Autophagy promotes Braf ^V600E-driven lung tumorigenesis by preserving mitochondrial metabolism

Abstract

The role of autophagy in cancer is complex and context-dependent. Here we describe work with genetically engineered mouse models of non-small cell lung cancer (NSCLC) in which the tumor-suppressive and tumor-promoting function of autophagy can be visualized in the same system. We discovered that early tumorigenesis in Braf ^V600E -driven lung cancer is accelerated by autophagy ablation due to unmitigated oxidative stress, as observed with loss of Nfe2l2/Nrf2-mediated antioxidant defense. However, this growth advantage is eventually overshadowed by progressive mitochondrial dysfunction and metabolic insufficiency, and is associated with increased survival of mice bearing autophagy-deficient tumors. Atg7 deficiency alters progression of Braf ^V600E-driven tumors from adenomas (Braf ^V600E ; atg7^−/−) and adenocarcinomas (trp53^−/−; Braf ^V600E ; atg7^−/−) to benign oncocytomas that accumulated morphologically and functionally defective mitochondria, suggesting that defects in mitochondrial metabolism may compromise continued tumor growth. Analysis of tumor-derived cell lines (TDCLs) revealed that Atg7-deficient cells are significantly more sensitive to starvation than Atg7–wild-type counterparts, and are impaired in their ability to respire, phenotypes that are rescued by the addition of exogenous glutamine. Taken together, these data suggest that Braf ^V600E -driven tumors become addicted to autophagy as a means to preserve mitochondrial function and glutamine metabolism, and that inhibiting autophagy may be a powerful strategy for Braf ^V600E -driven malignancies.

The role of autophagy in cancer is complex and context dependent. Mice with allelic loss of the essential autophagy gene Becn1 are tumor prone and mosaic or liver-specific deletion of Atg5 or Atg7 produces benign hepatomas, suggesting a role for autophagy in tumor suppression. However, autophagy localizes to metabolically stressed, hypoxic regions of solid tumors where it enables tumor cell survival. Oncogene activation upregulates autophagy, required for the maintenance of functional mitochondria, survival during starvation, and tumor growth. In the absence of autophagy, defective mitochondria accumulate, triggering a reduction in mitochondrial respiration that is ultimately incompatible with proliferation and survival. This compromises stress tolerance, prompting us to label these tumors “autophagy-addicted” and to suggest that cancer cells may be particularly vulnerable to autophagy inhibition.

Atg7 Deficiency has Distinct Consequences for Tumor Establishment and Maintenance in Braf ^V600E -Driven Lung Tumors

To directly test the role of autophagy in Braf ^V600E-driven lung cancers, mice with Cre-activatable Braf ^V600E with and without conditional alleles of the essential autophagy gene Atg7 were generated, lung tumorigenesis was induced via intranasal administration of an adenovirus expressing Cre recombinase, and the resulting tumors were followed over time. Compound mice in which the tumor suppressor Trp53 was deleted in tumors were also generated to assess the consequences of Trp53 loss on the need for autophagy to sustain metabolism and tumorigenesis in the mouse lung.

Autophagy-competent tumors progress from hyperplasia to discrete adenomas, forming papillary adenomas (in the Trp53 intact model) or adenocarcinomas (in the Trp53 null model) after which point the mice rapidly succumb due to tumor burden. In contrast, in both the Trp53 intact and null models, autophagy ablation causes robust early tumor growth (between 3 and 5 wk post-Cre) that is associated with increased proliferation, yet by 10 wk post-Cre, the autophagy-deficient tumors display blunted growth. Importantly, the increased tumorigenesis in the mice bearing autophagy-deficient tumors is phenocopied by loss of the master regulator of antioxidant defense, Nfe2l2, indicating that the early tumor growth is driven by increased oxidative stress. Loss of both Nfe2l2 and Atg7 has no additive effect on tumor growth, suggesting that the early burst in tumor proliferation in both cases is caused by increased oxidative stress.

Histological examination of the autophagy-deficient tumors at later times (10 wk post-Cre and beyond) revealed the presence of oncocytes rather than papillary adenomas or adenocarcinomas. Oncocytomas are rare, predominately benign tumors characterized by the accumulation of defective mitochondria. Further analysis of these autophagy-deficient tumors demonstrated an accumulation of defective mitochondria by immunohistochemistry and electron microscopy. Thus, autophagy deficiency alters tumor cell fate from adenomas and adenocarcinomas to oncocytomas. Consistent with conversion to a benign phenotype, Atg7 deficiency extends life span of mice with Braf ^V600E-driven tumors independent of Trp53 status. Importantly, loss of Nfe2l2 in combination with Atg7 deficiency has no additive effect on survival, suggesting that the early burst in tumor proliferation is unable to support continued tumor growth.

Loss of Atg7 Impairs Mitochondrial Metabolism and Survival During Starvation

Having established that autophagy deficiency is associated with promotion of early but impairment of late tumor growth in 2 distinct genetically engineered mouse models of NSCLC, we turned our attention to identifying the mechanism underlying the defective tumorigenesis. Tumor-derived cell lines were isolated from the autophagy-competent and -deficient trp53^−/−; Braf ^{V600E /+} tumors 9 wk post-Cre and analyzed. Atg7-deficient cell lines are unable to survive starvation in Hank’s buffered saline solution and have reduced oxygen consumption rates indicative of impaired mitochondrial respiration. Addition of exogenous glutamine (and to a lesser extent sodium pyruvate), but not glucose or the reactive oxygen species scavenger N-acetyl cysteine, is sufficient to rescue survival during starvation, indicating that autophagy ablation renders TDCLs glutamine-dependent. The inability of N-acetyl cysteine to rescue this phenotype indicates that the failure to survive during starvation is not the result of increased reactive oxygen species levels, but rather is due to a metabolic defect. Indeed, addition of glutamine to starvation media is able to partially restore mitochondrial respiration in the autophagy-deficient TDCLs. Thus, Braf ^V600E -driven tumors become addicted to autophagy as a means to preserve mitochondrial function and glutamine metabolism, suggesting that strategies to inhibit autophagy may be particularly effective for these and other tumors.

Several important points came from this study. First, inhibiting autophagy at different stages of tumorigenesis has different outcomes, confirming the context-dependent role of autophagy in cancer. Second, we provide evidence that Braf-driven tumors are “autophagy-addicted” extending this designation further than RAS-driven tumors. Importantly, in both the Kras and Braf-driven NSCLCs, autophagy ablation extends life span and alters tumor cell fate to oncocytomas suggesting that autophagy deficiency may be a root cause of human oncocytomas and that autophagy inhibition and failure of mitochondrial quality control can convert aggressive cancers to a more benign disease. Finally, we provide evidence that autophagy may supply glutamine to rescue survival during starvation. However, it is likely that there are additional autophagy-supplied substrates that preserve the metabolism and growth of tumors, the identification of which will be important to increase our understanding of the phenomenon of “autophagy-addiction.”

Glossary

Abbreviations:

NSCLC

non-small cell lung cancer

TDCLs

tumor-derived cell lines