Hypoxia-Induced Modulation of Apoptosis and BCL-2 Family Proteins in Different Cancer Cell Types

Hypoxia plays an important role in the resistance of tumour cells to chemotherapy. However, the exact mechanisms underlying this process are not well understood. Moreover, according to the cell lines, hypoxia differently influences cell death. The study of the effects of hypoxia on the apoptosis induced by 5 chemotherapeutic drugs in 7 cancer cell types showed that hypoxia generally inhibited the drug-induced apoptosis. In most cases, the effect of hypoxia was the same for all the drugs in one cell type. The expression profile of 93 genes involved in apoptosis as well as the protein level of BCL-2 family proteins were then investigated. In HepG2 cells that are strongly protected against cell death by hypoxia, hypoxia decreased the abundance of nearly all the pro-apoptotic BCL-2 family proteins while none of them are decreased in A549 cells that are not protected against cell death by hypoxia. In HepG2 cells, hypoxia decreased NOXA and BAD abundance and modified the electrophoretic mobility of BIM_EL. BIM and NOXA are important mediators of etoposide-induced cell death in HepG2 cells and the hypoxia-induced modification of these proteins abundance or post-translational modifications partly account for chemoresistance. Finally, the modulation of the abundance and/or of the post-translational modifications of most proteins of the BCL-2 family by hypoxia involves p53-dependent and –independent pathways and is cell type-dependent. A better understanding of these cell-to-cell variations is crucial in order to overcome hypoxia-induced resistance and to ameliorate cancer therapy.     

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.     

Human embryonic stem cells express elevated levels of multiple pro-apoptotic BCL-2 family members

Two of the greatest challenges in regenerative medicine today remain (1) the ability to culture human embryonic stem cells (hESCs) at a scale sufficient to satisfy clinical demand and (2) the ability to eliminate teratoma-forming cells from preparations of cells with clinically desirable phenotypes. Understanding the pathways governing apoptosis in hESCs may provide a means to address these issues. Limiting apoptosis could aid scaling efforts, whereas triggering selective apoptosis in hESCs could eliminate unwanted teratoma-forming cells. We focus here on the BCL-2 family of proteins, which regulate mitochondrial-dependent apoptosis. We used quantitative PCR to compare the steady-state expression profile of all human BCL-2 family members in hESCs with that of human primary cells from various origins and two cancer lines. Our findings indicate that hESCs express elevated levels of the pro-apoptotic BH3-only BCL-2 family members NOXA, BIK, BIM, BMF and PUMA when compared with differentiated cells and cancer cells. However, compensatory expression of pro-survival BCL-2 family members in hESCs was not observed, suggesting a possible explanation for the elevated rates of apoptosis observed in proliferating hESC cultures, as well as a mechanism that could be exploited to limit hESC-derived neoplasms.     

The BCL-2 protein family,BH3-mimetics and cancer therapy

Escape from apoptosis is a key attribute of tumour cells and facilitates chemo-resistance. The ‘BCL-2-regulated'' or ‘intrinsic'' apoptotic pathway integrates stress and survival signalling to govern whether a cancer cell will live or die. Indeed, many pro-apoptotic members of the BCL-2 family have demonstrated tumour-suppression activity in mouse models of cancer and are lost or repressed in certain human cancers. Conversely, overexpression of pro-survival BCL-2 family members promotes tumorigenesis in humans and in mouse models. Many of the drugs currently used in the clinic mediate their therapeutic effects (at least in part) through the activation of the BCL-2-regulated apoptotic pathway. However, initiators of this apoptotic pathway, such as p53, are mutated, lost or silenced in many human cancers rendering them refractory to treatment. To counter such resistance mechanisms, a novel class of therapeutics, ‘BH3-mimetics'', has been developed. These drugs directly activate apoptosis by binding and inhibiting select antiapoptotic BCL-2 family members and thereby bypass the requirement for upstream initiators, such as p53. In this review, we discuss the role of the BCL-2 protein family in the development and treatment of cancer, with an emphasis on mechanistic studies using well-established mouse models of cancer, before describing the development and already recognised potential of the BH3-mimetic compounds.     

Direct and selective small-molecule activation of proapoptotic BAX

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.     

BH3分析技术研究进展及其应用

许多化疗药物发挥作用的重要方式是通过诱导线粒体介导的细胞凋亡.细胞凋亡在维持正常机体的内环境稳态中有重要作用,而在肿瘤细胞中,细胞凋亡的失调成为肿瘤细胞逃避化疗药物杀灭细胞作用的一道屏障. BCL-2家族蛋白在调节线粒体诱导的凋亡中处于中心地位,因此一项基于BCL-2家族蛋白的检测技术,BH3分析技术应运而生.该项技术的提出或许能为肿瘤的个性化治疗提供新的思路.本文重点综述BH3分析技术的原理,以及在肿瘤化疗药物选择和新型化疗药物开发中的应用.     

How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?

The mitochondrial pathway of apoptosis proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane permeabilization (MOMP). This process is controlled by the BCL-2 family, which is composed of both pro- and anti-apoptotic proteins. Although there is no disagreement that BCL-2 proteins regulate apoptosis, the mechanism leading to MOMP remains controversial. Current debate focuses on what interactions within the family are crucial to initiate MOMP. Specifically, do the BH3-only proteins directly engage BAX and/or BAK activation or do these proteins solely promote apoptosis by neutralization of anti-apoptotic BCL-2 proteins? We describe these models and contend that BH3-only proteins must perform both functions to efficiently engage MOMP and apoptosis.     

Neuronal cell life,death, and axonal degeneration as regulated by the BCL-2 family proteins

Axonal degeneration and neuronal cell death are fundamental processes in development and contribute to the pathology of neurological disease in adults. Both processes are regulated by BCL-2 family proteins which orchestrate the permeabilization of the mitochondrial outer membrane (MOM). MOM permeabilization (MOMP) results in the activation of pro-apoptotic molecules that commit neurons to either die or degenerate. With the success of small-molecule inhibitors targeting anti-apoptotic BCL-2 proteins for the treatment of lymphoma, we can now envision the use of inhibitors of apoptosis with exquisite selectivity for BCL-2 family protein regulation of neuronal apoptosis in the treatment of nervous system disease. Critical to this development is deciphering which subset of proteins is required for neuronal apoptosis and axon degeneration, and how these two different outcomes are separately regulated. Moreover, noncanonical BCL-2 family protein functions unrelated to the regulation of MOMP, including impacting necroptosis and other modes of cell death may reveal additional potential targets and/or confounders. This review highlights our current understanding of BCL-2 family mediated neuronal cell death and axon degeneration, while identifying future research questions to be resolved to enable regulating neuronal survival pharmacologically.Subject terms: Cell biology, Chemical tools, Neuroscience, Neurological disorders     

Apoptosis regulators

Over the last decade, a great deal of attention has been directed at elucidating the role of apoptosis regulators in governing survival decisions in neoplastic cells, particularly those of hematopoietic origin. A major focus of this work has involved investigation of the function of pro- and anti-apoptotic members of the BCL-2 family, and the relationship between these proteins and mitochondrial integrity. Currently, these proteins can be classified into two broad categories: those that modulate mitochondrial function and those that regulate the activation of caspases responsible for activation and execution of the apoptotic cascade. Within the first category, certain proteins (e.g., BCL-2, BCL-xL) act to preserve mitochondrial integrity by preventing loss of mitochondrial membrane potential and/or release of pro-apoptotic proteins such as cytochrome C into the cytosol. Other proapoptotic proteins (e.g., BAX, BAK, BIM) promote release of cytochrome C. These proteins are therefore primarily involved in regulation of the intrinsic, mitochondrial apoptotic pathway. Within the second category, proteins such as the inhibitors of apoptosis proteins (e.g., XIAP) or FLIP block the activation of caspases, particularly those involved in engagement of the receptor-related, extrinsic apoptotic pathway. Cross-talk between the intrinsic and extrinsic pathways exists. For example, the BH3-domain only protein BID is cleaved by the activation of pro-caspase-8 through the extrinsic pathway, and translocates to the mitochondrion to promote cytochrome C release. Apoptosis is also regulated by various signal transduction pathways, possibly through post-translational modifications in BCL-2 family proteins. For example, phosphorylation of BCL-2 through a JNK-dependent mechanism has been postulated to contribute to apoptosis induced by the taxane class of cytotoxic agents. Finally, attempts to modulate apoptotic pathways with small molecules have recently received much attention. For example, small molecule inhibitors of BCL-2 or mimetics of SMAC/DIABLO, which opposes the actions of XIAP, have recently been shown to promote the antineoplastic activity of conventional cytotoxic agents. It is likely that an improved understanding of apoptosis regulation will lead to new insights into neoplastic transformation, and may also provide important leads for the development of novel antileukemic strategies.     

Multiple Functions of BCL-2 Family Proteins

BCL-2 family proteins are the regulators of apoptosis, but also have other functions. This family of interacting partners includes inhibitors and inducers of cell death. Together they regulate and mediate the process by which mitochondria contribute to cell death known as the intrinsic apoptosis pathway. This pathway is required for normal embryonic development and for preventing cancer. However, before apoptosis is induced, BCL-2 proteins have critical roles in normal cell physiology related to neuronal activity, autophagy, calcium handling, mitochondrial dynamics and energetics, and other processes of normal healthy cells. The relative importance of these physiological functions compared to their apoptosis functions in overall organismal physiology is difficult to decipher. Apoptotic and noncanonical functions of these proteins may be intertwined to link cell growth to cell death. Disentanglement of these functions may require delineation of biochemical activities inherent to the characteristic three-dimensional shape shared by distantly related viral and cellular BCL-2 family members.     

Apoptosis and oncogenesis: give and take in the BCL-2 family

The mitochondrial pathway of apoptosis constitutes one of the main safeguards against tumorigenesis. The BCL-2 family includes the central players of this pathway that regulate cell fate through the control of mitochondrial outer membrane permeabilization (MOMP), and important progress has been made in understanding the dynamic interactions between pro-apoptotic and anti-apoptotic BCL-2 proteins. In particular, recent studies have delineated a stepwise model for the induction of MOMP. BCL-2 proteins are often dysregulated in cancer, leading to increased survival of abnormal cells; however, recent studies have paradoxically shown that apoptosis induction can under some circumstances drive tumor formation, perhaps by inducing compensatory proliferation under conditions of cellular stress. These observations underline the complexity of BCL-2 protein function in oncogenesis.     

B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins

The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these “BH3 mimetics” in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.     

Fatty acid synthase (FASN) regulates the mitochondrial priming of cancer cells

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-X_L/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-X_L-selective inhibitors such as {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540"}}S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.Subject terms: Cancer metabolism, Lipid signalling     

MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction

The BCL-2 family of proteins plays a major role in the control of apoptosis as the primary regulator of mitochondrial permeability. The pro-apoptotic BCL-2 homologues BAX and BAK are activated following the induction of apoptosis and induce cytochrome c release from mitochondria. A second class of BCL-2 homologues, the BH3-only proteins, is required for the activation of BAX and BAK. The activity of both BAX/BAK and BH3-only proteins is opposed by anti-apoptotic BCL-2 homologues such as BCL-2 and MCL-1. Here we show that anti-apoptotic MCL-1 inhibits the function of BAX downstream of its initial activation and translocation to mitochondria. Although MCL-1 interacted with BAK and inhibited its activation, the activity of MCL-1 against BAX was independent of an interaction between the two proteins. However, the anti-apoptotic function of MCL-1 required the presence of BAX. These results suggest that the pro-survival activity of MCL-1 proceeds via inhibition of BAX function at mitochondria, downstream of its activation and translocation to this organelle.     

Molecular docking studies of anti-apoptotic BCL-2, BCL-XL, and MCL-1 proteins with ginsenosides from Panax ginseng

Anti-apoptotic proteins such as BCL-2, BCL-XL and MCL-1 bind with pro-apoptotic proteins to induce apoptosis mechanism. BCL-2 family proteins are key regulators of apoptosis process. Over expression of these anti-apoptotic proteins lead to several cancers by preventing apoptosis. A number of studies revealed that ginseng derivatives reduce tumor growth. Ginseng, the most valuable medicinal herb found in eastern Asia belongs to Araliaceae family. In this study, docking simulations were performed for anti-apoptotic proteins with several ginsenosides from Panax ginseng. Our finding shows ginsenosides Rf, Rg1, Rg3 and Rh2 have more binding affinity with BCL-2, BCL-XL and MCL-1 and other ginsenosides also interact with each anti-apoptotic proteins. Therefore, ginseng derivatives represent a novel class of potent inhibitors and could be used for cancer chemotherapy.     

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis¹. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)¹. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues².In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)^3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation^7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members^7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization¹⁰. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)¹⁰. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)¹¹. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.     

Prostaglandin A2 activates intrinsic apoptotic pathway by direct interaction with mitochondria in HL-60 cells

HL-60 cells treated by prostaglandin (PG) A₂ showed characteristics of apoptosis such as accumulation of hypodiploid and annexin V positive cells, condensed and fragmented nuclei, cytochrome c (Cyt C) release from mitochondria and activation of caspase-1, -2, -3, -7 and -9. PGA₂-induced cell death was rescued by inhibitors of caspase-9 and -3, but PGA₂-induced Cyt C release was not prevented by caspase inhibitors. During Cyt C release by PGA₂, mitochondrial transmembrane potential was maintained and mitochondrial permeability transition pore was not formed. In addition, anti-apoptotic BCL-2 family proteins like BCL-2 and BCL-XL, and ROS scavengers including ascorbic acid and 2,2,6,6-tetramethyl-1-piperidinyloxy were not able to inhibit Cyt C release as well as apoptosis by PGA₂. Finally, it was shown that PGA₂-induced Cyt C release in vitro from purified mitochondria in the absence of cytosolic components. Furthermore, thiol-containing compounds such as N-acetylcysteine, l-cysteine and monothioglycerol prevented Cyt C release, and hence induction of apoptosis. Taken together, these results suggest that PGA₂ activates intrinsic apoptotic pathway by directly stimulating mitochondrial outer membrane permeabilization to release Cyt C, in which thiol-reactivity of PGA₂ plays a pivotal role.     

BCL-2 in the crosshairs: tipping the balance of life and death

The discovery of B-cell lymphoma-2 (BCL-2) over 20 years ago revealed a new paradigm in cancer biology: the development and persistence of cancer can be driven by molecular roadblocks along the natural pathway to cell death. The subsequent identification of an expansive family of BCL-2 proteins provoked an intensive investigation of the interplay among these critical regulators of cell death. What emerged was a compelling tale of guardians and executioners, each participating in a molecular choreography that dictates cell fate. Ten years into the BCL-2 era, structural details defined how certain BCL-2 family proteins interact, and molecular targeting of the BCL-2 family has since become a pharmacological quest. Although many facets of BCL-2 family death signaling remain a mechanistic mystery, small molecules and peptides that effectively target BCL-2 are eliminating the roadblock to cell death, raising hopes for a medical breakthrough in cancer and other diseases of deregulated apoptosis.     

Putative tumor suppressor Lats2 induces apoptosis through downregulation of Bcl-2 and Bcl-x(L)

Lats2, also known as Kpm, is the second mammalian member of the novel Lats tumor suppressor gene family. Recent studies have demonstrated that Lats2 negatively regulates the cell cycle by controlling G1/S and/or G2/M transition. To further understand the role of Lats2 in the control of human cancer development, we have expressed the protein in human lung cancer cells by transduction of a replication-deficient adenovirus expressing human Lats2 (Ad-Lats2). Using a variety of techniques, including Annexin V uptake, cleavage of PARP, and DNA laddering, we have demonstrated that the ectopic expression of human Lats2 induced apoptosis in two lung cancer cell lines, A549 and H1299. Caspases-3, 7, 8, and 9 were processed in the Ad-Lats2-transduced cells; however, it was active caspase-9, not caspase-8, that initiated the caspase cascade. Inhibitors specific to caspase-3 and 9 delayed the onset of Lats2-mediated apoptosis. Western blot analysis revealed that anti-apoptotic proteins, BCL-2 and BCL-x(L), but not the pro-apoptotic protein, BAX, were downregulated in Ad-Lats2-transduced human lung cancer cells. Overexpression of either Bcl-2 or Bcl-x(L) in these cells lead to the suppression of Lats2-mediated caspase cleavage and apoptosis. These results show that Lats2 induces apoptosis through downregulating anti-apoptotic proteins, BCL-2 and BCL-x(L), in human lung cancer cells.