DEDD, a novel tumor repressor, reverses epithelial-mesenchymal transition by activating selective autophagy

Abstract

Metastasis is the spread of cancer cells from their primary location to other parts of the body. Metastatic cancer is responsible for most cancer deaths. Increasing evidence indicates that epithelial-mesenchymal transition (EMT), a crucial developmental program, contributes to control cancer invasion and metastasis. We recently reported that death effector domain-containing DNA-binding protein (DEDD), a key effector molecule for cell death signaling receptors, attenuates EMT and acts as an endogenous suppressor of tumor growth and metastasis. We found that DEDD physically interacts with the class III PtdIns 3-kinase complex containing PIK3C3 and BECN1, which controls critical aspects of autophagy; this interaction activates autophagy and induces the autophagy-mediated lysosomal degradation of SNAI/Snail and TWIST, two master inducers of the EMT process. Further study reveals that the DEDD-PIK3C3 interaction can support the stability of PIK3C3 to maintain autophagic activity and promote the degradation of SNAI and TWIST. Our finding indicates that DEDD is a prognostic marker and a potential therapeutic target for the prevention and treatment of cancer metastasis. Moreover, regulation of the DEDD-PIK3C3 interaction may serve as an entry point to translate modifiers of this interaction into clinical endpoints.

Metastasis is a complex and multistep process, which has been divided into two phases, namely, physical translocation of a tumor cell from the primary tumor to a distant tissue to seed, and colonization of disseminated tumor cells in distant organs. Epithelial-mesenchymal transition, first recognized as a feature of embryogenesis, is characterized by loss of cell adhesion, repression of CDH1/E-cadherin expression, and increased cell mobility. It is well known that the EMT process is involved in the two-phase events in cancer to promote metastasis. Moreover, recent studies demonstrate that the activation of the EMT can promote noncancer stem cells (non-CSCs) to enter into a CSC-like state, with the cell obtaining self-renewal and tumor-initiating ability.

The death-effector domain-containing DEDD is a member of a family of death effector domain-containing proteins that is comprised of seven proteins including FADD, CFLAR/c-FLIP, PEA15, CASP8, CASP10, DEDD1 and DEDD2. DEDD plays an important role in FAS/CD95-mediated apoptosis in response to the activation of TNFA/TNF-α through activating CASP3 in the cytoplasm or CASP6 in the nucleus as a scaffold protein. Recent studies found that DEDD can suppress the activity of CDK1-CCNB1/cyclin B1 complexes and maintain RPS6KB1/S6K1 activity, suggesting that DEDD participates in the regulation of the cell cycle and inhibits cell mitosis. Our previous work indicates that DEDD attenuates TGFB/TGF-β1-SMAD3 signaling through interacting with SMAD3 to suppress the TGF-β1-SMAD3-mediated invasion in cancer cells. Moreover, a recent study reports that DEDD is indispensable for the establishment of an adequate uterine environment to support early pregnancy in mice. These findings suggest that DEDD is involved in the regulation of embryonic developmental programs including EMT.

In the current study, we have demonstrated that the expression level of DEDD in breast and colon cancers correlates conversely with the poor prognosis of these cancers. Moreover, the expression level of DEDD is negatively correlated with the invasive phenotype of breast cancer cell lines. In metastatic MAD-MB-231 cells with DEDD expression lost, ectopic expression of DEDD stabilizes and activates PIK3C3 through a physical interaction with this protein, leading to the autophagy-lysosome dependent degradation of SNAI and TWIST, two master inducers of EMT, to attenuate the EMT process and the metastatic phenotype in these cancer cells (Fig. 1). In contrast, in non-metastatic MCF-7 cells with normal DEDD expression, silencing DEDD promotes the degradation and inactivation of the PIK3C3, leading to an attenuation of autophagy activity and the autophagy-lysosome dependent degradation pathway. Hence, accumulation of SNAI and TWIST in these cancer cells promotes the EMT process and results in the acquisition of the most central traits of the CSCs.

Figure 1. Model of DEDD-activated autophagic lysosomal degradation of SNAI and TWIST. DEDD binds directly to the class III PtdIns3K (BECN1-containing) core complex to stabilize PIK3C3 and promote the interaction of this enzyme with BECN1, which then localizes to the phagophore and facilitates recruitment of other autophagy-related proteins. Ubiquitinated SNAI-TWIST binds SQSTM1 and is then transported to the phagophore. The autophagosomes containing SNAI-TWIST are subsequently delivered to the lysosome for degradation. Inhibition of these two master modulators of the EMT results in a reversal of the EMT and inhibits invasion and metastasis of breast cancer.

Autophagy, a self-catabolic process that maintains intracellular homeostasis and prolongs cell survival under stress, is generally thought to be a double-edged sword in the regulation of tumor progression. Autophagy may inhibit metastasis by restricting necrosis and infiltration of prometastatic inflammatory cells, maintaining dormancy of disseminated tumor cells for extended periods of time. Conversely, autophagy may promote metastasis via anoikis resistance, and promoting the survival of isolated dormant cells. Our studies highlight a new role for autophagy in the metastatic process. DEDD reverses the EMT by activating the autophagy-lysosome degradation of SNAI and TWIST. These proteins are generally considered to be degraded by the ubiquitin-proteasome system. However, we present new evidence that alteration of autophagy can regulate their degradation. Inhibiting autophagy with the PIK3C3 inhibitor 3-MA can prolong the half-life of SNAI-TWIST, as well as narrow the difference in their degradation rates in MCF-7 cells expressing control or DEDD shRNA. Changing the expression of BECN1 or PIK3C3 by overexpression or knockdown, respectively, also regulates the expression of SNAI-TWIST and the EMT.

The next question is, how is SNAI-TWIST degraded by an autophagy-dependent pathway? Selective autophagy is a specialized type of autophagy, which specifically targets protein aggregates, organelles, and intracellular pathogens. Recently, accumulation of the selective autophagy receptor SQSTM1/p62 in autophagy-deficient cells was directly linked to tumorigenesis. The accumulation of SQSTM1 in autophagy-deficient cells inhibits the degradation of cancer-relevant proteins whose expression level is primarily regulated by the ubiquitin-proteasomal pathway (i.e., TP53/p53 and CTNNB1/β-catenin). In our studies, the degradation of SNAI-TWIST is associated with the clearance of SQSTM1 when autophagy is activated. By contrast, inhibiting autophagy results in the accumulation of SQSTM1 and increases the amounts of SNAI-TWIST in cells. These findings indicate that SQSTM1 plays a key role in regulating the autophagy-associated degradation of SNAI-TWIST.

The PIK3C3-BECN1 complex plays a central role in the initiation and maturation of autophagosomes. We find that DEDD interacts with PIK3C3 or BECN1. However, silencing DEDD results in a marked decrease in the degradation half-life of PIK3C3 but not BECN1, indicating that the DEDD-PIK3C3 interaction plays a crucial role in maintaining the stability of the PIK3C3-BECN1 complex. Recently, a series of investigations have shown that the antiapoptotic BCL2 protein inhibits BECN1-dependent autophagy through an interaction via its BH3 domain with BECN1. Does the interaction of DEDD-BECN1 influence autophagy like that of BCL2 with BECN1? The respective interaction domains of DEDD with BECN1-PIK3C3 will be uncovered in our following work to reveal more details of DEDD-regulated autophagy.

Lv Q, Wang W, Xue J, Hua F, Mu R, Lin H, Yan J, Lv X, Chen X, Hu ZW. DEDD Interacts with PI3KC3 to Activate Autophagy and Attenuate Epithelial-Mesenchymal Transition in Human Breast Cancer. Cancer Res. 2012;72:3238–50.