Here, we demonstrate that ECM detachment induces autophagy in various nontumorigenic epithelial cell lines and in primary human epithelial cells. We also discover that ATG depletion results in both enhanced cleaved caspase-3 during anoikis and reduced clonogenic viability upon reattachment. Remarkably, matrix-detached cells still exhibit autophagy when apoptosis is blocked by Bcl-2 expression, and ATG depletion impairs the clonogenic survival of detached Bcl-2–expressing cells. Thus, autophagy promotes epithelial cell survival during anoikis, including detached cells harboring antiapoptotic lesions.
Anoikis serves as an important mechanism to maintain tissue homeostasis by killing cells that have lost contact with an underlying basement membrane (Gilmore, 2005
). However, evidence indicates that the detachment of nonmalignant cells also triggers antiantiapoptotic signals, such as nuclear factor-κB and inhibitor of apoptosis protein family members; these antiapoptotic mechanisms presumably delay the onset of apoptosis and allow cells to survive if they are able to reestablish ECM contact in a timely manner (Yan et al., 2005
; Liu et al., 2006
). Our results indicate, similar to these antiapoptotic pathways, the induction of autophagy also contributes to cell viability during ECM detachment.
Overall, our results resemble other circumstances where autophagy protects cells exposed to various forms of duress, including nutrient deprivation, growth factor withdrawal, ER stress, and ischemia (Boya et al., 2005
; Lum et al., 2005
; Degenhardt et al., 2006
; Ogata et al., 2006
; Kouroku et al., 2007
). Autophagy may promote cell survival by a number of mechanisms, such as 1) generating nutrients and energy to sustain starving or stressed cells, 2) degrading toxic proteins, or 3) protecting cells from oxidative stress by sequestering damaged mitochondria (Debnath et al., 2005
; Levine and Yuan, 2005
). Further studies are required to identify the precise mechanisms through which detachment-induced autophagy promotes cell survival. Moreover, in addition to regulating survival, autophagy may direct other critical biological functions during matrix detachment; for example, autophagy suppresses both DNA damage and chromosomal instability in response to metabolic stress (Karantza-Wadsworth et al., 2007
; Mathew et al., 2007
Because integrin engagement is critical for the proper transduction of growth factor receptor mediated signals, we hypothesized that detachment-induced autophagy results from reduced growth factor receptor signaling. Importantly, in human mammary epithelial cells, ECM contact is required for both EGFR expression and the activation of critical downstream signals (Reginato et al., 2003
). However, we demonstrate that even though enforced EGFR expression during anoikis can restore certain downstream survival-promoting signals, such as ERK activation, it is not sufficient to prevent detachment-induced autophagy. Thus, we postulate that other cell adhesion regulated stress pathways also direct detachment-induced autophagy. We have identified multiple signals during ECM detachment that may positively regulate autophagy, including AMPK activation and the phosphorylation of eIF2α; in ongoing studies, we are trying to dissect how these signals, either individually or in combination, contribute to detachment-induced autophagy.
Remarkably, a recent study demonstrates that integrin engagement, most notably α3β1, is actually required to sustain autophagy in starved prostate epithelial cells (Edick et al., 2007
). In this study, the observed reductions in autophagy were based exclusively on measurements of GFP-LC3 puncta; thus, it remains unknown whether the reductions in punctate GFP-LC3 observed upon integrin blockade are actually due to decreased autophagosome formation versus increased LC3-II turnover in the lysosome (Tanida et al., 2005
). Alternatively, detachment-induced autophagy may be cell type or context dependent. Importantly, whereas human mammary epithelial cells die after 24–48 h of detachment, certain epithelial cells, notably primary mouse mammary epithelial cells and rat intestinal epithelial cells, perish within a few hours following substratum detachment; thus, one can speculate that detachment-induced autophagy will not contribute to the viability of cells that undergo rapid anoikis (Gilmore, 2005
Protection from anoikis is thought to promote filling of the normally hollow lumen in glandular epithelial structures, a hallmark of early epithelial cancers, such as carcinomas in situ (Debnath and Brugge, 2005
; Gilmore, 2005
). Although our previous work suggested that cells undergo autophagy as they die during 3D morphogenesis, two results in this study argue against a direct role for type 2 death during lumen formation. First, the acute pharmacological inhibition of autophagy during morphogenesis enhances luminal cell death. Second, the knockdown of ATG5 or ATG7, two proteins critical for early autophagosome formation, enhances luminal apoptosis during MCF-10A 3D morphogenesis and fails to elicit long-term luminal filling, even when combined with apoptosis inhibition. Our results are consistent with recent studies in E1A immortalized mouse mammary cells lacking one allele of Beclin; when grown in 3D culture, cells with reduced Beclin exhibit increased lumen formation compared with wild-type controls (Karantza-Wadsworth et al., 2007
). Interestingly, in embryoid bodies derived from cells lacking atg5
, apoptotic cell corpses fail to clear during cavitation, because autophagy is critical for the presentation of signals, such as phosphatidylserine, that mediate the phagocytic clearance of apoptotic cells (Qu et al., 2007
). Accordingly, the increased numbers of apoptotic cells that we observe when autophagy is inhibited during lumen formation could arise from defective corpse clearance. However, over longer times, we do not observe reduced clearance in ATG5 or ATG7-depleted structures; rather, these structures form hollow lumens at rates similar to controls. Clearance may ultimately proceed during 3D morphogenesis because we have only reduced these ATGs rather than completely eliminated these proteins in acini. Nonetheless, recent work indicates that inhibition of autophagy, a late stage event observed in MCF7 carcinoma cells during anoikis, does not prevent phagocytic engulfment (Petrovski et al., 2007
). Importantly, during both 3D lumen formation and ECM detachment, we have observed the robust induction of autophagy in Bcl-2–expressing cells, indicating that autophagy can proceed independently of apoptosis in ECM-detached cells, rather than merely serve as a secondary clearance mechanism to remove cells undergoing apoptosis (Debnath et al., 2002
). Further delineating the respective roles of autophagy in luminal cell survival versus the phagocytic clearance of dying cells during lumen formation remains a topic for future investigation.
Several autophagy regulators are down-regulated in human cancers, including BECN-1/ATG6
, which is monoallelically deleted in 40–75% of breast, prostate, and ovarian tumors, and Death Associated Protein Kinase 1
, which is methylated in many tumors (Liang et al., 1999
; Inbal et al., 2002
). The importance of autophagy as a tumor suppressor is further supported by the development of tumors in becn-1
+/− mice (Qu et al., 2003
; Yue et al., 2003
). In contrast, because autophagy has well-established cytoprotective functions, it can promote the survival of tumor cells exposed to stresses such as hypoxia, nutrient limitation, and chemotherapy (Ogier-Denis and Codogno, 2003
; Jin et al., 2007
). Both of these opposing functions may be relevant to cancer progression and treatment. Our results broach the possibility that autophagy contributes to the survival of oncogenic cells lacking appropriate matrix contact. Indeed, the ability to survive in the absence of normal ECM is considered a critical feature of metastasis, because cancer cells in the bloodstream or secondary tissue sites are either deprived of matrix or exposed to foreign matrix components (Chambers et al., 2002
). Accordingly, we are currently investigating how oncogenic pathways regulate autophagy during ECM detachment and determining whether autophagy contributes to the survival and expansion of oncogene-expressing cells undergoing anoikis and in the lumens of 3D structures.