Therapeutics, Targets, and Chemical Biology

GUCY2C Signaling Opposes the AcuteRadiation-Induced GI SyndromePeng Li1, Evan Wuthrick2, Jeff A. Rappaport3, Crystal Kraft3, Jieru E. Lin3,Glen Marszalowicz3, Adam E. Snook3, Tingting Zhan4, Terry M. Hyslop5, andScott A.Waldman3

Abstract

High doses of ionizing radiation induce acute damage toepithelial cells of the gastrointestinal (GI) tract,mediating toxicitiesrestricting the therapeutic efficacy of radiation in cancer andmorbidity and mortality in nuclear disasters. No approved pro-phylaxis or therapy exists for these toxicities, in part reflectingan incomplete understanding of mechanisms contributing tothe acute radiation-induced GI syndrome (RIGS). Guanylatecyclase C (GUCY2C) and its hormones guanylin and uroguanylinhave recently emerged as one paracrine axis defending intestinalmucosal integrity against mutational, chemical, and inflammatoryinjury. Here, we reveal a role for the GUCY2C paracrine axis incompensatory mechanisms opposing RIGS. Eliminating GUCY2Csignaling exacerbated RIGS, amplifying radiation-induced mortal-

ity, weight loss, mucosal bleeding, debilitation, and intestinaldysfunction. Durable expression of GUCY2C, guanylin, and uro-guanylin mRNA and protein by intestinal epithelial cells waspreserved following lethal irradiation inducing RIGS. Oral deliveryof the heat-stable enterotoxin (ST), an exogenous GUCY2C ligand,opposed RIGS, a process requiring p53 activation mediated bydissociation from MDM2. In turn, p53 activation prevented celldeath by selectively limitingmitotic catastrophe, but not apoptosis.These studies reveal a role for the GUCY2C paracrine hormoneaxis as a novel compensatory mechanism opposing RIGS, andthey highlight the potential of oral GUCY2C agonists (Linzess;Trulance) to prevent and treat RIGS in cancer therapy and nucleardisasters. Cancer Res; 77(18); 5095–106. �2017 AACR.

IntroductionExposure to radiation in the context of terrorist attacks or

natural disasters produces death within about 10 days reflectingtoxicity to the gastrointestinal (GI) tract, constituting the acuteradiation-induced GI syndrome (RIGS; refs. 1–3). In contrast toradiation-induced bone marrow toxicity, in which death can bepreventedbybonemarrow transplantation, there are noapprovedmanagement paradigms to prevent or treat RIGS (4). Importantly,radiotherapy remains a mainstay in the management of cancer, aleading cause of death worldwide. Radiotherapy destroys rapidlyproliferating cancer cells and, inevitably, normal tissues charac-terized by continuous regeneration programs, including hairfollicles, bone marrow, the GI tract as well as other glandularepithelia (5). In that context, dose-limiting toxicities of radiationdiscourage patients from completing therapy, restrict maximum

doses of radiation, which limits the efficacy of treatment, and canlead to chronic morbidity and mortality (5).

Inadequate management in part reflects an incompleteunderstanding of mechanisms underlying RIGS. Indeed, criticalmolecular mechanisms and cellular targets mediating epithelialtoxicity underlying RIGS remain controversial (6–14). Recentstudies suggest that p53 in intestinal epithelial cells principallycontrols radiation-induced GI toxicity in mice, independentlyof apoptosis (7). In that context, deletion of the intrinsicapoptotic pathway from intestinal endothelial or epithelialcells failed to protect mice from GI toxicity–related death(7). In contrast, tissue-specific targeted deletion of intestinalepithelial cell p53 exacerbates, while its overexpression rescues,RIGS in mice (7, 14). However, mechanisms underlying radi-ation-induced intestinal epithelial cell death and intestinalmucosa damage remain undefined (7).

GUCY2C is the intestinal receptor for the endogenousparacrinehormones guanylin (GUCA2A) and uroguanylin (GUCA2B) andthe heat-stable enterotoxins (ST) produced by diarrheagenicbacteria (15–17). This signaling axis plays a central role inmucosal physiology, regulating fluid and electrolyte secretion(15, 16), and in coordinating crypt-surface homeostasis, regulat-ing enterocyte proliferation, differentiation,metabolism, apopto-sis, DNA repair, and epithelial–mesenchymal cross-talk (18–20).Furthermore, this axis maintains the intestinal barrier, opposingepithelial injury induced by carcinogens, inflammation, andradiation, and its dysfunction contributes to the pathophysiologyof inflammatory bowel disease and tumorigenesis (19–30).Whilethe GUCY2C signaling axis has emerged as one guardian ofintestinal epithelial integrity, the role of this axis in responses tolethal radiation, and its utility as a therapeutic target to preventand treat RIGS remains undefined (22).

1Department of Pathology, Immunology and Laboratory Medicine, College ofMedicine, The University of Florida, Gainesville, Florida. 2Department of Radi-ation Oncology, The Ohio State University Comprehensive Cancer Center,Columbus, Ohio. 3Department of Pharmacology and Experimental Therapeutics,Thomas Jefferson University, Philadelphia, Pennsylvania. 4Divisions of ClinicalPharmacology and Biostatistics, Thomas Jefferson University, Philadelphia,Pennsylvania. 5Department of Biostatistics and Bioinformatics, Duke University,Durham, North Carolina.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Scott A. Waldman, Thomas Jefferson University, 1020Locust Street, 368 JAH, Philadelphia, PA 19107. Phone: 215-955-6086; Fax: 215-955-5680; E-mail: scott.waldman@jefferson.edu

doi: 10.1158/0008-5472.CAN-17-0859

�2017 American Association for Cancer Research.

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Here, we define a novel role for the GUCY2C paracrine hor-mone axis in compensatory responses opposing RIGS. Indeed,eliminating GUCY2C signaling amplifies radiation-induced GItoxicity. In that context, durable expression of GUCY2C,GUCA2A, andGUCA2BmRNAandprotein is preserved followinghigh doses of radiation that induce RIGS. Moreover, oral admin-istration of the GUYC2C ligand ST opposed RIGS through a p53-dependent mechanism associated with the rescue of intestinalepithelial cells selectively from mitotic catastrophe, but not fromapoptosis. These observations reveal a previously unrecognizedcompensatory mechanism to epithelial injury induced by high-dose radiation, involving signaling by the GUCY2C paracrine axisthat opposes RIGS. They highlight the potential for oral GUCY2C-targeted agents to prevent or treat RIGS in the setting of cancerradiotherapy or environmental exposure through nuclear acci-dent or terrorism. The opportunity to immediately translate theseapproaches is underscored by the recent regulatory approval oflinaclotide (Linzess) and plecanatide (Trulance), oral GUCY2Cligands that treat chronic constipation (31).

Materials and MethodsAnimal models

Mice with a targeted germline deletion of GUCY2C(Gucy2c�/�) are well-characterized, and were used after �14generations of backcrossing onto the C57BL/6 background(15, 16, 18–20, 26, 32). p53FL-vil-Cre-ERT2 mice were gener-ated by crossbreeding vil-Cre-ERT2 (provided by S. Robine,Institut Curie-CNRS, France) with p53FL transgenic mice(mixed FVB.129 and C57BL/6 backgrounds, kindly providedby Dr. Karen Knudsen, Thomas Jefferson University, Philadel-phia, PA). Biallelic loss of p53 in intestinal epithelial cells(p53int�/�) was induced by intraperitoneal administration oftamoxifen (75 mg tamoxifen/kg/day � 5 days) to F2 p53FL-vil-Cre-ERT2 and control littermate p53þ-vil-Cre-ERT2, and deletionconfirmed structurally by immunoblot analysis of phosphor-ylated p53 (pp53; Supplementary Fig. S1) and functionally byradiation-induced mortality (Supplementary Fig. S2). Allexperiments were carried out with mice that were between 2and 3.5 months old (mixed males and females) and all micewere on mixed genetic backgrounds as described above and inSupplementary Fig. S3. Where appropriate, age-matched andlittermate controls were utilized to minimize the effect ofgenetic backgrounds. C57BL/6 mice used in oral ST or controlpeptide supplementation studies were obtained from NIH(NCI-Frederick), whereas those used for GUCY2C and ligandexpression analysis were obtained from the Jackson Labora-tory. This study was approved by the Institutional AnimalCare and Use Committee of Thomas Jefferson University(protocol 01518).

Gamma irradiation–induced GI toxicityAnesthetized mice were irradiated with total-body gamma

irradiation (TBI) or with back limbs to tail and front limbs tohead shielded with lead covers for subtotal-body irradiation(STBI) with exposure of abdominal area (approximately 1 inch2

from xiphoid to pubic symphysis). Mice were irradiated with a137Cs irradiator (Gammacell 40) at a dose rate of approximately70 cGy/minute for different doses from8 to 25Gy/mice.Mice hadfree access to regular food and water before and after irradiation.The severity of GI toxicity was evaluated bymortality, debilitation

(untidy fur coats), body weight, visible diarrhea, fecal occultblood, stool formation, stool water accumulation, andhistopathology.

ST and control peptidesST1-18 and control peptide (CP; inactive ST analogue contains

the same primary amino acid sequence, but with cysteines atpositions 5, 6, 9, 10, 14, 17 replaced by alanine) were purchasedfrom Bachem Co. (customer order; purity > 99.0%). ST andcontrol peptides were resuspended in 1� PBS at a concentrationof 50 ng/mL. Mice were orally gavaged with 10 mg of CP or ST (in200-mL solution) using a feeding needle (catalog no. 01-208-88,Thermo Fisher Scientific; ref. 26) daily for 14 days before and14 days after irradiation. ST and CP were prepared by solid-phasesynthesis and purified by reverse-phase HPLC, their structureconfirmed bymass spectrometry by BachemCo. (customer order;purity > 99.0%), and their activities confirmed by quantifyingcompetitive ligand binding, guanylate cyclase activation andsecretion in the suckling mouse assay (16, 33).

ReagentsMcCoy 5A and DMEM containing 10% FBS and other

reagents for cell culture were obtained from Life Technologies.8-Bromoguanosine 30, 50-cyclic monophosphate (8-Br-cGMP), acell-permeant analogue of cGMP, was obtained from Sigma and500 mmol/L was used in all experiments (18, 20, 25, 26, 34).

Cell linesC57BL/6-derived EL4 lymphoma cells (lymphoblasts inmouse

thymus; thymoma), C57BL/6-derived B16 melanoma cells, andHCT116 (wild-type p53) human colon cancer cells, which lackGUCY2C (Supplementary Fig. S4; refs. 19, 34, 35), were obtainedfrom ATCC. Isogenic HCT116-p53–null cells were a gift fromDr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD;ref. 36). Cell lines were authenticated employing establishedtranscriptomic, proteomic, phenotypic, and functional character-istics. Cell stocks were refreshed after the earlier of 10 passages ortwo years, and cell lines were screened for mycoplasma contam-ination every 6monthswith theUniversalMycoplasmaDetectionKit (ATCC). Cell lines were obtained and used in 2010–2011.

Ectopic tumor seeding and growth measurementEL4 and B16 cells (104 cells per injection) were injected

subcutaneously in mouse flanks (EL4, left and B16, right). Tumorgrowth was measured once every 3 days and tumor volumewas calculated by multiplying 3 tumor dimensions. Nosignificant differences in tumor growth before and after subtotalbody irradiation was observed in mice treated with ST comparedwith CP.

Immunoblot analysesProtein was extracted from mouse small intestine and colon

mucosa in T-Per reagent (Pierce), or from in vitro cell lysates inLaemmli buffer, and supplemented with protease and phospha-tase inhibitors (Roche). Protein was quantified by immunoblotanalysis employing antibodies to: phosphorylated histone H2AX(catalog no. 2577, 1:200 dilution), phosphorylated p53 (catalogno. 9284, 1:200 dilution), cleaved caspase-3 (catalog no. 9579,1:200 dilution), Mdm2 (catalog no. 3521, 1:200 dilution), andGAPDH (catalog no. 2118, 1:200 dilution) from Cell Signaling

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Technology, phosphorylated histone H2AX (catalog no. 05-636,1:1,000 dilution) from Millipore, and p53 (catalog no. sc-126,1:1,000 dilution) from Santa Cruz Biotechnology. The antibodyto GUCY2C was validated previously (25, 26). Antisera toGUCA2A and GUCA2B were generously provided by Dr. MichaelGoy (University of North Carolina, Chapel Hill, NC; refs. 37, 38).Secondary antibodies conjugated to horseradish peroxidase werefrom Jackson Immunoresearch Laboratories. Staining intensity ofspecific bands quantified by densitometry was normalized to thatfor GAPDH using a Kodak imaging system. Average relativeintensity reflects the mean of at least three animals in each groupand themean of at least two independent experiments. Molecularweight markers (catalog no. 10748010, 5 mL per run, or catalogno. LC5800, 10 mL per run) for immunoblot analyses were fromInvitrogen. Secondary antibodies specific to light chains, includ-ing goat anti-mouse IgG (catalog no. 115-065-174) and mouseanti-rabbit IgG (catalog no. 211-062-171), were from JacksonImmunoresearch Laboratories for immunoblot analysis follow-ing immunoprecipitation.

ImmunoprecipitationProtein from 8–10 � 106 HCT116 cells was extracted in 1%

NP40 immunoprecipitation (IP) lysis buffer supplementedwith protease and phosphatase inhibitors and incubated withantibodies to Mdm2 (catalog no. 3521, 5 mg) from Cell Sig-naling Technology and p53 (catalog no. sc-126, 1 mg) fromSanta Cruz Biotechnology and protein A beads (Invitrogen)overnight followed by six washes. Precipitated proteins werecollected in Laemmli buffer (with 5% beta mercaptoethanol)supplemented with protease and phosphatase inhibitors(Roche) and quantified by immunoblot analysis employingantibodies to Mdm2 (catalog no. 3521, 1:200 dilution)from Cell Signaling Technology and p53 (catalog no. sc-126,1:1,000 dilution) from Santa Cruz Biotechnology. Mouse IgG(5 mg, catalog no. 10400C, Invitrogen) and rabbit IgG (5 mg,catalog no. 10400C, Invitrogen) were isotype controls forimmunoprecipitation.

IHC and immunofluorescenceAntigens were unmasked in paraffin-embedded sections

(5 mm) by heating at 100�C for 10 minutes in 10 mmol/Lcitric buffer, pH 6.0. In addition to those already described,antibodies to antigens probed here included: phosphorylatedhistone H2AX from Cell Signaling Technology (catalog no.2577, 1:200 dilution), or from Millipore (catalog no. 05-636, 1:1,000 dilution), cleaved caspase-3 from Cell SignalingTechnology (catalog no. 9579, 1:200 dilution), and b-cateninfrom Santa Cruz Biotechnology (catalog no. sc-7199, 1:50dilution). The antibody to GUCY2C (25, 26) and the antiserato GUCA2A and GUCA2B were described previously (37, 38).Fluorescent secondary antibodies were from Invitrogen. Tyra-mide signal amplification was used to detect GUCY2C andGUCA2A; secondary antibodies conjugated to horseradish per-oxidase were from Jackson Immunoresearch Laboratories (cat-alog nos. 115-035-206 and 111-036-046, 1:1,000 dilution),and fluorescein-conjugated tyramine was prepared from tyra-mine HCl (catalog no. T2879, Sigma) and NHS-fluorescein(catalog no. 46410, Thermo Scientific; ref. 39). Phosphorylatedhistone H2AX-positive cells were quantified in 200–1,000crypts per section per animal and positive cells normalizedto crypt number. Results reflect the means � SEM of at least

3 animals in each group. Immunofluorescence stains wereperformed in HCT116 and HCT116 p53-null cells using anti-bodies to the following antigens included: a/b-tubulin fromCell Signaling Technology (catalog no. 2148, 1:200 dilution)and g-tubulin from Abcam (catalog no. ab11317, 1:100 dilu-tion). Fluorescence images were captured with an EVOS FL autocell imaging system from Life Technologies-Thermo FischerScientific.

Cell treatment, irradiation, and colony formation assayHCT116 and HCT116 p53-null cells were plated in 6-well

dishes at 1 � 104 cells/well followed by treatment for 7 dayswith vehicle or cell-permeable cGMP (8-Br-cGMP, 500 mmol/L).Media containing different treatments were changed every otherday. After exposure to radiation (0–4 Gy), cells were trypsinizedand plated in 6-well dishes at different densities depending on thepotency of the treatments (104 cells/well for HCT116 exposed at0, 1, and 2 Gy; 4 � 104 cells/well for HCT116 p53-null exposedat 0, 1, and 2 Gy; 50 � 104 cells/well for HCT116 exposed at3 and 4 Gy; 2 � 106 cells/well for HCT116 p53-null exposed at 3and 4Gy). Cells were treated with vehicle or 8-Br-cGMP for 7 daysafter irradiation, then fixed and stained with 10%methylene bluein 70% ethanol. The number of colonies, defined as >50 cells/colony were counted, and the surviving fraction was calculated asthe ratio of the number of colonies in the treated sample to thenumber of colonies produced by cells that were not irradiated.Triplicates were used for each condition in three independentexperiments.

Anaphase bridge index and aneuploidyCells preconditioned with 8-Br-cGMP, or control cells, were

irradiated (5 Gy), then seeded on coverslips in 24-well plates (5�104 cells per well). Anaphase bridge index (ABI) and aneuploidywere quantified 2 days after irradiation.

ABI.Cells were fixed in 4% PFA and stained with DAPI. Anaphasecells were analyzed and abnormal anaphase cells were calculatedunder a fluorescence microscope. More than 200 anaphase cellswere analyzed in each treatment group in each independentexperiment. Any abnormal anaphase cells with anaphase bridgesor anaphase lag showing extended chromosome bridgingbetween two spindle poles were enumerated and the ABI wascalculated as percentage of abnormal anaphase cells over totalanaphase cells.

Aneuploidy. Cells were fixed in 4% PFA and stained with DAPI,and immunofluorescence stains were performed usinga/b-tubulin–specific antibodies and centromere-specific g-tubu-lin antibodies, detected with Alexa Fluor 555 or Alexa Fluor 488–labeled secondary antibodies from Invitrogen. Images wereacquiredwith a laser confocalmicroscope (Zeiss 510MandNikonC1 Plus, Thomas Jefferson University Bioimaging SharedResource), and 0.5-mmoptical sections in the z-axis were collectedwith a 100 � 1.3 NA oil immersion objective at room temper-ature. Iterative restoration was performed using LSM ImageBrower (Zeiss), and images represent three to four merged planesin the z-axis. Abnormal anaphase chromatidswere counted if cellscontained more than two centrosomes or two centrosomes locat-ed in the same direction to the spindle midzone separated fromkinetochores at the poles.

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Quantitative RT-PCR analysisTranscripts for GUCY2C, GUCA2A, and GUCA2B were quan-

tified by RT-PCR employing primers and conditions describedpreviously (25, 26).

125I-labeled ST bindingBinding of 125I-labeled ST to GUCY2C was performed as

described previously (33). Briefly, membranes were preparedfrom cells as described previously (33) and ST was iodinated(125I-Tyr4-ST) to a final specific activity of 2,000 Ci/mmol (33).Total binding was measured by counts per minute (CPM) in theabsence of unlabeled ST competition, whereas nonspecific bind-ingwasmeasured in the presence of 1� 10�5mol/L unlabeled ST.Specific binding was calculated by subtracting nonspecific bind-ing from total binding (33). Assays were performed at least intriplicate.

Statistical analysesStatistical significance was determined by unpaired two-tailed

Student t test unless otherwise indicated. Results represent means� SEM from at least 3 animals or three experiments performed intriplicate. Survival and disease-free survival were analyzed byKaplan–Meier analysis. Body weight was analyzed using a frailtymodel combining a segmented linear longitudinalmodel of bodyweight, a log-normal model for survival time, and a log-normalmodel for randombreak point for bodyweight (inflection point).Analyses of fecal occult blood and untidy fur were performed byCochran–Mantel–Hansel test. Colony formation was analyzedby a pairwise comparison in four treatments with isotherm slopesby linear regression.

ResultsSilencing GUCY2C exacerbates RIGS

A role for GUCY2C in opposing epithelial cell apoptosisinduced by low doses of ionizing radiation (22) suggested thatthis receptor may play a role in RIGS. Targeted germline deletionofGucy2c (Gucy2c�/�mice; refs. 15, 16, 18–20, 26, 32) acceleratedthe death of mice following exposure to a lethal dose (high dose,15 Gy) of total body irradiation (TBI; Fig. 1A). This dose ofradiation produced death by inducing RIGS, which could not berescued by bone marrow transplantation, in contrast to low dose(8 Gy) radiation, which produced the hematopoietic, but not theGI syndrome (Fig. 1B). Similarly, silencing GUCY2C signalingexacerbated acute GI toxicity quantified by diarrhea (Fig. 1C) anddecreased survival (Fig. 1D) following 18Gy sub-total abdominalirradiation (STBI), with bone marrow preservation by shielding.

Exacerbation of RIGS in the absence of GUCY2C signaling wasassociatedwith increased intestinal dysfunction, includingweightloss (Fig. 1E), intestinal bleeding (Fig. 1F), debilitation (untidyfur, Fig. 1G; ref. 40), and stool water accumulation (Fig. 1H;ref. 41). Silencing GUCY2C signaling amplified intestinal epithe-lial disruption, quantified as crypt loss, produced by STBI in smallintestine (Fig. 1I and J). Moreover, it created novel epithelialvulnerability in the colon, which is relatively resistant to RIGS(Fig. 1K and L; refs. 1–5). Together, these observations reveal thatthe GUCY2C signaling axis plays a compensatory role in mod-ulating mechanisms contributing to RIGS.

GI-toxic irradiation preserves durable expression of GUCY2Cand its paracrine hormones

A role for the GUCY2C paracrine hormone axis in compensa-tory mechanisms opposing RIGS is predicated on the persistenceof expression of the receptor and its hormones following highdoses of radiation. Indeed, GUCY2C mRNA and protein, char-acteristically expressed along the entire crypt–villus axis (17), wasdurably preserved following lethal TBI (Fig. 2A–C), a result that issimilar to other conditions disrupting epithelial integrity, includ-ing tumorigenesis (18–20, 25). Unexpectedly, GI-toxic TBI pre-served expressionofGUCA2A (Fig. 2D–F) andGUCA2B (Fig. 2G–I), in contrast to other modes of disrupting epithelial integrity,including tumorigenesis, inflammatory bowel disease, and met-abolic stress in which ligand expression is lost (18–21, 25, 26).Indeed, expression of GUCA2A, which is low in small intestine,was retained primarily in isolated epithelial cells, as describedpreviously (42). In contrast, expression of GUCA2B, which is thepredominant GUCY2C hormone in the small intestine, wasretained principally by differentiated epithelial cells in distal villi(42). Preservation of receptor and hormone expression wasdurable, and there were no significant differences in mRNA orprotein levels across the time course of injury response (Fig. 2;Supplementary Fig. S5). Moreover, preservation of hormoneexpressionwas independent ofGUCY2C expression (Supplemen-tary Fig. S6). These observations are consistent with a role for theGUCY2C paracrine hormone signaling axis in compensatoryresponses that oppose acute radiation-induced GI toxicity. More-over, the persistence of receptor expression across the continuumof injury response suggests the potential utility of GUCY2C as atherapeutic target to prevent RIGS.

GUCY2C activation by oral ligand rescues RIGS, but not extra-GI tumor responses to radiation

In wild-type mice, oral administration of ST, an exogenousGUCY2C ligand, reduced morbidity and mortality induced by

Figure 1.GUCY2C silencing amplifies RIGS. A, Gucy2c�/� mice are more susceptible to death, compared with Gucy2cþ/þ mice, induced by high dose (15 Gy)whole-body irradiation (TBI, Kaplan–Meier analysis, ��� , P < 0.001; n ¼ 34 Gucy2cþ/þ mice, n ¼ 39 Gucy2c�/� mice). B, Mortality from low dose (8 Gy)TBI reflected hematopoietic toxicity, which was abrogated by bone marrow transplantation (BMT). In contrast, mortality from high dose TBI (15 Gy)reflected both hematopoietic and GI toxicity, which could not be rescued by bone marrow transplantation [Kaplan–Meier analysis, ��� , P < 0.001 betweenGucy2cþ/þ mice (n ¼ 11) and Gucy2cþ/þ mice with bone marrow transplantation (n ¼ 5) following low dose (8 Gy) TBI; P < 0.05 (not significant)between Gucy2cþ/þ mice (n ¼ 21) and Gucy2cþ/þ mice with bone marrow transplantation (n ¼ 15) following high dose TBI]. Following 18 Gy STBI, Gucy2c�/�

mice were more susceptible to diarrhea (C; c2 test, two sided, �, P < 0.05), mortality (D; Kaplan–Meier analysis, �� , P < 0.01), weight loss (E; frailtymodel analysis, � , P < 0.05), intestinal bleeding (F; FOB, fecal occult blood; Cochran–Mantel–Haenszel test, � , P < 0.05), debilitation (G; untidy fur;Cochran–Mantel–Haenszel test, � , P < 0.05), and stool water accumulation (H; Loess smoothing curves with 95% confidence bands and comparison ofarea under curve, � , P < 0.05; dashed line, stool water content before irradiation). I–L, Gucy2c�/� mice were more susceptible to radiation-induced GIinjury in both small (I and J) and large (K and L) intestines quantified by crypt enumeration post irradiation (15 Gy TBI), compared with Gucy2cþ/þ mice(ANOVA, � , P < 0.05; �� , P < 0.01, n � 3 Gucy2cþ/þ and Gucy2c�/� mice, respectively, at each time point). Bars in low power images represent 500 mm,and in high power images 50 mm, in I and K. n ¼ 34 Gucy2cþ/þ mice, n ¼ 35 Gucy2c�/� mice in C–E and G; n ¼ 13 Gucy2cþ/þ mice, n ¼ 13 Gucy2c�/� mice inF; n ¼ 26 Gucy2cþ/þ mice and n ¼ 28 Gucy2c�/� mice in H.

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STBI, quantifiedby the incidence of diarrhea (Fig. 3A) and survival(Fig. 3B) respectively. Similarly, oral ST opposed STBI-inducedintestinal dysfunction, including weight loss (Fig. 3C), intestinalbleeding (Fig. 3D), debilitation (Fig. 3E), and stool water accu-mulation (Fig. 3F). Furthermore, oral ST rescued intestinal mor-phology and stool formation (Fig. 3G), and water reabsorptionassociated with preservation of normal histology (Fig. 3H) afterSTBI. In contrast, oral ST did not rescue RIGS in Gucy2c�/� mice(Supplementary Fig. S7). Furthermore, oral ST did not altertherapeutic radiation responses of radiation-sensitive thymomaor radiation-resistant melanoma (Fig. 3I). Moreover, chronic oralST was safe, without adverse pharmacologic effects like diarrhea(Fig. 3J) or growth retardation (Fig. 3K). These observationssupport the suggestion that GUCY2C signaling comprises a com-pensatory mechanism opposing RIGS that can be engaged byorally administered ligands. Indeed, GUCY2C ligands safely

protect intestinal epithelial cells specifically (26), without alteringtherapeutic responses to radiation of tumors outside the intestine.

GUCY2C signaling opposing RIGS requires p53GUCY2C signaling protects intestinal epithelial cells from

apoptosis induced by low-dose radiation (22). However, whilesilencing GUCY2C signaling increased basal levels of apoptosis insmall intestine, as demonstrated previously (18), it did not alterapoptosis associated with RIGS along the rostral–caudal axis ofthe intestine across the continuumof injury responses (Fig. 4A). Inthat context, p53 also opposes RIGS throughmechanisms that areindependent of apoptosis (Supplementary Fig. S8; ref. 7). Indeed,eliminating p53 phenocopied GUCY2C silencing, exacerbatingRIGS-related mortality (Fig. 1D; Supplementary Fig. S2). Further-more, activation of GUCY2C with oral ST improved survival inwild-type, but not p53int�/�, mice following STBI (Fig. 4B).

Figure 2.

The GUCY2C hormone axis is preserved inRIGS. A–F, TBI (15 Gy) did not significantlyalter the relative expression of GUCY2C (A),guanylin (GUCA2A; D), or uroguanylin(GUCA2B; G) mRNA or GUCY2C (B),GUCA2A (E), or GUCA2B protein in jejunumover time [H;n¼4–8per timepoint;P>0.05(not significant) for mRNA or protein byANOVA]. C, F, and I, Representativeimmunofluorescence images for theexpression of GUCY2C, GUCA2A, andGUCA2B before and 48 hours after 15 Gy TBI[green, GUCY2C; red, hormone (GUA2A,GUCA2B); blue, DAPI]. Scale bar, 50 mm.

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Moreover, GUCY2C activation opposed STBI-induced intestinaldysfunction quantified by body weight loss (Fig. 4C), intestinalbleeding (Fig. 4D), and debilitation (Fig. 4E) in wild-type, butnot in p53int�/�, mice. These observations suggest that theGUCY2C signaling axis opposes RIGS through a mechanismrequiring p53.

GUCY2C signaling opposing RIGS is associated withamplification of p53 responses

Consistent with a role for p53 in mediating the effects ofGUCY2C signaling on radiation-induced intestinal toxicity, oralST increased levels of phosphorylated p53 in mouse intestinalepithelial cells in RIGS induced by STBI (Fig. 4F). Recapitulating

these in vivo results, the GUCY2C second messenger, cGMP,increased total and phosphorylated p53 induced by radiation inHCT116 human colon carcinoma cells (Fig. 4G), an in vitromodelof intestinal epithelial cells that express wild-type p53, but notGUCY2C (Supplementary Fig. S4; refs. 19, 34, 35). Amplificationof p53 responses to radiation induced by cGMP signaling inHCT116 cells was associated with reduced interactions betweenp53 and the inhibitory protein Mdm2 (Fig. 4H).

GUCY2C signaling opposing RIGS is associated with p53-dependent rescue of mitotic catastrophe

Induction of GUCY2C signaling by oral ST opposed chromo-somal instability in intestinal epithelial cells following STBI,

Figure 3.

Oral ST selectively opposes RIGS.A–F,Gucy2cþ/þ mice preconditioned withoral ST for 14 days prior to andfollowing 18 Gy STBI (on day 0)exhibited a lower incidence of diarrhea(A) induced by STBI, compared withmice treated with control peptide (CP;Fisher exact test, two sided, � ,P < 0.05). Similarly, following 18 GySTBI, ST improved diarrhea-freesurvival (Kaplan–Meier survivalanalysis, �� , P < 0.01; B); weight lossand weight recovery (frailty modelanalysis, � , P < 0.05; C); fecal occultblood (FOB; D) and untidy fur(Cochran–Mantel–Haenszel test,� , P < 0.05; E); and stool water content(Loess smoothing curves with 95%confidence bands and comparison ofarea under curve, ��� , P < 0.001;dashed line, stool water contentbefore irradiation; F). G and H, STreduced radiation-induced intestinaldamage 15 days following STBI,reflected by gross morphology (G),with lower hyperemia, edema, andunformed stool, and histology (H),without disruption of normal crypt-villus architecture quantified by cryptdepth, stromal hypertrophy reflectedby intestinal transmural thickness andlymphocytic infiltration (Fisher exacttest, two sided, � , P < 0.05 in G; t test,two sided, ��� , P <0.001 inH; n¼ 4CP-treated mice, n ¼ 5 ST-treated mice).Scale bar in H, 100 mm. I, Oral ST didnot alter radiation responses bysubcutaneous thymoma or melanoma(t test, two sided,P>0.05; dashed line,original tumor size). J and K, Chronic(>2 weeks) oral ST did not producediarrhea (J) or growth retardation (P >0.05, ANOVA;K). InA–F and I–K, n¼ 9CP-treated mice, n ¼ 9 ST-treatedmice.

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Figure 4.

GUCY2C signaling amplifies p53 response to RIGS by disrupting p53–Mdm2 interaction. A, Silencing GUCY2C did not affect apoptosis induced by 15 GyTBI in small intestine and colon (t test, two sided, � , P < 0.05; Gucy2c�/� mice, n � 3 and Gucy2cþ/þ mice, n � 3 at each time point). Oral ST improveddiarrhea-free survival [Kaplan-Meier analysis, �� , P < 0.01 between p53intþ/þ mice treated with CP (n ¼ 17) or ST (n ¼ 16); P > 0.05 between p53int�/� micetreated with CP (n ¼ 11) or ST (n ¼ 11); B]; weight [frailty model analysis, � , P < 0.05 between p53intþ/þ mice treated with CP (n ¼ 17) or ST (n ¼ 16); P > 0.05between p53int�/� mice treated with CP (n ¼ 11) or ST (n ¼ 11); C]; and fecal occult blood (FOB; D) and untidy fur [Cochran–Mantel–Haenszel test, � , P < 0.05between p53intþ/þ mice treated with CP (n ¼ 17) or ST (n ¼ 16); P > 0.05 between p53int�/� mice treated with CP (n ¼ 11) or ST (n ¼ 11); E] following18 Gy STBI in p53intþ/þ, but not p53int�/�, mice. F,Oral ST promoted p53 phosphorylation in small intestine 7 days after 18 Gy STBI (t test, two sided, � , P < 0.05, n¼ 6in CP and n ¼ 6 in ST-treated mice). Scale bar, 50 mm. G, 8-Br-cGMP increased total and phosphorylated p53 in response to 5 Gy radiation in HCT116 humancolon carcinoma cells (n � 3 in each treatment group; � , P < 0.05; �� , P < 0.01, ANOVA). H, 8-Br-cGMP increased p53 activation in HCT116 cells in responseto 5 Gy radiation by disrupting p53–Mdm2 interaction, quantified by immunoprecipitation (IP) and Western blot (WB) with antibodies to p53 or Mdm2 (n � 3 ineach group; � , P < 0.05; �� , P < 0.01, ANOVA). Mouse and rabbit IgG was used as isotype control in H.

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reducing double-strand DNA breaks (Fig. 5A) and abnormalmitoses characteristically associatedwithmitotic catastrophe (Fig.5B). Similarly, chromosomal instability produced by irradiation,quantified by centrosome counts or the anaphase bridge index(43), was reduced in HCT116 cells treated with 8-Br-cGMP(Fig. 5C and D). In contrast, elimination of p53 (HCT116p53�/�) amplified chromosomal instability produced by irradi-ation, and this damage was insensitive to 8-Br-cGMP (Fig. 5C andD). Moreover, cell death by mitotic catastrophe reflecting radia-tion-induced aberrant mitosis was reduced by cGMP in parental,but not in p53�/�, HCT116 cells (Fig. 5E).

DiscussionRIGS reflects radiation-induced genotoxic stress in intestinal

epithelial cells (7–9, 11, 12, 14). Radiation produces DNA dam-age, directly and through reactive oxygen species (23), activatingp53 (7, 9, 14, 44). In turn, p53 mediates a bifurcated injuryresponse. Cells damaged beyond repair undergo caspase-depen-dent apoptosis initiated by p53 activation of PUMA (6, 9, 12, 13).Furthermore, in cells that can be rescued, p53 induces the expres-sion of p21, a key inhibitor of cyclin-dependent kinases thatregulates cell-cycle checkpoints (7, 9, 14). Inhibition of

Figure 5.

GUCY2C signaling requires p53 to oppose mitotic catastrophe. Oral ST reduced DNA double strand breaks (g-H2AX; t tests, two sided, ��� , P < 0.001,n ¼ 4 mice treated with CP, n ¼ 5 mice treated with ST; >200 crypts were examined in each mouse; A) and abnormal mitotic orientation [percentage ofmetaphase plates oriented nonorthogonally to the crypt-surface axis (B); t tests, two sided, � , P < 0.05; n ¼ 4 mice treated with CP, n ¼ 5 mice treated withST; �50 mitotic figures were evaluated in each mouse intestine] following 18 Gy STBI (red, b-catenin; green, g-H2AX; blue, DAPI). Scale bars, 50 mm.C, Radiation (5 Gy)-induced anaphase bridging, a marker of abnormal mitosis quantified by the ABI in wild-type (parental) and p53-null (p53�/�) HCT116human colon carcinoma cells, was reduced by pretreatment with a cell-permeant analogue of cGMP in a p53-dependent fashion. Representative imagesof ABI: i, normal mitosis without anaphase bridge; ii–iii, abnormal mitoses with anaphase bridges (c2 tests, two sided, � , P < 0.05; �100 cells in anaphasewere examined in each group; ns, not significant). D, Radiation (5 Gy)-induced aneuploidy, quantified by centrosome enumeration, in parental and p53-nullHCT116 cells, was reduced in a p53-dependent fashion by pretreatment with 8-Br-cGMP. Representative images of ploidy: i, normal diploidy; ii, abnormaldiploidy; iii, triploidy; iv, tetraploidy (red, a/b-tubulin; green, g-tubulin; purple, DAPI; c2 tests, two sided, ��� , P < 0.001, �200 mitotic cells were examined ineach group). E, Cytogenetic toxicity induced by increasing doses of radiation, quantified by colony formation, in parental and p53-null HCT116 cells wasreduced in a p53-dependent fashion by pretreatment with 8-Br-cGMP [pairwise comparison of isotherm slopes, � , P < 0.05: HCT116 cells treated with cGMPcompared with the three other groups including HCT116 cells treated with PBS, HCT116 p53-null cells treated with PBS or cGMP; P > 0.05 (nonsignificant)between any two of these three latter groups].

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proliferation associated with these checkpoints permits cells torepair damaged DNA (7–9, 12, 14, 45). However, p53 responsesare limited and cells with damaged DNA escape checkpointswithin days of irradiation, enter prematurely into the cell cyclewith damaged DNA, and undergo mitotic catastrophe (7, 9, 12,14, 46). In turn, this produces epithelial loss and mucositis,disrupting barrier function associated with fluid and electrolyteloss and infection, which are principle mechanisms of death fromRIGS (47). Here, we reveal an unexpected compensatory mech-anism that opposes this pathophysiology, involving theGUCY2Csignaling axis at the intersection of radiation injury andp53 responses.

GUCY2C is selectively expressed by intestinal epithelial cellsand activation by the endogenous hormones guanylin and uro-guanylin, or the diarrheagenic bacterial STs, increases intracellularcGMP accumulation (17). While there is evidence for GUCY2Csignaling in other tissues (32, 48), the effect of oral ST in ame-liorating RIGS in the current study is consistent with a primaryeffect on intestinal receptors, reflecting the absence of bioavail-ability of oral GUCY2C ligands (31). GUCY2C-cGMP signalingmodulates intestinal secretion, onemechanism bywhich bacteriainduce diarrhea, and the oral GUCY2C ligands linaclotide (Lin-zess) and plecanatide (Trulance) improve constipation andrelieve abdominal pain in patients with irritable bowel syndrome(31, 49). Furthermore, GUCY2C signaling regulates proliferationand DNA damage repair, processes that are canonically disruptedin RIGS (26). Indeed, signaling through the GUCY2C–cGMP axisinhibits DNA synthesis and prolongs the cell cycle, imposing aG1–S delay in part by regulating p21, key injury responses toradiation (18–20, 50). Furthermore, silencing GUCY2C increasesDNA oxidation and double strand breaks, amplifying mutationsinduced by chemical or genetic DNA damage, reflecting ROS andinadequate repair (20).Moreover, silencingGUCY2Cdisrupts theintestinal barrier (26), a key pathophysiologic mechanism con-tributing to RIGS (47). Conversely, GUCY2C ligands block thatdamage, enhancing barrier integrity and accelerating recoveryfrom injury (23, 24, 26, 27, 30). This role in promoting mucosalbarrier integrity supports GUCY2C as a therapeutic targetfor RIGS.

The current observations suggest a previously unrecognizedcompensatory mechanism opposing RIGS in which the paracrinehormones guanylin and uroguanylin activate GUCY2C-cGMPsignaling to defend the integrity of the intestinal epithelial barrier.In that model, paracrine hormone stimulation of the GUCY2C–cGMP signaling axis supports p53 responses to radiation injury bydisrupting interactions with Mdm2, a key regulator of responsesto genotoxic stress, which binds to the amino terminal of 18–19.p53, inhibiting its transactivation function and targeting it forproteasomal degradation (45, 51, 52). In turn, amplified p53responses contribute to resolving DNA damage, limiting mitoticcatastrophe (7). Beyond these compensatory responses, the dura-ble preservation of GUCY2C expression following high-doseirradiation across the rostral–caudal axis of the intestine and thecontinuum of injury responses offers an opportunity to target thisreceptor for mitigation of RIGS by oral GUCY2C hormoneadministration. Indeed, it creates the unique possibility of trans-forming RIGS from a syndrome of irreversible DNA damage toone that can be reversed or prevented by oral GUCY2C ligandsupplementation.

These studies stand in contradistinction to other models ofintestinal injury in which homeostasis is disrupted through

paracrine hormone loss silencing GUCY2C. Indeed, guanylin anduroguanylin are the most commonly lost gene products in spo-radic colorectal cancer and these hormones are lost at the earlieststages of neoplasia (29, 53, 54). Hormone loss silences theGUCY2C signaling axis and interrupts canonical homeostaticmechanisms that regulate the continuously regenerating intesti-nal epithelium and whose disruption is essential for tumorigen-esis (17–20, 25, 26, 34). Similarly, while obesity and colorectalcancer are associated, underlying mechanisms have remainedunclear. Recent studies revealed that overconsumption of calories,which is an essential mechanism contributing to obesity, pro-duces ER stress, leading to guanylin loss silencing the GUCY2Ctumor suppressor (25). Indeed, replacing guanylin suppressed bycalories eliminated tumorigenesis (25). Moreover, oral dextransulfate injures intestinal mucosa, producing inflammatory boweldisease (IBD), and silencing GUCY2C amplifies injury in IBD,increasingmortality inmice (24, 26, 30). Indeed, IBD is associatedwith GUCY2C paracrine hormone loss in humans (21). In thecontext of this emerging paradigm of intestinal epithelial injury,the present results demonstrating the preservation of paracrinehormone expression in the context of high-dose irradiation wasunexpected. However, they are consistent with a role for theGUCY2C paracrine hormone axis in compensatory mechanismsopposing RIGS.

Previous studies revealed that silencing GUCY2C amplifiedapoptosis induced by low doses of radiation (5 Gy; ref. 22).These radiation doses are belowGI-toxic levels that produce RIGSor bone marrow failure. Furthermore, silencing GUCY2C(Gucy2c�/� mice) did not alter the induction of apoptosis insmall or large intestine in RIGS, in contrast to those earlier studies(see Fig. 4A). Moreover, silencing GUCY2C recapitulated theeffects of eliminating p53 signaling (p53�/� mice), which alsohad no effect on apoptosis in small or large intestine in RIGS asreported previously (see Supplementary Fig. S8; ref. 7). In thecontext of the role of GUCY2C in optimizing p53 injuryresponses, reinforced by the ability of Gucy2c�/� mice to pheno-copy p53�/�mice (see Supplementary Fig. S2; ref. 7), the primarymechanism amplifying epithelial disruption in RIGS in theabsence of GUCY2C appears to be mitotic catastrophe, ratherthan apoptosis.

Targeting p53 directly to prevent and treat RIGS has beenuniquely challenging and treatments exploiting this mecha-nism have not yet emerged (7, 9, 12, 14). The therapeuticchallenge arises from the paradoxically opposite roles of p53 inRIGS and the radiation-induced hematopoietic syndrome, thetwo principal toxicities associated with radiation. Protection ofepithelial cells by p53 has made its activation a target to treatRIGS (7–9, 14, 46). In striking contrast, radiation toxicitymediated by p53 in bone marrow has made its inhibition atarget to treat the hematopoietic syndrome (8, 46, 55). Hence,p53 has remained an elusive target, requiring tissue-specificstrategies for appropriate directional regulation. The currentstudy provides insights into novel molecular mechanismsunderlying the pathophysiology of RIGS that can be readilytranslated into p53-targeted medical countermeasures to pre-vent and treat acute radiation-induced GI toxicity. Thus,GUCY2C has a narrow tissue distribution, selectively expressedby intestinal epithelial cells from the duodenum to the rectum(15–17). Furthermore, GUCY2C is anatomically privileged,expressed in lumenal membranes of those cells, directly acces-sible to oral agents but inaccessible to the systemic

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compartment (17, 31). Moreover, linaclotide (Linzess) andplecanatide (Trulance) are oral GUCY2C ligands recentlyapproved for the treatment of chronic constipation syndromes,with negligible oral bioavailability or bioactivity outside theGI tract (31). The anatomic privilege of GUCY2C coupledwith the compartmentalized activity of linaclotide and pleca-natide, confined only to the intestinal lumen, offers a uniquelytargeted approach to specifically engage p53-dependent mecha-nisms, compared with other available approaches, to preventand treat RIGS. In turn, this offers prophylactic and therapeuticsolutions to civilians, first-responders, and military personnelat risk from radiation disasters, like Chernobyl or Fukushima.Similarly, it provides a clinically tractable approach for targetedprevention of GI toxicity from abdominopelvic radiotherapyfor cancer, reducing dose-limiting toxicities and permittinggreater radiation fractions to be administered, without alteringthe therapeutic radiosensitivity of extra-intestinal tumors(see Fig. 3I; ref. 5).

Disclosure of Potential Conflicts of InterestS.A. Waldman is the Chair of the Data Safety Monitoring Board for

the Chart-1 Trial sponsored by Cardio3 Biosciences and the Chair (uncom-pensated) of the Scientific Advisory Board of Targeted Diagnostics &Therapeutics, Inc., which provided research funding that, in part, supportedthis work and has a license to commercialize inventions related to thiswork. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: P. Li, E.J. Wuthrick, J.E. Lin, A.E. Snook, S.A. WaldmanDevelopment of methodology: P. Li, E.J. Wuthrick, J.E. Lin, G. Marszalowicz,T.M. Hyslop, S.A. WaldmanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Li, E.J. Wuthrick, J. Rappaport, C.L. Kraft, J.E. Lin,G. MarszalowiczAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. Li, E.J. Wuthrick, J. Rappaport, C.L. Kraft, J.E. Lin,A.E. Snook, T. Zhan, T.M. Hyslop, S.A. WaldmanWriting, review, and/or revision of the manuscript: P. Li, E.J. Wuthrick,C.L. Kraft, J.E. Lin, G. Marszalowicz, A.E. Snook, T.M. Hyslop, S.A. WaldmanStudy supervision: S.A. Waldman

Grant SupportSupported by grants to S.A. Waldman from NIH (R01 CA204881,

R01 CA206026) and Targeted Diagnostic and Therapeutics Inc., and to theKimmelCancerCenter of Thomas JeffersonUniversity (P30CA56036). P. Li andJ.E. Lin were supported by NIH institutional award 32 GM08562 for Postdoc-toral Training in Clinical Pharmacology. J.E. Lin is the recipient of the YoungInvestigator Award from the American Society for Clinical Pharmacology andTherapeutics (ASCPT).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 27, 2017; revised June 30, 2017; accepted July 18, 2017;published online September 15, 2017.

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