a Investigation of glycine transporter GLYT1 in tumour cell proliferation

Abstract

Rapidly proliferating cancer cells consume significantly more glycine than slow growing cancer cells and rapidly proliferative normal cells. Extracellular glycine supports high growth rates of tumour cells, being utilised in purine and glutathione synthesis. Glutathione, the major cellular antioxidant, is involved in chemotherapy resistance and cancer progression. It was observed that glycine transport via a specific glycine transporter GLYT1 is utilised to maintain adequate glutathione levels under cellular stress. Additionally, GLYT1 is regulated by the transcription factor ATF4, itself upregulated under cellular stress and also linked to cancer progression. Thus, extracellular glycine may play an essential role in tumour cell survival and proliferation and inhibiting its uptake could be a possible target in association with current cancer treatments. To address this hypothesis the work described in this thesis evaluated the role of a specific glycine transporter, GLYT1, in supporting tumour cell proliferation utilising molecular and pharmacological approaches. A549, a non-small cell lung cancer (NSCLC) cell line, and HT29, a colorectal cancer cell line, were utilised as models of rapidly proliferating tumour cells. A498, a renal cancer cell line, and HUVEC, a human umbilical vein endothelial cell, were utilised as models of slow growing tumour cell and non-transformed fast growing cell line, respectively. Following GLYT1 knockdown (kd), tumour cells presented a significant reduction in glycine uptake and cell proliferation. When compared to control cells, rapidly proliferating GLYT1kd tumour cells showed a reduction of ~30% in cell number over time and a decrease of ~50% in DNA replication rate, whereas it had only a minor or no effect on the other cell lines. GLYT1kd downregulated expression of mRNA of other amino acid transporters able to transport glycine, PAT1 and SNAT2. GLYT1kd also reduced glutathione levels of all tumour cells. A reduction in mRNA levels of cystine transporter subunit, xCT, essential to maintain intracellular levels of cysteine, another component of glutathione, was also observed following GLYT1 downregulation. When cells were treated with a specific GLYT1 inhibitor, ALX-5407, in a pharmacological approach, a reduction in cell proliferation was observed after 96h treatment with a prolonged doubling time for the fast growing tumour cells, while A498 and HUVEC cells proliferation were not affected by the inhibitor, when compared to untreated cells. ATF4 effect on cell proliferation and glycine uptake was also evaluated. ATF4kd decreased significantly GLYT1 mRNA levels, but had only a mild influence on glycine uptake of all cell types analysed. There was a significant reduction in proliferation of ATF4kd tumour cells, ~40% for A549 and HT29 cells and 26% for A498 cell; ATF4kd in HUVEC cells increased cell proliferation by ~11%, when compared to control cells. ATF4 was also important to maintain intracellular levels of glutathione. ATF4kd dowregulated the transcription factors involved in stress response (ATF3, ATF5 and ATF6) suggesting a connection between them. Data from this work show that glycine uptake by GLYT1 is required to maintain proliferation rates of highly proliferative cancer cells, but not slowly proliferating and nontumour cells. This suggests that targeting GLYT1 may be a possible new approach to cancer treatment.