Autophagy is activated to maintain cellular energy homeostasis in response to nutrient starvation. However, autophagy is not persistently activated, which is poorly understood at a mechanistic level. Here, we report that turnover of FoxO1 is involved in the dynamic autophagic process caused by glutamine starvation. X-box-binding protein-1u (XBP-1u) has a critical role in FoxO1 degradation by recruiting FoxO1 to the 20S proteasome. In addition, the phosphorylation of XBP-1u by extracellular regulated protein kinases1/2 (ERK1/2) on Ser61 and Ser176 was found to be critical for the increased interaction between XBP-1u and FoxO1 upon glutamine starvation. Furthermore, knockdown of XBP-1u caused the sustained level of FoxO1 and the persistent activation of autophagy, leading to a significant decrease in cell viability. Finally, the inverse correlation between XBP-1u and FoxO1 expression agrees well with the expression profiles observed in many human cancer tissues. Thus, our findings link the dynamic process of autophagy to XBP-1u-induced FoxO1 degradation.

Autophagy is characterized by the sequestration of bulk cytoplasm, including damaged proteins and organelles, and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity, the mechanism is not yet clear. Here we report that cytosolic FoxO1, a forkhead O family protein, is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation, but this process was independent of the transcriptional activity of FoxO1. In response to stress, FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2), a NAD+-dependent histone deacetylase, and the acetylated FoxO1 bound to Atg7, an E1-like protein, to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy.

The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (Cdc37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in Cdc37 compromised the recruitment of client kinases to a complex comprising Cdc37 and heat shock protein 90 (Hsp90) but only modestly affected Cdc37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of Cdc37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-Cdc37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells.

Autophagy is a protein degradation process by which intracellular materials are recycled for energy homeostasis. However, the metabolic status and energy source of autophagy-defective tumor cells are poorly understood. Here, our data show that amino acid uptake from the extracellular environment is increased in autophagy-deficient cells upon glutamine deprivation. This elevated amino acid uptake results from activating transcription factor 4 (ATF4)-dependent upregulation of AAT (amino acid transporter) gene expression. Furthermore, we identify SIRT6, a NAD+-dependent histone deacetylase, as a corepressor of ATF4 transcriptional activity. In autophagy-deficient cells, activated NRF2 enhances ATF4 transcriptional activity by disrupting the interaction between SIRT6 and ATF4. In this way, autophagy-deficient cells exhibit increased AAT expression and show increased amino acid uptake. Notably, inhibition of amino acid uptake reduces the viability of glutamine-deprived autophagy-deficient cells, but not significantly in wild-type cells, suggesting reliance of autophagy-deficient tumor cells on extracellular amino acid uptake.

Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, and loss of autophagy has been linked to increased genome instability. Here, we report that loss of autophagy is coupled to reduced histone H2A ubiquitination after DNA damage. p62/SQSTM1, which accumulates in autophagy-defective cells, directly binds to and inhibits nuclear RNF168, an E3 ligase essential for histone H2A ubiquitination and DNA damage responses. As a result, DNA repair proteins such as BRCA1, RAP80, and Rad51 cannot be recruited to the sites of DNA double-strand breaks (DSBs), which impairs DSB repair. Moreover, nuclear-localized p62 increased the sensitivity of tumor cells to radiation both in vitro and in vivo, and this required its interaction with RNF168. Our findings indicate that autophagy-deficiency-induced p62 accumulation results in inhibition of histone ubiquitination and highlight the complex relationship between autophagy and the DNA damage response.