Watch thy neighbor:cancer is a communalaffairValerie M. Weaver* and PenneyGilbertDepartment of Pathology and Institute for Medicineand Engineering, University of Pennsylvania, PA19104, USA

*Author for correspondence (e-mail:vmweaver@mail.med.upenn.edu)

Journal of Cell Science 117, 1287-1290Published by The Company of Biologists 2004doi:10.1242/jcs.01137

SummaryMalignant transformation of anepithelium occurs within the contextof a dynamically evolving tissuestroma that is composed of multiplecell types surrounded by anextracellular matrix. Because stromal-epithelial interactions regulate tissuehomeostasis and can profoundlyinfluence tumorigenesis it has beenproposed that the stromalmicroenvironment is an epigenetictumor modifier that can eitherpositively or negatively regulate themalignant behavior of geneticallyaberrant cells. New work reported inthis issue of Journal of Cell Sciencenow provides compelling evidencethat alterations in the stroma arenecessary and also sufficient forinduction of malignant behavior bygenetically normal cells.

The multi-hit genetic model of cancermaintains that tumors arise through acombination of hereditary alterationsand accumulation of incremental andsequential acquired changes in thegenome of targeted cells. Central to thisreductionist paradigm is the concept thatcell transformation will ensue followingaccumulation of a sufficient numberof mutations, amplifications, and/ordeletions in key genes that are essentialfor tissue homeostasis. Malignancyarises because critical mutationstheoretically release target cells fromtheir normal growth and survivalconstraints and permit their invasion,growth and survival in the surroundingextracellular matrix (Kinzler andVogelstein, 1996). Loss of geneticheterozygosity, however, has beendetected in morphologically normal

lobules adjacent to breast cancers (Denget al., 1996), and promoter methylation(silencing) of tumor suppressors such asp16INK4a occurs in histologically normalhuman mammary epithelial tissue (Holstet al., 2003). Moreover, the rate of tumorpenetration for hereditary germlinemutations in tumor suppressors isvariable, and malignant transformationof benign lesions often takes yearsbefore a clinically diagnosed malignancyemerges. Once tumors have formed,their behavior is often erratic andstochastic such that some tumors growand invade aggressively, while others ofsimilar grade can experience extendedperiods of dormancy (reviewed in Ungerand Weaver, 2003). Such observationsare consistent with the idea thattumorigenesis is an indolent andinefficient process that is probably morecomplex than initially appreciated. Thisrealization has heightened appreciationof the role played by genetic modifiersand epigenetics in malignancy.

Epithelial tissues are multicellular, 3Dstructures that interact dynamically withmultiple cell types, such as fibroblasts,adipocytes, infiltrating immune cells andendothelial cells, within the context of aproteinaceous microenvironmentalnetwork called the extracellular matrix(Fig. 1A, right). The fidelity of tissuedevelopment, adult tissue remodelingand tissue homeostasis all depend uponthe strict maintenance of a complexspatial and temporal dialogue betweenthe epithelium and the cellular andacellular components of the tissuestroma. Perturbations in stromal-epithelial interactions result in loss oftissue homeostasis and induction ofpathologies such as malignancy. In fact,tumor development is associated withinduction of a ‘reactive or desmoplastic’response in the stroma that ischaracterized by proliferation andtransdifferentiation of fibroblasts,infiltration and activation ofinflammatory cells, induction ofangiogenesis and altered deposition anddegradation of the extracellular matrix.Indeed, the desmoplastic tumor stroma isstrikingly similar to the stroma found ina wound (Fig. 1A, left). Because thewound stroma by necessity promotesepithelial cell growth and migration andfosters angiogenesis to drive healing,Bissell and co-workers hypothesized and

thereafter experimentally demonstratedthat the desmoplastic tumor stroma orwounded microenvironment is a tumorpromoter. These and other similarobservations by investigators includingCunha, Chung and colleagues heraldedthe study of tumorigenesis as a tissue-based disease in which malignanttransformation is studied in the contextof the tissue microenvironment(reviewed in Kenny and Bissell, 2003).

Over the past decade the epitheliocentricview of tumorigenesis has slowlybeen supplanted in favor of thetissue microenvironment concept ofmalignancy, where tumor pathogenesisis viewed as a ‘tissue-phenomena’ linkedto alterations in stromal-epithelialinteractions (Bissell and Radisky, 2001;Kenny and Bissell, 2003; Unger andWeaver, 2003). Key to this ‘tumormicroenvironment’ perspective ofmalignancy is the idea that ‘initiated’genetically primed or mutant target cellsthat give rise to epithelial tumors pre-exist or are acquired within a tissue. Thetheory asserts that genetically primedresident cells have a low pre-dispositionto develop into a tumor and willprobably remain dormant unless anexogenous stimulus, such as factorsproduced by the activated stroma, altersthe kinetics of tumor progression topromote the probability of diseaseinception, through the creation of afavorable microenvironment. A keyassumption is that the activated stromaacts as an auxillary factor or ‘normalwound response’ against a backgroundof pre-existing genetically altered targettumor cells. In favor of this scenariois the following evidence: thedemonstration that abnormal stromalfibroblasts can promote carcinogenesisin genetically abnormal butnontumorigenic prostate epithelial cellsand fail to alter the behavior ofgenetically normal prostate cellssignificantly; the observation thatco-culture of oncogene-expressingmammary epithelial cells (MECs) withfibroblasts significantly enhancestheir tumorigenicity in vivo; thedemonstration that induction of areactive stroma in the mammarygland following γ-irradiation drivestumorigenesis of a genetically aberrantmammary epithelium; the fact thatfactors secreted by infiltrating immune

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cells drive malignant invasion ofgenetically abnormal tissues; andexperiments showing that tempering thedesmoplastic response significantlyreduces malignant transformation ofHPV16 transgenic keratinocytes in mice(reviewed in Unger and Weaver, 2003).

Interestingly, evidence also suggests thatgenetic mutations need not pre-exist inthe target cells that give rise to thetumors for malignant transformation of atissue to ensue. Instead, it is possible thattumors could arise through alteredstromal-epithelial interactions because

of pre-existing genetic mutations(familial) or acquired alterations instromal cells. For example, Moinfar andcolleagues found that distinct geneticalterations and loss of heterozygosity ispresent in a high proportion of DNAanalyzed from excised stromal tissue

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AdipocyteFibroblast

ECM

KEY

Malignanttransform ation

X

XX X

a b c d e f

A

B

CX

Epithelial cell

Immune cellBasement membrane/myoepithelial cellsMatrix metalloproteinase

NMUinsult

Fig. 1. (A) Malignant transformation of an epithelium occurs within the context of a three dimensional tissue that is accompanied by (1) fibroblast proliferationand transdifferentiation, (2) extracellular matrix deposition and remodeling, (3) increased matrix metalloproteinase expression and activity, (4) infiltration ofimmune cells, and (5) angiogenesis. The tumor microenvironment therefore is a dynamically evolving microenvironment that fosters tumor cell invasion, survivaland growth. (B) Diagram showing potential NMU targets in the mammary tissue in vivo. Although the epithelium has been classically viewed as the criticalmutagenic target of chemical carcinogens, cells within the stroma and the extracellular matrix may also constitute viable chemical targets. (C) Experimentalscheme used by Maffini and colleagues to test the tissue organization field theory of carcinogenesis. Animal manipulations included: (a) stromal fat pad treatedwith NMU and reconstituted with vehicle treated MECs, (b) stromal fat pad and MECs both treated with NMU, (c) normal stromal fat pad reconstituted withNMU-treated MECs, (d) stromal fat pad and MECs treated with vehicle only, (e) intact mammary gland treated with NMU, and (f) intact mammary gland treatedwith vehicle only.

adjacent to primary breast tumors inpatients (Moinfar et al., 2000). Thisfinding is consistent with studiesconducted several years ago by Schorand colleagues, who found thatfibroblasts isolated from the healthy‘normal’ relatives of patients withfamilial breast disease exhibited atumor-like phenotype (Haggie et al.,1987). This perspective is also in accordwith a recent report that Nf1heterozygosity in resident stromalfibroblasts, mast cells and perineurialcells is probably essential forneurofibroma formation (Zhu et al.,2002). Such provocative observationstherefore raise the possibility thatgenetic alterations present in stromalcells may contribute to or even drivemalignant transformation of epithelialcells by perturbing the normal stromal-epithelial dialogue. As such, the‘microenvironmental’ concept oftumorigenesis would benefit from beingexpanded to incorporate the possibilitythat genetic alterations in either theepithelial or the stromal cells could leadto altered stromal-epithelial interactionsand thereby promote tumor formation(Fig. 1B).

It has been proposed that carcinogenesisresults not from acquisition of keymutations in genes in epithelial orstromal cells, but rather is theconsequence of a loss or breakdown ofthe biological organization of the tissueinduced by perturbed stromal-epithelialinteractions or an aberrant tissuemicroenvironment. The ‘tissueorganization field theory’ asserts that thenormal ‘default’ behavior of a cell is notquiescence but rather is proliferation,and that to sustain tissue homeostasisand promote differentiation this behaviormust be restricted through cell-adhesion-dependent tissue organization. Theconcept further predicts that themolecules and pathways critical formaintaining tissue architecture, such ascell adhesion molecules, constitutetumor suppressors. Based upon thisparadigm it follows that loss of tissuearchitecture or dysfunction of celladhesion, which could be inducedby perturbing stromal-epithelialinteractions, will drive malignantbehavior of cells within a tissue, evenin the absence of primary geneticmutations. The theory further maintains

that restoration of tissue organizationshould be able to repress the malignantphenotype of genetically aberrant cells(Sonnenschein and Soto, 2000). Supportfor the latter prediction comes from earlyexperiments by Mintz and colleagues,who showed that tissue architecture canrepress the malignant phenotype ofundifferentiated embryonal carcinoma(EC) cells, and by Dolberg and Bissell,who reported that Rous sarcoma virusdoes not induce sarcomas indifferentiated tissues derived from anavian embryo (reviewed in Kenny andBissell, 2003). Additional evidenceis provided by studies employingimmortalized malignant MECs andRas-transformed keratinocytes whichdemonstrated that reformation of acell-adhesion-dependent ‘differentiated’tissue structure was sufficient to repressexpression of the malignant phenotypeof transformed cells both in culture andin vivo, despite the presence of multiplegenetic alterations (reviewed in Ungerand Weaver, 2003). However, it shouldbe noted that although theseobservations arguably support the tissueorganization field theory they do notrefute a role for genetic mutations intumorigenesis.

To test the validity of the ‘tissueorganization field theory’, a numberof years ago Werb and colleaguesengineered the luminal mammaryepithelium of mice to overexpressstromelysin-1 (a metalloproteinase thathas been shown to degrade ECMprotein), which predictably disruptednormal stromal-epithelial interactionsand perturbed tissue organization anddifferentiation. The experiments clearlyshowed that a desmoplastic stroma candrive malignant transformation of anepithelium; however, because the tumorlatency in the studies was long thepossibility that the mice developedtumors by acquiring the genetic ‘hits’deemed necessary for malignanttransformation could not be excluded.Indeed comparative genomichybridization (CGH) analysis of tumorDNA from these mice revealed thepresence of multiple geneticabnormalities, which suggest that eitherloss of tissue organization promotesgenetic instability, or alternativelypermits expression of pre-existing

or acquired genetic abnormalities(reviewed in Kenny and Bissell, 2003).

To test the tissue organization fieldtheory directly, Maffini and co-workershave now used an acute chemicalcarcinogen treatment to rapidly inducetumor formation, and a mammary glandepithelial reconstitution approach todistinguish between the contributionof stromal-epithelial interactions andgenetic mutations to malignancy(Maffini et al., 2004) (see pp. 1495-1502 in this issue). To explore the roleof genomic alterations in tumorigenesisthe investigators monitored both thestroma and epithelial tissue for evidenceof oncogenic Ras mutations. Usingthe tumor susceptible strain of Wistar-Furth rats and the well characterizedcarcinogen N-nitroso-methyl urea(NMU; which is a direct carcinogen thatdoes not require metabolic conversionfor DNA adduct formation and has avery short half-life) they surgicallycleared the epithelium from themammary fat pads of test rats. Afterrecovering from surgery the animalswere divided into four treatment groups,including two groups that received asingle NMU treatment to theirmammary fat pad stroma, and two othergroups that received vehicle only.Mammary gland reconstitution was thenperformed on all four treatment groupsusing MECs from primary cultures ofexplanted tissue of older maturelittermates that had been acutely treatedeither with vehicle or NMU. Theanimals were thereafter monitored fordevelopment of a normal mammaryductal tree or tumors (Fig. 1C). Controlanimals included one group of‘negative’, vehicle-treated animals andanother group of ‘positive’, NMU-treated, non-surgically manipulatedanimals (Fig. 1Ce,f). Interestingly, allof the animals that received NMUtreatment developed tumors, regardlessof whether or not the mammaryepithelium used to reconstitute theirmammary fat pad was treated withcarcinogen. Even more intriguing wasthe investigators observation that noneof the animals that received MECstreated with NMU in culture developedneoplastic lesions, unless their stromalfat pad had also received a prior bolusof NMU. Because NMU is a directcarcinogen such data suggest that the

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stroma might itself constitute animportant mutagenic target (Fig. 1B).Such an observation would accord withan expanded tissue microenvironmentview of malignancy.

Provocatively, however, in the studiesreported by Maffini and colleagues(Maffini et al., 2004) no correlationcould be established between NMU-induced Ras mutations in either themammary epithelium or stroma andtumor formation. Although it is possiblethat NMU treatment of the stromapromoted malignant behavior of thetissue by inducing novel, as-yet-unidentified mutations in the DNA of thestromal cells, it is also reasonable tosuggest that chemical treatment of thestroma per se induced the ‘malignantbehavior’ of the tissue. With regards tothe former possibility, exploitation of agenetic screening approach such as CGHarray analysis should help to identifyadditional candidate genetic changes.Addressing the latter question, however,will necessitate determining just whattype of stromal change is induced by thechemical treatment, exploring whetherother chemical mutagens act similarlyon the stroma, and most importantlydelineating just how such chemicalmodification of the stroma might operateto incite malignant behavior of a tissue.

The studies reported by Maffini andcolleagues provide tantalizing evidencein support of the ‘tissue organizationfield theory’ of malignancy (Maffini etal., 2004). However, these studies alsoraise several important questions noteasily resolved, not the least of which iswhat constitutes malignancy? From amorphological perspective a malignantlesion is defined by subjectivemacroscopic and microscopic criteriathat include histological evidence of aloss of normal tissue architecture, cellproliferation, invasion of the epitheliuminto the interstitial stroma, the presence

of well-defined nuclear changes in thecells (such as anaplasia, large andmultiple nucleoli, and chromatinasymmetry), as well as evidence oftumor metastasis. Thus from a strictlygross morphological perspective theneoplastic lesions obtained in the studiesreported by Maffini and colleagues doappear to qualify as bona fide tumors(Maffini et al., 2004). However, furtheranalyses are needed to clarify just howmalignant these lesions really are,including an assessment of their nuclearmorphology and an assay of theirmetastatic potential. Moreover, giventhat loss of tissue architecture could pre-dispose cells to genomic instability(Sternlicht et al., 1999), it will beimportant to characterize these tumorsgenetically, and to determine whetheror not they can be phenotypicallyreverted by transplantation into anormal mammary gland stromalmicroenvironment. In this regard, alltissues are chronically exposed toenvironmental mutagens (radiation,chemical) and therefore will probablyharbor some genetically mutant but‘dormant’ cells eventually. Indeed, thevery definition of genetically ‘normal’tissue is fast becoming dubious at best.Therefore, it is possible and probablethat the cells comprising the malignantlesions detected by Maffini andcolleagues are genetically abnormal(Maffini et al., 2004). The question thenbecomes what contribution would thesemutations make to the malignantbehavior of their tissue and howimportant is the desmoplastic stroma totheir phenotype? In fact, definingmalignancy in strictly non-genetic termsmay be difficult if not impossible,and from a practical perspective thetissue organization field theory ofcarcinogenesis might just converge orcollide with the somatic mutation theory.Regardless, based upon the everexpanding body of evidence supportingthe importance of stromal-epithelial

interactions and cell adhesion in tumorpathogenesis, it seems advisable if notimperative to study tumorigenesis as adisease that occurs within the context ofa dynamic microenvironment that isregulated by the spatial organization ofthe tissue.

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