Autophagy-deficient tumor cells rely on extracellular amino acids to survive upon glutamine deprivation

ABSTRACT

Macroautophagy/autophagy is a unique protein degradation process by which intracellular materials are recycled for energy homeostasis. However, the metabolic status and energy source of autophagy-defective tumor cells is poorly understood. Here in this study, we found ATF4-dependent amino acid transporter (AAT) gene expression and amino acid uptake were increased in autophagy-deficient cells under conditions of Gln deprivation. Notably, inhibition of amino acid uptake reduced the viability of Gln-deprived autophagy-deficient cells, but not significantly in wild-type cells, suggesting the reliance of autophagy-deficient tumor cells on extracellular amino acid uptake.

Autophagy is a unique protein degradation process by which intracellular materials are recycled for energy homeostasis. In contrast to the survival function of autophagy, autophagy defects that impair survival can promote tumorigenesis. However, the metabolic status and energy source of autophagy-defective tumor cells is poorly understood.

Glutamine is a key substrate required for anabolic growth of cancer cells. Increased consumption of glutamine results in selective depletion of glutamine in tumors compared with normal tissues. Therefore, the investigation of strategies that cells use to adapt to glutamine limitation reveals novel opportunities for cancer therapy. In our study, we found that the concentrations of several intracellular amino acids, including essential amino acids, increase under glutamine starvation [1]. More importantly, the increase was augmented in autophagy-deficient cells (atg3 or atg7 knockout cells). By using stable isotope-labeled leucine and glycine, we observed that upon glutamine starvation, autophagy deficiency enhances amino acid (both essential amino acids and non-essential amino acids) uptake during glutamine starvation, thus contributing to the increased concentration of intracellular free amino acids. Because the amino acids are consumed by different metabolic pathways to create energy, we quantified isotope incorporation in metabolites. The data indicate that autophagy-deficient cells utilize extracellular amino acids to create energy in the absence of Gln.

To identify the mechanism for increased amino acid uptake in autophagy-deficient cells, we tested the expression level of amino acid transporter genes, which mediate the uptake of amino acids. We found that, during glutamine starvation, several AAT genes are upregulated in autophagy-deficient cells compared with wild-type cells, including SLC6A9, SLC7A1, SLC7A5, SLC36A1, SLC36A4, SLC38A1 and SLC38A3. During amino acid-deprivation response, ATF4 has a key role in maintaining amino acid homeostasis and regulates a wide array of AAT genes by binding to the CEBP-ATF response element (CARE). Stable knocking-down of ATF4 blocks the induction of AAT gene expression under glutamine starvation even in autophagy-deficient cells. In addition, ATF4 is enriched at these gene promoters after glutamine starvation, and the enrichment of ATF4 is further enhanced in autophagy-deficient cells. Furthermore, ATF4 knockdown decreases the uptake of isotope-labeled amino acids and the concentration of intracellular amino acids, demonstrating that the transcriptional regulation of AAT genes by ATF4 controls the amino acid uptake in autophagy-deficient cells.

Then, we identified SIRT6, a NAD⁺-dependent histone deacetylase, as an interaction partner of ATF4. The interaction between ATF4 and SIRT6 suggests that SIRT6 might influence ATF4-dependent transcription. In response to glutamine starvation, SIRT6 is recruited to the promoters of AAT genes dependent on its interaction with ATF4. Several studies have shown that SIRT6 play its transcriptional co-repressor functions predominantly by deacetylating histone H3 lysine 9 and histone H3 lysine 56 (H3K56). Consistently, we found that SIRT6 is recruited to promoters of AAT genes to deacetylate H3K56, leading to ATF4 destabilization from target gene chromatin. In contrast, in autophagy-deficient cells, the recruitment of SIRT6 is abolished, indicating that genes bound by ATF4 recruit SIRT6 in an autophagy-dependent manner.

Next, we sought to examine the molecular mechanism for how autophagy regulates SIRT6 recruitment to ATF4 target genes. Dysregulation of autophagy results in prolonged NFE2L2 activation due to SQSTM1/p62 accumulation, which interrupts the interaction between NFE2L2 and KEAP1. We observed that the interaction between ATF4 and SIRT6 is repressed by NFE2L2. Additionally, NFE2L2 knockdown reduces AAT expression and amino acid uptake. Moreover, NFE2L2 knockdown significantly decreases binding of ATF4 to AAT gene promoters in an autophagy-deficient cell line. In contrast, glutamine starvation-induced occupancy of SIRT6 at AAT gene promoters increases after NFE2L2 is knocked down. Furthermore, NFE2L2, but not NFE2L2∆neh1,3 which cannot interact with ATF4, rescues the decreased binding of ATF4 and increased binding of SIRT6 at AAT gene promoters, demonstrating that NFE2L2 represses the recruitment of SIRT6 to CARE elements by competitive binding to ATF4. These results indicate that autophagy-deficiency-induced NFE2L2 accumulation disrupts the binding of SIRT6 and ATF4, resulting in the upregulation of AAT gene expression and amino acid uptake.

We next tested whether amino acid uptake results in the survival of autophagy-deficient cells upon glutamine withdrawal. Autophagy deficiency significantly decreases the percentage of viable cells in response to total amino acid deprivation, but not to glutamine deprivation, suggesting that extracellular amino acids are critical for autophagy-deficient cell survival. By using different combinations of amino acid deprivation, we found that Leu, Ile, Thr, Lys, Val, His, Gly and Tyr, which showed higher concentrations in autophagy-deficient cells than in wild-type cells, were important for the survival of autophagy-deficient cells under glutamine starvation.

In addition, we also utilized AAT inhibitors to specifically suppress amino acid transporter activity in HCT116 cells. Compared with wild-type cells, autophagy-deficient cells with AAT inhibition show significantly decreased viability following glutamine depletion, indicating the reliance of autophagy-deficient cells on extracellular amino acid uptake. Furthermore, tumor growth experiments in a mouse model also confirm the role of autophagy in cancer cell sensitivity to AAT inhibitors. Hence, our observations suggest that autophagy-deficient tumor cells rely on extracellular amino acids to survive upon glutamine deprivation (Figure 1).

Figure 1.

ATF4-dependent AAT gene expression and amino acid uptake are increased in autophagy-deficient cells under Gln deprivation. Amino acid uptake from the extracellular environment is increased in autophagy-deficient cells upon glutamine deprivation, resulting from ATF4-dependent upregulation of AAT gene expression. SIRT6, a NAD⁺-dependent histone deacetylase, is a corepressor of ATF4 transcriptional activity. In autophagy-deficient cells, activated NFE2L2 enhances ATF4 transcriptional activity by disrupting the interaction between SIRT6 and ATF4. In this way, autophagy-deficient cells exhibit increased AAT expression and show increased amino acid uptake.

Funding Statement

This study was supported by the National Key R&D Program of China (2017YFA0503900), National Natural Science Foundation of China (81472581, 81672712 and 81621063).

Disclosure statement

No potential conflict of interest was reported by the authors.