Molecular Aspects of Tumor Cell Migration and Invasion

66 Ann Ist Super Sanità 2010 | Vol. 46, No. 1: 66-80 DOI: 10.4415/ANN_10_01_09

Molecular aspects of tumor cell migration and invasion xper i ence e Giuseppina Bozzuto, Paola Ruggieri and Agnese Molinari Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Rome, Italy l i n c a c to

Summary. Cell migration and invasion are crucial steps in many physiological events. However, they are also implicated in the physiopathology of many diseases, such as cancer. To spread through the tissues, tumor cells use mechanisms that involve several molecular actors: adhesion receptor fami- est i ng lies, receptor tyrosine kinases, cytoskeleton proteins, adapter and signalling proteins interplay in a t complex scenario. The balance of cellular signals for proliferation and survival responses also regulates migratory behaviours of tumor cells. To complicate the scene of crime drug resistance players

n i m a l can interfere thus worsening this delicate situation. The complete understanding of this molecular a

jungle is an impossible mission: some molecular aspects are reviewed in this paper. Key words: neoplasms, neoplasm invasion, molecular markers. from

Riassunto (Aspetti molecolari della migrazione ed invasione delle cellule tumorali). La migrazione e l’invasione cellulare rappresentano momenti cruciali in molti eventi fisiologici: questi due processi, tuttavia, sono anche implicati nella fisiopatologia di varie malattie, tra cui i tumori. Per diffonder- ese a rch

r si attraverso i tessuti, le cellule tumorali ricorrono a meccanismi che vedono il coinvolgimento di diversi componenti cellulari: famiglie di molecole di adesione, recettori tirosinchinasi, proteine del citoscheletro, proteine di segnalazione intracellulare intervengono in un complesso scenario molecolare. Le vie di segnalazione regolanti i processi di sopravvivenza e proliferazione cellulare giocano un ruolo importante anche nei comportamenti migratori delle cellule tumorali. A complicare la scena del crimine, marcatori proteici della farmacoresistenza contribuiscono al conferimento di un fenotipo maggiormente aggressivo, peggiorando in tal modo una situazione già di per sé delicata. La comprensione completa di questa “giungla molecolare” è una missione impossibile: in questa rassegna verranno presi in considerazione alcuni degli aspetti molecolari. Parole chiave: neoplasie, invasione delle cellule tumorali, marcatori molecolari.

INTRODUCTION Each of these steps requires a distinct molecular pro- Cell migration and invasion are crucial steps in gram in which the modulation of the adhesive and many physiological events such as implantation of migratory as well as the cytoskeletal properties of the embryo, embryogenesis, morphogenesis, neurogen- disseminating tumor cells play essential roles. esis, angiogenesis, wound healing and inflammation Tumors can spread in a variety of channels/ways: [1, 2]. However, cell migration and invasion are also the most common pathway is tumor extending in implicated in the pathophysiology of many diseases, continuity beyond the organ or structure of origin, such as cancer. Indeed, the capacity to produce me- i.e. when it passes from the original organ to another tastases, very different among cancers, is the main fea- organ or vessel or cavity by continuity. Dissemination tures of malignant tumors and it is one of the main for contiguity occurs when tumor infiltrates tissue causes of death for cancer. This is due to the fact that spaces of non continuous adjacent structures. metastases are constituted by cells much more resist- The most common pathways for distant spread in ant, aggressive and efficient than those forming the the chest and abdomen are the lymphatics, blood primary tumor. vessels, and coelomic cavities. Cancer cells can dis- In the last years, major efforts have been undertak- seminate from the primary site via lymphatic routes en to understand the molecular mechanism underly- (“lymphatic metastases”) and by haematogenous ing the distinct steps of metastasis, which are (i) de- routes (“ab initio hematogeneous metastases”). tachment of tumor cells from the primary tumor, (ii) Secondary haematogenous dissemination of lym- invasion into surrounding tissue, (iii) intravasation phatic metastases also occurs from overt metastases into blood or lymphatic vessels, (iv) dissemination in to other distant sites. Coelomic cavities involved in the blood stream or the lymphatic system and, finally, tumor dissemination include the pleural space of the (v) extravasation and outgrowth at a secondary site. thoracic cavity and the peritoneal spaces of the ab-

Address for correspondence: Agnese Molinari, Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. E-mail: [email protected]. Migration and invasion of tumor cells 67

domen and pelvis. The most commonly involved is The cellular protrusions that initially recognize the peritoneum, which carries tumor cells in ascitic and bind to the ECM may be morphologically dif- fluid.T he distribution of intraperitoneal metastases ferent: lamellipodia, filopodia, pseudopodia and often corresponds to predictable flow patterns, the invadopodia [11], all contain filamentous actin. most classic example of which is seen with ovarian Indeed, the propulsion and elongation of pseu- cancer. With this tumor, or any tumor demonstrat- dopodia are driven by actin polymerization and by ing intraperitoneal spread, the paracolic gutters, filament assembly [9, 12]. Growing cell protrusions cul-de-sac, omentum, and liver surface are common reach and begin to bind to adjacent ECM by means sites of metastases. In the chest, pleural dissemina- of adhesion molecules such as integrins and cadher- tion typically spreads via gravitational forces and is ins: these molecules are involved in the formation of often seen in the lower thoracic cavity [3]. “focal contacts” complex composed of extracellular Each phase involved in the metastatic process re- ligands, tyrosine kinase receptors and cytoskeletal quires many specific molecular interactions between proteins. the tumor cells, the extracellular matrix (ECM) and the cells of the stroma. Liotta et al. [4] proposed the well-known three-steps theory for tumor cell inva- ADHESION MOLECULES sion: in the first step tumor cell adhere to specific The high degree of specificity that characterizes components of the matrix through cell surface re- both the cell recognition and the adhesion phe- ceptors; in the second step, the anchored tumor cell nomena requires the interaction between molecules secrets hydrolytic enzymes which can locally de- to constitute cell-cell or a ECM-cell bridges. These grade the matrix. The third step is represented by proteins are the so-named “adhesion molecules” the tumor cell migration through the matrix region or CAMs (cell-cell adhesion molecules), glycopro- modified by proteolysis. teins expressed on the cell surface. Most of CAMs To spread through the tissues, tumor cells use belongs to six protein families: cadherins, integrins, mechanisms that are similar but not identical to immunoglobulin superfamily, selectins, lymphocyte those used by normal cells during physiological homing receptors. CAMs are involved in many processes such as morphogenesis and migration of physiological and pathological processes, and it is immune cells [5]. Cell migration was firstly studied now well known that they can assume a key role in in fibroblasts, keratinocytes and myoblasts [6, 7]. the complex evolution of metastases [13]. Further studies showed that some basic strategies Cadherins (calcium-dependent adhesion mol- are also preserved in tumor cells. ecules) belong to a large family of transmembrane The cell migration through the tissues results from glycoproteins that mediate cell-cell adhesions in a continuous cycle of coordinated and interdepend- a calcium-dependent manner [14]. The epithelial ent steps that involve the cytoskeletal machinery [7, cadherin (E-cadherin) has been the first adhesion 5]. Migration begins when a cell responds to an ex- molecules to be discovered and characterized. Thus, ternal signal that leads to the polarization and ex- E-cadherin is the prototype member of cadherin tension of a “leading front” in the direction of the family and plays a fundamental role in the devel- movement. Then, the leading front binds to ECM opment and maintenance of adhesion between epi- proteins, and the cell body shrinks: a traction force thelial cells [15]. E-cadherin consists of an extracel- is thus generated, which determines the slow sliding lular domain, constituted of five cadherin repeats of the cell body behind the migrating front. (EC1, EC2, EC3, EC4 and EC5), a transmembrane In the first phase of the migration process the domain, and an intracellular domain that binds to extension of cell protrusions is driven by actin po- both P120 catenin and beta-catenin. It has been well lymerization. This reaction may be increased by the documented that tumors of epithelial origin partial- actin monomer addition to existing filaments or by ly, or totally, lose the expression of E-cadherin with the nucleation of new actin filaments byA rp2/3 (ac- the acquisition of a more aggressive phenotype [16]. tin-related protein 2/3) [8]. Arp2/3 is predominantly On the other hand, several studies have also shown regulated by the family of adapter proteins WASP the strong anti-invasive and anti-metastatic role of (Wilscott-Aldrich syndrome protein) and WAVE E-cadherin [17-19]. (WASP-family verprolin-homologous protein), The large family of integrins comprises a wide which act as a molecular platform for the forma- number of cellular receptors, heterodimeric trans- tion of the complex responsible for actin nucleation. membrane glycoproteins constituted of two subu- As described in the speculative model proposed by nits, the alfa chains associated with the beta chains Rohatgi et al., [9] Cdc42 and lipids (PI(4,5)P2) may through non-covalent bond [20]. Integrins are adhe- regulate actin assembly at membrane-proximal sites sion molecules essential in the intercellular interac- by recruitment and activation of the Arp2/3 complex tions and in the integration (hence the name) of cells via N-WASP-like proteins. Phosphoinositides bind with the extracellular environment. Both alfa and and activate the guanosine-nucleotide-exchange beta chains penetrate into the cell membrane giving factors (GEFs) that, in turn, regulate the activity of rise to the cytoplasmic domains essential for signal small GTPases [10] able to activate actin assembly transduction. The molecular mass of alfa subunits regulatory proteins. varies between 120 and 180 kDa, whereas that of the 68 Giuseppina Bozzuto, Paola Ruggieri and Agnese Molinari

beta subunits ranges from 90 to110 kDa. By differ- [23] demonstrated that a high expression of VLA2 ent combinations of 18 alfa chains and 8 beta chains and CD44 was associated with a high capability of are generated 24 distinct integrins [21]. Outside of producing metastases by renal carcinoma. In fact, the plasma membrane the alfa and beta subunits by analyzing the adhesion molecule expression in 37 protrude about 23 nm, and the NH2 terminal ends cell lines, isolated from nephrectomies, and the rela- of each chain are used to link the ECM. The main tive behaviour in the extra-renal stroma, a positive role of these adhesion molecules, in fact, is to medi- correlation was observed between invasive capacity ate cell-matrix and cell-cell interactions [22]. and level of expression of both the molecules on the The integrins are involved in many physiological cell membrane. A study published in 2006 also high- and pathological processes, including inflammation lighted the correlation between VLA2, VLA5 and and wound repair [23], proliferation, differentiation CD44 adhesion molecules, and tumor metastases and apoptosis [24]. In particular, they seem to have in human osteosarcoma cells [36]. Very important a crucial role in metastasis, by mediating the interac- is also the association of CD44 with ezrin, radixin, tion of tumor cells with the ECM [25]. These roles myosin (ERM) and merlin (moesin-ezrin-radixin- are possible thanks to the physical bond of adhesive like protein) proteins [37-39]. In particular, ERM contacts with the actin cytoskeleton, with the con- proteins are essential for the regulation of protein sequent activation of cytoplasmic pathways medi- movements in the plasma membrane, cells shape ated by different signal proteins such as Rho, Src, and cell migration [40, 41]. MAPKs and PKB [22]. Evidence of the connections with the cytoskeleton comes from a large number of studies conducted by electron microscopy and dem- INTERACTION WITH THE ECM onstrating the co-localization of integrins with the AND FORMATION OF FOCAL CONTACTS cytoskeletal structures [26]. The integrins bind to a The integrin family of heterodymeric transmem- wide range of matrix proteins, including laminin, fi- brane receptors play a particularly important role bronectin, trombospondin, vitronectin and various in the interaction with ECM and formation of “fo- types of collagen [27-29]. cal contacts” [24, 42]. Cells adhere to ECM via in- A widely used classification of integrin superfami- tegrin-mediated adhesions that link matrix to actin ly is based on the type of chain constituting the het- cytoskeleton. In cultured cells, integrin-based mo- erodimer. VLAs are integrins belonging to the beta1 lecular complexes form discrete morphological enti- subfamily, and consist of six heterodimers. They are ties of several types. Small (0.5-1 µm) “dot-like” or called “very late activation antigens” (VLAs) be- “point contacts” also known as “focal complexes” cause the first glycoproteins identified (VLA-1 and are localized at the edges of lamellipodia. Elongated VLA-2) were only expressed at a late stage after T- (3-10 µm in length) streak-like structures associated cell activation. The beta1 integrins are expressed on with actin- and myosin-containing filament bundles lymphocytes [20, 30], where they mediate the binding (stress fibers) are known as “focal contacts” or “fo- with the proteins of the ECM, playing an important cal adhesions” [43]. “Podosomes” and “invadopo- role in the extravasation and migration in tissues dia” are highly dynamic and specialized adhesive during the immune response [31]. In this subfamily structures, rich in focal contacts. They contribute to the beta1 subunit binds to 6 different alfa subunits, remodel the cytoskeleton and the matrix by control- leading to the integrin classification VLA1, VLA2, ling both the local turnover of focal contacts and VLA3, VLA4, VLA5, VLA6. VLA2 consists of the the degradation of ECM. alfa 2 and beta 1 subunits, binds to different types of Following the contact with specific ECM ligands, collagen (I-II-III-IV) and to laminin I. VLA5 (alfa5, integrins clusterize on the cell membrane, and recruit beta1) binds to fibronectin and to the adhesion mol- through their intracellular domain either adapter ecule L1CAM (L1 cell adhesion molecule). proteins or signal proteins. This leads to phospho- Another adhesion molecule widely expressed rylation and dephosphorylation signals within the on lymphocytes is CD44. This small molecule is a cell. In particular, cytoplasmic region of integrins membrane glycoprotein of Class I of 85-95 kDa. directly interacts with adapter proteins such as alpha CD44 is encoded by a single gene [32] but the het- actinin, tensin, talin and the signal protein FAK (fo- erogeneity of produced protein is partly generated cal adhesion kinase). All these proteins can in turn by post-transcriptional modifications [33], that dif- bind to other adapter proteins to recruit in focal fer with respect to both cell type and growth con- contacts actin ligands, such as vinculin, paxillin and ditions. This glycoprotein is able to bind to lam- alpha-actinin, which are all involved in the dynamic inin, fibronectin, collagen and, particularly, to- hy association with actin filaments. [43, 44]. aluronic acid, an important ECM component [34]. Assembly of focal contacts is regulated through The CD44 is a multifunctional receptor, not only various signaling pathways that include the phos- important in the context of the immunological re- phatidylinositol 3-phosphate (PI3K), the protein ki- sponse. Similar to integrins discussed above, CD44 nase C (PKC) and Rho family GTPases [44, 45]. was initially detected on the membrane of the im- The dynamics of focal cell-ECM adhesions is de- mune system cells. Its identification on other type of termined by the cyclic formation and destruction of cells has expanded its function [35]. Gilcrease et al. these structures, and both intracellular calpain pro- Migration and invasion of tumor cells 69

teinases and the ubiquitin-dependent proteasome increase of the actin-myosin complex contractility. system are involved in these regulatory mechanisms ROCK in cooperation with mDia1 can also stimu- [46-48]. late the formation of “stress fibers”. ROCK phos- Focal adhesions allow the cell to acquire a “mor- phorylates and activates LIMK1 which in turn in- phological polarization”, which results in a direc- hibits the depolymerisation of actin microfilaments tional motility. The generation of a protrusive force, [56]. Finally, mDia1 promotes actin polymerization produced by the asymmetric actin polymerization and microtubule stabilisation. It has been suggested on the leading edge of the cell, and the generation of that mDia1 also increases the rate of cytoskeletal re- a contractility force within the cell, through the in- organization [57]. teraction between actin filaments and myosin motor The increase of contractile tension induced by Rho system, contribute to the process of directional mi- results in the formation of numerous actin bundles gration. In some cases, during the processes of mi- associated with mature focal contacts. In contrast, gration and invasion, tumor cells are able to adopt ROCK and Rho inhibition leads to actin disassembly an “ameboid” mechanism of propulsion, involving and focal contacts processing in small focal complex- integrin-independent adhesions and actomyosin-de- es: this transformation is probably the consequence pendent expansion/contraction cycles. These “ame- of the decrease in actin-myosin tension [56]. boid” cells circumnavigate, rather than degrade, Rho and MLCK allow cells to separately control the ECM physical barrier [49]. In other cases, tu- the movements of cortical actin from the contrac- mor cells actively invade different tissues through a tion of actin filaments, located in the deeper layers turnover of adhesion molecules that allow the cell of the cytoplasm. Indeed, the contraction of actin to form contractility structures and degradate the filaments, controlled by myosin II, is induced pri- ECM [50]. marily by Rho and its effector ROCK [58, 59]. In contrast, the cortical actin network seems to be regulated by MLCK and not by Rho [60, 61]. CELL CONTRACTION: REGULATION Therefore, the Rho protein family plays a central OF ACTIN-MYOSIN COMPLEX role in regulating the movement of the cytoskeleton: Actin-myosin contraction promotes the cell short- the level of action of these proteins is regulated by ening along the long axis, and generate internal ten- the balance between a state of activation mediated sion in the direction of the focal contacts located on by the GEFs exchange factors (GTPase exchange the leading edge. Subsequently, the cell-substrate factors), which facilitate the replacement of GDP bonds preferentially dissolve at the rear edge of the with GTP, and a state of inactivation where GAPs cell, while the migration front, still attached, moves (GTPase-activating proteins) operate by attenuat- further forward [51, 52]. This induces the slow sliding the signal through the stimulated hydrolysis of ing of the cell rear edge forward. Organization, bound GTP [62]. assembly and the tension of the actin-myosin skel- Besides Rho, other proteins of the same group, Rac eton are controlled by different regulatory enzyme and CDC42, can play an important role in regulating systems. One of such systems directly controls the actin movements in migrating cells. Activated Rac myosin contractility, by modulating the light chain stimulates the “ruffling” of the membrane, which re- (myosin light chain, MLC) through the MLC kinase sults in the formation of the actin-rich cellular pro- activity (MLCK) and MLC phosphatase (MLCP) trusions lamellipodia, while activated CDC42 stim- counteraction. The activity of these enzymes, in ulates the actin polymerization and the formation of turn, is regulated by another set of enzymes, the the thin membrane protrusions filopodia [62]. The Rho-GTPases, belonging to the group of small activity of Rho GTPase also regulates other types GTPases (guanosine triphosphatases). This group of dynamic structures such as cadherin-dependent includes several members: Rho, Rac and CDC42 intercellular junctions, thus mediating the transition proteins [53-55]. from epithelial to mesenchymal phenotype. The epi- Targets of Rho protein are mDia1 (mammalian thelial-mesenchymal transition (EMT) is a frequent Diaphanous 1), LIMKs (LIM kinases), and Rho- event in the progression of various malignancies associated kinase (ROCK). mDia1 is a mammalian and is characterized by loss of expression of E-cad- homolog of Drosophila diaphanous and works as an herin and acquisition of expression of N-cadherin effector of the small GTPase Rho. LIM kinase-1 and vimentin. (LIMK1) and LIM kinase-2 (LIMK2) are actin- binding kinases that phosphorylate members of the ADF/cofilin family of actin binding and filament THE INVADOPODIA severing proteins. Rho-associated kinase (Rho-ki- The invadopodia are cellular protrusions rich in nase/ROCK/ROK) is a serine/threonine kinase and actin that can mediate the proteolysis of ECM [63- plays an important role in various cellular func- 65]. The molecular origin and the role of invadopo- tions. dia in particularly aggressive tumors such as glio- ROCK phosphorylates and inhibits the activity of mas, breast carcinomas and melanomas have been MLCP: this inhibition in turn results in increased recently discussed [63]. These subcellular organelles MLC phosphorylation and, consequently, in an have been observed for the first time in fibroblasts, 70 Giuseppina Bozzuto, Paola Ruggieri and Agnese Molinari

genetically modified by inserting v-Src oncogene, of ECM mediated by the invadopodia in various tu- which encodes for a protein with tyrosine kinase ac- mor cell lines. Tks5/Fish, thanks to its anchoring do- tivity. Following transformation, this protein is con- mains, binds to N-WASP, whereas AMAP1/ASAP1 stitutively activated and cells, grown on fibronectin, binds to cortactin: these associations are important form prominent protrusions with both adhesive and for the subsequent formation of invadopodia. degradation properties [66, 67]. The invadopodia have a diameter varying between 0.1 and 0.8 µm and their length can be up to 2 µm. Several markers are FOCUSED PROTEOLYSIS OF ECM: used for their identification: F-actin (filamentous THE METALLOPROTEINASES actin form), Arp2/3, N-WASP and cortactin, SH3- Degradation and remodeling of ECM are essen- proteins involved in various tumors [69-73]. tial stages of migration, invasion and metastasis of The maturation of invadopodia sees a four-steps cancer cells. These processes are primarily mediated process: (i) location of cortactin on the site of the by two types of proteolytic enzymes: the plasmino- protrusion formation; (ii) recruitment of MT1- gen activator system components and the matrix MMP metalloproteinase (membrane-type type 1 metalloproteinases (MMPs) [78]. MMP); (iii) degradation of the matrix; (iv) dissocia- The MMPs belong to a family of zinc-dependent tion of the cortactin from the cell membrane [70]. endopeptidases, highly conserved and structurally Several proteins are involved in the molecular related, capable of degrading many components of pathways responsible for the invadopodia forma- basement membrane and ECM [79]. The substrates tion, whose invasive properties are attributed mainly of MMPs include a wide variety of proteins such as to integrin-ligand binding (Figure 1) [69]. The mo- chemotactic molecules, adhesion molecules, protein- lecular cascade starts with the ECM-integrin link- ase inhibitors, cell surface receptors, blood coagula- age, together with the interaction of growth factors, tion factors, growth factors and growth factor-bind- such as the “epidermal growth factor” (EGF) and ing proteins. Studies on the activity of MMPs in vari- the “fibroblast growth factor” (FGF), with their re- ous cells and tissues, demonstrated the importance of ceptors. The recruitment and the activation of other these enzymes in many physiological (e.g. embryonic components occur downstream, outlining a complex development, bone resorption, angiogenesis) and molecular network in which many pathways are still pathological processes (e.g. rheumatoid arthritis, mul- unknown. Src is the first cytosolic protein involved, tiple sclerosis, tumor growth and metastasis) [80, 81]. and it is activated by phosphorylation. Following Human MMPs generally contain a signal pep- the activation mediated by activated Src, cortactin tide, a N-terminal pro-peptide domain, a catalytic binds to N-WASP, F-actin and Arp2/3 (these two domain that includes highly conserved zinc-binding molecules are also triggered by activated N-WASP), sites, and a hinge region followed by an hemopexin- and inhibits actin depolimerization. This coopera- like C-terminal domain (Figure 2a). tive action is essential to allow the actin assembly, They can be divided into different classes according physiological process essential in invadopodia for- to their sequence homology, substrate specificity, cel- mation. Experiments with RNA interference, nega- lular localization and structure (Figure 2b). MMP-2 tive mutants or inhibitory antibodies show that pro- and MMP-9 gelatinases present an additional domain teins such as cortactin [70, 72, 74], N-WASP [73, 75], inserted between the catalytic domain and the active AMAP1/ASAP1 (protein belonging to Arf GAP site domain. MT-MMPs may have an additional trans- family, and complexed with a peptidase acting as membrane domain site, either a glycosylphosphatidyli- GTPase) [76] and Tks5/Fish (an anchor protein with nositol anchor site (GPI) or a Ig-like domain that de- SH3-domains) [77] are necessary for the degradation termines the localization on the cell surface.

Fig. 1 | Molecular pathway underlying invadopodia formation and ECM degradation. GF and RTK indicate the growth factor and the relative receptor tyrosine kinase, respectively. Lines indicate an association between components; arrows an activation sequence. Migration and invasion of tumor cells 71

Fig. 2 | a) General structure of metalloproteinases. b) Classification of MMPs according to their structure. (Modified from: Lafleur MA, Handsley MM, Edwards DR. Metalloproteinases and their inhibitors in the angiogenesis. Expert Rev Mol Med 2003;5:1-39). © Cambridge University Press. Reproduced with kind permission. .

According to substrate five MMP subclasses have been ing for these endopeptidases, including cytokines, defined: collagenases (MMP-1, MMP-8, MMP-13), growth factors, hormones, oncogenes and tumor gelatinases (MMP-2, MMP-9), stromalysins (MMP-3, promoters [84, 85]. Cytokines and growth factors MMP-10, MMP-11), metalloelastases (MMP-12, are able to regulate the expression of metallopro- MMP-18, MMP-19), matrilysins (MMP-7), mem- teinases through the MAPKs pathway that includes brane-type-MMPs (MT-MMPs) [82, 83]. proteins such as ERK 1/2 (extracellular regulated The expression, secretion, and activity of MMPs kinase 1/2), JNK/SAPK1/2 (c-Jun N-terminal ki- are finely controlled in normal tissue. In particular, nase 1/2) and p38MAPK. Thus, the activation of the expression of MMPs is regulated at both tran- AP-1 and ETS transcription factors by the mitogen scriptional and post-transcriptional level. Several kinases is responsible of the maximum expression factors influence the transcription of genes cod- of MMPs [85]. 72 Giuseppina Bozzuto, Paola Ruggieri and Agnese Molinari

begins in the extracellular space by serine proteases, or by other members of the family of MMPs [86]. On the cell surface, the MT-MMPs (membrane-type MMPs) have been identified as potent physiological activators of several MMPs [82, 87]. The activity of MMPs is modulated by the family of “tissue inhibitors of metalloproteinases” (TIMPs) that are able to inhibit active MMPs after binding in their catalytic domain. In addition, TIMP-1 and TIMP-2 regulate the activation of some pro-MMPs by binding to their carboxy-terminal domains. In particular, TIMP-1 inhibits the activation of pro- MMP-9, while TIMP-2 binds and regulates the activation of pro-MMP-2 [88]. As shown in Figure 4, MT1-MMP can form a complex with TIMP-2, which subsequently serve as a receptor for pro-MMP-2. A second molecule of MT1-MMP adjacent to the complex, not inhibitor-linked can convert pro-MMP-2 in its active conformation [79, 89]. In particular, at Fig. 3 | “Cysteine switch” activation mechanism. a) A conserved cysteine-rich domain, located in the pro-domain, forms a bond with low concentrations, TIMP-2 promotes the formation the coordinated zinc ion located in the active site. b) The cleav- of a complex with pro-MMP-2 and MT1-MMP on age of the pro-domain leads to the breaking of zinc-cysteine bond the cell surface, thus leading to activation of MMP-2 followed by the amino-terminal pro-domain loss (Modified from: (Figure 4a). However, at high concentrations TIMP- Somerville RPT, Oblander SA, Apte SS. Matrix metalloprotein- 2 inhibits the activation (Figure 4b). ases: old dox with new tricks. Genome Biol 2003;4:216-26). The active MMPs may remain localized on the cell © BioMed Central. Reproduced with kind permission. surface through binding with membrane molecules, and this leads to a more focused ECM degradation. At post-transcriptional level, the biological activ- The expression of MMPs and TIMPs changes dur- ity of MMPs is regulated by their state of activation. ing the neoplastic transformation. A high secretion Many MMPs are secreted from cells in latent form as of MMPs by tumor cells has been demonstrated in zymogens (pro-MMPs). The pro-MMPs conversion many types of cancer [3, 90] and the imbalance be- in a functionally active form requires a more specific tween MMPs and their specific inhibitors seem to multi-stage process known as “cysteine switch” and play an important role in the tumor growth and in- that leads to the proteolytic removal of parts of the vasion [3, 78]. molecule (Figure 3). A highly conserved cysteine- rich domain, located in the prodomain, links to zinc ion in the active site (Figure 3a). The prodomain ROLE OF MITOGEN-ACTIVATED PROTEIN cleavage leads to the rupture of the zinc-cysteine (Zn- KINASES (MAPKs) IN THE PROCESSES C) bond, followed by the amino-terminal prodomain OF CELL MIGRATION AND INVASION loss (Figure 3b): in this way the active site becomes Many extracellular signals converge on the path- accessible. For many MMPs proteolytic activation way of proteins belonging to the serine/threonine

Fig. 4 | Mechanism of activation of pro-metalloproteinase 2 (pro-MMP-2). a) When present at low concentrations, TIMP-2 can form a complex with a molecule of MT1-MMP, which in turn will serve as a receptor for proMMP-2. A second molecule of unbound MT1- MMP, adjacent to the complex, can convert proMMP-2 in its active conformation. b) At high concentrations, TIMP-2 forms a complex with both proMMP-2 molecules, already bound to MT1-MMP, and with MT1-MMP free molecules thus inhibiting the activation of MMP-2 (Modified from: Lafleur MA, Handsley MM, Edwards DR. Metalloproteinases and their inhibitors in the angiogenesis. Expert Rev Mol Med 2003;5:1-39). © Cambridge University Press. Reproduced with kind permission. Migration and invasion of tumor cells 73

Fig. 5 | Activation pathways of mitogen-activated protein kinases (MAPKs).

kinase family and known as MAPKs. MAPKs play hypertrophy and cell differentiation, plays a key role an important role in cell proliferation, oncogenesis, in the migration of different cell types, [101-104]. differentiation, inflammation and response to stress p38 MAPK activity is stimulated by many growth [91], but several evidence suggest that these kinases factors, cytokines and chemotactic substances that is also essential for cell migration [92]. activate MEK3/6: this protein, in turn, phosphor- All MAPKs contain tyrosine-X-threonine motifs ylates and activates p38 MAPK [105]. p38 MAPK (where X is any amino acid) in the activation do- substrates are mainly MAPKAPK2/3 (a protein ac- main and are activated through a kinase cascade in tivated by either MAPK or MK), paxillin and cald- which MAPKKKs (MAPKs kinase kinase) activates esmon which, directly or through the phosphoryla- MAPKK (MAPKs kinase) which, in turn, activates tion of other proteins, lead to the reorganization of MAPK by the phosphorylation of threonine and actin and the formation of the “stress fibers” and tyrosine residues located in the activation domain adhesion structures, thus stimulating the directional (Figure 5). Based on differences in the activation migration of cells [106]. Studies on Drosophila em- domain, MAPK family can be subdivided into three bryos have shown that JNK is involved in the con- groups: kinases regulated by extracellular signals trol of actin cytoskeleton in the formation of filopo- (ERK1/2), which display a threonine-glutamine- dia and lamellopodia, and in the movement of cells tyrosine motif, p38 MAPK isoforms which have a during neural tube closure [107, 108]. It was also threonine-alanine-tyrosine motif, and JNK1/2/3 demonstrated that JNK determines the formation which present threonine-proline-tyrosin. of “stress fibers” and their accumulation in the lead- Numerous experimental observations have shown ing front of fibroblasts [109]. that ERK1/2 is directly involved in cell motility [93- 95]. Some growth factors and ECM components activate ERK through signalling pathways involving RELATIONSHIP BETWEEN DRUG Ras, Raf and MEK1/2. Once activated, ERK phos- RESISTANCE AND INVASION phorylates different substrates including MLCK, In the past studies on drug resistance and invasion calpain, paxillin and FAK [96-99]. generally proceeded along different research paths. The MLCK phosphorylation determines the focal Later the interest was focused on the possible rela- contact turnover and the formation of cell mem- tionship between the two phenomena. Currently, brane protrusions at the migrating front [60, 100]. many experimental evidence suggest a possible cor- Activated calpain, instead, interacts with the cy- relation between drug resistance instaurance and toskeleton proteins by promoting focal contact dis- acquisition of a highly aggressive phenotype. This assembly [95]. Finally, the phosphorylation of FAK relationship has been demonstrated by two obser- and paxillin by ERK may regulate the focal contact vations: (i) drug resistant tumor cells are more in- dynamics likely by affecting the interaction between vasive and metastatic, when compared with sensi- FAK and paxillin [97]. tive tumor cells, (ii) in some cases, most metastatic Recent studies have shown that p38 MAPK in- tumors show a greater resistance to chemotherapy volved in inflammation, apoptosis, cardiomyocyte [110]. The resistance of tumors to chemotherapy is a 74 Giuseppina Bozzuto, Paola Ruggieri and Agnese Molinari

multifactorial and complex phenomenon that often cells line. These data suggest a possible correlation involves different cellular mechanisms [111]. Two between metastasis and drug resistance mediated by types of resistance are known: an “intrinsic resist- P-gp [117]. An in vivo study conducted by Bradley et ance” that occurs in cells never exposed to chemo- al. [118] showed that different stages of tumor pro- therapy and an “acquired resistance” induced in re- gression displayed different levels of P-gp m-RNA sponse to chemotherapeutic treatment [112, 113]. expression. Surprisingly, 460 lung metastases were The main cellular mechanisms so far identified, examined and each metastasis was positive for P-gp. and that play a significant role in the phenomenon Moreover, in an in vitro study it was demonstrated of drug resistance can be classified into five groups: that human hepatoma cells rich in P-gp showed in- 1. drug-target interaction (target resistance); creased invasive properties, when compared to cells 2. alterations of drug activation, or inactivation, expressing low levels of P-gp [119]. Bates and col- by endogenous biochemical systems present in leagues [120] found that a CD44 isoform, normally tumor cell (metabolic resistance); involved in migration and invasion, coferred chem- 3. alteration of DNA repair; oresistance to colon carcinoma cells. Also a study of 4. alteration in the ability of cancer cell to respond lung carcinoma cells showed that the standard form to death (resistance to apoptosis); of CD44 is able to increase the expression of the 5. increased drug transport through cell membrane MRP2 transporter (belonging to the family of “ATP- (transport molecule-mediated resistance). After binding cassette”), resulting in the acquisition of a selection for resistance to a single drug, cancer drug resistance phenotype by NSCLC cell line [121]. cells may also show cross-resistance to other drugs, The human breast cancer drug resistant cell line structurally and functionally not related [114]. MCF-7 ADR which overexpress P-gp shows a differ- This phenomenon is known as “multidrug resist- ent invasive and metastatic potential when compared ance” (MDR) and could explain why combined to the parental line [122, 123] shown that the loss of treatments that involve the use of cytotoxic agents E-cadherin and the increase of N-cadherin during with different targets can be not efficacious. MDR the acquisition of the resistant drug phenotype cor- acquisition, induced by the treatment with a cyto- related with the increase in metastasizing capacity toxic agent, is generally associated with an increase of MCF-7 ADR cells. The variation in expression of of transport proteins that results in a reduced in- cadherin could be due to the control by twist tran- tracellular concentration of cytotoxic drug. scription factor. In fact, twist overexpression results The currently known active transporters that play in the epithelial-mesenchymal transition (EMT) an important role in MDR phenomenon are: mem- with the increase in cell motility and invasion. Many bers of the P-glycoprotein/TAP (ABCB-P-gp) sub- studies have shown that the transcriptional activa- family (Figure 6), members of the of MDR-associ- tion of the MDR1 transporter is regulated by the ated protein subfamily (ABCC1-MRP1), members MAPK pathway [124, 125], while others have shown of the MXR/BCRP (ABCG) subfamily and the lung that also Snail, a transcription factor that mediates resistance related protein (LRP). Except for LRP, all EMT, is regulated by the MAPK pathway as well belong to the superfamily of ABC transporters. [126]. Further studies on the involvement of P-gp If P-gp, MRP and LRP are three important mark- in the mechanisms of cell migration and invasion ers of drug resistance, immunohistochemical analy- have shown that treating MCF-7 ADR cells with the ses showed that they are also markers of invasion and transport substrates of the molecule an increase in metastasis [110, 115]. Moreover, a study on human the production of CD147, and MMP-2 and MMP-9 prostate carcinoma cell lines and its resistant vari- is achieved [127]. So the expression and activity of ants with different metastatic capacity [116] gave the drug resistance genes could simultaneously cross- following results: cultured, highly metastatic cells ex- activate genes that induce tumor metastasis. press higher levels of bFGF m-RNA, IL-8, MMP- Finally, malignant melanoma shows high levels of 2, MMP-9 and P-gp than poorly metastatic parental intrinsic drug resistance associated with a highly in-

Fig. 6 | Two-dimensional model of human P-glycoprotein based on the analysis of the amino acid sequence and its functional domains. ATP binding sites, phosphorylation sites, peptide linker, gly- cosylation sites and 12 transmembrane domains can be identified (Modified from: Di Pietro A, Dayan G, Conseil, G, Steinfels E, Krell T, Trompier D, Baubichon Cortay H, Jault J. P-glycoprotein-mediated resistance to chemotherapy in cancer cells using recombinant cytosolic domain to establish structure-function relationship. Braz J Med Biol Res 1999;32:925-39). Reproduced with kind permission. Migration and invasion of tumor cells 75

Fig. 7 | SEM observations of drug- sensitive (M14 WT) cell invasion through MatrigelTM. During invasion intense focused proteolysis is visible in sensitive cell samples, indeed the extracellular matrix appears digested around the cell. vasive phenotype. CD44, it is the major cell surface The association of Pgp with actin mediated by ERM receptor to hyaluronan, implicated in cell adhesion, family proteins was demonstrated in drug resistant metastasis and tumor progression [128, 129]. When human tumor cells. Such an association appeared to overexpressed in melanoma cells CD44 enhances be essential for the maintaining of the MDR pheno- the experimental metastatic potential and tumori- type [133]. Successively, a relationship between CD44 genicity [128], but its clinical significance in cutane- and the MDR1 transporter Pgp does exist in carci- ous melanoma is still unclear. noma cell lines [134]. In this study it was stated that CD44 proteins assemble intracellular complexes the expression of Pgp alone does not increase the mi- that are important in signal transduction: key play- gration potential and that it is the interaction of Pgp ers in this regulation are ezrin, radixin and moesin with CD44 that affects cell migration. (ERM) proteins. ERM proteins are involved in many In an our recent study [135], we investigated the physiological functions including regulation of actin role of the drug transporter P-glycoprotein (Pgp) cytoskeleton, control of cell shape, adhesion, motili- in the invasion potential of drug-sensitive (M14 ty and modulation of signal transduction pathways. WT, Pgp-negative) and drug-resistant (M14 ADR, In the phosphorylated form ERM proteins anchor Pgp-positive) human melanoma cells. In particu- CD44 to actin and support cell proliferation [130]. lar, co-immunoprecipitation experiments assessed Advances on ezrin involvement in the metastatic the association of Pgp with the adhesion molecule phenomenon has been recently reviewed [131]. Novel CD44 in multidrug resistant (MDR) melanoma molecular processes driven by ezrin activation in- cells, compared with parental ones. In MDR cells, clude: phagocytosis, acquisition of resistance to the two proteins colocalized in the plasma mem- chemotherapeutics and triggering of programmed brane as visualized by confocal microscopy and cell death signals. Federici et al., [132] highlight that immunoelectron microscopy on ultrathin cryosec- ezrin activity is mandatory for both the maintenance tions. MDR melanoma cells displayed a more in- of migratory and invasive capacity of tumors and vasive phenotype compared with parental cells, as conservation of the ability to feed through other cells. demonstrated by quantitative transwell chamber This occurs through the specific molecular association invasion assay. The Pgp molecule, after stimulation between ezrin and molecules involved in many activi- with specific antibodies, appeared to cooperate with ties of metastatic cells, such as CD44 and Lamp-1. CD44, through the activation of ERK1/2 and p38 Particularly, the molecular interaction between ezrin MAPK proteins. This activation led to an increase and Lamp-1 was never been described together with of metalloproteinase (MMP-2, MMP-3, and MMP- its importance in allowing Lamp-1 membrane locali- 9) mRNAs, and proteolytic activities, which are as- zation in metastatic cells. sociated with an increased invasive behavior. RNA 76 Giuseppina Bozzuto, Paola Ruggieri and Agnese Molinari

Fig. 8 | SEM observations of drug- resistant (M14 ADR) cell invasion through MatrigelTM. In the lower side of the filter, the invadopodia of resistant cells appeared to infiltrate between the stitches of MatrigelTM in the absence of focused proteolysis.

interference experiments further demonstrated Pgp Noteworthy, tumor acidity is also related to tumor involvement in migration and invasion of resistant cannibalism, a characteristic of malignancy and melanoma cells. A link was identified between MDR metastatic behaviour. Through this function meta- transporter Pgp, and MAPK signaling and invasion. static tumors feed off other cells, either dead or alive, Finally, differently from drug-sensitive (Pgp-nega- including the T lymphocytes that should kill them. tive) melanoma cells, which showed an “individual Experimental data have shown that cannibalism is mesenchimal” behaviour (Figure 7), Pgp-overex- increased in acidic culture conditions [137]. A new pressing melanoma cells adopted a ‘chain collective’ marker of malignancy with a specific role in tumor migration strategy reflecting potential high -meta cannibalism and in the establishment of metastatic static capacity (Figure 8). phenotype has been recently proposed [138]. Tumor Finally, new perspectives raise from a growing cannibalism has some similarities to the phagocytic body of literature data which suggest a key role of activity of Dictyostelium discoideum. Recently, ph- tumor acidic microenvironment in cancer develop- g1A has been described as a protein that is primarily ment, progression, and metastasis [136]. As reviewed involved in the phagocytic process of this microor- in Fais et al [136], V-ATPases play a crucial function ganism. The closest human homologue to phg1A in determining the acidification of tumor microen- is transmembrane 9 superfamily protein member 4 vironment and are overexpressed in many types of (TM9SF4). TM9SF4 is highly expressed in human metastatic cancers and positively correlated to their malignant melanoma cells deriving from metastatic invasion and metastasis. The promoting effect of V- lesions, whereas it is undetectable in healthy hu- ATPases on cancer invasion and metastasis mainly man tissues and cells. TM9SF4 is predominantly relies on their ability to maintain an acidic pH of expressed in acidic vesicles of melanoma cells, in extracellular microenvironment and very acidic which it co-localizes with the early endosome anti- luminal pH. This pathway is in turn related to the gens Rab5 and early endosome antigen 1. TM9SF4 activation, secretion, and cellular distribution of silencing induced marked inhibition of cannibal many proteases involved in the digestion of ECM. activity, which is consistent with a derangement of Molecular inhibition of V-ATPases by small inter- intracellular pH gradients, with alkalinization of fering RNA in vivo as well as a pharmacologic inhi- acidic vesicles and acidification of the cell cytosol. bition through proton pump inhibitors (PPIs) led to tumor cytotoxicity and marked inhibition of human Received on 16 October 2009. tumor growth in xenograft models. Accepted on 14 January 2010.

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