Comparative Tumorigenic Amino Acid Starvation and Integrated Stress Responses in Epithelial Vs. Mesenchymal HCC Cells with ASCT2 Or LAT1 Transporter Knockouts

Résumé du livre

The previously established roles of the amino acid glutamine have recently been expanded to include not only protein and nucleotide synthesis, as well as contributing significantly to energy production via mitochondrial hydrolysis of glutamine to glutamate, but also the role of this molecule in the reductive carboxylation of Îł-ketoglutarate resulting in carbon atoms used in cell membrane biosynthesis. Additionally, glutamine participates in maintaining glutathione homeostasis as well as regulation of anabolic signaling. Given its central role in anabolic processes, glutamine is preferentially transported into human cancer cells at a significant rate as compared to normal cells. This distinction has prompted terms such as "glutamine sinks" and "glutamine addiction" to be applied to cancer cells and has likewise led to increased research into therapeutic considerations of altered glutamine metabolism.In this study, human hepatocellular carcinoma (HCC) cell lines were deployed that have been genetically manipulated via CRISPR-Cas9 to have eliminated expression of ASCT2 or LAT1 plasma membrane transporters. ASCT2 and LAT1 expression is consistently enhanced in a broad spectrum of human cancer types, both of which transport glutamine. These ASCT2 and LAT1 knockout HCC lines, compared to the parent lines and non-silencing control, have been evaluated for their ability to form and grow in three-dimensional structures (an ostensible marker of tumorigenicity) using both soft agar and spinner flask modalities. Under conditions of three-dimensional growth, as in initial avascular tumor growth, areas of hypoxia and nutrient limitation evoke adaptive responses vital for cell survival, including the amino acid starvation response (AASR). Therefore, end-point assays were used to compare Amino Acid Starvation Responses (part of the Integrated Stress Responses (ISR)) of the transporter knockout lines versus control lines under monolayer and three-dimensional growth conditions (soft agar/ flask suspension formats).The results show that the CRISPR-Cas9 knockout of the ASCT2 or LAT1 transporters does not diminish the ability of HCC cells to form three-dimensional cellular aggregates. The knockout lines demonstrated significant growth, comparable to the control lines, in all modalities including monolayer plate growth, soft agar spheroid formation, and spinner flask aggregation. Additionally, western blot protein analysis of GCN2 and ATF4 expression (markers of the AASR/ ISR) were performed with inconclusive results for both the more differentiated epithelial HCC line HUH7 and the mesenchymal HCC line SK Hep. The expression of GCN2 and ATF4 varied between monolayer plate growth and three-dimensional aggregate formation without demonstrating definitive initiation of the Amino Acid Starvation Response. This outcome may suggest the involvement of compensatory amino acid transport during avascular growth. An additional consideration, which has been purported by similar studies, is that the effect of ASCT2 or LAT1 transporter suppression may be dependent on cancer cell type.