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ATG3 (autophagy-related 3) is an E2-like enzyme essential for autophagy; however, it is unknown whether it has an autophagy-independent function. Here, we report that ATG3 is a relatively stable protein in unstressed cells, but it is degraded in response to DNA-damaging agents such as etoposide or cisplatin. With mass spectrometry and a mutagenesis assay, phosphorylation of tyrosine 203 of ATG3 was identified to be a critical modification for its degradation, which was further confirmed by manipulating ATG3Y203E (phosphorylation mimic) or ATG3Y203F (phosphorylation-incompetent) in Atg3 knockout MEFs. In addition, by using a generated phospho-specific antibody we showed that phosphorylation of Y203 significantly increased upon etoposide treatment. With a specific inhibitor or siRNA, PTK2 (protein tyrosine kinase 2) was confirmed to catalyze the phosphorylation of ATG3 at Y203. Furthermore, a newly identified function of ATG3 was recognized to be associated with the promotion of DNA damage-induced mitotic catastrophe, in which ATG3 interferes with the function of BAG3, a crucial protein in the mitotic process, by binding. Finally, PTK2 inhibition-induced sustained levels of ATG3 were able to sensitize cancer cells to DNA-damaging agents. Our findings strengthen the notion that targeting PTK2 in combination with DNA-damaging agents is a novel strategy for cancer therapy.

Macroautophagy/autophagy is a unique protein degradation process by which intracellular materials are recycled for energy homeostasis. However, the metabolic status and energy source of autophagy-defective tumor cells is poorly understood. Here in this study, we found ATF4-dependent amino acid transporter (AAT) gene expression and amino acid uptake were increased in autophagy-deficient cells under conditions of Gln deprivation. Notably, inhibition of amino acid uptake reduced the viability of Gln-deprived autophagy-deficient cells, but not significantly in wild-type cells, suggesting the reliance of autophagy-deficient tumor cells on extracellular amino acid uptake.

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the autophagy regulation machinery has been widely studied, the key epigenetic control of autophagy process still remains unknown. Here we report that the methyltransferase EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) epigenetically represses several negative regulators of the MTOR (mechanistic target of rapamycin [serine/threonine kinase]) pathway, such as TSC2, RHOA, DEPTOR, FKBP11, RGS16 and GPI. EZH2 was recruited to these genes promoters via MTA2 (metastasis associated 1 family, member 2), a component of the nucleosome remodeling and histone deacetylase (NuRD) complex. MTA2 was identified as a new chromatin binding protein whose association with chromatin facilitated the subsequent recruitment of EZH2 to silenced targeted genes, especially TSC2. Downregulation of TSC2 (tuberous sclerosis 2) by EZH2 elicited MTOR activation, which in turn modulated subsequent MTOR pathway-related events, including inhibition of autophagy. In human colorectal carcinoma (CRC) tissues, the expression of MTA2 and EZH2 correlated negatively with expression of TSC2, which reveals a novel link among epigenetic regulation, the MTOR pathway, autophagy induction, and tumorigenesis.

The importance of autophagy in the DNA damage repair process is clear; however, the detailed molecular mechanism is still largely unknown. Here we found that DNA damage-induced histone H2A ubiquitination is suppressed in autophagy-deficient cells in a SQSTM1/p62 dependent manner. SQSTM1 binds and inhibits E3 ligase RNF168s activity, which is essential for H2A ubiquitination. As a result, several important factors for DNA repair cannot be recruited to the sites of DNA double-strand breaks (DSBs) in autophagy-deficient cells, leading to diminished DNA repair and increased sensitivity of cells to radiation.

Earlier studies have shown that macroautophagy is not a constitutively activated process, however, the mechanism of activation is not fully understood. Here, we report that autophagy is a dynamic process in cancer cells in response to glucose starvation. In addition, we determined that FOXO1 turnover is involved in the regulation of this dynamic process. X-box binding protein 1u (XBP1u) plays a critical role in FOXO1 degradation by recruiting FOXO1 to the 20S proteasome. Moreover, the phosphorylation of XBP1u by mitogen-activated protein kinases 1 and 3 (MAPK1/3, also known as ERK2/1) on serine residues 61 and 176 was found to be essential for the enhancement of the interaction between XBP1u and FOXO1. Thus, our findings support the hypothesis that the turnover of FOXO1 induced by MAPK1/3 and XBP1u is a critical factor regulating the autophagic process.

Although Beclin 1 and mTOR are considered to be the main molecules to modulate the autophagic process, searching for other autophagy-regulating molecules is still an ongoing challenge to scientists. Here we demonstrated that FoxO1, a forkhead O family protein, is a mediator of autophagy. Upon oxidative stress or serum starvation, endogenous FoxO1 was required for autophagy in human cancer cell lines, and this process was independent of FoxO1's transcriptional activity as well as mTOR or Beclin 1. In response to stress, FoxO1 dissociated from an NAD+-dependent histone deacetylase SIRT2, and FoxO1 thus became acetylated, and in turn bound to Atg7, an E1-like protein, to influence the autophagic process leading to cell death. In particular, cytosolic FoxO1 suppressed tumor xenograft growth in nude mice in an autophagy-dependent manner. Our studies provide evidence that cytosolic FoxO1-induced autophagy is associated with tumor suppression function.