Accumulation of p62 in response to metabolic stress is a striking phenotype of autophagy-defective tumors cells, suggesting defective protein quality control may contribute to tumorigenesis and that autophagy is the main mechanism by which tumor cells turnover p62. Moreover, the failure of autophagy-defective tumor cells to eliminate p62 was sufficient for tumorigenesis. Unlike brain tissue (Hara et al., 2006
; Komatsu et al., 2006
), there was no accumulation of polyubiquitinated proteins in autophagy-defective tumor cells. There may be tissue-specific differences in autophagy-mediated protein elimination or dilution of polyubiquitinated proteins through cell proliferation in tumor cells may prevent their accumulation, which is not possible in post-mitotic neurons. Alternatively, proteasome-mediated turnover of polyubiquitinated proteins may be elevated in tumor cells compared to neuronal tissues. Indeed, autophagy defects sensitized cancer cells to proteasome inhibitors, suggesting a compensatory function of the two protein degradation pathways.
The persistence of p62 and accumulation of ER chaperones and the oxidative protein folding machinery in autophagy-deficient cells and tumors indicated a defect in the management of protein turnover. The inability to degrade damaged or misfolded proteins through autophagy may increase the burden on the ER protein folding machinery necessitating its upregulation. Both p62 and GRp170 were dramatically upregulated in beclin1+/−
tissues as well as in spontaneous tumors, indicating that coping with unfolded proteins may be a biomarker for impaired autophagy that precedes tumor initiation. ER chaperone and PDI upregulation are common in human tumors (Goplen et al., 2006
; Ni and Lee, 2007
), and increased GRp170 expression is associated with poor prognosis in breast cancer (Tamatani et al., 2001
). Although the cause of ER chaperone accumulation in tumors was not known, chaperones are stress-responsive and provide a protective function by suppressing the accumulation of unfolded proteins that may be an important compensatory mechanism for autophagy-defective cells. Protein folding is a source of oxidative stress (Tu and Weissman, 2004
), particularly when cells are overburdened with damaged and unfolded proteins, in concordance with increased ROS in stressed beclin1+/−
Stressed autophagy-defective tumor cells accumulate damaged mitochondria as a potential additional source of oxidative stress. This accumulation of unfolded protein and protein aggregates and the persistence of damaged mitochondria may collectively lead to elevated ROS production in autophagy-defective cells. As ROS scavengers partially suppressed p62 accumulation and cell death in stressed beclin1+/−
tumor cells, the elevated oxidative stress may contribute to p62 induction, cell damage and death. Although oxidative stress and DNA damage arise through multiple genotoxic events (Halazonetis et al., 2008
), stress-mediated p62 accumulation in autophagy-defective cells was sufficient for ROS and DNA damage response induction that was prevented by knockdown of p62, establishing that that elevated oxidative stress was attributable directly to p62 accumulation. Enforced p62 expression induced ROS, suggesting a possible amplification loop where oxidative stress induces p62 accumulation, which in turn amplifies ROS generation. Thus, the inability of autophagy-defective tumor cells to eliminate p62 contributes to oxidative stress and likely to DNA damage. These observations are strikingly similar to the rescue of oxidative stress toxicity caused by atg7
deficiency with p62 deficiency in mouse liver (Komatsu et al., 2007
). In normal tissues, toxicity due to p62 accumulation resulting from autophagy defect may trigger cell death, whereas in checkpoint-defective tumor cells this instead may also result in enhancement of mutations, genome instability and tumor progression.
tumors displayed pronounced p62 and p62 aggregate accumulation and this p62 expression was sufficient to activate the DNA damage response and to enhance tumor growth. p62 expression, p62 aggregates, and Mallory-Denk bodies containing p62 are common in steatosis and in HCC and other cancers (Zatloukal et al., 2007
). Defects in autophagy may be a mechanism for sustained p62 accumulation and formation of Mallory-Denk bodies. As such, p62 accumulation is not merely a histologic marker for certain cancers, but rather, directly contributes to tumor growth. While the prevalence of autophagy defects in HCC is not yet known, mutations such as pten
loss that constitutively activates the PI3-kinase pathway and mTOR that inhibits autophagy are common (Wong and Ng, 2008
). Interestingly, pten
(Watanabe et al., 2005
, or atg7
deficiency (Komatsu et al., 2007
; Qu et al., 2003
; Yue et al., 2003
) produce liver steatosis in mice suggesting that suppression of autophagy and the resulting steatosis can lead to HCC.
How persistent p62 promotes oxidative stress and tumorigenesis appears to be related to its role as an adaptor protein regulating receptor signaling and the activation of NF-κB. p62 is also required for efficient oncogene activation in vitro
and p62 deficiency suppresses spontaneous lung tumorigenesis by K-ras
(Duran et al., 2008
). Thus, p62 had been identified as an oncoprotein in both loss- (Duran et al., 2008
) and gain-of-function situations (). p62 gain-of-function caused by defective autophagy altered NF-κB signal transduction pathways that regulate host defense. Whether p62 is upregulated and either sequestered in non-functional aggregates inhibiting signal transduction as indicated here, or is retained in an active state enhancing signal transduction, may be cell type- or stress-specific. In liver, defects in NF-κB canonical pathway activation promote tumorigenesis by stimulation of inflammation and activation of the non-canonical NF-κB pathway. However, this function of NF-κB may be tissue specific, and as NF-κB signaling also regulates anti-oxidant defense, suppression of NF-κB by p62 may explain increased oxidative stress in other autophagy-defective tissues. In conclusion, defects in autophagy promote a failure of protein and organelle quality control in tumors this leads to p62 accumulation resulting in perturbation of gene expression, increased oxidative stress, genome damage and tumorigenesis (). As p62 upregulation is common in liver tissues of individuals at risk and hepatocellular carcinomas in patients, this suggests that facilitating the clearance of p62 by promoting autophagy may be a strategy for cancer chemoprevention.