JOURNAL OF INTERFERON & CYTOKINE RESEARCH 27:543–552 (2007)© Mary Ann Liebert, Inc.DOI: 10.1089/jir.2007.0009

Transforming Growth Factor-� Signaling in Normal andMalignant Hematopoiesis

IRIS ISUFI,* MAHESH SEETHARAM,* LI ZHOU, DAVENDRA SOHAL, JOANNA OPALINSKA, PERRY PAHANISH, and AMIT VERMA

ABSTRACT

Transforming growth factor-� (TGF-�) is an important physiologic regulator of cell growth and differentia-tion. TGF-� has been shown to inhibit the proliferation of quiescent hematopoietic stem cells and stimulatethe differentiation of late progenitors to erythroid and myeloid cells. Insensitivity to TGF-� is implicated inthe pathogenesis of many myeloid and lymphoid neoplasms. Loss of extracellular TGF receptors and disrup-tion of intracellular TGF-� signaling by oncogenes is seen in a variety of malignant and premalignant states.TGF-� can also affect tumor growth and survival by influencing the secretion of other growth factors andmanipulation of the tumor microenvironment. Recent development of small molecule inhibitors of TGF-� re-ceptors and other signaling intermediaries may allow us to modulate TGF signaling for future therapeutic in-terventions in cancer.

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INTRODUCTION

TRANSFORMING GROWTH FACTORS-� (TGF-�s) are membersof a large superfamily of polypeptide growth factors that

control the growth, proliferation, differentiation, and apoptosisof cells of multiple lineages. They can exert either a positiveor a negative effect based on the concentration and isoform ofTGF-�, the developmental stage of the target cell, the local en-vironment in vivo, and serum factors in vitro. Because hema-topoiesis is finely regulated by cytokines, dysregulation ofTGF-� signaling can cause abnormalities in development ofcells of different lineages and can give rise to the developmentof various hematologic disorders and malignancies.

TGF-� SIGNALING CASCADE

There are three mammalian isoforms, TGF-�1, TGF-�2, andTGF-�3, of which TGF-�1 is the most abundant. TGF-� is syn-thesized as a prepropeptide and requires a two-step process ofproteolytic degradation to give rise to active TGF-�.1 MatureTGF-� is secreted into the extracellular matrix (ECM) as an in-active complex linked to its propeptide by noncovalent bondsand to a member of a family of high molecular weight proteins

called latent TGF-� binding proteins, or LTBPs.2,3 This pre-vents TGF-� from inadvertently binding to its receptors. Thereare several mechanisms of activation of latent TGF-�s, includ-ing those initiated by plasmin, metalloproteinases, and throm-bospondin.4–7

Once activated, TGF-�s mediate their actions via a well-es-tablished mechanism, by binding to three cell surface receptors,known as T�RI, T�RII, and T�RIII.8 The type III receptor (be-taglycan) presents ligand to the type II receptor, has no signal-ing role,9 and is not present on the cell surface of hematopoi-etic progenitors.10 The type I and type II receptors containserine/threonine kinases in their distinct intracellular domains.TGF-� recognizes several cysteine residues in the short extra-cellular domain of the type II receptor. Once bound to TGF-�,T�RII recruits T�RI into a heteromeric complex and transpho-sphorylates it.11 Transphosphorylation of T�RI activates itsprotein kinase activity, allowing it to propagate the signal down-stream.12,13

The TGF-� signal is propagated downstream of the recep-tors through a small family of structurally related proteinsknown as Smads (homologs of Drosophila mothers against dpp[decapentaplegic function in Drosophila melanogaster]).Smads may either activate or repress transcription by interact-ing with DNA binding transcription factors as well as via an

Albert Einstein College of Medicine, Bronx, NY 10461.*These authors contributed equally to this work.

REVIEW

intrinsic DNA binding capability.14–16 There are three distinctfunctioning subclasses of Smad proteins.17 The first group, theR-Smads (for receptor-activated) are phosphorylated by acti-vated T�RI at their carboxy-terminal.18 R-Smads includeSmad2 and Smad3.19 Phosphorylated Smad2 and Smad3 inter-act with the second group of Smads, the co-Smads (for com-mon partner), to form heterodimeric complexes, which translo-cate to the nucleus to regulate transcription. Smad4 is the mostcommon co-Smad.20 The third group is composed of the in-hibitory Smad6 and Smad7, which form stable complexes withactivated type I receptors and prevent phosphorylation of R-Smads.21 Smad7, which is activated by direct binding of Smad3and Smad4 to its promoter, serves as a TGF-�-induced nega-tive feedback loop.22

TGF-� may stimulate or inhibit cell proliferation, differen-tiation, motility, adhesion, or death depending on the type anddevelopmental state of a cell. Inhibition of cell proliferation isthe most common biologic effect and is mostly mediated viaeffects on cell cycle machinery.15 In addition to causing a G0/G1

cell cycle arrest in a variety of cell types,23–25 TGF-� also reg-ulates the expression of ECM proteins, including collagens andfibronectin. In addition to these two biologic processes, TGF-� can modulate a host of other processes in hematopoetic de-velopment and negatively regulate the pathogenesis of hema-tologic malignancies.

TGF-� IN NORMAL HEMATOPOIESIS

Hematopoiesis is the process during which pluripotent stemcells give rise to lineage-committed progenitors that differenti-ate into mature lymphoid and myeloid cells. Several membersof the TGF-� superfamily, including TGF-� bone morphogenicproteins (BMPs) and activin, are known to regulate the fate ofhematopoietic progenitor cells (HPCs). Each of these peptidegrowth factors mediates its effects through select members ofthe Smad signaling cascade, with considerable overlap andcross-talk between them. For example, BMPs can regulate thedevelopment of human hematopoietic stem cells (HSCs)through Smad 1 and Smad 5, although26 Smad5 may also beactivated by TGF-�. Suppression of Smad5 expression by an-tisense oligonucleotides against Smad5 mRNA has been shownto partially reverse inhibition of primitive HPCs by TGF-�,27

demonstrating cross-talk between BMP and TGF signaling.Studies have also shown that there is significant cross-talk be-tween the Smad pathway and several other signaling pathwaysin hematopoiesis. For example, TGF-� can signal through mi-togen-activated protein kinases (MAPKs), NF-�B, or phospha-tidylinositol 3-kinase/AKT (PI3K/AKT) pathways28–30 in he-matopoietic and other cell types.

In vitro murine and human studies demonstrateinhibitory effects of TGF on primitive progenitors

TGF-� has been shown to selectively inhibit the growth anddifferentiation of early HSCs, which are largely quiescent, butnot of more mature progenitors. In an early murine study, it in-hibited the growth and colony formation of interleukin-3 (IL-3) dependent bone marrow cells but had no effect on the proliferation and differentiation induced by later acting hema-topoietic growth factors, such as granulocyte colony-stimulat-

ing factor (G-CSF), granulocyte-macrophage CSF (GM-CSF),and erythropoietin (EPO).31

Studies in human hematopoietic cells demonstrated thatTGF-� selectively inhibited colony formation from earlyCD34�CD33� progenitors and did not affect more differenti-ated G-CSF-responsive cells.32–34 In some of these studies, thesame effects were observed with both TGF-�1 and TGF-�2,whereas in others, HPCs were shown to be much more sensi-tive to inhibition of colony formation and antiproliferative ef-fects of TGF-�1 than TGF-�2.32–34

As the cited studies used freshly aspirated unseparated bonemarrow cells, it was possible that the action of TGF-� on theseHPCs was indirectly due to its effects on other subpopulations.A later study demonstrated that TGF-� can inhibit the growthof single cell suspensions of primitive Thy�Lin� murine pro-genitors. Additionally, the study showed that TGF-�1 can alsoinhibit the growth of high proliferative potential colony-form-ing cells (HPP-CFC), the earliest HPCs measured in vitro.35

The effects of disrupting TGF-� signaling at a single cell levelwere also studied using a dominant negatively acting mutant ofTGF-� type II receptor. Adenoviral vector-mediated gene de-livery was used to transiently block TGF-� signaling, and thiswas shown to enhance the survival, proliferation, and growthkinetics of human HSCs with an HSC immunophenotype.36

Further evidence was obtained from a highly enriched popula-tion of Lin�, c-kit�, Sca1�, CD34� quiescent stem cells iso-lated from T�RI knockout mice that were cultured in serum-free medium with low stimulation (stem cell factor [SCF]alone). These T�RI null HSCs had a 2-fold increase in the num-ber of proliferating clones compared with control HSCs whenstimulated by active autocrine TGF-�1. These findings werelikewise consistent with an inhibitory effect of TGF-� on HSCproliferation in vitro.37

There are conflicting data about whether TGF-� is a com-mitted inducer of apoptosis or if its inhibitory effects on HPCsare reversible. It has been shown that TGF-� inhibits survivalof Lin�, Sca1� HPCs dormant in the G0/G1 phase of the cellcycle through induction of apoptosis as detected by an in situTdT assay.38 Conversely, another in vitro murine study sug-gested that TGF-�-induced inhibition of long-term repopulat-ing hematopoietic stem cells (LTR-HSC) via sustained down-regulation of bone marrow cell surface cytokine receptors wasreversible and not accompanied by programmed cell death.39

Other studies using human cells have confirmed this by show-ing that TGF-�1 is not apoptotic and can reversibly maintainprimitive human HPCs in quiescence. In these studies TGF-�1was also shown to upregulate expression of the CD34 mem-brane antigen, which is a marker of immature HSCs. These find-ings are important, as they point to a potential clinical utililityof TGF-�1 in maintaining pools of quiescent HSCs.

In vivo studies reveal bidirectional and lineage-specific control of hematopoiesis by TGF-�

The role of TGF-� in hematopoiesis has been studied in vivoeither through its direct administration or through the use ofmurine knockout models. Early studies demonstrated that lo-coregional administration of TGF-�1 into the femoral artery ofmice inhibited early HPC growth and differentiation in a time-dependent and dose-dependent manner. This effect was also re-versible.40 Subsequently, several in vivo studies were under-

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taken to directly assess the role of TGF-� signaling in hema-topoiesis.41,42 Homozygous deletion of TGF-� resulted in em-broyonic lethality with defective yolk sac vasculogenesis sec-ondary to abnormal endothelial differentiation and defectivehematopoiesis with a reduced yolk sac erythroid cell number.Whereas in vitro neutralization of TGF-� resulted in increasedhematopoietic cell growth and proliferation, in vivo, loss ofTGF-�1 function resulted in defective erythroid differentia-tion.43 Homozygous T�RII deletion causes an indistinguishablephenotype from that of TGF-�1 null mice, with defective vas-culogenesis and hematopoiesis.44 Mice lacking T�RI die as aresult of defects in vascular development of the yolk sac as well.In vitro, T�RI�/� endothelial cells have impaired migration andfibronectin production, which would explain the malformedvascular structures in vivo. To further study the role of defec-tive TGF-� signaling in hematopoiesis and HSC function invivo, T�RI was conditionally deleted in adult mice. Surpris-ingly, differentiation and maturation of hematopoietic cellswere normal in these adult mice. In vitro studies further showedthe size of the Lin�, c-kit�, Sca1�, CD34� quiescent stem cellpool isolated from these T�RI knockout mice was not af-fected.37 The authors determined this result to be due to thepresence of other negative regulators of stem cell activity thatmay have redundant or overlapping functions with TGF-�. Thisis possible, as HSCs require a tighter regulatory network withmore backup mechanisms than other cell types to minimize therisk of uncontrolled growth, leading to leukemia or premature

exhaustion of the stem cell pool.37 Thus, the role of TGF-�seems to be strongly contextual, and these apparent differencesbetween the in vitro and in vivo systems may come as a resultof the rich bone marrow environment filled with cytokines,overlapping signaling cascades, negative feedback loops, andECM components.

The role of TGF-� in the differentiation of committed HPCshas been clarified by many recent observations. It has been ob-served that TGF-�1 and TGF-�2 at low concentrations had astimulatory effect on day 7 human colony-forming units-GM(CFU-GM) induced by GM-CSF. The stimulatory effect wasexhibited in the form of increased number of granulocytecolonies, which points toward a role in progenitor cell differ-entiation.45 These observations were validated in murine mod-els, where low doses of TGF-� stimulated granulopoiesis pref-erentially.46 In erythropoiesis, TGF-�1 has also been shown tostimulate erythroid differentiation by triggering the conversionof early erythroid burst-forming units (BFU-E) into later BFU-E or CFU-E and by inducing hemoglobin synthesis in maturecells.47 A recent report showed that a nuclear protein, tran-scriptional intermediary factor 1� (TIF1�) mediates the differ-entiating effects of TGF-� in human hematopoiesis. TIF1�competes with Smad4 for Smad2/3 complex binding after TGF-� stimulation, and whereas Smad2/3-Smad4 complexes medi-ate the antiproliferative response, Smad2/3-TIF1� complexesmediate erythroid differentiation in response to TGF-�48

(Fig. 1).

TGF-� IN HEMATOLOGIC MALIGNANCIES 545

FIG. 1. TGF-� binding to its receptor complex initiates an intracellular signaling cascade that affects gene transcription in he-matopoiesis. The binding of TGF-� to its receptor leads to the phosphorylation of Smads2/3. The Smad4-Smad2/3 complex reg-ulates the antiproliferative effects of TGF-� on hematopoiesis, whereas the TIF1�/Smad2/3 complex regulates erythroid differ-entiation.

In contrast to such observations, TGF-�1 has been shown toinhibit both the early and the late stages of megakaryopoiesis.It inhibited all stages of the differentiation process dependingon the concentration and the combination of growth factorsused.49–51 Several of these growth, proliferation, and differen-tiation pathways affected by TGF-� in either an inhibitory orstimulatory way have physiologic relevance in the pathogene-sis of leukemias, myelodysplasia, and lymphoma.

TGF-� IN MALIGNANT HEMATOPOIESIS

Role of TGF-� in leukemia

Acute myeloid leukemia (AML) is characterized by malig-nant proliferation of leukemic cells, ultimately resulting in a sup-pression of the normal hematopoietic system. Several factorshave been implicated in contributing to the growth advantage ofthe malignant cells over their normal counterparts. Increased re-sponsiveness to stimulatory growth factors or insensitivity to in-hibitory growth factors can lead to transformation of cells and

render them resistant to normal homeostatic mechanisms.52 Insupport of these hypotheses, leukemic cell lines generally areless sensitive to TGF-�1 in vitro.25 Table 1 shows some of themany leukemia-specific mechanisms that have been describedto explain this loss of sensitivity to TGF-�.

As Smad family proteins are known critical components inthe TGF-� signaling pathway, mutations in the Smad 4 genehave been associated with the pathogenesis of AML.53 A mis-sense mutation, P102L, in the Smad4 MH1 domain causes dis-ruption of its DNA binding ability and disrupts TGF-�-medi-ated transcriptional activity. A second frame shift mutationresulting in termination in the MH2 domain, delta(483–552),prevents nuclear localization of Smad4 and thus disrupts TGFsignaling. Both these mutants also exert dominant negative ef-fects and can result in insensitivity of leukemic cells to TGF-�.53 Smad3 has been implicated in the pathogenesis of T cellleukemias. A relative reduction in Smad3 levels has been shownto impair the tumor suppressor ability of the TGF-� pathwayand can lead to oncogenic transformation in the presence of ad-ditional mutations. Loss of the p27 tumor suppressor gene incombination with one Smad3 allele results in spontaneous T

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TABLE 1. MECHANISMS THAT DISRUPT TGF-� SIGNALING IN HEMATOLOGIC MALIGNANCIES

Disease Mechanism Reference

Acute myeloid leukemia Missense mutation, P102L, in the Smad4 MH1 Imai et al.53

domain disrupts its DNA binding abilityFrame shift mutation in the Delta(483–552) Smad4

MH2 domain prevents its nuclear localizationAcute myeloid Oncoprotein AML/ETO physically associates with Jakubowiak et

leukemia with 8:21 Smads and interrupts TGF-�-mediated gene al.55

translocation transcriptionAcute promyelocytic c-PML binds to Smads and facilitates their association Lin et al.56

leukemia with 15:17 with SARAtranslocation PML-RAR� blocks phosphorylation of Smads2/3

Acute myeloid leukemia Evi-1 directly interacts with Smad3 to disrupt TGF Izutsu et al.57

and myelodysplasia signalingEvi-1 represses Smad-induced transcription by

recruiting C-terminal binding protein (CtBP) as acorepressor

Acute T cell Decreased Smad3 expression Wolfraim et al.54

lymphoblasticleukemia

HTLV-1 induced T cell Overexpression of tax prevents Smad2/3/4 complex Lee et al.58

leukemia formation and DNA bindingTax competes for Smad binding with transcriptional Mori et al.59

coactivator p300Burkitt’s lymphoma Downregulation of T�RII at RNA level Inman and Allday60

Hodgkin lymphoma Increased activity of TGF-� in nodular sclerosis type Gruss et al.61

Hsu et al.62

Non-Hodgkin’s Increased expression of T�RI, T�RII, T�RIII in Woszczyk et al.63

lymphoma high-grade lymphomasDecreased T�RI and T�RII mRNA levels in advanced Kadin et al.64

cutaneous T cell lymphomasLoss of surface T�RII due to defective trafficking in Capocasale et al.65

Sezary patientsT�RI gene deletion in anaplastic large cell lymphoma Schiemann et al.66

Multiple myeloma Loss of surface T�RI and T�RII due to defective Amoroso et al.67

trafficking in plasmacytomas Fernandez et al.68

TGF-� mediated upregulation of IL-6 and VEGF Urashima et al.69

Dankbar et al.70

cell leukemias in mice. Strikingly, nearly absent levels ofSmad3 protein were seen in a series of pediatric T cell leuke-mias compared with normal levels seen in B cell leukemias andnormal T cells. These low protein levels were seen in the pres-ence of normal Smad3 mRNA levels and in the absence of anygene mutations.54 Thus, the level of Smad3 was found to be adeterminant of the T cell response to TGF-�, and a reductionin Smad3 works synergistically with other known oncogenicpathways to promote T cell leukemogenesis.

Loss of sensitivity to TGF-�-induced growth arrest due todecreased TGF-� receptors has been described in leukemicblasts derived from myelodysplasia patients.71 AML secondaryto myelodysplasia carries a poorer prognosis and is associatedwith a higher rate of cytogenetic alterations compared with denovo AML. It is possible that the reduced sensitivity to TGFseen in secondary AML blasts may partly explain their refrac-toriness to present therapies.

Evi-1 is a zinc finger nuclear protein that is highly expressedin human myeloid leukemias and myelodysplastic syndromes(MDS). Evi-1 is also expressed at a very low level in a limitedstage of normal myeloid differentiation. Aberrant overexpres-sion of Evi-1 as a fusion transcript with AML-1 leads to blas-tic transformation in patients with chronic myelogenous leuke-mia (CML). It has been shown that Evi-1 may disrupt TGFsignaling by direct interactions with Smad3 protein.57,72 Addi-tionally, Evi-1 has been shown to repress Smad-induced tran-scription by recruiting C-terminal binding protein (CtBP) as acorepressor. These mechanisms have been postulated to con-tribute to Evi-1 mediated leukemogenesis.

The 8:21 chromosomal translocation leads to AML via for-mation of the fusion oncoprotein AML1/ETO. AML/ETO is achimeric transcription factor that represses the expression ofmany genes involved in myeloid cell development and leads toa maturation arrest. In addition to direct affects on transcrip-tion, the AML/ETO protein may also disrupt normal TGF-�signaling. Both AML1 and AML/ETO have been shown tophysically associate with Smads and participate in their nuclearlocalization. Even though wild-type AML1 stimulates TGF-�-mediated gene transcription, AML1/ETO interrupts it. Thus, in-sensitivity to TGF-�-mediated growth regulation may partly ex-plain the leukemogenic potential of AML1/ETO.55

The promyelocytic leukemia protein/retinoic acid receptor-� (PML/RAR�) fusion oncoprotein is formed by the recipro-cal chr15:17 translocation and leads to the pathogenesis of acutepromyelocytic leukemia (APL). This transcriptional repressoroncoprotein represses retinoic acid mediated transcription ofgenes necessary for myeloid development and causes a matu-ration arrest. Recent evidence suggests that this fusion proteindisrupts normal PML function in a dominant negative manner.The cytoplasmic form of PML (c-pml) is an essential modula-tor of TGF-� signaling and has been shown to directly bind toSmads and facilitate their association with the Smad anchor forreceptor activation (SARA) for their accumulation in the en-dosomes. Cells devoid of PML are resistant to growth arrestand apoptosis induced by TGF-�.56 Disrupted nuclear transportand phosphorylation of Smads2/3 are seen in PML null cellsand cause impaired induction of the TGF-� target genes. Thus,these phenomena contribute to the pathogenesis of APL.56

Human T cell lymphotropic virus type 1 (HTLV-1) infectionleads to an aggressive T cell leukemia and has been linked to

the overexpression of the Tax oncoprotein. Tax has been shownto interact with Smads2, 3, and 4 and interrupts their associa-tion with each other and their target DNA sequences.59 Tax alsocompetes for Smad binding with transcriptional coactivatorp300 and, thus, decreases the transcription of TGF-�-mediatedgenes, such as plasminogen activator.58 The decrease in TGF-� signaling likely provides the optimal conditions for develop-ment of aggressive, resistant T cell leukemias.

Role of TGF-� in myelodysplasia

MDS are a heterogeneous group of diseases characterized byineffective hematopoiesis, which leads to peripheral cytopeniasin the elderly.73 As hematopoietic failure is the cause of lowblood counts in MDS, investigators have implicated cytokinedysregulation as one of the etiologies of this syndrome. TGF-� is a myelosuppressive cytokine that has been found to be in-creased in MDS bone marrow plasma.74 Greater amounts ofbound TGF-� on the surface of progenitors have been observedby immunohistochemical examination of MDS bone mar-rows.75 Because TGF has been shown to cause a G0/G1 cell cy-cle arrest in primary human HPCs, it has been strongly impli-cated in the pathogenesis of MDS. Thus, signaling cascadesstimulated by TGF-� are potential therapeutic targets in MDS.TGF-� has been shown to strongly activate the p38 MAPK inprimary human hematopoietic cells.30 p38 MAPK is an evolu-tionary conserved serine/threonine kinase that is activated bymany inhibitory cytokines and mediates growth inhibitory re-sponses in HSCs. It has been shown that inhibition of thisMAPK pathway can reverse the growth inhibition mediated byTGF-� in primary human HPCs.76 Most interestingly, pharma-cologic inhibitors of p38 MAPK pathway can stimulate hema-topoiesis in primary MDS HPCs in vitro and lead to increasederythroid and myeloid colony formation.77 Ongoing clinicalstudies are investigating the efficacy of a selective p38a inhib-itor, SCIO-469, in the treatment of MDS. Preliminary resultsin a few patients demonstrate increases in red cells, white cells,and platelets, with acceptable toxicity. Current studies are eval-uating the potential of other TGF-� signaling inhibitors in MDSand similar cytokine-driven hematopoietic diseases.

Role of TGF-� in B cell biology and B cell lymphomas

Both B and T lymphocytes can synthesize and secrete TGF-�. B lymphocytes have receptors for TGF-� and upregulatethem upon activation. Exogenous TGF-� inhibits IL-2-depen-dent B cell proliferation and immunoglobulin secretion in vitro.Its inhibition of cell differentiation appears to be independentof its effect on cell proliferation, as increasing the presence ofIL-2 in the medium restores DNA synthesis but has no effecton immunoglobulin secretion.78 The effects on proliferation aremainly related to its ability to cause G1 cell cycle arrest directlyin normal and neoplastic B lymphocytes.78 TGF-� can also in-duce apoptosis of human B lymphocytes, but exposure to TGF-� alone is not sufficient for induction of apoptosis and may bedependent on paracrine and autocrine secretion of other cyto-kines.79 Thus, TGF-� affects the capacity of B lymphocytes toproliferate, differentiate, and undergo apoptosis.

The growth-inhibitory qualities of TGF-� have been exten-sively studied in the presence of Epstein-Barr virus (EBV) in-

TGF-� IN HEMATOLOGIC MALIGNANCIES 547

fection, as the EBV genome is found in several types of lym-phomas. Review of a cell line panel derived from lymphomasor in vitro EBV transformation showed that EBV-negative celllines are inhibited by TGF-�, whereas EBV-positive cell linesare not.80,81 Certain EBV-transformed B cell lines, such asCESS, also continue to constitutively express immunoglobulins(Igs) despite the presence of TGF-�.82 The exact mechanismby which EBV renders cells resistant to TGF-� has not beenfully determined, but it is thought to be linked to the down-regulation of both type I and type II TGF receptors.83,84 Geneexpression studies have shown that downregulation of receptorexpression may occur at the transcriptional level and rendersthese cells resistant to TGF-�-induced growth suppression andapoptosis.60,85 As downregulation of TGF-� receptors has beenobserved in lymphomas, strategies to increase receptor expres-sion can be potentially therapeutic in this disease.86 Treatmentof the human B cell lymphoma cell line RL, which does notconstitutively express receptors for TGF-�, with anti-Ig anti-bodies causes upregulation of TGF-� receptors and increasessensitivity to TGF-�.87 In fact, TGF-� has been shown to actadditively with anti-Ig to induce apoptosis in cell lines derivedfrom Burkitt’s lymphoma (BL).88 TGF-� treatment of BL celllines has been shown to lead to apoptosis that is caspase-8 de-pendent but is caspase-3 independent.89 Activation of caspase-8 results from TGF-�-mediated p38 MAPK phosphorylation.Inhibitors of this MAPK pathway prevent capsase-8 activationand apoptosis induced by late cell activation of TGF-�.90

Signaling through CD40 has a protective antiapoptotic effecton BL cells, and its dysregulation is hypothesized to be anothermechanism for lymphomagenesis.85 CD40 ligand rescue fromprogrammed cell death, however, is minimal when done in thepresence of TGF-� and anti-Ig, demonstrating the importantrole of TGF signaling in lymphoma biology.88 TGF receptorgene mutations have also been associated with some subtypesof lymphoma. Anaplastic large cell lymphoma, a very aggres-sive subtype of non-Hodgkins lymphoma (NHL), has been as-sociated with a deletion in the T�RI gene that causes loss ofT�RI expression.66

TGF-� also plays a role in indolent follicular lymphomascharacterized by the fusion of the antiapoptotic bcl2 gene withIg heavy chain promoter. Cell lines positive for chr14:18(Bcl2/IgH) gene translocation as well as primary follicular lym-phomas with the same translocation are found to overexpressSmad1.91 Smad1 is considered a member of the BMP signal-ing pathway and is phosphorylated by ALK1 receptor. In mostcell types, including normal B cells, smad1 is not phosphory-lated by TGF-�. In follicular lymphoma (FL) cells, Smad1could be phosphorylated by TGF-�, suggesting that aberrantTGF-� signaling may play a role in the pathogenesis of FL.92

TGF-� may also have a role as a prognostic factor in non-Hodgkin lymphomas. There is an indication that high-gradelymphomas are associated with increased overall activity ofTGF-�1 and increased expression of type I, II, and III recep-tors. This overactivation of the TGF-� pathway may lead to in-creased angiogenesis that supports tumor growth and to inhibi-tion of tumor-infiltrating lymphocytes that enables evasion ofimmunesurveillance.63 These data are consistent with thepleiotropic role of TGF in other cancer models, where it sup-presses the primary tumor but has been shown to encourage thedevelopment of metastases.93 TGF-�1 gene polymorphisms in-

fluence the disease course in non-Hodgkin lymphoma as well.The TGF-�1 high producer genotype is an independent risk fac-tor of NHL manifestation in two or more extranodal sites.94

Hodgkin lymphoma is a tumor composed of malignantReed-Sternberg (RS) cells in a background of multiple reac-tive normal cells, such as lymphocytes, plasma cells, histio-cytes, and fibroblasts. RS cells produce abundant cytokinesand growth factors, which contribute to their proliferation andto the impaired host immune response.61 TGF-� is one of thecytokines produced and secreted by RS cells and reactive Tcells.62 TGF-� activity has been demonstrated in severalcases of nodular sclerosis but not in other histologic types ofHodgkin disease. Nodular sclerosis is a common type ofHodgkin lymphoma characterized by the presence of fibroticbands in the lymph nodes. TGF-� was localized mainly atthe margins of collagen bands, blood vessels, and areas ofnecrosis correlating with the histologic findings of nodularsclerosis and consistent with its profibrotic role in othermodel systems. Thus, TGF-� may contribute to the progres-sion and etiology of Hodgkin disease.

TGF-� in T cell biology and T cell lymphomas

TGF-� plays an important role in normal T cell growth reg-ulation and proliferation. Mature T cells have receptors forTGF-�, which are upregulated upon T cell receptor activation.Addition of exogenous TGF-� to T cell cultures blocks IL-2receptor upregulation and inhibits IL-2-dependent cell prolifer-ation. This inhibitory effect is reversed with increased concen-trations of IL-2, indicating that a balance between TGF-� andIL-2 is necessary to control T cell proliferation. T cells can alsosynthesize TGF-� upon activation to control their degree of pro-liferation in response to antigenic stimuli.95 Tumors have beenshown to use TGF-� to apply this mechanism to escape im-mune surveillance.

Cutaneous T cell lymphoma (CTCL) is an indolent T cellmalignancy that preferentially affects the skin and frequentlyleads to an immunodeficient state. It has been shown that im-mature dendritic cells (DCs) may stimulate the proliferation ofCTCL clones by presenting antigens from apoptotic CTCLs andother T cells.96 Stimulated CTCL cells then secrete TGF-�,which further replenishes the pool of immature DCs by the dif-ferentiation of monocytes and CD34 precursors in the epider-mis into Langerhans cells.97,98 In this manner, TGF-� providesa continual stimulus for CTCL cells to proliferate. StimulatedCTCL cells also adopt a T-regulatory (Treg) cell phenotype(CTLA-4�, FoxP3�, CD25�), which acts to inhibit normal Tcells from responding to antigen stimulation, contributing to theimmunosuppression that accompanies the disease.99

Advanced aggressive CTCL cell lines have also been shownto be resistant to TGF-�-induced growth inhibition. These ad-vanced CTCLs have decreased TGF-� receptor type II mRNAlevels and have lost type I and II TGF-� receptor expression.64

Sezary syndrome is an aggressive form of CTCL where the ma-lignant cells invade the bloodstream. Loss of cell surface TGF-� receptors is also seen in Sezary syndrome.64 In these patients,despite the loss of surface T�RII, the intracellular pools ofT�RII are normal, which indicates that defective traffickingmay be responsible for resistance to the inhibitory effects ofTGF-� and growth of the neoplastic cells.65

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TGF-� in multiple myeloma

Most of the data on the effects of TGF-� on mature, fullydifferentiated B cells comes from the study of murine plasma-cytomas, which are tumors of immunoglobulin-secretingplasma cells. Primary plasmacytomas and established plasma-cytoma cell lines are resistant to the growth-inhibitory andapoptotic effects of TGF-�. Even though they have mRNA andproteins for type I and II TGF-� receptors, these receptors areabsent from the cell surface of these malignant cells, likely dueto defective trafficking.67 A novel mechanism has been pro-posed by which active TGF-�1 binds to intracellular T�RII,impeding its cell surface expression and resulting in insensi-tivity to extracellular growth inhibitory signals. Use of antisenseTGF-�1 mRNA disrupts this ligand-receptor complex fromforming, thus restoring T�RII cell surface pools.68

Even though primary myeloma cells are resistant to TGF-�-induced growth suppression, TGF-� may also directly par-ticipate in myelomagenesis by its effects on the tumor mi-croenvironment. TGF-� upregulates IL-6 secretion by bonemarrow stromal cells.69 IL-6 is a potent growth factor formyeloma cells and an inhibitor of plasma cell apoptosis. It isbelieved to act in a paracrine fashion, being produced by ad-herent cells in the bone marrow microenvironment. The high-est levels of IL-6 have been found in patients with aggressivemultiple myeloma (MM).100 IL-6 also stimulates myelomacells to secrete vascular endothelial growth factor (VEGF),which facilitates MM cell migration and stimulates BM an-giogenesis.70 The role of TGF-� in this process was shownby the efficacy of a novel small molecule T�RI inhibitor, SD-208, in a model of myeloma. The T�RI kinase inhibitor SD-208 was shown to inhibit the production of IL-6 and VEGFby bone marrow stromal cells and inhibit MM cell prolifera-tion in vitro.101 These results establish an important role forTGF-� in the pathogenesis of MM and shed some light ontopossible therapeutic targets.

DISCUSSION

TGF-� signaling plays an important role in maintaining nor-mal hematopoiesis. Disruption of TGF-� signaling by onco-proteins can lead to malignant transformation. Strategies basedon restoration of TGF-�-induced cell cycle arrest can form thebasis of antineoplastic interventions. On the other hand, in-creased TGF-� signaling can lead to hematopoietic failure andsuch diseases as myelodysplasia. TGF-� can also potentiatemyelomagenesis by stimulating autocrine and paracrine secre-tion of such growth factors as IL-6. In this context, inhibitorsof TGF-� receptors may have a therapeutic potential. Thus, acomprehensive molecular study of TGF-� signaling will leadto the development of novel therapies for cancer.

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Address reprint requests or correspondence to:Dr. Amit Verma

Albert Einstein College of MedicineChanin 302B

1300 Morris Park AvenueBronx, NY 10461

Tel: (718) 430-4091Fax: (718) 430-8702

E-mail: averma@aecom.yu.edu

Received 18 January 2007/Accepted 30 January 2007

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