Aquaporins - from ion channels to human cancers
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(Thesis)
Date
2023
Authors
Nourmohammadi, Saeed
Editors
Advisors
Yool, Andrea
Adelson, David
Adelson, David
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Thesis
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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.
School/Discipline
School of Biomedicine
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, School of Biomedicine, 2023
Provenance
This thesis is currently under Embargo and not available.