CD98 at the Crossroads of Adaptive Immunity and Cancer

Commentary 1373 CD98 at the crossroads of adaptive immunity and cancer

Joseph M. Cantor1 and Mark H. Ginsberg1,* Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA *Author for correspondence ([email protected])

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

Summary Adaptive 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 of rare antigen-specific lymphocytes to produce large, clonally expanded effector populations that neutralize pathogens. Rapid proliferation and 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 presence of CD98 in vertebrates. CD98 supports lymphocyte clonal expansion by amplifying integrin signals that enable proliferation and prevent apoptosis. These integrin-dependent signals can also provoke cancer development and invasion, anchorage-independence and the rapid proliferation 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 display propensity 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

Introduction receptor components (V, D and J) through the action of The crucial role of clonal expansion in vertebrate recombination-activating gene (RAG) enzymes (Hsu, 2009). adaptive immunity This recombination mechanism generates tremendous diversity Journal of Cell Science All multicellular eukaryotic organisms initiate immune responses (Kuby, 1997) because there are many alleles for each V, D or J after recognizing common pathogen-associated molecular gene segment [or variable lymphocyte receptor (VLR)-A and patterns (PAMPs) on the surface of invading microbes VLR-B gene regions (Saha et al., 2010; Herrin and Cooper, 2010) (Medzhitov and Janeway, 1997). Pattern recognition receptors in the case of agnathans (jawless fish), discussed below], with (PRRs), including toll-like receptors (TLRs), nucleotide random nucleotide addition by terminal deoxynucleotidyl oligomerization domain (NOD)-like receptors (NLRs) and transferase (TdT) also adding junctional diversity. retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs; Possessing such a large repertoire of randomly generated RIG-I is also known as DDX58), generate signals that activate antigen specificities has obvious benefits (Murphy et al., 2011). ‘innate’ immune cells (Kumar et al., 2011) to neutralize or First, the greater diversity of antigen receptors enables the destroy viruses and bacteria either through reactive chemicals neutralization of a correspondingly larger diversity of pathogens [reactive oxygen species (ROS), reactive nitrogen species (RNS) and makes evasion of their neutralizing effects more challenging etc.], complement or enzymatic digestion (lysozyme, etc.) (Tosi, for pathogens. Second, greater diversity combined with efficient 2005; Leclerc and Reichhart, 2004). This process must be deletion of self-reactive cells (Fig. 1) enables more specific repeated every time the organism is exposed to the same targeting of pathogens rather than host tissues. Third, increased pathogen. However, many pathogens that infect higher organisms specificity combined with expansion of pathogen-specific can overcome innate immunity and thus maintain infections that lymphocytes allows for formation of specific recall or result in serious damage to the host. ‘memory’ responses (Sprent and Surh, 2002). Maintaining a Vertebrates utilize an additional powerful weapon against few memory cells that rapidly expand in response to previously pathogens, namely, adaptive immunity. Adaptive immune experienced antigen conserves resources that would otherwise be responses provide long-lasting, flexible and specific protection expended in maintaining a large population of cells that to a wider variety of pathogens than innate immunity alone encompass the entire repertoire of recognition specificities (Boehm, 2011). Lymphocytes or lymphocyte-like cells are the (Murphy et al., 2011). The stimulation of this memory central adaptive immune cell. They each express unique antigen mechanism by vaccines underlies the protection that has saved receptors of a single specificity on their surface. As they develop millions of lives. For these reasons, the vertebrate immune from lymphoid progenitor cells in the bone marrow, lymphocytes system is usually viewed as more complex and potent than that of randomly rearrange gene segments that encode three antigen invertebrates with respect to diversity, specificity and memory 1374 Journal of Cell Science 125 (6)

(Boehm, 2011). These benefits depend on the rapid proliferation hypermutation of receptor genes to increase affinity for antigen, of lymphocytes that results in clonal expansion, which thus and thus can last up to 3 weeks (Murphy et al., 2011). Both B and T functions as a keystone of adaptive immunity (Fig. 1), allowing cell clonal expansion rely on the capacity of lymphocytes to vertebrates to take advantage of the striking specificity and accelerate from a resting state to sustained rapid proliferation. diversity of adaptive immunity, while conserving metabolic Several molecules and mechanisms assist in this process, including resources. growth cytokines or receptors (Nelson and Willerford, 1998; Clonal expansion requires resting lymphocytes to enter the cell Weaver et al., 2007), adhesion molecules (Mitchell and Williams, cycle rapidly upon receiving the appropriate activation signals 2010), anti-apoptotic proteins (Hildeman et al., 2007) and the from antigen receptors, co-receptors, co-stimulatory molecules and vertebrate-specific transmembrane protein CD98 (Cantor et al., cytokines. Extremely rapid cellular proliferation must then follow 2009; Cantor et al., 2011). (with doubling times of less than 5 hours) in order to differentiate and generate sufficient effector cells in time (typically in less 5 CD98: more than a lymphocyte activation marker days) (Abbas, 2003). For B cells, this rapid proliferation phase The CD98 heterodimer consists of a type II single-pass can also occur several times, with accompanying somatic transmembrane heavy chain (CD98hc, also known as 4F2

Spleen, lymph nodes Plasma Self-reactive B cells cells are destroyed IgG Memory B cell IgA B cell antigen receptors Diverse resting B cells light chain VJ rearrangement Pathogens IgM heavy chain VDJ rearrangement Free virus Extracellular bacteria Parasites Bone marrow Clonal expansion Virus-infected cells Tumor cells Lymphoid progenitor cell Intracellular bacteria Cytokine ‘help’ for B cells

Journal of Cell Science Antigen-presenting cell T cell antigen receptors (APC) Memory CD4+ T cell alpha chain VJ rearrangement CD4+ beta chain VDJ rearrangement ‘Helper’ T cells

Diverse resting T cellss CytokineCytokoki ‘help’ for CTL

Self-reactive T cells are destroyed CD8+ Cytotoxic (CTL) Thymus ‘killer’‘‘k T cellss Memory CD8+ T cell

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. CD98, cancer and immunity 1375

antigen heavy chain or FRP-1; encoded by the genes SLC3A2 and 2001; Mori et al., 2004; Ohgimoto et al., 1996; Suga et al., 1997; Slc3a2 for human and mouse, respectively) of ,80–85 kDa that Tsurudome and Ito, 2000). is disulfide-linked with a multi-pass light chain of ,40 kDa CD98 has two known biochemical functions (Fenczik et al., (Deves and Boyd, 2000) (Fig. 2). Well-conserved orthologues of 2001). The heavy chain binds to cytoplasmic tails of integrin-b CD98 are expressed in both jawed and jawless vertebrates but not chains (Miyamoto et al., 2003; Prager et al., 2007; Zent et al., in invertebrates (Uinuk-Ool et al., 2002). Heterodimeric amino 2000) and mediates adhesive signals that control cell spreading, acid transporters containing a single-pass transmembrane heavy survival and growth (Fenczik et al., 1997; Feral et al., 2005; chain have been found in Drosophila and in schistosomes, but Rintoul et al., 2002; Zent et al., 2000). The CD98 light chain can these do not appear to have all the functions of CD98 described be any one of six permease-type amino acid transporters and is below (Krautz-Peterson et al., 2007; Reynolds et al., 2009). bound to CD98hc by disulfide bond. The light chain functions in CD98hc was discovered in 1981 when anti-leukocyte monoclonal amino acid transport (Deves and Boyd, 2000; Verrey, 2003); antibodies were prepared under the theory that ‘‘lymphocyte some of the light chains have broad specificity, but the large differentiation antigens exist and reflect various neutral amino acid transporters LAT-1 and LAT-2 (encoded by immunoregulatory functions’’ (Haynes et al., 1981b). The 4F2 SLC7A6 and SLC7A8), which are the best-studied, have monoclonal antibody (mAb) bound to all monocytes, weakly to preference for importing certain essential amino acids, resting lymphocytes, but strongly to activated human B and T particularly leucine, isoleucine and arginine (LAT-1), in cells (Haynes et al., 1981b), and the 4F2 antigen comprises an exchange for glutamine (Bertran et al., 1992; Palacin, 1994; ,80–85-kDa heavy chain and a ,40-kDa light chain (Hemler Pineda et al., 1999; Torrents et al., 1998; Verrey, 2003; Verrey and Strominger, 1982). Mouse leukocytes express a similar et al., 2000). Indeed, through this nutrient function, CD98 can protein (Bron et al., 1986; Quackenbush et al., 1986), and the contribute to the survival and growth of many cell types (Cho human and mouse 4F2 antigens were given the systematic et al., 2004; Reynolds et al., 2007). Importantly, surface CD designation CD98, with 4F2 mAb recognizing CD98hc expression of the light chain is dependent on the presence of (Gottesdiener et al., 1988; Lumadue et al., 1987; Quackenbush et the heavy chain, whereas the isolated heavy chain can be al., 1987). Following its discovery, CD98 was used as an expressed without light chains (Cantor et al., 2009; Cantor et al., 2011; Mastroberardino et al., 1998; Teixeira et al., 1987). Thus, activation marker for both B and T cells during normal and CD98hc functions in amplifying integrin signaling and in the disease states (Hafler et al., 1985; Kehrl et al., 1984; Konttinen transport of amino acids; both of these functions can contribute to et al., 1985; Moretta et al., 1981; Patterson et al., 1984). CD98hc cell survival and proliferation. This Commentary will summarize was also designated ‘fusion regulatory protein’ (FRP-1) to reflect how CD98 enables vertebrate adaptive immunity through support its function in cell fusion events that lead to multinucleated giant of clonal expansion; however, CD98 also contributes to cells such as osteoclasts or in viral-induced syncitia (Mori et al., pathological clonal expansion in the form of cancer.

CD98 heterodimer CD98 in adaptive immunity Discovery of CD98 in immune cells

Journal of Cell Science Although CD98 was first identified in lymphocytes (Haynes et al., 1981b), its function in immunity has only recently been CD98 heavy chain (4F2 Ag, FRP-1) established. Early studies using antibody blockade and/or CD98 crosslinking suggested that CD98 could function in B and T cell activation, 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), C perhaps by acting as a type of co-stimulatory receptor. However,

S-S depending on the conditions used, the effects on proliferation varied (Gerrard et al., 1984). The role of CD98 in integrin

N signaling, 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; CD98hc region required for C N Palacin, 1994; Pineda et al., 1999; Torrents et al., 1998; Verrey, integrin signaling CD98 light chain (amino acid exchanger) 2003; Verrey et al., 2000), provoked studies that examined the biochemistry and functions of CD98 in lymphocytes upon Fig. 2. Schematic illustration of CD98. The CD98 heavy chain (known as selective deletion of the Slc3a2 gene followed by reconstitution CH98hc, 4F2 Ag or FRP-1) is encoded by the Slc3a2 gene in mice and with mutants that support only one of the biochemical functions SLC3A2 in humans. CD98hc is a type II transmembrane protein with a large, of CD98. heavily glycosylated extracellular domain, and a short transmembrane domain and cytoplasmic tail. The extracellular, or ecto-, domain was crystallized, and CD98 in T lymphocyte function a ribbon representation of the structure is shown (Fort et al., 2007). The CD98 To examine the function of CD98hc in adaptive immunity, our heterodimer is formed by disulfide bonds between the membrane-proximal section of the CD98hc extracellular domain and any one of at least six group generated a conditional knockout for CD98hc by flanking possible CD98 light chains (the amino acid transporters LAT-1 or LAT-2, part of the Slc3a2 gene with LoxP sites using homologous etc.). The integrin signaling function of CD98hc is dependent on the recombination in mouse embryonic stem cells (Feral et al., 2007), transmembrane and cytoplasmic domains. CD98 is an unusual protein in that and bred it with dLck-Cre mice (Zhang et al., 2005), in which Cre it combines adhesive signaling and amino acid transport functions. recombinase deletes CD98 in the late single-positive stage in 1376 Journal of Cell Science 125 (6)

both CD4+ and CD8+ T cell lineages (Cantor et al., 2011). the direct role of CD98 in B cell effector function (e.g. antibody CD98hc-null T cells populate secondary lymphoid tissue in secretion) cannot be examined directly. The apparently differing normal numbers, form immunological synapses with antigen- degrees by which T and B cells depend on CD98hc remain presenting cells (APC) normally and are activated by antigen to puzzling. One possibility is that the intensely competitive nature express activation markers. These cells have moderately impaired of B cell clonal expansion in germinal centers (Allen et al., 2007; homeostatic proliferation in vivo but exhibit markedly defective Pritchard-Briscoe et al., 1977) renders CD98-null B cell unable to polyclonal and monoclonal antigen receptor-driven proliferation compete for available anchorage or nutrients within the both in vitro and in vivo (Cantor et al., 2011). Effector functions environment of a germinal center. Nevertheless, taken together, such as cytokine secretion and cytotoxic target lysis appear to be these studies demonstrate that CD98hc is required for both normal. These data establish a crucial role for CD98hc in the humoral and cell-mediated adaptive immunity because it is clonal expansion stage of T cell immune responses and show that essential for clonal expansion (Fig. 3). CD98hc is required for T-cell-dependent adaptive immunity. Given that clonally expanding T cells would have increased Mechanism of action for CD98 in lymphocyte proliferation metabolic demands, one might expect amino acid transport to be Although the cellular role of CD98hc in lymphocytes has become the crucial function of CD98 in T cells. However, reconstitution fairly clear, the biochemical mechanism by which this protein of CD98hc-null T cells with CD98 mutants shows that the portion drives clonal expansion is not fully understood. There are at least of CD98hc required for amino acid transport is not absolutely three indications that the integrin signaling function of CD98hc required, but that the region that is involved in integrin signaling is required for rapid lymphocyte proliferation. First, CD98hc is crucial for T cell proliferation (Cantor et al., 2011). The mutants that exclusively support integrin signaling can importance of CD98hc in T cell immune responses is also seen in reconstitute proliferation, whereas those that only support autoimmune disease. Here, loss of T cell CD98 prevents disease amino acid transport do not. Second, CD98-null B cells show development in two models of T-cell-mediated autoimmunity, defective integrin functions in that they are unable to adhere type 1 diabetes (T1D) and multiple sclerosis (Cantor et al., 2011). firmly and spread on anti-aLb2-integrin-coated solid surfaces. This raises the possibility that CD98hc could serve as a More importantly, mitogen-induced early (,30 min) mitogen- therapeutic target for blocking inappropriate adaptive immune activated protein kinase (MAPK) signaling is intact in CD98-null responses. B cells as indicated by a normal initial wave of extracellular- On the basis of these recent observations, several questions can signal-regulated kinase (ERK1/2) phosphorylation. However, now be raised regarding CD98hc function in T cells, such as what loss of CD98hc abrogates sustained secondary phosphorylation are the effects an antigen-activated CD98-null T cell might have of ERK and downregulation of cyclin-dependent kinase (CDK) on the overall immune response? Cytokine secretion and the inhibitors. This loss of sustained ERK activation and capacity to differentiate to regulatory-type Foxp3+ T cells are downregulation of CDK inhibitors resembles the effects of the still intact in the absence of CD98hc (Cantor et al., 2011). The loss of integrin signals that support growth factor stimulation of ability of a regulatory T cell to suppress inflammatory T cell fibroblasts (Assoian and Schwartz, 2001; Schwartz and Assoian, responses might be less dependent on clonal expansion than the 2001; Walker and Assoian, 2005). These data raise the question

Journal of Cell Science effector functions of an inflammatory subset (Gavin and of which integrin ligands support B and T cell proliferation. One Rudensky, 2002; Takahashi et al., 1998; Taams et al., 1998). possibility is that there is a ligand-independent function of Thus, there is the potential to modulate the quality of an immune integrins in sustaining ERK signaling, which supports cell cycle response through interfering with CD98hc function. The second progression. Another possibility is that CD98hc-dependent question is how partial or transient loss of CD98hc might differ integrins recognize cell-associated ligands during cell–cell from its complete deletion. Another unresolved issue is whether interactions (Nguyen et al., 2008) that occur during lymphocyte T cells require CD98hc for secondary responses to the same localization and synapse formation within lymph nodes. Whereas pathogen. In other words, what is the function of CD98hc in morphological synapse formation appears grossly normal in memory CD4 or CD8 T cells? This might have implications in CD98-null T cells in vitro, it is possible that the quality and designing intervention strategies that target CD98hc. duration of contacts is defective in vivo, perhaps preventing T cell polarization (Chang et al., 2007). Additional hints that CD98 in B lymphocyte function CD98hc functions in cell–cell contacts comes from its Until recently, only one study had examined CD98 function in B documented roles in cell fusion, and in monocyte or B cell cells using inhibition with the 4F2 antibody, with confusing aggregation (Cho et al., 2001; Cho et al., 2004; Mori et al., 2001; results that included both inhibition and enhancement of B cell Mori et al., 2004; Ohgimoto et al., 1996; Suga et al., 1997; functions (Gerrard et al., 1984). To address this issue, our group Tsurudome and Ito, 2000). Further study is needed to understand genetically deleted CD98hc in B cells using CD19-Cre (Cantor the details of how CD98hc and integrins might cooperate to et al., 2009). Similar to the results in T cells, CD98hc is not support lymphocyte proliferation and adaptive immunity. required for B cell compartmentalization or their initial Given that highly conserved CD98 orthologs are present only activation, but is crucial for B cell clonal expansion (Cantor in vertebrates (Prager et al., 2007; Uinuk-Ool et al., 2002) and are et al., 2009). CD98-null B cells display strikingly reduced required for adaptive immunity, they should be found in all proliferation after stimulation with strong mitogens, and thus are adaptive immune systems. Jawless fish (agnathans), which have unable to differentiate into plasma cells. Consequently, mice an alternative adaptive immune system with a unique set of genes lacking CD98hc in B cells have strongly impaired antibody that form VLRs (Herrin and Cooper, 2010; Pancer et al., 2005) responses following immunization, and class-switching to IgG, a still rely on clonal expansion of lymphocyte-like cells (Mariuzza hallmark of mature antibody responses, is dramatically impaired. et al., 2010) and possess an unambiguous CD98hc orthologue Because CD98hc-null B cells lack antigen-driven proliferation, (Uinuk-Ool et al., 2002). This fact adds additional compelling CD98, cancer and immunity 1377

3 Differentiation/ effector function 5 Secondary (memory) 2 Proliferation response Cytokines

4 Contraction/ memory formation

CD4+ Antigen

APC Selective (> 30 days) expansion of Expansion of Ag-specific memory cells clone Target 1 Activation cell

CD8+

Cell death

Early MAPK Secondary signals MAPK signaling

CD98 requirement CD98?

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.

evidence for the absolute requirement of CD98hc for the ability abolishes its amino acid transport function (Shishido et al., 2000). of lymphocytes to mount adaptive immune responses. However, this mutation also affects the ability of CD98hc to bind integrins (Kolesnikova et al., 2001), making this result difficult to CD98 and invasive cancers: the turbocharger interpret. Moreover, a later study using CD98hc chimeras, in gone awry which its amino acid transport is separated from its integrin

Journal of Cell Science CD98 expression in tumors signaling function (Fenczik et al., 2001), has shown that the The capacity for rapid proliferation, especially without firm CD98 domain that is involved in integrin signaling is required for anchorage, can be dangerous in malignant cells, and a large body transforming Chinese hamster ovary (CHO) cells (Henderson of evidence implicates CD98 in cancer (Table 1). Many tumors et al., 2004) and for the development of embryonic stem cell express CD98hc, and its expression correlates with poor teratomas in mice (Feral et al. 2005). Taken together, these prognosis in B cell lymphomas (Holte et al., 1987; Holte et al., genetic studies show that CD98hc expression is important, and 1989; Salter et al., 1989). Furthermore, nearly all studies that that its overexpression is sufficient for cellular transformation. have examined the expression of CD98hc or CD98 light chains in solid tumors show that their expression is correlated with CD98 blockade in tumors progressive or metastatic tumors (Esteban et al., 1990; Kaira The third line of evidence for the importance of CD98 in cancer et al., 2009a; Kaira et al., 2009b; Kaira et al., 2009c; Kaira et al., comes from a number of studies that used CD98 inhibitors in a 2008; Kaira et al., 2009d; Kobayashi et al., 2008; Nawashiro variety of tumor cells; these have resulted in the inhibition of et al., 2002; Nawashiro et al., 2006; Oleinik et al., 2005; cellular proliferation and tumor growth both in vitro and in vivo Powlesland et al., 2009; Shennan et al., 2003; Xiao et al., 2005). (Imai et al., 2010; Nawashiro et al., 2006; Oda et al., 2010; Shennan and Thomson, 2008; Yamauchi et al., 2009). One CD98 depletion and overexpression studies in cancer particular study used anti-CD98 antibodies specific for the heavy Genetic modulation of CD98 expression in human cell lines chain and showed that the growth of human tumor cells is and in animal models has established a causal link between CD98 inhibited in vitro (Yagita et al., 1986). Overall, these studies and cancer; CD98 promotes transformation and tumor growth indicate that CD98 might constitute a target for therapeutic (Feral et al., 2005; Ohkawa et al., 2011). Furthermore, CD98 intervention in cancer. In fact, a microRNA that modulates CD98 overexpression drives both anchorage independence and expression during intestinal epithelial differentiation was recently tumorigenesis (Nguyen et al., 2011; Hara et al., 1999; identified, raising the possibility of an additional approach to Henderson et al., 2004; Shishido et al., 2000), and the degree alter CD98 expression (Nguyen et al., 2010). of transformation correlates with the level of CD98hc present in the cells (Hara et al., 1999). An early report showed that the CD98 amino acid transport in cancer transforming ability of CD98hc is lost in a mutant that ablates the The mechanism by which CD98 promotes tumorigenesis is an ability to form disulfide bonds with the light chain and thus important area of current research. Significant questions remain 1378 Journal of Cell Science 125 (6)

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+; few (Haynes et al., 1981a) normal T cells are Bladder cancer cells (transitional epithelium) Anti-CD98 antibody treatment inhibits proliferation (Yagita et al., 1986) of human tumor cells B cell lymphomas CD98 expression on lymphomas can identify patients (Holte et al., 1987; Holte et al., 1989; Salter with poor prognosis et al., 1989; Powlesland et al., 2011) Oral squamous cell carcinoma CD98 histological expression pattern within a tumor (Esteban et al., 1990; Kim et al., 2004) is a significant indicator of progression and metastasis Childhood acute lymphoblastic leukemia CD98 expression level correlates with duration (Taskov et al., 1996) of complete remission (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 increased (Nawashiro et al., 2002) in astrocytic cancers Human glioblastoma High LAT1 expression correlates with poor survival; (Nawashiro et al., 2006) (and rat C6 glioma model) LAT1 inhibition decreases glioma tumor cell growth Breast cancer cell lines LAT-1 inhibitors suppress tumor cell growth and (Shennan and Thomson, 2008; Shennan et al., metabolism 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 may (Xiao et al., 2005) be a tumor biomarker Breast tissue RNA Expression of CD98-encoding mRNAs correlates (Esseghir et al., 2006) with poor prognosis Multiple CD98hc and LAT-1 expression is higher in metastatic (Kaira et al., 2008) than primary tumor sites (Mouse) Glioma cells Overexpression of CD98 light chain LAT-1 (Kobayashi et al., 2008) enhances tumor growth rate in vivo Pulmonary tumors CD98 expression predicts poor prognosis (Kaira et al., 2009a; Kaira et al., 2009b; Kaira et al., 2009c) Renal cell cancer CD98 histological expression correlates directly (Prager et al., 2009) with malignancy grade Squamous cell carcinoma lines CD98 light chain LAT-1 inhibitor improves efficacy (Yamauchi et al., 2009) of cisplatin treatment Non-small cell lung cancer LAT-1 inhibitor BCH reduces viability of tumor cells (Imai et al., 2010) ex vivo Chicken cell line and HeLa cells Genetic disruption or inhibition of LAT-1 blocks (Ohkawa et al., 2011) tumor cell growth in vitro and in vivo Journal of Cell Science

unanswered, such as whether CD98hc allows tumor growth by et al., 2010). Thus, it appears probable that high expression levels boosting the transport of nutrients or by augmenting integrin of CD98hc could allow for rapid proliferation of lymphocytes and signaling, and if so, how would each of these roles be fulfilled. The tumors by stimulating the exchange of glutamine for limiting rapidly dividing tumor cell has intense metabolic demands, often EAAs (Fig. 4, left-hand panel). under conditions of diminishing nutrient availability within a solid tumor. CD98hc could therefore support tumor growth through its CD98 integrin signaling function in cancer association with the amino acid transporter light chain and the CD98hc can also contribute to transformation by amplifying subsequent increased supply of amino acids. Under conditions of integrin signaling that results in reduced anchorage dependence. limited amino acid availability, CD98 light chains such as LAT-1 Integrins themselves have been implicated in invasive cancers are upregulated and their expression can be dysregulated in tumor because the ‘outside-in’ signals they transduce control cell cells (Campbell et al., 2000). LAT-1 and LAT-2 are amino acid proliferation and survival (Huck et al., 2010; Lahlou et al., 2007; exchangers and can import essential amino acids (EAA), such as Desgrosellier and Cheresh, 2010; Harburger and Calderwood, leucine, isoleucine and arginine in exchange for the export of 2009). Integrins act as mechanotransducers that sense the glutamine that has been imported by other transporters such as stiffness of the extracellular matrix (ECM). Increased ECM through system A transporters (Verrey, 2003). Furthermore, this stiffness drives tumor progression through clustering of integrins, CD98-mediated exchange of glutamine in the presence of EAAs is which leads to adhesive signaling through focal adhesion kinase a rate-limiting step in the activation of mammalian target of (FAK) and phosphatidylinositol 3-kinase (PI3K) (Levental et al., rapamycin (mTOR) (Nicklin et al., 2009), which regulates cell 2009). These signals can also direct the proliferation of micro- growth and proliferation, providing another pathway through metastases in the lungs through b1 integrin (Shibue and which CD98 could stimulate tumor growth. Cancer cells are Weinberg, 2009). In a softer matrix, such as one with larger heavily dependent on glutamine, not merely as an ingredient for amounts of flexible ECM components (elastin, etc.), high nucleotide and amino acid biosynthesis, but also to promote expression of CD98hc could permit integrin signaling that increased uptake of EAAs through this exchange mechanism would promote cell growth and survival. Thus, the expression (Wise and Thompson, 2010). Interestingly, T lymphocytes are level of CD98hc would fine-tune the capacity of the cell to sense another cell type that is particularly dependent on glutamine (Carr the compliance of the matrix. Indeed, CD98hc is important in CD98, cancer and immunity 1379

CD98 amino acid transport in cancer CD98 integrin signaling in cancer

CD98hc CD98hc Glutamine Essential Integrin amino acids

S-S S-S

Unidirectional CD98 light chain CD98 light chain transporter (LAT-1, LAT-2, etc.) (LAT-1, LAT-2, etc.) Glutamine Leucine AKT mTOR p130CAS activation FAK

Autophagy

Survival Survival Anchorage-independence Growth Metastasis

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.

allowing a cell to exert force on the ECM through integrin-driven (Fogelstrand et al., 2009; Nguyen et al., 2011). Vertebrates

Journal of Cell Science RhoA signaling (Feral et al., 2007), which is a key element in the have stratified squamous epithelium containing rapidly-dividing sensing of stiffness (Samuel et al., 2011). keratinocytes, which express high levels of CD98hc (Fernandez- Anchorage independence (i.e. independence of integrin signals Herrera et al., 1989; Lemaitre et al., 2005; Lemaitre et al., 2011; provided by adhesion to ECM) is another hallmark of cellular Patterson et al., 1984). The role of CD98hc in pathologic states in transformation, and CD98hc is important for anchorage these tissues requires further study. independence (Feral et al., 2005). CD98hc co-immunoprecipitates with integrins (Henderson et al., 2004; Rintoul et al., 2002), and Conclusions binds to the b1andb3 integrin cytoplasmic domains in a purified Here, we have summarized the central importance of rapid system (Prager et al., 2007). Overexpression or crosslinking of proliferation for clonal expansion in the vertebrate-specific CD98hc stimulates FAK and AKT phosphorylation (Cai et al., adaptive immune system. CD98hc enables lymphocyte clonal 2005; Rintoul et al., 2002) (Fenczik et al., 1997), and deletion of expansion and adaptive immune responses, probably in part CD98hc impairs integrin signaling (Feral et al., 2005). This integrin through its effects on integrin signaling. CD98hc is also signaling function of CD98hc is dependent on the transmembrane important in cancer, which is probably mediated through and cytoplasmic domains (Fenczik et al., 2001), the same region that increased transport of amino acids and/or by boosting integrin is crucial for lymphocyte proliferation (Cantor et al., 2009; Cantor signals that allow growth in soft ECM or without anchorage. et al., 2011) and for cellular transformation caused by overexpression Importantly, clonal expansion is the nexus at which CD98 acts in of CD98hc (Henderson et al., 2004). CD98hc can thus regulate adaptive immunity, and clonal expansion of tumor cells is also a integrin signaling, and integrins are crucial for tumor progression central feature of many cancers. CD98hc and the integrins that through mechanotransduction and control of anchorage-independent bind to it are vertebrate-specific, as is adaptive immunity, and it growth. Therefore CD98hc might control tumorigenesis by is noteworthy that vertebrates appear to be particularly prone to governing integrin signaling (Fig. 4, right-hand panel). developing invasive and metastatic cancers. This confluence of CD98hc might also contribute to other pathologies. For adaptive immunity, and the expression of CD98hc and CD98- example, in addition to autoimmunity disorders (Cantor et al., binding integrins (Prager et al., 2007), coincident with increased 2011), CD98hc overexpression could lead to other adaptive invasive cancer, in vertebrates, suggests that CD98hc provides immunopathologies. Arterial restenosis and intestinal villous vertebrates with the benefit of adaptive immunity, but that this inflammation are dependent on CD98hc in animal models comes with a price: increased susceptibility to invasive cancers. 1380 Journal of Cell Science 125 (6)

One could speculate that other vertebrate-specific genes that are Esteban, F., Ruiz-Cabello, F., Concha, A., Perez Ayala, M., Delgado, M. and Garrido, F. (1990). Relationship of 4F2 antigen with local growth and metastatic involved in clonal expansion must exist and are maintained potential of squamous cell carcinoma of the larynx. Cancer 66, 1493-1498. because of the survival benefits they provide through adaptive Fenczik, C. A., Sethi, T., Ramos, J. W., Hughes, P. E. and Ginsberg, M. H. (1997). immunity. Such genes might, like those encoding CD98, be Complementation of dominant suppression implicates CD98 in integrin activation. Nature 390, 81-85. upregulated in activated lymphocytes and could be therapeutic Fenczik, C. A., Zent, R., Dellos, M., Calderwood, D. A., Satriano, J., Kelly, C. and targets for autoimmunity diseases or cancer. Going forwards, it is Ginsberg, M. H. (2001). Distinct domains of CD98hc regulate integrins and amino key for this field to define the specific molecular mechanisms by acid transport. J. Biol. Chem. 276, 8746-8752. Feral, C. C., Nishiya, N., Fenczik, C. A., Stuhlmann, H., Slepak, M. and Ginsberg, which CD98 contributes to adaptive immunity and cancer. M. H. (2005). CD98hc (SLC3A2) mediates integrin signaling. Proc. Natl. Acad. Sci. Second, the dramatic effects of CD98 loss on tumors and on USA 102, 355-360. Feral, C. C., Zijlstra, A., Tkachenko, E., Prager, G., Gardel, M. L., Slepak, M. and lymphocyte clonal expansion demands that we make efforts to Ginsberg, M. H. (2007). CD98hc (SLC3A2) participates in fibronectin matrix define the therapeutic potential of CD98 inhibition in cancer and assembly by mediating integrin signaling. J. Cell Biol. 178, 701-711. autoimmune diseases. Fernandez-Herrera, J., Sanchez-Madrid, F. and Diez, A. G. (1989). 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