2 International Journal of Cell BiologyMetastatic invasion is generally the ﬁnal phase of cancerprogression, and it involves formation of new blood vesselseither by neovasculogenesis, in which endothelial cell precur-sors (angioblasts) migrate to the tumor site and diﬀerentiateand assemble into primitive blood vessels, or by angiogenesisin which we observe sprouting of new blood vessels frompreexisting ones, or their longitudinal bifurcation, in thetumor . The invasive tumor cells migrate through thesenewly formed blood vessels to other sites such as lung andliver brain and this leads to the death of tumor-bearingpatients or animals as the case may be.Based on available evidence, the entire process of cancerformation can be divided into four diﬀerent stages: initiation,progression, epithelial mesenchymal transition (EMT), andmetastasis (see Figure 1). At the initiation stage, a normalcell acquires oncogenic properties mainly through geneticalterations, which lead to changes in cell structure, adhesionproperties, and response to signals from ECM proteins. Inthe second stage, transformed cells respond to cues from thealtered environmental conditions and acquire properties ofadhesion-independent growth and colonization. The thirdstage is also referred to as a transitional or the EMT stage,and, in this stage, the fully transformed cells begin to exhibitmesenchymal gene expression patterns which induce themto invade into the neighbouring tissue and enter into bloodcirculation . The fourth and prominent stage is metastasisin which the invasive mesenchymes like cells move fromthe primary site and colonize in a new location. This stagespreads the disease into diﬀerent parts of the body andinvolves several alterations in the adhesion properties of cells.From all earlier observations, it is clear that the celladhesion in transformed cells plays an important role inall four stages of cancer formation. This paper highlightsrecent studies done on the integrin-mediated interaction oftransformed cells with the ECM and discusses its role incancer progression.2. ECM Components and PropertiesOver the past two decades, research in the ﬁeld of cancer biol-ogy has focussed extensively on the role of ECM constituentsduring cancer progression. These molecules comprise thecell’s microenvironment, and they can aﬀect the mechanicaland biophysical properties of cells as well as that of the ECMsuch as its mechanics, geometry, and topology .In some tissues, mainly of epithelial origin, ECMconstituents are present in the basement membrane thatdeﬁnes the boundaries of that tissue. In this location, theorganization of these components is diﬀerent than in thematrix. In the basement membrane, we notice moleculessuch as collagens, proteoglycans, laminins, and ﬁbronectinsassociate strongly with certain carbohydrate polymers andgenerate a membrane-like structure which facilitates theformation of a framework of cells and ECM constituents. Speciﬁc domains in ECM proteins that are createdby partial gene duplication and exon shuﬄing during theprocess of evolution  play a critical role in keeping thecells attached to the ECM and the basement membrane andinitiating signalling cascades in the cells.Inside the cells, the ECM-induced signaling pathwaysare transmitted mainly through integrin molecules that aretransmembrane multifunctional ECM receptors. Integrin-mediated signaling in association with many cofactors, forexample, cytokines, growth factors, and intracellular adaptermolecules, can signiﬁcantly aﬀect diverse cell processes suchas cell cycle progression, migration, and diﬀerentiation. Theinterplay between the biophysical properties of the cell andECM establishes a dynamic, mechanical reciprocity betweenthe cell and the ECM in which the cell’s ability to exertcontractile stresses against the extracellular environment bal-ances the elastic resistance of the ECM to that deformation[4, 9]. The ECM in association with the available growthfactors activates a sequence of reactions with a complexnetwork of proteases, sulfatases, and possibly other enzymesto liberate and activate various signalling pathways in ahighly speciﬁc and localized fashion. The maintenance ofECM homeostasis therefore involves a tight balance betweenbiosynthesis of ECM proteins, their 3D organization, cross-linking, and degradation.3. Modulation of ECM-GeneratedSignaling in CancerDuring cancer progression, we observe signiﬁcant changesin the structural and mechanical properties of ECM con-stituents. It has been reported that changes in matrixstiﬀness, which oﬀers resistance to cell traction forces and also inﬂuences “shape dependence” in cells, cancontribute actively to the tumor formation . Dereg-ulation of cell shape and alterations in the interactionswith the ECM are considered as important hallmarks ofcancer cells. These changes in ECM homeostasis can bebrought about by the properties of tumor cells themselvesor by the secretions of other surrounding cells such asﬁbroblasts, macrophages, and leukocytes . Integrin ECMinteractions are signiﬁcantly modulated by crosstalk withseveral other signal-generating molecules, some of them arereceptor molecules on the cell surface whereas others arepresent in the cytoplasm as adaptor proteins and actin-binding proteins . These signaling crosstalks in whichintegrin molecules lie at the center are very useful forthe transition of transformed cells to metastatic cells .Integrin-generated ECM remodelling is further controlled bythe localization and activity of proteases . One exampleof such an integrin-directed cancer progression is seenin breast cancers, where adhesion-independent mammaryepithelial cells secrete laminin-5 and luminal cells secretelaminin-1. This leads to aberrant polarity in cells, causingupregulation of metalloproteinases (MMPs, such as MMP9)and induction of tumor invasion and metastasis [18–20].4. Integrins: Its Ligands and SignallingIntegrins are heterodimeric cell-surface receptors that medi-ate adhesion to ECM and immunoglobulin superfam-ily molecules. At least 24 distinct integrin heterodimersare formed by the combination of 18 α-subunits and
International Journal of Cell Biology 3ECMEndothelial cells with basement membrane(a) (b) (c)(d) (e)Normal cellsTumor cellsFibroblastBlood vesselImmune inﬂammatory cellsPericyteFigure 1: Various steps in tumor initiation and progression where panel (a) represents initiation of tumor by transforming normal cells,panel (b) shows the modulation of ECM proteins allowing transformed cells to multiply, panel (c) shows progression of cancer by replacingnormal cells, panel (d) represents the invasion, where cancer cells migrate into the blood stream by modulating ECM and cell adhesionmolecules, and panels (e) shows metastasis where the cancer cells are localized at diﬀerent sites enabling angiogenesis.8 β-subunits. Speciﬁc integrin heterodimers preferentiallybind to distinct ECM proteins like laminin, collagen IV,ﬁbronectin, and so forth. The level of integrin expressionon the cell surface dictates the eﬃciency of cell adhesionand migration on diﬀerent matrices. While some integrinsselectively recognise primarily a single ECM protein ligand(e.g., α5β1 recognises primarily ﬁbronectin), others canbind several ligands (e.g., integrin αvβ3 binds vitronectin,ﬁbronectin, ﬁbrinogen, denatured or proteolysed collagen,and other matrix proteins). Several integrins recognise thetripeptide Arg-Gly-Asp (e.g., αvβ3, α5β1, αIIbβ3), whereasothers recognise alternative short peptide sequences (e.g.,integrin α4β1 recognises EILDV and REDV in alternativelyspliced CS-1 ﬁbronectin). Inhibitors of integrin functioninclude function-blocking monoclonal antibodies, peptideantagonists, and small molecule peptide mimetics matrix[21–23].The positioning of integrin receptors acts as a directbridge between the extracellular matrix and the internalcell cytoskeleton by transducing key intracellular signalsby associating with the clusters of kinases and adaptorproteins in focal adhesion complexes. Integrins thus act asmediators in transmitting diﬀerent signals from “inside out”(intracellular to extracellular) and “outside in” (extracellularto intracellular) between ECM to cells and vice versa.Through these pathways, ECM proteins are able to controlproapoptotic and antiapoptotic cascades by regulating theactivity of caspase 8 and caspase 3 [24–26]. ECM-integrininteractions thus determine the balance of apoptotic and cellsurvival signals and maintain the homeostasis of organs andtissues. Although integrins lack kinase activity, by inter- andintramolecular clustering, they recruit and activate kinases,such as focal adhesion kinases (FAKs) and src family kinases(sFKs) to a focal adhesion complex. In addition to scaﬀoldingmolecules, such as p130 CRK-associated substrate (p130CAs;also known as BCAR1), integrins also couple the ECMto actin cytoskeleton by recruiting cytoskeletal proteins,including talin, paxillin, α-actinin, tensin, and vinculin.Additionally, they form a ternary complex consisting of anintegrin-linked kinase, PINCH, and parvin to regulate manyscaﬀolding and signalling functions required for integrin-mediated eﬀects on cell migration and survival .5. Integrin Expression and Signalling inCancer ProgressionAlthough anchorage-independent growth is a hallmark ofmalignant transformation, integrins expression levels andactivity are an important role in diﬀerent steps of tumour
4 International Journal of Cell Biologyprogression including initiation . Higher expressionof α3, α5, α6, αv, β1, β4, α6β4, α9β1, αvβ5, and αvβ3integrins is directly correlated with the progression of thedisease . Several epithelial cell tumors showed the alteredα6β4, α6β1, αvβ5, α2β1, and α3β1 integrin expression .Integrin recruitment to membrane microdomains has beenshown to be regulated by tetraspanins and crucially regulateintegrin function in tumour cells . Recent studies havedemonstrated that cell signalling generated by growth factorsand oncogenes in transformed cells requires collaborationwith speciﬁc integrins, especially during tumour initiation.In tumor cells, several survival signals are upregulated uponintegrin ligation, which includes increased expression of BCl-2 or FlIP (also known as CFlAR), activation of the PI3K-AKTpathway or nuclear factor-κB (nF-κB) signaling, and/or p53inactivation .Invasive cancer cells evacuate from the primary site andmigrate to the secondary site by the process of tissue invasionand cell migration. Integrin-mediated pathways involvingfocal adhesion kinase (FAK) and src family kinase (SFK)signaling play a major role in this. In order to survive inthe new location and to withstand the stressful conditions ofhypoxia, nutrient deprivation, and inﬂammatory mediatorsthe migratory cells increase the blood supply to themselvesby neoangiogenesis. This is achieved by increased expressionof αvβ3 and αvβ5 integrins and by deposition of provisionalmatrix proteins such as vitronectin, ﬁbrinogen, von Wille-brand factor, osteopontin, and ﬁbronectin in the tumourmicroenvironment. Interaction between these moleculesplays a critical role in the process of generating new bloodvessels in the newly formed tumor site [31, 32].Integrins found on tumour-associated normal cells,such as the vascular endothelium, perivascular cells, ﬁbrob-lasts, bone-marrow-derived cells, and platelets, also have aprofound eﬀect in tumor progression via integrin-mediatedpathways. A summary of these has been given in Table 1 [33–47].6. Genetic and Chromosomal Aberrations atthe Onset of CancerNeoplasia occurs when cells are exposed to cancer-promoting substances that cause single or multiple prema-lignant genetic/epigenetic changes which may coalesce toform a large lesion. These genetic changes may have neutral,deleterious, or advantageous eﬀects on the proliferation ofa clone or clones of cells. Neutral or deleterious geneticchanges may result in stagnation or cell death, whereasthe cell receiving advantageous events may result in higherproliferation, recruitment of blood vessels to the developingtumors, and gain the ability to metastasize . The model ofBraakhuis et al., 2004 , advanced this idea by suggestingthat initial genetic alterations occur in stem cells, forminga patch and expanding ﬁeld of cells with the originaland subsequent genomic and or chromosomal alterations.Then, clonal selection of one or more cells within thisﬁeld of preneoplastic cells leads to the development of acarcinoma. There is a considerable cytogenetic variabilityamong cells reﬂecting heterogeneity due to clonal evolutionwithin the original tumor . Initial heterogeneity or cell-to-cell diﬀerences in cancer are due to cytoskeletal alter-ations which result in defecting chromosomal segregationand lead to karyotypic variations during mitosis, causingchromosomal aberrations, for example, NUMA1 gene at11q13, which results in multipolar spindles, leading todaughter cells that diﬀer from each other and their mothercells . Structural chromosome alterations also occurdue to deletions, translocations, isochromosomes, dicentricchromosomes, and endoduplicated chromosomes. The gainor ampliﬁcation of chromosomal segments is driven by morethan one gene . Structural rearrangements involving thecleavage and fusion of centromeres from participating chro-mosomes, also referred to as Robertsonian translocations,are the most frequently observed alterations. Chromosomalaberrations identiﬁed with the help of cytogenetic methodsincluding FISH, cCGH, or aCGH showed the gain of theentire long arm of chromosome 3 which ampliﬁes theEGFR gene in SCCHN , 8q24 gain to amplify MYC andPTK2 in primary tumors, 11q13 ampliﬁcations to amplifycyclin D1 gene, loss of 3q14 causes deletion of fragile siteFRA3B/FHIT, necessary to protect cells from accumulationof DNA damage . Aberrations mainly in chromosome13 and also involving chromosomes 6, 11, 12, and 17 areassociated with B-CLL .7. Anchorage-Independent TumorModel SystemThe wide range of in vivo tumor models like syngeneic,human tumor xenograft, orthotopic, metastatic, and genet-ically engineered mouse models is available from the basisof the compounds selected and treatments that go intoclinical testing of patients . The ability to exhibitanchorage-independent cell growth (colony-forming capac-ity in semisolid media) has been considered to be funda-mental in cancer biology because it has been connected withtumor cell aggressiveness in vivo such as tumorigenic andmetastatic potentials and also utilized as a marker for invitro transformation. Although multiple genetic factors foranchorage-independence have been identiﬁed, the molecularbasis for this capacity is still largely unknown [56, 57].During the process of in vitro tumorigenesis, various onco-genes with distinct pathways have been shown to transformanchorage-dependent cells to anchorage-independent cells[5, 57]. For example, transfer of c-Myc (a transcriptionfactor), v-Src (a tyrosine kinase), or H-Ras (a smallGTPase) into spontaneously immortalized mouse embryonicﬁbroblasts (MEFs) provides the cells an ability to grow inan anchorage-independent manner [56, 57]. Anchorage-independent multicellular spheroids made by Ewing tumorcell lines were more closely related to primary tumors withrespect to cell morphology, cell-cell junctions, proliferativeindex, and kinase activation .However, changes in ECM and cell adhesion molecularinteraction and genetic variations were observed till thedate only with the primary or secondary tumors. We have
International Journal of Cell Biology 5Table 1: Various integrins in association with diﬀerent ligands to induce diﬀerent signaling pathways in generation of tumor and metastasis.Integrin type Interacting ECM protein Activated signaling cascade Tumor/metastasis Referenceα3β1 LamininMMP9 and oncogenic Ras,VEGF, FAK-paxillin signalingcascadeInvasion in keratinocytes,Induces angiogenesis,Human hepatoma cells[33, 34]α6β1 LamininUrokinase plasminogen activatorand MMP-2, PI3Kinase, SrcTumor invasion in pancreaticcells[35, 36]α7β1 Laminin Rho-A signaling cascade Invasion in breast cancer α2β1, α1β1, α10β1,αIIβ1Collagen FAK and src signalingInvasion of melanoma cells,cancer progression, and invasionof lung adenocarcinoma[38, 39]αvβ1, αvβ6, αvβ3Vitronectin, syndican,thromospondin-1MMP9, urokinase signaling,MEK/Erk/NF-κB, PKCa, FAKMetastatic breast Cancer,pancreatic, cervical, colon,lung/liver metastasis[40–42]α9β1 CCN3, osteopontinSrc, P130 Cas, Rac, NOSsignalingMetastatic potential αIIb3, αvb3 Von Willebrand factorInteracts withthrombospondin-1 and inducesVEGF/FGF signalingBreast cancer [44, 45]α5 FibronectinFAK, ERK, PI-3 K, ILK, andnuclear factor-kappa B -Metastatic lung and cervicalcancer[46, 47]αLβ2Intercellular cell adhesionmoleculesBreast cancer developed a cellular model system by using normal, adherentrat ﬁbroblast cell lines. These cells lose their cytoskeletalorganization and speciﬁcity to ﬁbronectin as α5β1 integrinsare constantly recycled between cytoplasm and plasma. Inthe drastic unfavorable stressful conditions, the mechanical,phenotypic, and genetic characteristics are altered/modiﬁedto sustain their identity [5, 26]. We observed that this cellularmodel system represents a tumor with all characteristicsof cancer described by Hanahan and Weinberg 2011 ashallmarks of cancer.The cells during the nonadhesion process can evadefrom cell death by caspase 3 interaction with unligated α5β1integrins inducing resistance to integrin-mediated death(IMD) and also gain the ability to metastatise. Mutationalchanges mainly with 2;6 Robertsonian’s translocation andactivated Ras, FAK, and PKC provide self-suﬃcient growthsignals potential for uncontrollable growth of the cells(Figure 2). Upregulated Spp1, MMP3, Egfr, Rb1, Ddit3,Egln3, Vegfa, Stc1, Hif1a, MMP3, and altered pathwayslike glycolysis/gluconeogenesis and hypoxia (Pfkm, HK2,Pdk1, Adh1, aldh3a1, and Slc2a) lead the cells to invade,metastasize, and sustain angiogenesis. We observed anotherphenomenon of dediﬀerentiation by gaining the stem-cell-like and multidrug resistance properties by expressing Cd133and ABCG-2 when the cells are exposed to unfavorablecondition .The anchorage-independent cellular model system rep-resents a multicentric tumor model system apprehendedwith genes related to tumor progression, angiogenesis, andmetastasis (Figure 2). It is very advantageous, convenient,and possible model system to study the eﬀect of variouscancer-mediated drugs at the initial stage itself for the properdiagnosis.8. Integrin Signalling as a Target inCancer TreatmentSeveral studies showed the correlation of integrin inhibitionat any point of its action will lead to the inhibition oftumor progression . Therefore, integrins are focusedpharmacologically in the treatment and prevention of cancer.Antagonists of these integrins suppress cell migration andinvasion of primary and transformed cells by inducingapoptosis in primary cells could block tumor angiogenesisand metastasis. Recycling integrins present on the surface ofendothelial cells are targeted in the blood stream by exposingto the circulating drugs and agents . Various antibod-ies, cyclic peptides, disintegrins, and peptidomimetics aremeant to bind the targeted integrins to prevent integrinligation. cRGD, cyclic arginine-glycine-aspartic acid; RGDK,arginine-glycine-aspartic acid-lysine; TRAIL, tumour necro-sis factor-related apoptosis-inducing ligand are being usedas antagonists integrins to hit the integrin ligand function.The function of upregulated αvβ3 integrin can be blocked byfunction-blocking monoclonal antibodies, such as LM 609. The human αv integrin speciﬁc monoclonal antibodyCnTo 95, which targets both αvβ3 and αvβ5 integrinsto induce endothelial apoptosis, also had antitumour andantiangiogenic eﬀects in xenograft tumour models .Cilengitide, inhibitor of both αvβ3 and αvβ5 integrinsand volociximab, a function-blocking monoclonal antibodyagainst integrin α5β1, inhibits angiogenesis and impedestumour growth [61, 62]. αvβ3 is targeted by various ther-apeutic antibodies like LM609, vitaxin, humanized mousemonoclonal derived from LM609, CNTO 95, humanizedIgG1, c7E3, chimeric mouse human, 17E6, mouse mono-clonal antibodies to inhibit tumour growth, and angiogenesis
6 International Journal of Cell BiologyOncogenesHypoxiaGeneticinstability DedifferentiationTumorformationMetastasis/angiogenesisAlteredmetabolic pathways(Gluconeogenesis)Rb1, Rbpp9, Fos, Junb2; 6 RobertsoniantranslocationAldh3a1, Aldh6a1,Aldh3b1, Egfr, Eno2VEGF, STC1, MMP3Adh1, Adh2, Aldh3a1, Hk2PfkmHif1α, STC1, Vhl,FOSABCG2, CD133Transformed cellsTumour initiationTumor progressionFigure 2: This ﬁgure shows the hallmark characteristics of transformed cells at initial stages.in tumour xenografts and preclinical studies . Theprognostic α5β1 integrins in ovarian cancer can be targetedeﬀectively both in vitro and in vivo by using speciﬁc antibod-ies. Cell-mediated α5β1 adhesion can be blocked with a smallmolecule antagonist (SJ479), a ﬁbronectin-derived peptidesin prostate, and colon cancer models [64, 65]. Recent activityis extended to detect tumors and angiogenesis and deliver thedrugs to the site of cancer by coupling integrin antagonists toa paramagnetic contrast agent or radionuclide in rabbit andmouse tumour models [66, 67].9. ConclusionNormal cells lead to the transformation when exposed toadverse conditions such as anchorage independence andeﬀects are found to be similar to the eﬀect of carcinogensand mutagens. These cells could alter the ECM and other celladhesion molecules by showing altered integrins expressionon their surface and are associated with diﬀerent kinds ofgrowth factors and oncogene. ECM-integrin interactionsalong with other growth factors provide the diversiﬁedanchorage-independent signals to the transformed cells toprogress as cancers, metastasis, and angiogenesis. Severaltumours are sustained with diversiﬁed integrins found tobe speciﬁc to the tumour-host microenvironment. In future,these integrins can be targeted at the initial stages of cancerby using integrin antagonists to minimize the growth oftumour and metastasis.AcknowledgmentThis work was supported by Grant no. GAP0220 fromDepartment of Science and Technology, Government ofIndia, to G. Pande.References T. Tian, S. Olson, J. M. Whitacre, and A. Harding, “The originsof cancer robustness and evolvability,” Integrative Biology, vol.3, no. 1, pp. 17–30, 2011. D. Hanahan and R. A. Weinberg, “Hallmarks of cancer: thenext generation,” Cell, vol. 144, no. 5, pp. 646–674, 2011. C. Coghlin and G. I. Murray, “Current and emerging conceptsin tumour metastasis,” Journal of Pathology, vol. 222, no. 1, pp.1–15, 2010. C. M. Nelson and M. J. Bissell, “Modeling dynamic reciprocity:engineering three-dimensional culture models of breast archi-tecture, function, and neoplastic transformation,” Seminars inCancer Biology, vol. 15, no. 5, pp. 342–352, 2005. J. Rajeswari, R. Kapoor, S. Pavuluri et al., “Diﬀerential geneexpression and clonal selection during cellular transformationinduced by adhesion deprivation,” BMC Cell Biology, vol. 11,article 93, pp. 1–13, 2010.
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