evading apoptosis in cancer

TICB-985; No. of Pages 14

Evading apoptosis in cancerKaleigh Fernald1 and Manabu Kurokawa1,2

1 Department of Pharmacology and Toxicology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA2 Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA

Review

Carcinogenesis is a mechanistically complex and vari-able process with a plethora of underlying geneticcauses. Cancer development comprises a multitude ofsteps that occur progressively starting with initial drivermutations leading to tumorigenesis and, ultimately,metastasis. During these transitions, cancer cells accu-mulate a series of genetic alterations that confer on thecells an unwarranted survival and proliferative advan-tage. During the course of development, however, can-cer cells also encounter a physiologically ubiquitouscellular program that aims to eliminate damaged orabnormal cells: apoptosis. Thus, it is essential that cancercells acquire instruments to circumvent programmed celldeath. Here we discuss emerging evidence indicating howcancer cells adopt various strategies to override apopto-sis, including amplifying the antiapoptotic machinery,downregulating the proapoptotic program, or both.

Regulation of apoptosis in normal and cancer cellsApoptosis is a cellular suicide program that organismshave evolved to eliminate unnecessary or unhealthy cellsfrom the body in the course of development or followingcellular stress. It involves a series of cellular events thatultimately leads to activation of a family of cysteine pro-teases called caspases. In response to various apoptoticstimuli, ‘initiator’ caspases (caspase-2, -8, -9, or -10) areactivated. Initiator caspases, in turn, cleave and activatethe zymogenic forms of ‘executioner’ caspases (e.g., cas-pase-3 or -7), resulting in the proteolytic cleavage of specificcellular substrates and, consequently, cell death (Figure 1).In this regard, the cleavage (activation) of executionercaspases is a hallmark of apoptosis. There are two routesto apoptosis: extrinsic and intrinsic. In the extrinsic path-way, initiator caspase-8 and -10 are activated through theformation of a death-inducing signal complex (DISC) inresponse to the engagement of extracellular ligands [e.g.,Fas, tumor necrosis factor (TNF)] by cell surface receptors(Figure 1). In the intrinsic pathway of apoptosis, mitochon-drial outer membrane permeabilization (MOMP) is in-volved; MOMP triggers the release of a group ofproapoptotic proteins, including cytochrome c and secondmitochondria-derived activator of caspases (SMAC), fromthe mitochondrial intermembrane space to the cytoplasm(Figure 1). In the cytoplasm, cytochrome c binds to theadaptor protein apoptotic protease-activating factor 1(APAF-1) to form a caspase-9-activating complex called

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� 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2013.07.006

Corresponding author: Kurokawa, M. ([email protected]).Keywords: BH3; caspase; MOMP; phosphorylation; ubiquitination.

the apoptosome (Figure 1). SMAC augments cytochromec-induced caspase activation by binding and neutralizingX-linked inhibitor of apoptosis protein (XIAP), an inhibitorof caspase-3, -7, and -9 (Figure 1).

The balance between pro- and antiapoptotic B cell lym-phoma 2 (BCL-2) family proteins is a fundamental determi-nant for the initiation of MOMP. The BCL-2 family proteinsare classified based on the presence of shared blocks ofsequence homology, termed BCL-2 homology (BH). Theproapoptotic BCL-2 family members, which promoteMOMP, include BH3-only proteins [e.g., Bcl2-interactingmediator of cell death (BIM), BH3-interacting domain deathagonist (BID), and Bcl2-associated agonist of cell death(BAD), which share only a single block, the BH3 domain,of BCL-2 homology] and multi-BH domain proteins [e.g.,Bcl2-associated protein X (BAX) and Bcl2 antagonist/killer(BAK), which share BH1–BH3 domains]. Following cytotox-ic or genotoxic stress, BH3-only proteins are activated andpromote oligomerization of BAX (or BAK), resulting inMOMP, whereas prosurvival members [e.g., BCL-2, B celllymphoma extra large (BCL-XL), and induced myeloid leu-kemia cell differentiation protein (MCL-1), which contain allfour BH domains] counteract this process by sequesteringproapoptotic family members (Figure 1). Importantly, theinteractions between pro- and antiapoptotic BCL-2 familyproteins are determined with specific binding specificities[1,2]; for instance, BIM interacts with all six antiapoptoticBCL-2 family proteins as well as BAX and BAK [3], whereasphorbol-12-myristate-13-acetate-induced protein 1 (NOXA)binds to MCL-1 and BFL-1/A1 only.

Unlike normal cells, cancer cells are under constantstress, including oncogenic stress, genomic instability,and cellular hypoxia. In response to such apoptotic stimuli,the intrinsic pathway of apoptosis would normally beactivated. Yet cancer cells can often avoid this cellularresponse by disabling the apoptotic pathways. Notably,mouse genetic models have shown that genetic inactiva-tion of a BH3-only protein or a caspase can not only lead toresistance to certain proapoptotic stimuli but also acceler-ate tumor formation in mice [4–6]. Moreover, forced ex-pression of antiapoptotic BCL-2 family proteins cansignificantly augment tumor development induced by anoncogene, such as MYC, although overexpression of anantiapoptotic BCL-2 family protein alone may not resultin tumor formation similar to that seen with an oncogenealone [7–10]. Thus, it is strongly suggested that inhibitionof apoptosis plays a critical role in cancer cell survival andtumor development.

Cancer cells can modulate apoptotic pathways tran-scriptionally, translationally, and post-translationally. In

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BIMPUMABADNOXABMFHRKBIK

MOMP

SMAC

XIAP

Apoptosis

Apoptosome

PIDDosomeBCL-2BCL-XLMCL-1BCL-WBFL-1/A1BCL-B

BAX, BAK, BOK

Caspase-2

Caspase-8, -10

Cytochrome c

APAF-1

Caspase-9

Caspase-3, -7

BCL-2BCL-XLMCL-1BCL-WBFL-1/A1BCL-B

BIMPUMABADNOXABMFHRKBIK

BID → tBIDBID → tBID

DISC

An�apopto�c BCL-2 family

BH3-only proteins

BAX, BAK, BOK

Mul�-BH proteins

C

Apoptosome

pp

TRENDS in Cell Biology

Figure 1. Apoptosis is triggered in response to internal or external stimuli. The

intrinsic pathway to apoptosis is initiated by activation of BH3-only protein.

Among the BH3-only proteins, expression of which is often transcriptionally

induced following an apoptotic stimulus, BID is unique in that it is activated by

caspase cleavage (by caspase-2 or -8/10); cleaved BID is termed tBID. BH3 proteins

trigger the activation of multi-BH domain proteins by direct binding or by

inhibition of antiapoptotic BCL-2 family proteins. Once activated, multi-BH domain

proteins form oligomers that induce MOMP. MOMP prompts the proapoptotic

proteins SMAC and cytochrome c to be released to the cytoplasm, where

cytochrome c induces formation of the apoptosome. The initiator caspase-9

activated within the apoptosome initiates the activation of executioner caspase-3

and -7, resulting in apoptosis. Of note, caspase-2 is activated via formation of the

PIDDosome, a complex comprising the adaptor PIDD and RAIDD. However, despite

identification of this complex, the precise mechanism of caspase-2 activation

remains elusive. In addition, only a few cellular substrates of caspase-2, including

BID, have been identified. Colored triangles denote that the indicated gene or gene

product may be transcriptionally (blue), translationally (red), or post-translationally

(green) up- or downregulated in cancer cells (see text and Table 1).

Review Trends in Cell Biology xxx xxxx, Vol. xxx, No. x


some cases, cancer cells may escape apoptosis by increasingexpression of antiapoptotic genes or by decreasing expres-sion of proapoptotic genes. Alternatively, they may inhibitapoptosis by stabilizing or destabilizing anti- or proapopto-tic proteins, respectively. Moreover, cancer cells may alsoprevent apoptosis by changing the functions of anti- orproapoptotic proteins through post-translational modifica-tions such as phosphorylation. Importantly, these mecha-nisms are not mutually exclusive and cancer cells mayemploy one or multiple mechanisms to evade apoptosis.

2

In this review, we highlight the core antiapoptotic machin-ery that cancer cells may employ for survival, focusing ourdiscussion on transcriptional, translational, and post-trans-lational modifications to evade apoptosis (note that recentstudies suggest that cancer metabolism can also modulatethis machinery to protect cells from apoptosis [11,12]; how-ever, this is outside the scope of this discussion).

Transcriptional/translational regulationGenomic and epigenomic abnormalities are a characteris-tic of cancer cells. This includes gene copy number ampli-fication, gene deletion, gene silencing by DNA methylation,and activation (or inactivation) of transcription factors thathave an impact on the expression of apoptotic regulators[13]. In addition, miRNAs negatively control gene expres-sion by targeting the 30 untranslated region (30-UTR) ofmRNAs, although they could function as tumor suppres-sors or oncogenes, depending on the target messengers[14]. Thus, transcriptional and translational alterationscan be a means for cancer cells to directly raise the thresh-old for apoptotic induction at gene expression levels. In thissection, we discuss how cancer cells may increase or blockthe expression of anti- or proapoptotic gene products,respectively (see Table 1 for loss or amplification of apo-ptotic genes identified in cancer tissues).

Inducing antiapoptotic protein expression

To overcome stress signals, cancer cells frequently over-express antiapoptotic proteins, especially antiapoptoticBCL-2 family proteins. Cancer cells prevent MOMP byamplification of antiapoptotic BCL-2 family proteins. Con-sequently, inhibition of one or multiple antiapoptotic BCL-2 family proteins causes apoptosis in cancer cells but not inhealthy normal cells – it is often said that cancer cells areprimed for death or addicted to antiapoptotic BCL-2 familyproteins. Overexpression of antiapoptotic BCL-2 familyproteins is often associated with poor prognosis, recur-rence, and resistance to cancer therapeutics [15–17]. TheBCL-2 gene was originally found in B cell follicular lym-phoma, where genetic translocation resulted in constitu-tive expression of BCL-2 [18–20] (Table 1). Amplification ofthe BCL-XL gene has also been reported in various can-cers, including lung and giant-cell tumor of bone [21–23](Table 1). Likewise, frequent amplification of the MCL-1gene has been reported in breast and lung cancer samples[23,24] (Table 1).

In normal cells, MCL-1 transcription is induced uponcytokine and growth factor stimulation [25,26], whereas itssynthesis is inhibited in response to various cellular stres-ses, including DNA damage [27–29]. In particular, it wasshown that the transcription factor E2F1, known to controlproliferation and apoptosis, could repress MCL-1 geneexpression by binding directly to the MCL-1 promoter[30], resulting in increased apoptosis. Alternatively, thetranscription factors cAMP response element-binding pro-tein (CREB) and signal transducer and activator of tran-scription 3 (STAT3), which are known to activate genesresponding to prosurvival stimuli, can transactivate theMCL-1 gene [25,27]. Importantly, whereas these signalingpathways operate to maintain cellular homeostasis innormal cells, they are often deregulated in cancer cells.

Table 1. Cancer and apoptotic regulators

Loss or amplification

of the apoptotic geneaKinase

(phosphatase)

Siteb Outcome of

phosphorylation

Cancer connection

to phosphorylationc

Caspase-2 CK2 Ser157 Suppression of caspase activity

[122]

TRAIL resistance

Esophageal cancer cells

Colon cancer cells

Glioma cells

CaMKII

(PP1)

Ser164 Suppression of caspase activity

[123]

Metabolically regulated

CDK1/CYCLIN B1 Ser340 Suppression of caspase activity

[124]

Suppressing mitotic cell

death

Caspase-3 PKCd ND Enhancement of caspase activity

[125]

p38 MAPK

(PP2A)

Ser150 Suppression of caspase activity

[126]

Caspase-6 ARK5 Ser257 Suppression of caspase activity

[197]

FasL resistance

Colorectal cancer cells

Caspase-7 PAK2 Ser30

Thr173 Ser239

Suppression of caspase activity

[127]

Breast cancer cells

Caspase-8 ND Tyr310 SHP1 binding required for

dephosphorylation of Tyr397

and Tyr465

[128]

RSK2 Thr263 Ubiquitination and degradation

[198]

FasL resistance

p38 MAPK Ser364 Suppression of caspase activity

[126]

CDK1/CYCLIN B1 Ser387 Suppression of caspase activity

[199]

FasL resistance

Breast cancer

Colon cancer cells

SRC

LYN

(SHP1)

Tyr397

(=Tyr380)


[128,129]

Colon cancer

LYN

(SHP1)

Tyr465 Suppression of caspase activity

[128]

Caspase-9 PKA Ser99

Ser183

Ser195

No effects

[130]

CDK1

DYRK1A

ERK1/2

p38 MAPK

(PP1a)

Thr125 Suppression of caspase activity

[131–136]

Suppressing mitotic cell

death

PKCz Ser144 Suppression of caspase activity

[137]

c-ABL Tyr153 Promoting caspase activation

[138]

Promoting DNA damage-

induced apoptosis

AKT Ser196

(human specific)d


[139]

Prostate cancer cells

Colon cancer cells

BID CK1

CK2

Thr58

Ser61

Ser64

(mouse)

Prevents cleavage by caspase-8

[121]

ATM

ATR

Ser61

Ser64

Ser78

(mouse)

S-phase arrest in response to

DNA damage

[140]

BIMe <Loss>

Chronic myeloid leukemia

and non-small cell lung

cancer [60]

Cutaneous T cell lymphoma

[141]

ERK Ser55

Ser65

Ser73

Ser100

(mouse)

Inactivation

[4,116]

Under the control of

cytokine interleukin (IL)-3

JNK

p38 MAPK

Ser65

(mouse, rat)

Activation

[114,115,117]

ERK1/2

JNK

Ser69 Ubiquitination and degradation

by promoting phosphorylation

at Ser93/Ser94/Ser98 by RSK

[99,101,142]

Non-small cell lung

cancer cells

Chronic myeloid

leukemia cells

T cell acute lymphoblastic

leukemia cells



3

Table 1 (Continued )



(phosphatase)

Siteb Outcome of

phosphorylation

Cancer connection

to phosphorylationc

PKA Ser83

(mouse)

Prevents degradation

[143]

RSK1/2 Ser93

Ser94

Ser98

SCFb-TrCP-mediated

ubiquitination and degradation

following phosphorylation at

Ser69

[101]

Non-small cell lung

cancer cells

JNK Thr112

(mouse)

Activation

[4]

PUMA <Loss>

Breast, hepatocellular, non-

small cell lung,

medulloblastoma, ovarian,

renal cancers [23]

IKK Ser10 Ubiquitination and degradation

[144,145]


cytokine IL-3

BAD IKK Ser26

(mouse)

Inactivation

[146]

TNFa resistance

PAK1 Ser111

(mouse)

Inactivation

[147]

Malignant peripheral

nerve sheath tumor cells

Lung cancer cells

PAK1

PAK4

PAK5

PKA

PKCi

RAF1

RSK1

Ser112

(mouse)

Inactivation

[147–155]

Malignant peripheral

nerve sheath tumor cells

Lung cancer cells


cytokine IL-3

AKT

PKCi

p70S6K

Ser136

(mouse)

Inactivation

[112,155,156]

Lung cancer cells


insulin-like growth factor 1

PKA

PKCi

RSK1

Ser155

(mouse)

Inactivation

[155,157,158]

Lung cancer cells

NOXA CDK5 Ser13 Inactivation

[159]

Metabolically regulated

T cell leukemia cells

BMF JNK

ERK2

Ser74 Release BMF from dynein light

chain complexes (homologous

to BIML Thr56)

[160,161]

ERK2 Ser77 Inactivation

[161]

Melanoma cells

BIK ERK1/2 Thr124 Ubiquitination and degradation

[162]

Lung cancer cells

Colon cancer cells

BAX <Loss>

Colon cancer

[45]

PKA Ser60 Activation

[163]

GSK3 Ser163 Activation [163,164]

ERK1/2 Thr167 Inactivation via interaction with

PIN1

[165]

AKT

PKCz

(PP2A)

Ser184 Inactivation

[166–168]

Lung cancer cells

JNK

p38 MAPK

ND Activation

[169,170]

Hepatoma cells

BAK Kinase unidentified

(PTPN2)

(PTPN5)

(PTPN23)

Tyr108 Inactivation

[171]

Fibrosarcoma cells

Colon cancer cells

Kinase unidentified

(PP2A)

Ser117 Inactivation

[172]

Colon cancer cells

BCL-XL <Amplification>

Non-small cell lung cancer

Pancreas cancer [21]

Giant-cell tumor of bone

[22]

Breast cancer

Ovarian cancer

[23]

PLK3 Ser49 Cell cycle (G2) arrest

[173]

CDK1/CYCLIN B

PLK1

JNK2

Ser62 Cell cycle (G2) arrest

Inactivation

[174,175]



4

Table 1 (Continued )



(phosphatase)

Siteb Outcome of

phosphorylation

Cancer connection

to phosphorylationc

BCL-2 <Amplification>

B cell follicular lymphoma

[18–20]

ERK1/2

PKC

Ser70 Activation

[176,177]


cytokine IL-3

JNK1 Ser70 Inactivation at G2/M phase

[178]

ERK1/2

(MKP-3)

Thr 56

Thr 74

Ser 87

Protects against TNF-induced

degradation

[179,180]

CDK1/CYCLIN B ND Inactivation

[174]

MCL-1 <Amplification>

Lung cancer [23,24]

Breast cancer

Dedifferentiated

liposarcoma, esophageal

adenocarcinoma,

hepatocellular, melanoma,

ovarian, prostate, renal

[23]

CDK1

CDK2

JNK1

Ser64 Enhances binding to BIM,

NOXA, BAK

[181]

TRAIL resistance

Cholangiocarcinoma

cells

CDK1/CYCLIN B Thr92 APC/CCDC20-mediated


[89]

JNK

p38 MAPK

CK2

Ser121 SCFFBW7-mediated


[91]

Ovarian cancer

Non-small-cell lung

cancer

GSK3

JNK

p38 MAPK

CK2

Ser155

Ser159

Thr163

Ubiquitination and degradation

mediated by TRIM17, SCFb-TrCP,

and SCFFBW7

Inhibits USP9X binding

[81–85,90,91,97]

Breast cancer cells

Ovarian cancer

Non-small-cell lung

cancer

T cell acute

lymphoblastic leukemia

Follicular lymphomas

Diffuse large B cell

lymphomas

Multiple myeloma


cytokine IL-3

ERK1/2 Thr163 Prevents degradation

[86,87]

Hepatoma cells

Burkitt lymphoma cells

APAF-1 <Loss>

Melanoma

[68–70],

Colorectal cancer [71,72],

Gastric cancer [73]

Bladder cancer [74,75]

<Amplification>

Glioblastoma/

Medulloblastoma/

Astrocytoma

[182]

RSK1/2 Ser268 Inhibition

[118]

Prostate cancer cells

XIAP <Amplification>

Cervical cancer

[38]

Acute myeloid leukemia

[183]

Squamous cell cancer

[184]

Rectal cancer

[185]

AKT Ser87 Prevents ubiquitination and

degradation

[186]

Ovarian cancer cells

TBK1

IKKeSer430 Autoubiquitination and

degradation

[187]

aConfirmed at copy number, mRNA, or protein level in cancer tissues.

bHuman sites unless specified.

cDemonstrated or suggested in cancer cell lines or cancer tissues.

dNot conserved in other species.

eBIMEL.



In particular, because the phosphatidylinositol 3-kinase(PI3K)/AKT pathway is frequently hyperactivated in can-cer cells by mutations and other mechanisms, transcrip-tional regulation of this pathway, such as the activation ofCREB or STAT3, could promote MCL-1 expression incertain cancer cells (Figure 2). In this regard, it was also

shown that MCL-1 mRNA translation could be facilitatedby mammalian target of rapamycin complex 1 (mTORC1),a downstream target of PI3K/AKT signaling, in a mouselymphoma model (Figure 2) [31].

miRNAs also play a role in the regulation of antiapop-totic proteins. miR-15 and miR-16 inhibit BCL-2 mRNA

5

Oncogenic kinases(e.g.,HER2, EGFR, BCR-ABL)

MEKPI3K

ERKAKT

BIM

FOXOs

mTORC1STAT3CREB

MCL-1

Transcrip�ontransla�on

Protein stability

PUMA

Transcrip�on

Transcrip�onProtein instabilityProtein func�ons

TKI

GSK3

ERKAKT

BIM

FOXOs

CL-1

Protein stability

PUMA

Transc

Transcrip�onProtein instabi lityProtein f unc�on sSK3 T crip�on

Protein stability


Figure 2. Oncogenic kinase signaling regulates MCL-1, BIM, and PUMA. Oncogenic kinases regulate BIM, PUMA, and MCL-1 by changing gene expression, protein stability,

and protein functions by activating two major prosurvival pathways: AKT and ERK. Prosurvival and proapoptotic pathways are denoted in blue and red, respectively.



translation [32]. Likewise, miR-29b, miR-101, and miR-193a-3p suppress MCL-1 mRNA translation, making cellssusceptible to apoptosis [33–35] – note that the role of miR-29b in cell death may be complex because it also targetsseveral BH3-only proteins, inhibiting apoptosis in neu-rons [36]. Expression of miR-15 and miR-16 is reduced inchronic lymphocytic leukemia [32], whereas expression ofmiR-101 is diminished in hepatocellular carcinoma [35],suggesting a possible mechanism to evade apoptosis bythese cancers. Lastly, two recent studies have shown thatexpression of miR-7 and miR-24, which inhibit XIAPmRNA, is suppressed in various cancer cells includingcervical and lung cancer cells, resulting in elevated XIAPprotein levels and blocked caspase activation [37,38]. Itwould be interesting to fully investigate whether expres-sion of these miRNAs is altered in various cancer tissuesand whether their expression patterns are associated withprognostic outcomes.

Suppressing proapoptotic protein expression

In normal cells, genotoxic and cytotoxic stress can induceexpression of proapoptotic genes, particularly some of theBH3-only proteins that trigger apoptosis. In cancer cells,this mechanism is often nullified by mutation and silencingof key apoptotic genes.

In normal cells, p53 is a critical transcription factor thatmodulates the expression of an array of genes known totrigger apoptosis following various cellular stresses. Giv-en the fact that TP53 encoding p53 is the most frequentlymutated, or inactivated, tumor suppressor gene in cancer(�50%) [39,40], apoptotic pathways induced by this gene,including BAX, p53 upregulated modulator of apoptosis(PUMA), NOXA, and APAF-1, may be widely affected incancer cells [41–44] – note that frameshift mutations inthe BAX gene have also been reported in colon cancer,which is likely to be independently of p53 [45] (Table 1).Interestingly, p53 could also activate BAX (and BAK) in a

6

transcription-independent manner in response to DNAdamage and metabolic changes [46–48]. Thus, loss ofp53 in cancer cells will elevate the threshold for MOMPby limiting BAX expression and reducing the likelihood ofBAX oligomerization.

Transcription of the BH3-only protein BIM is inducedupon growth factor withdrawal and other apoptotic stimuli[5,49]. Various transcription factors have been implicatedin BIM expression, including FOXOs [49–51], RUNX3 [52],AP-1 [53], and E2F1 [54]. Among the transcription factors,FOXOs are of particular interest because their activity canbe suppressed by two major prosurvival kinases, AKT [55]and ERK [56–59]. Thus, in cancer cells with high levels ofAKT or ERK, BIM expression is suppressed; conversely,agents that directly or indirectly inhibit AKT or ERK couldtrigger expression of BIM (Figure 2).

BIM plays an important role in cancer cells, especially,when apoptosis is induced by tyrosine kinase inhibitors(TKIs) [5,59]. Because AKT and ERK are key effectorsdownstream of oncogenic kinases [e.g., epidermal growthfactor receptor (EGFR), human epidermal growth factorreceptor 2 (HER2), and BCR-ABL], inhibition of the tyro-sine kinase could result in suppression of AKT and ERKactivity, resulting in the expression of BIM (Figure 2).Notably, a recent study showed that BIM RNA levelscan predict clinical responsiveness to targeted cancer ther-apies such as EGFR, HER2, and PI3K inhibitors; higherBIM RNA levels detected before the inhibitor treatmentsare associated with better clinical outcomes [59]. Addition-ally, a BIM deletion polymorphism has been reported incertain human populations and is significantly associatedwith innate resistance to TKI therapies in chronic myeloidleukemia and non-small cell lung cancer [60] (Table 1).Therefore, BIM induction is critical for the efficacy of TKItherapies and BIM RNA levels may be used as a biomarkerto predict cancer patients’ prognosis and susceptibility toTKI therapies.



A large somatic copy number analysis identified anotherBH3-only family member, PUMA, as one of the proapoptoticBCL-2 family genes that are most frequently deleted incancer (the study identified two genes and the other deletedgene was BOK) [23] (Table 1). PUMA was originally discov-ered as a p53 and p73 target gene [42,61]. However, FOXOscan also transactivate PUMA independently of p53 in re-sponse to growth factor withdrawal or TKI treatments [5,62](Figure 2). In this regard, a recent study has shown thatPUMA may be implicated in apoptosis triggered by FOXOsfollowing inhibition of the PI3K/AKT pathway, whereas BIMmay be primarily under the control of the ERK pathway inHER2-positive breast cancer and EGFR-mutant lung cancer(as described below, BIM is also regulated at the protein levelby ERK); for full execution of apoptosis, inhibition of bothpathways by TKIs (i.e., expression of both PUMA and BIM)may be required in these cancer cells [5]. This finding shedslight on an underappreciated role of PUMA in TKI-inducedapoptosis. Because BIM is also known to be regulated byFOXOs, how PUMA and BIM are independently regulatedremains to be fully elucidated (Figure 2).

In addition to the signaling pathways that impedeactivation of BAX and BAK, a defect in the pathwayfollowing cytochrome c release could provide cancer cellswith an additional survival advantage independent of BAXand BAK inhibition (Figure 1). Expression of some caspasegenes (caspase-2, 7, -8, and -9) is transcriptionally regulat-ed in response to E2F1 or p53 [63–65]; in cancer cells wherethese pathways are inactivated, expression of these cas-pases could be compromised, resulting in protectionagainst apoptosis. The APAF-1 gene is transactivated byp53 [43,44] and E2F1 [44,66] and its mRNA translation isinitiated through an internal ribosome entry site (IRES)[67]. Loss of APAF-1 expression has been reported inmelanoma [68–70], colorectal cancer [71,72], gastric cancer[73], and bladder cancer [74,75] and its loss is often associ-ated with poor clinical outcomes. Studies show that loss ofAPAF-1 expression in cancer cells can be secondary topromoter methylation [68,72,73,75] and loss of heteroge-neity at the APAF-1 locus [68–70,73] (Table 1).

Collectively, it is evident that the transcriptional net-work is deregulated in cancer cells, which has a significantimpact on the expression of apoptotic regulators and,consequently, contributes to tumor development and resis-tance to therapeutic agents.

Post-translational regulationVarious apoptotic regulators are modulated not only bytranscriptional/translational machinery but also by post-translational modifications, including ubiquitination andphosphorylation. Protein ubiquitination is implicated inprotein stability and is dynamically regulated through theubiquitin/proteasome pathway [76], whereas phosphoryla-tion can change protein functions [77,78]. It should benoted, however, that phosphorylation can also influenceubiquitination of the target protein by providing a dockingsite for an E3 ubiquitin ligase (phosphodegron).

The ubiquitin/proteasome pathway involves a series ofbiochemical events mediated by E1 ubiquitin-activatingenzymes, E2 ubiquitin-conjugating enzymes, and E3ubiquitin ligases [79]. In normal cells, expression levels

of an antiapoptotic protein with a short half-life (e.g., MCL-1) may readily decline by inhibition of transcription ortranslation following an apoptotic stimulus. By contrast,an apoptotic signal may prevent constant degradation of aproapoptotic protein (e.g., BIM), resulting in the accumu-lation of that protein. Importantly, cancer cells may dereg-ulate this process by enhancing the degradability of specificproapoptotic proteins or by promoting the stability ofantiapoptotic proteins. This may also be the case whencancer cells acquire resistance to a therapeutic agent. Itwas recently shown that HER2-amplified breast cancercells modulate the stabilities of pro- and antiapoptoticproteins, both upstream and downstream of MOMP, whenacquiring resistance to the HER2 inhibitor lapatinib; con-sequently, the resistant cancer cells exhibit strong protec-tion against cell death induced by HER2 inhibition [80].

Here we discuss how the stability of some apoptoticregulators (MCL-1, BIM, and BAX) is controlled in normalcells and how this stability may be altered in cancer cells,contributing to apoptotic protection. Then, we highlightsome examples where phosphorylation plays a role inaltering the functions of apoptotic regulators.

Stabilizing MCL-1

Whereas BCL-2 and BCL-XL proteins are relatively stable,MCL-1 protein is characterized by its rapid turnover[28,81]. As described above, MCL-1 transcription is stimu-lated, at least in part, by prosurvival factors such ascytokines. Hence, once its transcription is suppressed fol-lowing apoptotic stimuli, MCL-1 protein levels quicklydrop, potentially facilitating MOMP. Importantly, emerg-ing evidence strongly suggests that the rapid turnover ofMCL-1 protein may be abrogated in some cancer cells. Todate, five E3 ligases have been discovered to ubiquitinateMCL-1 for degradation, including the most recently iden-tified tripartite motif containing17 (TRIM17), which facil-itates MCL-1 degradation during neuronal apoptosis [82].It should be noted, however, that MCL-1 protein could alsobe degraded in a ubiquitination-independent manner [83],although the precise mechanism remains elusive.

Phosphorylation plays a key role in the stability of MCL-1 protein. Phosphorylation of MCL-1 at T163 followed byphosphorylation at S155/S159 targets the protein for pro-teasomal degradation, which is mediated by the E3 ligaseSkp-, Cullin-, F-box-containing complex with b-transducinrepeat-containing protein (SCFb-TrCP) [81,84]. The primingphosphorylation site T163 is a target of multiple kinases(Figure 3). An initial study identified glycogen synthasekinase 3 (GSK3) as a kinase that phosphorylates thisresidue; however, a more recent study demonstrated thatc-Jun N-terminal kinase (JNK) primarily phosphorylatesT163; in both cases, T163 phosphorylation will triggersubsequent phosphorylation of S159 (and then S155) byGSK3 [81,85]. Collectively, phosphorylation at S155/S159/T163 serves as a degron sequence for SCFb-TrCP (Table 1and Figure 3).

Paradoxically, it has also been reported that phosphor-ylation at T163 by ERK can lead to marked MCL-1 stabili-zation [86,87] (Table 1). How can we reconcile theseconclusions? Is the mechanism of MCL-1 degradationsimply cell-type specific? We speculate that it may be

7

P

AKT

Stabiliza�on

GSK3

SCFβ-TrCP

SCFFBW7

TRIM17

Degrada�on

Low AKT

S155S159

T163

S155S159

T163

S155S159

T163

S155S159

T163

MCL-1

PPPGSK3

JNKp38 MAPKCK2GSK3

Normal cells(A)

(B) Cancer cells

P

MCL-1

ERKP

High AKT

MCL-1

MCL-1


Figure 3. MCL-1 stability is controlled by multiple kinases. (A) MCL-1 can be

phosphorylated at T163 by several kinases including GSK3. AKT inhibits GSK3.

Thus, at low levels of AKT, phosphorylation by GSK3 sequentially occurs at S155/

S159, targeting MCL-1 for degradation by the E3 ligases SCFb-TrCP, SCFFBW7, and

TRIM17. (B) In cancer cells with high levels of AKT and ERK, ERK is likely to be the

main kinase that phosphorylates MCL-1. Phosphorylation does not continue to

S155/S159 and MCL-1 is stabilized.



phosphorylation of S159 that determines the fate of MCL-1protein following T163 phosphorylation. It is known thatGSK3 activity is negatively regulated by AKT, which iscommonly upregulated in cancer cells; therefore, in cancercells with high AKT and ERK activity, MCL-1 may bephosphorylated at T163 but not at S155 and S159, whichpromotes stabilization of MCL-1 (Figure 3). Supportingthis, it is reported that the abundance of MCL-1 protein isassociated with low GSK3 activity in breast cancer tissues[88]. By contrast, in normal cells, cellular stress signals,such as growth factor deprivation, would lead to inactiva-tion of AKT (and activation of JNK), which elicits serialphosphorylation events at S155/S159/T163; this wouldresult in degradation of MCL-1 protein (Figure 3). It shouldbe noted, however, that there is at least one additional E3ligase, F-box/WD repeat-containing protein 7 (SCFFBW7),that also regulates MCL-1 stability depending on thephosphorylation status of T163 (see below). Thus, the

8

regulation of MCL-1 protein stability mediated by thisphosphorylation motif is apparently complex and themechanism remains to be fully elucidated.

A failure in the cell cycle can trigger apoptosis in normalcells. In particular, prolonged mitotic arrest can lead toactivation of the intrinsic apoptotic pathway. For thisreason, some microtubule inhibitors that induce mitoticarrest by inhibiting mitotic spindle assembly (e.g., pacli-taxel) are used clinically to treat various cancers. However,the emergence of cancer cells with intrinsic or acquiredresistance to this type of chemotherapeutic agents is anunresolved clinical problem. Three independent studiesdescribed the mechanism of MCL-1 degradation duringprolonged mitotic arrest, suggesting a possible role forMCL-1 stabilization in resistance to mitotic apoptosis.These studies have shown that, in addition to the mecha-nisms described above, two E3 ligases, anaphase-promot-ing complex/cyclosome with cell-division cycle protein 20(APC/CCDC20) and SCFFBW7, target MCL-1 for destructionduring mitosis [89–91]. APC/CCDC20 ubiquitination ofMCL-1 is mediated by phosphorylation at T92 by cyclin-dependent kinase 1 (CDK1)/CYCLIN B and the D-boxmotif during mitotic arrest [89]. Ubiquitination of MCL-1 by SCFFBW7 is mediated by two phosphodegrons on MCL-1 (S121/E125 and S159/T163), of which S159/T163 appearsto play a major role [90,91]. Interestingly, JNK, p38, andcasein kinase 2 (CK2) phosphorylate both degrons duringmitotic arrest [91], although GSK3 phosphorylation ofMCL-1 at S159/T163 may be sufficient to target MCL-1for SCFFBW7-mediated degradation [90] (Table 1).Because,unlike CDK1/CYCLIN B, these kinases are not directlycontrolled by the cell cycle, the mechanism of mitoticphosphorylation of MCL-1 remains undetermined. In ad-dition, it is unknown whether APC/CCDC20 and SCFFBW7

compete for the mitotic degradation of MCL-1 or workindependently. From the clinical viewpoint, it is interest-ing to note that mutations or loss of FBW7 (a substrate-binding component of the multisubunit E3 complexSCFFBW7) has been reported in various cancer types[92–94]. Moreover, cancer cells with low FBW7 levels haveelevated MCL-1 protein and exhibit intrinsic resistance toABT-737, a small-molecule inhibitor of BCL-2, BCL-XL,and BCL-W [90] (Box 1).

A HECT-domain containing E3 ligase, HUWE1 [alsoknown as MCL-1 ubiquitin ligase E3 (MULE)], also targetsMCL-1 for proteasomal degradation through a pathwaythat does not require MCL-1 phosphorylation by GSK3; thebinding between MCL-1 and HUWE1 is mediated throughthe BH3 domains on each protein [95]. Interestingly, it hasbeen reported that ERK activity directly or indirectlyreduces the interaction between MCL-1 and HUWE1,although the detailed molecular mechanism remains tobe elucidated [96]. It would be interesting to test whetherthe aforementioned T163 phosphorylation by ERK is im-plicated in the MCL-1–HUWE1 interaction.

Deubiquitinating enzymes (DUBs) are negative regula-tors of the ubiquitination-mediated protein degradation,because DUBs can stabilize their substrate proteins byremoving polyubiquitin chains. Ubiquitin-specific pepti-dase 9 X-linked (USP9X) reverses ubiquitination ofMCL-1 [97]. As opposed to SCFb-TrCP and SCFFBW7, the

Box 1. BH3 mimetics and cancer therapy

The binding between pro- and antiapoptotic BCL-2 family proteins is

mediated through a hydrophobic groove on the antiapoptotic

proteins and a BH3 domain. Several small-molecule inhibitors, so-

called BH3 mimetics (e.g., ABT-737, ATB-263, ATB-199), have been

engineered to bind specifically to the hydrophobic pocket, thereby

antagonizing the function of antiapoptotic BCL-2 family proteins.

The initial inhibitor of this class, ABT-737, and its orally bioavailable

analog ABT-263, both of which inhibit BCL-2 and BCL-XL, were a

preclinical success [188,189]. Nonetheless, the clinical success was

hampered by thrombocytopenia caused by BCL-XL inhibition. To

circumvent this problem, a BCL-2-specific inhibitor, ABT-199, was

recently developed and is now being tested in clinical trials [190].

Another challenge in BCL-2/BCL-XL inhibitors is that cancer cells can

acquire resistance to this class of inhibitors by overexpressing MCL-

1 [191]. Recently, two groups independently identified MCL-1

inhibitors through gene-expression-based or biochemical screen-

ings: Golub and colleagues identified compounds that suppress

MCL-1 transcription [192], whereas Walensky and colleagues

discovered a small molecule, MIM1, that binds to the BH3-domain

binding pocket of MCL-1 [193] – these inhibitors may overcome

resistance to BCL-2/BCL-XL inhibitors. Of note, the short protein

half-life of MCL-1 makes MCL-1-addicted cancer cells more suscep-

tible to chemotherapy than cancer cells depending on BCL-2 or BCL-

XL for survival [8]. However, given the multiple mechanisms of

MCL-1 stabilization in cancer cells (see text), it is assumed that this

approach may not work in all MCL-1-addicted cancer cells. For

instance, for cancer cells with stabilized MCL-1 protein, agents that

directly inhibit MCL-1 protein (e.g., MIM1) or promote MCL-1 protein

degradation may be an option, whereas for cancer cells with

enhanced MCL-1 mRNA levels, a transcriptional inhibitor may be

more effective.



binding of USP9X to MCL-1 is negatively regulated byGSK3 phosphorylation of MCL-1 at S155/S159/T163, sug-gesting that USP9X may directly counteract MCL-1 deg-radation controlled by SCFb-TrCP and SCFFBW7. USP9Xand MCL-1 protein expression levels were positively cor-related in several cancer types, including follicular lym-phomas [97]. Elevated USP9X transcription was alsoassociated with poor prognosis in multiple myelomas [97].

Destroying proapoptotic proteins

As cancer cells can attenuate MCL-1 degradation, theymay also suppress accumulation of proapoptotic proteinssuch as BIM. As with MCL-1, BIM is also characterized byits short protein half-life. BIM stability is regulated byERK phosphorylation at S69; phosphorylation of this sitepromotes BIM degradation through the ubiquitin/protea-some pathway [98,99]. A transgenic mouse study demon-strated that replacement of the ERK phosphorylation siteswith Ala prolongs BIM protein half-life and increasesapoptotic cell death in response to serum starvation [4].SCFb-TrCP, c-CBL, and TRIM2 are the E3 ligases responsi-ble for ERK-dependent BIM degradation [100–102]. Nota-bly, SCFb-TrCP-mediated BIM ubiquitination requiresphosphorylation of the degron sequence containing S93/S94/S98 by ribosomal S6 kinase (RSK), which is promotedby ERK phosphorylation at S69 [101] (Table 1). WhetherBIM ubiquitination by c-CBL and TRIM2 may also requireRSK phosphorylation remains elusive. Lastly, the ubiquitinE3 ligase complex ElonginB/C–Cullin2–CIS is also impli-cated in BIM ubiquitination and degradation [103]. BIMubiquitination mediated by ElonginB/C–Cullin2–CIS is en-hanced following paclitaxel treatment and a defect in this

ubiquitination pathway may confer resistance to this agentin cancer cells [103].

BAX is a central component of apoptosis because itsactivation is directly linked to MOMP (Figure 1). Thus,causing degradation of BAX protein may be a ‘last-minute’safeguard for cancer cells to prevent apoptosis. BAX pro-tein mainly localizes in the cytoplasm. On genotoxic orcytotoxic stress, BAX translocates to the mitochondrialouter membrane, forms oligomeric structures, and triggersMOMP [104]. A recent study has shown, however, that thesubcellular localization of BAX protein may be in a moredynamic equilibrium between the cytoplasm and the mi-tochondrial outer membrane [105]. Although the detailedmechanism of how the equilibrium may change followingapoptotic stimuli remains elusive, the BAX equilibriumhypothesis suggests there may be a mechanism that pre-vents activation of BAX on the mitochondrial outer mem-brane. Interestingly, it is known that BCL-XL can block themitochondrial translocation of BAX by redirecting BAXback to the cytoplasm [106]. Moreover, BAX degradationoccurs on the mitochondrial outer membrane followingapoptotic stimuli [107,108]. BAX is a protein with a shorthalf-life [109,110] and its rapid degradation has beenassociated with poor clinical outcomes and high tumorgrades in chronic lymphocytic leukemia and prostate can-cer [109]. The E3 ligase IBR domain-containing 2(IBRDC2), also known as p53RFP or RNF144B, was re-cently identified and shown to ubiquitinate BAX specifi-cally on the mitochondrial outer membrane [108].Overexpression of IBRDC2, however, did not block apopto-sis triggered by actinomycin D or staurosporine treatment[108], suggesting that there may be additional factors (e.g.,cofactors or other ligases) involved in this process.

Altering protein functions

In addition to modulating docking sites for E3 ligases andsubsequent ubiquitin-mediated protein degradation, phos-phorylation can also alter the function of apoptotic regu-lators. In particular, phosphorylation by prosurvivalkinases (e.g., AKT and ERK) often results in suppressionof the proapoptotic function of the target protein, whereasphosphorylation by proapoptotic kinases (e.g., GSK3 andJNK) can lead to activation of proapoptotic proteins orinhibition of antiapoptotic proteins (see Table 1 for acomprehensive list of known phosphorylation sites of apo-ptotic regulators). For instance, the proapoptotic activity ofthe BH3-only protein BAD is inhibited by several prosur-vival kinases, including PAK1 and AKT [111–113]. Like-wise, ERK phosphorylation of BIM prevents theinteraction between BIM and BAX, thereby inhibitingapoptosis, whereas phosphorylation of BIM by JNK orp38 MAP kinase promotes the proapoptotic activity ofBIM by enhancing binding to antiapoptotic BCL-2 familyproteins [4,114–117]. As described above, ERK also pro-motes suppression of BIM expression and degradation ofBIM protein [4,98,99] (Figure 2), further highlighting acritical role for ERK in BIM inhibition.

Prosurvival kinases also negatively regulate apoptoticpathways downstream of mitochondrial cytochrome c re-lease (Figure 1). Activation of APAF-1 in response tocytochrome c is inhibited via phosphorylation of APAF-1

9

Box 2. Outstanding questions

� Do cancer cells simply depend on the net abundance of

prosurvival BCL-2 family proteins relative to the total BH3-only

proteins? Alternatively, may the regulation of a particular BCL-2

family member selectively play a role in survival in certain

cancer types? How do the functional redundancy and

specificity of prosurvival BCL-2 family proteins affect cancer

cell survival?

� Cancer cells employ multiple mechanisms to silence the apoptotic

machinery. Letai and colleagues have developed a functional

assay, called BH3 profiling, to gauge the dependency of cancer

cells on antiapoptotic BCL-2 family proteins. Although BH3

profiling made a successful breakthrough [194], is there any other

way to predict which pathways are altered in particular cancer

cells?

� Abundance of MCL-1 is one of the mechanisms for resistance to a

BCL-2/BCL-XL inhibitor in cancer (Box 1). If a BCL-2/BCL-XL

inhibitor is used concomitantly with an MCL-1 inhibitor, can we

cure all cancers? Alternatively, will cancer cells eventually acquire

resistance to the combination of a BCL-2/BCL-XL inhibitor and an

MCL-1 inhibitor? If so, how?

� Why is the pathway downstream of mitochondrial cytochrome c

release often inhibited in cancer cells in addition to the inhibition

of MOMP [195]?

� Failure to activate caspases in response to an apoptotic stimulus

can result in caspase-independent cell death [196]. Does caspase-

independent cell death play a role in cancer cell survival?



by RSK [118], a kinase often amplified in prostate cancer[119] (Table 1). Leukemic tyrosine kinases, such as BCR-ABL, also inhibit APAF-1 oligomerization by modulatingthe phosphorylation status of the APAF-1 inhibitorHSP90b [120]. In addition to APAF-1, caspases are directtargets of various kinases; activation of caspases can besuppressed by direct phosphorylation (Table 1). Further-more, it is known that phosphorylation can protect somecaspase substrates from proteolytic cleavage [78]. Forexample, phosphorylation by CK1 or CK2 protects BIDfrom caspase-8, thereby blocking the activation of thisBH3-only protein and subsequent cytochrome c releasefrom mitochondria [121] (Table 1).

In summary, post-translational modifications play acritical role in the regulation of apoptosis by modulatingprotein stabilities and functions.

Concluding remarksTo survive and proliferate, cancer cells need to overridenumerous barriers that otherwise would cause apoptosis.It is conceivable that cancer cells employ multiple mecha-nisms instead of a single pathway. Given the multiplicity ofantiapoptotic mechanisms utilized by cancer cells, it is spec-ulated that agents that can globally alter cancer’s apoptoticresistance by targeting multiple pathways may provide uswith the most valuable therapeutic benefits in the future ofcancer treatment. To target cancer cells most efficiently andselectively, we must further elucidate the mechanisms ofhow they evade the apoptotic program (Box 2).

AcknowledgmentsThe authors are grateful to Leta Nutt, Joseph Opferman, MarisaBuchakjian, and Matthew Ung for critical reading and feedback on themanuscript. This work was supported by an NCI Career DevelopmentAward R00 CA140948 (to M.K.) and a cancer center core grant P30CA23108 (to the Norris Cotton Cancer Center).

10

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evading apoptosis in cancer

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