Cell demise inhibited: Unexpected liaisons between mitochondria and IκΒα

ABSTRACT

IκΒα (the protein product of NFKBIA gene) has widely been considered a pro- apoptotic factor due to its ability to inhibit the anti-apoptotic transcription factor NFκB. Our findings indicate that IκΒα also exerts a strong anti-apoptotic activity at the outer mitochondria membrane (OMM). This function we uncovered is distinct from its ability to sequester and inhibit NFκB. IκΒα instead binds to voltage dependent anion channel 1 (VDAC1) and Hexokinase 2 (HK2), stabilizes this complex and prevents mitochondria outer membrane permeabilisation (MOMP) and apoptosis.

Author’s View

Mitochondria, apart from being the cell’s powerhouse, are the organelle that cells use to self-destruct¹. Many signal transduction pathways channel their signals to mitochondria to cause rapture of the outer mitochondrial membrane (OMM) and ultimately destroy the cell. NFκB is a transcription factor activated by several extracellular and intracellular stimuli². Among the pleotropic functions of NFκB is therapy resistance and apoptosis inhibition via the upregulation of several anti- apoptotic target genes that accumulate at mitochondria and protect cells from apoptosis-inducing signals. One of the NFκB target genes is NFKBIA (gene name for the IκΒα protein), which is rapidly degraded to allow the activation of the transcription factor. IκΒα participates in a negative feedback loop to ensure tight regulation of NFκB activation via rapid transcription and translation that allows the removal of NFκB from the nucleus. NFκB is deregulated in multiple diseases including cancer, in which it is often constitutively activated. In many tumors NFκB is activated through an IκΒα-independent manner allowing transcription of several anti-apoptotic genes and NFKBIA³.

Earlier reports have shown that NFκB and IκΒα are found at mitochondria but their effects on mitochondrial apoptosis were not investigated⁴. We showed that IκΒα inhibits apoptosis at the outer mitochondrial membrane through a novel, NFκB-independent interaction with VDAC1 (voltage dependent anion channel 1)⁵. IκΒα was found to partially overlap with the OMM in all cell lines studied, but in cell lines with constitutively active NFκB the overlap was complete (Figure 1). All IκΒα present in these cells resided at the mitochondria, an observation that motivated us to investigate its function in mitochondrial-mediated apoptosis.

Figure 1.

Mechanisms for apoptosis inhibition by IκΒα at mitochondria. In normal and early tumor samples IκΒα binds to hexokinase II (HK2, commonly known as HK2) and associates with Voltage Dependent Anion Channel 1 (VDAC1) but the interaction is not stable. Upon apoptosis pro-apoptotic molecules (eg. Bax) bind to VDAC1, induce Mitochondrial Outer Membrane Permeabilisation (MOMP), release cytochrome c (Cyt-C) and eventually apoptosis (lower left). In malignant and transformed tissues, IκΒα is accumulated at mitochondria and stabilizes the VDAC1-HK2 complex which in turns prevents pro-apoptotic molecules to induce MOMP (lower right).

In order to test if IκΒα can alter the sensitivity of cells exposed to several apoptosis signals, we employed a tightly controlled system of RNAi-mediated reduction of IκΒα in cells in which IκΒα is only present at mitochondria (PC3,

MDA-MB-231, HCT-116). We also reconstituted IκΒα in different compartments in cells lacking its expression (3T3 IκΒα^−/−) to probe its compartment-specific functions. Only IκΒα residing at the OMM was able to inhibit apoptosis induced by several stimuli (chemotherapeutics, oxidizers, Ca⁺² scavengers or genetic insults). The signal was so specific that IκΒα targeted in the inter-membrane space of mitochondria was incapable of inhibiting apoptosis. Moreover, a highly similar IKB family member, IκΒβ (I kappa B beta, NFKBIB), was dispensable for apoptosis inhibition. We tested if this ability of IκΒα was connected to NFκB. Through transcriptional and reporter assays we confirmed that manipulation of IκΒα did not affect NFκB target gene expression, confirming that constitutive activation of NFκB transcriptional activity in these cells is IκΒα-independent. Moreover, we verified that manipulation of IκΒα using mutants that affect its ability to inhibit or activate NFκB were unable to affect NFκB status but retained the ability to inhibit specific apoptosis signaling converging at mitochondria.

MOMP (mitochondrial outer membrane permeabilisation) is the point of no return for the cell, since this allows pro-apoptotic molecules like Bax, Bim, Bak, Bid to rapture the membrane and release cytochrome c. The rapture of the OMM is mediated by direct interaction of these molecules with the membrane, through the induction of permeability transition pore or through interaction with VDAC1⁶. While the mechanism underlying this event is still poorly characterized, the importance of VDAC1 as an apoptosis mediator is well established. Anti- apoptotic proteins like Bcl-2, Bcl_XL or Hexokinase 2 (HK2) inhibit MOMP by interacting with VDAC and prevent attachment of Bax. IκΒα was found to interact with VDAC1 and HK2. The interaction was specific to IκΒα, since the highly similar IκΒβ was not able to interact with VDAC1. Importantly, we were able to show that IκΒα’s ability to regulate MOMP, cytochrome c release and apoptosis was through the stabilization of the anti-apoptotic complex between VDAC1 and HK2.

The role of HK2 as the mediator of the first step in glycolysis is well characterized. HK2 is overexpressed in several tumors⁷ and recent reports show that its expression is critical for cancer cell survival⁸. The current focus on HK2 is on its catalytic ability to maintain and enhance the Warburg effect however HK2 has been shown to posses a strong anti-apoptotic effect when is localized to mitochondria. A well-characterized pathway, namely the mTOR-AKT (mammalian Target Of Rapamycin – Protein Kinase B-also known as AKT) pathway, is altered in many tumors and its activation is mediated by oncogenes, through loss of PTEN (phosphatase and tensin homolog) or through mutations in the PI3 kinase (Phosphatidylinositol-4,5-bisphosphate 3-kinase) genes. HK2 has been shown to be overexpressed in several tumors but AKT mediates the recruitment of HK2 at the mitochondria⁹. At this locale, HK2 accesses available ATP and enhances the glycolytic pathway but also prevents MOMP through interaction with VDAC. We found that IκΒα can stabilize the complex between VDAC1 and HK2, thereby inhibiting apoptosis by preventing the attachment of anti-apoptotic molecules such as Bax (Figure 1).

Due to tumor heterogeneity, a universal approach for cancer therapy has not been successful so far. However, mitochondria represent a target that can potentially be used by cancer therapies since they are mediators of the Warburg effect and apoptosis¹⁰°, which represent two of the major hallmarks of cancer. Enhanced cancer metabolism through HK2, apoptosis inhibition at mitochondria and constitutive activation of NFκB are events that coexist in many tumors. Our findings therefore shed light on a previously unexplored mechanism of IκΒα and provide a novel therapeutic strategy in cancer through impairment of apoptosis inhibition. Such an approach would include dissociation of IκΒα from the mitochondria, resulting in increased sensitivity of tumor cells to apoptotic stimuli.

Funding Statement

This funding was supported by Evangelos Pazarentzos

Disclosure statement

No potential conflict of interest was reported by the author(s).