Autophagy promotes mammalian survival by suppressing oxidative stress and p53.

Autophagy captures intracellular components and delivers them to lysosomes for degradation and recycling. Conditional autophagy deficiency in adult mice causes liver damage, shortens life span to 3 mo due to neurodegeneration, and is lethal upon fasting. As autophagy deficiency causes p53 induction and cell death in neurons, we sought to test whether p53 mediates the lethal consequences of autophagy deficiency. Here, we conditionally deleted Trp53 (p53 hereafter) and/or the essential autophagy gene Atg7 throughout adult mice. Compared with Atg7Δ/Δ mice, the life span of Atg7Δ/Δp53Δ/Δ mice was extended due to delayed neurodegeneration and resistance to death upon fasting. Atg7 also suppressed apoptosis induced by p53 activator Nutlin-3, suggesting that autophagy inhibited p53 activation. To test whether increased oxidative stress in Atg7Δ/Δ mice was responsible for p53 activation, Atg7 was deleted in the presence or absence of the master regulator of antioxidant defense nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2-/-Atg7Δ/Δ mice died rapidly due to small intestine damage, which was not rescued by p53 codeletion. Thus, Atg7 limits p53 activation and p53-mediated neurodegeneration. In turn, NRF2 mitigates lethal intestine degeneration upon autophagy loss. These findings illustrate the tissue-specific roles for autophagy and functional dependencies on the p53 and NRF2 stress response mechanisms.

Control of p53-dependent transcription and enhancer activity by the p53 family member p63.

Transcriptional activation by p53 provides powerful, organism-wide tumor suppression. We hypothesized that the local chromatin environment, including differential enhancer activities, contributes to various p53-dependent transcriptional activities in different cell types during stress-induced signaling. In this work, using ChIP-sequencing, immunoblotting, quantitative PCR, and computational analyses across various mammalian cell lines, we demonstrate that the p53-induced transcriptome varies by cell type, reflects cell type-specific activities, and is considerably broader than previously anticipated. We found that these molecular events are strongly influenced by p53's engagement with differentially active cell type-specific enhancers and promoters. We also observed that p53 activity depends on the p53 family member tumor protein p63 in epithelial cell types. Notably, we demonstrate that p63 is required for epithelial enhancer identity, including enhancers used by p53 during stress-dependent signaling. Loss of p63, but not p53, caused site-specific depletion of enhancer-associated chromatin modifications, suggesting that p63 functions as an enhancer maintenance factor in epithelial cells. Additionally, a subset of epithelial-specific enhancers depends on the activity of p63 providing a direct link between lineage determination and enhancer structure. These results suggest that a broad, cell-intrinsic mechanism controls p53-dependent cellular stress response through differential regulation of cis-regulatory elements.

Elevated p62/SQSTM1 determines the fate of autophagy-deficient neural stem cells by increasing superoxide.

Autophagy plays important roles in many biological processes, but our understanding of the mechanisms regulating stem cells by autophagy is limited. Interpretations of earlier studies of autophagy using knockouts of single genes are confounded by accumulating evidence for other functions of many autophagy genes. Here, we show that, in contrast to Fip200 deletion, inhibition of autophagy by deletion of Atg5, Atg16L1, or Atg7 does not impair the maintenance and differentiation of postnatal neural stem cells (NSCs). Only Fip200 deletion, but not Atg5, Atg16L1, or Atg7 deletion, caused p62/sequestome1 aggregates to accumulate in NSCs. Fip200 and p62 double conditional knockout mice demonstrated that p62 aggregate formation triggers aberrant superoxide increases by impairing superoxide dismutase functions. By comparing the inhibition of autophagy by deletion of Atg5, Atg16L1, or Atg7 with Fip200 deletion, we revealed a critical role of increased p62 in determining the fate of autophagy-deficient NSCs through intracellular superoxide control.

Autophagy is required for glucose homeostasis and lung tumor maintenance.

UNLABELLED: Macroautophagy (autophagy hereafter) recycles intracellular components to sustain mitochondrial metabolism that promotes the growth, stress tolerance, and malignancy of lung cancers, suggesting that autophagy inhibition may have antitumor activity. To assess the functional significance of autophagy in both normal and tumor tissue, we conditionally deleted the essential autophagy gene, autophagy related 7 (Atg7), throughout adult mice. Here, we report that systemic ATG7 ablation caused susceptibility to infection and neurodegeneration that limited survival to 2 to 3 months. Moreover, upon fasting, autophagy-deficient mice suffered fatal hypoglycemia. Prior autophagy ablation did not alter the efficiency of non-small cell lung cancer (NSCLC) initiation by activation of oncogenic Kras(G12D) and deletion of the Trp53 tumor suppressor. Acute autophagy ablation in mice with preexisting NSCLC, however, blocked tumor growth, promoted tumor cell death, and generated more benign disease (oncocytomas). This antitumor activity occurred before destruction of normal tissues, suggesting that acute autophagy inhibition may be therapeutically beneficial in cancer. SIGNIFICANCE: We systemically ablated cellular self-cannibalization by autophagy in adult mice and determined that it is dispensable for short-term survival, but required to prevent fatal hypoglycemia and cachexia during fasting, delineating a new role for autophagy in metabolism. Importantly, acute, systemic autophagy ablation was selectively destructive to established tumors compared with normal tissues, thereby providing the preclinical evidence that strategies to inhibit autophagy may be therapeutically advantageous for RAS-driven cancers.

Autophagy sustains mitochondrial glutamine metabolism and growth of BrafV600E-driven lung tumors.

UNLABELLED: Autophagic elimination of defective mitochondria suppresses oxidative stress and preserves mitochondrial function. Here, the essential autophagy gene Atg7 was deleted in a mouse model of BrafV600E-induced lung cancer in the presence or absence of the tumor suppressor Trp53. Atg7 deletion initially induced oxidative stress and accelerated tumor cell proliferation in a manner indistinguishable from Nrf2 ablation. Compound deletion of Atg7 and Nrf2 had no additive effect, suggesting that both genes modulate tumorigenesis by regulating oxidative stress and revealing a potential mechanism of autophagy-mediated tumor suppression. At later stages of tumorigenesis, Atg7 deficiency resulted in an accumulation of defective mitochondria, proliferative defects, reduced tumor burden, conversion of adenomas and adenocarcinomas to oncocytomas, and increased mouse life span. Autophagy-defective tumor-derived cell lines were impaired in their ability to respire and survive starvation and were glutamine-dependent, suggesting that autophagy-supplied substrates from protein degradation sustains BrafV600E tumor growth and metabolism. SIGNIFICANCE: The essential autophagy gene Atg7 functions to promote BrafV600E-driven lung tumorigenesis by preserving mitochondrial glutamine metabolism. This suggests that inhibiting autophagy is a novel approach to treating BrafV600E-driven cancers.

Autophagy suppresses progression of K-ras-induced lung tumors to oncocytomas and maintains lipid homeostasis.

Macroautophagy (autophagy hereafter) degrades and recycles proteins and organelles to support metabolism and survival in starvation. Oncogenic Ras up-regulates autophagy, and Ras-transformed cell lines require autophagy for mitochondrial function, stress survival, and engrafted tumor growth. Here, the essential autophagy gene autophagy-related-7 (atg7) was deleted concurrently with K-ras(G12D) activation in mouse models for non-small-cell lung cancer (NSCLC). atg7-deficient tumors accumulated dysfunctional mitochondria and prematurely induced p53 and proliferative arrest, which reduced tumor burden that was partly relieved by p53 deletion. atg7 loss altered tumor fate from adenomas and carcinomas to oncocytomas-rare, predominantly benign tumors characterized by the accumulation of defective mitochondria. Surprisingly, lipid accumulation occurred in atg7-deficient tumors only when p53 was deleted. atg7- and p53-deficient tumor-derived cell lines (TDCLs) had compromised starvation survival and formed lipidic cysts instead of tumors, suggesting defective utilization of lipid stores. atg7 deficiency reduced fatty acid oxidation (FAO) and increased sensitivity to FAO inhibition, indicating that with p53 loss, Ras-driven tumors require autophagy for mitochondrial function and lipid catabolism. Thus, autophagy is required for carcinoma fate, and autophagy defects may be a molecular basis for the occurrence of oncocytomas. Moreover, cancers require autophagy for distinct roles in metabolism that are oncogene- and tumor suppressor gene-specific.

Pramanicin analog induces apoptosis in human colon cancer cells: critical roles for Bcl-2, Bim, and p38 MAPK signaling.

Pramanicin (PMC) is an antifungal agent that was previously demonstrated to exhibit antiangiogenic and anticancer properties in a few in vitro studies. We initially screened a number of PMC analogs for their cytotoxic effects on HCT116 human colon cancer cells. PMC-A, the analog with the most potent antiproliferative effect was chosen to further interrogate the underlying mechanism of action. PMC-A led to apoptosis through activation of caspase-9 and -3. The apoptotic nature of cell death was confirmed by abrogation of cell death with pretreatment with specific caspase inhibitors. Stress-related MAPKs JNK and p38 were both activated concomittantly with the intrinsic apoptotic pathway. Moreover, pharmacological inhibition of p38 proved to attenuate the cell death induction while pretreatment with JNK inhibitor did not exhibit a protective effect. Resistance of Bax -/- cells and the protective nature of caspase-9 inhibition indicate that mitochondria play a central role in PMC-A induced apoptosis. Early post-exposure elevation of cellular Bim and Bax was followed by a marginal Bcl-2 depletion and Bid cleavage. Further analysis revealed that Bcl-2 downregulation occurs at the mRNA level and is critical to mediate PMC-A induced apoptosis, as ectopic Bcl-2 expression substantially spared the cells from death. Conversely, forced expression of Bim proved to significantly increase cell death. In addition, analyses of p53-/- cells demonstrated that Bcl-2/Bim/Bax modulation and MAPK activations take place independently of p53 expression. Taken together, p53-independent transcriptional Bcl-2 downregulation and p38 signaling appear to be the key modulatory events in PMC-A induced apoptosis.

Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis.

Autophagy is a catabolic pathway used by cells to support metabolism in response to starvation and to clear damaged proteins and organelles in response to stress. We report here that expression of a H-ras(V12) or K-ras(V12) oncogene up-regulates basal autophagy, which is required for tumor cell survival in starvation and in tumorigenesis. In Ras-expressing cells, defective autophagosome formation or cargo delivery causes accumulation of abnormal mitochondria and reduced oxygen consumption. Autophagy defects also lead to tricarboxylic acid (TCA) cycle metabolite and energy depletion in starvation. As mitochondria sustain viability of Ras-expressing cells in starvation, autophagy is required to maintain the pool of functional mitochondria necessary to support growth of Ras-driven tumors. Human cancer cell lines bearing activating mutations in Ras commonly have high levels of basal autophagy, and, in a subset of these, down-regulating the expression of essential autophagy proteins impaired cell growth. As cancers with Ras mutations have a poor prognosis, this "autophagy addiction" suggests that targeting autophagy and mitochondrial metabolism are valuable new approaches to treat these aggressive cancers.