MCL-1 regulates the balance between autophagy and apoptosis

BCL-2 homologues lie at the interface between apoptosis and autophagy, regulating these two critical cellular pathways. However, the mechanisms controlling their coordinate regulation and the consequences on cellular survival are not fully understood. We recently showed that MCL-1 is a critical regulator of autophagy in cell lines and neurons. Our findings indicate that activation of apoptosis and autophagy is controlled in a developmentally regulated manner. In addition, the fact that MCL-1 null neurons die in an autophagy-dependent manner suggests that while a basal level of autophagy is required for neuronal survival, its sustained activation may be detrimental. This could have major implications for the treatment of neurodegenerative diseases using strategies involving activation of autophagy to clear protein aggregates from the brain.     

MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL-2 homologue MCL-1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL-1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL-1 levels regulate activation of autophagy. Furthermore, deletion of MCL-1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin-1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL-1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.     

MCL-1ES Induces MCL-1L-Dependent BAX- and BAK-Independent Mitochondrial Apoptosis

MCL-1 (myeloid cell leukemia-1), a member of the BCL-2 family, has three splicing variants, antiapoptotic MCL-1L, proapoptotic MCL-1S, and MCL-1ES. We previously reported cloning MCL-1ES and characterizing it as an apoptotic molecule. Here, we investigated the molecular mechanism by which MCL-1ES promotes cell death. MCL-1ES was distinct from other proapoptotic BCL-2 members that induce apoptosis by promoting BAX or BAK oligomerization, leading to mitochondrial outer membrane permeabilization (MOMP), in that MCL-1ES promoted mitochondrial apoptosis independently of both BAX and BAK. Instead, MCL-1L was crucial for the apoptotic activity of MCL-1ES by facilitating its proper localization to the mitochondria. MCL-1ES did not interact with any BCL-2 family proteins except for MCL-1L, and antiapoptotic BCL-2 members failed to inhibit apoptosis induced by MCL-1ES. The BCL-2 homology 3 (BH3) domain of MCL-1ES was critical for both MCL-1ES association with MCL-1L and apoptotic activity. MCL-1ES formed mitochondrial oligomers, and this process was followed by MOMP and cytochrome c release in a MCL-1L-dependent manner. These findings indicate that MCL-1ES, as a distinct proapoptotic BCL-2 family protein, may be useful for intervening in diseases that involve uncontrolled MCL-1L.     

Evaluation and critical assessment of putative MCL-1 inhibitors

High levels of BCL-2 family proteins are implicated in a failed/ineffective apoptotic programme, often resulting in diseases, including cancer. Owing to their potential as drug targets in cancer therapy, several inhibitors of BCL-2 family proteins have been developed. These primarily target specific members of the BCL-2 family, particularly BCL-2 and BCL-X_L but are ineffective against MCL-1. Major efforts have been invested in developing inhibitors of MCL-1, which is commonly amplified in human tumours and associated with tumour relapse and chemoresistance. In this report, the specificity of several BCL-2 family inhibitors (ABT-263, UCB-1350883, apogossypol and BH3I-1) was investigated and compared with putative MCL-1 inhibitors designed to exhibit improved or selective binding affinities for MCL-1 (TW-37, BI97C1, BI97C10, BI112D1, compounds 6 and 7, and MCL-1 inhibitor molecule (MIM-1)). ABT-263, BI97C1, BI112D1, MIM-1 and TW-37 exhibited specificity in inducing apoptosis in a Bax/Bak- and caspase-9-dependent manner, whereas the other agents showed no killing activity, or little or no specificity. Of these inhibitors, only ABT-263 and UCB-1350883 induced apoptosis in a BCL-2- or BCL-X_L-dependent system. In cells that depend on MCL-1 for survival, ABT-263 and TW-37 induced extensive apoptosis, suggesting that at high concentrations these inhibitors have the propensity to inhibit MCL-1 in a cellular context. TW-37 induced apoptosis, assessed by chromatin condensation, caspase processing and phosphatidylserine externalisation, in a BAK-dependent manner and in cells that require MCL-1 for survival. TW-37-mediated apoptosis was also partly dependent on NOXA, suggesting that derivatives of TW-37, if engineered to exhibit better selectivity and efficacy at low nanomolar concentrations, may provide useful lead compounds for further synthetic programmes. Expanded medicinal chemistry iteration, as performed for the ABT series, may likewise improve the potency and specificity of the evaluated MCL-1 inhibitors.     

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.     

Co-targeting MCL-1 and ERK1/2 kinase induces mitochondrial apoptosis in rhabdomyosarcoma cells

The RAS/MEK/ERK genetic axis is commonly altered in rhabdomyosarcoma (RMS), indicating high activity of downstream effector ERK1/2 kinase. Previously, we have demonstrated that inhibition of the RAS/MEK/ERK signaling pathway in RMS is insufficient to induce cell death due to residual pro-survival MCL-1 activity. Here, we show that the combination of ERK1/2 inhibitor Ulixertinib and MCL-1 inhibitor S63845 is highly synergistic and induces apoptotic cell death in RMS in vitro and in vivo. Importantly, Ulixertinib/S63845 co-treatment suppresses long-term survival of RMS cells, induces rapid caspase activation and caspase-dependent apoptosis. Mechanistically, Ulixertinib-mediated upregulation of BIM and BMF in combination with MCL-1 inhibition by S63845 shifts the balance of BCL-2 proteins towards a pro-apoptotic state resulting in apoptosis induction. A genetic silencing approach reveals that BIM, BMF, BAK and BAX are all required for Ulixertinib/S63845-induced apoptosis. Overexpression of BCL-2 rescues cell death triggered by Ulixertinib/S63845 co-treatment, confirming that combined inhibition of ERK1/2 and MCL-1 effectively induces cell death of RMS cells via the intrinsic mitochondrial apoptotic pathway. Thus, this study is the first to demonstrate the cytotoxic potency of co-inhibition of ERK1/2 and MCL-1 for RMS treatment.     

Crosstalk Between Bak/Bax and mTOR Signaling Regulates Radiation-Induced Autophagy

Bax and Bak, act as a gateway for caspase-mediated cell death. mTOR, an Akt downstream effector, plays a critical role in cell proliferation, growth and survival. The inhibition of mTOR induces autophagy, whereas apoptosis is a minor cell death mechanism in irradiated solid tumors.

We explored possible alternative pathways for cell death induced by radiation in Bax/Bak^-/- double knockout (DKO) MEF cells and wild-type cells, and we compared the cell survival: the Bax/Bak^-/- cells were more radiosensitive than the wild-type cells. The irradiated cells displayed an increase in the pro-autophagic proteins ATG5-ATG12 and Beclin-1.

These results are surprising in the fact that the inhibition of apoptosis resulted in increasing radiosensitivity; indicating that perhaps autophagy is the cornerstone in the cell radiation sensitivity regulation. Furthermore, irradiation up-regulates autophagic programmed cell death in cells that are unable to undergo Bax/Bak-mediated apoptosis. We hypothesize the presence of a phosphatase—possibly PTEN, an Akt/mTOR negative regulator that can be inhibited by Bax/Bak. This fits with our hypothesis of Bax/Bak as a down-regulator of autophagy.

We are currently conducting experiments to explore the relationship between apoptosis and autophagy. Future directions in research include strategies targeting Bax/Bak in cancer xenografts and exploring novel radiosensitizers targeting autophagy pathways.

Addendum to:

Autophagy for Cancer Therapy through Inhibition of Proapoptotic Proteins and mTOR Signaling

K.W. Kim, R.W. Mutter, C. Cao, J.M. Albert, M. Freeman, D.E. Hallahan and B. Lu

J Biol Chem 2006; Epub ahead of print     

MCL-1 is essential for survival but dispensable for metabolic fitness of FOXP3+ regulatory T cells

FOXP3⁺ regulatory T (Treg) cells are essential for maintaining immunological tolerance. Given their importance in immune-related diseases, cancer and obesity, there is increasing interest in targeting the Treg cell compartment therapeutically. New pharmacological inhibitors that specifically target the prosurvival protein MCL-1 may provide this opportunity, as Treg cells are particularly reliant upon this protein. However, there are two distinct isoforms of MCL-1; one located at the outer mitochondrial membrane (OMM) that is required to antagonize apoptosis, and another at the inner mitochondrial membrane (IMM) that is reported to maintain IMM structure and metabolism via ATP production during oxidative phosphorylation. We set out to elucidate the relative importance of these distinct biological functions of MCL-1 in Treg cells to assess whether MCL-1 inhibition might impact upon the metabolism of cells able to resist apoptosis. Conditional deletion of Mcl1 in FOXP3⁺ Treg cells resulted in a lethal multiorgan autoimmunity due to the depletion of the Treg cell compartment. This striking phenotype was completely rescued by concomitant deletion of the apoptotic effector proteins BAK and BAX, indicating that apoptosis plays a pivotal role in the homeostasis of Treg cells. Notably, MCL-1-deficient Treg cells rescued from apoptosis displayed normal metabolic capacity. Moreover, pharmacological inhibition of MCL-1 in Treg cells resistant to apoptosis did not perturb their metabolic function. We conclude that Treg cells require MCL-1 only to antagonize apoptosis and not for metabolism. Therefore, MCL-1 inhibition could be used to manipulate Treg cell survival for clinical benefit without affecting the metabolic fitness of cells resisting apoptosis.Subject terms: Disease genetics, Immune cell death     

MCL-1V, a novel mouse antiapoptotic MCL-1 variant, generated by RNA splicing at a non-canonical splicing pair

Myeloid cell leukemia-1 (MCL-1) that belongs to BCL-2 family is essential for survival of hematopoietic stem cells. It is upregulated in various types of cancer and promotes cancer cell metastasis. It is known that human MCL-1 gene undergoes differential splicing and yields three mRNAs encoding antiapoptotic MCL-1L and proapoptotic MCL-1S and MCL-1ES. However, no MCL-1 variants have been reported in mouse cells. We report here a new splicing variant of mouse Mcl-1, Mcl-1V, that is expressed in a variety of mouse normal and tumor cell lines and tissues. Comparative sequence analysis of the full-length Mcl-1 and Mcl-1V cDNAs suggested that Mcl-1V mRNA results from splicing within the first coding exon of Mcl-1 gene at a non-canonical donor-acceptor pair. MCL-1V lacks 46 amino acid residues within the N-terminal region of MCL-1. It localizes in mitochondria and inhibits anoxia- and anticancer drug-induced apoptosis as potent as MCL-1, and decayed less rapidly than MCL-1 in the cells undergoing apoptosis. Collectively, our results show that mouse cells ubiquitously express antiapoptotic MCL-1V that may play a role in mitochondrial cell death.     

Paclitaxel-Induced Apoptosis Is BAK-Dependent,but BAX and BIM-Independent in Breast Tumor

Paclitaxel (Taxol)-induced cell death requires the intrinsic cell death pathway, but the specific participants and the precise mechanisms are poorly understood. Previous studies indicate that a BH3-only protein BIM (BCL-2 Interacting Mediator of cell death) plays a role in paclitaxel-induced apoptosis. We show here that BIM is dispensable in apoptosis with paclitaxel treatment using bim^−/− MEFs (mouse embryonic fibroblasts), the bim^−/− mouse breast tumor model, and shRNA-mediated down-regulation of BIM in human breast cancer cells. In contrast, both bak ^−/− MEFs and human breast cancer cells in which BAK was down-regulated by shRNA were more resistant to paclitaxel. However, paclitaxel sensitivity was not affected in bax^−/− MEFs or in human breast cancer cells in which BAX was down-regulated, suggesting that paclitaxel-induced apoptosis is BAK-dependent, but BAX-independent. In human breast cancer cells, paclitaxel treatment resulted in MCL-1 degradation which was prevented by a proteasome inhibitor, MG132. A Cdk inhibitor, roscovitine, blocked paclitaxel-induced MCL-1 degradation and apoptosis, suggesting that Cdk activation at mitotic arrest could induce subsequent MCL-1 degradation in a proteasome-dependent manner. BAK was associated with MCL-1 in untreated cells and became activated in concert with loss of MCL-1 expression and its release from the complex. Our data suggest that BAK is the mediator of paclitaxel-induced apoptosis and could be an alternative target for overcoming paclitaxel resistance.     

Proteinase-activated Receptor 2 (PAR2) Decreases Apoptosis in Colonic Epithelial Cells

Mucosal biopsies from inflamed colon of inflammatory bowel disease patients exhibit elevated epithelial apoptosis compared with those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate inflammatory bowel disease, but treatments that specifically target apoptotic pathways are lacking. Proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor activated by trypsin-like serine proteinases, is expressed on intestinal epithelial cells and stimulates mitogenic pathways upon activation. We sought to determine whether PAR2 activation and signaling could rescue colonic epithelial (HT-29) cells from apoptosis induced by proapoptotic cytokines that are increased during inflammatory bowel disease. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspase-3, -8, and -9, poly(ADP-ribose) polymerase, and the externalization of phosphatidylserine after treatment of cells with IFN-γ and TNF-α. Knockdown of PAR2 with siRNA eliminated the anti-apoptotic effect of 2f-LI and increased the sensitivity of HT-29 cells to cytokine-induced apoptosis. Concurrent inhibition of both MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. 2f-LI was found to increase phosphorylation and inactivation of pro-apoptotic BAD at Ser¹¹² and Ser¹³⁶ by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic MCL-1. Simultaneous knockdown of both BAD and MCL-1 had minimal effects on PAR2-induced survival, whereas single knockdown had no effect. We conclude that PAR2 activation reduces cytokine-induced epithelial apoptosis via concurrent stimulation of MEK1/2 and PI3K but little involvement of MCL-1 and BAD. Our findings represent a novel mechanism whereby serine proteinases facilitate epithelial cell survival and may be important in the context of colonic healing.     

GX15-070 (obatoclax) overcomes glucocorticoid resistance in acute lymphoblastic leukemia through induction of apoptosis and autophagy

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies including acute lymphoblastic leukemia (ALL). The BCL-2 family has an essential role in regulating GC-induced cell death. Here we show that downregulation of antiapoptotic BCL-2 family proteins, especially MCL-1, enhances GC-induced cell death. Thus we target MCL-1 by using GX15-070 (obatoclax) in ALL cells. Treatment with GX15-070 in both dexamethasone (Dex)-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases the Annexin V-positive population, which is indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from the BAK/MCL-1 complex following GX15-070 treatment. Consistently, downregulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, downregulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Downregulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy.     

Beclin 1-independent autophagy contributes to apoptosis in cortical neurons

Neuronal autophagy is enhanced in many neurological conditions, such as cerebral ischemia and traumatic brain injury, but its role in associated neuronal death is controversial, especially under conditions of apoptosis. We therefore investigated the role of autophagy in the apoptosis of primary cortical neurons treated with the widely used and potent pro-apoptotic agent, staurosporine (STS). Even before apoptosis, STS enhanced autophagic flux, as shown by increases in autophagosomal (LC3-II level, LC3 punctate labeling) and lysosomal (cathepsin D, LAMP1, acid phosphatase, β-hexasominidase) markers. Inhibition of autophagy by 3-methyladenine, or by lentivirally-delivered shRNAs against Atg5 and Atg7, strongly reduced the STS-induced activation of caspase-3 and nuclear translocation of AIF, and gave partial protection against neuronal death. Pan-caspase inhibition with Q-VD-OPH likewise protected partially against neuronal death, but failed to affect autophagy. Combined inhibition of both autophagy and caspases gave strong synergistic neuroprotection. The autophagy contributing to apoptosis was Beclin 1-independent, as shown by the fact that Beclin 1 knockdown failed to reduce it but efficiently reduced rapamycin-induced autophagy. Moreover the Beclin 1 knockdown sensitized neurons to STS-induced apoptosis, indicating a cytoprotective role of Beclin 1 in cortical neurons. Caspase-3 activation and pyknosis induced by two other pro-apoptotic stimuli, MK801 and etoposide, were likewise found to be associated with Beclin 1-independent autophagy and reduced by the knockdown of Atg7 but not Beclin 1. In conclusion, Beclin 1-independent autophagy is an important contributor to both the caspase-dependent and -independent components of neuronal apoptosis and may be considered as an important therapeutic target in neural conditions involving apoptosis.     

Rapamycin and Interleukin-1β Impair Brain-derived Neurotrophic Factor-dependent Neuron Survival by Modulating Autophagy

The mammalian target of rapamycin (mTOR) pathway has multiple important physiological functions, including regulation of protein synthesis, cell growth, autophagy, and synaptic plasticity. Activation of mTOR is necessary for the many beneficial effects of brain-derived neurotrophic factor (BDNF), including dendritic translation and memory formation in the hippocampus. At present, however, the role of mTOR in BDNF''s support of survival is not clear. We report that mTOR activation is necessary for BDNF-dependent survival of primary rat hippocampal neurons, as either mTOR inhibition by rapamycin or genetic manipulation of the downstream molecule p70S6K specifically blocked BDNF rescue. Surprisingly, however, BDNF did not promote neuron survival by up-regulating mTOR-dependent protein synthesis or through mTOR-dependent suppression of caspase-3 activation. Instead, activated mTOR was responsible for BDNF''s suppression of autophagic flux. shRNA against the autophagic machinery Atg7 or Atg5 prolonged the survival of neurons co-treated with BDNF and rapamycin, suggesting that suppression of mTOR in BDNF-treated cells resulted in excessive autophagy. Finally, acting as a physiological analog of rapamycin, IL-1β impaired BDNF signaling by way of inhibiting mTOR activation as follows: the cytokine induced caspase-independent neuronal death and accelerated autophagic flux in BDNF-treated cells. These findings reveal a novel mechanism of BDNF neuroprotection; BDNF not only prevents apoptosis through inhibiting caspase activation but also promotes neuron survival through modulation of autophagy. This protection mechanism is vulnerable under chronic inflammation, which deregulates autophagy through impairing mTOR signaling. These results may be relevant to age-related changes observed in neurodegenerative diseases.     

IL-32γ Delays Spontaneous Apoptosis of Human Neutrophils through MCL-1, Regulated Primarily by the p38 MAPK Pathway

IL-32γ is a multifunctional cytokine involved in various inflammatory and auto-immune diseases in which neutrophils can affect the evolution of these diseases. To persist at inflammatory sites, neutrophils require inhibition of their rapid and constitutive apoptosis, an inhibitory effect that phlogogenic cytokines support. To date, the effects of IL-32γ on neutrophils remain unknown. We demonstrate that IL-32γ delays, in a dose-dependent manner, the spontaneous apoptosis of human blood neutrophils by activating mainly p38 MAPK through rapid p38 phosphorylation. PI3-K and ERK1/2 MAPK are also involved, but to a lesser extent. Most of cytokines that induce retardation of neutrophil apoptosis activate the expression of MCL-1 at both mRNA and protein levels. IL-32γ added to human blood neutrophils in vitro is associated with sustained levels of MCL-1 protein. This effect in neutrophils corresponds to a decrease of MCL-1 protein degradation without any effect on MCL-1 mRNA levels. The sustained levels of MCL-1 induced by IL-32γ are only abrogated by the p38β MAPK inhibitor SB202190. Additionally, IL-32γ induces a reduction in caspase 3 activity in neutrophils. In conclusion, IL-32γ affects human blood neutrophils in vitro by increasing their survival, suggesting that this cytokine could have profound effects on the deleterious functions of neutrophils in several diseases.     

BID-Deficient Breast Cancer MCF-7 Cells as a Model for the Study of Autophagy in Cancer Therapy

The BH3-only death factors share just the short BH3 domain with the other Bcl-2 family subclasses. With the exception of BID, which might also bind to BAX, they are thought to act by binding to and neutralizing Bcl-2 like survival factors.Camptothecin (CPT)-induced apoptosis in breast cancer MCF-7 cells is associated with activation of cathepsin B and aggregation of BAX and BID on mitochondria. BID knock down protects cancer cells against apoptosis and induces autophagy, manifested with increased expression of Beclin1 and MAP1LC3. The compensatory increase in the concentration of Hrk (another member of the BH3-only protein family) and its co-localization with BCL-2 on organelles in BID(-) breast cancer cells has also been observed. Nonetheless, Hrk is not able to substitute for BID in triggering apoptosis. Its role in autophagy induction is also doubtful, since MAP1LC3 expression was equally high in BID(-)Hrk(-) and BID(-)Hrk(+) breast cancer cells exposed to CPT. We conclude that BID can serve as a molecular switch between apoptosis and autophagy. BID(+) and BID(-) breast cancer MCF-7 cells could be considered to be a useful model for the study of the molecular interdependences between apoptosis and autophagy and the role of both processes in cancer therapy.

Addenda to:

Cathepsins and BID are Involved in the Molecular Switch between Apoptosis and Autophagy in Breast Cancer MCF-7 Cells Exposed to Camptothecin

M. Lamparska-Przybysz, B. Gajkowska and T. Motyl

J Physiol Pharmacol 2005; 56:159-79     

Breast cancer dependence on MCL-1 is due to its canonical anti-apoptotic function

High levels of the anti-apoptotic BCL-2 family member MCL-1 are frequently found in breast cancer and, appropriately, BH3-mimetic drugs that specifically target MCL-1’s function in apoptosis are in development as anti-cancer therapy. MCL-1 also has reported non-canonical roles that may be relevant in its tumour-promoting effect. Here we investigate the role of MCL-1 in clinically relevant breast cancer models and address whether the canonical role of MCL-1 in apoptosis, which can be targeted using BH3-mimetic drugs, is the major function for MCL-1 in breast cancer. We show that MCL-1 is essential in established tumours with genetic deletion inducing tumour regression and inhibition with the MCL-1-specific BH3-mimetic drug {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 significantly impeding tumour growth. Importantly, we found that the anti-tumour functions achieved by MCL-1 deletion or inhibition were completely dependent on pro-apoptotic BAX/BAK. Interestingly, we find that MCL-1 is also critical for stem cell activity in human breast cancer cells and high MCL1 expression correlates with stemness markers in tumours. This strongly supports the idea that the key function of MCL-1 in breast cancer is through its anti-apoptotic function. This has important implications for the future use of MCL-1-specific BH3-mimetic drugs in breast cancer treatment.Subject terms: Cancer models, Cell biology, Genetics     

PIWIL2 interacting with IKK to regulate autophagy and apoptosis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies and cause of death from cancer in China. Previous studies showed that autophagy and apoptosis inhibition are critical for the survival of ESCC cells. However, the underlying mechanisms remain to be clarified. Recently, we found that PIWIL2, a novel cancer testis protein, is highly expressed in ESCC and associated with high T-stage and poor 5-year survival rate in patients. Our further study showed that PIWIL2 can directly bind to IKK and promote its phosphorylation, leading to phosphorylation of IκB and subsequently nuclear translocation of NF-κB for apoptosis inhibition. Meanwhile, PIWIL2 competitively inhibits binding of IKK to TSC1, and thus deactivate mTORC1 pathway which suppresses ULK1 phosphorylation and initiation of autophagy. The mouse xenograft model suggested that PIWIL2 can promote ESCC growth in an IKK-dependent manner. This present work firstly revealed that PIWIL2 can play a role in regulating autophagy and apoptosis, and is associated with poor prognosis in ESCC patients, providing novel insights into the roles of PIWIL2 in tumorigenesis.Subject terms: Macroautophagy, Prognostic markers, Cancer genetics