This study demonstrates a relationship between ATRA-induced degradation of PML-RARα fusion protein and autophagy activation, which may contribute to myeloid cell differentiation. In addition, the present study establishes human p62 as a direct mediator of ATRA-induced degradation of RARα and provides mechanistic insight into the role of p62 in regulating cell differentiation.
Autophagy is an evolutionarily conserved lysosomal self-digestion process essential for cellular homeostasis and survival. As an adaptive response, it protects organisms against a wide range of pathologies including cancer, infection, neurodegeneration, heart disease and aging.6–8
Cell differentiation is often associated with decreased cell growth, indicating an altered rate of macromolecule synthesis and degradation. A recent study demonstrated that autophagy is an important event for megakaryocytic differentiation of the chronic myelogenous leukemia K562 cell line.26
Knockdown of the autophagy genes LC3
and beclin 1
by specific siRNAs impairs phorbol ester PMA and p38 inhibitor SB202190-mediated megakaryocytic differentiation.26
Similarly, Beclin 1 is required for Vitamin D3-induced autophagy and differentiation in HL-60 cells.13
In this study, we demonstrated that autophagy mediated the effects of ATRA on myeloid cell differentiation, as: (1) Inhibition of autophagy by shRNAs that target essential autophagy genes such as ATG1, ATG5
decreased ATRA-induced differentiation in HL-60 and NB4 cells; (2) an autophagy inhibitor (3-MA) inhibited ATRA-induced myeloid cell differentiation whereas an autophagy inducer (rapamycin) promoted myeloid cell differentiation. Moreover, autophagy also is required for differentiation of nonhematologic cells and tissues, such as adipose mass and neuroblastoma.27,28
Together, these findings indicate that an appropriate level of autophagic activity is important for cell differentiation.
Furthermore, we demonstrate that autophagy promoted myeloid cell differentiation activities potentially via controlling degradation of PML-RARα. A hallmark of the ATRA response in APL is PML-RARα degradation, which subsequently promotes cell differentiation.2–5
Previous studies indicate that the proteasome and caspase pathways are involved in the degradation of PML-RARα, although neither is sufficient for complete degradation.2–5
The current data show that ATRA not only directly targets PML-RARα fusions to the proteasomal and caspase-mediated pathways, but also triggers an autophagy-dependent degradation of PML-RARα. Knockdown or pharmacological inhibition of autophagy inhibited PML-RARα degradation, whereas induction of autophagy using rapamycin promoted ATRA-induced degradation of PML-RARα. Rapamycin, a lipophilic, macrolide antibiotic, induces autophagy by inactivating mTOR.29
Others have demonstrated that the activity of mTOR regulates differentiation of memory CD8 T-cell,30
regulatory T cells31
in vivo and in vitro. Moreover, inactivation of mTOR by RAD001 potentiates the ability of MS-275, a synthetic benzamide histone deacetylase inhibitor, to induce differentiation of HL60 and NB4 cells.33
Recent study consistently finds that both ATRA and arsenic trioxide (ATO) induce autophagy via the mTOR pathway in APL cells and that autophagic degradation contributes therapy-induced proteolysis of PML-RARα.15
These results suggest that mTOR signaling is important for autophagy and myeloid cell differentiation.
Our results also demonstrated that autophagy promoted PML-RARα degradation through an interaction between p62 and RARα. p62 is an adaptor protein that binds ubiquitin and regulates signaling cascades through ubiquitination;34
it may regulate the activation of NFκB in response to upstream signals.34
More recently, a critical role for p62 in macroautophagic removal of intracellular protein aggregates has also been proposed.16
Studies involving the cellular depletion of p62 have indicated a critical role for its association with LC3 and aggregate proteins to facilitate correct formation of the autophagosome.16,35
p62 bodies are found both as membrane-free protein aggregates (sequestosomes) and as membrane-confined autophagosomal and lysosomal structures in the cytosol or nucleus.35,36
Although most of the PML-RARα fusion protein is localized to the nucleus, significant levels of PML-RARα are also found in the cytoplasm.37
Moreover, a recent study demonstrated that the localization of the large phosphoinositide-binding protein ALFY to nuclear PML bodies is dependent on p62 in HeLa cells.38
In the current study, p62-mediated degradation of PML-RARα links the ubiquitin-proteasome and autophagy-lysosome pathways, the two main routes utilized by cells to degrade intracellular proteins.
Collectively, the current study describes a direct link between autophagy and ATRA-induced degradation of PML-RARα, both of which target myeloid cell differentiation.26
Based upon the work reported here and previous evidence,13,26
we propose a more nuanced conceptual model incorporating autophagic processes and myeloid cell differentiation (). In this model, ATRA inhibits the mTOR pathway and activates the Atg1-PI3KC3-Atg5-dependent autophagy pathway, which promotes autophagosome formation. Furthermore, the interaction between p62 and PML-RARα regulates degradation of PML-RARα. Inhibition of p62 impaired the degradation of PML-RARα during cell differentiation. In contrast, inhibition of autophagy results in an accumulation of p62,35
which, through a negative feedback mechanism, inhibits the activity of the proteasome as well as degradation of PML-RARα.25
Thus, autophagy plays an important role in regulating degradation of the PML-RARα oncoprotein and myeloid cell differentiation by p62.