Antiapoptotic Bcl-2 Protein Mcl-1 Research Articles

Abstract 280: MCL-1 Matrix Promotes Mitochondrial Fusion and Protects Against TAC Induced Hypertrophy

The anti-apoptotic BCL-2 protein MCL-1 is highly expressed in the myocardium, and we have previously found that deletion in cardiomyocytes leads to mitochondrial dysfunction and rapid development of heart failure. MCL-1 exists both in the outer mitochondrial membrane (MCL-1 OM ) and in the mitochondrial matrix (MCL-1 Matrix ). Because Mcl-1 -/- mice lack both MCL-1 Matrix and MCL-1 OM , the functional role of each form remains unknown. While studies have implicated MCL-1 OM in regulating apoptosis, very little is known about the functional role of MCL-1 Matrix . Here, we show that mitochondria are responsive to alterations in cellular metabolism, and culturing mouse embryonic fibroblasts (MEFs) in “oxidative” media containing galactose or acetoacetate led to a significant increase in the MCL-1 Matrix /MCL-1 OM ratio and elongation of mitochondria. We confirmed that MCL-1 Matrix overexpression promoted mitochondrial fusion and reduced maximal mitochondrial respiration under baseline conditions. Interestingly, the elongated mitochondria were initially protected against FCCP-induced fission and mitophagy. However, prolonged exposure to FCCP resulted in abrogation of MCL-1 Matrix import. Accumulation of MCL-1 Matrix on the outer membrane led to its degradation and subsequent induction of mitophagy. Furthermore, to investigate its functional role in the heart, we generated cardiac specific MCL-1 Matrix (αMHC-MCL-1 Matrix ) transgenic mice. The mice show no differences in cardiac structure and function compared to wild-type litter mates at 3 months. However, ultrastructural analysis revealed the presence of enlarged mitochondria in myocytes from αMHC-MCL-1 Matrix mice. Finally, we subjected WT and αMHC-MCL-1 Matrix mice to transverse aortic constriction (TAC) and we found that αMHC-MCL-1 Matrix mice had reduced heart weight to body weight (HW/BW) ratio and decreased expression of hypertrophy markers ANF, BNP, and MHC-β at two weeks post-TAC compared to WT. Thus, our studies indicate that cardiac overexpression of MCL-1 Matrix promotes mitochondrial enlargement in myocytes and alleviates stress placed on the heart during pressure overload.

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR.

Targeting the intrinsic apoptosis pathway as a strategy for melanoma therapy

Melanoma drug resistance is often attributed to abrogation of the intrinsic apoptosis pathway. Targeting regulators of apoptosis is thus considered a promising approach to sensitizing melanomas to treatment. The development of small-molecule inhibitors that mimic natural antagonists of either antiapoptotic members of the BCL-2 family or the inhibitor of apoptosis proteins (IAPs), known as BH3- or SMAC-mimetics, respectively, are helping us to understand the mechanisms behind apoptotic resistance. Studies using BH3-mimetics indicate that the antiapoptotic BCL-2 protein MCL-1 and its antagonist NOXA are particularly important regulators of BCL-2 family signaling, while SMAC-mimetic studies show that both XIAP and the cIAPs must be targeted to effectively induce apoptosis of cancer cells. Although most solid tumors, including melanoma, are insensitive to these mimetic drugs as single agents, combinations with other therapeutics have yielded promising results, and tests combining them with BRAF-inhibitors, which have already revolutionized melanoma treatment, are a clear priority.

Increased leukocyte survival and accelerated onset of lymphoma in the absence of MCL-1 S159-phosphorylation

The antiapoptotic BCL-2 protein MCL-1, which opposes mitochondrial outer membrane permeabilization, was shown to have a crucial role in the survival of hematopoietic cells. We have previously shown that, upon loss of phosphatidylinositol 3-kinase signaling, S159 of MCL-1 is phosphorylated by glycogen synthase kinase-3 (GSK-3), earmarking MCL-1 for enhanced ubiquitylation and degradation. In this study, we introduced MCL-1(wt) or the phosphorylation-deficient mutant MCL-1(S159A) in mouse BM cells, followed by adoptive transfer to recipient mice. Mice expressing MCL-1(S159A) exhibited significantly elevated white blood cell and lymphocyte counts, whereas no effect was observed on the distribution of T and B lymphocyte subsets or the numbers of monocytes, red blood cells or platelets. Expression of MCL-1(S159A) in Eμ-Myc transgenic bone marrow significantly accelerated the onset of disease, and these mice displayed increased spleen weights compared with Eμ-Myc/MCL-1(wt) mice. Our data demonstrate that the absence of MCL-1 S159 phosphorylation provides a survival advantage for hematopoietic cells in vivo and facilitates oncogenesis.

Beyond Cell Death – Antiapoptotic Bcl-2 Proteins Regulate Migration and Invasion of Colorectal Cancer Cells In Vitro

Migration and invasion of malignant cells are prerequisites for cancer progression and metastasis. The Bcl-2 family of proteins consists of about 25 members and has been extensively studied in the context of apoptosis. Despite the fact that small molecules targeting Bcl-2 proteins have already entered clinical trials, very few studies investigated a role of antiapoptotic Bcl-2 proteins beside cell death in the context of metastasis. The aim of this study was to dissect a potential role of the antiapoptotic Bcl-2 proteins Mcl-1, Bcl-2 and Bcl-xL on migration and invasion of colorectal cancer cells independent of their cell death control function. We used migration and invasion assays as well as three dimensional cell cultures to analyze colorectal cancer cell lines (HT29 and SW480) after siRNA mediated knockdown or overexpression of Mcl-1, Bcl-2 or Bcl-xL. We observed neither spontaneous cell death induction nor impaired proliferation of cells lacking Mcl-1, Bcl-2 or Bcl-xL. In contrast, knockdown of Mcl-1 led to increased proliferation. Strikingly, we demonstrate a profound impairment of both, migration and invasion, of colorectal cancer cells after Mcl-1, Bcl-2 or Bcl-xL knockdown. This phenotype was completely revised in cells overexpressing Mcl-1, Bcl-2 or Bcl-xL. The most pronounced effect among the investigated proteins was observed for Bcl-2. The data presented indicate a pivotal role of Mcl-1, Bcl-2 and Bcl-xL for migration and invasion of colorectal cancer cells independent of their known antiapoptotic effects. Thus, our study illustrates novel antitumoral mechanisms of Bcl-2 protein targeting.

The Bcl-2 proteins Noxa and Bcl-xL co-ordinately regulate oxidative stress-induced apoptosis

Because the detailed molecular mechanisms by which oxidative stress induces apoptosis are not completely known, we investigated how the complex Bcl-2 protein network might regulate oxidative stress-induced apoptosis. Using MEFs (mouse embryonic fibroblasts), we found that the endogenous anti-apoptotic Bcl-2 protein Bcl-xL prevented apoptosis initiated by H(2)O(2). The BH3 (Bcl-2 homology 3)-only Bcl-2 protein Noxa was required for H(2)O(2)-induced cell death and was the single BH3-only Bcl-2 protein whose pro-apoptotic activity was completely antagonized by endogenous Bcl-xL. Upon H(2)O(2) treatment, Noxa mRNA displayed the greatest increase among BH3-only Bcl-2 proteins. Expression levels of the anti-apoptotic Bcl-2 protein Mcl-1 (myeloid cell leukaemia sequence 1), the primary binding target of Noxa, were reduced in H(2)O(2)-treated cells in a Noxa-dependent manner, and Mcl-1 overexpression was able to prevent H(2)O(2)-induced cell death in Bcl-xL-deficient MEF cells. Importantly, reduction of the expression of both Mcl-1 and Bcl-xL caused spontaneous cell death. These studies reveal a signalling pathway in which H(2)O(2) activates Noxa, leading to a decrease in Mcl-1 and subsequent cell death in the absence of Bcl-xL expression. The results of the present study indicate that both anti- and pro-apoptotic Bcl-2 proteins co-operate to regulate oxidative stress-induced apoptosis.

Abstract 2012: MAP kinase activation and suppression of anti-apoptotic Bcl-2 family members are associated with induction of apoptotic death in melanoma cells following down-regulation of glutathione S-transferase P1

Abstract Introduction. The multifunctional protein, glutathione S-transferase P1, GSTP1, is frequently overexpressed in malignant melanoma and has been associated with increased tumor growth and resistance to therapy and poor overall patient survival. A major cellular function of GSTP1 is as a regulator of cell signaling mediated through its ability to interact with and regulate key signaling proteins, notably, the MAP kinases. In a previous study, we showed that RNA interference-mediated GSTP1 downregulation in GSTP1-overexpressing melanoma cells decreased cell growth and increased the levels of both spontaneous and drug-induced apoptosis. Here, we examined the molecular mechanisms underlying these observations, particularly, those involving alterations in MAP kinase signaling and pro-and anti-apoptotic members of the Bcl2 family of proteins. Methods. Exponentially growing cells of two human melanoma cell lines A375 and DM6 were infected with a lentiviral vector carrying GSTP1-targeted short hairpin RNA (shRNA) targeting. A heterogeneous stable population was isolated by puromycin selection. Alternatively, GSTP1 was knocked down by transfection of the cells with GSTP1-targeted siRNA. The cells were confirmed as GSTP1-ve by PCR and western blotting. At various time-points post GSTP1 suppression, the cells were analyzed for the level of apoptosis by flow cytometry (FCM) and by measuring the level of caspase 3/7 mediated cleavage of the peptide substrate, Ac-DEVD-AFC. Extracts were prepared from control and GSTP1-knockdown cells and analyzed for total and phosphorylated MAP kinases (P38, ERK, and JNK) and for proteins of the Bcl-2 family. Results. There was a near-complete suppression of GSTP1 transcripts and protein in both melanoma cell lines following shRNA-lentiviral infection or at 72 hrs post-siRNA transfection. Over 7 days, there was a steady increase in the level of apoptosis in the cells with GSTP1 suppression, as evidenced by an increase in the pre-G1 population by FCM or in caspase 3/7 activity. This was associated with a significant increase in the level of phosphorylation of the MAP kinases, Erk1/2 and JNK1/2. Following GSTP1 suppression, we also observed a significant decrease in the pro-survival anti-apoptotic Bcl-2 proteins Mcl-1, Bcl-XL, and Bcl-2 with little to no change in pro-apoptotic Bcl2 proteins Bad, Bim and Bid. Conclusion. These results suggest that GSTP1 is obligate for growth and survival of of GSTP1-expressing melanoma cells and that suppression of GSTP1 expression in these cells induces an apoptotic response that involves activation of the MAP Kinases, ERK and JNK, and suppression of anti-apoptotic Bcl-2 proteins. Supported by NIH grants RO1 CA 153050, RO1CA127872, RO1 CA 112519, P50CA108786 and P30-CA14236. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2012. doi:1538-7445.AM2012-2012

Noxa induces apoptosis in oncogene-expressing cells through catch-and-release mechanism operating between Puma and Mcl-1

Tumor suppressor p53 induces apoptosis by transcriptional induction of Noxa and Puma, which encode the proapoptotic BH3-only member of the Bcl-2 family proteins. In the p53-mediated tumor surveillance system, p53 induces apoptosis or replicative senescence in oncogene-expressing cells, resulting in elimination of such cells. In this context, we previously found that Noxa and Puma synergistically induce apoptosis. Here, we found the adenovirus oncogene E1A to induce p53-dependently expression of Puma, but not Noxa. The induced Puma associates with antiapoptotic Bcl-2 protein Mcl-1, accompanied by accumulated Mcl-1 protein on mitochondria. Moreover, E1A also reduces expression of the antiapoptotic Bcl-2 protein Bcl-XL. In contrast, the DNA-damaging agent adriamycin induces Noxa expression in E1A-expressing cells. Interestingly, Mcl-1 knockdown itself induced apoptosis in E1A-expressing MEFs. Furthermore, Noxa displaced Puma’s association with Mcl-1, accompanied by Mcl-1 degradation and apoptosis induction by activating mitochondrial apoptotic executers Bax and Bak. These results suggest that p53-induced apoptosis in oncogene-expressing cells is regulated by differential induction and sequential activation of Noxa and Puma. Accumulated Puma by oncogene enhances susceptibility to apoptosis through “catch” in mitochondria by Mcl-1. Subsequently, in response to DNA-damage, Noxa efficiently induces apoptosis by “release” of Puma from Mcl-1.

TRAIL-induced apoptosis of hepatocellular carcinoma cells isaugmented by targeted therapies

To analyze the effect of chemotherapeutic drugs and specific kinase inhibitors, in combination with the death receptor ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL), on overcoming TRAIL resistance in hepatocellular carcinoma (HCC) and to study the efficacy of agonistic TRAIL antibodies, as well as the commitment of antiapoptotic BCL-2 proteins, in TRAIL-induced apoptosis. Surface expression of TRAIL receptors (TRAIL-R1-4) and expression levels of the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) were analyzed by flow cytometry and Western blotting, respectively. Knock-down of MCL-1 and BCL-x(L) was performed by transfecting specific small interfering RNAs. HCC cells were treated with kinase inhibitors and chemotherapeutic drugs. Apoptosis induction and cell viability were analyzed via flow cytometry and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TRAIL-R1 and -R2 were profoundly expressed on the HCC cell lines Huh7 and Hep-G2. However, treatment of Huh7 and Hep-G2 with TRAIL and agonistic antibodies only induced minor apoptosis rates. Apoptosis resistance towards TRAIL could be considerably reduced by adding the chemotherapeutic drugs 5-fluorouracil and doxorubicin as well as the kinase inhibitors LY294002 [inhibition of phosphoinositol-3-kinase (PI3K)], AG1478 (epidermal growth factor receptor kinase), PD98059 (MEK1), rapamycin (mammalian target of rapamycin) and the multi-kinase inhibitor Sorafenib. Furthermore, the antiapoptotic BCL-2 proteins MCL-1 and BCL-x(L) play a major role in TRAIL resistance: knock-down by RNA interference increased TRAIL-induced apoptosis of HCC cells. Additionally, knock-down of MCL-1 and BCL-x(L) led to a significant sensitization of HCC cells towards inhibition of both c-Jun N-terminal kinase and PI3K. Our data identify the blockage of survival kinases, combination with chemotherapeutic drugs and targeting of antiapoptotic BCL-2 proteins as promising ways to overcome TRAIL resistance in HCC.

Importance of Bak for celecoxib-induced apoptosis

The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib constitutes the prototype of pro-apoptotic agents acting through the intrinsic death pathway in a Bcl-2 independent manner. To gain further insight into celecoxib-mediated apoptosis regulation at the level of the mitochondria we tested in how far the crucial pro-apoptotic Bcl-2 proteins Bak and Bax were involved using clones of the Bax-deficient Jurkat T-lymphoma cell model either expressing Bak (Jurkat Bak positive) or being negative for Bak (Jurkat Bak negative), or overexpressing Bcl-2 (Jurkat Bcl-2). Celecoxib induced substantial apoptosis in Jurkat Bak-positive cells. Overexpression of Bcl-2 had only limited protective effects. However, loss of Bak-expression conferred almost complete resistance of Jurkat cells to celecoxib-induced apoptosis. Neither enhanced celecoxib-concentrations nor prolonged incubation times were sufficient to normalize apoptotic rates upon celecoxib-treatment in these Bak/Bax-negative cells. In line with that observation, siRNA-mediated silencing of Bak in the Bak-positive Jurkat cells largely reduced the extent of celecoxib-induced cell death. Interestingly, in celecoxib-sensitive Bak-positive cells, celecoxib-treatment resulted in down-regulation of the anti-apoptotic Bcl-2 protein Mcl-1 which may contribute to celecoxib-mediated activation of Bak-dependent apoptosis. Taken together our data clearly show for the first time the functional relevance of Bak for celecoxib-induced apoptosis in Bax-deficient Jurkat T-lymphoma cells.

Respiratory Syncytial Virus Inhibits Granulocyte Apoptosis through a Phosphatidylinositol 3-Kinase and NF-κB-Dependent Mechanism

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease in children. It is associated with increased neutrophil numbers in the airway. In this study, we assessed whether this ssRNA virus can directly influence granulocyte longevity. By culturing RSV with granulocytes, it was observed that virus delays both constitutive neutrophil and eosinophil apoptosis. Using pharmacological inhibitors, the RSV-induced delay in neutrophil apoptosis was found to be dependent on both PI3K and NF-kappaB, but not p38 MAPK or MEK1/MEK2 activation. Using blocking Abs and a reporter cell line, we were able to exclude TLR4 as the receptor responsible for mediating RSV-induced delay in neutrophil apoptosis. The antiapoptotic effect was abrogated by preincubation with the lysosomotropic agent chloroquine, indicating the requirement for endolysosomal internalization. Furthermore, addition of ssRNA, a ligand for the intracellular TLR7/TLR8, also inhibited neutrophil apoptosis, suggesting that intracellular TLRs could be involved in induction of the antiapoptotic effect. Using the BioPlex cytokine detection assay (Bio-Rad), we found that IL-6 was present in supernatants from RSV-exposed neutrophils. IL-6 was found to inhibit neutrophil apoptosis, suggesting that there is an autocrine or paracrine antiapoptotic role for IL-6. Finally, RSV treatment of neutrophils resulted in increased expression of the antiapoptotic Bcl-2 protein Mcl-1. Taken together, our findings suggest involvement of multiple intracellular mechanisms responsible for RSV-induced survival of granulocytes and point toward a role for intracellular TLRs in mediating these effects.