DAMP-mediated autophagy contributes to drug resistance

Abstract

Damage-associated molecular pattern molecules (DAMPs) are cellularly derived molecules that can initiate and perpetuate immune responses following trauma, ischemia and other types of tissue damage in the absence of pathogenic infection. High mobility group box 1 (HMGB1) is a prototypical DAMP and is associated with the hallmarks of cancer. Recently we found that HMGB1 release after chemotherapy treatment is a critical regulator of autophagy and a potential drug target for therapeutic interventions in leukemia. Overexpression of HMGB1 by gene transfection rendered leukemia cells resistant to cell death; whereas depletion or inhibition of HMGB1 and autophagy by RNA interference or pharmacological inhibitors increased the sensitivity of leukemia cells to chemotherapeutic drugs. HMGB1 release sustains autophagy as assessed by microtubule-associated protein 1 light chain 3 (LC3) lipidation, redistribution of LC3 into cytoplasmic puncta, degradation of p62 and accumulation of autophagosomes and autolysosomes. Moreover, these data suggest a role for HMGB1 in the regulation of autophagy through the PI3KC3-MEKERK pathway, supporting the notion that HMGB1-induced autophagy promotes tumor resistance to chemotherapy.

Drug resistance, intrinsic or acquired, is a challenge for most chemotherapeutic agents in tumor therapy. Many mechanisms of drug resistance are well recognized, such as those due to drug export transporters (e.g., permeability-glycoprotein), altered drug target sites, more effective DNA repair mechanisms, altered pharmacokinetics, resistance to apoptosis and resistant tumor stem cells. Recent studies focus on the role of autophagy, a process by which cytoplasmic components including macromolecules (e.g., nucleic acids, proteins, carbohydrates and lipids) and organelles (e.g., mitochondria, peroxisomes and endoplasmic reticulum) are degraded by the lysosome, in drug resistance (Fig. 1A). However, the mechanisms by which autophagy regulates drug resistance are still largely unknown. Recently, we reported that DAMPs such as HMGB1 contribute to chemotherapy resistance though the upregulation of autophagy in leukemia.

Figure 1.

DAMP-mediated autophagy contributes to drug resistance. (A) Many mechanisms of drug resistance are well recognized, such as those due to drug export transporters, altered drug target sites, more effective DNA repair mechanisms, altered pharmacokinetics, downregulation of apoptosis, resistant tumor stem cells and upregulation of autophagy. (B) HMGB1 modulates drug resistance by regulating autophagy. HMGB1 is released in tumor cells after chemotherapy-induced cytotoxicity. The released HMG B1 functions as an activator of autophagy which increases drug resistance through the PI3KC3-MEK-ERK pathway. Knockdown by RNA interference (RNAi) or pharmacological inhibition (e.g., 3-MA and U0126) of the PI3KC3-MEK-ERK pathway reverses the resistance of leukemia cells to chemotherapy.

HMGB1 is Involved in Drug Resistance

Unlike other DAMPs, HMGB1 has various unique roles in cancer. HMGB1 expression is greater in tumor cells than normal surrounding epithelium in a large variety of human neoplasms including lymphoma, melanoma and cancers of the breast, cervix, colon, liver, lung and pancreas. Moreover, serum levels of HMGB1 are significantly increased in patients with cancer such as acute lymphocytic leukemia. We and others, have found that release of HMGB1 from leukemia cells is a universal event during exposure to chemotherapy such as vincristine, adriamycin, cytosine arabinoside, arsenic trioxide, etoposide, camptothecin, staurosporine and cycloheximide. Importantly, we have demonstrated that HMGB1-neutralizing antibodies, potential HMGB1 release inhibitors (e.g., quercetin) and knockdown of HMGB1 by RNA interference (RNAi) increase the sensitivity of leukemia cells to these chemotherapeutic agents. In contrast, overexpression of HMGB1 by gene transfection renders leukemia cells resistant to cell death. Moreover, pretreatment with exogenous HMGB1 protein increases drug resistance in these leukemia cells, supporting a potential prosurvival role for HMGB1 in cells exposed to chemotherapy.

Autophagy is Required for HMGB1-Mediated Drug Resistance

Recently, interest in autophagy has been renewed among oncologists as various types of cancers undergo autophagy following most anticancer therapies. Autophagy confers stress tolerance, limits damage and sustains viability under adverse conditions. The mammalian autophagy gene beclin 1, an ortholog of autophagy-related (ATG) gene 6 in yeast, was cloned in 1998. Beclin 1 is important for the localization of autophagic proteins to phagophore assembly sites, which are required for the initiation of the formation of the autophagasome. Our recent study suggests that endogenous HMGB1 is a novel Beclin 1-binding protein active in autophagy. To explore the potential mechanism of HMGB1-mediated drug resistance, we suppressed the expression of Beclin 1 in leukemia cells. Knockdown of beclin 1 reverses exogenous HMGB1- induced drug resistance. Moreover, pharmacological inhibition of autophagy with bafilomycin A₁, a potent and specific inhibitor of vacuolar H⁺-ATPase, also reverses HMGB1-mediated drug resistance, suggesting that autophagy is required for HMGB1-mediated chemotherapy resistance in leukemia cells. Since leukemia cell activation by HMGB1 results in the release of proinflammatory cytokines and chemokines, further studies must explore whether autophagy also mediates these functions of HMGB1.

HMGB1 Directly Induces Autophagy in Leukemia Cells

In addition to its nuclear functions, HMGB1 has been identified as a ‘danger signal’ that can wake up several cellular anti-injury mechanisms. Autophagy is an adaptive mechanism used to mitigate cell stress. To investigate whether HMGB1 is a direct activator of autophagy, we detected widely used markers of autophagy including accumulation of LC3-II and degradation of p62. Consistent with increased autophagic flux following treatment with HMGB1, there is a time-dependent decrease in the level of p62 by immunoblotting. Moreover, LC3 accumulation following HMGB1 treatment is exaggerated in leukemia cells after treatment with the lysosomal protease inhibitor E64d and pepstatin A. Furthermore, we found that HMGB1 increases the interaction of Beclin 1/class III phosphatidylinositol 3-kinase (PI3KC3) and suppresses interaction of Beclin 1/Bcl-2, suggesting that HMGB1 promotes vesicle nucleation. In addition, HMGB1 promotes Atg12-Atg5-Atg16 complex formation and enhances the accumulation of the Atg12-Atg5-Atg16 complex with LC3, suggesting that HMGB1 contributes to phagophore membrane elongation and autophagosome formation. Importantly, electron microscopy reveals that leukemia cells exhibit more autophagosomes and autolysosomes after HMGB1 treatment. HMGB1 activates cells through the differential engagement of multiple surface receptors including the receptor for advanced glycation end-products (RAGE) and toll like receptors (TLRs). A recent study indicates that RAGE RNAi abolishes HMGB1-induced autophagy in pancreatic cancer cells (Fig. 1B). In contrast TLR4 is required for pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharideinduced autophagy in macrophages, suggesting that the induction of autophagy by DAMPs or PAMPs may have a different receptor-dependent pathway.

PI3KC3-MEK-ERK Pathway is Required for HMGB1-Mediated Autophagy

PI3KC3 plays a pleiotropic role in autophagy and protein sorting pathways. We found that suppression of expression of PI3KC3 by RNAi or the use of the PI3KC3 inhibitor 3-methyladenine (3-MA) significantly inhibits HMGB1-induced autophagy, suggesting that PI3KC3 is required for HMGB1-induced autophagy. Interestingly, inhibition of PI3KC3 blocks HMGB1-induced phosphorylation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2). Extracellular signal-regulated kinase (MEK) functions as an immediate upstream activator of ERK. The tight interaction between PI3KC3 and ERK has previously been demonstrated to regulate inflammation and protein synthesis. The requirement of MEK-ERK for HMGB1-induced cytokine production has also been demonstrated previously, but little was known about their involvement in HMGB1-mediated autophagy. Indeed, we have shown that inhibition of the MEK-ERK pathway via knockdown of MEK by RNAi or MEK inhibitors (e.g., U0126), attenuates HMGB1-induced autophagy as well as drug resistance. Consistently, knockdown of PI3KC3 and MEK decreases HMGB1-induced autophagosome formation based on electron microscopy analysis. These data indicate that HMGB1 induces autophagy through the PI3KC3-MEK-ERK pathway (Fig. 1B).

Conclusion

Mammalian organisms have evolved diverse systems to recognize certain molecules (e.g., DAMPs) as ‘danger signals’ and respond quickly to life-threatening events, including infection, injury and genetic and metabolic stress. Autophagy is a lysosomal degradation pathway that is essential for survival, differentiation, development and homeostasis. Our study demonstrates that HMGB1, the best characterized DAMP, is released into the tumor microenvironment as a critical regulator of autophagy to increase drug resistance during tumor therapy. These findings provide insight into how ‘danger signals’ stimulate intrinsic cellular stress responses to antagonize effects of chemotherapy. Thus, targeting HMGB1 and autophagy signaling may be a novel approach to improve the effectiveness of anti-cancer agents.

Punctum to: Liu L, Yang M, Kang R, Wang Z, Zhao Y, Yu Y, Xie M, Yin X, Livesey KM, Lotze MT, Tang D, Cao L. HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells. Leukemia. 2010 doi: 10.1038/leu.2010.225. In press.