Abstract
The purpose of this study is to provide an increased understanding of the molecular mechanisms responsible for mammalian polyamine transport, a process that has been a long-standing ‘black box’ for the polyamine field. Here, we describe how ATP13A3, a P-type ATPase, functions as a polyamine transporter in response to different polyamine stimuli and polyamine-targeted therapies in highly proliferating pancreatic cancer cells. We assessed the expression, cellular localization and the response of the human ATP13A3 protein to polyamine treatments in different pancreatic cancer cell lines using Western blot and immunofluorescence microscopy. Using CRISPR mutagenesis and radiolabeled polyamine uptake assays, we investigated the role of ATP13A3 protein in polyamine transport. Highly metastatic cancer cells with high polyamine import express higher levels of the full-length ATP13A3 compared to cells with slow proliferation and low import activity. Highlighting its role in polyamine trafficking, the localization of ATP13A3 is altered in the presence of polyamine stimuli and polyamine-targeted therapies in these cells. Using CRISPR mutagenesis, we demonstrate that the first membrane-associated domain of this protein is critical and indispensable for its function as a spermidine and spermine transporter in cells. Further analysis of existing databases revealed that pancreatic cancer patients with high expression of ATP13A3 have decreased overall survival consistent with the role of intracellular polyamines in supporting tumor growth. Our studies shed light on the mysterious polyamine transport process in human cells and clearly establishes ATP13A3 as an intrinsic component of the spermidine and spermine transport system in humans.
Highlights
The purpose of this study is to provide an increased understanding of the molecular mechanisms responsible for mammalian polyamine transport, a process that has been a long-standing ‘black box’ for the polyamine field
Our prior study showed that different human pancreatic cancer cell lines exhibited different rates of radiolabeled SPD (3H-SPD) uptake under DFMO pressure and characterized these cell lines, L3.6pl, Panc[1], and AsPC-1 cells as high, medium, and low polyamine importers, respectively[15]
Our studies reveal a mechanistic understanding of how ATP13A3 functions in polyamine transport in mammalian cells
Summary
The purpose of this study is to provide an increased understanding of the molecular mechanisms responsible for mammalian polyamine transport, a process that has been a long-standing ‘black box’ for the polyamine field. Different pancreatic cancer cell lines demonstrate preferential expression of the ATP13A3 protein and exhibit distinct cytosolic and plasma membrane localization patterns in the presence of polyamine stimuli and polyamine-targeted therapies. CRISPR mutagenesis revealed that the first membrane-associated domain of the ATP13A3 protein is critical for its function as a polyamine transporter, for spermidine and spermine import into cells. This discovery provides an important first step in understanding the role of proteins involved in the polyamine transport pathway and provides a defined target for combination therapies that seek to starve cancers of their polyamine pro-growth factors[9]
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