BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

Regulation of interplay between autophagy and apoptosis in the diabetic heart

Diabetes induces cardiomyocyte apoptosis and suppresses cardiac autophagy, indicating that the interplay between autophagy and apoptotic cell death pathways is important in the pathogenesis of diabetic cardiomyopathy. The potential mechanism, however, remains unknown. We recently reported that diabetes depresses AMP-activated protein kinase (AMPK) activity, inhibits MAPK8/JNK1-BCL2 signaling, and promotes the interaction between BECN1 and BCL2. Concomitantly, diabetes induces cardiomyocyte apoptosis and suppresses cardiac autophagy. Activation of AMPK directly phosphorylates MAPK8, which mediates BCL2 phosphorylation and subsequent BECN1-BCL2 dissociation, leading to restoration of cardiac autophagy, protection against cardiac apoptosis, and ultimately improvement in cardiac structure and function. We conclude that dissociation of BCL2 from BECN1 through activation of MAPK8-BCL2 signaling may be an important mechanism by which AMPK activation restores autophagy, protects against cardiac apoptosis, and prevents diabetic cardiomyopathy.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

Attenuation of TNFSF10/TRAIL-induced apoptosis by an autophagic survival pathway involving TRAF2- and RIPK1/RIP1-mediated MAPK8/JNK activation

Although it is known that tumor necrosis factor-related apoptosis-inducing ligand (TNFSF10/TRAIL) induces autophagy, the mechanism by which autophagy is activated by TNFSF10 is still elusive. In this report, we show evidence that TRAF2- and RIPK1-mediated MAPK8/JNK activation is required for TNFSF10-induced cytoprotective autophagy. TNFSF10 activated autophagy rapidly in cancer cell lines derived from lung, bladder and prostate tumors. Blocking autophagy with either pharmacological inhibitors or siRNAs targeting the key autophagy factors BECN1/Beclin 1 or ATG7 effectively increased TNFSF10-induced apoptotic cytotoxicity, substantiating a cytoprotective role for TNFSF10-induced autophagy. Blocking MAPK8 but not NFκB effectively blocked autophagy, suggesting that MAPK8 is the main pathway for TNFSF10-induced autophagy. In addition, blocking MAPK8 effectively inhibited degradation of BCL2L1/Bcl-xL and reduction of the autophagy-suppressing BCL2L1–BECN1complex. Knockdown of TRAF2 or RIPK1 effectively suppressed TNFSF10-induced MAPK8 activation and autophagy. Furthermore, suppressing autophagy inhibited expression of antiapoptosis factors BIRC2/cIAP1, BIRC3/cIAP2, XIAP and CFLAR/c-FLIP and increased the formation of TNFSF10-induced death-inducing signaling complex (DISC). These results reveal a critical role for the MAPK8 activation pathway through TRAF2 and RIPK1 for TNFSF10-induced autophagy that blunts apoptosis in cancer cells. Thus, suppression of MAPK8-mediated autophagy could be utilized for sensitizing cancer cells to therapy with TNFSF10.     

Co-Inhibition of BCL-W and BCL2 Restores Antiestrogen Sensitivity through BECN1 and Promotes an Autophagy-Associated Necrosis

BCL2 family members affect cell fate decisions in breast cancer but the role of BCL-W (BCL2L2) is unknown. We now show the integrated roles of the antiapoptotic BCL-W and BCL2 in affecting responsiveness to the antiestrogen ICI 182,780 (ICI; Fulvestrant Faslodex), using both molecular (siRNA; shRNA) and pharmacologic (YC137) approaches in three breast cancer variants; MCF-7/LCC1 (ICI sensitive), MCF-7/LCC9 (ICI resistant), and LY2 (ICI resistant). YC137 inhibits BCL-W and BCL2 and restores ICI sensitivity in resistant cells. Co-inhibition of BCL-W and BCL2 is both necessary and sufficient to restore sensitivity to ICI, and explains mechanistically the action of YC137. These data implicate functional cooperation and/or redundancy in signaling between BCL-W and BCL2, and suggest that broad BCL2 family member inhibitors will have greater therapeutic value than targeting only individual proteins. Whereas ICI sensitive MCF-7/LCC1 cells undergo increased apoptosis in response to ICI following BCL-W±BCL2 co-inhibition, the consequent resensitization of resistant MCF-7/LCC9 and LY2 cells reflects increases in autophagy (LC3 cleavage; p62/SQSTM1 expression) and necrosis but not apoptosis or cell cycle arrest. Thus, de novo sensitive cells and resensitized resistant cells die through different mechanisms. Following BCL-W+BCL2 co-inhibition, suppression of functional autophagy by 3-methyladenine or BECN1 shRNA reduces ICI-induced necrosis but restores the ability of resistant cells to die through apoptosis. These data demonstrate the plasticity of cell fate mechanisms in breast cancer cells in the context of antiestrogen responsiveness. Restoration of ICI sensitivity in resistant cells appears to occur through an increase in autophagy-associated necrosis. BCL-W, BCL2, and BECN1 integrate important functions in determining antiestrogen responsiveness, and the presence of functional autophagy may influence the balance between apoptosis and necrosis.     

BECN1 and BIM interactions with MCL-1 determine fludarabine resistance in leukemic B cells

The purine analog fludarabine (Fd) is an essential therapeutic for chronic lymphocytic leukemia (CLL). Innate or acquired resistance to Fd is a significant clinical problem and is largely mediated by increased expression of BCL-2 family members. The antiapoptotic BCL-2 family proteins inhibit both apoptosis and autophagy, therefore, downregulation of antiapoptotic BCL-2 family proteins and enhanced autophagy must coexist in cells dying in response to an apoptosis inducing therapeutic. However, in the drug-resistant cells that have an increased dependence on antiapoptotic proteins, whether autophagy is also inhibited remains unclear. Here, we examined the role of the BCL-2 family in regulating cell death and autophagy in leukemic cell lines and their derivative isogenic Fd-resistant (FdR) cells. MCL-1 degradation following Fd treatment freed the proapoptotic effectors BIM and BECN1, thus leading to cell death-associated autophagy in Fd-sensitive cells. However, in FdR cells, low BIM expression and BECN1 sequestration by MCL-1 prevented cell death. Consistently, in sensitive cells inhibition of apoptosis using siBIM and of both the early-phase autophagy nucleation steps by siBECN1, shATG7 or 3-methyladenine and the late-phase autophagy by shLAMP2, significantly reduced Fd-induced cell death. Paradoxically, FdR cells were addicted to basal autophagy, which was dependent on AMP-activated protein kinase (AMPK) but not BECN1. Moreover, in FdR cells, inhibition of autophagy by shLAMP2, but not siBECN1, enhanced cell death. The BH3-mimetic obatoclax released BIM and BECN1 from MCL-1 in Fd-sensitive and BECN1 from MCL-1 in FdR cells, and was effective at killing both Fd-sensitive and - resistant leukemic cells, including primary CLL cells. Therefore, a differential regulation of autophagy through BECN1 and AMPK signaling in Fd-sensitive and - resistant cells determines the different possible outcomes of autophagy inhibition. These findings suggest effective means to overcome Fd resistance by induction of BIM-dependent apoptosis and activation of BECN1-dependent autophagy.     

Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.     

BAX-BAK1-independent LC3B lipidation by BH3 mimetics is unrelated to BH3 mimetic activity and has only minimal effects on autophagic flux

Inhibition of prosurvival BCL2 family members can induce autophagy, but the mechanism is controversial. We have provided genetic evidence that BCL2 family members block autophagy by inhibiting BAX and BAK1, but others have proposed they instead inhibit BECN1. Here we confirm that small molecule BH3 mimetics can induce BAX- and BAK1-independent MAP1LC3B/LC3B lipidation, but this only occurred at concentrations far greater than required to induce apoptosis and dissociate canonical BH3 domain-containing proteins that bind more tightly than BECN1. Because high concentrations of a less-active enantiomer of ABT-263 also induced BAX- and BAK1-independent LC3B lipidation, induction of this marker of autophagy appears to be an off-target effect. Indeed, robust autophagic flux was not induced by BH3 mimetic compounds in the absence of BAX and BAK1. Therefore at concentrations that are on target and achievable in vivo, BH3 mimetics only induce autophagy in a BAX- and BAK1-dependent manner.     

Bim inhibits autophagy by recruiting beclin 1 to microtubules

Bim is a proapoptotic BH3-only Bcl-2 family member.?In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in?vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites.     

Aβ1-42 monomers or oligomers have different effects on autophagy and apoptosis

The role of autophagy and its relationship with apoptosis in Alzheimer disease (AD) pathogenesis is poorly understood. Disruption of autophagy leads to buildup of incompletely digested substrates, amyloid-β (Aβ) peptide accumulation in vacuoles and cell death. Aβ, in turn, has been found to affect autophagy. Thus, Aβ might be part of a loop in which it is both the substrate of altered autophagy and its cause. Given the relevance of different soluble forms of Aβ1-42 in AD, we have investigated whether monomers and oligomers of the peptide have a differential role in causing altered autophagy and cell death. Using differentiated SK-N-BE neuroblastoma cells, we found that monomers hamper the formation of the autophagic BCL2-BECN1/Beclin 1 complex and activate the MAPK8/JNK1-MAPK9/JNK2 pathway phosphorylating BCL2. Monomers also inhibit apoptosis and allow autophagy with intracellular accumulation of autophagosomes and elevation of levels of BECN1 and LC3-II, resulting in an inhibition of substrate degradation due to an inhibitory action on lysosomal activity. Oligomers, in turn, favor the formation of the BCL2-BECN1 complex favoring apoptosis. In addition, they cause a less profound increase in BECN1 and LC3-II levels than monomers without affecting the autophagic flux. Thus, data presented in this work show a link for autophagy and apoptosis with monomers and oligomers, respectively. These studies are likely to help the design of novel disease modifying therapies.     

BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy

Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-X_L is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-X_L may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.     

PAWR-mediated suppression of BCL2 promotes switching of 3-azido withaferin A (3-AWA)-induced autophagy to apoptosis in prostate cancer cells

An active medicinal component of plant origin with an ability to overcome autophagy by inducing apoptosis should be considered a therapeutically active lead pharmacophore to control malignancies. In this report, we studied the effect of concentration-dependent 3-AWA (3-azido withaferin A) sensitization to androgen-independent prostate cancer (CaP) cells which resulted in a distinct switching of 2 interrelated conserved biological processes, i.e. autophagy and apoptosis. We have observed 3 distinct parameters which are hallmarks of autophagy in our studies. First, a subtoxic concentration of 3-AWA resulted in an autophagic phenotype with an elevation of autophagy markers in prostate cancer cells. This led to a massive accumulation of MAP1LC3B and EGFP-LC3B puncta coupled with gradual degradation of SQSTM1. Second, higher toxic concentrations of 3-AWA stimulated ER stress in CaP cells to turn on apoptosis within 12 h by elevating the expression of the proapoptotic protein PAWR, which in turn suppressed the autophagy-related proteins BCL2 and BECN1. This inhibition of BECN1 in CaP cells, leading to the disruption of the BCL2-BECN1 interaction by overexpressed PAWR has not been reported so far. Third, we provide evidence that pawr-KO MEFs exhibited abundant autophagy signs even at toxic concentrations of 3-AWA underscoring the relevance of PAWR in switching of autophagy to apoptosis. Last but not least, overexpression of EGFP-LC3B and DS-Red-BECN1 revealed a delayed apoptosis turnover at a higher concentration of 3-AWA in CaP cells. In summary, this study provides evidence that 3-AWA is a strong anticancer candidate to abrogate protective autophagy. It also enhanced chemosensitivity by sensitizing prostate cancer cells to apoptosis through induction of PAWR endorsing its therapeutic potential.     

AMPK regulates autophagy by phosphorylating BECN1 at threonine 388

Macroautophagy/autophagy is a conserved catabolic process that recycles cytoplasmic material during low energy conditions. BECN1/Beclin1 (Beclin 1, autophagy related) is an essential protein for function of the class 3 phosphatidylinositol 3-kinase (PtdIns3K) complexes that play a key role in autophagy nucleation and elongation. Here, we show that AMP-activated protein kinase (AMPK) regulates autophagy by phosphorylating BECN1 at Thr388. Phosphorylation of BECN1 is required for autophagy upon glucose withdrawal. BECN1^T388A, a phosphorylation defective mutant, suppresses autophagy through decreasing the interaction between PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and ATG14 (autophagy-related 14). The BECN1^T388A mutant has a higher affinity for BCL2 than its wild-type counterpart; the mutant is more prone to dimer formation. Conversely, a BECN1 phosphorylation mimic mutant, T388D, has stronger binding to PIK3C3 and ATG14, and promotes higher autophagy activity than the wild-type control. These findings uncover a novel mechanism of autophagy regulation.     

S100A8通过自噬促进B细胞淋巴瘤耐药性的机制研究

B细胞淋巴瘤是起源于淋巴造血系统的恶性肿瘤,是由分化过程中淋巴细胞恶性转化的复杂过程引起的。B细胞淋巴瘤细胞的耐药性是制约B细胞淋巴瘤治疗的关键因素。自噬是细胞成分降解和再循环的重要细胞生物学过程,近年来其与肿瘤耐药性的相关性受到越来越多的关注。S100A8是钙结合蛋白S100家族的重要成员,其在淋巴瘤的的耐药调控中发挥重要作用,但具体机制尚不清楚。在该研究中,以人Burkitt淋巴瘤细胞Daudi、人B淋巴瘤细胞SUDHL-4和人套细胞淋巴瘤细胞JeKo-1为研究对象,揭示了B细胞淋巴瘤细胞的耐药性与S100A8的表达水平密切相关,且S100A8可以显著激活淋巴瘤细胞的自噬进程。慢病毒感染稳定敲低S100A8的表达后,一方面,淋巴瘤细胞内质网和线粒体内的BNIP3蛋白的表达水平显著降低,自噬受到抑制;另一方面,自噬起始复合物BECN1-PI3KC3的表达显著降低,而BCL2与BECN1的结合显著增多,进而抑制细胞自噬。     

DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.     

Conformational flexibility of BECN1: Essential to its key role in autophagy and beyond

BECN1 (Beclin 1), a highly conserved eukaryotic protein, is a key regulator of autophagy, a cellular homeostasis pathway, and also participates in vacuolar protein sorting, endocytic trafficking, and apoptosis. BECN1 is important for embryonic development, the innate immune response, tumor suppression, and protection against neurodegenerative disorders, diabetes, and heart disease. BECN1 mediates autophagy as a core component of the class III phosphatidylinositol 3‐kinase complexes. However, the exact mechanism by which it regulates the activity of these complexes, or mediates its other diverse functions is unclear. BECN1 interacts with several diverse protein partners, perhaps serving as a scaffold or interaction hub for autophagy. Based on extensive structural, biophysical and bioinformatics analyses, BECN1 consists of an intrinsically disordered region (IDR), which includes a BH3 homology domain (BH3D); a flexible helical domain (FHD); a coiled‐coil domain (CCD); and a β‐α‐repeated autophagy‐specific domain (BARAD). Each of these BECN1 domains mediates multiple diverse interactions that involve concomitant conformational changes. Thus, BECN1 conformational flexibility likely plays a key role in facilitating diverse protein interactions. Further, BECN1 conformation and interactions are also modulated by numerous post‐translational modifications. A better structure‐based understanding of the interplay between different BECN1 conformational and binding states, and the impact of post‐translational modifications will be essential to elucidating the mechanism of its multiple biological roles.     

SLC9A3R1 stimulates autophagy via BECN1 stabilization in breast cancer cells

Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.     

Regulation of BECN1-mediated autophagy by HSPB6: Insights from a human HSPB6S10F mutant

HSPB6/Hsp20 (heat shock protein family B [small] member 6) has emerged as a novel cardioprotector against stress-induced injury. We identified a human mutant of HSPB6 (HSPB6^S10F) exclusively present in dilated cardiomyopathy (DCM) patients. Cardiac expression of this mutant in mouse hearts resulted in remodeling and dysfunction, which progressed to heart failure and early death. These detrimental effects were associated with reduced interaction of mutant HSPB6^S10F with BECN1/Beclin 1, leading to BECN1 ubiquitination and its proteosomal degradation. As a result, autophagy flux was substantially inhibited and apoptosis was increased in HSPB6^S10F-mutant hearts. In contrast, overexpression of wild-type HSPB6 (HSPB6 WT) not only increased BECN1 levels, but also competitively suppressed binding of BECN1 to BCL2, resulting in stimulated autophagy. Indeed, preinhibition of autophagy attenuated the cardioprotective effects of HSPB6 WT. Taken together, these findings reveal a new regulatory mechanism of HSPB6 in cell survival through its interaction with BECN1. Furthermore, Ser10 appears to be crucial for the protective effects of HSPB6 and transversion of this amino acid to Phe contributes to cardiomyopathy.