CD98 at the crossroads of adaptive immunityand cancer

Joseph M. Cantor1 and Mark H. Ginsberg1,*Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA

*Author for correspondence (mhginsberg@ucsd.edu)

Journal of Cell Science 125, 1373–1382� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.096040

SummaryAdaptive immunity, a vertebrate specialization, adds memory and exquisite specificity to the basic innate immune responses present in

invertebrates while conserving metabolic resources. In adaptive immunity, antigenic challenge requires extremely rapid proliferation ofrare antigen-specific lymphocytes to produce large, clonally expanded effector populations that neutralize pathogens. Rapid proliferationand resulting clonal expansion are dependent on CD98, a protein whose well-conserved orthologs appear restricted to vertebrates. Thus,

CD98 supports lymphocyte clonal expansion to enable protective adaptive immunity, an advantage that could account for the presenceof CD98 in vertebrates. CD98 supports lymphocyte clonal expansion by amplifying integrin signals that enable proliferation and preventapoptosis. These integrin-dependent signals can also provoke cancer development and invasion, anchorage-independence and the rapidproliferation of tumor cells. CD98 is highly expressed in many cancers and contributes to formation of tumors in experimental models.

Strikingly, vertebrates, which possess highly conserved CD98 proteins, CD98-binding integrins and adaptive immunity, also displaypropensity towards invasive and metastatic tumors. In this Commentary, we review the roles of CD98 in lymphocyte biology and cancer.We suggest that the CD98 amplification of integrin signaling in adaptive immunity provides survival benefits to vertebrates, which, in

turn, bear the price of increased susceptibility to cancer.

Key words: CD98, Cell adhesion, Cell proliferation, Immunity, Integrin

IntroductionThe crucial role of clonal expansion in vertebrate

adaptive immunity

All multicellular eukaryotic organisms initiate immune responses

after recognizing common pathogen-associated molecular

patterns (PAMPs) on the surface of invading microbes

(Medzhitov and Janeway, 1997). Pattern recognition receptors

(PRRs), including toll-like receptors (TLRs), nucleotide

oligomerization domain (NOD)-like receptors (NLRs) and

retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs;

RIG-I is also known as DDX58), generate signals that activate

‘innate’ immune cells (Kumar et al., 2011) to neutralize or

destroy viruses and bacteria either through reactive chemicals

[reactive oxygen species (ROS), reactive nitrogen species (RNS)

etc.], complement or enzymatic digestion (lysozyme, etc.) (Tosi,

2005; Leclerc and Reichhart, 2004). This process must be

repeated every time the organism is exposed to the same

pathogen. However, many pathogens that infect higher organisms

can overcome innate immunity and thus maintain infections that

result in serious damage to the host.

Vertebrates utilize an additional powerful weapon against

pathogens, namely, adaptive immunity. Adaptive immune

responses provide long-lasting, flexible and specific protection

to a wider variety of pathogens than innate immunity alone

(Boehm, 2011). Lymphocytes or lymphocyte-like cells are the

central adaptive immune cell. They each express unique antigen

receptors of a single specificity on their surface. As they develop

from lymphoid progenitor cells in the bone marrow, lymphocytes

randomly rearrange gene segments that encode three antigen

receptor components (V, D and J) through the action of

recombination-activating gene (RAG) enzymes (Hsu, 2009).

This recombination mechanism generates tremendous diversity

(Kuby, 1997) because there are many alleles for each V, D or J

gene segment [or variable lymphocyte receptor (VLR)-A and

VLR-B gene regions (Saha et al., 2010; Herrin and Cooper, 2010)

in the case of agnathans (jawless fish), discussed below], with

random nucleotide addition by terminal deoxynucleotidyl

transferase (TdT) also adding junctional diversity.

Possessing such a large repertoire of randomly generated

antigen specificities has obvious benefits (Murphy et al., 2011).

First, the greater diversity of antigen receptors enables the

neutralization of a correspondingly larger diversity of pathogens

and makes evasion of their neutralizing effects more challenging

for pathogens. Second, greater diversity combined with efficient

deletion of self-reactive cells (Fig. 1) enables more specific

targeting of pathogens rather than host tissues. Third, increased

specificity combined with expansion of pathogen-specific

lymphocytes allows for formation of specific recall or

‘memory’ responses (Sprent and Surh, 2002). Maintaining a

few memory cells that rapidly expand in response to previously

experienced antigen conserves resources that would otherwise be

expended in maintaining a large population of cells that

encompass the entire repertoire of recognition specificities

(Murphy et al., 2011). The stimulation of this memory

mechanism by vaccines underlies the protection that has saved

millions of lives. For these reasons, the vertebrate immune

system is usually viewed as more complex and potent than that of

invertebrates with respect to diversity, specificity and memory

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(Boehm, 2011). These benefits depend on the rapid proliferation

of lymphocytes that results in clonal expansion, which thus

functions as a keystone of adaptive immunity (Fig. 1), allowing

vertebrates to take advantage of the striking specificity and

diversity of adaptive immunity, while conserving metabolic

resources.

Clonal expansion requires resting lymphocytes to enter the cell

cycle rapidly upon receiving the appropriate activation signals

from antigen receptors, co-receptors, co-stimulatory molecules and

cytokines. Extremely rapid cellular proliferation must then follow

(with doubling times of less than 5 hours) in order to differentiate

and generate sufficient effector cells in time (typically in less 5

days) (Abbas, 2003). For B cells, this rapid proliferation phase

can also occur several times, with accompanying somatic

hypermutation of receptor genes to increase affinity for antigen,

and thus can last up to 3 weeks (Murphy et al., 2011). Both B and T

cell clonal expansion rely on the capacity of lymphocytes to

accelerate from a resting state to sustained rapid proliferation.

Several molecules and mechanisms assist in this process, including

growth cytokines or receptors (Nelson and Willerford, 1998;

Weaver et al., 2007), adhesion molecules (Mitchell and Williams,

2010), anti-apoptotic proteins (Hildeman et al., 2007) and the

vertebrate-specific transmembrane protein CD98 (Cantor et al.,

2009; Cantor et al., 2011).

CD98: more than a lymphocyte activation marker

The CD98 heterodimer consists of a type II single-pass

transmembrane heavy chain (CD98hc, also known as 4F2

Lymphoid progenitor cell

Memory B cell

IgG

IgA

IgM

Diverse resting B cells

Self-reactive B cellsare destroyed

Pathogens

Free virusExtracellular bacteriaParasites

Virus-infected cellsTumor cellsIntracellular bacteria

Thymus

Bone marrow

Diverseresting T cells

MemoryCD4+ T cell

‘Helper’ T cells

Self-reactive T cells are destroyed

CD4+

CD8+ Cytotoxic(CTL)

‘killer’ T cells

Antigen-presenting cell (APC)

Cytokine ‘help’ for B cells

Cytokine ‘help’ for CTL s

‘‘k

CCyyyyyyyyyyyyytttttttokkooookokoo ii

s MemoryCD8+ T cell

B cell antigen receptors

light chain VJ rearrangement

heavy chain VDJ rearrangement

T cell antigen receptors

alpha chain VJ rearrangement

beta chain VDJ rearrangement

Clonalexpansion

Spleen, lymph nodes Plasmacells

Infected host cell

Fig. 1. Vertebrate adaptive immunity. Antigen diversity in jawed vertebrates is generated by somatic recombination of the V, D and J regions (variable,

diversity and joining immunoglobulin gene regions) in both immunoglobulin (Ig, B-cell receptor) and TCR (T-cell receptor) chains. After their development in

bone marrow (and thymus for T cells), the clones that express self-reactive antigen receptors are deleted. Very few mature resting B and T cells are maintained for

each specific antigen (indicated with different colors), thus conserving valuable metabolic resources. Upon antigen exposure in the periphery, the appropriate

lymphocyte clone(s) are expanded quickly through rapid proliferation to generate large numbers of antigen-specific effector cells (clonal expansion, illustrated by

the green triangles). Effector B cells secrete soluble receptors (antibodies, i.e. IgG, IgA and IgM) that can neutralize or eliminate extracellular pathogens.

Cytotoxic (CD8+) T cells scan host cells for the presence of virus or intracellular bacteria, killing any cells that are infected. Helper (CD4+) T cells secrete

cytokines that boost both cytotoxic T cells and effector B cells for efficient class-switched antibody responses. Most effector cells die after pathogen clearance,

leaving only small numbers of memory B and T cells that are poised for a more rapid clonal expansion upon a secondary exposure (illustrated by the pink

triangles). Thus, adaptive immunity utilizes clonal expansion to provide long-lasting specific protection against a wide variety of pathogens while minimizing

nutrient cost to the host. IgG, immunoglobulin c; IgA, immunoglobulin a; IgM, immunoglobulin m.

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antigen heavy chain or FRP-1; encoded by the genes SLC3A2 and

Slc3a2 for human and mouse, respectively) of ,80–85 kDa that

is disulfide-linked with a multi-pass light chain of ,40 kDa

(Deves and Boyd, 2000) (Fig. 2). Well-conserved orthologues of

CD98 are expressed in both jawed and jawless vertebrates but not

in invertebrates (Uinuk-Ool et al., 2002). Heterodimeric amino

acid transporters containing a single-pass transmembrane heavy

chain have been found in Drosophila and in schistosomes, but

these do not appear to have all the functions of CD98 described

below (Krautz-Peterson et al., 2007; Reynolds et al., 2009).

CD98hc was discovered in 1981 when anti-leukocyte monoclonal

antibodies were prepared under the theory that ‘‘lymphocyte

differentiation antigens exist and reflect various

immunoregulatory functions’’ (Haynes et al., 1981b). The 4F2

monoclonal antibody (mAb) bound to all monocytes, weakly to

resting lymphocytes, but strongly to activated human B and T

cells (Haynes et al., 1981b), and the 4F2 antigen comprises an

,80–85-kDa heavy chain and a ,40-kDa light chain (Hemler

and Strominger, 1982). Mouse leukocytes express a similar

protein (Bron et al., 1986; Quackenbush et al., 1986), and the

human and mouse 4F2 antigens were given the systematic

CD designation CD98, with 4F2 mAb recognizing CD98hc

(Gottesdiener et al., 1988; Lumadue et al., 1987; Quackenbush et

al., 1987). Following its discovery, CD98 was used as an

activation marker for both B and T cells during normal and

disease states (Hafler et al., 1985; Kehrl et al., 1984; Konttinen

et al., 1985; Moretta et al., 1981; Patterson et al., 1984). CD98hc

was also designated ‘fusion regulatory protein’ (FRP-1) to reflect

its function in cell fusion events that lead to multinucleated giant

cells such as osteoclasts or in viral-induced syncitia (Mori et al.,

2001; Mori et al., 2004; Ohgimoto et al., 1996; Suga et al., 1997;Tsurudome and Ito, 2000).

CD98 has two known biochemical functions (Fenczik et al.,2001). The heavy chain binds to cytoplasmic tails of integrin-bchains (Miyamoto et al., 2003; Prager et al., 2007; Zent et al.,2000) and mediates adhesive signals that control cell spreading,survival and growth (Fenczik et al., 1997; Feral et al., 2005;

Rintoul et al., 2002; Zent et al., 2000). The CD98 light chain canbe any one of six permease-type amino acid transporters and isbound to CD98hc by disulfide bond. The light chain functions inamino acid transport (Deves and Boyd, 2000; Verrey, 2003);

some of the light chains have broad specificity, but the largeneutral amino acid transporters LAT-1 and LAT-2 (encoded bySLC7A6 and SLC7A8), which are the best-studied, have

preference for importing certain essential amino acids,particularly leucine, isoleucine and arginine (LAT-1), inexchange for glutamine (Bertran et al., 1992; Palacin, 1994;

Pineda et al., 1999; Torrents et al., 1998; Verrey, 2003; Verreyet al., 2000). Indeed, through this nutrient function, CD98 cancontribute to the survival and growth of many cell types (Cho

et al., 2004; Reynolds et al., 2007). Importantly, surfaceexpression of the light chain is dependent on the presence ofthe heavy chain, whereas the isolated heavy chain can beexpressed without light chains (Cantor et al., 2009; Cantor et al.,

2011; Mastroberardino et al., 1998; Teixeira et al., 1987). Thus,CD98hc functions in amplifying integrin signaling and in thetransport of amino acids; both of these functions can contribute to

cell survival and proliferation. This Commentary will summarizehow CD98 enables vertebrate adaptive immunity through supportof clonal expansion; however, CD98 also contributes to

pathological clonal expansion in the form of cancer.

CD98 in adaptive immunityDiscovery of CD98 in immune cells

Although CD98 was first identified in lymphocytes (Haynes et al.,1981b), its function in immunity has only recently beenestablished. Early studies using antibody blockade and/or CD98

crosslinking suggested that CD98 could function in B and T cellactivation, proliferation or effector functions (Diaz et al., 1997;Freidman et al., 1994; Gerrard et al., 1984; Komada et al., 2006;

Nakao et al., 1993; Spagnoli et al., 1991; Warren et al., 1996),perhaps by acting as a type of co-stimulatory receptor. However,depending on the conditions used, the effects on proliferation

varied (Gerrard et al., 1984). The role of CD98 in integrinsignaling, cell survival and proliferation in non-lymphoid cells(Fenczik et al., 1997; Feral et al., 2005; Rintoul et al., 2002; Zent

et al., 2000), and in amino acid transport (Bertran et al., 1992;Palacin, 1994; Pineda et al., 1999; Torrents et al., 1998; Verrey,2003; Verrey et al., 2000), provoked studies that examined thebiochemistry and functions of CD98 in lymphocytes upon

selective deletion of the Slc3a2 gene followed by reconstitutionwith mutants that support only one of the biochemical functionsof CD98.

CD98 in T lymphocyte function

To examine the function of CD98hc in adaptive immunity, ourgroup generated a conditional knockout for CD98hc by flankingpart of the Slc3a2 gene with LoxP sites using homologous

recombination in mouse embryonic stem cells (Feral et al., 2007),and bred it with dLck-Cre mice (Zhang et al., 2005), in which Crerecombinase deletes CD98 in the late single-positive stage in

CD98 heavy chain (4F2 Ag, FRP-1)

CD98 light chain(amino acid exchanger)

S-S

N

CN

CD98 heterodimer

CD98hc region required for integrin signaling

C

Fig. 2. Schematic illustration of CD98. The CD98 heavy chain (known as

CH98hc, 4F2 Ag or FRP-1) is encoded by the Slc3a2 gene in mice and

SLC3A2 in humans. CD98hc is a type II transmembrane protein with a large,

heavily glycosylated extracellular domain, and a short transmembrane domain

and cytoplasmic tail. The extracellular, or ecto-, domain was crystallized, and

a ribbon representation of the structure is shown (Fort et al., 2007). The CD98

heterodimer is formed by disulfide bonds between the membrane-proximal

section of the CD98hc extracellular domain and any one of at least six

possible CD98 light chains (the amino acid transporters LAT-1 or LAT-2,

etc.). The integrin signaling function of CD98hc is dependent on the

transmembrane and cytoplasmic domains. CD98 is an unusual protein in that

it combines adhesive signaling and amino acid transport functions.

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both CD4+ and CD8+ T cell lineages (Cantor et al., 2011).CD98hc-null T cells populate secondary lymphoid tissue in

normal numbers, form immunological synapses with antigen-presenting cells (APC) normally and are activated by antigen toexpress activation markers. These cells have moderately impairedhomeostatic proliferation in vivo but exhibit markedly defective

polyclonal and monoclonal antigen receptor-driven proliferationboth in vitro and in vivo (Cantor et al., 2011). Effector functionssuch as cytokine secretion and cytotoxic target lysis appear to be

normal. These data establish a crucial role for CD98hc in theclonal expansion stage of T cell immune responses and show thatCD98hc is required for T-cell-dependent adaptive immunity.

Given that clonally expanding T cells would have increasedmetabolic demands, one might expect amino acid transport to bethe crucial function of CD98 in T cells. However, reconstitutionof CD98hc-null T cells with CD98 mutants shows that the portion

of CD98hc required for amino acid transport is not absolutelyrequired, but that the region that is involved in integrin signalingis crucial for T cell proliferation (Cantor et al., 2011). The

importance of CD98hc in T cell immune responses is also seen inautoimmune disease. Here, loss of T cell CD98 prevents diseasedevelopment in two models of T-cell-mediated autoimmunity,

type 1 diabetes (T1D) and multiple sclerosis (Cantor et al., 2011).This raises the possibility that CD98hc could serve as atherapeutic target for blocking inappropriate adaptive immune

responses.

On the basis of these recent observations, several questions cannow be raised regarding CD98hc function in T cells, such as whatare the effects an antigen-activated CD98-null T cell might have

on the overall immune response? Cytokine secretion and thecapacity to differentiate to regulatory-type Foxp3+ T cells arestill intact in the absence of CD98hc (Cantor et al., 2011). The

ability of a regulatory T cell to suppress inflammatory T cellresponses might be less dependent on clonal expansion than theeffector functions of an inflammatory subset (Gavin and

Rudensky, 2002; Takahashi et al., 1998; Taams et al., 1998).Thus, there is the potential to modulate the quality of an immuneresponse through interfering with CD98hc function. The secondquestion is how partial or transient loss of CD98hc might differ

from its complete deletion. Another unresolved issue is whetherT cells require CD98hc for secondary responses to the samepathogen. In other words, what is the function of CD98hc in

memory CD4 or CD8 T cells? This might have implications indesigning intervention strategies that target CD98hc.

CD98 in B lymphocyte function

Until recently, only one study had examined CD98 function in Bcells using inhibition with the 4F2 antibody, with confusingresults that included both inhibition and enhancement of B cell

functions (Gerrard et al., 1984). To address this issue, our groupgenetically deleted CD98hc in B cells using CD19-Cre (Cantoret al., 2009). Similar to the results in T cells, CD98hc is not

required for B cell compartmentalization or their initialactivation, but is crucial for B cell clonal expansion (Cantoret al., 2009). CD98-null B cells display strikingly reduced

proliferation after stimulation with strong mitogens, and thus areunable to differentiate into plasma cells. Consequently, micelacking CD98hc in B cells have strongly impaired antibody

responses following immunization, and class-switching to IgG, ahallmark of mature antibody responses, is dramatically impaired.Because CD98hc-null B cells lack antigen-driven proliferation,

the direct role of CD98 in B cell effector function (e.g. antibodysecretion) cannot be examined directly. The apparently differing

degrees by which T and B cells depend on CD98hc remainpuzzling. One possibility is that the intensely competitive natureof B cell clonal expansion in germinal centers (Allen et al., 2007;Pritchard-Briscoe et al., 1977) renders CD98-null B cell unable to

compete for available anchorage or nutrients within theenvironment of a germinal center. Nevertheless, taken together,these studies demonstrate that CD98hc is required for both

humoral and cell-mediated adaptive immunity because it isessential for clonal expansion (Fig. 3).

Mechanism of action for CD98 in lymphocyte proliferation

Although the cellular role of CD98hc in lymphocytes has becomefairly clear, the biochemical mechanism by which this proteindrives clonal expansion is not fully understood. There are at least

three indications that the integrin signaling function of CD98hcis required for rapid lymphocyte proliferation. First, CD98hcmutants that exclusively support integrin signaling can

reconstitute proliferation, whereas those that only supportamino acid transport do not. Second, CD98-null B cells showdefective integrin functions in that they are unable to adhere

firmly and spread on anti-aLb2-integrin-coated solid surfaces.More importantly, mitogen-induced early (,30 min) mitogen-activated protein kinase (MAPK) signaling is intact in CD98-nullB cells as indicated by a normal initial wave of extracellular-

signal-regulated kinase (ERK1/2) phosphorylation. However,loss of CD98hc abrogates sustained secondary phosphorylationof ERK and downregulation of cyclin-dependent kinase (CDK)

inhibitors. This loss of sustained ERK activation anddownregulation of CDK inhibitors resembles the effects of theloss of integrin signals that support growth factor stimulation of

fibroblasts (Assoian and Schwartz, 2001; Schwartz and Assoian,2001; Walker and Assoian, 2005). These data raise the questionof which integrin ligands support B and T cell proliferation. One

possibility is that there is a ligand-independent function ofintegrins in sustaining ERK signaling, which supports cell cycleprogression. Another possibility is that CD98hc-dependentintegrins recognize cell-associated ligands during cell–cell

interactions (Nguyen et al., 2008) that occur during lymphocytelocalization and synapse formation within lymph nodes. Whereasmorphological synapse formation appears grossly normal in

CD98-null T cells in vitro, it is possible that the quality andduration of contacts is defective in vivo, perhaps preventing Tcell polarization (Chang et al., 2007). Additional hints that

CD98hc functions in cell–cell contacts comes from itsdocumented roles in cell fusion, and in monocyte or B cellaggregation (Cho et al., 2001; Cho et al., 2004; Mori et al., 2001;

Mori et al., 2004; Ohgimoto et al., 1996; Suga et al., 1997;Tsurudome and Ito, 2000). Further study is needed to understandthe details of how CD98hc and integrins might cooperate tosupport lymphocyte proliferation and adaptive immunity.

Given that highly conserved CD98 orthologs are present onlyin vertebrates (Prager et al., 2007; Uinuk-Ool et al., 2002) and arerequired for adaptive immunity, they should be found in all

adaptive immune systems. Jawless fish (agnathans), which havean alternative adaptive immune system with a unique set of genesthat form VLRs (Herrin and Cooper, 2010; Pancer et al., 2005)

still rely on clonal expansion of lymphocyte-like cells (Mariuzzaet al., 2010) and possess an unambiguous CD98hc orthologue(Uinuk-Ool et al., 2002). This fact adds additional compelling

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evidence for the absolute requirement of CD98hc for the abilityof lymphocytes to mount adaptive immune responses.

CD98 and invasive cancers: the turbochargergone awryCD98 expression in tumors

The capacity for rapid proliferation, especially without firmanchorage, can be dangerous in malignant cells, and a large body

of evidence implicates CD98 in cancer (Table 1). Many tumorsexpress CD98hc, and its expression correlates with poor

prognosis in B cell lymphomas (Holte et al., 1987; Holte et al.,1989; Salter et al., 1989). Furthermore, nearly all studies that

have examined the expression of CD98hc or CD98 light chains insolid tumors show that their expression is correlated with

progressive or metastatic tumors (Esteban et al., 1990; Kairaet al., 2009a; Kaira et al., 2009b; Kaira et al., 2009c; Kaira et al.,

2008; Kaira et al., 2009d; Kobayashi et al., 2008; Nawashiroet al., 2002; Nawashiro et al., 2006; Oleinik et al., 2005;

Powlesland et al., 2009; Shennan et al., 2003; Xiao et al., 2005).

CD98 depletion and overexpression studies in cancer

Genetic modulation of CD98 expression in human cell lines

and in animal models has established a causal link between CD98and cancer; CD98 promotes transformation and tumor growth

(Feral et al., 2005; Ohkawa et al., 2011). Furthermore, CD98overexpression drives both anchorage independence and

tumorigenesis (Nguyen et al., 2011; Hara et al., 1999;Henderson et al., 2004; Shishido et al., 2000), and the degree

of transformation correlates with the level of CD98hc present inthe cells (Hara et al., 1999). An early report showed that the

transforming ability of CD98hc is lost in a mutant that ablates theability to form disulfide bonds with the light chain and thus

abolishes its amino acid transport function (Shishido et al., 2000).However, this mutation also affects the ability of CD98hc to bind

integrins (Kolesnikova et al., 2001), making this result difficult to

interpret. Moreover, a later study using CD98hc chimeras, in

which its amino acid transport is separated from its integrinsignaling function (Fenczik et al., 2001), has shown that the

CD98 domain that is involved in integrin signaling is required for

transforming Chinese hamster ovary (CHO) cells (Henderson

et al., 2004) and for the development of embryonic stem cellteratomas in mice (Feral et al. 2005). Taken together, these

genetic studies show that CD98hc expression is important, and

that its overexpression is sufficient for cellular transformation.

CD98 blockade in tumors

The third line of evidence for the importance of CD98 in cancer

comes from a number of studies that used CD98 inhibitors in avariety of tumor cells; these have resulted in the inhibition of

cellular proliferation and tumor growth both in vitro and in vivo

(Imai et al., 2010; Nawashiro et al., 2006; Oda et al., 2010;Shennan and Thomson, 2008; Yamauchi et al., 2009). One

particular study used anti-CD98 antibodies specific for the heavy

chain and showed that the growth of human tumor cells is

inhibited in vitro (Yagita et al., 1986). Overall, these studiesindicate that CD98 might constitute a target for therapeutic

intervention in cancer. In fact, a microRNA that modulates CD98

expression during intestinal epithelial differentiation was recently

identified, raising the possibility of an additional approach toalter CD98 expression (Nguyen et al., 2010).

CD98 amino acid transport in cancer

The mechanism by which CD98 promotes tumorigenesis is an

important area of current research. Significant questions remain

Selectiveexpansion ofAg-specific

clone

2 Proliferation

3 Differentiation/effector function

Targetcell

Celldeath

Antigen

1 Activation

APC

Cytokines

CD98requirement

4 Contraction/memory formation

(> 30 days) Expansion ofmemory cells

5 Secondary (memory)response

CD98?

CD4+CD8+

Early MAPKsignals

SecondaryMAPK signaling

Fig. 3. The role of CD98hc in cell-mediated adaptive immunity. Upon immune challenge, a rare antigen-specific naive T cell (shown in blue) is activated by

peptide antigen (Ag) on an antigen-presenting cell (APC) and rapidly proliferates to expand into a large number of antigen-specific effector T cells. After

expansion and differentiation, effector CD4+ ‘helper’ cells secrete cytokines to direct the adaptive immune response, and CD8+ cytotoxic ‘killer’ T cells kill

infected target cells. After pathogen clearance, effector T cells die to leave only a few ‘memory’ T cells that are primed for a rapid secondary response. Following

secondary exposure to the same antigen, memory T cells re-expand to eliminate the pathogen. CD98hc is required for rapid clonal expansion, but appears

dispensable for antigen recognition, activation and effector functions. Whether or not CD98hc is necessary for secondary expansion is an open question.

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unanswered, such as whether CD98hc allows tumor growth by

boosting the transport of nutrients or by augmenting integrin

signaling, and if so, how would each of these roles be fulfilled. The

rapidly dividing tumor cell has intense metabolic demands, often

under conditions of diminishing nutrient availability within a solid

tumor. CD98hc could therefore support tumor growth through its

association with the amino acid transporter light chain and the

subsequent increased supply of amino acids. Under conditions of

limited amino acid availability, CD98 light chains such as LAT-1

are upregulated and their expression can be dysregulated in tumor

cells (Campbell et al., 2000). LAT-1 and LAT-2 are amino acid

exchangers and can import essential amino acids (EAA), such as

leucine, isoleucine and arginine in exchange for the export of

glutamine that has been imported by other transporters such as

through system A transporters (Verrey, 2003). Furthermore, this

CD98-mediated exchange of glutamine in the presence of EAAs is

a rate-limiting step in the activation of mammalian target of

rapamycin (mTOR) (Nicklin et al., 2009), which regulates cell

growth and proliferation, providing another pathway through

which CD98 could stimulate tumor growth. Cancer cells are

heavily dependent on glutamine, not merely as an ingredient for

nucleotide and amino acid biosynthesis, but also to promote

increased uptake of EAAs through this exchange mechanism

(Wise and Thompson, 2010). Interestingly, T lymphocytes are

another cell type that is particularly dependent on glutamine (Carr

et al., 2010). Thus, it appears probable that high expression levels

of CD98hc could allow for rapid proliferation of lymphocytes and

tumors by stimulating the exchange of glutamine for limiting

EAAs (Fig. 4, left-hand panel).

CD98 integrin signaling function in cancer

CD98hc can also contribute to transformation by amplifying

integrin signaling that results in reduced anchorage dependence.

Integrins themselves have been implicated in invasive cancers

because the ‘outside-in’ signals they transduce control cell

proliferation and survival (Huck et al., 2010; Lahlou et al., 2007;

Desgrosellier and Cheresh, 2010; Harburger and Calderwood,

2009). Integrins act as mechanotransducers that sense the

stiffness of the extracellular matrix (ECM). Increased ECM

stiffness drives tumor progression through clustering of integrins,

which leads to adhesive signaling through focal adhesion kinase

(FAK) and phosphatidylinositol 3-kinase (PI3K) (Levental et al.,

2009). These signals can also direct the proliferation of micro-

metastases in the lungs through b1 integrin (Shibue and

Weinberg, 2009). In a softer matrix, such as one with larger

amounts of flexible ECM components (elastin, etc.), high

expression of CD98hc could permit integrin signaling that

would promote cell growth and survival. Thus, the expression

level of CD98hc would fine-tune the capacity of the cell to sense

the compliance of the matrix. Indeed, CD98hc is important in

Table 1. Studies examining the role of CD98hc in cancers

Cell type Observations Reference(s)

Cutaneous T cell lymphoma (CTCL) All malignant CTCL T cells are CD98+; fewnormal T cells are

(Haynes et al., 1981a)

Bladder cancer cells (transitional epithelium) Anti-CD98 antibody treatment inhibits proliferationof human tumor cells

(Yagita et al., 1986)

B cell lymphomas CD98 expression on lymphomas can identify patientswith poor prognosis

(Holte et al., 1987; Holte et al., 1989; Salteret al., 1989; Powlesland et al., 2011)

Oral squamous cell carcinoma CD98 histological expression pattern within a tumoris a significant indicator of progression and metastasis

(Esteban et al., 1990; Kim et al., 2004)

Childhood acute lymphoblastic leukemia CD98 expression level correlates with durationof complete remission

(Taskov et al., 1996)

(Mouse) fibroblast (3T3 cell line) CD98 overexpression leads to transformation (Hara et al., 1999; Henderson et al., 2004;Shishido et al., 2000)

Astrocytic neoplasms CD98 light chain (LAT-1) expression is increasedin astrocytic cancers

(Nawashiro et al., 2002)

Human glioblastoma(and rat C6 glioma model)

High LAT1 expression correlates with poor survival;LAT1 inhibition decreases glioma tumor cell growth

(Nawashiro et al., 2006)

Breast cancer cell lines LAT-1 inhibitors suppress tumor cell growth andmetabolism

(Shennan and Thomson, 2008; Shennan et al.,2003)

(Mouse) Embryonic stem cell teratomas Genetic deletion of CD98hc blocks tumor growth (Feral et al., 2005)

Blood proteins during lung cancer High soluble CD98 and two other blood proteins maybe a tumor biomarker

(Xiao et al., 2005)

Breast tissue RNA Expression of CD98-encoding mRNAs correlateswith poor prognosis

(Esseghir et al., 2006)

Multiple CD98hc and LAT-1 expression is higher in metastaticthan primary tumor sites

(Kaira et al., 2008)

(Mouse) Glioma cells Overexpression of CD98 light chain LAT-1enhances tumor growth rate in vivo

(Kobayashi et al., 2008)

Pulmonary tumors CD98 expression predicts poor prognosis (Kaira et al., 2009a; Kaira et al., 2009b; Kairaet al., 2009c)

Renal cell cancer CD98 histological expression correlates directlywith malignancy grade

(Prager et al., 2009)

Squamous cell carcinoma lines CD98 light chain LAT-1 inhibitor improves efficacyof cisplatin treatment

(Yamauchi et al., 2009)

Non-small cell lung cancer LAT-1 inhibitor BCH reduces viability of tumor cellsex vivo

(Imai et al., 2010)

Chicken cell line and HeLa cells Genetic disruption or inhibition of LAT-1 blockstumor cell growth in vitro and in vivo

(Ohkawa et al., 2011)

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allowing a cell to exert force on the ECM through integrin-driven

RhoA signaling (Feral et al., 2007), which is a key element in the

sensing of stiffness (Samuel et al., 2011).

Anchorage independence (i.e. independence of integrin signals

provided by adhesion to ECM) is another hallmark of cellular

transformation, and CD98hc is important for anchorage

independence (Feral et al., 2005). CD98hc co-immunoprecipitates

with integrins (Henderson et al., 2004; Rintoul et al., 2002), and

binds to the b1 and b3 integrin cytoplasmic domains in a purified

system (Prager et al., 2007). Overexpression or crosslinking of

CD98hc stimulates FAK and AKT phosphorylation (Cai et al.,

2005; Rintoul et al., 2002) (Fenczik et al., 1997), and deletion of

CD98hc impairs integrin signaling (Feral et al., 2005). This integrin

signaling function of CD98hc is dependent on the transmembrane

and cytoplasmic domains (Fenczik et al., 2001), the same region that

is crucial for lymphocyte proliferation (Cantor et al., 2009; Cantor

et al., 2011) and for cellular transformation caused by overexpression

of CD98hc (Henderson et al., 2004). CD98hc can thus regulate

integrin signaling, and integrins are crucial for tumor progression

through mechanotransduction and control of anchorage-independent

growth. Therefore CD98hc might control tumorigenesis by

governing integrin signaling (Fig. 4, right-hand panel).

CD98hc might also contribute to other pathologies. For

example, in addition to autoimmunity disorders (Cantor et al.,

2011), CD98hc overexpression could lead to other adaptive

immunopathologies. Arterial restenosis and intestinal villous

inflammation are dependent on CD98hc in animal models

(Fogelstrand et al., 2009; Nguyen et al., 2011). Vertebrates

have stratified squamous epithelium containing rapidly-dividing

keratinocytes, which express high levels of CD98hc (Fernandez-

Herrera et al., 1989; Lemaitre et al., 2005; Lemaitre et al., 2011;

Patterson et al., 1984). The role of CD98hc in pathologic states in

these tissues requires further study.

ConclusionsHere, we have summarized the central importance of rapid

proliferation for clonal expansion in the vertebrate-specific

adaptive immune system. CD98hc enables lymphocyte clonal

expansion and adaptive immune responses, probably in part

through its effects on integrin signaling. CD98hc is also

important in cancer, which is probably mediated through

increased transport of amino acids and/or by boosting integrin

signals that allow growth in soft ECM or without anchorage.

Importantly, clonal expansion is the nexus at which CD98 acts in

adaptive immunity, and clonal expansion of tumor cells is also a

central feature of many cancers. CD98hc and the integrins that

bind to it are vertebrate-specific, as is adaptive immunity, and it

is noteworthy that vertebrates appear to be particularly prone to

developing invasive and metastatic cancers. This confluence of

adaptive immunity, and the expression of CD98hc and CD98-

binding integrins (Prager et al., 2007), coincident with increased

invasive cancer, in vertebrates, suggests that CD98hc provides

vertebrates with the benefit of adaptive immunity, but that this

comes with a price: increased susceptibility to invasive cancers.

CD98 amino acid transport in cancer CD98 integrin signaling in cancer

S-S

CD98hc

CD98 light chain (LAT-1, LAT-2, etc.)

AKTp130CAS

FAK

� Survival� Anchorage-independence

� Metastasis

mTORactivation

� Survival� Growth

Essentialamino acids

Glutamine

Glutamine

Unidirectionaltransporter

Autophagy

S-S

Integrin

CD98hc

CD98 light chain (LAT-1, LAT-2, etc.)

Leucine

Fig. 4. Roles of CD98 in cancer. CD98 has two main biochemical functions, amino acid transport and integrin signaling; both appear important in tumor growth

and metastasis. Left panel: CD98 amino acid transport in cancer. LAT-1 and LAT-2, the most commonly studied CD98 light chains, are system L bi-directional

transporters of large neutral amino acids. Unidirectional transporters (such as system A or N transporters) import the non-essential amino acid glutamine into a

rapidly growing cell. LAT-1 or LAT-2 can then export glutamine in exchange for importing EAAs. EAAs then activate the mTOR pathway, blocking

autophagy and promoting cell growth and protein synthesis. Right panel: CD98 integrin signaling in cancer. CD98hc interacts with integrins and mediates

integrin-dependent signals that promote tumorigenesis. CD98-dependent integrin signaling through proteins such as AKT, p130CAS (also known as BCAR1) and

focal adhesion kinase (FAK) allow tumor cell survival and proliferation without anchorage.

CD98, cancer and immunity 1379

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One could speculate that other vertebrate-specific genes that are

involved in clonal expansion must exist and are maintained

because of the survival benefits they provide through adaptive

immunity. Such genes might, like those encoding CD98, be

upregulated in activated lymphocytes and could be therapeutic

targets for autoimmunity diseases or cancer. Going forwards, it is

key for this field to define the specific molecular mechanisms by

which CD98 contributes to adaptive immunity and cancer.

Second, the dramatic effects of CD98 loss on tumors and on

lymphocyte clonal expansion demands that we make efforts to

define the therapeutic potential of CD98 inhibition in cancer and

autoimmune diseases.

FundingWork in our laboratories was supported by grants from the NationalInstitutes of Health [grant numbers HL31950, HL 57900, AR 27214,and HL 078784 to M.H.G.]. J.M.C. is supported by the MultipleSclerosis Society and an NIH K01 grant [grant numberK01DK090416]. Deposited in PMC for release after 12 months.

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