Abstract
Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. Here, we analyse the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. We show that independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, our data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention.
Highlights
During cancer growth, tumour cell adaptation is driven by adverse microenvironmental conditions such as hypoxia and starvation.[1]
By using an inducible proton-assisted amino-acid transporter 4 (PAT4) short hairpin RNA (shRNA) knockdown in HCT116 colorectal cancer cells, we find that PAT4 responds to two rapidly metabolised, non-essential amino acids, glutamine and serine,[26,27] to drive rapamycin-resistant, Mechanistic target of rapamycin complex 1 (mTORC1)-mediated cell proliferation
Validation of a novel PAT4 monoclonal antibody We generated a highly specific mouse monoclonal antibody against PAT4. Staining with this antibody revealed that PAT4 was localised to an asymmetric perinuclear region in formalin-fixed, paraffin-embedded 786-O renal cancer cells, which express high PAT4 levels, and lost in 786-O cells transfected with PAT4 small interfering RNA (Figures 1a and b)
Summary
Tumour cell adaptation is driven by adverse microenvironmental conditions such as hypoxia and starvation.[1]. How changes in mTOR structure[10] or mTOR regulators modulate rapamycin sensitivity remains of considerable interest
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