Autophagy, which occurs at low basal levels in virtually all cells to perform homeostatic functions, such as protein and organelle turnover, is rapidly upregulated when cells need to generate intracellular nutrients and energy during starvation, growth factor withdrawal, or in conditions of high bioenergetic demand. Such upregulation of autophagy also occurs when cells are preparing to undergo structural remodeling, such as during developmental transitions, or when cells need to eliminate damaging cytoplasmic components in some poor environmental conditions, such as oxidative stress, infection or protein aggregate accumulation.42
The regulation of autophagy overlaps closely with signaling pathways that regulate tumorigenesis.12–14,42
In the present study, we demonstrated that the induction of the PML-RARα protein could significantly upregulate constitutive autophagic activation in leukemic U937 cells, as evidenced by the increase of LC3-positive punctate structures, the elevation of LC3-II and Beclin 1 protein levels, the decrease of p62 protein, and the accumulation of cytoplasmic AV-like structures based on TEM. This phenomenon could also be seen in two other nonleukemic cell lines upon the transient expression of PML-RARα. These results suggest that the upregulating effect of PML-RARα fusion protein on autophagy was independent of the cellular context. Besides the PML-RARα fusion protein, rare APL cases also express other fusion proteins due to different chromosome translocations, which always involve the RAR
α gene in chromosome 17. The in vitro and transgenic mice analysis showed that all these fusion proteins are critical for the pathogenesis of APL.23,43
Therefore, we also investigated the potential effect of the two other APL-related fusion proteins, PLZF-RARα and NPM-RARα, on autophagy. Unlike PML-RARα, unexpectedly, these two fusion proteins have no significant effect on constitutive autophagy formation, as seen in multiple assays for autophagy. We then investigated whether the enhancing effect of PML-RARα on autophagy activation is restricted to the in vitro conditions by using leukemic cells derived from the MRP8 PML-RAR
α transgenic mice. Transplanted APL-like cells that had infiltrated into the BM and spleen also showed increased constitutive autophagic activation. All these data supported the hypothesis that PML-RARα enhances autophagy generation.
Autophagy involves the delivery of the cytoplasmic cargo sequestered inside double-membrane vesicles to the lysosome, where the captured material, together with the inner membrane, is degraded.42
Our results demonstrated that the lysosomal protease inhibitors E64d and pepstatin A further increased PML-RARα-induced LC3-II accumulation and GFP-LC3 foci formation, indicating that the upregulation of autophagy by PML-RARα was not due to the alterations of an autolysosomal event. On the other hand, the inhibition of autophagy by 3-MA at the sequestration step could significantly reduce the increase of autophagy induced by PML-RARα, indicating that PML-RARα induces autophagy through the increase of autophagosome formation.
The observation that PML-RARα expression triggers mammalian cells to undergo autophagy led to the speculation that the alteration of autophagy-related signaling molecules possibly contributed to PML-RARα-enhanced autophagic activity. As widely reviewed in reference 5
, autophagy is regulated by a series of signaling molecules such as Atg7 and Vps34. Our results showed that there was no significant difference in the expression of Atg7 and Vps34 in the presence and absence of PML-RARα (data not shown). More recently, it has been demonstrated that an endoplasmic reticulum stress-related autophagic modulator, Grp78, is upregulated in more than 10% of AML patients with a favorable prognosis while Grp78va, a variant form of Grp78, is increased in several human leukemic cells and leukemia patients.44,45
However, Grp78 expression was also not significantly affected by the transient expression of PML-RARα in U2
OS and HEK293T cells (data not shown). On the other hand, PML-RARα expression simultaneously increased the accumulation of LC3-I and LC3-II protein, but had no effect on its mRNA, implying the modulation of PML-RARα on LC3 is independent of its transcriptional level. But the exact mechanism of how PML-RARα increases LC3-I protein level remains to be further investigated.
Considering that the promotion of autophagic activity was only specifically produced by PML-RARα, but not by PLZF-RARα or NPM-RARα, we extrapolated that the PML-RARα-induced autophagic activity involves its PML motif. Previous studies show that PML interacts with mTOR and inhibits its activity by negatively regulating its association with Rheb, thus further sequestering mTOR in the nucleus under normal, and especially in hypoxic, conditions.46
Moreover, PML has also been found to interact with the Akt phosphatase, PP2a, thereby causing the dephosphorylation of Akt, which further inhibits mTOR activity.47
However, we found that the transient expression of PML did not influence autophagic activities ( and Figs. S1C and S5
), but it appeared to sequester part of the LC3 population within PML nuclear bodies, which deserves further investigation for its significance. The mTOR protein belongs to the phosphatidylinositol kinase-related kinase (PIKK) family and binds several proteins to form two distinct protein complexes, mTORC1 (mTOR complex 1) and mTORC2. Emerging evidence suggests that the inhibition of mTORC1 activity, which is targeted and regulated by several pathways such as nutrient starvation and reduced growth factor, is a crucial step for autophagy induction in eukaryotes.48
The growth factor signaling that regulates mTORC1 mainly involves the insulin/insulin-like growth factor-PI3K-Akt pathway, which negatively regulates autophagy induction. Indeed, our data showed that PML-RARα induction also reduced the activation of Akt with decreased phosphorylation of the direct downstream substrate of mTORC1. Although it remains to be further explored how PML-RARα inhibits Akt/mTOR activation, our results supported the notion that PML-RARα-induced constitutive autophagy activation may involve the Akt-mTOR signaling pathway.
All of the RARα-involving fusion proteins found so far can trigger leukemia with similar or distinct phenotypes in transgenic mice.23,49
However, they present different responses to ATRA and As2
in mouse models and human APL patients.49
For example, leukemia involving PML-RARα, but not PLZF-RARα, is responsive to ATRA or As2
treatment, which induces the proteolysis of PML-RARα, but not PLZF-RARα. PML-RARα destruction is essential for the induction of terminal differentiation and/or the eradication of leukemia-initiating cells of APL.18
Recently, Isakson et al. reported that both ATRA- and As2
-induced autophagy contributes significantly to both the basal turnover and the therapy-induced proteolysis of PML-RARα in APL cells. They also showed a correlation between autophagy and therapy-induced differentiation of APL cells. However, here we demonstrated that the autophagy induced by PML-RARα was not accompanied by the acceleration of fusion protein degradation. We extrapolated that the discrepancy was possibly due to difference between the overexpressed and endogenous expression of PML-RARα in APL cells. However, we did find the easy decay of this fusion protein in U937/PR9 cells following induced expression for a longer period of time (>24 h) as previously reported in reference 43
Coincidentally, the studies from two other groups also reported that autophagy is a critical mechanism for As2
-induced anti-leukemic effect and ATRA-induced cell differentiation by accelerating the degradation of PML-RARα and its interaction with p62.50,51
Although the suppression of beclin 1
expression did not compromise ATRA-induced leukemic cell differentiation (Fig. S7
), we found the fusion protein-induced autophagy was responsible for leukemic cell growth and apoptotic resistance when insulting with apoptotic inducers. Taken together, given the critical role of the PML-RARα oncoprotein in APL pathogenesis, this study extends our understanding of the important role of autophagy in the development and treatment of this disease.