targets for treatment strategies in diverse classes of cancers, but understanding of their full range of substrate permeabilities remains incomplete. Our primarily aim was to discover new classes of aquaporins that serve as dual water and ion channels and then provide better insights into the novel function of aquaporins, their mechanism of gating and signaling networks in human-related diseases such cancer. Using a combination of molecular biology, electrophysiology, and computational biology, we introduced the first unbiased screening method for ion channel activity across all 13 classes of human aquaporins, addressing a major gap in knowledge. Using known AQP ion channel, hAQP1, we optimized an assay which, unlike traditional electrophysiology methods, provides 1- an unbiased high-throughput screen of ion channel functionality of diverse phyla, 2- screening a large number of intracellular signals that might govern their activity and function, 3- mass-screening of drugs, and 4- a broad range of mutagenesis study of AQP ion channels shorter time frame. Strikingly, we found all hAQPs appear to have cation permeability, though to some different degree. Moreover, we noted that ion functionality of hAQPs, unlike most of other ion channels, is active from acidic to neutral pH values ( pH 5.0-7.4). Finally, following our study in chapter 4, we used a combination of wet and dry lab approaches to investigate the potential significance of hAQPs in cancer development. Using transcriptome analysis, we identified an association between AQP mRNA expression and cancer severity and their translational importance in patient tissue samples. As a result, we found AQP9, -7, -5 and -3 as the most promising prognostic marker among other hAQPs in common cancers. This was followed by unrevealing these four AQP ion permeability modulatory mechanism using our optimised yeast screening. In summary, this work augmented our understanding of the fundamental properties of natural compounds for cancer treatment and introduced a novel approach to dissecting their downstream targets in different hallmarks of cancer. Moreover, we further discovered a new set of AQP ion channels and revealed their potential prognostic values in cancer. Outcomes from this dissertation are likely to serve as a strong foundation for the future basic research and clinical innovation and shed more light on the significance of ion channels in cancer development and paved the way for developing an AQP-based therapy.