REVIEW

PDGF: the nuts and bolts of signalling toolbox

Ammad Ahmad Farooqi & Salman Waseem & Asma M. Riaz & Bilal Ahmed Dilawar &

Shahzeray Mukhtar & Sehrish Minhaj & Makhdoom Saad Waseem & Suneel Daniel &Beenish Ali Malik & Ali Nawaz & Shahzad Bhatti

Received: 28 May 2011 /Accepted: 7 July 2011 /Published online: 19 July 2011# International Society of Oncology and BioMarkers (ISOBM) 2011

Abstract PDGF is a growth factor and is extensivelyinvolved in multi-dimensional cellular dynamics. Itswitches on a plethora of molecules other than itsclassical pathway. It is engaged in various transitions ofdevelopment; however, if the unleashed potentials leadastray, it brings forth tumourigenesis. Conventionally, ithas been assumed that the components of this signallingpathway show fidelity and act with a high degree ofautonomy. However, as illustrated by the PDGF signaltransduction, reinterpretation of recent data suggests thatmachinery is often shared between multiple pathways,and other components crosstalk to each other throughmultiple mechanisms. It is important to note thatmetastatic cascade is an intricate process that we haveonly begun to understand in recent years. Many of theearly steps of this PDGF cascade are not readilytargetable in the clinic. In this review, we will unravelthe paradoxes with reference to mitrons and cellularplasticity and discuss how disruption of signalling cascadetriggers cellular proliferation phase transition and metas-tasis. We will also focus on the therapeutic interventionsto counteract resultant molecular disorders.

Keywords PDGF/PDGFR .Mitrons . Cancer . EGCG .

Sulforaphane . Curcumin

Introduction

PDGFs are the growth factors which have a broadspectrum of implications. These factors are involved inmultifaceted mechanistic details. Various activities of thecell are triggered ranging from organogenesis to repair.The amplitude of the signals determines the fate of thecell. In case of derailed or deregulated transduction,error-prone activities are instigated. Recent data suggestthat various isoforms of PDGF bind with differentialaffinities to two related tyrosine kinase receptors,denoted the PDGF α- and β-receptors. The binding ofligand to native receptor induces receptor dimerization,creating receptor homo- or heterodimers. Dimerizationconsequently results in receptor autophosphorylation andkinase activation. It is intriguing to note that receptorautophosphorylation serves to regulate the kinase activityand to generate scaffolds for downstream signallingcomponents [1–9].

Substantial fraction of information has been added inunderstanding signalling events that drive PDGF signaltransduction cascade. Various key players have been wellcharacterized; however, other aspects of the signallingmechanisms involved are still uncertain [10–12]. Thisreview emphasizes on the major determinants of PDGF-mediated transduction cascade and explores how disparatesignalling pathways synergize to regulate cellular activity.Thus, we devote the various sections of this multi-partreview to discussing the undeniable role of PDGF signal-ling in smooth muscle cell regulation, angiogenesis andrespective emerging therapeutic paradigms.

Furthermore, we bring the PDGF-signalling pathwayinto focus, chronicling key concepts and recent advanceswith respect to microRNA (miRNA) signalling in normaland cancerous cells.

A. A. Farooqi (*) : S. Waseem :A. M. Riaz : B. A. Dilawar :S. Mukhtar : S. Minhaj :M. S. Waseem : S. Daniel :B. A. Malik :A. Nawaz : S. Bhatti (*)Institute of Molecular Biology and Biotechnology (IMBB),The University of Lahore,1 km defence road,Lahore, Pakistane-mail: ammadahmad638@yahoo.come-mail: drshahzadbhatti@yahoo.com

Tumor Biol. (2011) 32:1057–1070DOI 10.1007/s13277-011-0212-3

Differential PDGFR expression in vascular smooth musclecells

It is worth mentioning that serum-starved human arterialand venous smooth muscle cells (SMCs) showed differ-ential proliferative responses to PDGF isoforms. Datasuggested that arterial SMCs were strongly stimulated byPDGF-AA. On the contrary, venous SMCs exhibitedenhanced responsiveness to PDGF-BB. This differentialresponse was accredited to divergence in PDGF receptorexpression. There was a 2.5-fold higher (P<0.05) densityof PDGF receptor-α (PDGF-Rα) and a 6.6-fold lower (P<0.05) density of PDGF-Rβ expression while drawing aparallel between arterial and venous SMCs. Treatment ofarterial SMCs with PDGF-AA resulted in a markedPDGF-Rα activation, escalated phosphorylation ofERK1/2 and Akt and subsequent activation of c-JunNH2-terminal kinase (JNK) and a considerable suppres-sion in expression of the cell cycle inhibitor p27(kip1).However, there was PDGF-Rβ activation and simulta-neous epidermal growth factor receptor (EGFR) trans-activation upon treatment with PDGF-BB in venous SMCs[13].

It has recently been documented that PDGF receptor(PDGFR)-β expression is upregulated in high-grade astro-cytomas. Kaplan–Meier analysis confirmed that the densityof SMA-Vs, the size of SMA-Vs and PDGFR-β expressionwere major prognostic factors [14].

PDGF crosstalks in smooth cell regulation

PDGF signalling orchestrates with various signalling whichnegatively regulate PDGF signal dissemination. But thereare various situations in which mutations in the effectors ofvarious signalling cascades potentiate PDGF transductioncascade. Considerable information unfolds a correlationbetween various effectors of Wnt signalling with PDGFtransduction cascade. It has recently been shown thathuman atherosclerotic coronary arteries showed noticeablyenhanced expression of mutant LRP6 (R611C) and coloc-alization with PDGFR-β. It is interesting to note that wild-type LRP6 associates with PDGFR-β and enhances itslysosomal degradation. In accordance with the sameapproach, another receptor that co-existed with PDGFR-βis integrin αvβ3. This receptor offered binding site fortenascin-C. Treatment of smooth muscle cells withtenascin-C and PDGF-BB resulted in an enhanced prolif-eration [15, 16]. A detailed analysis of mutations in Wntsignalling is necessary to evaluate collaboration or conniv-ance with PDGF signalling. Treatment of pulmonaryarterial smooth muscle cell (PASMC) with PDGF inducedGSK3ß inactivation. However, treatment with the PDGFRinhibitor, imatinib, attenuated PDGF-BB-mediated GSK3ß

phosphorylation. This highlights the fact that Wnt signal-ling is positively regulated by PDGF signalling. In supportof this interpretation, a recent documentation suggested thatPDGF-BB treatment resulted in the stimulation of Wnt2and Wnt4 mRNA in proliferating vascular smooth musclecells (VSMCs) (Fig. 1b) [17, 18]. Further investigations areimportant to explore more components of positive feedbackloop that drives cellular disorders

It has lately been explored that PDGFR-β crosstalkswith EGFR upon activation and undergoes cleavage bymolecular scissor (ADAM17). This enzyme is also in-volved in cleavage of EGF from cell surface [19].

It is nonetheless recently investigated that PDGFRassociates with various receptors which potentiates anddrives derailed activities of core biological systems. In thefollowing section, we discuss the types of receptors thatresult in integration of linear transduction cascades.Moreover, we also summarize cytoplasmic effectors ofvarious signalling paradigms which coordinate or antago-nize PDGF-mediated signal communication.

It is intriguing to explore that adaptor protein FRS2is necessary for fibroblast growth factor receptor 1(FGFR1)-mediated phenotypic modulation and suppres-sion of VSMC smooth muscle alpha-actin (SMA) geneexpression. PDGF-BB and FGF2 act synchronously totrigger cell proliferation and downregulate SMA andSM22alpha in VSMC. PDGF-BB modulates tyrosinephosphorylation of FGFR1, and this phosphoproteomeactivity is mediated by PDGF receptor-beta. FRS2 co-exists with PDGFRbeta in a complex that requiresFGFR1, and both the extracellular and the intracellulardomains of FGFR1 are required for association withPDGFRbeta, whereas the cytoplasmic domain of FGFR1is required for FRS2 association with the FGFR1-PDGFRbeta complex. Abrogation of FRS2 in VSMC byRNA interference suppressed PDGF-BB-mediated down-regulation of SMA and SM22alpha; however, PDGF-BB-mediated cell proliferation or ERK activation was notdisrupted [20].

Data suggest that there is a tight interaction betweenPDGF and NRP as NRP1 and PDGFR-α co-localised inVSMCs. HCASMC migration induced by PDGF-BB andPDGF-AA was inhibited by NRP1 silencing and byadenoviral overexpression of an NRP1 mutant deficient inintracellular domain (Ad.NRP1ΔC) [21].

FGFR2 stimulates the expression of EGFR andPDGFRalpha via activation of PKCalpha-dependent AP-1transcriptional activity. Furthermore, Cbl-mediated degra-dation of EGFR is inhibited by enhanced association of Cblwith sprout which is triggered by FGFR. It is understand-able that deregulated molecular crosstalks between activat-ed FGFR2, EGFR and PDGFRalpha functionally contributeto the cellular disorders [22]. Mammalian target of

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rapamycin (mTOR) signalling is activated in PDGFRAmutants and in wild-type cases, which points towards thefact that mTOR or upstream mTOR inhibitors might offerexciting avenues [23].

Various regulators play a central role in the PDGF-mediated signal communication. In the subsequent section,we will discuss some effectors which are instrumental inPDGF signal transduction cascades. It seems obvious thatphosphatidylinositol 3-kinase (PI3K)-mediated activationof AKT is an important phosphorylation mechanism thatgoverns further downstream regulators. PI3K signalling isthe major initiator that further interconnects PDGF andinsulin signalling. It is important to reveal that modulatorsthat underlie actin rearrangements are regulated by PDGFsignalling. Detailed mechanistic insights in the next section

will enable us in interpreting the intricate network moreconceptually.

Mounting data suggest that PDGF signalling enhancesphosphorylation of protein kinases such as Akt, p70S6ki-nase and ERK1/2, which trigger VSMC proliferation. Thenon-receptor proline-rich tyrosine kinase 2 (PYK2) isactivated by a multiplicity of regulators and assembles acytoplasmic signalosome by crosstalks with PI3K andmitogen-activated protein kinase (MAPK) cascades. Inhi-bition of PYK2 attenuated PDGF-dependent signal trans-duction, which interrelated inhibition of AKT and ERK1/2but not p38 MAPK activation. It is becoming increasinglyapparent that PYK2 is an important upstream mediator inPDGF-dependent signalling cascades that regulate VSMCproliferation [24] (Fig. 1).

Fig. 1 a Crosstalks of PDGFRβand FGFR. b Association ofvarious types of receptors withPDGFR. Co-receptor andvarious cytoplasmic effectors ofWnt signalling associate withPDGFR in cellular disorders andstimulate the expression ofvarious target genes(Wnt2/Wnt4)

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Additionally, PDGF enhanced IRS-1/IRS-2 serine phos-phorylation and downregulated IRS-2 expression in aspatio-temporal manner. Outstandingly, abrogation of PI 3-kinase and mTOR attenuated PDGF-modulated Akt andp70S6kinase phosphorylation. Furthermore, IRS-1 serinephosphorylation and IRS-2 downregulation was alsoblocked. Contrary to this, MEK1/ERK inhibitor (U0126)failed to inhibit PDGF-induced IRS-1 serine phosphoryla-tion and IRS-2 downregulation. It is intriguing to note thatPDGF-mediated IRS-1/IRS-2 dysregulation resulted in thedampening of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Purposefully, targeted inhibition of PDGFreceptor tyrosine kinase with imatinib reversed IRS-1/IRS-2dysregulation and retrieved insulin receptor signalling [25].The integration of these multiple layers of new proteins willhave a major impact in translational oncology, leading tosignificant breakthroughs in rational drug design.

It has lately been explored that PDGF induced migrationin rat airway smooth muscle cells by increasing WASP andArp2/3 protein levels along with actin reorganization.However, treatment of cells with Slit2-N inhibited RASMcells migration by suppressing the expressions of WASPand Arp2/3 and consequent actin rearrangement [26]

It has lately been found that SMCs cultured in fibrinhydrogels have a more robust chemotactic response toPDGF-BB compared with FGF-2 [27]. Growth factors(GFs) have remarkable interactions with nonproteoglycanextracellular matrix proteins. In accordance with thisassumption, 12th–14th type three repeats of fibronectin(FN III12-14) were shown to have a higher affinity forPDGF. It is noteworthy that sprouting of human smoothmusclecell spheroids was significantly improved after administrationof FN III12-14 as a carrier of growth factors [28].

Impact of PDGFR fusions on cellular signalling

There are various instances in which there is a genomicrearrangement that results in the formation of fusion/chimericprotein. Focusing on the same concept, a research groupdocumented that occasionally, chimeric receptor, containingthe extracellular domain of hPDGFRbeta, gets fused to thetransmembrane and intracellular regions of discoidin domainreceptor (DDR1). Although chimeric receptors, which arecomposed of various combinations of intracellular regionsfrom DDR1 and TrkA (with the extracellular domain ofhPDGFRbeta), depict ligand (PDGF)-inducible receptorresponses. The DDR1 is characterized by a discoidin I motifin the extracellular domain, a juxtamembrane segment and akinase domain that is 45% similar in structure to that of theNGF receptor, TrkA [29]. Consistently, TEL-PDGFRbeta(TPbeta, also called ETV6-PDGFRB) and FIP1L1-PDGFRalpha (FPalpha) are fusion proteins which circum-vent Cbl-mediated degradation. Ubiquitination of TPbeta and

FPalpha was significantly suppressed compared to that ofwild-type receptors in cells expressing hybrid receptors [30].However, it has lately been documented that Lnkmimetic drugs might provide a novel therapeutic strategyas it is a negative regulator of PDGFR signalling, and itcan bind to the FIP1L1-PDGFRA fusion protein andattenuate signal dissemination [31]. Moreover, fusiononcoproteins (KANK1-PDGFRB) are involved in activatingSTAT independent of JAK. Additionally, phosphorylation ofphospholipase C-ERK1 and ERK2was triggered by this fusedoncoprotein [32].

Role of PDGF in different metastatic and angiogeniccascades

To ensure the correct balance between vascular mainte-nance, division and differentiation, several regulatorymechanisms that modulate the PDGF signal at many stagesof the pathway have been described. Here, we discuss thenature of the PDGF signal and evaluate the catalogue ofmechanisms that adjust it, demonstrating how the finemodulation of PDGF signalling in this context can result inprecise and robust control of vascular remodelling.

It is worth mentioning that quiescent vascular endothelialcells insensitive to PDGF-BB stimulation are re-sensitizedafter stimulation with FGF-2, which transcriptionally switcheson PDGF receptor expression in the activated endothelial cells[33]. Another interesting feature is that PDGF-BB triggeredMAP kinase activity and cell motility of isolated lymphaticendothelial cells. In vivo, PDGF-BB enhanced the growth oflymphatic vessels. Enforced expression of PDGF-BB inmurine fibrosarcoma cells induced tumour lymphangio-genesis, leading to enhanced metastasis in lymph nodes [34].

It is an intriguing feature that PDGFR signallingpotentiated epithelial mesenchymal transition (EMT) andcaused survival in murine and human mammary carcinomacell lines. Moreover, TGF-beta enhanced metastasis ofmammary tumours, induced EMT and escalated PDGFRsignalling [35]. PDGF and PDGFR-α were overexpressedin thymoma, especially in type B2 and B3, in the tumourepithelial cells [36]. Combinatorial blockade of PDGF andVEGFR might offer exciting avenues in management ofadvanced-stage mouse corneal neovascularization andintestinal-type/diffuse-type gastric carcinomas [37, 38].

Role of PDGF in breast cancer progression

Breast carcinogenesis arises from aberrant decisionmaking by cells concerning their survival or death,proliferation or quiescence, damage repair or bypass.These decisions are made by a plethora of growth factor-mediated molecular signalling networks that process

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information from outside and from within the breastcancer cell and commence responses that decide thecellular survival and invasive potential.

PDGF-D is found to be upregulated in invasive breastcancer cell lines; it also correlates with Notch-1 expressionand increases DNA binding activity of NFKB [39, 40].Furthermore phospholipase D is also an important memberof this molecular hierarchy including PDGF-D-inducedNFKB's activation. Two binding sites of NFKB arecritically important for transcriptional activation of PLD-1which is further involved in carcinogenesis [41]. Recentdata suggest that PDGF-BB is significantly higher inpatients with breast cancer compared to the benign breastdisease patients. It is therefore important that analysis ofangiogenesis markers in tumour and serum of breast cancerpatients using multiplex protein assay can enhance diagno-sis and prognosis in this disease [42]. On a similar note,another interesting piece of evidence is that human breastcancers express high levels of PDGF-D. Overexpression ofPDGF-D enhanced tumour growth and lymph nodemetastasis via uncontrolled cellular proliferation and inductionof CXCR4 expression. Abrogation of CXCR4 signallingcompromised PDGF-D-induced lymph node metastasis. Be-sides, enforced expression of PDGF-D increased perivascularcell coverage and normalized tumour blood vessels. Conse-quently, PDGF-D overexpression potentiated drug influx ofdoxorubicin and enhanced its treatment efficacy [43].

It is noteworthy that a considerable number of adiposetissue stem cells (ASCs) migrated towards the tumour-conditioned medium, and more importantly, migration ofhuman ASCs remarkably increased in response to increasedconcentrations of recombinant PDGF-BB. On the contrary,neutralizing antibodies to PDGF receptor-beta decreasedmigration of ASCs towards a breast cancer-conditionedmedium. These data advocate that tumour cell-derivedPDGF-BB is an imperative factor in governing themicroenvironment communication between tumour cellsand local tissue-resident stem cells [44].

MicroRNAs deal with PDGF: opposite sides of the same coin

MicroRNAs are major determinants in the post-transcriptionalcontrol of gene expression. After the classification ofhundreds of miRNAs, the challenge is now to comprehendtheir specific, biologically distinct and differential activity.Signalling pathways are important candidates for miRNA-modulated regulation owing to the sharp dose-sensitive natureof their implications and resultant effects. Undeniably,emerging verification suggests that miRNAs affect theresponsiveness of cells to signalling molecules such as PDGF.Nonetheless, miRNAs serve as nodes of intricate signallingnetwork that guarantee homeostasis and regulate cancer andmetastasis. This is illustrated in Fig. 2.

Growing evidence suggests that transcription of collagenis triggered by miR-29a. Another important facet is thatTGFbeta, PDGF-B or IL-4 reduced the levels of miR-29a innormal fibroblasts. However, abrogation of PDGF-B andTGFbeta pathways by treatment with imatinib recapitulatedthe levels of miR-29a [45].

PDGF is instrumental in cellular migration and oligo-dendrocyte differentiation. Interestingly, PDGF is transcrip-tionally regulated by mitrons which impact various cellularactivities.

It is also captivating to note that loss of miR-143/145results in the formation of podosomes, which are actin-richmembrane protrusions implicated in cellular migrationincluding SMCs. Consistent with the equivalent concept,PDGF modulates podosome formation in SMCs throughthe regulation of miR-143/145 expression. PDGF receptoralpha is the target gene of miR-143 [46]. Furthermore, miR-219 represses the expression of PDGFRalpha, whichnormally helps to promote oligodendrocyte precursor cellproliferation [47].

Another important miRNA believed to be involved inregulation of PDGF receptor is miR-9. It is an activation-induced regulator of PDGFR-β expression in cardiomyo-cytes that negatively regulates PDGFR-β expression uponligand stimulation through direct interaction with the 3′UTR of PDFGR-β [48]. There are various situations inwhich miRNA influences the PDGFR via intermediateproteins. In accordance with this conception, growth factor-induced miR-296 degrades the hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) mRNA, resultingin substantial decrease in levels of HGS and thus suppressingHGS-mediated degradation of the growth factor receptorPDGFRbeta [49]. Additionally, miRNA140 negatively regu-lates PDGF signalling during palatal development; however,disruption of PDGF signalling causes palatal clefting. ThePDGF receptor alpha (PDGFRα) 3′UTR contained amiRNA140 binding site functioning in the negative regula-tion of PDGFRα protein levels in vivo [50].

PDGF intracellular signalling pathways trigger geneexpression during differentiation by regulating microRNAexpression [51]. PDGF negatively regulates proteins andeffectors of TGF transduction cascade via mitrons. Concor-dant with the same approach, PDGF-BB induces microRNA-24 (miR-24), which in turn leads to downregulation ofTribbles-like protein-3 (Trb3). Suppression of Trb3 is tightlycorrelated with reduced expression of Smad proteins anddecrease in BMP and TGFbeta signalling [52]. Evidencesuggest that PDGF-D-induced EMT in prostate cancer,through suppression of miR-200. Therefore, miR-200 recon-stitution offers a promising approach for the treatment ofinvasive prostate cancer [53]. Various oncomirs are inducedby PDGF which potentiate uncontrolled cellular division. Inthis context, miR-221 is transcriptionally induced upon

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PDGF treatment in primary VSMCs and enhances cancerprogression [54].

PDGF's dynamics with LRP

PDGF signal transduction has emerged as a governing bodyof biological processes and propagation of information inthe cell. A wealth of information has been gained about thecentral regulation of PDGF pathway in endocytic traffick-ing and protein degradation

Lipoprotein receptor-related protein (LRP) has beenacclaimed as a guardian of PDGF/PDGFR. It crosstalkswith a broad range of proteins which underlie suppressionof PDGF signalling. In support of this notion, factor VII-activating protease (FSAP) can inhibit neointima formationand VSMC proliferation by cleavage of PDGF-BB.Protease nexin-1 inhibits the enzymatic activity of FSAP.FSAP–inhibitor complex is internalized via LRP whichresults in neutralization of its effect on PDGF-BB-mediated

VSMC proliferation [55]. On a similar note, LRP chaper-ones PDGFR from degradation. Cells deficient in LRPshowed an enhanced degradation rate of PDGFR; however,cells reconstituted for LRP restored the levels of PDGFR[56]. Activated PDGFR undergoes tyrosine phosphoryla-tion and consequently interacts with a variety of signallingmolecules, including PI3K as regulation of PI3 kinase byPDGFRbeta is necessary for vascular integrity [57].Captivatingly, MAPK is another downstream substratethat is documented to be modulated by PDGFR, andLRP abrogation resulted in suppression of MAPK-mediated signalling [58]. However, there is a documenta-tion that distinctly highlights the fact that abrogation ofLRP results in an enhanced accumulation of effectors ofTGF signalling in the nucleus and a robust expression ofPDGFRβ [59]. It appears as if it opens another avenue forinvestigation of transcriptome profiling in LRP-deficientenvironment. Diagrammatic representation of the activitiesis shown in Fig. 3.

Fig. 2 Involvement of PDGF in mediating transcriptional responses via miRNA

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PTP, PDGF and PKC: referee between two wrestlers

It is significant to note that there is a push and pull betweentwo diametrically opposed enzyme groups in regulatingphosphoproteome. The overexpression, hyperactivation orsuppression of these two proteins dictate endocytosis orrecycling of PDGFR, PKC and T cell protein-tyrosinephosphatase (TC-PTP) works in an anti-parallel manner;PTP attenuates recycling and enhances endocytosis. Con-versely, PKC is involved in the recycling of the receptor viaRab4a [60]. PKC and PTP are two opposing enzymes thatimpact phosphoproteome and resultant cellular activities.Concordant with the same concept, low molecular weightphosphotyrosine phosphatase (LMW-PTP) is involved inplatelet-derived growth factor-mediated cellular activities.In the forthcoming section, we bring to the limelight thefactors which underpin suppression and activation of PTP.

Evidence supports the notion that LMW-PTP docked toand dephosphorylated Tyr-857 of activated PDGFR, thusinhibiting the cell [61]. Another aspect in concordance withthe regulation was documented by a research group whosuggested increment in neointima formation upon PTPabrogation [62]. An additional aspect is that loss of TC-PTPrecapitulates PDGF beta receptor on the cellular surface viarapid recycling, which displays differential traffickingpatterns of PDGF receptor family members. Analysis byconfocal microscopy has shown that in TC-PTP ko mouseembryonic fibroblasts (MEFs), activated PDGF beta receptors

co-existed with Rab4a. Rab4a is a marker for retrieval ofendocytosed entities on the cell membrane. In agreement withthis, cells reconstituted for a dominant-negative Rab4a escalat-ed clearance rate of cell surface receptors on TC-PTP ko MEFs[63]. Analogously, hydrogen peroxide suppressed the activityof PTP [64]. Fascinatingly, hypoxia suppressed expression ofvarious PTPs (T cell PTP, density-enhanced phosphatase-1,PTP1B and SH2 domain-containing phosphatase-2), ensuingin reduced PTP activity. Hypoxia downregulates expressionand activity of PDGFR-β antagonizing PTPs in a HIF-1α-dependent manner [65]. Recent data suggest that lipidperoxides are earlier unrecognized mediators of oxidation ofPTPs. Nonetheless, it is a vital pathway for control of receptortyrosine kinase signalling, which might also underlie diseasesassociated with increased lipid peroxidation [66]. Hypoxiaand hypoxia/reoxygenation (H/R) escalates generation ofreactive oxygen species (ROS). Candidate molecular targetsof ROS are the catalytic site cysteine of PTPs [67, 68].Superoxide dismutase 1 (SOD1) is copper/zinc enzyme foundin the cytoplasm that converts superoxide into hydrogenperoxide and molecular oxygen. In addition, SOD1 inhibitioncauses the downregulation of the PDGF receptor [69].

PDGF and dorsal ruffle formation

The plasma membrane is the supramolecular assembly thatundergoes highly synchronized protrusions and invagina-tions that support the configuration of dorsal ruffles. These

Fig. 3 Effectors involved invarious PDGF-mediated cellularactivities

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modellings are triggered by highly conserved actin–mem-brane machinery. These transient surface membrane dis-tortions are discrete. Their role is to help the cell as itmigrates, attaches and invades.

PDGF modulates dorsal ruffle formation in coordinationwith various regulators. In this framework, mammalian actin-binding protein-1 (mAbp1) is localised to dorsal ruffles and isrequired for PDGF-mediated dorsal ruffle formation. Addi-tionally, mAbp1 directly interacts with the actin regulatoryprotein WASp-interacting protein (WIP) through its SH3domain as this interaction between mAbp1 and WIP issignificant in regulating dorsal ruffle formation [70]. Anotherprotein that is central in dorsal ruffle formation is Gab1,located downstream to PDGFR. Gab1 associates constitu-tively with the actin-nucleating factor N-WASP. FollowingPDGFR activation, Gab1 recruits Nck, an activator of N-WASP, resulting in assembly of signalosome, localised todorsal ruffles. Organization of dorsal ruffles involves interac-tion between Gab1 and Nck and also requires functional N-WASP [71]. Particularly, activated Rac1 mediates membranedorsal ruffle formation in response to PDGF. Similarly, Abl-interactor-1 and betaPIX, a guanine nucleotide exchangefactor for Rac1, localise at these Rac1-induced actinstructures and occupy central positions in the induction ofmembrane dorsal ruffling in response to PDGF in fibroblasts[72]. Similarly, a pool of PDGF-activated Src family ofprotein-tyrosine kinases (SFKs) that was insensitive tomembrane cholesterol depletion was found in non-caveolaefractions. Non-caveolin SFK activation was connected to thecompetence of PDGF to induce F-actin conformationalreorientations leading to dorsal ruffle formation [73]. On asimilar note, direct and specific interaction between palladinand Eps8 facilitates rapid and transient remodelling of theactin cytoskeleton, which drives the formation of highlydynamic membrane protrusions after treatment with PDGF[74]. Some proteins suppress the PDGF-mediated dorsalruffle formation. In concordance with this notion, cdc-42interacting protein 4 (CIP4) was observed to suppressPDGFR which blunted ruffle formation. However, knock-down of CIP4 recapitulated dorsal ruffle formation andcellular migration [75].

It is intriguing to note that PDGF induced a threefoldincrease in the proportion of PP2A activity regulated bycholesterol. On the contrary, cholesterol depletion inhibiteddorsal ruffle formation, decreased PP2A levels and increasedthe Hsp27-P to Hsp27 ratio. The documentation highlights thefact that Hsp27 is dephosphorylated by PP2A in dorsal ruffles,in non-caveolar lipid raft microdomains [76].

PDGF interaction with ATM and hetronuclear ribonucleoprotein

PDGF triggered interaction between hnRNP-K and themRNA-encoding myosin regulatory light-chain (MRLC)-

interacting protein (MIR). MIR is an E(3)-ubiquitin ligasethat is documented to be involved in degradation of MRLC.This sequentially rapidly increased MIR expression and ledto ubiquitination and proteasome-mediated degradation ofMRLC and subsequent actin rearrangements [77]. Recentdata suggest that hnRNP-K modulates the expression ofVEGF [78]. A detailed analysis is necessary to pinpoint theinvolvement of PDGF in stimulating the expression ofVEGF via hnRNP-K. Furthermore, attenuation of hnRNP-K suppressed the levels of active MEK and ERK whichalso confirms the fact that hnRNP-K potentiates the “MEK-and ERK-based linear transduction cascade [79].

Emerging evidence points towards an interaction betweenataxia telangiectasia mutated (ATM) and PDGF. It is ofparticular interest that PDGF beta receptor abrogationseverely compromised H2O2-induced ATM activation, indi-cating that ATM lies downstream to the PDGF beta receptorin this signalling cascade [80]. Furthermore, it has recentlybeen cited that PDGFR mediates cytoplasmic activation ofATM [81]. The evidence suggests that a complex barcodeunderlies the heterogeneous response of ATM towardsPDGF/PDGFR.

PDGF and non-invasive EMT

It is compelling to note that correct establishment andmaintenance of cell polarity are decisive for normal cellularphysiological activities. On the other hand, loss of cellpolarity is one of the hallmarks of cancer. In the upcomingsection, we will focus on identifying the stages of tumouraldevelopment that are affected by deregulation of PDGFlinear or integrated signal transduction cascade. Asymmet-ric division is a well-acclaimed regulatory mechanism thattriggers cell numbers and differentiation.

An imperative aspect of PDGF signalling is that it isinvolved in EMT but simultaneously involved in resistingcellular migration, which is an aspect of non-invasivetumour. It is convincing to note that TGFR independentPDGF transduction cascade was instrumental in EMT. EMTwas observed in both SMAD competent and deficient cells,which proves that PDGF-mediated EMT is irrespective ofTGF signalling [82]. The novel findings also show thatPDGF had a role in non-invasive EMT; in this mechanism,there is again no correlation between TGFβ/Smad signal-ling with that of PDGF. The phenomenon was confirmed inmesothelial cells by an increased SNAIL and decreased E-cadherin expression with presence of epithelial and mesen-chymal markers [83]. Scientists have started unveiling thelinks between cell polarity and asymmetric cell division inthe context of cancer. Consistent with same approach,apical–basal polarity and cell–cell adhesion are tightlyinterconnected. Therefore, how loss of cell polarity inepithelial cells may promote epithelial mesenchymal tran-

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sition and metastasis needs a detailed investigation.Altogether, it is obvious that PDGF signalling is contribu-tory in loss of epithelial cell polarity and may have animportant role in both the initiation of tumourigenesis andin later stages of tumour development.

PDGF as a major challenge in translational oncology

Recent structural data support the idea that PDGFR is asophisticated machine with multiple signalling outputs. Mis-representation of growth factor signalling is the most impera-tive prerequisite in tumour progression. PDGF signallingmodulates tumour progression by a tumour cell-autonomousmechanism or through tumour–stroma interaction. Doubtlessly,it has either a tumour-suppressing or tumour-promotingfunction depending on cellular context. Many cellularcontext-dependent factors tightly maintain the equilibrium ofPDGF signalling and contribute to the regulation of PDGF-induced cell responses.

Keeping in view some advances in the past few years,the investigation for rational drug design for advancedcancer is far from over. Existing data clearly reveal that thedesigning of new drugs will have minimal, if any,probability of success if it is not guided by comprehensiveknowledge of disease biology. On the other hand, usingbiologic agents to target key molecular pathways, such asthose modulated by PDGF/PDGFR family members, maybe effectual. Without a doubt, there is a necessity toevaluate the efficacy of chemotherapeutic drugs, miRNAand phytonutrients alone and in combinations to explore thepositive results achieved in various phases of clinical trialsin patients that support this approach. Many new anti-PDGF molecules are now under evaluation for thetherapeutic intercession of cancer, but hitherto efforts toidentify reliable predictive factors from phase I and II trialshave produced unconvincing results. This corroborates thesuggestion that refining patient selection is critical to takefull advantage of the benefit of targeted agents, tocircumvent significant toxicities and for the developmentof alternative therapeutic approaches in patients who havenonresponsive disease.

There is a remarkable expression profile of PDGF-α andPDGF-β in endometrial stromal sarcoma [84]. PDGFRactivity is deregulated in human glioblastoma that results inthe activation of downstream kinases such as PI3K, Akt andmTOR [85]. Recent data suggest that synergistic adminis-tration of perifosine and CCI-779 is effective in themanagement of the disease. On a similar note, it has beenrecently documented that imatinib is not efficient in themanagement of glioblastoma [86]. However, some com-pounds have efficacy and offer therapeutic potential. In thiscontext, 2,3,4′,5-tetrahydroxystilbene-2-O-β-d-glucoside(TSG) is suggested to be effective in inhibition of PDGF-

BB-stimulated VSMC proliferation via cell cycle arrest[87]. Similarly, 1,4-naphthoquinone derivative also hasinhibitory effects [88]. Furthermore, benzylideneacetophe-none analogues are also effective in the suppression ofPDGF signal transduction cascade [89].

Mounting evidence signifies the fact that multiprongedapproach is more potent and effective in the clinicalmanagement. In support of this notion, two tyrosine kinaseinhibitors, imatinib and vatalanib, are documented toincrease the effects of paclitaxel on PDGF-BB tumours.Consistent with the same concept, imatinib and carboplatinhave enhanced efficiency in suppressing VEGF, PDGF andPDGF-Rα/ß expression in head and neck squamous cellcarcinoma [90, 91]. Docetaxel has been currently acclaimedas an inhibitor of PDGF; however, there are controversialfindings, and future research is necessary to unveilopportunities and challenges in the standardization oftherapy [92, 93]. Bevacizumab has been used in combinationwith (PDGF aptamer, AX102) and displayed therapeuticefficacy [94].

Recently, there is a paradigm shift in the molecularmedicine. To enhance penetration of doxorubicin, it wasconjugated to pPB-HSA through an acid-sensitive hydra-zone linkage. Results suggested that treatment with Dox-HSA-pPB considerably suppressed the C26 tumour growthin mice whereas free doxorubicin-treated mice had lowerresponse to the therapy [95]. Furthermore, pazopanib is asmall-molecule inhibitor of PDGF receptors [96, 97].Baicalin, an herb-derived flavonoid compound, has beenrecently shown in inducing growth arrest of PDGF-stimulated VSMCs [98]. Cediranib is a potent therapeuticintervention for inhibition of VEGF receptor (VEGFR)-2and VEGFR-3 tyrosine kinases. However, recently, itsefficacy has been evaluated in tumour cell lines having anenhanced expression of PDGFR α and PDGFRβ. Further-more, in vivo, ligand-induced PDGFR-β phosphorylationin murine lung tissue was inhibited by 55% followingtreatment with cediranib. Similarly, rat glial tumour xeno-grafts in mice displayed suppression of ligand-inducedphosphorylation of both PDGFR-α and PDGFR-β by 46%to 61% with cediranib [99].

Recently, Affibody molecules have occupied top slot inthe spotlight. They comprise a class of very small bindingproteins that are highly appropriate for in vivo imagingapplications and that can be selected to exclusivelyrecognize a target protein. PDGFRβ-specific Affibodymolecules are effective as they accumulated aroundtumoural blood vessels in a model of spontaneous insuli-noma, confirming a prospective for in vivo targeting [100].

On a similar note, single-chain antibody fragments(scFvs) targeting PDGFRbeta and VEGF-A have currentlygained attention for superior stability. The scFvs were fusedto both termini of human Fc to engineer a bispecific,

Tumor Biol. (2011) 32:1057–1070 1065

tetravalent molecule. This molecule has shown potentactivity, binds both targets concurrently and is stable inserum [101].

Accumulating data points towards that disruption inPDGF signalling and primary dysfunction in the tumourmicroenvironment, in addition to epithelial dysfunction,can be fundamental for carcinogenesis. These currentfindings make a convincing case for a multiprongedapproach for targeting the microenvironment for cancerchemoprevention. It is believed that further investigationswill unravel mechanistic insights of pathophysiology ofthe microenvironment and new approaches to control itwith chemopreventive agents. It is interesting to note thatmicroenvironment of a cancer is an integral part of itsanatomy and physiology, and functionally, one cannotentirely dissociate this microenvironment from what haveusually been called ‘cancer cells’. There is a need foreffective clinical implementation of this knowledge inpreventive strategies.

Impelling evidence points towards the fact that duringhepatic fibrogenesis, suppression of peroxisome proliferator-activated receptor-gamma (PPAR gamma) resulted insimultaneous activation of PDGF and epidermal growthfactor (EGF) signalling in hepatic stellate cells (HSCs).However, curcumin restored the activities of PPARgamma and concomitant suppression of PDGF and EGFsignal transduction cascade.It is also documented to beinvolved in downregulation of PDGF-BB, PDGFRbeta inHSCs [102, 103].

Another attention-grabbing feature is that anthocyaninspresenting a hydroxyl residue at position 3′ suppress PDGF(AB)-induced VEGF expression by inhibiting p38 MAPKand JNK in VSMCs [104]. On a similar note, resveratrolconsiderably suppressed PDGF-stimulated c-Src and Aktkinase activation. It suppressed PDGFR phosphorylation atthe PI 3 kinase and Grb-2 binding sites tyrosine-751 andtyrosine-716, respectively, and remarkably escalated theactivity of PTP1B. This phosphatase is involved indephosphorylation of PDGF-stimulated phosphorylation attyrosine-751 and tyrosine-716 on PDGFR with concomitantreduction in Akt and Erk1/2 kinase activity [105]. Recentdata highlight the fact that piceatannol (a metabolite ofresveratrol) suppressed PI3K activity more effectively thanresveratrol [106]. Similarly, pterostilbene, a natural dime-thylated analogue of resveratrol, suppressed PDGF-BB-mediated phosphorylation of Akt kinase [107].

It is attractive to interpret that lycopene inhibited PDGF-AA-induced SMC and fibroblast migration in a concentration-dependent manner. Mounting data suggest that lycopene-binding region exists within PDGF and is located at loop 2region [108]. Consistent with same line, lycopene restrictsPDGF-BB-induced cellular migration through inhibition ofPI3K/Akt, ERK and p38 activation [109, 110].

Conclusion

In the quest to recognize PDGF signalling, great strideshave been made to comprehend how these proteins controltheir downstream targets. However, scores of mechanismsby which these proteins generate linear or integratedcascades remain obscure.

With an addition of substantial fraction of elucidations tothe pre-existing understandings of PDGF, it is now evidentthat an integrated network of proteins triggers the dynamicsof the cell. Another thing that cannot be overlooked is thecrosstalks of two linear transduction cascades. This inte-grated framework works with striking synergy duringtumour development. It is noteworthy that in cancertherapy, the identification of novel and potent PDGFR/EGFR inhibitors with preferred kinase inhibitory spectrumthat provides superior antitumour efficacy, although withmanageable side effects and toxicities, will continue to bethe key for success. Additionally, interest in targeting PDGFsignalling for intervention of cancer will surely addsufficient information into the existing pool of concepts ofclinical management. On a similar note, clinical successesof these agents are unquestionably based on the broadeninglandscape of knowledge on the molecular mechanisms thatunderpin the development and progression of a malignantphenotype. It is necessary to revisit the existing web ofproteins with reference to PDGF signalling to tailor someeffective clinical outcomes.

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