Roles of the multifunctional glycoprotein, emmprin (basigin; CD147), in tumour progression

Summary Emmprin (basigin;CD147) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of malignant tumour cells. Emmprin is involved in numerous physiological and pathological systems and exhibits several molecular and cellular characteristics, but a major function of emmprin is stimulation of synthesis of several matrix metalloproteinases. In tumours, emmprin most likely stimulates matrix metalloproteinase production in stromal fibroblasts and endothelial cells as well as in tumour cells themselves by a mechanism involving homophilic interactions between emmprin molecules on apposing cells or on neighbouring cells after membrane vesicle shedding. Membrane-associated cofactors, including caveolin-1 and annexin II, regulate emmprin activity. Emmprin induces angiogenesis via stimulation of VEGF production, invasiveness via stimulation of matrix metalloproteinase production and multidrug resistance via hyaluronan-mediated up-regulation of ErbB2 signaling and cell survival pathway activities. Although the detailed mechanisms whereby it regulates these numerous phenomena are not yet known, it is clear that emmprin is a major mediator of malignant cell behavior.


Introduction
The progression of cells to malignancyi sc haracterizedb y emergence of several properties, includingself-sufficiencywith respect to growth-promotingsignals, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicativep otential, invasiveness, and metastatic potential (1).Acentral creed in cancer research over the past fewd ecades has been thata cquisition of these propertiesistriggered by genetic mutations of oncogenesa nd tumour suppressor genesw ithin developingt umour cells.However,both old and newresearch has also implicated tumour-stroma interactions in each of the critical stepsin cancer progression. Forexample, sustained angiogenesis, which arisesfrom tumour-host interactions, has beenadded to the 'canonical' listo fp ropertiesc haracteristic of developingt umours (1).The stromal compartment of atumour contains avarietyof host cells,i ncluding endothelial cells,fibroblasts, and inflammatoryc ells and it is becoming increasingly appreciated that these host-derivedc ells infiltratei nto tumour tissue, interact with tumour cells,and aresubsequentlyconscripted by tumour cells to produce an arrayo fs oluble and insoluble factorst hat stimulate tumour angiogenesis, growth, and metastasis. Accumulating evidencesuggests aprominent rolefor emmprin in mediating interactions both betweent umour cells themselves and betweent umour cells and "hijacked" host stromal cells to promote anumber of eventsduring cancerprogression. One of the importantand most studied functions of emmprin is its role in induction of matrix metalloproteinase (MMP) production via cell interactions -thus the derivation of its name: extracellular matrix metalloproteinase inducer (2).
(2), it wasrenamed emmprin. On cloning of emmprin cDNA, it becameapparent that emmprin is identicaltohuman basigin and the M6 antigen present on membranes of leukocytes from patients with rheumatoid arthritis. Mouse emmprin (basigin) wasdemonstrated to have the same MMP stimulatoryeffect as its humancounterpart(27).
Similart oo therm emberso ft he Ig superfamily, emmprin forms homo-oligomers in a cis -dependent mannerinthe plasma membrane; the N-terminal Ig-likedomain is necessary and sufficient foroligomerization,probably throughhydrophobic interactions (33). TheMMP-inducing function of emmprin in part involves the moleculeacting as acounter-receptor for itself (30). This homophilicc ounter-receptor binding activity of emmprin requiresthe N-terminal Ig domain, butinthis case interaction is in a trans manner. Inhibition of this homophilic interaction interferes with MMP production and MMP-dependent invasion in tumour cells (30). Emmprin also interactswith integrins a3b1and a6b1 (34),probably via the N-terminal Ig domain (35).Asubset of emmprin molecules, separate from that whichassociateswith integrin, associateswith caveolin-1 in lipidrafts. The secondIg domain is requiredfor lipidraft association. Over-expression of caveolin-1 causes adecreaseinclustering of emmprinonthe cell surfacea nd decreasedi nduction of MMP-1, therebyc ontributing to the onco-suppressive effectsofcaveolin-1(35). Association with caveolin-1 also prevents formation of highlyglycosylated formsofemmprin and consequentlyblocks emmprin aggregation and activity (32). Annexin II alsointeracts with emmprin and is requiredf or its activity in stimulating MMP production (H.G uo et al, submitted for publication). This is of interest in viewofthe finding that at least twoothercellsurface-associated proteases, plasminogen activator (36)and cathepsin B(37), interactwith annexin II suggesting acoordinating function for annexin II in assembling proteasesatthe cell surface. In addition to stimulating production of MMPs, emmprin also binds MMP-1 and retains it at the cell surface, an arrangement that maypromote turnoverofpericellularcollagen (38).

Roleofemmprin in cancer
Regulationoftumour growth, invasionand angiogenesis The pathological consequences of elevatedemmprin expression in tumour growth and invasion were directlydemonstrated using emmprin-overexpressing cancer cells. MDA-MB-436 human breast cancerc ells are normally slow-growing cells when they are implantedi nto nude mice. However, when these cells are transfected with emmprin, theyadopt amore aggressive phenotype, exhibiting both acceleratedgrowthand increased invasiveness (67).MMP-2 and MMP-9 expression wasincreased in the emmprin-enhanced tumours. An ew observation arising out of this and other studies( 30) wast he realization that emmprin stimulates MMP production in tumour cells themselves in addition to stromal cells.This most likelyoccursvia homophilic interactionsbetween emmprin molecules on adjacent cells (30), as described in the previous section. However, it is likelythat MMP production in homotypicc ancer celli nteractions also requires the cytoplasmic domain of emmprin (68). Using MDA-MB-231 humanbreast cancer cellsengineeredtoexpress different levels of emmprin, the potentialrole of increased expression of emmprin in tumours wasfurther elucidated (69).Inboth in vitro and in vivo studiesusing these cells, emmprin wasfound to increase production of VEGFinthe tumour cells. In vivo,increased emmprin expression acceleratedtumour growth, accompaniedbyenhanced tumour angiogenesis partiallyd ue to as ignificant upregulation of VEGF and MMPs in both tumour and stromalcompartments. Co-localization of mouse (i.e.host) emmprin, VEGF and MMP to angiogenic blood vessels suggested direct involvement of these molecules in tumoura ngiogenesis, supporting a newparadigm in whichtumour cell surfaceemmprin plays akey roleinregulating tumour angiogenesis and growth (69).

Emmprin and signal transduction
The abnormallyhigh levels of emmprin in cancer cells have recently beenattributedtodysregulation of EGFR signaling. Amphiregulin, acting through interaction with EGFR, promotes tumour progression through emmprin-induced increases in production of MMPs by fibroblasts and endothelial cells (83).The signaling eventsdownstreamofemmprin interactionsthat result in stimulation of MMP production aren ot yete stablished but MAPK p38 hasbeen implicated in induction of MMP-1production (84)and activation of 5-lipoxygenaseand phospholipase A2 in MMP-2 production (80).I nvestigation of the mechanism wherebyemmprin promotestumour growth in vivo (67) ledto in vitro experiments demonstrating that emmprin inducesanchorage-independent growth (85), ap henomenon that is characteristic of malignant cancer cells and thatreflectsresistancetoanoikis,i.e. apoptosis caused by celldetachment fromextracellular matrix. Further investigation showedt hat emmprin stimulates cell survivalp athway signaling, including phosphorylation of Akt, Erkand FA K. These effects of emmprinwere shown to depend on stimulation of production of hyaluronan, apericellular polysaccharide (85,86). The increase in anti-apoptoticsignaling in turnleads to increased multidrug resistance and is dependent on hyaluronan-induced ErbB2 and cellsurvivalsignaling pathways (86,87). Interestingly, it hasbeen shown thatmultidrug resistant cancer cells express increased amounts of emmprin, and as aconsequence producehigherlevelsofMMP-1, MMP-2, and MMP-9 (22).Although it hasnot yetbeen fullyestablished,the stimulatoryeffectsofemmprin on MMP production and invasivenessmay also be mediatedthrough hyaluronan-induced signaling (88).Inaddition, adisruptive rolefor emmprin in calcium mobilization through G-protein sensitive pathwaysh as been demonstrated in tumour cells, and it wassuggestedthat this effect of emmprinenhances the metastatic potential of hepatoma cells (59).I nt he light of these findings, Metuximab, am urine HAb18 F(ab')2 fragment specific for emmprin (59) (also known as LICARTIN), has beendeveloped in the iodine 131 -labeledform and is currentlybeing testedfor safety and clinicalefficacyinhepatocellularcarcinoma.