Regulation of V-ATPases in Amino Acid Sensing and Function of Subunit a Isoforms in Breast Cancer Growth and Metastasis
Collins, Michael.
2021
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Thesis (Ph.D.)--Tufts University, 2021.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Michael Forgac.
Committee: Peter Juo, Brent Cochran, and Philip Hinds.
Keyword: Cellular biology.
Vacuolar H+-ATPases (V-ATPases) are evolutionarily ancient enzymes that function in many physiological and disease processes. V-ATPase-dependent ... read moreproton transport governs the lumenal pH of various intracellular compartments, and is essential to processes such as membrane traffic, autophagic flux, and the coupled transport of small molecules. V-ATPase activity is dynamically controlled in vivo by the reversible dissociation of its component V1 and V0 domains, in a process termed regulated assembly. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes through an unknown signaling pathway. To identify the signaling pathways that control amino acid dependent changes in lysosomal V-ATPase activity, we began by using pharmacological inhibitors of kinases important in controlling cellular metabolism. We find that inhibitors of cAMP-dependent protein kinase (PKA), AMP-activated protein kinase (AMPK), and Ser/Thr kinase Akt, all block amino acid-dependent changes in lysosomal V-ATPase activity. However, while CRISPR-mediated gene disruption confirmed the importance of Akt in controlling the amino acid starvation response, PKA and AMPK were found to be dispensable. Furthermore, cells expressing only Akt1 or Akt3 respond normally to amino acid starvation, while cells expressing only Akt2 show a greatly diminished increase in lysosomal V-ATPase activity. These Akt-dependent changes in lysosomal V-ATPase activity appear to be mediated by V-ATPase assembly, as cells expressing only Akt1 display a normal increase in V-ATPase assembly upon amino acid starvation, while cells expressing only Akt2 display no change. These results show that Akt is required for controlling the rapid response of lysosomal V-ATPase activity and assembly to changes in amino acid availability, and that this response depends on specific Akt isoforms. While V-ATPases are localized intracellularly in all eukaryotes, they also function at the plasma membrane of certain cell types, including cancer cells. Membrane targeting of the V-ATPase is controlled by isoforms of subunit a, and we have previously shown that isoforms a3 and a4 are particularly important for the migration and invasion of several breast cancer cell lines in vitro. In order to test the hypothesis that a isoforms are important for in vivo metastasis, we first used CRISPR/Cas9 to selectively disrupt each a isoform in the 4T1-12B model of murine metastatic breast cancer. We find that in 4T1-12B cells, only a4 is essential for in vitro migration and invasion. Furthermore, only disruption of a4 significantly reduced plasma membrane V-ATPase localization and the extracellular acidification rate (ECAR). Finally, by implanting CRISPR-modified clones orthotopically in immunocompetent mice, we find that a4 is important for the growth and survival of 4T1-12B tumors in vivo. BALB/c mice bearing a4−/− 4T1-12B allografts had significantly smaller tumors than mice in the control group, as determined both by caliper measurement and bioluminescence imaging. These studies reveal that isoform a4 is critical for the invasiveness of certain breast cancer cells, and highlight CRISPR/Cas9 as an important tool for studying the role of individual V-ATPase subunit genes in vivo.read less - ID:
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