Tumor-Promoting and -Suppressive Roles of Autophagy in the Same Mouse Model of Braf^V600E-Driven Lung Cancer

Autophagy is a highly conserved cellular process in which cellular components or pathogens are sequestered in autophagosomes and delivered to lysosomes for degradation. Dysregulation of autophagy has been associated with a number of human diseases, including cancer, although the underlying mechanisms are still not well understood (1). The first hint of a potential role of autophagy in cancer came from the observation of frequent monoallelic deletions of the mammalian autophagy gene Beclin1 in sporadic human breast cancers and ovarian cancers. Indeed, studies using mouse models showed that haploinsufficiency in Beclin1 promoted spontaneous malignancies, including lung and liver cancers and lymphomas (2). Furthermore, in a series of elegant studies, White and colleagues have shown that blockade of autophagy in various apoptosis-defective tumor cells led to increased neoplastic transformation caused by the accumulation of p62/SQSTM1, damaged mitochondria and reactive oxygen species (ROS), and increased DNA damage, providing significant mechanistic insights into the tumor-suppressive functions of autophagy (1).

Since these early studies, mouse models with conditional knockout of other autophagy genes in different tissues have been generated, but surprisingly, with the exception of benign liver tumors in the mosaic deletion of Atg5 or liver-specific deletion of Atg7 (1, 3), these did not result in the development of malignancies as one might expect based on results from the Beclin1 haploinsufficiency model. Moreover, although they are associated with susceptibility to various diseases, mutations of other autophagy genes have not been linked directly to human cancers, raising the interesting possibility that only with specific autophagy genes and/or only under particular cellular contexts does autophagy function as a suppressor for tumor initiation. In contrast to the earlier studies, emerging data suggested a paradoxical role of autophagy in promoting the growth and progression of various cancers based on studies both in human cancer cells and new mouse models (1). Elevated autophagy has been observed in various cancers under basal conditions and in tumors treated with chemotherapy or irradiation. More importantly, inhibition of autophagy could sensitize tumor cells to the cytotoxic effects of chemotherapy and ionizing irradiation to benefit cancer treatment. In Ras-transformed cells, autophagy has been shown to facilitate tumor cell survival and proliferation through its role in maintaining aerobic glycolysis and the tricarboxylic acid cycle for cellular energy levels (1, 4). Consistent with these data from cancer cell lines and xenograft models in nude mice, a protumorigenic role of autophagy has been shown directly in a recent study using mouse models of breast cancer in animals with intact immune functions (5). Despite these recent advances, the paradoxical role of autophagy in both suppressing and promoting cancer development and progression as well as the molecular mechanisms associated with each remain major challenges in the field.

In this issue of Cancer Discovery, Strohecker and colleagues (6) describe an innovative approach to addressing these challenges by examining the effects of inactivating the key autophagy gene Atg7 at different stages of the oncogenic process in a mouse model of Braf^V600E-driven lung cancer. They showed that deletion of Atg7 accelerated tumor development initially, but suppressed tumor progression in later stages, converting adenomas to oncocytomas and increasing mouse survival (Fig. 1). Thus, these studies reconcile the paradoxical roles of autophagy in cancer development and progression in the same in vivo model, providing novel insights into our understanding of the context-dependent role of autophagy in cancer.

More importantly, Strohecker and colleagues (6) delved deeper into deciphering the mechanisms by which autophagy deficiency affected cancer development and progression differentially using the unique mouse models as well as tumor cells derived from these mice. On the basis of previous data from cancer cell lines (1), one potential mechanism of the accelerated tumorigenesis resulting from Atg7 knockout is a deficiency of Atg7-null tumor cells in removing dysfunctional mitochondria and consequent accumulation of excess ROS, which can promote tumorigenesis through increased DNA damage and gene mutations. However, the authors also explored the alternative possibility of a role for NRF2 in mediating the tumorigenic effect of Atg7 deletion in these cells. This is based on a very interesting recent report that activating a ROS-detoxification program by increasing NRF2 expression contributed to tumorigenesis in tumor models driven by classical oncogenes, such as Kras^G12D, Braf^V619E, and Myc (7). In addition to activation by oxidative stress to regulate the transcription of antioxidant-defense genes, NRF2 has been shown recently to be regulated by p62 through its interaction with KEAP1, an inhibitor of NRF2. p62 interacts with KEAP1 through a KEAP1 interaction region, which leads to the release (from KEAP1 binding in the cytoplasm) and activation (in the nucleus) of NRF2 (8). Indeed, the authors observed increased Nrf2 accumulation in the nucleus, which is correlated with p62 accumulation in Atg7-deficient tumor cells, raising the possibility that activated NRF2 might contribute to the increased tumorigenicity of these cells. Nevertheless, deletion of Nrf2 did not rescue the phenotype of Atg7-deficient tumors, suggesting that the upregulation of NRF2 and its targets for antioxidant defense mechanism are unlikely to be major players in the increased tumorigenesis observed in the Atg7-deficient tumors.

The observation of an inhibitory effect on tumor growth and progression at late stages upon Atg7 deletion is consistent with recent findings in a breast cancer model (5) and similar to a recent article by the same group showing that Atg7 deletion also suppressed progression of oncogenic Kras-induced lung cancer cells, converting them to oncocytomas (9). However, it is very interesting to note that the current studies identified a critical role of glutamine dependency, rather than defective fatty acid oxidation as shown in the previous studies in Kras-induced tumors, in the suppression of Braf^V600E-induced cancer. The authors found that autophagy-deficient Braf^V600E tumor cells displayed a significant decrease in survival upon starvation in vitro and in nude mice, and, strikingly, the addition of glutamine, but not glucose, was sufficient to rescue starvation-induced cell death. In contrast, addition of the ROS scavenger NAC (N-acetyl-l-cysteine) did not rescue the survival deficiency of Atg7-null tumor cells, suggesting that increased ROS and oxidative stress (responsible for the increased tumorigenesis of autophagy inhibition in early stages) is not involved in the decreased tumor growth and progression at late stages. Along with the previous findings, the current study supports the notion that autophagy may promote tumor progression through different mechanisms in various cellular contexts (e.g., tumors driven by different oncogenic events and/or in different tumors) and may influence cancer development and progression through differential mechanisms. Nevertheless, future studies will be necessary to clarify the molecular and cellular basis for such differences.

Although the data are compelling that defective autophagy is responsible for the observed phenotypes in the Atg7-null mouse model, it should be noted that ATG7 has been shown to regulate p53 to control cell cycle and death during metabolic stress in an autophagy-independent manner (10). Future studies will be necessary to evaluate whether such an autophagy-independent mechanism may also contribute to the observed phenotype during early and/or late stages in this study. Indeed, given that almost all autophagy genes are involved in cellular processes other than autophagy, a major challenge in autophagy research now is to determine whether phenotypes observed in this and various other gene knockout mouse models are due to disruption of autophagy per se or possibly due to the loss of other, autophagy-independent functions of these genes (11). Future development of mouse models that specifically disrupt autophagy function by knockin of mutant alleles of autophagy genes may resolve these critical questions. Likewise, generation of inducible knockout mouse models enabling autophagy to be selectively blocked at different stages of tumorigenesis will be very helpful in dissecting the role of autophagy functions in cancer in vivo.

Other remaining puzzles to be resolved include the mechanisms underlying the transition from stimulation of tumorigenesis in early stages to inhibition of tumor growth and progression in late stages in Atg7-null tumor cells. Although the increased ROS production could induce more DNA damage for the initial increase in tumorigenesis, the authors suggested that this is unlikely to be a major player given that the increased tumor growth is transient, but that other oncogenic signaling pathways activated by ROS may be responsible. An alternative possibility is that the changes (either increased mutations or other activated signaling pathways) may persist, but the glutamine deficiency caused by mitochondrial malfunction could become a more dominant defect in later stages to inhibit tumor growth in Atg7-null tumors. Either of these possibilities remains to be explored.

Despite the need to resolve these and other mechanistic issues, the current study further highlights the critical role of autophagy in maintaining mitochondrial function to sustain tumor cell growth in vivo. We anticipate that effective and novel treatments targeting autophagy and the metabolic pathways it controls in “autophagy-addicted” cancers will be developed on the basis of this and other related studies. It will be also interesting to determine whether the autophagy-dependent glutamine-addiction mechanism observed here is specific to oncogenic BRAF-driven lung cancers or can be extended or generalized to other cancers with BRAF mutation and/or tumors driven by other oncogenic mutations for potential new therapies based on this exciting study.

No potential conflicts of interest were disclosed.

J.L. Guan is supported by NIH grants (primarily CA150926 for work related to this review).