intestinal gucy2c prevents tgf-b secretion coordinating ... · erik s. blomain 1, satish rattan2,...

Molecular and Cellular Pathobiology

Intestinal GUCY2C Prevents TGF-b Secretion CoordinatingDesmoplasia and Hyperproliferation in Colorectal Cancer

Ahmara V. Gibbons1,4, Jieru E. Lin1, Gilbert W. Kim1, Glen P. Marszalowicz3, Peng Li1,5, Brian A. Stoecker1,Erik S. Blomain1, Satish Rattan2, Adam E. Snook1, Stephanie Schulz1, and Scott A. Waldman1

AbstractTumorigenesis is a multistep process that reflects intimate reciprocal interactions between epithelia and

underlying stroma. However, tumor-initiating mechanisms coordinating transformation of both epithelial andstromal components are not defined. In humans andmice, initiation of colorectal cancer is universally associatedwith loss of guanylin and uroguanylin, the endogenous ligands for the tumor suppressor guanylyl cyclaseC (GUCY2C), disrupting a network of homeostatic mechanisms along the crypt-surface axis. Here, we reveal thatsilencing GUCY2C in human colon cancer cells increases Akt-dependent TGF-b secretion, activating fibroblaststhrough TGF-b type I receptors and Smad3 phosphorylation. In turn, activating TGF-b signaling inducesfibroblasts to secrete hepatocyte growth factor (HGF), reciprocally driving colon cancer cell proliferation throughcMET-dependent signaling. Elimination of GUCY2C signaling in mice (Gucy2c�/�) produces intestinal desmo-plasia, with increased reactive myofibroblasts, which is suppressed by anti-TGF-b antibodies or genetic silencingof Akt. Thus, GUCY2C coordinates intestinal epithelial–mesenchymal homeostasis through reciprocal paracrinecircuits mediated by TGF-b and HGF. In that context, GUCY2C signaling constitutes a direct link between theinitiation of colorectal cancer and the induction of its associated desmoplastic stromal niche. The recentregulatory approval of oral GUCY2C ligands to treat chronic gastrointestinal disorders underscores the potentialtherapeutic opportunity for oral GUCY2C hormone replacement to prevent remodeling of themicroenvironmentessential for colorectal tumorigenesis. Cancer Res; 73(22); 6654–66. �2013 AACR.

IntroductionTumorigenesis reflects an intimate collaboration between

cancer cells and the supporting microenvironmental stroma(1–3). Genetic changes underlying malignant transformationcorrupt circuits regulating epithelial–mesenchymal cross-talkthat are essential for normal tissue organization, homeostasis,and regeneration (4). This reprogramming drives the coevo-lution of epithelial cells and their supporting microenviron-ment, producing reactive stroma that allows tumor cells toacquire the hallmarks of cancer (2, 5, 6).

The dynamic changes in cancer-associated stroma resemblethe desmoplastic reaction of wound healing, characterized byextracellular mesenchymal remodeling with increased matrixdeposition and matrix metalloproteinase (MMP) activity (4).

Tumor-associated desmoplasia is supported primarily by thetransformation of quiescent host fibroblasts into myofibro-blasts (1, 2, 7, 8). Although normal stroma contains fewfibroblasts, there is an increase in activated myofibroblastswithin the reactive stroma surrounding tumors (8). Inductionof the myofibroblastic phenotype in quiescent fibroblasts issupported by the secretion of TGF-b by cancer cells (1, 3, 9).Reciprocally, myofibroblasts play an essential role in solidtumors by producing paracrine and juxtacrine factors, includ-ingHGF, that drive normal epithelial cells toward acquiring themalignant phenotype (1, 5, 6, 10–14).

Paracrine cross-talk between epithelia and mesenchyme isessential to cancer progression and metastasis (15), but theprecise signaling mechanisms linking tumor-initiating eventsto desmoplastic induction remain incompletely defined. Forexample the cell type coordinating epithelial andmesenchymalreprogramming at the initiation of transformation remainsunknown (2, 8, 16). Thus, dissecting the individual stromal andepithelial contributions would define the paracrine signalingloops initiating and supporting carcinogenesis and providenew targets for tumor prevention and therapy.

Guanylyl cyclase C (GUCY2C) is the intestinal isoform fromthe family of guanylyl cyclase transmembrane receptorenzymes that signal by producing cyclic guanosine 30,50—monophosphate (cGMP; ref. 17). GUCY2C was first identifiedas the intestinal epithelial receptor regulating fluid and elec-trolyte transport in the secretory diarrhea induced by bacterialenterotoxins (ST; ref. 17). Discovery of the endogenous

Authors' Affiliations: Departments of 1Pharmacology and ExperimentalTherapeutics, and 2Medicine, Thomas Jefferson University; 3School ofBiomedical Engineering, Science and Health Systems, Drexel University,Philadelphia; 4Department of Ophthalmology, University of Pittsburgh,Pittsburgh, Pennsylvania; and 5Department of Pathology, University ofUtah, University Hospital, Salt Lake City, Utah

Note: A.V. Gibbons and J.E. Lin contributed equally to this article.

Corresponding Author: Scott A. Waldman, Department of Pharmacologyand Experimental Therapeutics, Thomas Jefferson University, 132 South10th Street, 1170 Main, Philadelphia, PA 19107. Phone: 215-955-6086;Fax: 215-955-5681; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-0887

�2013 American Association for Cancer Research.

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paracrine hormones uroguanylin, in the small intestine, andguanylin, in the colorectum (18), revealed a role for GUCY2C inepithelial cell dynamics and the homeostatic balance of pro-liferation, metabolism, and differentiation that organizes thecrypt-surface axis in the intestine (19–23).Notably, guanylin and uroguanylin are the most commonly

lost gene products in intestinal tumorigenesis in animals andhumans, and their expression is universally silenced at theearliest stages of transformation (24–27). Furthermore, elim-inating GUCY2C amplifies the tumorigenesis induced by car-cinogens or gene mutations in mice (20, 21). Indeed, silencingGUCY2C activates the AKT-mTOR signaling axis, whichimparts cell-autonomous characteristics of transformation tointestinal epithelial cells, encompassing accelerated prolifer-ation, reprogramming of metabolism from oxidative phos-phorylation to aerobic glycolysis, and impaired DNA damagerepair (19–23). Moreover, oral administration of GUCY2Chormone attenuates the colorectal tumorigenesis induced byadenomatous polyposis coli (APC) mutations in mice (25).GUCY2C has emerged as a novel intestinal tumor suppressorwhose silencing through hormone-loss universally contributesto the initiation and progression of colorectal cancer.In the heart, the natriuretic peptide receptor guanylyl

cyclase A isoform (NPR1) is expressed in cardiomyocytes andestablishes a homeostatic balance with the mesenchymalcompartment (28, 29). Silencing cardiomyocyte NPR1 andcGMP signaling produces cardiac hypertrophy and elevatedinterstitial fibrosis (28, 29). In fact, increases in growthand fibrosis are disproportionate to and perhaps even inde-pendent of increases in blood pressure or volume, suggestingthat NPR1 acts through local, paracrine pathways to suppresshypertrophy and desmoplasia (28, 29). The paracrine mechan-isms mediating this cardiomyocyte–mesenchymal cross-talkremain incompletely characterized. However, based on thisrole of NPR1 and cGMP signaling in the heart, the present studyexplored whether the tumor suppressor GUCY2C and cGMPhad homologous functions in the intestine, namely the main-tenance of epithelial–mesenchymal homeostasis whose cor-ruption contributes to tumor-associated desmoplasia.

Materials and MethodsAnimal modelsGucy2c�/� wild-type littermate mice on the C57BL/6

background (generation 14) were bred, maintained, geno-typed, and functionally characterized in accordance with theThomas Jefferson University Animal Care and Use guidelines(19–21). Akt1þ/�Gucy2cþ/þ and Akt1þ/�Gucy2c�/�were bredfrom Akt1þ/�Gucy2cþ/�, genotyped, and functionally char-acterized as described previously in accordance with theThomas Jefferson University Animal Care and Use guidelines(21). Where indicated, animals received an intraperitonealdose (0.5 mg/kg) of immunoglobulin G (IgG; InnovativeResearch) or TGF-b–specific monoclonal antibody purifiedfrom the 1D11.16.8 hybridoma cell line (American TypeCulture Collection, ATCC) twice weekly for 10 weeks. Thisregimen inhibits downstream canonical TGF-b signaling inintestine in C57BL/6 mice (30).

Cell culture and fibroblast activationT84 and Caco2 human colon cancer cells were maintained

and cultured in DMEM-F12 and Dulbecco's Modified EagleMedium (Cellgro) media supplemented with 10% FBS (21).CCD-18Co normal human intestinal fibroblasts were culturedand maintained in Eagle Minimum Essential Medium (ATCC).In studies of fibroblast induction, T84 or Caco2 cells werecultured in Minimum Essential Medium (MEM; Cellgro) with-out FBS for 48 hours. Then, completed growth media wereconditioned by cancer cells for another 48 hours, includingeither the GUCY2C ligand, ST (1 mmol/L), or vehicle, PBS (21,22). Conditioned media were centrifuged at maximum speedfor 5minutes to precipitate cancer cells and then dilutedwith 2parts of Opti-MEM GlutaMAX (Invitrogen) to obtain a 1:3dilution before applying to fibroblasts for 24 hours. Notably,T84 and Caco2 cells express GUCY2C, which mediates canon-ical downstream cGMP signaling upon ligand binding (19–21).In contrast, CCD-18Co do not express GUCY2C and are unre-sponsive to ligands targeting that receptor.

Antibody treatment, TGF-b receptor inhibitors, andsiRNA transfection

In experiments exploring the role of cancer cell TGF-b infibroblast activation, T84 or Caco2 cell–conditioned mediawere treated with a neutralizing antibody to TGF-b (30 ng/mL)or control IgG (30 ng/mL). In some experiments, TGF-b pro-duced by colon cancer cells was depleted from conditionedmedia with neutralizing antibody to TGF-b (30 ng/mL) fol-lowed by immunoprecipitation with Dynabeads Protein G(Invitrogen). Cancer cell-derived TGF-b removed by immuno-precipitation was replaced with recombinant purified TGF-b(rTGF-b; R&D Systems) at a concentration (10 ng/mL) corre-sponding to that produced by T84 cells, quantified by ELISA(R&D Systems). In experiments using TGF-b receptor inhibi-tors, colon cancer cells were treated with PBS or 1 mmol/L STfor 48 hours before the conditioned media was transferred toCCD-18Co cells pretreated for 0.5 hours with the TGF-b type Ireceptor inhibitor SB-505124 (50 nmol/L; Sigma-Aldrich). SB-505124 treatment continued in the conditioned media forthe indicated times. In studies using siRNA, colon cancer cellswere transfected with TGF-b–targeted or control siRNA (100nmol/L; ABI) by Amaxa Electroporation (Lonza) 48 hoursbefore use in conditioned media transfer experiments, topermit adequate time for suppression of TGF-b expression.

Akt activation and inhibition in cancer cellsT84 were infected with adenovirus-expressing siAKT1 and

GFP control for inhibition experiments, or adenovirus-expres-sing wtAKT1 and myrAKT1 for activation experiments, at 50%confluence using multiplicity of infections (MOI) of 100 at 48hours before GUCY2C activation during starvation (21). Then,cells were washed and incubated in complete media with 1mmol/L STor PBS (control) for 48 hours. The conditionedmediawere transferred tofibroblasts after dilution as described earlier.

HGF production by fibroblasts and cMET activationMedia conditioned by T84 cells with and without activation

of GUCY2C signaling by 1 mmol/L ST or PBS (control) for 48

GUCY2C Prevents Desmoplasia in Colorectal Cancer

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hours were used to induce fibroblast activation of CCD-18Cocells following incubation for 24 hours. Then, fibroblast-con-ditioned media were transferred to HCT116 human coloncancer cells, which do not express GUCY2C and possessinactivating mutations in the TGF-b receptor (21, 31), andproliferation was quantified using 3H-thymidine (21–23).HCT116 cells are particularly useful for quantifying the effectsof fibroblast-derived HGF on colon cancer cell proliferationin the absence of confounding by GUCY2C or TGF-b ligands. Insome experiments, media sequentially conditioned by humancancer cells and then by fibroblasts were treated with aneutralizing antibody to HGF or control IgG (20 mg/mL; R&DSystems). Where indicated, HGF produced by fibroblasts wasdepleted from conditioned media with neutralizing antibodyto HGF (20 mg/mL) followed by immunoprecipitation withDynabeads Protein G (Invitrogen). Fibroblast-derived HGFdepleted by immunoprecipitation was replaced with recom-binant purified HGF (rHGF; 2.5 ng/mL; R&D Systems). In someexperiments, HCT116 cells were pretreated with cMet KinaseInhibitor III (500 nmol/L; EMD Millipore), an inhibitor ofcMET, the canonical receptor for HGF (32), for 5 minutesbefore incubations with conditioned media.

Immunoblot analyses and immunofluorescenceProtein was extracted from cells and tissues in M-PER

reagent supplemented with protease and phosphatase inhibi-tors (Thermo Scientific) and quantified by immunoblot anal-ysis using antibodies to procollagen I (Sigma-Aldrich), a-SMAand MMP-9 (Biomol), and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) and prolyl hydroxylase-b (Abcam).Other antibodies were from Cell Signaling Technology. Anti-a-smooth muscle actin (a-SMA; Sigma-Aldrich), anti-collagenI, and anti-b-catenin (Santa Cruz Biotechnology) antibodieswere used to stain fibroblasts and epithelial cells, respectively(1, 33). Expression levels were quantified by estimating theintegrated density of the protein of interest and normalizing tothe integrated density of the nucleus (40,6-diamidino-2-phe-nylindole, DAPI). Stained tissues were quantified in 5 to 15crypt units in 5 to 10 sections in each mouse.

Masson trichrome stainTissue collected frommice were deparaffinized and stained

with Weigert iron hematoxylin solution and Biebrich scarlet-acid fuchsin, placed in phosphomolybdic/polyphosphotungs-tic acid solution, and counterstained with Aniline blue (Dako)orMasson Trichrome Stain Kit (Sigma-Aldrich). The bottom ofcrypts was identified by the presence of Paneth cells. In Figure1, the interstitial thickness was calculated as area of the bluestain divided by the length of the underlying muscle layer,quantified in Image J. In Figure 6, the interstitial thicknessdisplays the shortest distance between the bottom of the cryptand themuscle layer. In both cases, pixels were converted backtometric dimensions. Quantification represents data from 5 to10 crypt units in 5 to 10 sections per mouse.

Hydroxyproline assayTissuewas freeze-dried, homogenized, and hydrolyzed in 0.5

mL of 5 mol/L HCl for 16 hours at 116�C. Samples were dried,

dissolved in distilled water, and hydroxyproline quantifiedusing a colorimetric demethylmethylene blue assay (Sigma-Aldrich; ref. 34).

Collagen contractility assayCCD-18Co cells and Type I Collagen (Sigma-Aldrich) dis-

solved in acetic acid (0.1%) was used to access functionaldifferences in contractility between quiescent fibroblasts andfibroblasts (35). After solutions were neutralized with NaOH(1 mmol/L), cells were suspended in the collagen, plated, andcancer cell–conditioned media was added (35).

Quantitative reverse transcriptase PCRTotal RNA was subjected to one-step reverse transcriptase

(RT)-PCR using TaqMan EZ RT-PCR Core Reagents and spe-cific primer/probes for TaqMan Gene Expression Assays in anABI 7000 (Applied Biosystems; ref. 36). Relative expression wascalculated using the 2�DDCt method with GAPDH or b-actin asreference (36).

Electron microscopyIntestinal samples were collected, processed, fixed, and

embedded as previously described (21). Ninety-nanometersections were imaged with a JEOL-1010 electron microscopeusing 80 kV of acceleration.

Statistical analysesStatistical significance was determined by unpaired two-

tailed Student t test. Unless otherwise indicated, results rep-resent means� SEM from 3 animals or more or 3 experimentsor more, performed in triplicate.

ResultsGUCY2C in epithelial cells opposes desmoplasia

The absence of epithelial GUCY2C signaling in Gucy2cknockout mice (Gucy2c�/�) produced submucosal hypertro-phy (Fig. 1A), with canonical characteristics of desmoplasia (1,2, 5, 7). Specifically, stromal expansion was associated withincreased deposition of the matrix components tenascin C(Fig. 1B, top left), fibronectin (Fig. 1B, bottom), and collagen I(Fig. 1B, top right and bottom right), which is a hallmark ofmalignancy-associated desmoplasia (37). Also, absence of epi-thelial GUCY2C signaling increased submucosal fibroblastcontent, particularly in the pericryptal sheath (Fig. 1C1 and2), an anatomic location driving mesenchymally-directed epi-thelial cell proliferation (8, 38). Furthermore, these cells wereidentified as fibroblasts, reflected by increased transcription(Fig. 1D) and translation (Fig. 1E) of canonical markers offibroblast activation (1, 2, 5, 7). Thus, this submucosal hyper-trophy in Gucy2c�/� mice recapitulated the desmoplasticreaction in human colorectal cancer.

Moreover, in vitro studieswith human colon cancer cells andfibroblasts further demonstrated that fibroblast activation ismodulated by GUCY2C signaling. Media conditioned by T84human colon cancer cells in the presence or absence ofGUCY2C-activating ligand (ST) were collected and used totreat CCD-18Co human fibroblasts. T84 cells express GUCY2C,

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which mediates canonical downstream cGMP signaling uponligand binding (20–22). In contrast, CCD-18Co cells do notexpress GUCY2C and are unresponsive to ligands targetingthat receptor. As quantified by activated fibroblast numbers(Fig. 2A),matrix synthesis (Fig. 2B), and contraction of collagen(Fig. 2C; refs. 1, 7, 8, 35), conditioned media from T84 cellswithout ST treatment produced fibroblast activation, whichwas markedly reduced by ST.

Epithelial GUCY2C regulates fibroblast activationthrough TGF-b secretionTGF-b is an established mediator of fibroblast activation

and desmoplasia whose secretion characterizes the entiredevelopmental continuum of solid tumors in animals andhumans (1, 2, 7, 8, 11). GUCY2C activation by ST did not altertranscription of TGF-b, but markedly increased TGF-b reten-

tion and decreased TGF-b secretion, by cancer cells (Fig. 3Aand B). In a series of conditioned-media experiments, mediaconditioned by T84 human colon cancer cells were collectedand used to treat CCD-18Co humanfibroblasts. Reducing TGF-bmRNA expression and protein secretion in cancer cells withsiRNA (Fig. 3A) mimicked ST-induced GUCY2C signaling andopposed fibroblast activation, including collagen contractilityand translation of biomarkers of activation, by conditionedmedia (Fig. 3C and D). Inhibiting fibroblast TGF-b type Ireceptors with TGF-b receptor inhibitor SB-505124 also mim-icked ST-induced GUCY2C signaling and opposed fibroblastactivation by conditionedmedia (Fig. 3E). Furthermore, cancercell–conditioned media activated fibroblast TGF-b receptorsignaling through phosphorylation of the TGF-b signalingtarget Smad-3, and GUCY2C signaling in cancer cells blockedthat effect (Fig. 3F).

Figure 1. Silencing epithelialGUCY2C promotes intestinaldesmoplasia andstromal fibroblastactivation in mice. A1, Massontrichrome stain revealssubmucosal hypertrophy inGucy2c�/�, compared with wild-type Gucy2cþ/þ mice, associatedwith increased interstitial thickness(A, bottom) and matrix deposition(B, top), including tenascin (B, topleft). C, fibronectin (B, middle left),quantified by immunofluorescence(green, fibronectin; red, b-catenin;blue, DAPI; bottom of crypt isdetermined by presence ofb-catenin in nucleus, arrows) andcollagen, quantified byhydroxyproline analysis (B, topright), and direct visualization byelectron microscopy (n �3 mice;B, bottom right). Fibroblastswere visualized by a-SMAimmunohistochemistry (C1) andquantified in 10 sectionspermouseand 10 crypts per section (N ¼ 5mice; C2). Fibroblast activation inGucy2c�/�, compared withGucy2cþ/þ mice, was associatedwith increased expression ofmRNA (Col, collagen I; HGF,hepatocyte growth factor; D) andprotein (ProCol I, procollagen I;TIMP-1, tissue inhibitor ofmetalloproteinase-1; E) markers offibroblasts. Unless otherwiseindicated, data represent means ofN ¼ 5 � SEM. E, statistics reflectcomparisons with Gucy2cþ/þ.�, P < 0.05; ��, P < 0.01.






In addition, a neutralizing antibody to secreted TGF-breduced both the fibroblast activation (Fig. 4A) and theSmad-3 phosphorylation (Fig. 4B) induced by cancer cell–conditioned media. The effects of neutralizing antibodies onfibroblast activation and TGF-b signaling were abrogatedwhen TGF-b was reintroduced by adding rTGF-b (Fig. 4A andB). To reinforce these in vitro findings, in vivo studies wereconducted in which Gucy2c�/� mice were treated with TGF-b–neutralizing antibodies. Antibody administration reversedthe submucosal desmoplasia (Fig. 4C), reducing interstitialthickness (Fig. 4C), total collagen deposition (Fig. 4D), Smad-3phosphorylation (Fig. 4E), and fibroblast activation (Fig. 4F).These data reveal that GUCY2C mediates an efferent mucosalparacrine limb that modulates epithelial TGF-b secretion andfibroblast activation through TGF-b receptors and Smad-3phosphorylation, and thereby shapes the submucosal micro-environment to oppose the desmoplastic reaction.

GUCY2C regulates epithelial TGF-b secretion andfibroblast activation through Akt

GUCY2C regulates epithelial cell homeostasis and sup-presses tumorigenesis through the Akt signaling pathway(21). Notably, studies have also demonstrated that Akt regu-lates the secretion of TGF-b in mechanisms remodeling thestroma microenvironment (39). In vitro, elimination of AKT

expression with siRNA (siAkt) mimicked ST-induced GUCY2Csignaling, and replicated the effects of ST on cancer cellsecretion of TGF-b (Fig. 5A) and fibroblast activation (Fig.5B). Conversely, constitutive activation of AKT by myristoyla-tion (myrAKT; ref. 21) mimicked the absence of GUCY2Csignaling, and blocked the reverting effects of ST, leading tothe promotion of TGF-b secretion (Fig. 5C). Moreover, reduc-ing AKT expression through gene disruption in vivo subduedthe stromal remodeling produced by silencing GUCY2C (Fig.5D). These observations support a model in which, as in otherhomeostatic processes like proliferation and metabolism (19–23), the regulation of epithelial TGF-b secretion, fibroblastactivation, and stromal remodeling by GUCY2C is mediatedthrough epithelial AKT signaling.

GUCY2C reciprocally regulates fibroblast-dependentepithelial cell hyperproliferation

Beyond the contribution of efferent mucosal signals tosubmucosal fibroblasts, mechanisms by which this microen-vironmental reprogramming reciprocally contributes to anafferent paracrine loop modulating epithelial hyperprolifera-tion remain incompletely defined (1, 2, 7, 8). To investigate thisafferent loop, we conducted a series of in vitro experimentsusing sequentially conditioned media: media conditioned byhuman colon cancer cells were collected and used to activatehuman fibroblasts; and the media conditioned by these acti-vated fibroblasts were collected and applied to human coloncancer cells.

In one study, CCD-18Cohumanfibroblastswere activated byTGF-b at different concentrations, and the conditioned mediawere collected. Application of this fibroblast-conditionedmedia drove proliferation of human colon cancer cells, asquantified by 3H-thymidine incorporation, in a TGF-b concen-tration–dependent manner (Fig. 6A). Similarly, colon cancercell–conditioned media activated fibroblasts, which, in turn,produced conditioned media that reciprocally drove the pro-liferation of HCT116 human colon cancer cells, that do notexpress GUCY2C or TGF-b receptors (Fig. 6B). However, theability of fibroblasts to drive epithelial proliferation was elim-inated by pretreating epithelial cell–conditioned media withTGF-b neutralizing antibodies (Fig. 6B). Furthermore, whentreated with media from cancer cells incubated with theGUCY2C ligand ST, fibroblasts failed to drive epithelial pro-liferation (Fig. 6B). Exogenous supplementation of fibroblastswith rTGF-b reconstituted the fibroblast-conditioned media'scapacity to induce epithelial proliferation, abrogating theeffects of TGF-b neutralizing antibody or GUCY2C signaling(Fig. 6B). These results reveal that GUCY2C signaling regulatesthe epithelial secretion of TGF-b, which not only activatesfibroblasts, but also induces them to release factors thatreciprocally feed back to drive epithelial cell proliferation.

Desmoplasticfibroblast activation is associatedwith expres-sion of the mitogen HGF (Fig. 1D), a mediator of microenvi-ronmental influences on epithelial cells, including stromalinduction of epithelial proliferation (1, 2, 7, 8, 11, 40). Inagreement with previous studies, HGF induced HCT116human colon cancer cells to proliferate in a concentration-dependent fashion (Fig. 6C). Furthermore, pretreatment of

Figure 2. Activation of GUCY2C signaling prevents fibroblast activation.CCD-18Co human fibroblastswere incubatedwithmedia conditioned byT84 or Caco 2 human colon cancer cells, which both express GUCY2C,pretreated with or without 1 mmol/L ST for 48 hours and fibroblastactivation was quantified by enumeration of fibroblast activation bya-SMA immunofluorescence (red, a-SMA; blue, DAPI; A), immunoblotanalysis of fibroblast markers (Pro-Col I, procollagen I; B), fibroblastcontraction of collagen gels (red circle, contracted gel area; C). Datarepresent means of N ¼ 3 experiments � SEM in A–C. Data in Crepresent the percentage reduction in gel surface area induced bycontraction. �, P < 0.05; ��, P < 0.01.

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cancer cells with ST reduced HGF mRNA and protein produc-tion from fibroblasts that were activated by TGF-b secretedfrom colon cancer cells, (Fig. 6D). In turn, those fibroblast-conditioned media activated cMET, the canonical receptor forHGF (32), in HCT116 cells. This effect was eliminated by

pretreating fibroblast-conditioned media with HGF-neutraliz-ing antibody, an effect reversed by rHGF (Fig. 6E). Moreover,ST-activated GUCY2C signaling in epithelial cells reduced theirability to induce fibroblasts to drive colon cancer cell prolif-eration (Fig. 6B and F). This effect was replicated by adding

Figure 3. TGF-bmediates GUCY2C-dependent colon cancer cell regulation of fibroblast activation in vitro. A, effect of TGF-b1-specific siRNAs (siTGF1 andsiTGF2) on TGF-b1 mRNA (qRT-PCR) expression (top) and secretion (ELISA; bottom) by T84 cells in the presence or absence of ST stimulation (1 umol/L; 48hours) of GUCY2C. B, effect of GUCY2C, induced byST (1 mmol/L ST; 48 hours), to regulate TGF-b secretion, quantified by ELISA, in T84 human colon cancercell. C, effect of TGF-b1–specific siRNAson the ability of T84 cells pretreatedwithPBSorST (1mmol/LST; 48 hours) to conditionmediaquantifiedby fibroblastactivation (top) by confocal microscopy of a-SMA immunocytochemistry (activated fibroblasts) and fibroblast contraction of collagen gels (relativecontractility; bottom). D, effect of TGF-b1–specific siRNA on the ability of T84 cells pretreated with PBS or ST (1 mmol/L ST; 48 hours) to condition media,quantified by fibroblast PHD ora-SMA expression by immunoblot analysis (none, no siRNA; CTR, control, Scrambled siRNA; siTGF, TGF-b1 siRNA). E, effectof SB-505124, a selective inhibitor of TGF-b type I receptors, on the ability of CCD-18Co human fibroblasts to be activated by mediaconditioned by T84 human colon cancer cells pretreated with PBS (control) or the GUYC2C ligand ST. F, Smad3 phosphorylation in fibroblasts activatedby colon cancer cell–conditioned media (tSmad3, total Smad 3; pSmad3, phosphorylated Smad3). Data represent means of n �3 experiments � SEM.�, P < 0.05; ��, P < 0.01; NS, P > 0.05. NS, not significant.






HGF-neutralizing antibodies or an inhibitor of cMET to thefibroblast-conditioned media and blocked by rHGF (Fig. 6F).These observations suggest a model in which GUCY2C med-iates an efferent paracrine circuit regulating the epithelialsecretion of TGF-b, and the consequent activation of fibro-blasts. In turn, TGF-b ultimately induces a reciprocal afferentparacrine circuit in which the activated stromal fibroblastsproduceHGF, which promotes cMET-dependent epithelial cellproliferation (Fig. 7).

DiscussionUndergoing continuous cycles of regeneration, the intestinal

crypt-surface axis is dynamic. GUCY2C signaling organizesthe spatiotemporal tissue pattern, maintaining homeostasisalong this axis (19–23). Specifically, GUCY2C controls epithelial

cell proliferation by regulating the expression of criticalmediators that slow the cell cycle (19–23). Furthermore,GUCY2C and cGMP signaling establish cell fate by promot-ing the differentiation of epithelial cells in the secretorylineage, which includes Paneth and goblet cells (19). Theseeffects define the balance between the relative sizes ofproliferating and differentiated cell compartments along thecrypt-surface axis (19–23). Moreover, GUCY2C is key inmatching the appropriate metabolic program to the func-tional demands along this axis. Namely, low GUCY2C sig-naling in the crypt favors glycolysis to support rapidlyproliferating transit-amplifying cells (21). Conversely, robustGUCY2C signaling in the villus/surface compartment shiftsthe metabolic program to mitochondrial oxidative phos-phorylation, the canonical source for ATP generation indifferentiated cells (21).

Figure 4. TGF-b eliminates fibroblast activation in vitro and desmoplasia induced by silencing epithelial cell GUCY2C in mice. Media conditioned by T84human colon cancer cells incubated with PBS (control) or ST (1 mmol/L; 48 hours) were treated with control IgG or neutralizing anti-TGF-bmonoclonal antibody.In some experiments, T84 cell–derived TGF-b bound to neutralizing antibody was depleted using Protein G immunoaffinity beads and replaced withrTGF-b. Processed media were incubated with CCD-18Co human fibroblasts followed by quantification of fibroblast activation (A) by a-SMAimmunocytochemistry and phosphorylation (B) of Smad3 by immunoblot analysis. Colon cancer cell–conditioned media were treated with: IgG, control;TGF-b Ab, anti-TGF-b antibody (30 mg/mL); rTGF-b, depletion of TGF-b-anti-TGF-b complexes with protein G immunoaffinity beads followed byreplacement with recombinant TGF-b (10 ng/mL). C–F,Gucy2cþ/þ and Gucy2c�/� mice were treated with IgG or anti-TGF-b monoclonal antibody (0.5 mg/kg)intraperitoneally twice weekly for 10weeks to neutralize soluble TGF-b followed by quantification of intestinal matrix deposition (C), collagen content (D), Smad3phosphorylation (E), and fibroblast activation (F). Data represent means of n�4 experiments� SEM. �, P < 0.05; ��, P < 0.01; NS, P > 0.05. NS, not significant.

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The present study expands the homeostatic role for GUCY2Cbeyond these cell-autonomous functions to coordinating theparacrine cross-talk between the epithelial and mesenchymalcompartments that is essential for normal tissue patterning(1, 3, 40). Indeed, it suggests a model in whichmucosal GUCY2Ccoordinates bidirectional epithelial–mesenchymal cross-talk inthe intestine (Fig. 7). In this model, GUCY2C signaling in theepithelium opposes an efferent mucosal paracrine loop bysuppressing the epithelial secretion of TGF-b, the activation ofsubmucosal fibroblasts, and the desmoplastic reaction. In turn,the suppression of fibroblast activation by GUCY2C-regulated

paracrine signaling reduces HGF, silencing an afferent loop thatdrives epithelial cell proliferation. Notably, this GUCY2C func-tion in the intestinal mucosa recapitulates that of the relatedguanylyl cyclase isoformNPR1 in cardiomyocytes (28, 29). NPR1coordinates mesenchymal homeostasis in the heart, and itssilencing produces cardiac fibrosis that is independent ofincreases in blood pressure (28, 29). Targeted NPR1 knockout(Npr1�/�) produces local extracellular matrix remodeling inparacrine pathways involving increased TGF-b (28, 29).

The discovery that GUCY2C underlies spatiotemporal pat-terning along the crypt-surface axis expands its role in the

Figure 5. GUCY2C regulatesepithelial cell TGF-b secretion,fibroblast activation, anddesmoplasia through AKT. A,TGF-bprotein secretion fromcoloncancer cells was quantified byELISA. Cells were infected byadenovirus deliveringGFP (control)or siAKT (siRNA targeting AKT).The effect of siAKT was confirmedby immunoblotting for AKTphosphorylated at ser473 (pAKT).B, effect of siAkt on the abilityof T84 cells pretreated with PBS orST (1 mmol/L ST; 48 hours) tocondition media quantified bymyofibroblast activation by a-SMAimmunofluorescence (red, a-SMA;blue, DAPI; green, phospho-smad3). C, TGF-bprotein secretionfrom cells infected with adenovirusdeliveringwild-typeAKT (wtAKT) ormyristoylated AKT (myrAKT).Effect ofmyrAKTwas confirmed byimmunoblotting for pAKT. D,Masson trichrome stain forquantification of submucosalmatrix thickness. A–C, datarepresent means of n �3experiments � SEM. D, each pointrepresents 1 mouse. �, P < 0.05;��, P < 0.01; NS, P > 0.05. NS, notsignificant.






Figure6. MyofibroblastHGFproduction is controlled byepithelial cellGUCY2Candmediates afferent circuits regulatingcoloncancer cell proliferation. A, TGF-b induces CCD-18Co human fibroblasts to produce paracrine substances in conditioned media driving T84 human colon cancer cell proliferation in aconcentration-dependent fashion, quantified by 3H-thymidine incorporation. B, media conditioned by T84 human colon cancer cells incubated withPBS (control) or ST (1 mmol/L; 48 hours) were treatedwith control IgG or neutralizing anti-TGF-bmonoclonal antibody, with or without replacementwith rTGF-b. These media conditioned by T84 cells and processed as described were incubated with CCD-18Co human fibroblasts. Media sequentially conditioned byT84 cells and fibroblasts were then incubated with HCT116 human colon cancer cells that do not express GUCY2C or TGF-b receptors, followed byquantification of proliferation by 3H-thymidine incorporation. [Before conditioning by fibroblasts, T84 cell–conditioned media were treated with: IgG, control;TGF-b Ab, anti-TGF-b antibody (10 mg/mL); rTGF-b, depletion of TGF-b by immunoprecipitation followed by replacement with rTGF-b (5 ng/mL).]C, HGF induces HCT116 human colon cancer cell proliferation in a concentration-dependent fashion, quantified by 3H-thymidine incorporation. D, ST(1 mmol/L; 48 hours) pretreatment attenuates media conditioned by T84 colon cancer cells to induce HGF mRNA (left) and protein (right) expression in CCD-18Co human fibroblasts, quantifiedbyRT-PCR andELISA, respectively. E, HGF secretedbyCCD-18Co human fibroblasts activated bymedia conditioned byT84 human colon cancer cells treated with PBS or ST (1 mmol/L; 48 hours) activates cMET in HCT116 human colon cancer cells. cMET activation wasquantified by immunoblotting for phosphorylation of cMET at Tyr1234/1235. [Before addition to HCT116 cells, CCD-18Co cell-conditioned media weretreated with: IgG, control; HGF neutralizing antibody (HGF nAb; 20 mg/mL); rHGF, depletion of HGF by immunoprecipitation followed by replacementwith rHGF (1 ng/mL).] F, HGF secreted by CCD-18Co human fibroblasts activated bymedia conditioned by T84 human colon cancer cells treated with PBS orST (1mmol/L; 48 hours) inducesHCT116 colon cancer cell proliferation through cMET. [Before addition toHCT116 colon cancer cells, conditionedmediaweretreatedwith: IgG, control; HGFAb, anti-HGF neutralizing antibody (20mg/mL); rHGF, depletion of HGF-anti-HGF complexes by immunoprecipitation followedby replacement with recombinant HGF (2.5 ng/mL).] In some experiments, cMET was inhibited by cMet Kinase Inhibitor III (500 nmol/L; cMET Inhibitor).Processed media were then incubated with HCT116 human colon cancer cells, followed by quantification of proliferation by 3H-thymidine incorporation.Data in 6A, 6B, 6C, and 6Fare representative of 3 ormore experiments. Data inDandE representmeansofn�3 experiments�SEM.P<0.05; ��,P<0.01;NS,P > 0.05. NS, not significant.

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homeostatic maintenance of the intestinal mucosae (19, 22).Moreover, it highlights the emergence of GUCY2C as a tumorsuppressor universally silenced in the initiation and progres-sion of colorectal cancer (20, 21, 23). The endogenous ligandsfor GUCY2C, guanylin and uroguanylin, are the most com-monly lost gene products in intestinal tumorigenesis and theseligands are lost at the earliest stages of transformation (24–27).Loss of paracrine ligands silences GUCY2C, which, in turn,activates AKT and mTOR, well-established oncogenes drivingtumorigenesis in many tissues (21). Indeed, eliminatingGUCY2C signaling amplifies intestinal tumorigenesis inducedby carcinogens or gene mutations (20).Silencing GUCY2C establishes a number of cell-autonomous

mechanisms underlying epithelial cell tumorigenesis (6). Thus,the cell cycle is accelerated, expanding the proliferating cryptcompartment (19–23). Furthermore, metabolic programmingis shifted from mitochondrial oxidative phosphorylation toaerobic glycolysis characterizing the Warburg metabolic phe-notype (21). Moreover, genomic instability is accelerated byoverproduction of reactive oxygen species and exacerbated bya decrease in the ability to repair DNA damage (20, 21). Indeed,eliminating GUCY2C signaling induces Apc loss of heterozy-gosity in Apcmin mice and the acquisition of mutations inb-catenin in mice treated with the carcinogen AOM, encom-passing the genomic changes underlying nearly all sporadicintestinal tumorigenesis in humans (20). The present studyexpands the role of GUCY2C as a tumor suppressor whosesilencing not only corrupts cell-autonomous pathways regu-lating epithelial transformation, but also epithelial–mesenchy-mal paracrine circuits underlying fibroblast activation anddesmoplasia, a process that universally contributes to tumor-igenesis in most tissues (2, 5, 6).Although interactions between tumor cells and the desmo-

plastic stroma are essential for cancer progression, invasion,metastasis, and the evolution of therapeutic resistance, therelative contributions of each compartment to tumor initiationremain undefined (1, 2, 5, 16). The oncogenomic model ofcancer suggests that tumors initiate and evolve by accumu-lating mutations, which alter critical signaling pathways reg-

ulating cell-autonomous homeostatic functions, which ulti-mately leads to genomic instability and acquisition of invasiveand metastatic phenotypes (6, 41–43). In this model, themesenchymal compartment is dependent and reactive andshaped by the epithelial tumor to support cancer evolution. Analternate hypothesis suggests that, in some cases, stroma couldbe the pathophysiologic initiator of tumorigenesis in depen-dent normal epithelial cells. Mice inwhich TGF-b receptors aresilenced selectively in stromal fibroblasts develop neoplasia inthe prostate and stomach (10, 44). Similarly, genetic models ofHGF overexpression in fibroblasts are characterized by mam-mary tumorigenesis (45). In addition, mutations of tumorsuppressor genes in stromal fibroblasts drive the developmentof prostate tumors (46). This mechanistic debate about whichcompartment "leads" transformation has not yet been resolved(2, 5). The present study, in the context of GUCY2C silencing asone of the earliest changes underlying intestinal neoplasia (24–27), suggests that epithelial cell transformation leads tumorinitiation in intestine by corrupting cell-autonomous (19–23)and non–cell-autonomous homeostatic pathways, includingthe promotion of desmoplasia in colorectal cancer.

Beyond this question of compartmental hierarchy, therelationship between canonical networks whose corruptioninitiates neoplasia, and reciprocal paracrine signaling axesorganizing epithelial–mesenchymal cross-talk producing des-moplasia, remains undefined. For example, nearly all sporadiccolorectal tumors harbor mutations in the WNT signalingpathway, primarily in APC (about 80%) or b-catenin (about15%), which drive cell-autonomous changes in epithelia(41, 43, 47). However, the precise linkages between corruptedWNT signaling pathways underlying tumorigenesis andmechanisms coordinating desmoplasia characterizing colo-rectal cancer remain unknown. The present study offers amechanistic explanation for coordination of epithelial trans-formation and the development of the desmoplastic reactionin colorectal cancer. Indeed, silencing GUCY2C in epithelialcells seems to be one of the earliest universal changes incolorectal carcinogenesis (24–27), with the ability to promotegenomic instability and mutations in WNT signaling pathway

Figure 7. Model of GUCY2Csignaling effects on tumormicroenvironment. GUCY2Chormones, guanylin, anduroguanylin secreted by normalepithelial cells (blue) maintain ahomeostatic balance between themucosa and fibroblasts inunderlying stroma, mediated byepithelial cell GUCY2C. Silencingof GUCY2C through loss of ligandexpression, a universal early eventin tumorigenesis, inducestransforming epithelial cells (pink)to secrete TGF-b (purple bubbles),which activates fibroblasts. In turn,activated fibroblasts have theability to promote tumor cellproliferation byHGF (pink bubbles).






intermediates (20). In this model, silencing the GUCY2C tumorsuppressor early in transformation corrupts cell-autonomousfunctions that are essential for transformation, includingproliferation, metabolism, and DNA damage and repair (19–23).Moreover, the present study reveals that silencingGUCY2Csimultaneously initiates AKT-dependent paracrine signalingthrough increased epithelial cell TGF-b secretion, activatingfibroblasts that reshape the mesenchymal matrix compart-ment, producing the desmoplastic reaction promoting colo-rectal tumor invasion and progression.

There continues to be intense interest in elucidating theprecise compartmental contributions, and their sequence,underlying epithelial–mesenchymal cross-talk contributing toneoplasia (16). Deconvoluting this spatiotemporal signalingsequence could provide therapeutic targets that disrupt themutually reinforcing intercompartmental cycle essential fortumor initiation, progression, invasion, and metastasis (1, 2, 4,5, 8, 16). This challenge is underscored by the incompletelyunderstood role of TGF-b in tumorigenesis, sometimesreferred to as the "TGF-b Paradox" (11, 48, 49). On the onehand, TGF-b has potent antiproliferative properties in epithe-lial cells, and silencing tumor cell TGF-b receptors bymutationis a key genomic event mediating progression of colorectaltumorigenesis (41). On the other hand, TGF-b is one of themost potent drivers of quiescent fibroblast transformation tothe fibroblast phenotype, although the precise compartmentproducing this cytokine driving desmoplasia remains uncer-tain (1, 2, 5, 8). Moreover, the activity of TGF-b on the stromacan promote tumor initiation, progression, and metastasis(50). Similarly, although HGF, which drives epithelial prolifer-ation through cMET, is produced byfibroblasts in desmoplasia,the events inducing that production remain uncharacterized inmost tumors (1, 2, 5, 8). In that context, the present studyprovides insight into the spatiotemporal sequence of signalinginteractions coordinating events that drive tumor initiationand progression in epithelia and stroma in colorectal cancer. Itsuggests a model in which the earliest stages of tumorigenesisinitiate, at least in part, by silencing GUCY2C, reflecting loss ofligand expression (24–27). Eliminating cGMP signaling inintestinal epithelial cells not only drives cell-autonomouschanges coincident with transformation (19–23), but alsoamplifies epithelial secretion of TGF-b, creating an efferentsignal driving fibroblast activation and desmoplasia in adja-cent stroma. In turn, fibroblasts activated by epithelial cellTGF-b produce HGF, creating a reciprocal afferent cytokinecircuit with the potential to signal back to the epithelialcompartment through cMET. In more advanced stages oftransformation, characterized by loss of TGF-b signalingand its cell-cycle suppression in epithelia (41, 48, 49), thepotential of this reciprocal afferent HGF circuit is realized,further amplifying proliferation driving the progression oftumorigenesis.

Epithelial cell GUCY2C signaling is one component of aprogram establishing and maintaining the intestinal crypt-surface axis through continuous waves of physiologic regen-eration. Beyond this role in normal tissue patterning, loss ofguanylin and uroguanylin at the earliest stages of prema-lignancy suggests a novel model of intestinal tumorigenesis,

initiating as a disease of tissue-specific paracrine hormoneinsufficiency (24–27). The resultant silencing of the GUCY2Ctumor suppressor produces maladaptive amplification ofsurvival circuits producing DNA damage and mutations con-tributing to the cell-autonomous basis of colorectal cancer(19–23). Moreover, these changes in epithelial transformationare coordinated with simultaneous changes in cytokine sig-naling mediating the desmoplastic reaction that, reciprocally,drives epithelial proliferation. This unique model of intestinaltransformation as a paracrine hormone deficiency coordinat-ing epithelial–mesenchymal transformation has substantialimplications for the prevention and treatment of colorectalcancer. Indeed, GUCY2C is overexpressed by intestinal tumorscompared with normal adjacent tissues (36). Early loss ofhormones with compensatory overexpression of receptorsoffers a unique therapeutic window preceding the corruptionof epithelial survival pathways and stromal reactivity to pre-vent maladaptive reprogramming and the evolution of cancerthrough oral GUCY2C hormone replacement therapy (25).These considerations highlight the potential translationalopportunities for oral GUCY2C paracrine hormone supple-mentation as a novel strategy that could contribute to theprevention of colorectal cancer, underscored by the recentFDA approval and commercial availability of GUCY2C ligandsto treat chronic constipation (51).

Disclosure of Potential Conflicts of InterestS.A.Waldman received commercial research grant fromTargetedDiagnostics

& Therapeutics, Inc. and other commercial research support from Merck. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A.V. Gibbons, J.E. Lin, P. Li, E.S. Blomain, S. Rattan,A.E. Snook, S. Schulz, S.A. WaldmanDevelopment of methodology: A.V. Gibbons, J.E. Lin, G.P. Marszalowicz,E.S. Blomain, S. Rattan, S. Schulz, S.A. WaldmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.V. Gibbons, J.E. Lin, G.W. Kim, G.P. Marszalowicz,B.A. Stoecker, E.S. Blomain, S. RattanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.V. Gibbons, J.E. Lin, G.W. Kim, E.S. Blomain,S. Rattan, A.E. Snook, S. Schulz, S.A. WaldmanWriting, review, and/or revision of the manuscript: A.V. Gibbons, J.E. Lin,G.W. Kim, G.P. Marszalowicz, E.S. Blomain, S. Rattan, A.E. Snook, S. Schulz,S.A. WaldmanAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A.V. Gibbons, J.E. Lin, P. LiStudy supervision: A.V. Gibbons, J.E. Lin, P. Li, S. Schulz, S.A. Waldman

Grant SupportThese studies were supported by grants from the NIH (R01 CA75123, R01

CA95026, RC1 CA146033, P30 CA56036, and R01 CA170533), the PennsylvaniaDepartment of Health (SAP #4100059197 and SAP #4100051723), and TargetedDiagnostic and Therapeutics Inc. The Pennsylvania Department of Healthspecifically disclaims responsibility for any analyses, interpretations, or conclu-sions. A.V. Gibbons was supported by an NIH Minority Supplement (R01-CA095026-04S1). J.E. Linwas supported byNIH institutional award T32GM08562for Postdoctoral Training in Clinical Pharmacology and is the recipient of theYoung Investigator Award from the American Society for Clinical Pharmacologyand Therapeutics (ASCPT). G.W. Kim is the recipient of the Graduate Award forIntegrative Research in Pharmacology from the American Society for Pharma-cology and Experimental Therapeutics (ASPET). P. Li was enrolled in the NIH-supported institutional K30 Training Program In Human Investigation (K30HL004522) and was supported by NIH institutional award T32 GM08562 forPostdoctoral Training in Clinical Pharmacology. A.E. Snook is the recipient of theMeasey Foundation Fellowship.

Gibbons et al.





The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 29, 2013; revised July 31, 2013; accepted August 25, 2013;published OnlineFirst October 1, 2013.

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2013;73:6654-6666. Published OnlineFirst October 1, 2013.Cancer Res Ahmara V. Gibbons, Jieru E. Lin, Gilbert W. Kim, et al. Desmoplasia and Hyperproliferation in Colorectal Cancer

Secretion CoordinatingβIntestinal GUCY2C Prevents TGF-

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