Clinging to life cell to matrix adhesion and cell survival

Cancer and Metastasis Reviews24:425–439,2005.

C 2005Springer Science+Business Media,Inc.Manufactured in The Netherlands.

Clinging to life:cell to matrix adhesion and cell survival

Peter J.Reddig and Rudy L.Juliano

Department of Pharmacology,School of Medicine,University of North Carolina,Chapel Hill,NC27599

Key words:anoikis,integrin,Bim,Bax,PI3-kinase,FAK

Summary

Cell to matrix adhesion regulates cellular homeostasis in multiple ways.Integrin attachment to the extracellular matrix mediates this regulation through direct and indirect connections to the actin cytoskeleton,growth factor receptors,and intracellular signal transduction cascades.Disruption of this connection to the extracellular matrix has deleterious effects on cell survival.It leads to a speci?c type of apoptosis known as anoikis in most non-transformed cell types.Anchorage independent growth is a critical step in the tumorigenic transformation of cells.Thus,breaching the anoikis barrier disrupts the cell’s defenses against transformation.This review examines recent investigations into the molecular mechanisms of anoikis to illustrate current understanding of this important process.

1.Introduction

Adhesion of cells to the extracellular matrix stimulates

signal transduction cascades that have been shown to

impinge on cell growth,differentiation,and cell death

[1–4].In particular,numerous investigations have de-

lineated the important role of cell adhesion in cell

survival and apoptosis and have been reviewed else-

where[5–8].This review will focus on recent inves-

tigations into the regulation of cell death and survival

through signals regulated by adhesion of cells to the

extracellular matrix.

1.1.Molecular regulation of apoptosis

To understand the role of adhesion in cell survival,a

brief introduction to the mechanisms of apoptosis as

currently understood is required.Apoptosis removes

excess cells from tissues during normal growth and

development.During apoptosis,proteases precisely

dismantle the apoptotic cell through the digestion of

key structural and signaling proteins.Additionally,the

chromosomes themselves are torn asunder by nucle-

ase digestion.The cell is eventually broken into small

membrane enclosed particles that are phagocytosed

by neighboring cells or professional phagocytes

[9–11].

Caspases are the cysteine proteases that cleave at

conserved aspartic acids and are critical to apopto-

sis.The apoptotic caspases are separated into a hi-

erarchy of initiators(caspase-2,-8,-9,and-10)and

executioners(caspase-3,-6,and-7).These molecules

exist as zymogens in the cell with very low activ-

ity.Their activity can be induced by treatment of

the cell with ligands for death receptors or cellular

stress.The initiator caspases exist in the cell as inactive

monomers of large and small subunits.Dimerization of

these subunits in multi-protein complexes stimulates

their activation through conformational alterations

[12,13].

Cellular stress or death receptor initiated cell death

primarily uses caspase-9and caspase-8,respectively,

to start the caspase cascade.Clustering of initiator cas-

pases stimulates their activation and the subsequent

stimulation of the caspase cascade.Cleavage of the

initiator caspases is no longer postulated to be neces-

sary for their activation.The activation of caspase-9is

facilitated by its association with apoptotic protease

activating factor1(Apaf-1)bound to cytochrome c

in a structure denoted the apoptosome.Dimerization

also appears to be the important event for the acti-

vation of the caspase-8zymogen in the multi-protein

death inducing signaling complex(DISC)(see below).

Executioner caspases exist in the cytosol as inactive

dimers.The limited proteolysis of their inter-domain

426Reddig and Juliano

linkers,usually by initiator caspases,leads to their ac-tivation through conformational changes[9,13–15]. Receptor mediated activation of apoptosis,also known as the extrinsic pathway,leads to the asso-ciation of a death adapter protein with the receptor and the recruitment of a caspase-8monomer to the adapter protein forming the death inducing signaling complex,DISC.In the case of the Fas death receptor, after binding of Fas ligand(Fas-L),the adapter FADD (Fas-associated death domain)binds to Fas through death domains on the two molecules.FADD recruits procaspase-8monomers in a multiprotein complex to allow dimerization and activation of procaspase-8. Caspase-8can also cleave Bid to form tBid stimu-lating the release of cytochrome c from the mito-chondria and ampli?cation of this caspase cascade [9,16].

A central event in cell stress induced apoptosis,the intrinsic pathway,is the mitochondrial outer membrane permeablization and the release of mitochondrial pro-teins like cytochrome c.Cytosolic cytochrome c can in-teract with Apaf-1to activate caspase-9.This activates caspase-3and stimulates the caspase cleavage cascade. This cascade is initiated by the pro-apoptotic,BH3-only family of proteins Bad,Bim,Bmf,Bid,Noxa, and Puma.These proteins stimulate the translocation of homo-oligomerized pro-apoptotic proteins of the Bax family(Bax,Bak,and Bok)to the mitochondrial outer membrane.This leads to the mitochondrial outer mem-brane permeablization and release of cytochrome c and other pro-apoptotic factors.This disruption may result from intrinsic pore forming activity of the Bax pro-teins or their interaction with mitochondrial channel proteins such as the voltage dependent anion channel. The Bcl-2family of proteins(Bcl-2,Bcl-x L,Bcl-w, Mcl-1,and A1/B?-1)is structurally related to the Bax and BH3-only families and prevents disruption of the mitochondrial outer membrane and apoptosis[10,11, 17–19].

The precise interplay between these proteins and their function remains controversial.The original model of Bcl-2of inhibiting Bax function through het-erodimerization is no longer favored because of the dif?culty in detecting this complex under physiolog-ical conditions.Bcl-2proteins may block apoptosis by maintaining mitochondrial membrane integrity and preventing pore formation though an unde?ned mech-anism.Additionally,Bcl-2family proteins can bind BH3-only proteins and may act as rheostat for BH3-only protein levels.Thus,the level of Bcl-2proteins would determine the threshold for BH3-only activation of Bax family proteins[10,11,17–19].

Bax family proteins stimulate the release of cy-tochrome c by a conformational change that stimulates homo-oligomerization.This conformational change in Bax proteins may help them to induce pore formation and permeablization of the mitochondrial outer mem-brane,loss of the inner mitochondrial membrane po-tential,and release of cytochrome c.The signal for the activation of Bax family proteins and their interaction with the mitochondrial outer membrane remains ill de-?ned.One mechanism for Bax activation may be its interaction with a proteolytically cleaved form of the BH3-only protein Bid,t-Bid[10,11,17–19].

1.2.Anoikis

Anoikis is a speci?c type of apoptosis caused by de-tachment of a cell from its supportive matrix and was originally described in MDCK cells by Frisch and Fran-cis[20].Early work suggested that the JNK/SAPK signaling was important for the induction of anoikis. However,later work pointed to a more prominent role for other signaling molecules like PI3-kinase,FAK,and the Ras/MAPK cascade in the regulation of anoikis[7]. Recent work has further delineated the role of these and other signaling molecules and that of the apoptotic reg-ulatory proteins themselves in adhesion regulated cell survival.

2.BH3-only,Bax,and Bcl-2proteins and anoikis 2.1.Bim and anoikis

BH3-only proteins appear to act as sensors of cellu-lar stress and have no intrinsic ability to induce cell death on their own.Two members of this family Bim and Bmf appear to be important sensors of changes in the actin cytoskeleton.The Bim isoforms Bim EL and Bim L bind to LC8,also known as cytoplasmic dynein light chain(DLC1),a component of the microtubule-associated dynein motor complex.These proteins are sequestered in the dynein complex until an apoptotic stimuli induces their release[21].The BH3only pro-tein Bmf binds to dynein light chain-2localizing it to the myosin V motor.Treatment of cells with cytocha-lasin D or induction of anoikis initiates the release of Bmf from the cytoskeleton[22].Several studies have recently investigated the role Bim plays in anoikis.

Clinging to life:cell to matrix adhesion and cell survival427

It has been previously demonstrated that activation of the epidermal growth factor receptor(EGFR)and the subsequent stimulation of the Erk/MAPK cascade were capable of suppressing anoikis.For example,the introduction of a Raf estrogen receptor fusion protein (Raf-ER)suppressed anoikis in MCF-10A cells and CCL39lung?broblasts cells upon activation of Raf [23,24].Furthermore,the survival of MCF-10A cells in suspension depended on the autocrine production of EGFR ligands HB-EGF,TGFα,and amphiregulin[24]. The Erk/MAPK cascade directly leads to the phospho-rylation of Bim EL which can inhibit its function and lead to its degradation[25,26].

Several recent studies have examined the connec-tion between the EGFR-Erk/MAPK survival path-way,Bim,and anoikis.One investigation found that during anoikis in immortalized mammary breast ep-ithelial cells(MCF-10A),all three Bim isoforms Bim EL,Bim L,and Bim S were elevated.In adher-ent cells,EGFR repressed Bim expression through the Erk/MAPK pathway.Suppression of Bim ex-pression by hyper-activation of the EGFR/Erk/MAPK signaling pathway by over expression of EGFR or constitutively active MAPK components blocked anoikis.Conversely,the loss ofβ1integrin engage-ment in detached cells repressed expression of the EGFR and increased Bim expression.Thus,the neg-ative regulation of Bim expression by adhesion and EGFR signaling were implicated in the suppression of anoikis in breast epithelial cell lines(Figure1) [27].

An additional investigation found that suspension culture of the MCF-10A cells enhanced the expression of the Bim EL,and Bim L[28].Again,activation of the Erk/MAPK cascade suppressed anoikis.Erk activation stimulated phosphorylation and the proteasome depen-dent degradation of Bim EL.These alterations were not observed for Bim L.Furthermore,the elimination of Bim from MCF-10A cells by RNAi partially blocked anoikis.The relevance of the phosphorylation to the suppression of anoikis was indeterminate in this study since a phosphorylation resistant mutant of Bim EL still induced apoptosis(Figure1)[28].

The cells used in the previous studies to examine Bim’s role in anoikis were immortalized breast epithe-lial cell lines.Interestingly,examination of the same phenomena in primary breast epithelial cells or a breast epithelial cell line with a more normal phenotype(FSK-7)gave different results.Detachment from substrate in the absence of growth factors induced dephosphoryla-tion of Bim EL and Erk.However,in the presence of a cocktail of growth factors,2%fetal calf serum,5ng/ml EGF,and5μg/ml insulin,Bim EL and Erk phosphory-lation and protein levels were maintained in detached cells and cell death still occurred within4–8hours.In a parallel examination of MCF-10A cells,the status of Bim EL and Erk phosphorylation also did not corre-late with survival in suspension.The MCF-10A cells were able to survive independently of adhesion status and were only dependent on growth factor signals for survival.

In spite of no detectable role in anoikis in normal breast epithelial cells,Bim EL was important for EGF dependent survival.Suppression of Bim EL by RNAi in-hibited apoptosis in response to EGF withdrawal with no effect on anoikis.The absence of growth factors induced dephosphorylation of Bim EL and Erk and in-creased the level of Bim EL.Conversely,treatment of adherent cells with epidermal growth factor stimulated Bim EL phosphorylation in a MEK-Erk dependent man-ner and inhibited apoptosis.

These results,in contrast to the other investigations, indicate that adhesion is critical for survival of breast epithelial cells,but anoikis can occur independently of Bim and Erk.However,in agreement with the other studies,Bim senses changes in the epidermal growth factor dependent survival signals with the stimulation of the Erk/MAPK cascade being needed for Bim EL and Erk phosphorylation and inhibition of apoptosis (Figure1)[29].

2.2.Bax

The proapoptotic protein Bax contributes to the induc-tion of apoptosis by translocating to the outer mito-chondrial membrane,inducing its permeablization,and allowing the release apoptotic factors like cytochrome c[11,17].During anoikis in mammary epithelial cells, Bax translocation to the mitochondria depends on the loss of survival signals from FAK.As in other cell death programs,Bax’s movement to the mitochondria dur-ing anoikis correlates with it undergoing an activating, conformational change.The Bax translocation is inde-pendent of caspase activation and cytochrome c release. Detachment of cells from the extracellular matrix stim-ulates the translocation of Bax to the mitochondria in about?fteen minutes.However,the cells do not die immediately,rather death occurs after several hours in suspension.If cells are re-plated quickly enough,Bax

428Reddig and Juliano

Figure1.Suppression of apoptosis and anoikis in breast epithelial cells.(A).Attachment to the extracellular matrix and stimulation of growth factor signaling cascades suppresses the activity of apoptotic factors.EGF binding to the EGFR stimulates Erk/MAPK signaling,which negatively regulates Bim by phosphorylation and stimulating its degradation.This prevents Bim from antagonizing Bcl-2function or stimulating Bax activation.Inhibition of Bim suppresses disruption of the mitochondria and the induction of cell death.The adhesion survival signals suppress anoikis in a Bim dependent and independent manner depending on the time in suspension and the cell type used(B).Detachment from the matrix or growth factor deprivation shuts down these signaling cascades.Some investigations have found that suspension culture actively down regulates EGFR adding to the suppression of survival signals.Once survival signals are suppressed the levels of Bim increase in its dephosphorylated form.This leads to activation of Bax,inhibition of Bcl-2,and cell death.

Clinging to life:cell to matrix adhesion and cell survival429

will exit the mitochondria and the cells will remain viable[30,31]

The temporal discrepancy between Bax mitochon-drial translocation and execution of cell death during anoikis was examined to determine how this related to spatial and conformational changes in Bax.In viable, anchored cells,Bax exists in the cytoplasm as an in-active monomer.During anoikis,Bax translocates to the mitochondria in the inactive https://www.360docs.net/doc/0f1352536.html,ing a con-formation sensitive antibody,the activating conforma-tional change in Bax was observed only for Bax in the mitochondrial membrane.As observed for other apoptotic events,the mitochondrial Bax oligomerized with other Bax monomers.The activated Bax formed clusters just prior to loss of mitochondrial membrane potential and the release of cytochrome c.These events were independent of caspase activation.Interestingly, suspended cells commit to the apoptotic pathway be-fore Bax clustering,loss of membrane potential,or the release of cyotchrome c,but after the localization of Bax to the mitochondria.Thus,an unde?ned event oc-curs that commits cells to apoptosis that appears to be independent of Bax clustering,loss of mitochon-drial membrane potential,and release of cytochrome c [32].

2.3.Bid

As described above,Bid is a BH3only protein that is cleaved to form truncated t-Bid during death receptor mediated apoptosis.Death receptors signals for cleav-age of caspase8and t-Bid have been implicated in the regulation of anoikis[33–35].In contrast,studies of anoikis in mammary epithelial cells found that activa-tion of caspase-8and cleavage of Bid were not nec-essary for anoikis[30].Interestingly,during anoikis in mammary epithelial cells,Bid was found to translocate to the mitochondria as a full-length protein.Cleavage of Bid was not necessary for anoikis.The movement to the mitochondria by Bid was independent of caspase-8activation and translocation of Bax or Bcl-2family proteins to the mitochondria.Suppression of FAK and PI3-kinase signals with dominant negative mutants also stimulated the translocation of Bid to the mitochon-dria.Thus,full length Bid appears to be important for anoikis and suppression of the FAK/PI3-kinase survival signals by cell detachment stimulates its apoptotic ac-tivity[36].2.4.Bit1

Adhesion to the extracellular matrix through certain integrins likeα5β1can stimulate Bcl-2expression [37,38].To identify new modulators of anoikis,Jan et https://www.360docs.net/doc/0f1352536.html,ed a cDNA library screen to identify genes which could rescue integrin stimulated Bcl-2expres-sion in cells harboring an integrinα5subunit lacking its cytoplasmic domain[39].Surprisingly,a pro-apoptotic protein that inhibited Bcl-2transcription was isolated and named Bit1,Bcl-2inhibitor of transcription.Bit1 is a unique mitochondrial protein with no identi?able homologues and potently induces apoptosis when ex-pressed in the cytoplasm.This activity is speci?cally repressed by adhesion to?bronectin throughα5β1.The original cDNA clone may have been mutated in such a way that allowed it to act like a dominant negative and allowing it to rescue Bcl-2expression.

During anoikis,Bit1translocates from the mitochon-dria to the cytoplasm and interacts with AES,a mem-ber and negative regulator of the Groucho/TLE family of transcriptional regulators.The interaction of Bit1 and AES initiates cell death independently of caspase activation,Bcl-2or Bcl-x L levels,or PI3-kinase ac-tivity.This suggests that Bit1acts downstream or in parallel to these pathways,likely at the level of Bcl-2 expression.Supporting this idea,the transcriptional regulator TLE1,which binds AES,blocks Bit1in-duced apoptosis when over-expressed.The induction of apoptosis positively correlates with TLE1’s abil-ity to stimulate Bcl2expression and Bit1to inhibit it.Bit1may be key mediator of the adhesion sur-vival signal with adhesion being the only upstream survival signal found to repress its death inducing activity when ectopically placed in the cytoplasm [39].

3.PI3-kinase

The generation of3 phosphorylated phosphoinositides (PI3,4P&PI3,4,5P)is mediated by the enzyme PI3-kinase.These phosphorylated phosphoinositides stimulate the recruitment of PKB/Akt to the mem-brane through its PH domain where PDK-1/PRK-2 phosphorylation of PKB/Akt at Thr-308and Ser-473 activates PKB/Akt.Activated PKB/Akt mediates cell survival through phosphorylation of several substrates including Bad and pro-caspase-9and inhibiting their function.PI3-kinase and the subsequent activation of

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Figure 2.Suppression of anoikis through PI3-kinase signaling.Upon stimulation with growth factors and adhesion PI3-kinase stimulates the generation of the phosphoinositide PIP 3.PIP 3binds to PKB/Akt helping to recruit it to the membrane where PDK-1can phosphorylate PKB/Akt on threonine 308.The activated TrkB receptor stimulates this pathway for suppression of anoikis.Adhesion can stimulate PKB/Akt phosphorylation on serine-473through ILK-1activation.PP2A associated with β1integrins negatively regulates PI3-kinase signaling by dephosphorylation of serine 473and turning off this survival pathway.Cdc42stimulates the PI3-kinase/PKB/Akt pathway through Rac.Rac and PI3-kinase may function in a positive feedback loop that stimulates this survival pathway.For cell survival,active PKB/Akt suppresses the function of pro-apoptotic proteins and stimulates survival proteins.PKB/Akt directly phosphorylates Bad,pro-caspase 9,and DAP-3to stimulate survival by inhibiting their function.PKB/Akt also stimulates the adhesion mediated increase in survivin levels.

PKB/Akt play a central role in regulation of adhesion,mediated survival signals [7,40].Recent investigations have further delineated these connections.

In a study to identify amino acids in the integrin β1cytoplasmic domain that speci?cally regulate sur-vival signaling,tryptophan 775on the β1cytoplasmic domain was found to be important for stimulation of PKB/Akt activity [41].Substitution of this tryptophan on the β1cytoplasmic domain with an alanine speci?-cally inhibits PKB/Akt activity and decreases cell sur-vival in cells expressing this mutant.Mutation of this tryptophan does not signi?cantly interfere with inte-grin activation,binding to αsubunits,talin,vinculin,or α-actinin,localization to focal adhesion,or activa-tion of PI3-kinase.

Instead,β1was found to selectively recruit a subpopulation of protein phosphatase 2A (PP2A)to β1cytoplasmic domain complexes.PP2A selectively

dephosphorylated PKB/Akt and reduced integrin me-diated survival signaling.The inhibitory nature of this mutant β1arises from an increase in the activity of bound PP2A,not the amount,and a subsequent de-crease in PKB/Akt phosphorylation on serine 473.The presence of PP2A associated with β1may allow for rapid suppression of PKB/Akt activity in response to changes in adhesion status (Figure 2)[41].

In cells derived from strati?ed squamous epithelia,the selective activation of PI3-kinase via differential ex-pression of integrin subunits selectively regulates cell survival.In normal strati?ed squamous epithelia the αv integrin subunit normally pairs with the β5subunit as a receptor for vitronectin.During hyper-proliferation or in transformed cells the αv pairing with β5is re-placed with αv paired with β6.The switch to αv β6confers protection from anoikis in squamous epithe-lial cells.The increased adhesion independent survival

Clinging to life:cell to matrix adhesion and cell survival431

potentiated byαvβ6results from its ability to stimulate

PI3-kinase in suspension whileαvβ5cannot.Replac-

ing the cytoplasmic domains ofβ6with that ofβ5

suppressed its survival phenotype and ability to acti-

vate PI3-kinase;thus,localizing these functions to the β6cytoplasmic domain.Therefore,selective integrin isoform activation of PI3-kinase confers differential

survival phenotypes and this could be important for

cellular transformation[42].

Another connection in the web of PI3-kinase inter-

actions regulating anoikis was found to be the GTP-

binding,death associated protein-3(DAP-3).DAP-

3is a pro-apopptotic protein with mitochondrial and

cytoplasmic pools that regulates cell death mediated

by death receptor ligands like TNF-α.Suppression of

DAP3expression in HEK293cells inhibited anoikis.

Cell detachment stimulated association of DAP3with

FADD and increased caspase-8activity.Expression of

an active Akt inhibited the DAP-3/FADD association,

caspase-8activation,and anoikis in suspended cells.

The direct phosphorylation of DAP3by Akt mediated

Akt’s ability to stimulate survival in suspended cells.

Additionally,adhesion of suspended cells to vitronectin

increased survival,Akt activation,and phosphorylation

of DAP3.Thus,DAP3appears to be an important Akt

target in adhesion mediated survival signaling[43].

Another target of the PI3-kinase pathway important

for survival may be the IAP family protein survivin.It

is not expressed in most adult tissues,but is elevated in

numerous human cancers and is associated with a poor

prognosis[19].The regulation of survivin by adhesion

was examined in prostate cancer cell lines.Adhesion of

a malignant cancer cell line to?bronectin increased the

expression of survivin and resistance to TNF-αinduced

apoptosis.A dominant negative survivin blocked the

protective effect of adhesion to?bronectin.Conversely,

a wild type survivin imparted resistance to apoptosis to

cells cultured in suspension.This elevation of survivin

levels only occurred in the tumorigenic cell types exam-

ined.The survivin and?bronectin dependent increase

in survival was dependent on theβ1A integrin isoform

and PI3-kinase signaling(Figure2)[44].

The importance of the PI3-kinase/AKT survival-

signaling cascade in anoikis was reinforced in a screen

for suppressors of anoikis in rat intestinal epithelial

cells(RIE).TrkB,a neurotrophic tyrosine kinase re-

ceptor,was found to potently suppress anoikis in RIE

cells in the presence or absence of its ligand,brain-

derived neurotrophic factor.Elevation of TrkB levels

in RIE cells disrupted epithelial organization,stim-ulated cell aggregation,and enhanced the prolifera-tion of spheroids in suspension.The TrkB survival signal appeared to be speci?c for anoikis because it could not suppress apoptosis in serum deprived?brob-lasts overexpressing c-Myc.TrkB activated the PI3-kinase cascade and this activation was necessary for its suppression of anoikis and was independent of the cell aggregation.Rac,p70S6-kinase,and MEK,(down-stream effectors of PI3-kinase)were not necessary for its survival function.The importance of TrkB’s sup-pression of anoikis was con?rmed by its ability to form metastatic tumors alone or in combination with its lig-and.TrkB and its ligand are overexpressed in several human tumors types.These?ndings support the im-portance of anoikis,TrkB,and PI3-kinase signaling in tumorigenic transformation of cells(Figure2)[45]. The individual Akt isoforms may have unique and non-overlapping roles in anchorage dependent cell sur-vival.In intestinal epithelial cells Akt-1activation by cell adhesion throughβ1integrin,FAK,and PI3-kinase is critical for cell survival in all states of enterocyte differentiation.However,Akt-2is not regulated by PI3-kinase and cannot replace PKB/Akt in survival signaling[46].

Although important in adhesion dependent survival signaling,the PI3-kinase/Akt survival pathway can be circumvented.In an examination of anoikis in a panel of breast cancer cell lines,Akt activation did not strongly correlate with cell survival.Even pharmacological in-hibition of PI3-kinase did not impart anoikis sensitivity to cells with high PKB/Akt phosphorylation levels[47]. Clearly,PI3-K has a central role in anoikis.The iden-ti?cation of novel modes of regulation of its activity and substrates in relation to anoikis adds to our knowledge of this pathway.Further investigation into the precise interplay of these events and their deregulation in dis-ease will be required for a complete understanding of PI3-kinase regulation of anchorage dependent survival.

4.ILK

The integrin linked kinase(ILK)binds to theβ1cy-toplasmic domain and is regulated by growth factor receptors and integrins in cooperation with PI3-kinase. ILK activity is important for cell survival.Substantial evidence indicates that ILK can enhance phosphory-lation of PKB/Akt on Ser-473that,in combination with Thr-308phosphorylation by PDK-1,is essen-tial for PKB/Akt activation[40,48].However,recent

432Reddig and Juliano

investigations suggest that ILK may not be the media-tor of the Ser-473phosphorylation of PKB/Akt[49]. To de?nitively show that ILK is the mediator of PKB/Akt phosphorylation,ILK was knocked out in cultured cells using both RNAi inhibition of ILK ex-pression and Cre/Lox conditional gene disruption.The elimination of ILK expression blocked Ser-473phos-phorylation of PKB/Akt.The loss of ILK in cells also elevated the levels of apoptosis.Thus,ILK has an im-portant role in the Ser-473phosphorylation of PKB/Akt and cell survival.However,whether ILK directly medi-ates this phosphorylation or regulates the activity of an intermediary kinase is yet to be determined(Figure2) [50].

ILK interacts with the calponin homology domain-containing integrin-linked kinase-binding protein(CH-ILKBP)in a ternary complex with PINCH.CH-ILKBP has also been identi?ed as actopaxin orα-parvin.This trimeric complex of proteins localizes to focal adhe-sions[51–53].Investigations of CH-ILKBP in ILK sig-naling found it to be important for cell survival. Suppression of CH-ILKBP with RNAi increased the level of apoptosis without disruption of focal adhe-sions or ILK focal adhesion localization.This increased apoptotic activity positively correlated with a loss of phosphorylation of Thr-308and Ser-473of PKB/Akt and inhibition of its activation.The impairment of PKB/Akt activation resulted from impaired membrane localization in the absence of CH-ILKBP.Arti?cially targeting PKB/Akt to the membrane permitted its ac-tivation in the absence of CH-ILKBP.The Erk/MAPK and p38/MAPK pathways did not signi?cantly con-tribute to survival signaling mediated by CH-ILKBP and PKB/Akt.Thus,membrane targeting of PKB/Akt by CH-ILKBP plays an important role in cell survival (Figure2)[54].

The other member of this dynamic trio,PINCH, also appears to play an important role in cell survival. The depletion of PINCH-1with RNAi led to inhibi-tion of cell spreading and induction of apoptosis.This increased apoptosis positively correlated with the in-hibition of PKB/Akt phosphorylation of Thr-308and Ser-473.Again,depletion of ILK suppressed Ser-473 phosphorylation.However,an active PKB/Akt could not block the apoptosis induced by RNAi suppression of PINCH-1or ILK-1.Thus,PINCH-1and ILK-1may have functions downstream or in parallel to PKB/Akt activation that are important for cell survival.

The loss of PINCH-1stimulated the proteasomal degradation of ILK-1and CH-ILKBP.Expression of the PINCH-1homologue,PINCH-2,restored the lev-els of ILK-1and CH-ILKBP.However,the reduction in PKB/Akt activation and cell viability was not rescued by the presence of PINCH-2.Thus,the degradation of ILK-1and CH-ILKBP was not necessary for the re-duced viability in the absence of PINCH-1.However, caspase inhibition blocked the apoptotic response me-diated by PINCH-1reduction.Thus PINCH-1may be a conduit for survival signals from PKB/Akt or other parallel pathways[55].

Activation of PKB/Akt depends on ILK and its as-sociated proteins.Membrane association of PKB/Akt mediated by CH-ILKBP appears to be a prereq-uisite for PKB/Akt activation.Once at the mem-brane PKB/Akt depends on the presence of ILK-1and PINCH-1,in addition to other proteins,for activation via phosphorylation.However,activation of PKB/Akt is not the only critical function of PINCH-1and ILK-1given that arti?cially activated PKB/Akt did not sustain cell viability in their absence. Thus,this complex of integrin-associated proteins has a pivotal role in the maintenance of cell viability (Figure2).

5.Focal adhesion kinase(FAK)

FAK plays a central role in transmitting adhesion de-pendent signals and is important for adhesion de-pendent survival signaling.FAK interacts with sev-eral effector proteins,like the p85catalytic subunit of PI3-kinase,Src,Shc,and Cas,in adherent cells to signal.Importantly,anoikis can be prevented by ex-pression of constitutively active FAK mutants.The disruption of FAK signaling in cancer cells by vari-ous techniques induces cell death after the induction of cell rounding and detachment from the substra-tum.FAK survival signaling depends on the PI3-kinase signaling cascade.Cell death in the absence of FAK survival signals depends on the presence of p53 [7,56].

Recent investigations have further delineated the in-timate relationship between FAK and survival signal-ing.FAK was found to directly interact with RIP,a death domain containing serine/threonine kinase[57]. RIP contains a death domain that mediates the inter-action with other death domain proteins in the death receptor complex and regulates death/survival signal-ing through regulation of NF-κB[58,59].Induction of apoptosis in breast cancer cells with the protein

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kinase inhibitor,staurosporine,stimulated the dephos-phorylation and degradation of FAK and a reduction in RIP levels.The destruction of FAK in response to this apoptotic stimulus depended on the presence of RIP because the RIP null?broblasts treated with stau-rosporine exhibited little change in FAK phosphory-lation or protein levels and a minimal apoptotic re-sponse.RIP also appears to play an important role in mediating pro-apoptotic signals in response to disrup-tion of adhesion and FAK dependent survival signals. The absence of RIP suppresses cell death induced by suspension culture or the disruption of FAK signaling with the FAK’s carboxy terminus.Again,the absence of RIP prevented changes in FAK phosphorylation, levels,and localization.Thus,RIP transmits apop-totic signals to FAK in response to various stimuli and may be a critical component of the survival-signaling network,especially adhesion dependent survival [57].

Another focal contact protein,vinculin,was also recently implicated in the regulation of cell survival through modulation of FAK signaling.Vinculin binds to talin,actin,and paxillin and is an important link in the connection between integrins and the actin cy-toskeleton[60–63].The ablation of the vinculin gene in F9embryonic carcinoma cells confers resistance to several apoptotic stimuli including serum deprivation, suspension culture,and camptothecin treatment.Ex-pression of the hinge-tail domain of vinculin,which contains the paxillin binding site,in vinculin null cells restores the apoptotic response to serum withdrawal and cell suspension.

Somewhat paradoxically,the vinculin null cells ex-hibit elevated Erk activation after detachment from the substratum while vinculin positive cells do not.Ad-ditionally,the absence of vinculin allowed elevated tyrosine phosphorylation and interaction of FAK and paxillin.Re-expression of vinculin in the null cells dis-rupted the FAK/paxillin interaction and blocked the activation of Erk in detached cells and restored the apoptotic response.

Vinculin and FAK interact with paxillin through sim-ilar paxillin LD motifs[64].The absence of vinculin en-hances the FAK/paxillin survival signaling to Erk while elevation of vinculin disrupts this signal.Vinculin’s absence also enhances motility in F9cells[65].This investigation demonstrates that the enhanced motility results from activation of the same FAK/paxillin/Erk signaling cascade that inhibits apoptosis.Thus,differ-ential FAK protein-protein interactions are important for regulation of FAK survival and motility signaling [66].

6.Cleavage of signaling molecules

Protein cleavage is a hallmark of apoptosis.The ac-tivation of the caspase cleavage cascade is central to most programmed cell death.Cleavage of signaling molecules like FAK,Src,and paxillin can be impor-tant for the progression of the apoptotic cascade[67–69].The cleavage of FAK by caspases shuts down its survival signal and releases FAK’s carboxy-terminus. FAK’s carboxy-terminus contains the focal adhesion targeting domain and is inhibitory to FAK signaling, thereby enhancing the apoptotic effect of cleaving FAK [7].

CAS,an SH2/SH3adaptor protein,which binds FAK and transmits integrin signals,undergoes cleavage dur-ing apoptosis.In etoposide treated cells CAS is cleaved by caspase-3.This cleavage disrupts the CAS-paxillin interaction,CAS cellular localization,and focal adhe-sion architecture[70].The cleavage of CAS produces a31-kDa carboxy terminal fragment of CAS.Expres-sion of this31-kDa CAS fragment induces apoptosis in HeLa cells.Interaction of this fragment with the E2A transcription factor via a helix-loop-helix motif translocated it to the nucleus.This interaction with E2A blocked E2A’s ability to stimulate p21Waf1/Cip1and this repression contributes to the apoptotic response [71].

The induction of anoikis in MDCK cells also led to CAS and FAK cleavage as well as dephosphory-lation of CAS.Caspases and calpain mediated this early event.Again,the carboxy-terminus CAS cleavage product initiated apoptosis when overexpressed in cells resistant to anoikis[72,73].HEF-1,a CAS family pro-tein,also is cleaved by caspases during apoptosis and anoikis releasing a similar,toxic carboxy-terminal frag-ment[74,75].Thus,like FAK,the disruption of CAS controlled signaling by its cleavage and generation of inhibitory fragments could lead to the suppression of survival signals in non-adherent cells.

7.Rho GTPases

The Rho GTPases Rac1and Cdc42are important com-ponents of the adhesion dependent signaling network. Integrin engagement regulates the activation of Rac and Cdc42and their translocation to the membrane

434Reddig and Juliano

where they can interact with effectors like the p21 activated kinases[76,77].Given this connection,one might expect a connection between Rac and anoikis. This connection was found in studies of MDCK anoikis.Expression of an activated Rac mutant sup-pressed anoikis in non-adherent MDCK cells while a dominant negative mutant inhibited anoikis.Sup-pression of anoikis by active Rac was dependent on Rac activation of the p38-MAPK and PI3-kinase path-ways,but not the Erk-MAPK or NF-κB pathways [78].

Similarly,expression of an activated Cdc42protein inhibits anoikis in MDCK cells.This effect of Cdc42 depended on its ability to activate PI3-kinase and Rac. Cdc42activation of Erk,JNK,or p38MAPK kinase pathways in suspension was not important for Cdc42 regulation of anoikis.Dominant negative Rac blocked Cdc42activation of PI3-kinase and PI3-kinase suppres-sion of anoikis.Thus,Rac and PI3-kinase activation may act in a positive feedback loop downstream of Cdc42survival signaling.Disruption of the actin cy-toskeleton with Latrunculin B permitted anoikis in cells with active Cdc42and Rac,but this treatment did not alter the activation of PI3-kinase.Thus,Cdc42and Rac potentially regulate survival through PI3-kinase depen-dent and independent pathways(Figure2)[79].

The engagement of integrins with their ligands is im-portant for signal transduction as well as cell survival. As described,alterations in mitochondrial integrity and the release of various mitochondrial constituents are important for the induction of apoptosis.Werner and Werb connected these two phenomena,but the signal was used to induce gene expression not apoptosis[80]. It was demonstrated that disruption of cell shape using an integrinα5β1interfering antibody could induce the production of reactive oxidant species (ROS)through activation of the small GTPase Rac. The source of the Rac stimulated ROS was found to be the mitochondrial respiratory chain after de-polarization of the mitochondrial membrane.This ROS production led to the stimulation of an Nf-κB response element and the collagenase-1promoter. Bcl-2overexpression inhibited this signaling path-way,but it had no dependence on caspase activa-tion[80].While tied to control of gene expression in this work,this induction of ROS release from the mitochondria by alterations in integrin engagement could also be important for similar pathways regulating apoptosis.8.Matrix adhesion,DNA damage,and cell death The cellular response to DNA damage is cell cycle ar-rest or apoptosis.A central regulator of this response is the transcription factor p53.In the presence of cellular stress like DNA damage,the levels of p53in the cell are elevated.Subsequently p53stimulates expression of numerous growth arrest and pro-apoptotic genes like p21waf1/cip1and bax,respectively[81,82].An interest-ing relationship exists between cell adhesion and the cellular response to DNA damaging agents in some transformed,anchorage independent cells.

Recent studies found that some cell types capable of anchorage independent growth,when grown in sus-pension,did not undergo apoptosis in response to DNA damaging insults.However,when the cells were ad-herent they became sensitive to DNA toxic insults and apoptosis.The levels of p53are regulated,in part,by Mdm2stimulated degradation of p53.The tumor sup-pressor p19Arf suppresses Mdm2function by seques-tration of Mdm2to the nucleoli[82].In the resistant cells,the levels of p53and p19Arf declined in sus-pended cells.The p19Arf decline temporally preceded the decline in p53levels.The reduction in p53levels required the presence of Mdm2.The forced expression of p19Arf restored p53’s stability and cellular sensitiv-ity to DNA damaging agents in suspended cells.This demonstrates the importance of p19Arf/Mdm2in the decline of p53levels in suspended cells.Thus,the adhe-sion status of some cancer types may be important for their responsiveness to DNA damaging therapies[83]. The anchorage dependent apoptosis induced by DNA damaging agents also has a p53independent com-ponent.This pathway depends on the activity of the c-Abl tyrosine kinase.In the absence of p53,mouse embryonic?broblasts still exhibited adhesion depen-dent apoptosis in response to genotoxins that was sup-pressed upon culturing in suspension.However,the disruption of c-Abl signaling by inhibitors and gene deletion blocked the adherent cell apoptotic response in the absence of p53.Genotoxin treated cells had an adhesion dependent elevation of c-Abl kinase activity. This elevation of kinase activity positively correlated with the elevation of the p53homologue p73in p53null ?broblasts.Inhibition of c-Abl kinase activity blocked the p73increase.Interestingly,in wild type embry-onic?broblasts,reduced adhesion to a substrate,not only complete detachment,also decreased the apop-totic response to genotoxic insult.These results could

Clinging to life:cell to matrix adhesion and cell survival435

be important to cancer therapies based on genotoxic agents.The level of cellular adhesiveness and the cel-lular genotype could determine the cellular responsive-ness to therapy[84].

9.Tissue architecture

The three dimensional(3D)architecture of the cell and tissue environment also contributes to the viability of the cell in the face of apoptotic stimuli.A recent inves-tigation by Weaver et al.,demonstrated the importance of this in mammary epithelium.When malignant and non-malignant mammary epithelial cells were grown in standard two-dimensional cell culture environments, both underwent apoptosis in response to extrinsic and intrinsic apoptotic stimuli equally well.The culture of the non-malignant mammary epithelial cells in recon-stituted basement membrane(rBM)allowed the for-mation of3D,polarized organoids(acini).These po-lar acini were resistant to all forms of apoptotic stim-uli tested.The malignant cells did not form organized structures during culture with the rBM and exhibited no resistance to apoptotic stimuli.However,if the ma-lignant cells were forced to form the acini they also became resistant to apoptotic stimuli.Similarly,dis-ruption of the acini by blocking cadherin function sen-sitized the non-malignant cells to the noxious stimuli. This resistance to apoptotic stimulus was independent of the proliferative status of the cell.The proper polar structure of the acini induced by laminins in the rBM, not simply the3D culture,was critical for increased survival since collagen gel cultures could not support the formation of acini or enhanced resistance to apop-tosis.The proper formation of hemi-desmosomes by the integrinα6β4and the maintenance of NF-κB ac-tivity were demonstrated to be important for mediation of this apoptotic resistance in the mammary cell acini. The inhibition of the apoptotic response byα6β4regu-lated cellular polarization in3D rBM culture correlates well with the poor prognosis of tumor cells expressing α6β4and BM proteins[85].

10.Conclusions

Adhesion plays a central role in cell survival.Several of the proteins central to the transmission of adhesion sig-nals are also important for survival.In particular,PI3-kinase,which has a central role in other mechanisms of apoptosis,plays a critical role in adhesion-mediated survival.PI3-kinase activity and signaling are modu-lated by adhesion,in part,through FAK,the ILK/CH-ILKBP/PINCH complex,Rac,and PP2A and it regu-lates the function of caspase-9,Bad,survivin,Rac,and DAP-3.FAK,in its active,full length form,anchors the adhesion survival signaling with its ability to scaffold numerous focal adhesion protein interactions,like PI3-kinase.Disruption of FAK function through its degra-dation or by competition with other proteins promotes anoikis.Alterations in the levels or activities of effec-tors of FAK and PI3-kinase,like the Rho GTPases and CAS,also modulate the cell’s response to disruption of adhesion signals.Thus,reinforcing the importance of these signaling pathways for cell survival.

Proteins known to regulate apoptosis during other cell stress situations also mediate anoikis with some twists.Bax translocation to the mitochondria and its oligomerization occurred during anoikis.However,an unde?ned event that condemns the cell to death occurs subsequent to Bax’s association with the mitochon-dria,but prior to the other canonical events of intrinsic apoptosis.What this event is will be important area of investigation.

The role of BH3-only proteins in anoikis is inter-esting and controversial.Bim levels and its phospho-rylation status regulate survival in breast epithelial cells.The EGFR/Raf/MAPK negatively regulates Bim through its phosphorylation and depression of Bim lev-els.Two investigations suggest that cell suspension re-presses the EGFR/Raf/MAPK signal,thus freeing Bim to perform its death function in anoikis.In both cases, hyperactivation of the EGFR/Raf/MAPK can repress Bim function and block anoikis.Other investigators did not observed the adhesion dependent regulation of Bim during anoikis,only observing the EGFR/Raf/MAPK modulation of Bim independent of adhesion status. These differences may result from differences in the time courses used for induction of anoikis and the cell types used.Clari?cation of Bim’s relevance to anoikis will be important to the understanding of this process. The BH3-only protein Bid can regulate death in a novel manner during anoikis.In contrast to earlier re-ports describing Bid cleavage downstream of death re-ceptor and caspase-8activation during anoikis[33,35], intact Bid was found to mediate anoikis in mammary epithelial cells.This function of Bid did not require the death receptor-signaling pathway.However,the studies of RIP and DAP-3support the importance of the death receptor pathway and its components in anoikis.The differences in these studies may again stem from the

436Reddig and Juliano

use of different cell lines.This could point to impor-tant cell type differences in the regulation of anoikis. The general applicability of either model awaits further investigation.

The acquired ability to survive independently of ad-hesion is an important hurdle for cellular transforma-tion to a tumorigenic phenotype.The suppression of anoikis after the ecotopic expression of the oncogenic TrkB nicely illustrates this concept.Additionally,adhe-sion status also affects cellular responsiveness to toxic insult.This effect appears to be complex with adhesion of some anchorage independent cells types increas-ing their sensitivity to DNA damaging agents,while the incorporation of cells,irregardless of their malig-nancy status,into three dimensional organoids imparts resistance to toxic insult.The understanding of the dif-ferential response to these agents in relation to adhe-sion status will have important implication for cancer therapy.

Acknowledgments

This work was supported by NIH grants to RL Juliano. References

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