Abstract
The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner.
Highlights
Sprouty-related EVH1 domain (Spred) proteins function as negative regulators of growth factors, cytokines and chemokine-induced ERK activation by binding to Ras and Raf-1 [1,2,3].Spreds (Spred1, 2 and 3) contain an N-terminal Ena/VASP homology 1 domain (EVH1), a central c-Kit binding domain (KBD) and a C-terminal cysteine-rich Sproutyrelated domain (SPR) that is shared with the Sprouty proteins [1, 4]
Using clone formation assays, we showed that infection with adenoviruses expressing Myc-tagged Spred2 (Ad-Spred2) results in the significant inhibition of colony formation in human cervical carcinoma cells (HeLa) and A549 cells compared to control virus (Figure 1A), consistent with our previous work and others that Spred2 suppresses tumor cell growth [2, 8, 16]
To investigate whether apoptosis is involved in Spred2-induced tumor cell growth inhibition, HeLa cells infected with Ad-Spred2 were analyzed by flow cytometry using Annexin V and propidium iodide (PI) double-staining
Summary
Sprouty-related EVH1 domain (Spred) proteins function as negative regulators of growth factors, cytokines and chemokine-induced ERK (extracellular-regulated kinase) activation by binding to Ras and Raf-1 [1,2,3].Spreds (Spred and 3) contain an N-terminal Ena/VASP homology 1 domain (EVH1), a central c-Kit binding domain (KBD) and a C-terminal cysteine-rich Sproutyrelated domain (SPR) that is shared with the Sprouty proteins [1, 4]. Sprouty-related EVH1 domain (Spred) proteins function as negative regulators of growth factors, cytokines and chemokine-induced ERK (extracellular-regulated kinase) activation by binding to Ras and Raf-1 [1,2,3]. Several studies, including our previous work, have reported that the EVH1 and SPR domains www.impactjournals.com/oncotarget play critical roles in the Spred-mediated inhibitory effect on ERK activation [1, 3, 5,6,7,8,9], the KBD region is dispensable [4]. In vitro and in vivo models ectopically expressing Spreds led to a decrease in cancer cell proliferation. This may be due to reduced ERK/MAPK activity [2, 16]. The underlying mechanism by which Spreds suppress tumor growth remains to be elucidated
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