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Current View of the Role of Transforming Growth Factor β 1 in Skin Carcinogenesis

  • Allen Guanqun Li
    Affiliations
    Department of Otolaryngology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Dermatology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA
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  • Shi-Long Lu
    Affiliations
    Department of Otolaryngology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Dermatology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA
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  • Gangwen Han
    Affiliations
    Department of Otolaryngology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Dermatology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA
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  • Molly Kulesz-Martin
    Affiliations
    Department of Dermatology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA
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  • Xiao-Jing Wang
    Correspondence
    3710 SW US Veterans Hospital Rd, PVMC Bldg 103, Rm F-221, Mail code R&D46, Portland, OR 97239, USA.
    Affiliations
    Department of Otolaryngology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Dermatology, Oregon Health & Science University, Portland, Oregon, USA

    Department of Cell & Developmental Biology, Oregon Health & Science University, Portland, Oregon, USA
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      Previously, we have shown that transforming growth factor β 1 (TGFβ1) overexpression in suprabasal epidermis suppresses skin carcinogenesis at early stages, but promotes tumor invasion at later stages. To elucidate the role of TGFβ1 overexpression in naturally occurring human squamous cell carcinomas (SCC), we screened TGFβ1 expression patterns in human skin SCC samples and found that TGFβ1 was overexpressed with two distinct patterns: either predominantly in suprabasal layers or throughout tumor epithelia including basal proliferative cells. To determine the effect of TGFβ1 overexpression in basal keratinocytes, we generated transgenic mice expressing wild-type TGFβ1 in basal keratinocytes and hair follicles using the K5 promoter (K5.TGFβ1wt). Surprisingly, these mice developed a severe inflammatory skin disorder. Inflammation was also observed in head and neck tissue when TGFβ1 transgene expression was inducibly expressed in head and neck epithelia in our gene-switch-TGFβ1 transgenic mice. Given the importance of inflammation in cancer development, our data suggest that TGFβ1-induced inflammation may override its tumor-suppressive effect even at early stages of skin carcinogenesis. This notion is further suggested by our recent study that Smad3 knockout mice were resistant to skin chemical carcinogenesis at least in part via abrogation of endogenous TGFβ1-induced inflammation.

      Keywords

      ALK
      activin receptor-like kinase
      Id
      inhibitor of differentiation
      IFN-γ
      interferon-γ
      IL
      interleukin
      KGF
      keratinocyte growth factor
      MCP
      monocyte-chemotactic protein
      MIP
      macrophage inflammatory protein
      MMP
      matrix metalloproteinase
      R-Smads
      receptor-specific Smads
      SBE
      Smad-binding element
      SCC
      squamous cell carcinomas
      TGFβ
      transformation growth factor β
      TGFβRI
      type I TGFβ receptor
      TGFβRII
      type II TGFβ receptor
      TNF-α
      tumor necrosis factor-α
      VEGF
      vascular endothelial growth factor

      Transforming Growth Factor β (TGFβ) Signaling

      TGFβ represents a family of pluropotent cytokines that consist of three isoforms in mammals including TGFβ1, -2, and -3, with TGFβ1 being the predominant one in most tissue types including the skin (
      • Wang X.J.
      Role of TGFbeta signaling in skin carcinogenesis.
      ;
      • Li A.G.
      • Koster M.I.
      • Wang X.J.
      Roles of TGFbeta signaling in epidermal/appendage development.
      ). TGFβ1 plays a pivotal role in the maintenance of tissue homeostasis by regulating cell growth, differentiation, extracellular matrix deposit, immune/inflammatory responses, and angiogenesis (for recent reviews, see
      • Derynck R.
      • Zhang Y.E.
      Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
      ;
      • Siegel P.M.
      • Massague J.
      Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer.
      ). TGFβ1 is synthesized and secreted in a latent form (TGFβ1wt), in which the N-terminal latency-associated peptide (LAP) remains non-covalently bound to the C-terminal mature TGFβ1. The mature TGFβ1 is cleaved from the LAP following proteolysis and becomes a functionally active form (TGFβ1act) (
      • Lawrence D.A.
      Identification and activation of latent transforming growth factor beta.
      ). Constitutive activation of TGFβ1 has been achieved by two site-specific mutations in the LAP, Cys-223→Ser, and Cys-225→Ser (TGFβ1S223/225), which prevent the LAP from binding to the mature TGFβ1 (
      • Brunner A.M.
      • Marquardt H.
      • Malacko A.R.
      • Lioubin M.N.
      • Purchio A.F.
      Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins.
      ). TGFβ functions through a signaling cascade that is elicited when TGFβ ligands (TGFβ) bind to the specific membrane-bound kinase type I and type II TGFβ receptor complex (TGFβRI, TGFβRII, Figure 1, for recent reviews, see
      • Miyazawa K.
      • Shinozaki M.
      • Hara T.
      • Furuya T.
      • Miyazono K.
      Two major Smad pathways in TGF-beta superfamily signalling.
      ;
      • Derynck R.
      • Zhang Y.E.
      Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
      ;
      • Reguly T.
      • Wrana J.L.
      In or out? The dynamics of Smad nucleocytoplasmic shuttling.
      ;
      • Shi Y.
      • Massague J.
      Mechanisms of TGF-beta signaling from cell membrane to the nucleus.
      ). Although several subtypes of TGFβRI have been identified, to date, only one TGFβRII has been identified, which is essential for TGFβ binding and for assembly of the TGFβRI–TGFβRII complex (
      • Derynck R.
      • Zhang Y.E.
      Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
      ). When TGFβ binds to a TGFβRI–TGFβRII complex, the classic TGFβRI, also known as activin receptor-like kinase-5 (ALK5), phosphorylates two receptor-specific Smads (R-Smads); Smad2 and Smad3. Phosphorylated Smad2 and Smad3 form heteromeric complexes with the common Smad, Smad4, and translocate into the nucleus to regulate TGFβ-responsive genes (
      • Derynck R.
      • Zhang Y.E.
      Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
      ). Smad2/Smad3 activation mediates TGFβ1-induced growth inhibition on epithelial and endothelial cells (
      • Goumans M.J.
      • Lebrin F.
      • Valdimarsdottir G.
      Controlling the angiogenic switch: A balance between two distinct TGF-b receptor signaling pathways.
      ) and growth promotion on mesenchymal cells (such as fibroblasts) (
      • Pittelkow M.R.
      Growth factors in cutaneous biology and disease.
      ). Another TGFβRI, ALK1, has been recently identified to be preferentially expressed in endothelial cells Figure 1. Activated ALK1 phosphorylates and activates two other R-Smads, Smad1 and Smad5, which positively regulate endothelial cell proliferation and migration (
      • Goumans M.J.
      • Lebrin F.
      • Valdimarsdottir G.
      Controlling the angiogenic switch: A balance between two distinct TGF-b receptor signaling pathways.
      ). In this context, endoglin, a transmembrane glycoprotein that is also mainly expressed by endothelial cells, may function as an accessory molecule to facilitate TGFβ ligand and receptor interaction (Figure 1,
      • Cheifetz S.
      • Bellon T.
      • Cales C.
      • Vera S.
      • Bernabeu C.
      • Massague J.
      • Letarte M.
      Endoglin is a component of the transforming growth factor-beta receptor system in human endothelial cells.
      ;
      • Yamashita H.
      • Ichijo H.
      • Grimsby S.
      • Moren A.
      • ten Dijke P.
      • Miyazono K.
      Endoglin forms a heteromeric complex with the signaling receptors for transforming growth factor-beta.
      ). Two inhibitory Smad proteins, Smad6 and Smad7, can be rapidly induced by TGFβ, which then block TGFβ signaling by competing with R-Smads for interaction with the activated type I receptors, thereby inhibiting R-Smad phosphorylation by competing with Smad4 for heteromeric complex formation with phosphorylated R-Smads or by recruiting ubiquitin ligases to the activated receptors to induce their degradation via proteosomal and lysosomal pathways Figure 1 (
      • Imamura T.
      • Takase M.
      • Nishihara A.
      • Oeda E.
      • Hanai J.
      • Kawabata M.
      • Miyazono K.
      Smad6 inhibits signalling by the TGF-beta superfamily.
      ;
      • Afrakhte M.
      • Moren A.
      • Jossan S.
      Induction of inhibitory Smad6 and Smad7 mRNA by TGF-beta family members.
      ;
      • Schiffer M.
      • Schiffer L.E.
      • Gupta A.
      • Shaw A.S.
      • Roberts I.S.
      • Mundel P.
      • Bottinger E.P.
      Inhibitory smads and tgf-beta signaling in glomerular cells.
      ).
      Figure thumbnail gr1
      Figure 1Schematic of transforming growth factor β 1 (TGFβ1)-elicited signal transduction pathway. In most cell types such as keratinocytes and fibroblasts, the classic type I TGFβ receptor (TGFβRI), activin receptor-like kinase-5 (ALK5), is expressed and its activation upon TGFβ1 interacting with type II TGFβ receptor (TGFβRII) leads to phosphorylation of two receptor-specific Smads (R-Smads); Smad2 and Smad3 (pSmad2/3). Endothelial cells express both ALK5 and ALK1. Upon activation of ALK1, it phosphorylates another two R-Smads; Smad1 and Smad5 (pSmad1/5). The phosphorylated R-Smads then bind to Smad4 to form a protein complex that undergoes a nuclear translocation to regulate different downstream TGFβ1-responsive genes by binding to the Smad-binding element (SBE). Typically, expression of plasminogen activator inhibitor (PAI)-1 is mediated by pSmad2/3, whereas pSmad1/5 is responsible for the expression of inhibitor of differentiation (Id)-1.

      Paradoxical Role of TGFβ1 in Skin Carcinogenesis

      TGFβ1 is overexpressed in both early and late stages in many human cancer types (
      • Reiss M.
      Transforming growth factor-beta and cancer: A love–hate relationship?.
      ;
      • Gold L.I.
      The role for transforming growth factor-beta (TGF-beta) in human cancer.
      ). Studies have shown that TGFβ1 plays a dual role in skin carcinogenesis. When TGFβ1 transgene is targeted primarily in the suprabasal/differentiated layers of the epidermis in transgenic mice, TGFβ1 overexpression inhibits papilloma formation at early stages but promotes tumor progression, metastasis, and epithelial–mesenchymal transition at later stages of skin carcinogenesis (
      • Cui W.
      • Fowlis D.J.
      • Cousins F.M.
      • Duffie E.
      • Bryson S.
      • Balmain A.
      • Akhurst R.J.
      Concerted action of TGF-beta 1 and its type II receptor in control of epidermal homeostasis in transgenic mice.
      ,
      • Cui W.
      • Fowlis D.J.
      • Bryson S.
      • Duffie E.
      • Ireland H.
      • Balmain A.
      • Akhurst R.J.
      TGFbeta1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice.
      ;
      • Weeks B.H.
      • He W.
      • Olson K.L.
      • Wang X.J.
      Inducible expression of transforming growth factor beta1 in papillomas causes rapid metastasis.
      ). The earlier tumor-suppressive role of TGFβ1 is attributed to its growth-inhibitory effect on keratinocytes, whereas the later tumor promotion role is associated with its effects on loss of epithelial cell adhesion, extracellular matrix remodeling, and enhanced angiogenesis (
      • Weeks B.H.
      • He W.
      • Olson K.L.
      • Wang X.J.
      Inducible expression of transforming growth factor beta1 in papillomas causes rapid metastasis.
      ). The current view of the role of TGFβ1 in carcinogenesis is that its tumor-suppressive effect is dominant at early stages, whereas its tumor-promoting effect is dominant at later stages of carcinogenesis (
      • Roberts A.B.
      • Wakefield L.M.
      The two faces of transforming growth factor beta in carcinogenesis.
      ). Recent studies, however, have begun to challenge this point of view. When TGFβ1 transgene is expressed in proliferative keratinocytes of the epidermis and hair follicles using either a keratin K5 or K14 promoter, the preneoplastic transgenic skin exhibits many pathological alterations required for tumor promotion. These include severe inflammation, angiogenesis, basement membrane degradation, and surprisingly, epidermal hyperproliferation as a result of inflammation and angiogenesis (
      • Liu X.
      • Alexander V.
      • Vijayachandra K.
      • Bhogte E.
      • Diamond I.
      • Glick A.
      Conditional epidermal expression of TGFbeta 1 blocks neonatal lethality but causes a reversible hyperplasia and alopecia.
      ;
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). These data suggest that TGFβ1 overexpression may have a tumor-promoting effect even at the early stages of skin carcinogenesis if it is overexpressed in proliferative cells of the epidermis/tumor epithelia. Interestingly, when we performed in situ hybridization to examine TGFβ1 expression patterns in human skin squamous cell carcinomas (SCC), we observed two patterns with a similar ratio of occurrence: either predominantly in suprabasal layers or throughout tumor epithelia including basal proliferative cells Figure 2. The epidermis adjacent to SCC exhibited TGFβ1 expression patterns similar to the corresponding tumors Figure 2. These two TGFβ1 expression patterns did not correlate with the differentiation states of SCC. To date, it is not clear whether the specific locations of TGFβ1 expression differentially affect tumor progression.
      Figure thumbnail gr2
      Figure 2In situ hybridization of transforming growth factor β 1 (TGFβ1) in human skin squamous cell carcinomas (SCC) and adjacent epidermis. The dotted lines delineate the boundary between the basal layer of the epidermis and the dermis of skin adjacent to SCC, and between tumor epithelia and the stroma. (A, C) TGFβ1 transcripts were predominant in the suprabasal layers of the epidermis (A) and tumor epithelia (C). (B, D) TGFβ1 transcripts were also found to be present throughout the adjacent epidermis (B) and tumor epithelia (D) (left lobule of the SCC) or predominantly in the basal layer of tumor epithelia (right lobule of the SCC). The dermis and tumor stroma also express a weaker level of TGFβ1. (E, F) A hematoxylin & eosin (H&E) section (E) shows a typical SCC lobule surrounded by the stroma, and the sense probe control of in situ hybridization (F) displays little background staining. The bar represents 40 μm for all sections.

      Role of TGFβ1 in Skin Inflammation

      TGFβ1 has both positive and negative effects on the regulation of inflammation and immune response, depending on the state of differentiation of the cells and the cytokine milieu (
      • Wahl S.M.
      Transforming growth factor beta: The good, the bad, and the ugly.
      ;
      • Letterio J.J.
      • Roberts A.B.
      Regulation of immune responses by TGF-beta.
      ;
      • Ludviksson B.R.
      • Seegers D.
      • Resnick A.S.
      • Strober W.
      The effect of TGF-beta1 on immune responses of naive versus memory CD4+Th1/Th2 T cells.
      ;
      • Prud'homme G.J.
      • Piccirillo C.A.
      The inhibitory effects of transforming growth factor-beta-1 (TGF-beta1) in autoimmune diseases.
      ). The anti-inflammatory and immunosuppressive effect of TGFβ1 has been excessive as TGFβ1 knockout mice spontaneously develop multifocal inflammation and autoimmune disorders (
      • Shull M.M.
      • Ormsby I.
      • Kier A.B.
      Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease.
      ;
      • Kulkarni A.B.
      • Huh C.G.
      • Becker D.
      Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death.
      ). These knockout mice, however, do not develop any skin inflammatory phenotypes, suggesting that the action of TGFβ1 in the context of inflammation may vary depending on different tissue types. Further characterization of TGFβ1 knockout mice revealed a lack of Langerhans cells in TGFβ1 knockout epidermis (
      • Borkowski T.A.
      • Letterio J.J.
      • Farr A.G.
      • Udey M.C.
      A role for endogenous transforming growth factor beta 1 in Langerhans cell biology: The skin of transforming growth factor beta 1 null mice is devoid of epidermal Langerhans cells.
      ,
      • Borkowski T.A.
      • Letterio J.J.
      • Mackall C.L.
      A role for TGFbeta1 in Langerhans cell biology. Further characterization of the epidermal Langerhans cell defect in TGFbeta1 null mice.
      ), and a dysfunction of dermal dendritic cells in terms of trafficking antigens to regional lymph nodes (
      • Hemmi H.
      • Yoshino M.
      • Yamazaki H.
      Skin antigens in the steady state are trafficked to regional lymph nodes by transforming growth factor-beta1-dependent cells.
      ). These findings highlight the role of TGFβ1 in the maturation of skin-homing dendritic cells, which is crucial for triggering inflammatory and immune response upon various stimuli.
      Using a transgenic approach, we have shown that K5.TGFβ1wt transgenic mice, with the TGFβ1 expression level similar to the peak level during cutaneous wound healing in normal mice, spontaneously develop skin phenotypes that are strikingly similar to psoriasis in humans (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). By 3 mo of age, K5.TGFβ1wt skin developed focal erythematous plaques with a scaly appearance, which are similar to psoriatic plaques in humans Figure 3. As skin inflammation progresses, the entire K5.TGFβ1wt skin becomes scaly and erythematous, the gross appearance of which closely resembles psoriatic erytheroderma in humans Figure 3.
      Figure thumbnail gr3
      Figure 3Comparison of skin phenotypes in K5.TGFβ1wt mice (A, C, E) and psoriatic lesions in humans (B, D, F). The focal erythematous plaques with a scaly appearance in a 3-mo-old K5.TGFβ1wt mouse (A) resembled human psoriatic plaques (B). As the disease progressively worsens, K5.TGFβ1wt mice develop generalized scaly erythemas (C), reminiscent of psoriatic erythroderma in humans (D). Histology shows that K5.TGFβ1wt skin (E) has a morphology similar to human psoriasis lesion (F). (A) and (C) were reproduced from our previous work (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ) with the permission of the Nature Publishing Group. (B), (D), and (F) were reproduced from Figure 43-1-B and Figure 43-10-A, respectively, of
      • Christophers E.
      • Mrowietz U.
      Psoriasis.
      with the permission of the McGraw-Hill Companies. TGFβ1, transforming growth factor β 1.
      Prior to the development of overt psoriatic lesions, K5.TGFβ1wt transgenic mice remain macroscopically indistinguishable from non-transgenic littermates until about 1 mo of age. Skin inflammation and angiogenesis, however, have already been noticeable histologically as early as day 10 post-partum (p.p.). Gradually, inflammatory cell infiltration becomes profound at day 17 Figure 4 and the neovascularization reaches a peak around day 24 p.p. (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ), prior to the development of epidermal hyperplasia. Interestingly, aberrant K6 expression in the interfollicular epidermis, usually a marker for hyperplastic epidermis, is observed in day 17 transgenic skin Figure 4. This dynamic of pathological changes in K5.TGFβ1wt skin suggests that epidermal hyperplasia is the secondary effect of inflammation and angiogenesis. When we cultured transgenic keratinocytes in the absence of other cell types, transgenic keratinocytes underwent growth arrest (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). This result further indicates that transgenic keratinocytes by themselves are not sufficient to overcome the growth-inhibitory effect by TGFβ1. It is thus likely that the inflammatory cytokines (e.g., interleukin 1 (IL-1)), chemokines (e.g., macrophage inflammatory protein (MIP-2)), growth factors (e.g., keratinocyte growth factor (KGF)), and angiogenic factors (e.g., vascular endothelial growth factor (VEGF)) produced by inflammatory cells and/or hyperproliferative fibroblasts Figure 4, together with growth regulators (e.g., amphiregulin) produced by keratinocytes themselves (autocrine effect), are able to provide strong mitogenic signals and to override TGFβ1-induced growth inhibition on the epidermis in vivo (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). Previous transgenic models overexpressing genes encoding the above-mentioned molecules individually in keratinocytes have also shown epidermal hyperproliferation to varying degrees (
      • Schon M.P.
      Animal models of psoriasis—what can we learn from them?.
      ). Interestingly, in vivo epidermal hyperproliferation in K5.TGFβ1wt mice does not require a loss of TGFβ responsiveness in keratinocytes, which often occurs in tumors cells as transgenic keratinocytes attest a normal TGFβ/Smad signaling process (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ).
      Figure thumbnail gr4
      Figure 4Inflammation and angiogenesis precedes epidermal hyperplasia in K5.TGFβ1wt skin. N, non-transgenic littermates, Tg, transgenic mice. The bar in the first panel of (A) represents 40 μm for all sections. (A) Transgenic mice do not show epidermal hyperlasia at day 17 as compared with non-transgenic littermates. A K14 antibody was used as a counterstaining for immunofluorescence. Skin samples were harvested from day 17 transgenic mice and non-transgenic littermates with 0.125 mg per g BrdU injected 2 h prior to sacrifice. Hematoxylin & eosin (H&E) and immunofluorescence demonstrated that the epidermal thickness and the number of BrdU-labeled keratinocytes (short arrow in BrdU staining panel) in day 17 transgenic epidermis were similar to those of non-transgenic littermates. Transgenic skin exhibited an aberrant K6 expression in the interfollicular epidermis in addition to the normal staining pattern in hair follicle epithelia. Note the scattered BrdU+fibroblasts (long arrows in BrdU staining panel) in transgenic but not in non-transgenic dermis. Neovascularization is obvious (open arrows in H&E panel) in transgenic skin. (B) Day 17 transgenic skin exhibits prominent inflammatory cell infiltration. Hematoxylin was used as a counterstaining. Immunohistochemistry revealed significantly increased CD45+ cells, CD4+ lymphocytes (mainly in the dermis), CD8+ lymphocytes (predominantly in the epidermis), BM8+ macrophages, CD103+ cells (exclusively in the epidermis), Ly-6G+ granulocytes, and mast cells in transgenic skin in comparison with non-transgenic skin. The dotted lines delineate the boundary between the epidermis and the dermis. Representative inflammatory cells are shown in the epidermis (red arrows) and the dermis (black arrows). The figure is reproduced from our previous work (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ) with the permission of the Nature Publishing Group, with the exception of Ly-6G staining in (B). TGFβ1, transforming growth factor β 1.
      Psoriasis-like phenotypes in adult K5.TGFβ1wt mice are characterized by prominent epidermal hyperplasia, massive inflammatory cell infiltration, and increased angiogenesis (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ). The numerous inflammatory cells in transgenic skin include T cells (with CD4+ cells mainly in the dermis and CD8+ cells predominantly in the epidermis), monocytes/macrophages, neutrophils, as well as mast cells fitting the scope of leukocytes that respond toward the strong chemotactic effect of TGFβ1 (
      • Wahl S.M.
      • Hunt D.A.
      • Wakefield L.M.
      • McCartney-Francis N.
      • Wahl L.M.
      • Roberts A.B.
      • Sporn M.B.
      Transforming growth factor type beta induces monocyte chemotaxis and growth factor production.
      ;
      • Wahl S.M.
      Transforming growth factor beta (TGF-beta) in inflammation: A cause and a cure.
      ,
      • Wahl S.M.
      Transforming growth factor beta: The good, the bad, and the ugly.
      ,
      • Wahl S.M.
      TGF-beta in the evolution and resolution of inflammatory and immune processes. Introduction.
      ;
      • Bonifati C.
      • Ameglio F.
      Cytokines in psoriasis.
      ). The skin inflammatory phenotypes are associated with an upregulation in a variety of genes encoding inflammatory cytokines (e.g., IL-1, IL-2, tumor necrosis factor α (TNF-α), and interferon (IFN)-γ), chemokines (e.g., MIP-2 (murine counterpart of IL-8), monocyte-chemotactic protein (MCP)-1, and IFN-induced protein-10), growth regulators (e.g., insulin-like growth factor, amphiregulin, and KGF), matrix metalloproteinases (MMP) (e.g., MMP-2, -3, and -9), and angiogenic factors (e.g., VEGF) and their receptors (e.g., Flt-1 and Flk-1) (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ), all of which have been reported to be unregulated in human psoriatic lesions (
      • Christophers E.
      The immunopathology of psoriasis.
      ;
      • Cook P.W.
      • Piepkorn M.
      • Clegg C.H.
      • Plowman G.D.
      • DeMay J.M.
      • Brown J.R.
      • Pittelkow M.R.
      Transgenic expression of the human amphiregulin gene induces a psoriasis-like phenotype.
      ;
      • Christophers E.
      • Mrowietz U.
      Psoriasis.
      ;
      • Bonifati C.
      • Ameglio F.
      Cytokines in psoriasis.
      ). Our data suggest that TGFβ1 functions as an initiator or coordinator during psoriasis development by inducing or recruiting multiple pro-inflammatory molecules produced by various cell types. This complex cascade is depicted in Figure 5. Consistent with our study, recent clinical studies have reported that increased TGFβ1 levels in psoriatic lesions and sera of psoriatic patients correlate with disease severity or duration (
      • Flisiak I.
      • Chodynicka B.
      • Porebski P.
      • Flisiak R.
      Association between psoriasis severity and transforming growth factor beta(1) and beta (2) in plasma and scales from psoriatic lesions.
      ).
      Figure thumbnail gr5
      Figure 5Mechanisms of transforming growth factor β 1 (TGFβ1)wt overexpression-induced psoriasis-like phenotypes. The underlined events are essential for the development of psoriasis-like phenotypes in K5.TGFβ1wt mice. TGFβ1 itself is a potent chemoattractant for leukocytes, and can recruit them to the skin. Matrix metalloproteinases (MMP) are upregulated in keratinocytes and mediate basement membrane (BM) degradation. This facilitates TGFβ1 secretion into the dermis where it stimulates fibroblast proliferation and further enhances leukocyte infiltration. Growth factors produced by hyperproliferative fibroblasts feed back to the overlying keratinocytes, causing epidermal hyperproliferation and hyperplasia. In addition to angiogenic factors (e.g., vascular endothelial growth factor (VEGF)) produced by keratinocytes and inflammatory cells, TGFβ1 itself directly stimulates angiogenesis. Furthermore, interleukin (IL)-1 release by impaired keratinocytes initiates the inflammatory cascade in the skin, thus leading to the production of an array of inflammatory cytokines (especially the primary pro-inflammatory cytokines, IL-1 and tumor necrosis factor α (TNF-α)), chemokines, and other inflammatory mediators that result in deteriorated and unresolved inflammation. Together, these events confer a progressive psoriasis-like skin inflammatory disorder.

      TGFβ1-Associated Inflammation in Skin Carcinogenesis

      Inflammation plays a paradoxical role in cancer development. Inflammatory cells produce cytokines and chemokines, which initiate cancer immunity (
      • Coussens L.M.
      • Werb Z.
      Inflammation and cancer.
      ). On the other hand, many inflammatory cytokines and chemokines also stimulate cell proliferation and angiogenesis, which facilitate tumor growth (
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      Inflammation and cancer.
      ;
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      • Caux C.
      Chemokines in cancer.
      ). In addition to inflammatory cells, virtually all cells, including many tumor cell types, express inflammatory cytokines, chemokines, and their receptors (
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      Inflammation and cancer.
      ;
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      • Caux C.
      Chemokines in cancer.
      ). Certain chemokines and their receptors promote cellular transformation and mediate metastasis formation via a mechanism similar to chemokine-mediated leukocyte transport (
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      • Caux C.
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      ;
      • Sparmann A.
      • Bar-Sagi D.
      Ras-induced interleukin-8 expression plays a critical role in tumor growth and angiogenesis.
      ). With respect to skin cancer, paradoxical effects of inflammation on cancer development have also been reported. Psoriasis patients with chronic skin inflammation do not show increased skin cancer development from the psoriatic plaques (
      • Nickoloff B.J.
      The skin cancer paradox of psoriasis: A matter of life and death decisions in the epidermis.
      ). In contrast, an interesting report provided evidence for an association between inflammation and progression of actinic keratoses to SCC in humans (
      • Berhane T.
      • Halliday G.M.
      • Cooke B.
      • Barnetson R.S.
      Inflammation is associated with progression of actinic keratoses to squamous cell carcinomas in humans.
      ). SCC arising in connection with chronic venous leg ulcers also appear aggressive and have a high propensity to metastasize (
      • Baldursson B.T.
      • Hedblad M.A.
      • Beitner H.
      • Lindelof B.
      Squamous cell carcinoma complicating chronic venous leg ulceration: A study of the histopathology, course and survival in 25 patients.
      ). In experimental skin cancer, wounding, which elicits an inflammatory response, has long been known to enhance cancer development (
      • Wang X.J.
      • Greenhalgh D.A.
      • Lu X.R.
      • Bickenbach J.R.
      • Roop D.R.
      TGF alpha and v-fos cooperation in transgenic mouse epidermis induces aberrant keratinocyte differentiation and stable, autonomous papillomas.
      ), and most classical tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA), induce inflammation (
      • Patamalai B.
      • Burow D.L.
      • Gimenez-Conti I.
      • Zenklusen J.C.
      • Conti C.J.
      • Klein-Szanto A.J.
      • Fischer S.M.
      Altered expression of transforming growth factor-beta 1 mRNA and protein in mouse skin carcinogenesis.
      ). Conversely, in a human papillomavirus oncogene-induced skin carcinogenesis model, mice devoid of pro-inflammatory CD4+ T cells exhibit a lower incidence of tumors and a delayed neoplastic progression (
      • Daniel D.
      • Meyer-Morse N.
      • Bergsland E.K.
      • Dehne K.
      • Coussens L.M.
      • Hanahan D.
      Immune enhancement of skin carcinogenesis by CD4+ T cells.
      ). In addition, anti-inflammation drugs, e.g., non-sterol anti-inflammatory drugs, exert both chemopreventive and therapeutic effects on UV-induced skin cancer in mice (
      • Fischer S.M.
      Is cyclooxygenase-2 important in skin carcinogenesis?.
      ;
      • Fischer S.M.
      • Conti C.J.
      • Viner J.
      • Aldaz C.M.
      • Lubet R.A.
      Celecoxib and difluoromethylornithine in combination have strong therapeutic activity against UV-induced skin tumors in mice.
      ).
      To assess the role of TGFβ1-associated inflammation in carcinogenesis, we turned our attention to head and neck SCC (HNSCC). In comparison with skin SCC, HNSCC more frequently develop from the site of chronic inflammation (e.g., oral lichen planus) and have a much worse prognosis (
      • Coussens L.M.
      • Werb Z.
      Inflammation and cancer.
      ;
      • Philpott M.
      • Ferguson L.R.
      Immunonutrition and cancer.
      ). We found that TGFβ1 protein is increased in about 80% of human HNSCC, ranging from a 1.5- to 7.5-fold increase in comparison with the endogenous TGFβ1 in normal epithelia (
      • Lu S.L.
      • Reh D.
      • Li A.G.
      • Woods J.
      • Corless C.L.
      • Kulesz-Martin M.
      • Wang X.J.
      Overexpression of transforming growth factor beta1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation.
      ). Moreover, TGFβ1 is overexpressed at the preneoplastic lesions adjacent to HNSCC, relative to uninvolved mucosa from the same patient (
      • Lu S.L.
      • Reh D.
      • Li A.G.
      • Woods J.
      • Corless C.L.
      • Kulesz-Martin M.
      • Wang X.J.
      Overexpression of transforming growth factor beta1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation.
      ), suggesting that TGFβ1 overexpression is an early event during HNSCC development. To further determine whether early-stage TGFβ1 overexpression directly induces inflammation, we induced TGFβ1 transgene expression in head and neck epithelia in our keratinocyte-specific gene-switch transgenic mice. As shown in Figure 6, this system consists of a transactivator (K5.GLp65) transgenic line and a downstream target line (tata.TGFβ1). In K5.GLp65 transgene, the transactivation domain of nuclear factor-κB (NF-κB) subunit p65 is fused to the yeast GAL4 transcription factor and the truncated ligand-binding domain of the progesterone receptor (ΔPR). The ΔPR does not bind to endogenous progesterone, but binds to progesterone antagonists, such as RU486, which initiates a nuclear translocation of the p65 fusion protein and activates the target gene. The promoter of the target transgene tata.TGFβ1 has been specifically engineered to contain four copies of the yeast GAL4-binding element, which can be recognized by the GAL4 protein within the p65 fusion protein (
      • Lu S.L.
      • Reh D.
      • Li A.G.
      • Woods J.
      • Corless C.L.
      • Kulesz-Martin M.
      • Wang X.J.
      Overexpression of transforming growth factor beta1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation.
      ). TGFβ1 overexpression in head and neck epithelia or the epidermis can be induced upon topical application of RU486 to bigenic mice (K5.GLp65/tata.TGFβ1) Figure 6. Similar to the effect of TGFβ1 overexpression in the epidermis (
      • Li A.G.
      • Wang D.
      • Feng X.H.
      • Wang X.J.
      Latent TGFbeta1 overexpression in keratinocytes results in a severe psoriasis-like skin disorder.
      ), we found that TGFβ1 transgene induction in oral mucosa also results in inflammation, increased angiogenesis, and subsequent epithelial hyperproliferation (
      • Lu S.L.
      • Reh D.
      • Li A.G.
      • Woods J.
      • Corless C.L.
      • Kulesz-Martin M.
      • Wang X.J.
      Overexpression of transforming growth factor beta1 in head and neck epithelia results in inflammation, angiogenesis, and epithelial hyperproliferation.
      ). This result argues whether TGFβ1 overexpression at early stages of HNSCC development would provide any tumor-suppressive effect.
      Figure thumbnail gr6
      Figure 6Schematic of gene-switch-TGFβ1wt transgenic mice. (A) The gene-switch transgenic system contains two transgenic lines; the transactivator line (K5.GLp65) and the target line (tata.TGFβ1). Driven by a K5 promoter, the transactivator line overexpresses a fusion protein including the yeast transcription factor GAL4 protein, the truncated ligand-binding domain of progesterone receptor (ΔPR), and the transactivation domain of NF-κB subunit p65. The transgene of the target line cannot be expressed by itself, as the TGFβ1 cDNA is under the control of a minimal tata promoter sequence that is fused with four copies of yeast GAL-binding element. TGFβ1 overexpression can be induced only in the squamous epithelia (e.g., oral mucosa or dorsal skin) of bigenic mice by progesterone antagonists such as RU486. (B). RU486 binds to ΔPR and leads the p65 fusion protein to translocate to the nucleus, where it binds to the promoter of the target transgene and activates the transcription of TGFβ1.
      In the two-stage skin carcinogenesis model, TPA rapidly induces TGFβ1 expression in keratinocytes (
      • Patamalai B.
      • Burow D.L.
      • Gimenez-Conti I.
      • Zenklusen J.C.
      • Conti C.J.
      • Klein-Szanto A.J.
      • Fischer S.M.
      Altered expression of transforming growth factor-beta 1 mRNA and protein in mouse skin carcinogenesis.
      ). Therefore, if inflammation is a direct effect of endogenous TGFβ1 overexpression, it may contribute greatly to the tumor promotion effect of TGFβ1. This notion is supported by our recent finding that mice lacking Smad3, the major intracellular mediator for TGFβ1-mediated signaling, exhibit a resistance to cancer formation during the two-stage chemical carcinogenesis experiment (
      • Li A.G.
      • Lu S.L.
      • Zhang M.X.
      • Deng C.
      • Wang X.J.
      Smad3 knockout mice exhibit a resistance to skin chemical carcinogenesis.
      ). In this study, we applied 7,12-dimethylbenz[a]anthracene-initiation and TPA-promotion protocol to homozygous and heterozygous Smad3 knockout (Smad3-/-, Smad3+/-) mice, and wild-type (Smad3+/+) mice. Both Smad3-/- and Smad3+/- mice showed a significant reduction in papilloma formation and malignant conversion in comparison with wild-type mice (
      • Li A.G.
      • Lu S.L.
      • Zhang M.X.
      • Deng C.
      • Wang X.J.
      Smad3 knockout mice exhibit a resistance to skin chemical carcinogenesis.
      ). These data suggest that impaired TGFβ signaling affects both early (benign tumor formation) and late (tumor progression and metastasis) stages of skin carcinogenesis. In other words, TGFβ1 may promote tumor development even in the earlier stage of skin carcinogenesis. The resistance of Smad3 knockout mice to skin carcinogenesis is attributed to the reduction of AP-1 family members that are TGFβ responsive genes and play an important role in tumor promotion, and reduced inflammation in Smad3 knockout skin/tumors as compared with wild-type skin/tumors (
      • Li A.G.
      • Lu S.L.
      • Zhang M.X.
      • Deng C.
      • Wang X.J.
      Smad3 knockout mice exhibit a resistance to skin chemical carcinogenesis.
      ). In particular, Smad3 knockout papillomas contain fewer tumor-associated macrophages, which correlates with a reduction in expression of IL-1, a primary inflammatory cytokine, and of MCP-1, a strong chemoattractant for monocytes/macrophages, as compared with wild-type papillomas. Consistently, Smad3 knockout tumors exhibit decreased activation of the NF-κB pathway, the central signaling controlling tumor-associated inflammation (
      • Li A.G.
      • Lu S.L.
      • Zhang M.X.
      • Deng C.
      • Wang X.J.
      Smad3 knockout mice exhibit a resistance to skin chemical carcinogenesis.
      ). Thus, this study suggests that, at early stages of skin carcinogenesis, TGFβ1 overexpression induced by TPA may have a tumor-promoting effect, via activation of AP-1 family members in keratinocytes and inflammation in the stroma, both of which may require wild-type Smad3.
      In summary, our recent studies suggest that the effect of TGFβ1 on carcinogenesis largely depends on its cell-specific expression pattern. When TGFβ1 is overexpressed in proliferative keratinocytes, it induces profound inflammation that is sufficient to override its growth-inhibitory effect. Thus, our studies instigate future investigation of the role of TGFβ1 overexpression-associated inflammation in cancer development and its underlying molecular mechanisms.

      ACKNOWLEDGMENTS

      We thank the Molecular Profiling Resource of the Departments of Dermatology and Otolaryngology at Oregon Health & Science University for providing skin SCC and HNSCC samples, and surgeons in both departments for collecting human tissue samples. The original work in this laboratory was supported by NIH grants CA79998, CA87849, CA10549, and DE15953 to X.-J. W.

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