Macroautophagy is dispensable for growth of KRAS mutant tumors and chloroquine efficacy

Abstract

Macroautophagy is a key stress-response pathway that can suppress or promote tumorigenesis depending on the cellular context. Notably, Kirsten rat sarcoma (KRAS)-driven tumors have been reported to rely on macroautophagy for growth and survival, suggesting a potential therapeutic approach of using autophagy inhibitors based on genetic stratification. In this study, we evaluated whether KRAS mutation status can predict the efficacy to macroautophagy inhibition. By profiling 47 cell lines with pharmacological and genetic lossof-function tools, we were unable to confirm that KRAS-driven tumor lines require macroautophagy for growth. Deletion of autophagyrelated 7 (ATG7) by genome editing completely blocked macroautophagy in several tumor lines with oncogenic mutations in KRAS but did not inhibit cell proliferation in vitro or tumorigenesis in vivo. Furthermore, ATG7 knockout did not sensitize cells to irradiation or to several anticancer agents tested. Interestingly, ATG7-deficient and -proficient cells were equally sensitive to the antiproliferative effect of chloroquine, a lysosomotropic agent often used as a pharmacological tool to evaluate the response to macroautophagy inhibition. Moreover, both cell types manifested synergistic growth inhibition when treated with chloroquine plus the tyrosine kinase inhibitors erlotinib or sunitinib, suggesting that the antiproliferative effects of chloroquine are independent of its suppressive actions on autophagy.M acroautophagy is a catabolic pathway that shuttles cytoplasmic components via double-membrane vesicles (autophagosomes) into lysosomes for degradation and recycling. Autophagosome formation and elongation are facilitated by ubiquitin-like molecules such as MAP1LC3A/B (herein referred to as "LC3") and its homologs which are directly conjugated to phosphatidylethanolamine (PE), a reaction which requires the ubiquitin E1-like activity of autophagy-related 7 (ATG7), the E2-like activity of ATG3, and the E3-like activity of the ATG5-ATG12-ATG16L1 complex (1). Autophagy cargo receptors such as p62/ SQSTM1 bind both LC3 and ubiquitinated cargo, enabling cargo recruitment into autophagosomes and delivery to lysosomes (2, 3).Basal levels of macroautophagy control cellular homeostasis by clearing misfolded proteins or damaged organelles (4, 5). Upon starvation, macroautophagy can be induced above basal levels to supply the cell with nutrients (6, 7). This prosurvival function of macroautophagy is also used by cancer cells under conditions of metabolic stress (8). However, the role of autophagy in cancer is complex and context dependent, because the pathway has been reported to have tumor-suppressing as well as tumor-promoting properties (9-11). Liver-specific deletion of ATG7 results in increased formation of liver tumors through the activation of the Nrf2 pathway (12). Furthermore, the essential autophagy component beclin-1 inhibits tumorigenesis of breast carcinoma cells, and monoallelic deletion of beclin-1 is associated with an enhanced risk of breast cancer (13-15). I...

Targeting CD47 and Autophagy Elicited Enhanced Antitumor Effects in Non–Small Cell Lung Cancer

“…To further increase the antitumor effects of targeting CD47 by SIRPaD1-Fc in NSCLC, we examined probable mechanisms of resistance to SIRPaD1-Fc treatment. Autophagy, an important mechanism of tumor therapy resistance, is increasingly regarded as a target for synergistic antitumor therapy (41)(42)(43). We report here that targeting CD47 could trigger autophagy in NSCLC cells.…”

Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells

“…6F). Although human cancer cell lines can be selected to tolerate autophagy deficiency (Eng et al 2016), our findings reveal that up-regulation of glutamine flux to aspartate and nucleotide synthesis is one compensatory adaptation to loss of autophagy. Therefore, inhibiting autophagy, glutamine-mediated metabolic pathways, mitochondrial respiration, or nucleotide synthesis to deplete nucleotide pools is a potential therapeutic strategy for Kras-driven lung cancers.…”

“…In contrast, deletion of essential autophagy genes in human cancer cell lines by genome editing does not impair tumor growth in immune-compromised mice (Eng et al 2016). This suggests that the immune system may suppress growth of autophagy-deficient tumors or that there is selection for compensatory mechanisms that overcome autophagy deficiency.…”

Genetics and biology of pancreatic ductal adenocarcinoma

“…1; Guo et al 2011;Kim et al 2011;Lock et al 2011;Wei et al 2011;Yang et al 2011;Guo and White 2013). However, a recent study has called into question the role of autophagy in promoting oncogenic KRAS-driven tumor cell lines (Eng et al 2016). This discrepancy with numerous prior studies may relate to several experimental issues, including the use of short-term in vitro growth assays not mimicking the in vivo situation as well as the possible emergence of autophagy-independent clones through the selective pressures of gene editing.…”