PERK-dependent regulation of MDA-7/IL-24-induced autophagy in primary human glioma cells

Glioblastoma multiforme is one of the most lethal malignancies, with very low 5 year survival rates (<1%).¹ The mda-7 gene (recently renamed Interleukin 24, IL-24) was isolated from human melanoma cells induced to undergo terminal differentiation by treatment with fibroblast interferon and mezerein.² The protein expression of MDA-7/IL-24 is decreased in advanced melanomas, with nearly undetectable levels in metastatic disease.^2-4 This novel cytokine is a member of the interleukin-10 (IL-10) gene family.^5-12 Enforced expression of MDA-7/IL-24, by use of a recombinant adenovirus Ad.mda-7, inhibits the growth and kills a broad spectrum of cancer cells, without exerting deleterious effects in normal human epithelial or fibroblast cells.^9-14 Considering its potent cancer-specific apoptosis-inducing ability and tumor growth-suppressing properties in human tumor xenograft animal models, mda-7/IL-24 was evaluated in a Phase I clinical trial in patients with advanced cancers.^10,11,15 This study indicates that Ad.mda-7 injected intra-tumorally was safe, and with repeated injection, significant clinical activity was evident.

The apoptotic pathways by which Ad.mda-7 causes cell death in tumor cells are not fully understood, and current evidence suggests an inherent complexity and an involvement of proteins important for the onset of growth inhibition and apoptosis, including Bcl-X_L Bcl-2, Bax and APO2/TRAIL.^9-14 In melanoma cell lines, but not in normal melanocytes, Ad.mda-7 infection induces a significant decrease in both Bcl-2 and Bcl-X_L levels, with only a modest upregulation of Bax and Bak expression.¹⁶ These data support the hypothesis that Ad.mda-7 enhances the ratio of pro-apoptotic to anti-apoptotic proteins in cancer cells, thereby facilitating induction of apoptosis.^9-14,16,17 The ability of Ad.mda-7 to induce apoptosis in DU145 prostate cancer cells, which does not produce Bax, indicates that MDA-7/IL-24 can also mediate apoptosis in tumor cells by a Bax-independent pathway.^9-12 In prostate cancer cells, overexpression of either Bcl-2 or Bcl-X_L protects cells from Ad.mda-7-induced toxicity in a cell type-dependent fashion.¹⁸ Thus MDA-7/IL-24 lethality seems to occur by multiple distinct pathways in different cell types. More recently, MDA-7/IL-24 toxicity has been linked to alterations in endoplasmic reticulum stress signaling.¹⁹ In these studies, MDA-7/IL-24 physically associates with BiP/GRP78 and inactivates the protective actions of this ER-chaperone protein. In addition to virus-administered mda-7/IL-24, delivery of this cytokine as a bacterially-expressed GST fusion protein, GST-MDA-7, retains cancer-specific killing, selective ER localization and induces similar signal transduction changes in cancer cells. We have noted that high concentrations of GST-MDA-7 or infection with Ad.mda-7 kill rodent and human glioma cells.^20-23 However, the precise mechanisms by which Ad.mda-7 and GST-MDA-7 modulates cell survival in non-established human glioma cells are presently unknown.

A GST-MDA-7 concentration that causes profound toxicity ∼72 h after exposure in glioma cells correlates with strong activation of JNK1-3. This treatment nearly abolishes ERK1/2 signaling. Multiple studies using a variety of cytokine and toxic stimuli document that JNK1-3 activation in astrocytes, neurons and transformed versions of these cells can trigger cell death.²⁴ The balance between the readouts of ERK1/2 and JNK1-3 signaling may represent a key homeostatic mechanism that regulates cell survival versus cell death processes.²⁵ GST-MDA-7—induced JNK1-3 signaling is PERK-dependent and causal in Bax activation, with loss of Bax expression reducing GST-MDA-7—induced cell killing. GST-MDA-7—induced suppression of ERK1/2 signaling is also found to be PERK-dependent. These findings argue that a form of ER stress signaling may be a primary mediator of GST-MDA-7-induced toxicity in primary malignant glioma cells (Fig. 1). Our studies go on to demonstrate that MDA-7/IL-24 must simultaneously induce multiple pathways of mitochondrial dysfunction to provoke tumor cell killing, including activation of Bax and Bak, cleavage of BID, dephosphorylation of BAD, and dephosphorylation and increased expression of BIM. In parallel, MDA-7/IL-24 also reduces expression of mitochondrial protective proteins such as Bcl-X_L and Mcl-1. Activation of death receptor-caspase 8 signaling was not involved in any GST-MDA-7—stimulated death processes in GBM cells.

GST-MDA-7 activated a PERK-dependent pathway to initiate mitochondrial dysfunction that requires JNK activation downstream of PERK. Although cell killing is reduced in PERK^-/- cells, GST-MDA-7 toxicity is still evident and other ER stress regulatory proteins as well as other sensors of the unfolded protein response, e.g., activating transcription factor 6 (ATF6), inositol-requiring enzyme1 (IRE1), PKR, HRI and GCN2, may mediate the toxic response of GST-MDA-7.²⁶ Prior studies implicate MDA-7/IL-24 as a protein that associates with and activates PKR.²⁷ As PERK and PKR are proteins with structural similarities, it is possible that PKR and PERK represent MDA-7/IL-24 targets in the regulation of eIF2α phosphorylation and transformed cell survival. Matsuzawa et al. implicate a TRAF2-ASK1-JNK cascade downstream of IRE1 in ER-stress responses in multiple cell types, and based on our data PERK-dependent signaling can also feed into this survival regulatory process.²⁸

GST-MDA-7 causes cell killing in part via a PERK-dependent mechanism, PERK is a sensor of ER stress, and MDA-7/IL-24 has been previously shown to bind to a regulatory chaperone of PERK, namely BiP/GRP78. Therefore, we explored whether GST-MDA-7 altered intracellular vacuolization of cells and specifically whether GST-MDA-7 could cause the formation of autophagic vesicles. Using a plasmid expressing a GFP-tagged form of LC3 GST-MDA-7 caused vacuolization of GFP-LC3 in multiple GBM cell types within 12-24 h, at a time prior to measurable cell killing. Expression of a dominant negative PERK protein, knockdown of ATG5 or Beclin 1 protein expression, or overexpression of the MDA-7/IL-24 binding partner BiP/GRP78, suppresses vesicle formation and protects GBM cells from GST-MDA-7 toxicity.^29-32 3-methyladenine can suppress autophagic vesicle formation and incubation of GBM cells with this agent also suppresses GFP-LC3 -containing vesicle formation and protects cells from GST-MDA-7 toxicity. Our data strongly argue that GST-MDA-7 promotes GBM and transformed cell death and one of the earliest manifestations of GST-MDA-7-induced cellular dysfunction is the formation of autophagic vesicles.

Increased expression of HSP70 has been shown by several groups to stabilize endosomes and to promote cell survival in response to noxious stresses, including ER stress.^33,34 In our analyses, we demonstrate that GST-MDA-7 variably causes early, and definitively causes later, suppression of HSP70 protein levels that correlate with increasing amounts of autophagic vacuolization in glioma cells; overexpression of HSP70 blocks the formation of GFP-LC3 vesicles and significantly suppresses GST-MDA-7 toxicity. Many laboratories are attempting to generate small molecule HSP70 inhibitors and it will be of interest to determine whether MDA-7 lethality will be enhanced by any such putative HSP70 inhibitory drug.

In summary, in transformed cells GST-MDA-7 induces multiple pro-apoptotic pathways to promote cell death. In primary human GBM cells, activation of the JNK1-3 pathway represents a key nodal signal, downstream of PERK and the induction of a toxic form of autophagy in promoting the activation of multiple pro-apoptotic proteases and causing mitochondrial dysfunction. From our studies, it is clear that the downstream effectors are complex, but the defining events in MDA-7/IL-24-promoted lethality of GBM cells involve a shift in the balance between anti-apoptotic and pro-apoptotic signals eliciting mitochondrial dysfunction uniquely in the context of cancer cells.