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Wounds That Will Not Heal

Pervasive Cellular Reprogramming in Cancer
  • Jung S. Byun
    Affiliations
    Cancer Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
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  • Kevin Gardner
    Correspondence
    Address correspondence to Kevin Gardner, M.D., Ph.D., Genetics Branch, National Cancer Institute/Department of Health and Human Services/NIH/Center for Cancer Research, Bldg 41, Room D305, Bethesda, MD 20892.
    Affiliations
    Cancer Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
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Open AccessPublished:February 25, 2013DOI:https://doi.org/10.1016/j.ajpath.2013.01.009
      There has been an explosion of articles on epithelial-mesenchymal transition and other modes of cellular reprogramming that influence the tumor microenvironment. Many controversies exist and remain to be resolved. The interest of the pathologists in the molecular and functional parallels between wound healing and the developing tumor stroma has its earliest origin in the writings of Rudolph Virchow in the 19th century. Since then, most of the focus has been primarily on the dynamics of the extracellular matrix; however, new interest has been redirected toward deciphering and understanding the enigmatic, yet elegant, plasticity of the cellular components of the proliferating epithelia and stroma and how they are reciprocally influenced. Citing several examples from breast cancer research, we will trace how these perspectives have unfolded in the pages of The American Journal of Pathology and other investigative journals during the past century, their impact, and where the field is headed.
      In 1858, Rudolph Virchow first proposed his irritation theory for cancer.
      • Virchow R.
      Die Cellularpathologie in Ihrer Begründung auf Physiologische und Pathologische Gewebelehre.
      • Alter N.M.
      Mechanical irritation as etiologic factor of cancer: clinical observation.
      This concept was based on the observation that neoplastic lesions often develop at sites of chronic irritation. Virchow concluded that irritation of any type, including mechanical, chemical, or thermal, was “the essential factor of neoplastic tissue proliferation.”
      • Alter N.M.
      Mechanical irritation as etiologic factor of cancer: clinical observation.
      ,pp 511 Through an astute synthesis of these general observations with the microscopic finding that foci of irritation or abnormal excitation were invariably associated with a reactive process characterized by infiltration of inflammatory cells, Virchow later proposed his more celebrated concept that there is a causal link between inflammation and cancer.
      • Virchow R.
      Die Cellularpathologie in Ihrer Begründung auf Physiologische und Pathologische Gewebelehre.
      • Alter N.M.
      Mechanical irritation as etiologic factor of cancer: clinical observation.
      More than a century later, Dvorak colorfully coined the phrase that cancer was “a wound that does not heal,”
      • Dvorak H.F.
      Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.
      implying that the cellular and biochemical processes associated with wound healing are similar to those involved in the growth and development of tumor stroma. However, one of the earliest written recognitions of this similarity appeared, in 1924, in the Journal of Medical Research, the immediate predecessor of The American Journal of Pathology, in an article submitted by Montrose T. Burrows, entitled “Studies on Wound Healing: I ‘First Intention’ Healing of Open Wounds and the Nature of the Growth Stimulus in the Wound and Cancer.” Although, unlike Virchow, Burrows dismissed the possible significance of the role played by the infiltrating lymphocytes, he did recognize the critical importance of the relationship between the different fixed cells of epithelium and connective tissue in the generation of growth stimuli during the wound response. This led him to suggest that “cancer may be nothing more than a break in the balance” between these two populations. This notion was later reiterated by Haddow
      • Haddow A.
      Molecular repair, wound healing, and carcinogenesis: tumor production a possible overhealing?.
      in 1972. Notwithstanding its noted resemblance to cancer growth and invasion, the regenerative processes associated with wound healing have been a favorite topic of investigation by experimental pathologists for more than a century.
      • Akaiwa H.
      A quantitative study of wound healing in the rat, I: cell movements and cell layers during wound healing.
      • Loeb L.
      A comparative study of the mechanism of wound healing.
      A consistent and recurrent theme has been that the healing wound response can be characterized by three important factors: epithelial movements, cell proliferation, and contraction (or remodeling).
      • Akaiwa H.
      A quantitative study of wound healing in the rat, I: cell movements and cell layers during wound healing.
      • Dvorak H.F.
      Rous-Whipple Award Lecture: how tumors make bad blood vessels and stroma.
      It is generally accepted that wound healing is a sequential process that can be separated into three overlapping phases in which the appearance, growth, and differentiation of specific constituents have many similarities to developing tumor stroma. They include the following: i) inflammation, ii) proliferation, and iii) maturation. These processes differ in cancer and wound healing at the level of regulation, where there is lost control of multiple cellular, molecular, and biochemical processes that characterize each step.

      The Phases of Wound Healing and Their Similarity to Tumor Stroma Growth

      The word inflammation is derived from the Latin inflammatio, which means to ignite or set a fire. Although the role of nucleated cellular components of inflammation is the focus of modern cell biology, the most immediate and pronounced components of inflammation are the leakage of serum components, red blood cells, and platelets into the extracellular space of the connective tissue to initiate clot formation. Precipitated by disrupted vasculature integrity by either physical or biological injury, spilled plasma components are exposed to numerous thrombogenic elements in the extravascular space, thus initiating the clotting cascade via thrombin-cleaved conversion of leaked fibrinogen to fibrin, through the action of tissue factor, thromboplastin, combined with factor VII.
      • McKay D.G.
      Participation of components of the blood coagulation system in the inflammatory response.
      • Kumar V.
      • Abbas A.K.
      • Aster J.C.
      • Robbins S.L.
      Robbins Basic Pathology.
      The cleaved fibrin monomers then polymerize to form a fibrin gel that is stabilized by the cross-linking activity of factor XIIIa.
      • Kumar V.
      • Abbas A.K.
      • Aster J.C.
      • Robbins S.L.
      Robbins Basic Pathology.
      In combination with other components leaked from the vasculature, such as fibronectin, the fibrin gel forms a provisional matrix along which the other fixed cellular components of the epithelium and connective tissue can begin to wander.
      • Kumar V.
      • Abbas A.K.
      • Aster J.C.
      • Robbins S.L.
      Robbins Basic Pathology.
      • Clark R.A.
      • Lanigan J.M.
      • DellaPelle P.
      • Manseau E.
      • Dvorak H.F.
      • Colvin R.B.
      Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization.
      • Dvorak H.F.
      • Senger D.R.
      • Dvorak A.M.
      Fibrin as a component of the tumor stroma: origins and biological significance.
      At the same time, leaked platelets become activated by interaction with extracellular matrix (ECM) components in the extravascular space, including collagen and von Willebrand’s factor, to initiate the primary hemostatic process. The platelets degranulate, liberating a variety of factors, including fibrinogen, fibronectin, platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), histamine, epinephrine, and serotonin, that enhance the clotting cascade and, in combination with components of complement activation, attract a variety of cellular components.
      • Kumar V.
      • Abbas A.K.
      • Aster J.C.
      • Robbins S.L.
      Robbins Basic Pathology.
      • Markiewski M.M.
      • Lambris J.D.
      The role of complement in inflammatory diseases from behind the scenes into the spotlight.
      In addition to amplifying platelet activation and aggregation to form the primary hemostatic plug, these secreted factors serve to recruit a succession of cell types, including neutrophils, followed by mast cells, monocytes, and fibroblasts, into the wound area.
      • Dvorak H.F.
      Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.
      • Kumar V.
      • Abbas A.K.
      • Aster J.C.
      • Robbins S.L.
      Robbins Basic Pathology.
      • Schäfer M.
      • Werner S.
      Cancer as an overhealing wound: an old hypothesis revisited.
      In normal wound healing, hemostasis reaches completion, the extravasation subsides, and the inflammatory phase resolves with the replacement of the highly active provisional fibrin/fibronectin matrix with collagen. However, in tumor stroma, the lost integrity of the vasculature is not the result of mechanical, chemical, or biological injury; rather, it is the result of dramatic increases in the permeability of the vasculature due to elevated levels of vascular endothelial growth factor (VEGF), alias vascular permeability factor, secreted by the tumor cells.
      • Dvorak H.F.
      Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.
      • Folkman J.
      Tumor angiogenesis: therapeutic implications.
      • Senger D.R.
      • Galli S.J.
      • Dvorak A.M.
      • Perruzzi C.A.
      • Harvey V.S.
      • Dvorak H.F.
      Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid.
      The persistent fibrin and fibronectin deposition provides a sustained impetus that drives continuous recruitment, proliferation, and developmental differentiation in both the stromal and epithelial compartments.
      • Dvorak H.F.
      Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.
      • Haddow A.
      Molecular repair, wound healing, and carcinogenesis: tumor production a possible overhealing?.
      • Dvorak H.F.
      Rous-Whipple Award Lecture: how tumors make bad blood vessels and stroma.
      • Schäfer M.
      • Werner S.
      Cancer as an overhealing wound: an old hypothesis revisited.
      The proliferation phase of wound healing is marked by a robust increase in new tissue and expansion of the cellular mass. Under the influence of VEGF, new vessel formation occurs by increasing proliferation and migration of endothelial cells. PDGF induces the migration and proliferation of both fibroblasts and pericytes, the subendothelial cells that maintain capillary integrity.
      • Dvorak H.F.
      Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.
      • Haddow A.
      Molecular repair, wound healing, and carcinogenesis: tumor production a possible overhealing?.
      • Dvorak H.F.
      Rous-Whipple Award Lecture: how tumors make bad blood vessels and stroma.
      • Schäfer M.
      • Werner S.
      Cancer as an overhealing wound: an old hypothesis revisited.
      Entering monocytes begin to proliferate and differentiate into macrophages. Through this process, what is known as granulation tissue, the newly revascularized matrix of the wound forms, followed by hypertrophy and increased migration of the neighboring epithelium. Thus, the proliferation phase can be characterized by a massive and extensive phenotypic reprogramming of nearly all of the cellular components. In each case, this reprogramming is altered and/or exaggerated in the tumor stroma, changes that ultimately promote tumor progression.
      • Dvorak H.F.
      Tumors: wounds that do not heal: similarities between tumor stroma generation and wound healing.
      • Haddow A.
      Molecular repair, wound healing, and carcinogenesis: tumor production a possible overhealing?.
      • Dvorak H.F.
      Rous-Whipple Award Lecture: how tumors make bad blood vessels and stroma.
      • Schäfer M.
      • Werner S.
      Cancer as an overhealing wound: an old hypothesis revisited.
      We will examine the role and function of some of the major components.

      Tumor-Associated Macrophages

      Monocytes that are recruited to the wound quickly differentiate into macrophages and are the most highly represented cell type in the healing wound and tumor stroma.
      • Mantovani A.
      • Locati M.
      Orchestration of macrophage polarization.
      • Gordon S.
      • Martinez F.O.
      Alternative activation of macrophages: mechanism and functions.
      In the case of breast cancer, tumor-associated macrophages (TAMs) have been known to compose as much as 50% of the tumor mass.
      • Solinas G.
      • Germano G.
      • Mantovani A.
      • Allavena P.
      Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation.
      This cellular system represents one of the most plastic components in both wound and tumor stroma, with a wide range of phenotypes whose programs are highly dependent and responsive to the microenvironment.
      • Mantovani A.
      • Locati M.
      Orchestration of macrophage polarization.
      • Gordon S.
      • Martinez F.O.
      Alternative activation of macrophages: mechanism and functions.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Ojalvo L.S.
      • King W.
      • Cox D.
      • Pollard J.W.
      High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors.
      • Stout R.D.
      • Watkins S.K.
      • Suttles J.
      Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages.
      • Mantovani A.
      • Germano G.
      • Marchesi F.
      • Locatelli M.
      • Biswas S.K.
      Cancer-promoting tumor-associated macrophages: new vistas and open questions.
      • Mosser D.M.
      • Edwards J.P.
      Exploring the full spectrum of macrophage activation.
      Although there is significant disagreement about the different functional macrophage phenotypes, it is generally accepted that macrophages can be polarized into two generally distinct phenotypes.
      • Mantovani A.
      • Locati M.
      Orchestration of macrophage polarization.
      • Gordon S.
      • Martinez F.O.
      Alternative activation of macrophages: mechanism and functions.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      The macrophages frequently found in early healing wounds are the M1 phenotype. M1 macrophages are typically perceived as having tissue-destructive attributes that are polarized under the control of cytokines secreted by type 1 helper T-cell lymphocytes (eg, interferon-γ and tumor necrosis factor-α), in which they express high levels of major histocompatibility complex class II receptors and secrete IL-12 and IL-23 to fill a major role in combating infection by virus and other intracellular pathogens.
      • Mantovani A.
      • Locati M.
      Orchestration of macrophage polarization.
      • Gordon S.
      • Martinez F.O.
      Alternative activation of macrophages: mechanism and functions.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Stout R.D.
      • Watkins S.K.
      • Suttles J.
      Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages.
      • Bingle L.
      • Brown N.J.
      • Lewis C.E.
      The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.
      Major transcriptional programs associated with M1 macrophages are driven by Stat1 with broadened amplification through NF-κB networks.
      • Mantovani A.
      • Locati M.
      Orchestration of macrophage polarization.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Schmieder A.
      • Michel J.
      • Schonhaar K.
      • Goerdt S.
      • Schledzewski K.
      Differentiation and gene expression profile of tumor-associated macrophages.
      M2 macrophages represented a second alternatively reprogrammed class of activated cells that often have attributes that are considered wound resolving and increase in prominence at the later stages of wound healing.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Stout R.D.
      • Watkins S.K.
      • Suttles J.
      Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages.
      • Bingle L.
      • Brown N.J.
      • Lewis C.E.
      The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.
      • Schmieder A.
      • Michel J.
      • Schonhaar K.
      • Goerdt S.
      • Schledzewski K.
      Differentiation and gene expression profile of tumor-associated macrophages.
      This polarized phenotype is generally directed by cytokine signaling from type 2 helper T-cell lymphocytes (IL-4 and IL-13). M2 macrophages typically show low expression of major histocompatibility complex class II complexes and characteristically express stabilin-1 and arginase-1, which may function in building components of the ECM.
      • Mantovani A.
      • Locati M.
      Orchestration of macrophage polarization.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Schmieder A.
      • Michel J.
      • Schonhaar K.
      • Goerdt S.
      • Schledzewski K.
      Differentiation and gene expression profile of tumor-associated macrophages.
      The transcription networks that predominate in M2 macrophages are Stat3 and Stat6, with support pathways that use MYC (alias c-myc), peroxisome proliferator-activated receptor-γ, and C/EBP-β. They secrete IL-17, high levels of IL-10, and low levels of IL-12 and, thus, are thought to have a role in dampening the immune response.
      • Mosser D.M.
      • Edwards J.P.
      Exploring the full spectrum of macrophage activation.
      Notably, M2 macrophages also commonly secrete VEGF, TGF-β, EGF, prostaglandin E2, and matrix metalloproteinase (MMP)-9 and are, therefore, thought to play a significant role in angiogenesis and ECM remodeling in wounds and the tumor.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Schmieder A.
      • Michel J.
      • Schonhaar K.
      • Goerdt S.
      • Schledzewski K.
      Differentiation and gene expression profile of tumor-associated macrophages.
      As previously mentioned, the distinctions between M1 and M2 are blurred and there is ample evidence suggesting a significant role for phenotype switching in response to tissue and tumor microenvironment.
      • Stout R.D.
      • Watkins S.K.
      • Suttles J.
      Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages.
      Tumor-secreted chemokine ligand 2 and monocyte colony-stimulating factor play a prominent role in the accumulation of TAMs, in which the M2 phenotype is thought to play a significant role in promoting tumor invasion and metastasis.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Stout R.D.
      • Watkins S.K.
      • Suttles J.
      Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages.
      • Bingle L.
      • Brown N.J.
      • Lewis C.E.
      The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.
      Although there are conflicting reports, several studies have shown that the presence and amount of TAMs, particularly of the M2-like phenotype, is a poor prognostic factor in carcinoma of the breast and many other types of cancer.
      • Bingle L.
      • Brown N.J.
      • Lewis C.E.
      The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.
      • Mukhtar R.A.
      • Nseyo O.
      • Campbell M.J.
      • Esserman L.J.
      Tumor-associated macrophages in breast cancer as potential biomarkers for new treatments and diagnostics.
      In fact, recent studies suggest a role for TAMs in the racial/ethnic differences in breast cancer survival.
      • Martin D.N.
      • Boersma B.J.
      • Yi M.
      • Reimers M.
      • Howe T.M.
      • Yfantis H.G.
      • Tsai Y.C.
      • Williams E.H.
      • Lee D.H.
      • Stephens R.M.
      • Weissman A.M.
      • Ambs S.
      Differences in the tumor microenvironment between African-American and European-American breast cancer patients.
      • Mukhtar R.A.
      • Moore A.P.
      • Nseyo O.
      • Baehner F.L.
      • Au A.
      • Moore D.H.
      • Twomey P.
      • Campbell M.J.
      • Esserman L.J.
      Elevated PCNA+ tumor-associated macrophages in breast cancer are associated with early recurrence and non-Caucasian ethnicity.
      Moreover, the interaction between stromal macrophages and tumor is highly responsive to changes in the tumor microenvironment, and can respond to alterations in the tumor stroma secondary to events as diverse as cytotoxic chemotherapy, hormonal treatment, and local tissue events that induce hypoxia.
      • Sica A.
      • Larghi P.
      • Mancino A.
      • Rubino L.
      • Porta C.
      • Totaro M.G.
      • Rimoldi M.
      • Biswas S.K.
      • Allavena P.
      • Mantovani A.
      Macrophage polarization in tumour progression.
      • Stout R.D.
      • Watkins S.K.
      • Suttles J.
      Functional plasticity of macrophages: in situ reprogramming of tumor-associated macrophages.
      • Mantovani A.
      • Germano G.
      • Marchesi F.
      • Locatelli M.
      • Biswas S.K.
      Cancer-promoting tumor-associated macrophages: new vistas and open questions.
      • Schmieder A.
      • Michel J.
      • Schonhaar K.
      • Goerdt S.
      • Schledzewski K.
      Differentiation and gene expression profile of tumor-associated macrophages.
      • Talks K.L.
      • Turley H.
      • Gatter K.C.
      • Maxwell P.H.
      • Pugh C.W.
      • Ratcliffe P.J.
      • Harris A.L.
      The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages.

      The Role of Myofibroblasts in Tumor Stroma

      The most commonly recognized fixed or tissue-resident components of connective tissue are loosely referred to as fibroblasts. However, the fibroblast is, by far, one of the most phenotypically dynamic and enigmatic cellular components of the wound healing and tumor stroma.
      • Eddy R.J.
      • Petro J.A.
      • Tomasek J.J.
      Evidence for the nonmuscle nature of the “myofibroblast” of granulation tissue and hypertropic scar: an immunofluorescence study.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Prunotto M.
      • Desmouliere A.
      • Varga J.
      • De Wever O.
      • Mareel M.
      • Gabbiani G.
      Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.
      • Kalluri R.
      • Zeisberg M.
      Fibroblasts in cancer.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      The origin of reprogrammed fibroblasts and fibroblast-derived cells is diverse.
      • Eddy R.J.
      • Petro J.A.
      • Tomasek J.J.
      Evidence for the nonmuscle nature of the “myofibroblast” of granulation tissue and hypertropic scar: an immunofluorescence study.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      After inflammatory injury, fibroblasts are activated to migrate and proliferate in the wound area through interaction with the provisional fibrin matrix, where they begin to proliferate and initiate a differentiation program that drives the acquisition of secretory and contractile properties of myofibroblasts. This is marked by increased expression of α-smooth muscle actin and increased synthesis of collagen types I and III
      • Kalluri R.
      • Zeisberg M.
      Fibroblasts in cancer.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      ; however, the spectrum of gene expression in myofibroblasts is highly diverse and specific to anatomical locations.
      • Chang H.Y.
      • Chi J.T.
      • Dudoit S.
      • Bondre C.
      • van de Rijn M.
      • Botstein D.
      • Brown P.O.
      Diversity, topographic differentiation, and positional memory in human fibroblasts.
      The proliferation and reprogramming of fibroblast to myofibroblast is driven by the ED-A splice variant of fibronectin; TGF-β secreted by platelets, macrophages, and normal and malignant epithelia; and changes in the mechanoregulatory properties of ECM stiffness.
      • Tomasek J.J.
      • Gabbiani G.
      • Hinz B.
      • Chaponnier C.
      • Brown R.A.
      Myofibroblasts and mechano-regulation of connective tissue remodelling.
      In addition to influencing TGF-β availability, the mechanical properties of the ECM also have significant influence on myofibroblast differentiation through the regulation of focal adhesion and activation of the focal adhesion kinase signaling. Thus, ECM stiffness drives myofibroblast differentiation by a variety of mechanisms. Developing myofibroblasts have a twofold higher contractile activity than normal fibroblasts; throughout, the wound process begins to assume the mechanical load through growth and maturation of its adherence with the ECM via focal adhesion complexes linked to cytoskeleton to transduce signals that further promote the myofibroblast transcriptional program.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Prunotto M.
      • Desmouliere A.
      • Varga J.
      • De Wever O.
      • Mareel M.
      • Gabbiani G.
      Recent developments in myofibroblast biology: paradigms for connective tissue remodeling.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      • Tomasek J.J.
      • Gabbiani G.
      • Hinz B.
      • Chaponnier C.
      • Brown R.A.
      Myofibroblasts and mechano-regulation of connective tissue remodelling.
      In normal healing, once the ECM has taken over the normal mechanical load, the myofibroblast undergoes massive apoptosis.
      • Grinnell F.
      • Zhu M.
      • Carlson M.A.
      • Abrams J.M.
      Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue.
      However, the constant remodeling of the ECM of the tumor stroma, driven in part by the continuous TGF-β, fibroblast growth factor, and PDGF stimulation and matrix reshaping by MMPs secreted by TAMs, platelets, and tumor epithelia, provides a milieu in which the myofibroblast population persists.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      • Kalluri R.
      • Zeisberg M.
      Fibroblasts in cancer.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      • Labelle M.
      • Begum S.
      • Hynes R.O.
      Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis.
      Recently, another novel mode of myofibroblast activation has been identified, in which secreted exosome components, derived from tumor cells, drive myofibroblast reprogramming under the influenced TGF-β signaling.
      • Cho J.A.
      • Park H.
      • Lim E.H.
      • Lee K.W.
      Exosomes from breast cancer cells can convert adipose tissue-derived mesenchymal stem cells into myofibroblast-like cells.
      • Webber J.
      • Steadman R.
      • Mason M.D.
      • Tabi Z.
      • Clayton A.
      Cancer exosomes trigger fibroblast to myofibroblast differentiation.
      • Di Vizio D.
      • Morello M.
      • Dudley A.C.
      • Schow P.W.
      • Adam R.M.
      • Morley S.
      • Mulholland D.
      • Rotinen M.
      • Hager M.H.
      • Insabato L.
      • Moses M.A.
      • Demichelis F.
      • Lisanti M.P.
      • Wu H.
      • Klagsbrun M.
      • Bhowmick N.A.
      • Rubin M.A.
      • D’Souza-Schorey C.
      • Freeman M.R.
      Large oncosomes in human prostate cancer tissues and in the circulation of mice with metastatic disease.
      Myofibroblasts are recruited from a diverse array of precursors, and each source plays a significant and distinct role in driving tissue- and organ-specific fibrotic pathological conditions, ranging from the formation of reactive tumor stroma to hepatic, pulmonary, and renal fibrosis.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      • Kalluri R.
      • Zeisberg M.
      Fibroblasts in cancer.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      • Zeisberg M.
      • Bonner G.
      • Maeshima Y.
      • Colorado P.
      • Muller G.A.
      • Strutz F.
      • Kalluri R.
      Renal fibrosis: collagen composition and assembly regulates epithelial-mesenchymal transdifferentiation.
      • Kizu A.
      • Medici D.
      • Kalluri R.
      Endothelial-mesenchymal transition as a novel mechanism for generating myofibroblasts during diabetic nephropathy.
      In addition to tissue-resident fibroblasts, these sources include fibrocytes (bone marrow–derived fibroblast precursors distinguished by the presence of multiple hematopoietic surface markers
      • Bianchetti L.
      • Barczyk M.
      • Cardoso J.
      • Schmidt M.
      • Bellini A.
      • Mattoli S.
      Extracellular matrix remodelling properties of human fibrocytes.
      • Bucala R.
      • Spiegel L.A.
      • Chesney J.
      • Hogan M.
      • Cerami A.
      Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.
      ), local and bone marrow–derived mesenchymal stem cells,
      • Mishra P.J.
      • Glod J.W.
      • Banerjee D.
      Mesenchymal stem cells: flip side of the coin.
      pericytes (the support layer of small vessels),
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • Hudson T.E.
      • Nowlin B.T.
      • Bonventre J.V.
      • Valerius M.T.
      • McMahon A.P.
      • Duffield J.S.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      • Goritz C.
      • Dias D.O.
      • Tomilin N.
      • Barbacid M.
      • Shupliakov O.
      • Frisen J.
      A pericyte origin of spinal cord scar tissue.
      • Verbeek M.M.
      • Otte-Holler I.
      • Wesseling P.
      • Ruiter D.J.
      • de Waal R.M.
      Induction of alpha-smooth muscle actin expression in cultured human brain pericytes by transforming growth factor-beta 1.
      • Gressner A.M.
      Transdifferentiation of hepatic stellate cells (Ito cells) to myofibroblasts: a key event in hepatic fibrogenesis.
      • Qin L.
      • Han Y.P.
      Epigenetic repression of matrix metalloproteinases in myofibroblastic hepatic stellate cells through histone deacetylases 4: implication in tissue fibrosis.
      smooth muscle cells,
      • Coen M.
      • Gabbiani G.
      • Bochaton-Piallat M.L.
      Myofibroblast-mediated adventitial remodeling: an underestimated player in arterial pathology.
      endothelial cells,
      • Kizu A.
      • Medici D.
      • Kalluri R.
      Endothelial-mesenchymal transition as a novel mechanism for generating myofibroblasts during diabetic nephropathy.
      • Zeisberg E.M.
      • Potenta S.E.
      • Sugimoto H.
      • Zeisberg M.
      • Kalluri R.
      Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.
      • Piera-Velazquez S.
      • Li Z.
      • Jimenez S.A.
      Role of endothelial-mesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders.
      and normal and malignant epithelial cells.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      • Kizu A.
      • Medici D.
      • Kalluri R.
      Endothelial-mesenchymal transition as a novel mechanism for generating myofibroblasts during diabetic nephropathy.
      Once activated, myofibroblast cells secrete a constellation of substances that promote tumor growth, invasion, and metastasis, including MMPs (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and MMP-14
      • Lu P.
      • Takai K.
      • Weaver V.M.
      • Werb Z.
      Extracellular matrix degradation and remodeling in development and disease.
      ), tissue inhibitor of metalloproteinases, IL-1, IL-6, IL-8, TGF-β, EGF, basic fibroblast growth factor, and hepatocyte growth factor, in addition to collagens I and III and fibronectin.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      • Tomasek J.J.
      • Gabbiani G.
      • Hinz B.
      • Chaponnier C.
      • Brown R.A.
      Myofibroblasts and mechano-regulation of connective tissue remodelling.
      During the initial stages after myofibroblast activation, collagen III is the main secreted collagen that provides a substrate for continued migration and support for developing granulation tissue formation and angiogenesis. Recently, myofibroblast activation has been divided into two phases, one characterized by reprogrammed acquisition of contractile cytoskeletal features and referred to as protomyofibroblast.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      • Tomasek J.J.
      • Gabbiani G.
      • Hinz B.
      • Chaponnier C.
      • Brown R.A.
      Myofibroblasts and mechano-regulation of connective tissue remodelling.
      Mechanical stress and TGF-β drive further reprogramming of protomyofibroblasts to express α-smooth muscle actin in the stress fibers and contractile apparatus and, thus, become true contractile myofibroblasts.
      • Hinz B.
      • Phan S.H.
      • Thannickal V.J.
      • Galli A.
      • Bochaton-Piallat M.L.
      • Gabbiani G.
      The myofibroblast: one function, multiple origins.
      This activation is facilitated by broad roles played by transmembrane integrin in the activation of latent TGF-β associated with the ECM.
      • Nishimura S.L.
      Integrin-mediated transforming growth factor-beta activation, a potential therapeutic target in fibrogenic disorders.
      • Sheppard D.
      Integrin-mediated activation of latent transforming growth factor beta.
      Myofibroblasts also secrete high levels of glycosylated proteins, including glycosaminoglycan and proteoglycans.
      • Kalluri R.
      • Zeisberg M.
      Fibroblasts in cancer.
      • Otranto M.
      • Sarrazy V.
      • Bonté F.
      • Hinz B.
      • Gabbiani G.
      • Desmoulière A.
      The role of the myofibroblast in tumor stroma remodeling.
      • Tomasek J.J.
      • Gabbiani G.
      • Hinz B.
      • Chaponnier C.
      • Brown R.A.
      Myofibroblasts and mechano-regulation of connective tissue remodelling.
      • Tomasek J.J.
      • McRae J.
      • Owens G.K.
      • Haaksma C.J.
      Regulation of alpha-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic CArG element and the transforming growth factor-beta1 control element.
      One of these components, hyaluronan, acts in a feedback mechanism to enhance TGF-β–induced myofibroblast activation.
      • Simpson R.M.
      • Meran S.
      • Thomas D.
      • Stephens P.
      • Bowen T.
      • Steadman R.
      • Phillips A.
      Age-related changes in pericellular hyaluronan organization leads to impaired dermal fibroblast to myofibroblast differentiation.
      • Webber J.
      • Jenkins R.H.
      • Meran S.
      • Phillips A.
      • Steadman R.
      Modulation of TGFbeta1-dependent myofibroblast differentiation by hyaluronan.
      • Meran S.
      • Luo D.D.
      • Simpson R.
      • Martin J.
      • Wells A.
      • Steadman R.
      • Phillips A.O.
      Hyaluronan facilitates transforming growth factor-beta1-dependent proliferation via CD44 and epidermal growth factor receptor interaction.
      Interestingly, recent studies have found that a dominant population of reprogrammed myofibroblasts shows decreased expression of caveolin-1 linked to a functional inactivation of Rb and consequent up-regulated expression of Rb/E2F controlled gene networks.
      • Sotgia F.
      • Del Galdo F.
      • Casimiro M.C.
      • Bonuccelli G.
      • Mercier I.
      • Whitaker-Menezes D.
      • Daumer K.M.
      • Zhou J.
      • Wang C.
      • Katiyar S.
      • Xu H.
      • Bosco E.
      • Quong A.A.
      • Aronow B.
      • Witkiewicz A.K.
      • Minetti C.
      • Frank P.G.
      • Jimenez S.A.
      • Knudsen E.S.
      • Pestell R.G.
      • Lisanti M.P.
      Caveolin-1-/- null mammary stromal fibroblasts share characteristics with human breast cancer-associated fibroblasts.
      • Martinez-Outschoorn U.E.
      • Pavlides S.
      • Whitaker-Menezes D.
      • Daumer K.M.
      • Milliman J.N.
      • Chiavarina B.
      • Migneco G.
      • Witkiewicz A.K.
      • Martinez-Cantarin M.P.
      • Flomenberg N.
      • Howell A.
      • Pestell R.G.
      • Lisanti M.P.
      • Sotgia F.
      Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.
      These caveolin-deficient myofibroblasts were found to produce high levels of hepatocyte growth factor and TGF-β.
      • Sotgia F.
      • Del Galdo F.
      • Casimiro M.C.
      • Bonuccelli G.
      • Mercier I.
      • Whitaker-Menezes D.
      • Daumer K.M.
      • Zhou J.
      • Wang C.
      • Katiyar S.
      • Xu H.
      • Bosco E.
      • Quong A.A.
      • Aronow B.
      • Witkiewicz A.K.
      • Minetti C.
      • Frank P.G.
      • Jimenez S.A.
      • Knudsen E.S.
      • Pestell R.G.
      • Lisanti M.P.
      Caveolin-1-/- null mammary stromal fibroblasts share characteristics with human breast cancer-associated fibroblasts.
      • Li T.
      • Sotgia F.
      • Vuolo M.A.
      • Li M.
      • Yang W.C.
      • Pestell R.G.
      • Sparano J.A.
      • Lisanti M.P.
      Caveolin-1 mutations in human breast cancer: functional association with estrogen receptor alpha-positive status.

      Endothelial Cell and Pericyte Reprogramming

      Major components of the formation of granulation tissue during wound healing and the tumor angiogenesis are the endothelial cells and pericytes. For comprehensive reviews, we refer to several outstanding articles on angiogenesis published in The American Journal of Pathology and elsewhere.
      • Dvorak H.F.
      Rous-Whipple Award Lecture: how tumors make bad blood vessels and stroma.
      • Folkman J.
      Tumor angiogenesis: therapeutic implications.
      • Carmeliet P.
      • Jain R.K.
      Molecular mechanisms and clinical applications of angiogenesis.
      However, as previously stated, the increased proliferation of both endothelial cells and pericytes, associated with wound healing and reactive tumor stroma, is subject to reprogramming at many different levels. Endothelial transitions to myofibroblast-like phenotypes have been observed in several tissues and are associated with loss of cellular adhesion molecules in response to TGF-β and the acquisition of expression at α-smooth muscle actin and collagen type I, sometimes in association with tumors at the invasive front.
      • Kizu A.
      • Medici D.
      • Kalluri R.
      Endothelial-mesenchymal transition as a novel mechanism for generating myofibroblasts during diabetic nephropathy.
      • Zeisberg E.M.
      • Potenta S.E.
      • Sugimoto H.
      • Zeisberg M.
      • Kalluri R.
      Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.
      • Piera-Velazquez S.
      • Li Z.
      • Jimenez S.A.
      Role of endothelial-mesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders.
      • Virag J.I.
      • Murry C.E.
      Myofibroblast and endothelial cell proliferation during murine myocardial infarct repair.
      • Zeisberg E.M.
      • Potenta S.
      • Xie L.
      • Zeisberg M.
      • Kalluri R.
      Discovery of endothelial to mesenchymal transition as a source for carcinoma-associated fibroblasts.
      Despite the known role of pericytes in angiogenesis,
      • Ribatti D.
      • Nico B.
      • Crivellato E.
      The role of pericytes in angiogenesis.
      • Raza A.
      • Franklin M.J.
      • Dudek A.Z.
      Pericytes and vessel maturation during tumor angiogenesis and metastasis.
      the reprogramming of pericytes to acquire fibroblast features is only beginning to be explored. It is best understood in studies of renal fibrosis
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • Hudson T.E.
      • Nowlin B.T.
      • Bonventre J.V.
      • Valerius M.T.
      • McMahon A.P.
      • Duffield J.S.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      • Goritz C.
      • Dias D.O.
      • Tomilin N.
      • Barbacid M.
      • Shupliakov O.
      • Frisen J.
      A pericyte origin of spinal cord scar tissue.
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • Duffield J.S.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      • Morikawa S.
      • Baluk P.
      • Kaidoh T.
      • Haskell A.
      • Jain R.K.
      • McDonald D.M.
      Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.
      and, most notably, in liver fibrosis, in which the hepatic stellate cells, the pericytes of liver, are reprogrammed to myofibroblasts primarily through the action of TGF-β secreted from the Kupffer cells (resident macrophages) and platelets.
      • Gressner A.M.
      Transdifferentiation of hepatic stellate cells (Ito cells) to myofibroblasts: a key event in hepatic fibrogenesis.

      Epithelial Reprogramming in Wound Healing and the Tumor Stroma

      The most irretrievable event that leads to a terminal prognosis in patients with cancer is the progression to stage IV disease, the presence of distant metastasis.
      • Sugarbaker E.V.
      Some characteristics of metastasis in man.
      • Weil R.J.
      • Palmieri D.C.
      • Bronder J.L.
      • Stark A.M.
      • Steeg P.S.
      Breast cancer metastasis to the central nervous system.
      • Valastyan S.
      • Weinberg R.A.
      Tumor metastasis: molecular insights and evolving paradigms.
      The reprogramming of malignant cells through epithelial-mesenchymal transition represents the first step in the metastatic cascade. In this first step, a cancer cell must acquire the ability to invade the surrounding tissue. After invasion and breach of the basement membrane, it must gain access to the circulation through intravasation of blood or lymphatic vessels. Once in the circulation, the tumor cell must survive as a single cell until transported to distant tissues, where it must exit the vasculature or extravasate into the surrounding tissue to survive, proliferate, and reprogram to reacquire epithelial characteristics so that it may mature and colonize locally as a metastatic tumor.
      • Valastyan S.
      • Weinberg R.A.
      Tumor metastasis: molecular insights and evolving paradigms.
      The reacquisition of mesenchymal features after metastasis is referred to as mesenchymal-epithelial transition, a process that is often coupled with epithelial-mesenchymal transition (EMT) during embryonic development and plays a critical role in the development and seeding of metastatic disease.
      • Valastyan S.
      • Weinberg R.A.
      Tumor metastasis: molecular insights and evolving paradigms.
      • Gunasinghe N.P.
      • Wells A.
      • Thompson E.W.
      • Hugo H.J.
      Mesenchymal-epithelial transition (MET) as a mechanism for metastatic colonisation in breast cancer.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      • Polyak K.
      • Weinberg R.A.
      Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits.
      The reprogramming events during EMT are highly contextual; thus, the form and extent of this process are critically linked to the microenvironment.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Klymkowsky M.W.
      • Savagner P.
      Epithelial-mesenchymal transition: a cancer researcher’s conceptual friend and foe.
      • Scheel C.
      • Weinberg R.A.
      Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.
      This dynamic reprogramming is stimulated by a wide variety of agents, including TGF-β, bone morphogenetic proteins, Wnt signaling, fibroblast growth factors, hepatocyte growth factor, PDGF, Notch and Sonic Hedge Hog signaling, VEGF, inflammation, hypoxia, obesity, oxidative stress, and external agents (eg, smoking, UV irradiation, and alcohol).
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Nagathihalli N.S.
      • Massion P.P.
      • Gonzalez A.L.
      • Lu P.
      • Datta P.K.
      Smoking induces epithelial-to-mesenchymal transition in non-small cell lung cancer through HDAC-mediated downregulation of E-cadherin.
      EMT is a normal physiological process that was first described as a transdifferentiation event associated with tissue and organ morphogenesis during embryonic development.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Scheel C.
      • Weinberg R.A.
      Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.
      • Bolender D.L.
      • Markwald R.R.
      Epithelial-mesenchymal transformation in chick atrioventricular cushion morphogenesis.
      The primary features of EMT are a loss of epithelial tethering to neighboring cells and the basement membrane. This occurs in combination with a consequent increase in the mesenchymal features, characterized by loss of polarity, increased mobility and migration, and resistance to anoikis.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      • Klymkowsky M.W.
      • Savagner P.
      Epithelial-mesenchymal transition: a cancer researcher’s conceptual friend and foe.
      • Scheel C.
      • Weinberg R.A.
      Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.
      At the cellular level, these changes are defined by loss of various junctional complexes common to epithelial cells, including desmosomes, adherens junctions, tight junctions, and gap junctions. Accordingly, there is a reciprocal increase in acquisition of mesenchymal molecular features, including a switch from epithelial keratin to increased production of the intermediate filaments containing vimentin, the appearance of N-cadherin at the membrane surface, and the secretion of fibronectin and MMPs capable of digesting the basement membrane.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Klymkowsky M.W.
      • Savagner P.
      Epithelial-mesenchymal transition: a cancer researcher’s conceptual friend and foe.
      • Scheel C.
      • Weinberg R.A.
      Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.
      Integrins are a family of heterodimeric transmembrane proteins, composed of α and β subunits, that play a vast role in maintaining and regulating cellular attachments and interactions with the ECM.
      • Hood J.D.
      • Cheresh D.A.
      Role of integrins in cell invasion and migration.
      There are 18 known α subunits and 8 β subunits capable of forming 25 different cell adhesion complexes, each specific for different components of the ECM (eg, laminin, fibronectin, and vitronectin), as dictated by cell type, condition, and spatial orientation.
      • Hood J.D.
      • Cheresh D.A.
      Role of integrins in cell invasion and migration.
      The role of integrins in the wound healing response is complex, with shifting repertoires of receptors that influence cell type–specific adhesion with, and migration through, the continuously remodeled provisional matrix of the tumor stroma.
      • Koukoulis G.K.
      • Virtanen I.
      • Korhonen M.
      • Laitinen L.
      • Quaranta V.
      • Gould V.E.
      Immunohistochemical localization of integrins in the normal, hyperplastic, and neoplastic breast: correlations with their functions as receptors and cell adhesion molecules.
      • Zutter M.M.
      • Mazoujian G.
      • Santoro S.A.
      Decreased expression of integrin adhesive protein receptors in adenocarcinoma of the breast.
      Thus, the integrin repertoire will likely reflect different reprogrammed states within both the tumor cell and the other cellular components of the tumor stroma at different stages of invasion, intravasation, metastasis, extravasation, and recolonization of distant tissues after mesenchymal-epithelial transition. Just as cellular events inside the cell can influence the type and strength of adhesion with the ECM, integrin interactions with ECM components can transduce signals that broadly influence cellular attributes and behavior, including cell shape, stress fiber formation, and cell motility. Thus, throughout EMT, the integrins are involved in a variety of inside-out and outside-in signaling events that respond to and direct phenotypic changes.
      • Hood J.D.
      • Cheresh D.A.
      Role of integrins in cell invasion and migration.
      Two major mediators of the outside-in influences of the integrins are the integrin-associated kinases: focal adhesion kinase and integrin-linked kinase.
      • Hood J.D.
      • Cheresh D.A.
      Role of integrins in cell invasion and migration.
      • Oloumi A.
      • McPhee T.
      • Dedhar S.
      Regulation of E-cadherin expression and beta-catenin/Tcf transcriptional activity by the integrin-linked kinase.
      Integrin clustering induces activation of focal adhesion kinase, which influences cellular proliferation, survival, and motility. Focal adhesion kinase is expressed at higher levels in invasive tumors than benign or preneoplastic tissue, where it is thought to play a broad role in mediating morphogenesis and position-dependent survival during gland formation and stem cell maintenance.
      • Hood J.D.
      • Cheresh D.A.
      Role of integrins in cell invasion and migration.
      • Luo M.
      • Guan J.L.
      Focal adhesion kinase: a prominent determinant in breast cancer initiation, progression and metastasis.
      Engagement of the ECM by integrins leads to rapid and dynamic activation of integrin-linked kinase, which has broad influences on transcriptional programs that influence EMT, including transcriptional down-regulation of E-cadherin and the up-regulation of mesenchymal markers, such as vimentin.
      • Oloumi A.
      • McPhee T.
      • Dedhar S.
      Regulation of E-cadherin expression and beta-catenin/Tcf transcriptional activity by the integrin-linked kinase.
      Loss of E-cadherin results in furthering mesenchymal reprogramming by altering the distribution of β-catenin to the cytoplasm and nucleus to activate the Wnt targets. Finally, integrins have profound influences on the composition of the ECM through dynamic interactions and regulation of proteases that refashion the ECM and, therefore, participate in the constant remodeling of the tumor stroma.
      • Hood J.D.
      • Cheresh D.A.
      Role of integrins in cell invasion and migration.
      • van der Pluijm G.
      • Sijmons B.
      • Vloedgraven H.
      • van der Bent C.
      • Drijfhout J.W.
      • Verheijen J.
      • Quax P.
      • Karperien M.
      • Papapoulos S.
      • Lowik C.
      Urokinase-receptor/integrin complexes are functionally involved in adhesion and progression of human breast cancer in vivo.
      Although EMT has been heavily studied during the past few years, there still remains controversy among pathologists about its relevance to cancer. Much of this controversy centers on how rigidly EMT should be defined.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      • Klymkowsky M.W.
      • Savagner P.
      Epithelial-mesenchymal transition: a cancer researcher’s conceptual friend and foe.
      Recent efforts to circumscribe these concepts led to the defining of three different types of EMT (types I to III).
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      Type I is associated with embryonic development and organ formation and does not involve fibrosis or invasive attributes. Type I EMT generates cells that maintain the plasticity necessary to undergo the kinetically coordinated rounds of EMT and mesenchymal-epithelial transition necessary for tissue formation and organ development. Type II EMT is associated with wound healing, tissue regeneration, and organ fibrosis, linked to a self-limited inflammatory process. Type II EMT can lead to pathological fibrosis if inflammation persists. Type III is the type of EMT seen in malignant epithelial cells through altered genetic and epigenetic regulation. The entry and extent of neoplastic cell participation in EMT is heterogeneous.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      A central feature of the EMT-like changes seen in cancer is the high degree of variability and plasticity. Type III EMT is not, in the strictest sense, a completed transdifferentiation, as is thought to occur in organ fibrosis.
      • Zeisberg E.M.
      • Potenta S.E.
      • Sugimoto H.
      • Zeisberg M.
      • Kalluri R.
      Fibroblasts in kidney fibrosis emerge via endothelial-to-mesenchymal transition.
      • Kalluri R.
      • Weinberg R.A.
      The basics of epithelial-mesenchymal transition.
      Perhaps it is best to think of EMT in the context of cancer as a dedifferentiated process, or state, that is variable and highly plastic, rather than a well-defined terminal event.
      A variety of genetic and epigenetic programs, driven by a diverse array of pleiotropic transcription factors and transcriptional regulators, control the phenotypic transition during EMT.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Scheel C.
      • Weinberg R.A.
      Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.
      These include transcriptional regulators that have been well established as EMT transcription factors, including ZEB1,
      • Aigner K.
      • Dampier B.
      • Descovich L.
      • Mikula M.
      • Sultan A.
      • Schreiber M.
      • Mikulits W.
      • Brabletz T.
      • Strand D.
      • Obrist P.
      • Sommergruber W.
      • Schweifer N.
      • Wernitznig A.
      • Beug H.
      • Foisner R.
      • Eger A.
      The transcription factor ZEB1 (deltaEF1) promotes tumour cell dedifferentiation by repressing master regulators of epithelial polarity.
      TWIST1,
      • Yang M.H.
      • Hsu D.S.
      • Wang H.W.
      • Wang H.J.
      • Lan H.Y.
      • Yang W.H.
      • Huang C.H.
      • Kao S.Y.
      • Tzeng C.H.
      • Tai S.K.
      • Chang S.Y.
      • Lee O.K.
      • Wu K.J.
      Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition.
      SNAI1 (snail),
      • Cano A.
      • Perez-Moreno M.A.
      • Rodrigo I.
      • Locascio A.
      • Blanco M.J.
      • del Barrio M.G.
      • Portillo F.
      • Nieto M.A.
      The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.
      E47,
      • Moreno-Bueno G.
      • Cubillo E.
      • Sarrio D.
      • Peinado H.
      • Rodriguez-Pinilla S.M.
      • Villa S.
      • Bolos V.
      • Jorda M.
      • Fabra A.
      • Portillo F.
      • Palacios J.
      • Cano A.
      Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail, Slug, and E47 factors in epithelial-mesenchymal transition.
      and SNAI2 (slug).
      • Bolos V.
      • Peinado H.
      • Perez-Moreno M.A.
      • Fraga M.F.
      • Esteller M.
      • Cano A.
      The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors.
      In addition to these DNA binding proteins, multiple co-activators and corepressors play major roles in the epigenetic regulation of EMT, including the histone demethylase, LSD1,
      • Lin T.
      • Ponn A.
      • Hu X.
      • Law B.K.
      • Lu J.
      Requirement of the histone demethylase LSD1 in Snai1-mediated transcriptional repression during epithelial-mesenchymal transition.
      the histone methyl-transferase, G9a,
      • Dong C.
      • Wu Y.
      • Yao J.
      • Wang Y.
      • Yu Y.
      • Rychahou P.G.
      • Evers B.M.
      • Zhou B.P.
      G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer.
      the histone acetyl-transferase, p300,
      • Mizuguchi Y.
      • Specht S.
      • Lunz 3rd, J.G.
      • Isse K.
      • Corbitt N.
      • Takizawa T.
      • Demetris A.J.
      Cooperation of p300 and PCAF in the control of microRNA 200c/141 transcription and epithelial characteristics.
      • Yokomizo C.
      • Yamaguchi K.
      • Itoh Y.
      • Nishimura T.
      • Umemura A.
      • Minami M.
      • Yasui K.
      • Mitsuyoshi H.
      • Fujii H.
      • Tochiki N.
      • Nakajima T.
      • Okanoue T.
      • Yoshikawa T.
      High expression of p300 in HCC predicts shortened overall survival in association with enhanced epithelial mesenchymal transition of HCC cells.
      the polycomb components, Bmi190 and SUZ12,
      • Iliopoulos D.
      • Lindahl-Allen M.
      • Polytarchou C.
      • Hirsch H.A.
      • Tsichlis P.N.
      • Struhl K.
      Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated repression required for the formation and maintenance of cancer stem cells.
      and NADH-regulated transcriptional corepressor C-terminal binding protein 1/2.
      • Chinnadurai G.
      CtBP, an unconventional transcriptional corepressor in development and oncogenesis.
      • Di L.J.
      • Fernandez A.G.
      • De Siervi A.
      • Longo D.L.
      • Gardner K.
      Transcriptional regulation of BRCA1 expression by a metabolic switch.
      • Di L.J.
      • Byun J.S.
      • Wong M.M.
      • Wakano C.
      • Taylor T.
      • Bilke S.
      • Baek S.
      • Hunter K.
      • Yang H.
      • Lee M.
      • Zvosec C.
      • Khramtsova G.
      • Cheng F.
      • Perou C.M.
      • Ryan Miller C.
      • Raab R.
      • Olopade O.I.
      • Gardner K.
      Genome-wide profiles of CtBP link metabolism with genome stability and epithelial reprogramming in breast cancer.
      An unexpected characteristic associated with EMT is the acquisition of stem cell–like traits that confer properties of enhanced self-renewal to carcinoma cells undergoing EMT.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Scheel C.
      • Weinberg R.A.
      Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.
      This occurs because there is a significant overlap between the regulatory networks that control EMT and those pathways that are important for self-renewal. EMT transcription factors also repress pathways that inhibit or suppress the acquisition of stem cell traits.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Scheel C.
      • Weinberg R.A.
      Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links.
      For example, ZEB1 inhibits miR-200, a microRNA family that represses stem cell traits. In addition, multiple self-renewal pathways associated with promoting stemness, such as Wnt/B-catenin signaling, also function to stabilize EMT transcription factors, such as SNAI1.
      • Yook J.I.
      • Li X.Y.
      • Ota I.
      • Hu C.
      • Kim H.S.
      • Kim N.H.
      • Cha S.Y.
      • Ryu J.K.
      • Choi Y.J.
      • Kim J.
      • Fearon E.R.
      • Weiss S.J.
      A Wnt-Axin2-GSK3beta cascade regulates Snail1 activity in breast cancer cells.
      The concept of cancer stem cells still remains controversial,
      • Sell S.
      On the stem cell origin of cancer.
      as previously stated for EMT. A perhaps more palatable conceptual way to think about stem cell–like cancer cells is to consider or define them as a highly plastic dedifferentiated state possessing a threshold level of stem cell–like traits, instead of a defined discrete cellular population.
      • Nieto M.A.
      • Cano A.
      The epithelial-mesenchymal transition under control: global programs to regulate epithelial plasticity.
      • Mani S.A.
      • Guo W.
      • Liao M.J.
      • Eaton E.N.
      • Ayyanan A.
      • Zhou A.Y.
      • Brooks M.
      • Reinhard F.
      • Zhang C.C.
      • Shipitsin M.
      • Campbell L.L.
      • Polyak K.
      • Brisken C.
      • Yang J.
      • Weinberg R.A.
      The epithelial-mesenchymal transition generates cells with properties of stem cells.

      The Tumor Microenvironment: Dynamic Interfaces for Cross Talk in the Extracellular Space

      Throughout this review, we have emphasized the role played by various cellular components in the tumor stroma. However, the extracellular space represents a complicated and dynamic mix of growth factors, cytokines, chemokines, and metabolic intermediates that are secreted, shed, or spilled into the extracellular space in paracrine and autocrine patterns that actively induce migration, differentiation, and proliferation across each cellular component in the healing wound or reactive tumor stroma. The levels and diversity of the signaling components are dependent on the composition of cells and the composition of the secreted and deposited connective tissue, which continue to evolve in response to the protease secretion by tumor and stromal cells. This remodeling changes the mechanical properties of the stroma and can have profound influence on the latency of a variety of growth factors that depend on interaction with distinct components of the extracellular connective tissue to increase or decrease the potency of ligand-receptor interactions. Thus, the stromal interface with the tumor represents a highly dynamic and pleiotropic space where molecular information undergoes continuous exchange between epithelia, macrophages, myofibroblasts, endothelial cells, and pericytes during tumor growth and progression.

      Breast Cancer, Wound Healing, and the Postpartum Breast

      Reproductive history has long been known to have a significant impact on breast cancer incidence and outcome.
      • Clavel-Chapelon F.
      Differential effects of reproductive factors on the risk of pre- and postmenopausal breast cancer: results from a large cohort of French women.
      • Phipps A.I.
      • Chlebowski R.T.
      • Prentice R.
      • McTiernan A.
      • Wactawski-Wende J.
      • Kuller L.H.
      • Adams-Campbell L.L.
      • Lane D.
      • Stefanick M.L.
      • Vitolins M.
      • Kabat G.C.
      • Rohan T.E.
      • Li C.I.
      Reproductive history and oral contraceptive use in relation to risk of triple-negative breast cancer.
      • Sellers T.A.
      • Kushi L.H.
      • Potter J.D.
      • Kaye S.A.
      • Nelson C.L.
      • McGovern P.G.
      • Folsom A.R.
      Effect of family history, body-fat distribution, and reproductive factors on the risk of postmenopausal breast cancer.
      Early menarche, low parity, and older age at first pregnancy have all been associated with increased breast cancer risk.
      • Clavel-Chapelon F.
      Differential effects of reproductive factors on the risk of pre- and postmenopausal breast cancer: results from a large cohort of French women.
      • Phipps A.I.
      • Chlebowski R.T.
      • Prentice R.
      • McTiernan A.
      • Wactawski-Wende J.
      • Kuller L.H.
      • Adams-Campbell L.L.
      • Lane D.
      • Stefanick M.L.
      • Vitolins M.
      • Kabat G.C.
      • Rohan T.E.
      • Li C.I.
      Reproductive history and oral contraceptive use in relation to risk of triple-negative breast cancer.
      • Sellers T.A.
      • Kushi L.H.
      • Potter J.D.
      • Kaye S.A.
      • Nelson C.L.
      • McGovern P.G.
      • Folsom A.R.
      Effect of family history, body-fat distribution, and reproductive factors on the risk of postmenopausal breast cancer.
      These observations have been traditionally interpreted to indicate that a woman’s cumulative lifetime exposure to estrogen and, therefore, lifetime number of ovulatory cycles was a major positive determinant of estrogen receptor–positive breast cancer, thereby explaining the protection provided by pregnancy.
      • Clavel-Chapelon F.
      Differential effects of reproductive factors on the risk of pre- and postmenopausal breast cancer: results from a large cohort of French women.
      However, women with pregnancy-associated breast cancer have a higher mortality than patients without pregnancy-associated breast cancer.
      • Johansson A.L.
      • Andersson T.M.
      • Hsieh C.C.
      • Cnattingius S.
      • Lambe M.
      Increased mortality in women with breast cancer detected during pregnancy and different periods postpartum.
      Moreover, in women diagnosed with estrogen receptor–negative or triple-negative breast cancers (tumors negative for estrogen receptor, progesterone receptor, and the human EGF receptor 2), the reverse is true. Nulliparity is protective, whereas high parity is associated with increased risk and mortality.
      • Phipps A.I.
      • Chlebowski R.T.
      • Prentice R.
      • McTiernan A.
      • Wactawski-Wende J.
      • Kuller L.H.
      • Adams-Campbell L.L.
      • Lane D.
      • Stefanick M.L.
      • Vitolins M.
      • Kabat G.C.
      • Rohan T.E.
      • Li C.I.
      Reproductive history and oral contraceptive use in relation to risk of triple-negative breast cancer.
      This association was particularly strong in women of African heritage.
      • Millikan R.C.
      • Newman B.
      • Tse C.K.
      • Moorman P.G.
      • Conway K.
      • Dressler L.G.
      • Smith L.V.
      • Labbok M.H.
      • Geradts J.
      • Bensen J.T.
      • Jackson S.
      • Nyante S.
      • Livasy C.
      • Carey L.
      • Earp H.S.
      • Perou C.M.
      Epidemiology of basal-like breast cancer.
      The apparent paradox is readily explained if one considers that, after lactation, the involuting postpartum breast must undergo a massive wound healing response.
      • McDaniel S.M.
      • Rumer K.K.
      • Biroc S.L.
      • Metz R.P.
      • Singh M.
      • Porter W.
      • Schedin P.
      Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis.
      • O’Brien J.
      • Lyons T.
      • Monks J.
      • Lucia M.S.
      • Wilson R.S.
      • Hines L.
      • Man Y.G.
      • Borges V.
      • Schedin P.
      Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species.
      • Lyons T.R.
      • O’Brien J.
      • Borges V.F.
      • Conklin M.W.
      • Keely P.J.
      • Eliceiri K.W.
      • Marusyk A.
      • Tan A.C.
      • Schedin P.
      Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2.
      The involution of lactating mammary epithelium involves cycles of fibrin and fibronectin deposition and degradation, active turnover of the ECM by released MMPs, and active recruitment of inflammatory components that amplify the wound healing response, thereby providing a microenvironment that promotes tumor growth and invasion.
      • McDaniel S.M.
      • Rumer K.K.
      • Biroc S.L.
      • Metz R.P.
      • Singh M.
      • Porter W.
      • Schedin P.
      Remodeling of the mammary microenvironment after lactation promotes breast tumor cell metastasis.
      • O’Brien J.
      • Lyons T.
      • Monks J.
      • Lucia M.S.
      • Wilson R.S.
      • Hines L.
      • Man Y.G.
      • Borges V.
      • Schedin P.
      Alternatively activated macrophages and collagen remodeling characterize the postpartum involuting mammary gland across species.
      Thus, high parity may increase the risk of triple-negative breast cancers secondary to increased production and exposure of mammary epithelia to the wound healing response after involution of the lactating breast. Interestingly, several studies have found that prolonged breastfeeding is protective against triple-negative breast cancers.
      • Millikan R.C.
      • Newman B.
      • Tse C.K.
      • Moorman P.G.
      • Conway K.
      • Dressler L.G.
      • Smith L.V.
      • Labbok M.H.
      • Geradts J.
      • Bensen J.T.
      • Jackson S.
      • Nyante S.
      • Livasy C.
      • Carey L.
      • Earp H.S.
      • Perou C.M.
      Epidemiology of basal-like breast cancer.
      The reason remains obscure, but some investigators have conjectured that it may be the result of a more tapered wound healing response and inflammation with gradual weaning.
      • Kobayashi S.
      • Sugiura H.
      • Ando Y.
      • Shiraki N.
      • Yanagi T.
      • Yamashita H.
      • Toyama T.
      Reproductive history and breast cancer risk.
      However, recent studies by Sotgia et al
      • Sotgia F.
      • Casimiro M.C.
      • Bonuccelli G.
      • Liu M.
      • Whitaker-Menezes D.
      • Er O.
      • Daumer K.M.
      • Mercier I.
      • Witkiewicz A.K.
      • Minetti C.
      • Capozza F.
      • Gormley M.
      • Quong A.A.
      • Rui H.
      • Frank P.G.
      • Milliman J.N.
      • Knudsen E.S.
      • Zhou J.
      • Wang C.
      • Pestell R.G.
      • Lisanti M.P.
      Loss of caveolin-3 induces a lactogenic microenvironment that is protective against mammary tumor formation.
      might provide an additional and alternative interpretation. By using a mouse model of Cav-3–deleted mice, which undergoes pronounced precocious lactation, they were able to show that a lactogenic environment was protective against tumor formation.
      • Sotgia F.
      • Casimiro M.C.
      • Bonuccelli G.
      • Liu M.
      • Whitaker-Menezes D.
      • Er O.
      • Daumer K.M.
      • Mercier I.
      • Witkiewicz A.K.
      • Minetti C.
      • Capozza F.
      • Gormley M.
      • Quong A.A.
      • Rui H.
      • Frank P.G.
      • Milliman J.N.
      • Knudsen E.S.
      • Zhou J.
      • Wang C.
      • Pestell R.G.
      • Lisanti M.P.
      Loss of caveolin-3 induces a lactogenic microenvironment that is protective against mammary tumor formation.
      How and why this occurs and whether the prodifferentiation environment of the lactating breast reduces the size of the breast epithelial progenitor population at risk for transformation will be an important question to address in the future.

      Epigenetic Reprogramming and Cellular Plasticity in the Healing Wound and Tumor Stroma

      During the wound healing process and the evolution of the reactive tumor stroma, each of the cellular constituents responds to specific environmental clues to undergo significant stable, yet reversible, acquisition or loss of specific cellular features that can be inherited by subsequent cellular generations in the absence of alterations in DNA sequence. Similarly, epigenetic changes represent potentially reversible covalent changes to chromatin, through DNA methylation, histone acetylation, methylation, phosphorylation, and ubiquitylation, that are highly responsive to environmental influences and can be stably inherited by subsequent generations in the absence of changes to DNA sequence.
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome: biological and translational implications.
      These parallels indicate that epigenetic regulation is likely to be one of the most important modes of cellular phenotypic control during the wound healing response in cancer. However, despite the general role, epigenetic regulation is likely to play throughout wound healing and tumor stromal expansion; little is known about how cellular identity and the acquisition of epithelial and mesenchymal traits are controlled at the level of chromatin accessibility.
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome: biological and translational implications.
      A recent study provided one of the first assessments of epigenetic reprogramming on a genome scale. This was accomplished using non-transformed AML12 hepatocyte cells to profile large-scale epigenetic changes after TGF-β stimulation.
      • McDonald O.G.
      • Wu H.
      • Timp W.
      • Doi A.
      • Feinberg A.P.
      Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition.
      Although DNA methylation appears unchanged, there was a global reduction in histone H3 lysine 9 dimethylation, a known heterochromatin epigenetic mark, combined with an increase in histone H3 lysine 4 trimethylation, a known mark of active euchromatin, and an increase in histone H3 lysine 36 trimethylation, a mark of actively transcribing genes.
      • McDonald O.G.
      • Wu H.
      • Timp W.
      • Doi A.
      • Feinberg A.P.
      Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition.
      Surprisingly, most of these changes were located in large organized heterchromatin K9 modification domains that are normally situated at the periphery of the nucleus in association with the nuclear lamina.
      • Wen B.
      • Wu H.
      • Shinkai Y.
      • Irizarry R.A.
      • Feinberg A.P.
      Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells.
      Exposure to TGF-β was associated with decondensation of the peripheral heterochromatin in coordination with the histone changes. Notably, these changes were dependent on shifts in binding of the histone demethylase LSD1, implying a significant role for LSD1 in large-scale changes in chromatin during cellular reprogramming. Future genome-scale studies of this nature will be needed to profile the role of different epigenetic regulators in cellular reprogramming events, such as EMT, and how their activity may be modified by the microenvironment.
      This is particularly relevant given the growing consensus that many metabolic elements in the microenvironment may exert profound influences on epigenetic reprogramming. In fact, several normal products of carbohydrate metabolism have the potential to have significant effects on the epigenome, including well-known metabolites, such as acetyl-CoA, which could influence the level of histone acetylation through histone acetyl-transferases, such as p300.
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome: biological and translational implications.
      • Chalkiadaki A.
      • Guarente L.
      Sirtuins mediate mammalian metabolic responses to nutrient availability.
      NAD+, in combination with its reduced form, NADH, can have diverse influences on histone acetylation through the Sirtuin family of class III histone deacetylases and the NADH-binding class of dimeric C-terminal binding protein corepressors capable of recruiting class III histone deacetylases, histone methyl-transferases, histone demethylases, and DNA methyl-transferases.
      • Chinnadurai G.
      CtBP, an unconventional transcriptional corepressor in development and oncogenesis.
      • Di L.J.
      • Fernandez A.G.
      • De Siervi A.
      • Longo D.L.
      • Gardner K.
      Transcriptional regulation of BRCA1 expression by a metabolic switch.
      • Di L.J.
      • Byun J.S.
      • Wong M.M.
      • Wakano C.
      • Taylor T.
      • Bilke S.
      • Baek S.
      • Hunter K.
      • Yang H.
      • Lee M.
      • Zvosec C.
      • Khramtsova G.
      • Cheng F.
      • Perou C.M.
      • Ryan Miller C.
      • Raab R.
      • Olopade O.I.
      • Gardner K.
      Genome-wide profiles of CtBP link metabolism with genome stability and epithelial reprogramming in breast cancer.
      α-Ketoglutarate is a rapidly oxidized metabolite in the tricarboxylic acid cycle and is a substrate for both Jumonji C domain histone demethylases and the 10 to 11 translocation protein methylcytosine oxidases that play a role in histone demethylation and DNA demethylation, respectively.
      • Baylin S.B.
      • Jones P.A.
      A decade of exploring the cancer epigenome: biological and translational implications.
      • Xu W.
      • Yang H.
      • Liu Y.
      • Yang Y.
      • Wang P.
      • Kim S.H.
      • Ito S.
      • Yang C.
      • Xiao M.T.
      • Liu L.X.
      • Jiang W.Q.
      • Liu J.
      • Zhang J.Y.
      • Wang B.
      • Frye S.
      • Zhang Y.
      • Xu Y.H.
      • Lei Q.Y.
      • Guan K.L.
      • Zhao S.M.
      • Xiong Y.
      Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of alpha-ketoglutarate-dependent dioxygenases.
      Together, these metabolites and their epigenetic regulation represent a metabolic transduction cascade that can link metabolic parameters of both the tumor and microenvironment to epigenetic events important for cellular reprogramming in both wound healing and cancer.

      Concluding Remarks

      After nearly a century of research in the role of the microenvironment in wound healing and the formation of tumor stroma, the focus is shifting to understanding function and control at the cellular level. The tumor microenvironment is not just a stew of growth factors, cytokines, and extracellular enzymes that shape the ECM. Instead, it is an active and bustling harbor of many cells in dynamic communication with each other and undergoing shifts and changes in cellular identity (Figure 1). The newest frontier will be to define how the epigenome is shaped and sculpted within each cell type, during each cellular reprogramming event, and how the changing epigenomes within the tumor and stroma can act in synergy during tumor progression.
      Figure thumbnail gr1
      Figure 1Schematic diagram of the diverse cellular reprogramming that occurs during wound healing responses in the tumor stroma. Cellular processes are italicized. Indicated are reversible transitions between 1) epithelial cells and mesenchymal phenotypes, 2) M1 macrophages and M2 macrophages, 3) fibroblasts and myofibroblasts, 4) endothelial cells and myofibroblasts, and 5) pericytes and myofibroblasts. CCL, chemokine C-C motif ligand; FGF, fibroblast growth factor.

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