Adhesion to the extracellular matrix (ECM) is critical for epithelial tissue survival and function, because detachment from the ECM induces metabolic stress and programmed cell death via anoikis. Recently, it was shown that macroautophagy (hereafter called autophagy) is also induced by detachment from the ECM in different cell types, including human and murine epithelial cells and fibroblasts. Initiation of autophagy upon ECM detachment appears to be involved in cell survival, as inhibition of autophagy enhances cell death by anoikis.
The BMF (Bcl2 modifying factor) protein contains the BCL2 homology region 3, and thereby is a member of the BCL2 protein family. BMF is implicated in regulating cell death following detachment of cells from the ECM. During ECM detachment BMF mRNA is upregulated to allow anoikis in human breast epithelial cells, and suppressing BMF expression disrupts anoikis. BMF is attached to the cytoskeleton through the DYNLL2/DLC2 (dynein, light chain, LCB-type 2) that connects it with MYO5/myosin V and the actin filaments. Loss of attachment of a transformed cell line (MCF-7) releases BMF from the cytoskeleton, and the released BMF interacts with mitochondrial BCL2 to induce apoptosis.
We found that BMF also has a role as a regulator of autophagy. When BMF levels are suppressed in IFNγ-treated human or mouse airway epithelial cells, LC3-II conversion and BECN1 expression are increased. Furthermore, primary airway epithelial cells or embryonic fibroblasts from bmf−/− mice present more LC3-II and more autophagosome structures when analyzed by electron microscopy. The conclusion regarding the formation of autophagosomes is also supported by the observed change from a diffuse to punctate fluorescence of cherry-LC3 in bmf−/− cells. In contrast, restoring BMF levels in IFNγ-treated cells decreases the levels of LC3-II and BECN1, supporting the idea that BMF inhibits autophagy. We also demonstrated that BMF co-immunoprecipitates with BECN1 and BCL2, and that the interaction between BECN1 and BCL2 decreases in the absence of BMF, suggesting that BMF may inhibit initiation of autophagy by stabilizing the BECN1-BCL2 protein complex. However, the molecular mechanisms of this interaction, whether other proteins are part of this inhibitory complex, and whether other pathways impinge on this role of BMF remain to be elucidated.
Because BMF is involved in detachment-induced cell death, and our observations show that BMF regulates autophagy, together with the recent reports on autophagy being involved in ECM detachment and cell death, led us to postulate that detachment-induced autophagy may involve the BMF protein. Detachment from the ECM may cause the release of BMF from the cytoskeleton allowing the autophagy inhibitory complex BECN1-BCL2 to be released and BECN1 to localize to the ER to initiate autophagosome formation.
Other pathways that are initiated by ECM detachment may also be involved in inducing autophagy. Suppression of the PI3K-AKT-MTORC1 pathway activation is the major regulator of autophagy induction in detached mouse fibroblasts. However, in human mammary epithelial cells deprived of ECM contact, activation of the IKK complex (the catalytic subunits CHUK/IKKα and IKBKB/IKKβ, and the regulatory subunit IKBKG/IKKγ) is crucial in promoting autophagy and is independent of MTOR signaling. AMP-activated protein kinase (AMPK) is robustly activated during ECM detachment, but its role in activating autophagy after ECM detachment still needs to be verified. In this context, because BMF has been reported to be upregulated by AMPK activation, upregulated BMF following detachment from the ECM may lose its capacity to inhibit autophagy, because it is no longer attached to the cytoskeleton. This idea is also consistent with the upregulation of BMF mRNA to allow anoikis after ECM detachment in human breast epithelial cells. After cell detachment, there is also increased phosphorylation of EIF2S1/eIF2α (eukaryotic translation initiation factor 2, subunit 1 α, 35 kDa), a stress-regulated transcriptional suppressor that upregulates autophagy during nutritional starvation and ER stress. The various pathways may either occur simultaneously or may interact to enhance autophagy. For example, IKK-stimulated autophagy is controlled by the canonical AMPK-MTOR pathway and MAPK8/JNK1 activation in HeLa cells, although in certain cell types the IKK pathway induces autophagy independent of MTOR. All these pathways, including BMF released from the cytoskeleton, may participate in ECM detachment-induced autophagy depending on cell type and the nutritional or stressed conditions of the cells. Therefore, we can speculate that when BMF is released from the cytoskeleton in response to ECM detachment, BMF may interact with one or more autophagy-activating pathways. As autophagy is a survival mechanism that protects cells from dying after ECM detachment, the intensity of autophagy induction may determine why some cell types die within a few hours while others survive for several days following ECM detachment. We would propose that following detachment-induced expression, BMF would have a role in inhibiting autophagy or enhancing anoikis depending on whether BMF is attached to DYNLL2/DLC2 or is released into the cytoplasm (Fig. 1).
Figure 1. Proposed pathway for BMF involvement in anoikis and autophagy.
ECM detachment is a topic of high interest as the survival of oncogenic cells lacking matrix contact is considered to be a critical feature for metastasis. Therefore, detailed understanding of the pathways involved in regulating detachment-induced autophagy could help develop better therapies for a variety of cancers.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.