mda-7/IL-24 is a unique member of the IL-10 gene family, which displays a broad range of antitumor properties including induction of cancer-specific apoptosis. Adenoviral mediated delivery by Ad.mda-7 invokes an endoplasmic reticulum stress response that is associated with ceramide production and autophagy in some cancer cells. Here we report that Ad.mda-7-induced ER stress and ceramide production triggers autophagy in human prostate cancer cells, but not normal prostate epithelial cells, through a canonical signaling pathway that involves Beclin-1, atg5 and hVps34. Autophagy occurs in cancer cells at early times after Ad.mda-7 infection but a switch to apoptosis occurs by 48 hr post-infection. Inhibiting autophagy with 3-methyladenosine increases Ad.mda-7-induced apoptosis, suggesting that autophagy may be initiated first as a cytoprotective mechanism. Inhibiting apoptosis by overexpression of anti-apoptotic proteins Bcl-2 or Bcl-xL increased autophagy after Ad.mda-7 infection. During the apoptotic phase, the MDA-7/IL-24 protein physically interacted with Beclin-1 in a manner that could inhibit Beclin-1 function culminating in apoptosis. Conversely, Ad.mda-7 infection elicited calpain-mediated cleavage of the autophagic protein ATG5 in a manner that could facilitate switch to apoptosis. Our findings reveal novel aspects of the interplay between autophagy and apoptosis in prostate cancer cells that underlie the cytotoxic action of mda-7/IL-24, possibly providing new insights in the development of combinatorial therapies for prostate cancer.
mda-7/IL-24; protective autophagy; apoptosis; Beclin-1; atg5
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a unique member of the IL-10 gene family, displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis, and modulation of anti-tumor immune responses. Here we identify clusterin (CLU) as a MDA-7/IL-24 interacting protein in DU-145 cells and investigate the role of MDA-7/IL-24 in regulating CLU expression and mediating the antitumor properties of mda-7/IL-24 in prostate cancer. Ad.mda-7 decreased expression of soluble CLU (sCLU) and increased expression of nuclear CLU (nCLU). In the initial phase of Ad.mda-7 infection sCLU expression increased and CLU interacted with MDA-7/IL-24 producing a cytoprotective effect. Infection of stable clones of DU-145 prostate cancer cells expressing sCLU with Ad.mda-7 resulted in generation of nCLU that correlated with decreased cell viability and increased apoptosis. In the presence of mda-7/IL-24, sCLU-DU-145 cells displayed G2/M phase arrest followed by apoptosis. Similarly, Ad.mda-7 infection decreased cell migration by altering cytoskeleton in sCLU-DU-145 cells. Ad.mda-7-treated sCLU-DU-145 cells displayed a significant reduction in tumor growth in mouse xenograft models and reduced angiogenesis when compared to the vector control group. Tumor tissue lysates demonstrated enhanced nCLU generated from sCLU with increased apoptosis in the presence of MDA-7/IL-24. Our findings reveal novel aspects relative to the role of sCLU/nCLU in regulating the anticancer properties of MDA-7/IL-24 that may be exploited for developing enhanced therapies for prostate cancer.
MDA-7/IL-24; soluble clusterin; nuclear clusterin; G2/M arrest; apoptosis
Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors. In this study, we defined the proximal mechanisms by which Ad-mda-7 kills glioma cells. Key factors implicated included activation of the endoplasmic reticulum stress kinase protein kinase R–like endoplasmic reticulum kinase (PERK), Ca++ elevation, ceramide generation and reactive oxygen species (ROS) production. PERK inhibition blocked ceramide or dihydroceramide generation, which were critical for Ca++ induction and subsequent ROS formation. Activation of autophagy and cell death relied upon ROS formation, the inhibition of which ablated Ad.mda-7–killing activity. In contrast, inhibiting TRX induced by Ad.MDA-7 enhanced tumor cytotoxicity and improved animal survival in an orthotopic tumor model. Our findings indicate that mda-7/IL-24 induces an endoplasmic reticulum stress response that triggers production of ceramide, Ca2+, and ROS, which in turn promote glioma cell autophagy and cell death.
A natural BH3-mimetic, small molecule inhibitor of Bcl-2, (-)-gossypol, shows promise in ongoing Phase II-III clinical trials for human prostate cancer. Here we show that (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy via blocking Bcl-2—Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1 and activating the autophagic pathway. (-)-Gossypol-induced autophagy is Beclin1- and Atg5-dependent. Our results demonstrate for the first time that (-)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (-)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which would facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.
(-)-Gossypol; Bcl-2; Beclin1; Autophagy; Apoptosis
A natural BH3-mimetic, small-molecule inhibitor of Bcl-2, (−)-gossypol, shows promise in ongoing phase II and III clinical trials for human prostate cancer. In this study we show that (−)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, both in vitro and in vivo, but not in androgen-dependent (AD) cells with low Bcl-2 and sensitive to apoptosis. The Bcl-2 inhibitor induces autophagy through blocking Bcl-2–Beclin1 interaction, together with downregulating Bcl-2, upregulating Beclin1, and activating the autophagic pathway. The (−)-gossypol-induced autophagy is dependent on Beclin1 and Atg5. Our results show for the first time that (−)-gossypol can also interrupt the interactions between Beclin1 and Bcl-2/Bcl-xL at endoplasmic reticulum, thus releasing the BH3-only pro-autophagic protein Beclin1, which in turn triggers the autophagic cascade. Oral administration of (−)-gossypol significantly inhibited the growth of AI prostate cancer xenografts, representing a promising new regimen for the treatment of human hormone-refractory prostate cancer with Bcl-2 overexpression. Our data provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which will facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.
(−)-gossypol; Bcl-2; Beclin1; autophagy; apoptosis
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, apoptosis-inducing gene with broad-spectrum antitumor activity, making it an ideal candidate for a novel cancer gene therapy. A systemic and sustained antitumor immune response generated at the time of initial molecular-targeted therapy would provide additional clinical benefits in cancer patients, resulting in improved prevention of tumor recurrence. In this study, we explored the therapeutic efficacy of intratumoral delivery of a nonreplicating adenoviral vectors encoding mda-7/IL-24 (Ad.mda-7) and a secretable form of endoplasmic reticulum resident chaperone grp170 (Ad.sgrp170), a potent immunostimulatory adjuvant and antigen carrier. Intratumoral administration of Ad.mda-7 in combination with Ad.sgrp170 was more effective in controlling growth of TRAMP-C2 prostate tumor as compared to either Ad.mda-7 or Ad.sgrp170 treatment. Generation of systemic antitumor immunity was demonstrated by enhanced protection against subsequent tumor challenge and improved control of distant tumors. The combined treatments enhanced antigen and tumor-specific T-cell response as indicated by increased IFN-γ production and cytolytic activity. Antibody depletion suggests that CD8+ T-cell may be involved in the antitumor effect of the dual molecule-targeted therapies. Therefore, introducing immunostimulatory chaperone grp170 in situ strongly promotes the ‘immunogenic’ cell death when delivered to the mda-7/IL-24 induced apoptotic tumor cells, indicating that an improved anti-cancer efficacy may be achieved by concurrently targeting both tumor and immune compartments. Given multiple undefined antigens present endogenously within prostate cancer, these data provide a rationale for combining sgrp170-based vaccine strategy with mda-7/IL-24-targeted cancer therapy to induce durable systemic immunity.
mda-7/IL-24; cancer gene therapy; stress protein; chaperone; grp170; immunity
The interplay between a non-lethal autophagic response and apoptotic cell death is still a matter of debate in cancer cell biology. In the present study performed on human melanoma cells, we investigate the role of basal or stimulated autophagy in cisplatin-induced cytotoxicity, as well as the contribution of cisplatin-induced activation of caspases 3/7 and conventional calpains. The results show that, while down-regulating Beclin-1, Atg14 and LC3-II, cisplatin treatment inhibits the basal autophagic response, impairing a physiological pro-survival response. Consistently, exogenously stimulated autophagy, obtained with trehalose or calpains inhibitors (MDL-28170 and calpeptin), protects from cisplatin-induced apoptosis, and such a protection is reverted by inhibiting autophagy with 3-methyladenine or ATG5 silencing. In addition, during trehalose-stimulated autophagy, the cisplatin-induced activation of calpains is abrogated, suggesting the existence of a feedback loop between the autophagic process and calpains. On the whole, our results demonstrate that in human melanoma cells autophagy may function as a beneficial stress response, hindered by cisplatin-induced death mechanisms. In a therapeutic perspective, these findings suggest that the efficacy of cisplatin-based polychemotherapies for melanoma could be potentiated by inhibitors of autophagy.
The present studies focused on determining whether the autophagy-inducing drug OSU-03012 (AR-12) could enhance the toxicity of recombinant adenoviral delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) in glioblastoma multiforme (GBM) cells. The toxicity of a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7) was enhanced by OSU-03012 in a diverse panel of primary human GBM cells. The enhanced toxicity correlated with reduced ERK1/2 phosphorylation and expression of MCL-1 and BCL-XL, and was blocked by molecular activation of ERK1/2 and by inhibition of the intrinsic, but not the extrinsic, apoptosis pathway. Both OSU-03012 and expression of MDA-7/IL-24 increased phosphorylation of PKR-like endoplasmic reticulum kinase (PERK) that correlated with increased levels of autophagy and expression of dominant negative PERK blocked autophagy induction and tumor cell death. Knockdown of ATG5 or Beclin1 suppressed OSU-03012 enhanced MDA-7/IL-24-induced autophagy and blocked the lethal interaction between the two agents. Ad.mda-7-infected GBM cells secreted MDA-7/IL-24 into the growth media and this conditioned media induced expression of MDA-7/IL-24 in uninfected GBM cells. OSU-03012 interacted with conditioned media to kill GBM cells and knockdown of MDA-7/IL-24 in these cells suppressed tumor cell killing. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.
ROS; caspase; ER stress; CD95; cell death
Bcl-2 is a key dual regulator of autophagy and apoptosis, but how the level of Bcl-2 influences the cellular decision between autophagy and apoptosis is unclear. The natural BH3-mimetic (-)-gossypol preferentially induces autophagy in androgen-independent (AI) prostate cancer cells that have high levels of Bcl-2 and are resistant to apoptosis, whereas apoptosis is preferentially induced in androgen-dependent or -independent cells with low Bcl-2. (-)-Gossypol induces autophagy via blocking Bcl-2-Beclin 1 interaction at the endoplasmic reticulum (ER), together with downregulating Bcl-2, upregulating Beclin 1 and activating the autophagic pathway. Furthermore, (-)-gossypol-induced autophagy is Beclin 1- and Atg5-dependent. These results provide new insights into the mode of cell death induced by Bcl-2 inhibitors, which could facilitate the rational design of clinical trials by selecting patients who are most likely to benefit from the Bcl-2-targeted molecular therapy.
Bcl-2; (-)-gossypol; apoptosis; autophagy; Beclin 1
Ovarian cancer is the fifth most common cause of cancer-related death in women. Current interventional approaches, including debulking surgery, chemotherapy, and/or radiation have proven minimally effective in preventing the recurrence and/or mortality associated with this malignancy. Subtraction hybridization applied to terminally differentiating human melanoma cells identified melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), whose unique properties include the ability to selectively induce growth suppression, apoptosis, and radiosensitization in diverse cancer cells, without causing any harmful effects in normal cells. Previously, it has been shown that adenovirus-mediated mda-7/IL-24 therapy (Ad.mda-7) induces apoptosis in ovarian cancer cells, however, the apoptosis induction was relatively low. We now document that apoptosis can be enhanced by treating ovarian cancer cells with ionizing radiation (IR) in combination with Ad.mda-7. Additionally, we demonstrate that mda-7/IL-24 gene delivery, under the control of a minimal promoter region of progression elevated gene-3 (PEG-3), which functions selectively in diverse cancer cells with minimal activity in normal cells, displays a selective radiosensitization effect in ovarian cancer cells. The present studies support the use of IR in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in ovarian cancer, particularly in the context of tumors displaying resistance to radiation therapy.
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.
► Bim negatively regulates autophagy in cell culture and in vivo ► Bim inhibits autophagosome formation by interacting with Beclin 1 ► Bim inhibits autophagy by mislocalizing Beclin 1 from the ER to microtubules ► Starvation induces autophagy and dissociates the Bim-Beclin1 interaction
Autophagy and apoptosis are two important and interconnected stress-response mechanisms. However, the molecular interplay between these two pathways is not fully understood. To study the fate and function of autophagic proteins at the onset of apoptosis, we used a cellular model system in which autophagy precedes apoptosis. IL-3 depletion of Ba/F3 cells caused caspase (casp)-mediated cleavage of Beclin-1 and PI3KC3, two crucial components of the autophagy-inducing complex. We identified two casp cleavage sites in Beclin-1, TDVD133 and DQLD149, cleavage at which yields fragments lacking the autophagy-inducing capacity. Noteworthy, the C-terminal fragment, Beclin-1-C, localized predominantly at the mitochondria and sensitized the cells to apoptosis. Moreover, on isolated mitochondria, recombinant Beclin-1-C was able to induce the release of proapoptotic factors. These findings point to a mechanism by which casp-dependent generation of Beclin-1-C creates an amplifying loop enhancing apoptosis upon growth factor withdrawal.
autophagy; Beclin-1; PI3KC3; apoptosis; caspases; Ba/F3; IL-3
Melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that displays nearly ubiquitous cancer-specific toxicity, with no harmful effects toward normal cells or tissues. mda-7/IL-24 was cloned from human melanoma cells by differentiation induction subtraction hybridization (DISH) and promotes endoplasmic reticulum (ER) stress culminating in apoptosis or toxic autophagy in a broad-spectrum of human cancers, when assayed in cell culture, in vivo in human tumor xenograft mouse models and in a Phase I clinical trial in patients with advanced cancers. This therapeutically active cytokine also induces indirect anti-tumor activity through inhibition of angiogenesis, stimulation of an anti-tumor immune response, and sensitization of cancer cells to radiation-, chemotherapy- and antibody-induced killing.
mda-7/IL-24; apoptosis; autophagy; bystander antitumor activity; cancer terminator virus
Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), a cytokine belonging to the IL-10 family, selectively induces apoptosis in cancer cells without harming normal cells by promoting an endoplasmic reticulum (ER) stress response. The precise molecular mechanism by which the ER stress response culminates in cell death requires further clarification. The present study shows that in prostate carcinoma cells, the mda-7/IL-24-induced ER stress response causes apoptosis by translational inhibition of the antiapoptotic protein myeloid cell leukemia-1 (Mcl-1). Forced expression of Mcl-1 blocked mda-7/IL-24 lethality, whereas RNA interference or gene knockout of Mcl-1 markedly sensitized transformed cells to mda-7/IL-24. Mcl-1 downregulation by mda-7/IL-24 relieved its association with the proapoptotic protein Bak, causing oligomerization of Bak and leading to cell death. These observations show the profound role of the Bcl-2 protein family member Mcl-1 in regulating cancer-specific apoptosis induced by this cytokine. Thus, our studies provide further insights into the molecular mechanism of ER stress-induced cancer-selective apoptosis by mda-7/IL-24. As Mcl-1 is overexpressed in the majority of prostate cancers, mda-7/IL-24 might provide an effective therapeutic for this disease.
Previous studies showed that the human melanoma differentiation-associated gene-7 (mda-7), also known as interleukin-24 (IL-24), has potent antitumor activity against human and murine cancer cells. However, the majority of these studies were limited to in vitro testing. In the present study, we investigated the antitumor activity of mda-7/IL-24 against human ovarian cancer cells both in vitro and in vivo.
In vitro, treatment of ovarian cancer cells with an adenoviral vector carrying the mda-7 gene (Ad-mda7) resulted in inhibition of cell proliferation and induction of cell cycle arrest, leading to apoptosis. We did not observe inhibitory activity in Ad-mda7-treated normal cells. In vivo, treatment of subcutaneous tumor xenografts with Ad-mda7 resulted in significant tumor growth inhibition when compared with that in control groups (p < 0.001). Molecular analysis of ovarian tumor tissue lysates treated with Ad-mda7 showed that MDA-7 protein expression was associated with activation of the caspase cascade.
Our results show that treatment of ovarian cancer cells with mda-7/IL-24 results in growth suppression both in vitro and in vivo.
Apoptotic cell death is mediated by caspase activation. Autophagy involves the sequestration of cytoplasmic contents into autophagosomes for traffic to lysosomes for degradation. While autophagy is antiapoptotic, increased numbers of autophagosomes have been associated with forms of non-apoptotic cell death. Apoptosis and autophagy may be co-regulated in the same directions, as the anti-apoptotic Bcl-2 and Bcl-xL proteins negatively regulate autophagy by binding to Beclin 1 (mammalian Atg6), and pro-apoptotic BH3-only proteins may reverse this effect by displacing these interactions. Here we show that apoptosis can suppress autophagy. Apoptosis induced by the pro-apoptotic protein Bax reduced autophagy by enhancing caspase-mediated cleavage of Beclin 1 at D149. After cleavage, both N and C-terminal Beclin 1 fragments change their localisations and these fragments do not interact normally with Vps34, which is required for autophagy. The cleavage of Beclin 1 is a critical event whereby caspases inhibit autophagy, as a non-cleavable Beclin 1 mutant restored autophagy in cells overexpressing Bax.
apoptosis; autophagy; cleavage; caspase
The ubiquitin-proteasome system and macroautophagy (hereafter referred to autophagy) are two complementary pathways for protein degradation. Emerging evidence suggests that proteasome inhibition might be a promising approach for tumor therapy. Accumulating data suggest that autophagy is activated as a compensatory mechanism upon proteasome activity is impaired.
Autophagy activation was measured using acridine orange staining and LC3 transition. Cell viability and apoptosis were measured using MTT assay and flow cytometry, respectively. Beclin 1 expression vectors or shRNA against Beclin 1 (shBeclin 1) were transfected to investigate the role of Beclin 1 in autophagy activation and cytotoxicity of ovarian cancer cells induced by proteasome inhibitors.
Proteasome inhibitors suppressed proliferation and induced autophagy in ovarian cancer cells. Neither phosphoinositide 3-kinase (PI3K) inhibitors nor shRNA against Beclin 1 could abolish the formation of acidic vacuoles and the processing of LC3 induced by proteasome inhibitors. Moreover, Beclin 1 overexpression enhanced anti-proliferative effects of proteasome inhibitors in ovarian cancer cells.
For the first time, the current study demonstrated that proteasome inhibitors induced PI3K and Beclin 1-independent autophagy in ovarian cancer cells. In addition, this study revealed autophagy-independent tumor suppressive effects of Beclin 1 in ovarian cancer cells.
Proteasome inhibition; Beclin1; Ovarian cancer
Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.
cancer; homologous with E6-associated protein C-terminus (HECT); neural-precursor-cell-expressed developmentally down-regulated 4 (Nedd4); phosphoinositide; ubiquitination; VPS34; AIP4, atrophin-interacting protein 4; BH3, Bcl-2 homology 3; CCD, coiled-coil domain; CHX, cycloheximide; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; ECD, evolutionarily conserved domain; GST, glutathione transferase; HA, haemagglutinin; HECT, homologous with E6-associated protein C-terminus; HRP, horseradish peroxidase; Nedd4, neural-precursor-cell-expressed developmentally down-regulated 4; PI3K-III, class III PI 3-kinase; siRNA, small interfering RNA; TRAF6, tumour-necrosis-factor-receptor-associated factor 6; Ub, ubiquitin
Autophagy is an evolutionarily conserved process of cytoplasm and cellular organelle degradation in lysosomes. Autophagy is a survival pathway required for cellular viability during starvation; however, if it proceeds to completion, autophagy can lead to cell death. In neurons, constitutive autophagy limits accumulation of polyubiquitinated proteins and prevents neuronal degeneration. Therefore, autophagy has emerged as a homeostatic mechanism regulating the turnover of long-lived or damaged proteins and organelles, and buffering metabolic stress under conditions of nutrient deprivation by recycling intracellular constituents. Autophagy also plays a role in tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in many human ovarian, breast, and prostate cancers, and beclin1+/− mice are tumor-prone. We found that allelic loss of beclin1 renders immortalized mouse mammary epithelial cells susceptible to metabolic stress and accelerates lumen formation in mammary acini. Autophagy defects also activate the DNA damage response in vitro and in mammary tumors in vivo, promote gene amplification, and synergize with defective apoptosis to accelerate mammary tumorigenesis. Thus, loss of the prosurvival role of autophagy likely contributes to breast cancer progression by promoting genome damage and instability. Exploring the yet unknown relationship between defective autophagy and other breast cancer-promoting functions may provide valuable insight into the pathogenesis of breast cancer and may have significant prognostic and therapeutic implications for breast cancer patients.
autophagy; breast cancer; beclin1; DNA damage; genomic instability
beclin 1, the mammalian homologue of the yeast Atg6, is a key autophagy-promoting gene that plays a critical role in the regulation of cell death and survival of various types of cells. However, recent studies have observed that the expression of beclin 1 is altered in certain diseases including cancers. The causes underlying the aberrant expression of beclin 1 remain largely unknown. We report here that microRNAs (miRNAs), a class of endogenous, 22–24 nucleotide noncoding RNA molecules able to affect stability and translation of mRNA, may represent a previously unrecognized mechanism for regulating beclin 1 expression and autophagy. We demonstrated that beclin 1 is a potential target for miRNA miR-30a, and this miRNA could negatively regulate beclin 1 expression resulting in decreased autophagic activity. Treatment of tumor cells with the miR-30a mimic decreased, and with the antagomir increased, the expression of beclin 1 mRNA and protein. Dual luciferase reporter assay confirmed that the miR-30a binding sequences in the 3′-UTR of beclin 1 contribute to the modulation of beclin 1 expression by miR-30a. Furthermore, inhibition of beclin 1 expression by the miR-30a mimic blunted activation of autophagy induced by rapamycin. Our study of the role of miR-30a in regulating beclin 1 expression and autophagy reveals a novel function for miRNA in a critical cellular event with significant impacts in cancer development, progression and treatment, and in other diseases.
beclin 1; autophagy; microRNA; miR-30a; gene expression
Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.
chemoresistance; breast cancer; hypoxia; apoptosis; autophagy; taxol
Autophagy is an evolutionarily conserved stress response mechanism that often occurs in apoptosis-defective cancer cells and can protect against cell death. In this study, we investigated how apoptosis and autophagy affect each other in cancer cells in response to chemotherapeutic treatment. We found that specific ablation of the proapoptotic function of cytochrome c, a key regulator of mitochondria-mediated apoptosis, enhanced autophagy following chemotherapeutic treatment. Induction of autophagy required Beclin 1, and was associated with blockage of Beclin 1 cleavage by caspase 8 at two sites. To investigate the role of Beclin 1 cleavage in the suppression of autophagy and cell survival, a caspase-resistant mutant of Beclin 1 was knocked into HCT116 colon cancer cells. Beclin 1 mutant knock-in resulted in markedly increased autophagy and improved long-term cell survival after chemotherapeutic treatment, but without affecting apoptosis and caspase activation. Furthermore, Beclin 1 mutant tumors were significantly less responsive to chemotherapeutic treatment than wild-type tumors. These results demonstrate that chemotherapy-induced apoptosis inhibits autophagy at the execution stage subsequent to cytochrome c release through caspase 8-mediated cleavage of Beclin 1. If apoptosis fails to execute, autophagy is unleashed due to lack of Beclin 1 cleavage by caspases, and can contribute to cancer cell survival and therapeutic resistance. Therefore, Beclin 1 may be a useful target for inhibiting autophagy to sensitize chemotherapy.
Autophagy is an evolutionarily conserved process whereby cytoplasm and cellular organelles are degraded in lysosomes for amino acid and energy recycling. Autophagy is a survival pathway activated in response to nutrient deprivation and other stressful stimuli, such as metabolic stress and exposure to anticancer drugs. However, autophagy may also result in cell death, if it proceeds to completion. Defective autophagy is implicated in tumorigenesis, as the essential autophagy regulator beclin 1 is monoallelically deleted in human breast, ovarian and prostate cancers, and beclin 1+/− mice are tumor-prone. How autophagy suppresses tumorigenesis is under intense investigation. Cell-autonomous mechanisms, involving protection of genome integrity and stability, and a non-cell-autonomous mechanism, involving suppression of necrosis and inflammation, have been discovered so far. The role of autophagy in treatment responsiveness is also complex. Autophagy inhibition concurrently with chemotherapy or radiotherapy has emerged as a novel approach in cancer treatment, as autophagy-competent tumor cells depend on autophagy for survival under drug- and radiation-induced stress. Alternatively, autophagy stimulation and preservation of cellular fitness bymaintenance of protein and organelle quality control, suppression of DNA damage and genomic instability, and limitation of necrosis-associated inflammation may play a critical role in cancer prevention.
Autophagy; Tumorigenesis; Tumor suppression; Autophagy regulation; Cancer therapy; Autophagy modulation
Autophagy is often found in apoptosis-defective cancer cells and contributes to chemotherapy resistance. However, it is far from clear how the coordination of apoptosis and autophagy determines sensitivity of cancer cells to chemotherapy. Our recent study showed that Beclin 1, a key regulator of autophagy, is cleaved by caspase 8 at the execution stage of chemotherapy-induced and mitochondria-mediated apoptosis. Perturbation of Beclin 1 cleavage, by knock-in of a mutation, phenocopies the autophagy observed in apoptosis-defective cancer cells, and renders chemotherapy resistance in vitro and in vivo. These results demonstrate an important role of caspases in suppressing autophagy by cleaving autophagic machinery.
Beclin 1; caspase 8; autophagy; apoptosis; chemotherapy
The traditional Chinese medicine bufalin, extracted from toad’s skin, has been demonstrated to exert anticancer activities in various kinds of human cancers. The mechanisms of action lie in its capacity to induce apoptosis, or termed type I programmed cell death (PCD). However, type II PCD, or autophagy, participates in cancer proliferation, progression, and relapse, as well. Recent studies on autophagy seem to be controversial because of the dual roles of autophagy in cancer survival and death. In good agreement with previous studies, we found that 100 nM bufalin induced extensive HepG2 cell apoptosis. However, we also noticed bufalin triggered autophagy and enhanced Beclin-1 expression, LC3-I to LC3-II conversion, as well as decreased p62 expression and mTOR signaling activation in HepG2 cells. Blockage of autophagy by selective inhibitor 3-MA decreased apoptotic ratio in bufalin-treated HepG2 cells, suggesting a proapoptotic role of bufalin-induced autophagy. Furthermore, we investigated the underlying mechanisms of bufalin-induced autophagy. Bufalin treatment dose-dependently promoted AMPK phosphorylation while AMPK inhibition by compound C significantly attenuated bufalin-induced autophagy. Taken together, we report for the first time that bufalin induces HepG2 cells PCD, especially for autophagy, and the mechanism of action is, at least in part, AMPK-mTOR dependent.
hepatocellular carcinoma; bufalin; apoptosis; autophagy; AMPK; mTOR