Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate

Research Article

Gary D. Stoner,1 Alan A. Dombkowski,3 Rashmeet K. Reen,1 Daniela Cukovic,3 Sridevi Salagrama,3 Li-Shu Wang,2 and John F. Lechner2

1Department of Internal Medicine and 2Comprehensive Cancer Center, The Ohio State University College of Medicine, Columbus, Ohio and 3Institute of Environmental Health Sciences, Wayne State University, Detroit, Michigan

Abstract (4, 5). The former contains numerous chemopreventive agents, Our recent study identified 2,261 dysregulated genes in the including the isothiocyanates, and the latter contains high amounts esophagi of rats that received a 1-week exposure to the of polyphenols and ellagitannins. The most extensively studied carcinogen N-nitrosomethylbenzylamine (NMBA). We further isothiocyanate is phenylethyl isothiocyanate (PEITC), which reported that 1,323 of these genes were positively modulated owes its prevention properties, at least in part, to inhibition of to near-normal levels of expression in NMBA-treated animals carcinogen-activating cytochrome P450 enzymes and induction that consumed dietary phenylethyl isothiocyanate (PEITC), a of carcinogen-detoxifying phase II enzymes (6, 7). Regarding constituent of cruciferous vegetables. Herein, we report our the polyphenols, we originally found that a diet supplemented results with companion animals that were fed a diet with ellagic acid was preventative in the rat esophagus (8). Subse- containing 5% freeze-dried black raspberries (BRB) instead quently, we identified blackberries, raspberries, black raspberries of PEITC. We found that 462 of the 2,261 NMBA-dysregulated (BRB), strawberries, and cranberries as being exceptionally rich in genes in rat esophagus were restored to near-normal levels of ellagic acid, with dry weight concentrations ranging from 630 to A expression by BRB. Further, we have identified 53 NMBA- 1,500 g/g (9). Further analyses showed that the ellagic acid was dysregulated genes that are positively modulated by both most abundant in the pulp and seeds of berries, with little in the PEITC and BRB. These 53 common genes include genes juice. The pulp and seeds also contain many other known chemo- involved in phase I and II metabolism, oxidative damage, and preventive agents, including vitamins A, C, and E and folic acid, h a oncogenes and tumor suppressor genes that regulate apopto- calcium and selenium, -carotene, -carotene, lutein, gallic acid, sis, cell cycling, and angiogenesis. Because both PEITC and ferulic acid, p-coumaric acid, quercetin, several anthocyanins, h BRB maintain near-normal levels of expression of these 53 -sitosterol, stigmasterol, and kaempferol (4, 10, 11). Importantly, genes, their dysregulation during the early phase of NMBA- we also found that the active constituents can be concentrated induced esophageal cancer may be especially important in the 9- to 10-fold relative to the fresh berry by freeze drying (11). BRBs genesis of the disease. [Cancer Res 2008;68(15):6460–7] for the present study were obtained from a single Ohio farm, freeze dried, and ground into a powder as described (11). Typically, the Introduction berries are not grown organically; however, no pesticides or fungicides are applied to the berries within 10 days before harvest. This Worldwide, esophageal squamous cell carcinoma (ESCC) is the results in nondetectable levels of any pesticides or fungicides in the sixth most prevalent cancer in humans. Due to a lack of symptoms berry powder due to biodegradation of these compounds during during the early stages of the disease, ESCC is rarely detected until the 10-day period before harvest. it has metastasized (1). Tobacco products, alcohol, and inadequate The conversion of a normal cell into a tumorigenic cell is driven diet are primary causes of ESCC; thus, in addition to promoting by numerous molecular aberrations that arise over time that the lifestyle changes of smoking cessation and alcohol moderation, endow the ultimate cell with resistance to apoptosis, insensitivity there is a need to identify foods and food constituents that to growth-inhibitory signals, limitless replicative potential, sus- inhibit or prolong the onset of clinical disease (2, 3). The F344 rat tained angiogenesis, and tissue invasion/metastasis capabilities esophagus bioassay model (3), in which tumors can be induced (12). The time line of these events in the rat esophagus model by nitrosamine carcinogens such as N-nitrosomethylbenzylamine covers several months. We recently began delineating the genes (NMBA), has long been used to identify foods and food consti- dysregulated early in this carcinogenesis process and have reported tuents with anticarcinogenic activity. These studies have identified that treatment of rats with three s.c. injections (0.5 mg/kg body two dissimilar food groups, cruciferous vegetables and berries, to weight) of NMBA over 1 week led to the dysregulation of 2,261 be potent inhibitors of rat esophageal tumors induced by NMBA genes (13). We also reported that the expression of 1,323 of these genes was positively modulated if the animals were fed PEITC 2 weeks before and during the week of NMBA treatment. Herein, we have evaluated the esophagi of companion animals in the same Note: Supplementary data for this article are available at Cancer Research Online protocol whose diet was supplemented with 5% freeze-dried (http://cancerres.aacrjournals.org/). BRBs, instead of PEITC, for 2 weeks before and during the week Requests for reprints: Gary D. Stoner, Department of Internal Medicine, The Ohio State University College of Medicine, Innovation Centre, 2001 Polaris Parkway, the animals were treated with NMBA. We found that 462 of the Columbus, OH 43240. Phone: 614-293-3268; Fax: 614-293-5952; E-mail: gary.stoner@ NMBA-dysregulated genes were restored to near-normal levels of osumc.edu. I2008 American Association for Cancer Research. expression by BRB. Interestingly, we found that 53of the 2,261 doi:10.1158/0008-5472.CAN-08-0146 genes dysregulated by NMBA were expressed at control levels of

Cancer Res 2008; 68: (15). August 1, 2008 6460 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. BRB and PEITC Modulate Gene Expression in Rat Esophagus transcription if the animals received either PEITC or BRB. These NMBA-dysregulated genes affected by BRB. In our companion common genes include those involved in phase I and II meta- report (13), we noted that the expression of 2,261 genes was either bolism, oxidative damage, and oncogenes and tumor suppressor up-regulated or down-regulated more than z1.5-fold (P V 0.001) in genes that regulate apoptosis, cell cycling, and angiogenesis. the esophagi of rats treated for 1 week with NMBA, and PEITC Overall, our study has identified a unique collection of 53genes modulated 1,323 of these genes to near-normal levels of expression. whose early dysregulation may be especially important in the In contrast, only 462 of the NMBA-affected genes were modulated carcinogenesis of rat ESCC. by BRB. Of these, 203exhibited partially restored expression levels and 259 were expressed at control levels (F33%; see Fig. 2 and Materials and Methods Supplementary Table S1). Two hundred and fourteen of the 259 genes were up-regulated by NMBA and 45 were down-regulated. The procedures for determining the effects of dietary PEITC and BRB on gene expression in NMBA-treated rat esophagus have been described in We hypothesize that an important mechanism of chemoprevention detail in our companion report (13); thus, they are discussed only briefly by BRB is the correction of NMBA-dysregulated genes to homeo- here. Four- to 5-wk-old male Fischer F344 rats were randomized into three static levels of expression; thus, we have focused on understanding experimental groups of 18 animals each. Rats in group 1 received control the possible roles of the 259 genes returned to near-normal levels of AIN-76A diet. Those in group 2 were given AIN-76A + PEITC (5 Amol) and transcription by the BRB diet. Not all of these genes have known those in group 3were given AIN-76A + 5% BRB. After 2 wk, one half of the functions, as denoted by the DAVID program; however, known animals in group 1 received three s.c. injections of 20% DMSO/water among them were those involved in signal transduction, cell proli- (vehicle control) spaced every other day, and the other half received three feration, cell cycle progression, chromosome partitioning, inflam- s.c. injections of NMBA (0.5 mg/kg body weight in 20% DMSO/water). mation, differentiation, cell junctions, cytoskeleton, apoptosis, and Similarly, one half of the animals in groups 2 and 3were divided into PEITC angiogenesis (Table 1). As in our companion report (13), we also control and PEITC+NMBA subgroups and BRB control and BRB+NMBA subgroups, respectively. PEITC, at 5 Amol in the diet, reduces NMBA- observed effects of BRB on the expression of genes involved in induced tumors in the rat esophagus by f100% and BRB, at 5% of the diet, carcinogen metabolism and DNA adduct formation. These and causes an approximate 50% reduction in esophageal tumors (5). All rats other effects of BRB on gene expression in NMBA-treated rat were sacrificed 24 h after the last NMBA injection. The esophagus from esophagus are described below, in brief. each animal was excised, opened longitudinally, and cut into two parts. One NMBA-dysregulated DNA adduct formation genes normal- part was fixed in 10% buffered formalin for routine histopathology, and the ized by BRB and PEITC. Exposure of rats to NMBA causes an other was frozen in liquid nitrogen and stored at 80jC for analysis of elevation in O6-methylguanine adducts in esophageal DNA (3, 11). transcription changes using rat microarrays (41,000 transcripts). Triplicate Formation of O6-methylguanine adducts is considered to be an microarrays were completed for each of the six groups of nine rats for a important early event in NMBA-induced esophageal carcinogenesis total of 18 microarrays. because these adducts produce GC!AT transition mutations in Normalized data for each microarray were imported into Rosetta Resolver (14) for analysis (version 5.1.0.1.23; Rossetta Biosoftware). ANOVA the second base of codon 12 of the Hras1 oncogene, a gene that was performed on log ratios using the Rosetta Resolver error model and is activated early in esophageal tumorigenesis (18). Both BRB 6 weighting, as described by Weng and colleagues (15). One-way ANOVA was and PEITC have been shown to reduce O -methylguanine adduct used to identify genes with a significant treatment effect. A P value cutoff of levels in the esophagi of NMBA-treated rats (11, 19). In our com- 0.001 was used to determine statistical significance in each treatment panion report (13), we noted that the phase I enzymes, CYP2a2 and comparison. Statistically significant genes were further filtered to identify CYP3a13, were overexpressed in NMBA-treated rat esophagus, those with a minimum 1.5-fold change (16, 17). Gene ontology and pathway whereas their expression was normal in the esophagus of analysis of differentially expressed genes was done using the DAVID 4 NMBA+PEITC rats. In the present study, CYP2a2 expression was program. Microarray results were confirmed by comparison with mRNA also found to be near normal in BRB+NMBA–treated rat eso- levels obtained by quantitative reverse transcription-PCR (RT-PCR) using phagus; however, CYP3a13 remained up-regulated. Thus, our data selected gene-specific primer pairs for a total of eight genes as described in our companion report (13). For each of the eight genes evaluated, the suggest that CYP2a2 may be important in the bioactivation of RT-PCR results confirmed gene expression changes that were observed by NMBA in rat esophagus and that down-regulation of CYP2a2 by microarray. both BRB and PEITC may be responsible for the observed ability of these chemopreventives to inhibit O6-methylguanine adduct formation in NMBA-treated esophagus. Results and Discussion Phase II enzymes lower the level of DNA adducts because they Effect of BRB on esophageal histopathology. We previously inactivate carcinogens (and/or their metabolites) by conjugating reported that the cellular morphology of esophagi from rats treated them to acceptor molecules (e.g., glutathione, glucuronic acid, and with 5 Amol dietary PEITC alone or PEITC+NMBA was not signi- sulfates; ref. 6). The specifics of phase II enzymes involved in rat ficantly different from that of animals on control diet. In contrast, esophageal metabolism of NMBA have not been fully elucidated, the esophagi of rats treated with NMBA only exhibited significant although the formation of glucuronide conjugates has been hyperplasia and low-grade dysplasia with evident cytotoxicity and reported (20). As we previously reported (13), PEITC corrected marked infiltration of inflammatory cells. Essentially identical the NMBA-reduced expression levels of some glutathione S- results were observed in the esophagi of rats treated with BRB transferases (i.e., Gsta1, Gsta2, and Gstt2). In contrast, expression and BRB+NMBA [i.e., the esophagi from both the BRB alone and of these genes remained low in the esophagi of BRB+NMBA rats, BRB+NMBA animals were indistinguishable (P < 0.05) from the suggesting that overexpression of these phase II enzymes may not controls; Fig. 1]. be sentinel for the inactivation of NMBA in rat esophagus. In keeping with this interpretation is the finding that BRB also antagonized NMBA-induced up-regulation of Taldo1, a key trans- aldolase in the pentose phosphate pathway that provides the 4 http://david.niaid.nih.gov/ NADPH needed for reducing glutathione (21). www.aacrjournals.org 6461 Cancer Res 2008; 68: (15). August 1, 2008

Figure 1. Effect of BRB on NMBA-induced preneoplastic lesions in rat esophagus. Microscopic esophageal tissue sections (magnification, 100) stained with H&E. A, vehicle control esophagus. B, NMBA-treated esophagus (note extensive cytotoxicity and inflammatory response). C, BRB-treated esophagus. D, BRB+NMBA–treated esophagus.

NMBA-dysregulated inflammation genes normalized by phosphorylate MAPK14/p38-MAPK, and elevated expression of the BRB. Inducible nitric oxide synthase (iNOS) is overexpressed in Hras1 oncogene results in the accumulation of the active form of many human cancers, including ESCC (22). We have reported MAPK14/p38-MAPK in breast cancer cells (25). Apc1, which is that this radical generating enzyme is elevated in the esophagi of transcribed at increased levels in breast and colon cancers, has rats 15 and 25 weeks after NMBA treatment, whereas companion transforming capability, possibly through tyrosine dephosphoryla- rats that also received the BRB diet had normal levels of this tion of the EphA2 receptor (26). Apc1 also interacts with several enzyme (23). In contrast, iNOS was not elevated in the esophagi of other receptor tyrosine kinases and docking proteins, including rats in the present study. Cyclooxygenase-2 (COX-2) expression and platelet-derived growth factor receptor and h-catenin. It is enzyme activity are also up-regulated in human ESCC (24), with considered to be a negative regulator of growth factor–induced a corresponding increased level of prostaglandin E2 and associated cell proliferation, but in Hras1-transformed cells, its overexpression inflammatory processes. We have found that COX-2 was overex- increases cell proliferation (27). Thus, reducing NMBA up-regulated pressed in dysplastic lesions 15 and 25 weeks after NMBA treat- transcription of Map2k3 and Apc1 by BRB would antagonize ment (23), but the expression level of COX-2 in the esophagi the pro-oncogenic activities of activated Hras1. Other ras-related recovered from corresponding NMBA-treated animals in the NMBA-dysregulated genes are Rab1 and H2A histone family present study was not elevated. Thus, iNOS and COX-2 dysregu- member Z (H2afz). Rab1 is a member of the Ras oncogene super- lation does not seem to be involved with the early phase of NMBA- family and is up-regulated in tongue SCC (28). H2afz is interesting caused esophageal carcinogenesis and the chemopreventive effects because it is an adaptor protein that interacts preferentially with of BRB regarding the regulation of these genes are seemingly the active form of Hras1 and augments cell growth. Thus, several restricted to later stages of tumor development. genes associated with the ras signal transduction pathway were NMBA-dysregulated signal transduction genes normalized returned to homeostasis by the BRB diet. by BRB. Among the DAVID clustering of the genes dysregulated NMBA-dysregulated differentiation and morphogenesis by NMBA, listed in Table 1, are 37 genes involved in signal genes normalized by BRB. Eighteen transcripts were classified transduction. Of interest is the observation that several of these as cellular differentiation and morphogenesis genes (Table 1). Two genes are associated with ras activity. Among these are Map2k3 notable ones were Grem1 and the 14-3-3 family chaperone protein and Apc1, whose dysregulation affects Hras1 activity. As noted Ywhah. Grem1, which interacts with the Ywhah protein, encodes a above, f100% of rat esophageal tumors induced by NMBA have a secreted antagonist of the bone morphogenetic protein pathway, GC!AT transition mutation in the second base of codon 12 of the which in turn plays a crucial role in regulating the balance between Hras1 gene (18). Map2k3is a mitogen-activated protein kinase expansion and cell differentiation (29). In addition to its interaction (MAPK) kinase family member that is activated by stress to with Grem1, Ywhah mediates signal transduction via activation of

Cancer Res 2008; 68: (15). August 1, 2008 6462 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. BRB and PEITC Modulate Gene Expression in Rat Esophagus protein kinase C and calcium/calmodulin-dependent protein be to interfere with the formation of the Cdc42/Rac1 complex (34), kinase II. Loss of the chaperone activity of Ywhah may also play which promotes cell migration. Overall, the data support the a role in oxidative signaling underlying oxidative damage (29). conclusion that an important mechanism of BRB chemoprevention Another interesting transcript is sciellin, which encodes a precursor is correcting expression of NMBA-dysregulated cell junction, to the cornified envelope of terminally differentiated cells (30). Its adhesion, and motility genes. down-regulation by NMBA could disrupt the normal differen- NMBA-dysregulated apoptosis/cell death genes normalized tiation program of esophageal squamous cells and promote cell by BRB. Previous studies have shown that the BRB diet increased transformation. Thus, the observations that BRB restore the level of apoptosis/cell death in tumors and tumor cell lines (4). near-normal expression of Grem1, Ywhah, and sciellin could be The DAVID program analysis identified 14 genes (Table 1) that mechanisms by which the berries impede esophageal cell trans- were expressed at normal levels in the NMBA+BRB rats compared formation. with the NMBA animals. One is Hmox1; its increased expression is NMBA-dysregulated cell junction, adhesion, or motility associated with resistance to induction of apoptosis by oxidative genes normalized by BRB. Twenty transcripts were classified by stress caused by a wide range of chemical injuries (35). Another is the DAVID program as cell junction, adhesion, or motility genes Mcl1, a member of the Bcl-2 family, which was regulated to near- (Table 1). Among these is CD44 antigen, which encodes a cell normal levels by the BRB diet. Mcl1 protects mitochondrial inte- surface glycoprotein involved in cell-cell interactions, cell adhesion, grity though suppression of cytochrome c release (36). Therefore, and migration. The v6 alternate transcript of CD44 is up-regulated correction of Mcl1 transcription by BRB should augment apoptotic in human ESCC (31). Another is Actn4, which encodes a nonmuscle activity in NMBA-initiated cells. Expression of the gene Pip5k1a actinin and is localized to moving structures and is significantly (predicted) was also corrected by the BRB diet. Overexpression of elevated in cells exhibiting enhanced motility. The level of this this gene can rescue cells from stress-induced apoptosis mediated protein progressively increases from early- to late-stage ESSC (32). by the activation of extracellular signal-regulated kinase (ERK) 1/2 Another NMBA-caused overexpression normalized by BRB was signaling (37). Thus, appropriate regulation of apoptosis seems to moesin. Elevated moesin has been associated with oral squamous be a mechanism of BRB chemoprevention. cell carcinomas (33). The protein is a membrane-cytoskeleton NMBA-dysregulated angiogenesis genes normalized by BRB. linker in microvilli, ruffles, and cleavage furrows and thus plays a The BRB diet also normalized the expression of several genes the key role in cell morphology, adhesion, and motility. Lastly, the DAVID program clustered with angiogenesis activities that were up-regulation of Cdc42 by NMBA was also normalized by BRB. dysregulated by NMBA (Table 1). Among these was Klf5, which Thus, another possible chemopreventive mechanism of BRB may was down-regulated by NMBA. It is a Kruppel-like zinc finger

Figure 2. BRB modulates NMBA-induced changes in gene expression. Data points reflect mean value of the three replicate arrays. Y axis, normalized gene expression levels in which the expression value for each treatment of a gene was divided by the average of all treatments for that gene. www.aacrjournals.org 6463 Cancer Res 2008; 68: (15). August 1, 2008

Table 1. Gene ontology clusters of genes that were dysregulated by NMBA and adjusted to near-normal levels of transcription (F33%) by the 5% BRB diet in rat esophagus

Gene ontology/biological process No. genes Official symbol

Signal transduction 37 Up-regulated: Ambp, Ap3s1_predicted, Cacna1b, Cdc42, Cib2, Crk, Dusp5, Fst, Gabrd, Gng12, Grem1, Hmox1, Inhbb, Kifap3_predicted, Lamp2, Lepr, Lrba_predicted, Map2k3, Olfm3, Pnoc, Ppp2r2a, Ppp3ca, Prkar1a, Prkg2, Rab1, Rbbp6, Rgs17_predicted, RragB, Sdc1, Sh2d2a, Spsb3_predicted, Tg, Tgfa, Tmed2, Ttc1, Txnl1, Ube2c_predicted* Cell proliferation/cycle progression/ 19 Up-regulated: Bub1_predicted, Ccnc, Cdc42, Cops5, Eif4g2_predicted, chromosome partitioning H2afz, Kifap3_predicted, Klf10, LOC361237, LOC362587, Ndel1, Olfm3, Plk3, Ppp3ca, Prkar1a, Sh2d2a, Tgfa, Ube2c_predicted, Ywhah Response to stress/inflammation 19 Up-regulated: Adh6a_predicted, Akr1b8, Als2, Anxa2, Cyp2a2, Hmox1, Hspca, Map2k3, Mug1, Pcsk9, Ppp3ca, Rad23b, Top1, Txnl1 Down-regulated: Aldh3a2, Aox1, Hsd3b6, Pecr, Phgdh* Cellular differentiation/morphogenesis 18 Up-regulated: Anxa2, Cd44, Cdc42, Cldn1, Fst, Grem1, Inhbb, Klf10, Lepr, Mcl1, Ndel1, Olfm3, Pcsk9, Prkar1a, Slc30a1, Tgfa, Ywhah Down-regulated: Scel_predicted Cell junction/adhesion/motility 20 Up-regulated: Acp1, Actn4, Actr2, Ambp, Anxa2, Arpc2_predicted, Cd44, Cdc42, Cldn1, Crk, Cttn, Dsc3_predicted, Kifap3_predicted, Lamb3, Msn, Ndel1, Ppp2r2a, PVR, Sdc1 Down-regulated: Ahnak Cytoskeleton/actin 17 Up-regulated: Actn4, Actr2, Anxa2, Arpc2_predicted, Bub1_predicted, Cdc42, Crk, Cttn, Gng12, Hip1, Kifap3_predicted, Krt20, Msn, MYO10, Ndel1, Pls3, Tubb6 Apoptosis/cell death 14 Up-regulated: Eif4g2_predicted, Grem1, Hmox1, Hip1, Klf10, Mcl1, MGC72992, Miz1, Myd116, Pip5k1a_predicted, Prkg2, Rraga, Tgfa, Txnl1 Angiogenesis 6 Up-regulated: Anxa2, Cldn1, Fgfbp1, Tgfa Down-regulated: Ahnak, Klf5

*Up-regulated and down-regulated refer to the effects of NMBA treatment alone on gene expression in the rat esophagus.

transcription factor that modulates cell proliferation, differentia- carcinogens, and its dysregulation is corrected by a diet containing tion, cell cycle, apoptosis, and angiogenesis. Klf5 seems to be a indole-3-carbinol (41). Thus, Pls3 overexpression is antagonized by tumor suppressor for breast cancer (38). Thus, one mechanism of three different forms of chemopreventive diets (i.e., BRB, PEITC, BRB may be to restore expression of this tumor suppressor factor. and indole-3-carbinol) in both esophagus and lung rodent cancer Fgfbp1 was up-regulated by NMBA; it is a secreted protein thought models. to enhance fibroblast growth factor activity and drive tumor Other up-regulated genes that have been associated with cancer angiogenesis. Fgfbp1 is up-regulated early during wound healing of are Bub1, Crk, Map2k3, Psck9, PVR, Rab1, PDCD10, H2afz, and mouse and human skin as well as during the initiation of skin Cyp2a2. Map2k3, PVR, Rab1, H2afz, and Cyp2a2 are discussed neoplasia by chemical carcinogens (39). Thus, its normalization by above. Mutations in spindle checkpoint function kinase gene BRB should depress angiogenesis. Interestingly, previous studies Bub1 have been associated with aneuploidy (42). The oncogene Crk have shown that a 5% BRB diet down-regulates the expression is increased in several human cancers and its overexpression in levels of vascular endothelial growth factor-1 (VEGF-1) in the eso- cultured epithelial cells causes them to exhibit an altered morpho- phagus of rats when given for several weeks after treatment of rats logy, to proliferate in soft agar, and to grow as massive tumors in with NMBA (40). VEGF-1 was not overly expressed in the present nude mice (43). Psck9 is a Ca2+-dependent apoptosis-regulated study, indicating that this gene, along with iNOS and COX-2, does convertase that stimulates tumor cell proliferation, motility, and not seem to be involved in the early stages of tumor initiation in invasiveness (44). PDCD10 encodes a protein with similarity to the rat esophagus. proteins that participate in apoptosis. It interacts with MST4 Genes dysregulated by NMBA and normalized by PEITC or kinase to promote cell proliferation via modulation of the ERK BRB. Another purpose of our study was to identify gene expression pathway when overexpressed (45). Thus, returning all of these changes in NMBA-treated rat esophagus that were returned to genes to near-normal levels of expression might be mechanisms by homeostasis by both PEITC and BRB. We identified 53genes which PEITC and BRB impede esophageal cell transformation. modulated by both treatments (Table 2). These genes are of special Also among the 53genes are some that were not associated interest because they may be sentinel genes of NMBA-induced with a DAVID-identified cancer ontology. On closer review of the carcinogenesis and, as such, primary targets for chemoprevention. literature, however, they are of interest because they have activities One of the most interesting among these genes is Pls3. Its that, if dysregulated, could have carcinogenic effects. Several of overexpression increases cell proliferation and invasion of tumor these genes were up-regulated by NMBA. Among these is Adamts6_ cells, probably due to suppression of E-cadherin. Expression of this predicted, a member of a family of 20 genes involved in tissue gene is increased in the lung tissue of mice treated with organization during embryogenesis and angiogenesis. Some of the

Cancer Res 2008; 68: (15). August 1, 2008 6464 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. BRB and PEITC Modulate Gene Expression in Rat Esophagus proteins encoded by members of this family have matrix-degrading expression of genes with oncogenic or tumor suppressor activities. activity and may be involved in cell invasion (46). The gene Ddx5 is The transcript NS5A (hepatitis C virus) transactivated protein 9 a transcriptional coactivator and/or corepressor, depending on the (Ns5atp9) is the rat homologue of human KIAA0101 whose function context of the promoter and the transcriptional complex in which is unknown. However, it binds with proliferating cell nuclear it associates (47). Thus, its dysregulation may cause inappropriate antigen and is overexpressed in hepatocellular carcinomas (48).

Table 2. Genes that were significantly (>1.5-fold) dysregulated by NMBA and modulated back to control values by both BRB and PEITC

Accession Gene ID Primary sequence name NMBA vs control

BE116217 24161 Acp1 1.50 TC462241 361886 Adamts6_ predicted 1.71 XM_217560 302290 Ap3s1_ predicted 1.81 NM_053578 94170 Atp6v0e1 1.54 XM_342932 362614 Atpbd1b_ predicted 1.91 XM_215892 296290 Bpil1_ predicted 1.70 XM_215849 296137 Bub1_ predicted 1.58 XM_344255 364183 Cno 1.56 TC470302 54245 Crk 1.62 AW917841 60465 Cttn 1.58 NC_005100 24895 Cyp2a2 2.20 NM_001007613287765 Ddx5 1.56 CB546560 303608 Ftsj3 2.22 NM_001007731 361171 Golga7 1.55 NM_022674 58940 H2afz 1.50 BF290649 25460 Hmmr 1.74 NM_173128 286912 Krt20 1.81 XM_233763 298721 LOC298721 2.83 XM_214338 364558 LOC364558 1.97 ENSRNOT00000033720 678772 LOC678772 1.79 AI511259 679714 LOC679714 1.69 XM_213244 681124 LOC681124 2.60 ENSRNOT00000005240 683813 LOC683813 1.92 XM_217497 685792 LOC685792 1.66 BQ781225 303200 Map2k3 1.68 CB545171 494345 MGC72992 1.55 NM_001007678 304807 Mss4 1.98 NM_133546 171071 Myd116 1.69 AI230347 300795 Ns5atp9 1.60 NM_201418 300795 Ns5atp9 1.52 CB545486 298296 Pcsk9 2.33 XM_343776 81748 Pls3 2.10 AW141337 29673 Psma6 1.61 NM_017076 25066 PVR 1.73 NM_031090 81754 Rab1 1.77 BE117471 308968 Rbbp6 1.50 BE109962 294326 RGD1303003 1.59 TC475392 363213 RGD1304653_predicted 1.80 XM_343441 315863 RGD1306773 1.53 XM_213506 501731 RGD1561020_predicted 1.76 XM_228541 317241 Rnf12 2.13 NM_053972 117043 RragB 1.62 AI556201 64551 Sept7 1.68 NM_207605 310688 Sh2d2a 3.41 XM_220230 302981 Spsb3_ predicted 1.55 XM_233556 313620 Srrm1_ predicted 1.52 NM_012671 24827 Tgfa 1.63 NM_031722 65165 Tmed2 1.58 NM_001005529 287208 Ttc1 1.79 NM_080887 140922 Txnl1 1.74 XM_222627 304766 Ubxd2 1.68 NM_001008301 291796 Usp14 1.57 NM_019379 56042 Vdp 1.55

www.aacrjournals.org 6465 Cancer Res 2008; 68: (15). August 1, 2008

Transfection of this gene enhanced cancer cell growth and In summary, in this and our companion report (13), we have transformed NIH3T3 cells, whereas its inhibition caused attenua- found that a short exposure of the rat esophagus to NMBA causes tion of proliferation. dysregulation of 2,261 transcripts that affect a multitude of cellular Both BRB and PEITC diets normalized the expression of seven functions. Diets containing 5 Amol PEITC or 5% BRB restored genes that were down-regulated by NMBA. Of special note is 1,323 or 462 of these 2,261 transcripts to near-normal levels of Rbbp6, which encodes a 250-kDa ring finger-containing protein expression, respectively. Thus, as might be expected, there is a that is frequently up-regulated in human ESCC. This protein binds direct correlation between the number of transcripts modulated to to underphosphorylated but not phosphorylated retinoblastoma near-normal levels of expression and the inhibitory potential of protein (Rb). The phosphorylated Rb gene product binds the PEITC and BRB in this model because PEITC is a more potent nuclear transcription factor E2F and prevents its ability to function inhibitor of NMBA-induced rat esophageal tumorigenesis than in the S phase of the cell cycle (49). Thus, Rbbp6 competes with E2F BRB (3). In the present study, we discuss 53 transcripts that are for binding to the underphosphorylated form of Rb and high dysregulated by NMBA and restored to homeostatic levels of concentrations of the protein, as are found in human ESCC cells, expression by both BRB and PEITC. We speculate that these 53 would free E2F to stimulate cell proliferation. Rbbp6 also binds to genes are sentinel early changes in the process of NMBA-induced p53, thereby enhancing Mdm2-mediated ubiquitination and tumorigenesis in the rat esophagus, and future investigations will degradation of p53, leading to decreased apoptosis. Consequently, focus on elucidating their mechanistic roles. down-regulation of Rbbp6 by BRB or PEITC should slow cell growth and increase the rate of apoptosis. Another NMBA-dysregulated gene restored to normal levels of Disclosure of Potential Conflicts of Interest expression by BRB and PEITC was homologue of zebra fish ES1 No potential conflicts of interest were disclosed. (RGD1303003), which is the rat homologue of human C21orf33. This gene is required for the growth-inhibitory effect of all-trans retinoic acid on MCF-7 breast cancer cells (50). Its down-regulation Acknowledgments by NMBA suggests a mechanism whereby the carcinogen promotes Received 1/14/2008; revised 5/5/2008; accepted 5/7/2008. cancer by repressing terminal differentiation pathways. Mss4 Grant support: NIH grants RO1 CA103180 and R01CA96130 (G.D. Stoner). The protein (Mss4) binds to the membrane proximal conserved region microarray and bioinformatics work was facilitated by the Microarray and of a-integrin chains and regulates the activation of inactive pro– Bioinformatics Facility Core of the Environmental Health Sciences Center at Wayne State University (National Institute of Environmental Health Sciences Center grant P30 matrix metalloproteins. The active forms of metalloproteins are ES06639). important in tumor invasion and metastasis (51). Thus, it will be The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance interesting to determine if and how down-regulation of Mss4 by with 18 U.S.C. Section 1734 solely to indicate this fact. NMBA contributes to esophageal carcinogenesis. We thank Ronald Nines for his excellent technical assistance.

References administration of lyophilized black raspberries. Cancer 22. Matsumoto M, Furihata M, Kurabayashi A, Araki K, Res 2001;61:6112–9. Sasaguri S, Ohtsuki Y. Association between inducible 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, 12. Hanahan D, Weinberg RA. The hallmarks of cancer. nitric oxide synthase expression and p53status in Thun MJ. Cancer statistics, 2007. CA Cancer J Clin Cell 2000;100:57–70. human esophageal squamous cell carcinoma. Oncology 2007;57:43–66. 13. Reen RK, Dombkowski AA, Kresty LA, et al. Effects of 2003;64:90–6. 2. Engel LS, Chow WH, Vaughan TL, et al. Population phenylethyl isothiocyanate on early molecular events in 23. Chen T, Hwang H, Rose ME, Nines RG, Stoner GD. attributable risks of esophageal and gastric cancers. N-nitrosomethylbenzylamine-induced cytotoxicity in rat Chemopreventive properties of black raspberries in J Natl Cancer Inst 2003;95:1404–13. esophagus. Cancer Res 2007;67:6484–92. N-nitrosomethylbenzylamine-induced rat esophageal 3. Stoner GD, Gupta A. Etiology and chemoprevention of 14. Quackenbush J. Computational analysis of micro- tumorigenesis: down-regulation of cyclooxygenase-2, esophageal squamous cell carcinoma. Carcinogenesis array data. Nat Rev Genet 2001;2:418–27. inducible nitric oxide synthase, and c-Jun. Cancer Res 2001;22:1737–46. 15. Weng L, Dai H, Zhan Y, He Y, Stepaniants SB, Bassett 2006;66:2853–9. 4. Stoner GD, Chen T, Kresty LA, Aziz RM, Reinemann T, DE. Rosetta error model for gene expression analysis. 24. Liu JF, Jamieson G, Wu TC, Zhang SW, Wang QZ, Nines R. Protection against esophageal cancer in Bioinformatics 2006;22:1111–21. Drew P. Cyclooxygenase-2 expression in squamous cell rodents with lyophilized berries: potential mechanisms. 16. Harris MA, Clark J, Ireland A, et al. The Gene carcinoma of the esophagus. Dis Esophagus 2006;19: Nutr Cancer 2006;54:33–46. Ontology (GO) database and informatics resource. 350–4. 5. Stoner GD, Wang LS, Chen T. Chemoprevention of Nucleic Acids Res 2004;32:D258–61. 25. Shin I, Kim S, Song H, Kim HR, Moon A. H-Ras- esophageal squamous cell carcinoma. Toxicol Appl 17. Dennis G, Jr., Sherman BT, Hosack DA, et al. DAVID: specific activation of Rac-MKK3/6-p38 pathway: its Pharmacol 2007;224:337–49. Database for Annotation, Visualization, and Integrated critical role in invasion and migration of breast 6. Hecht SS. Inhibition of carcinogenesis by isothiocya- Discovery. Genome Biol 2003;4:3. epithelial cells. J Biol Chem 2005;280:14675–83. nates. Drug Metab Rev 2000;32:395–411. 18. Liston BW, Gupta A, Nines R, et al. Incidence and 26. Kikawa KD, Vidale DR, Van Etten RL, Kinch MS. 7. von Weymarn LB, Chun JA, Hollenberg PF. Effects of effects of Ha-ras codon 12 G!A transition mutations in Regulation of the EphA2 kinase by the low molecular benzyl and phenethyl isothiocyanate on P450s 2A6 and preneoplastic lesions induced by N-nitrosomethyl- weight tyrosine phosphatase induces transformation. 2A13: potential for chemoprevention in smokers. benzylamine in the rat esophagus. Mol Carcinog 2001; J Biol Chem 2002;277:39274–9. Carcinogenesis 2006;27:782–90. 32:1–8. 27. Ramponi G, Stefani M. Structure and function of the 8. Mandal S, Stoner GD. Inhibition of N-nitrosobenzyl- 19. Stoner GD, Morrissey DT, Heur Y-H, et al. Inhibitory low M r phosphotyrosine protein phosphatases. Biochim methylamine-induced esophageal tumorigenesis in rats effects of phenethyl isothiocyanate on N-nitrosobenzyl- Biophys Acta 1997;1341:137–56. by ellagic acid. Carcinogenesis 1990;11:55–61. methylamine carcinogenesis in the rat esophagus. 28. Shimada K, Uzawa K, Kato M, et al. Aberrant 9. Daniel EM, Krupnick AS, Heur YH, Blinzler JA, Nims Cancer Res 1991;51:2063–8. expression of RAB1A in human tongue cancer. Br J RW, Stoner GD. Extraction, stability and quantitation of 20. Reen RK, Nines R, Stoner GD. Modulation of Cancer 2005;92:1915–21. ellagic acid in various fruits and nuts. J Food Comp Anal N-nitrosomethylbenzylamine metabolism by black rasp- 29. Namkoong H, Shin SM, Kim HK, et al. The bone 1989;2:338–49. berries in the esophagus and liver of Fischer 344 rats. morphogenetic protein antagonist gremlin 1 is overex- 10. Stoner GD, Wang LS, Zikri N, et al. Cancer prevention Nutr Cancer 2006;54:47–57. pressed in human cancers and interacts with YWHAH with freeze-dried berries and berry components. Semin 21. Puskas F, Gergely P, Jr., Banki K, Perl A. Stimulation of protein. BMC Cancer 2006;6:74. Cancer Biol 2007;17:403–10. the pentose phosphate pathway and glutathione levels 30. Alibardi L, Toni M. Characterization of keratins and 11. Kresty LA, Morse MA, Morgan C, et al. Chemo- by dehydroascorbate, the oxidized form of vitamin C. associated proteins involved in the corneification of prevention of esophageal tumorigenesis by dietary FASEB J 2000;14:1352–61. crocodilian epidermis. Tissue Cell 2007;39:311–23.

Cancer Res 2008; 68: (15). August 1, 2008 6466 www.aacrjournals.org

31. Nozoe T, Kohnoe S, Ezaki T, Kabashima A, Maehara tumor suppressor role of the KLF5 transcription factor expression of antisense L-plastin gene. Am J Pathol 1999; Y. Significance of immunohistochemical over-expression in human breast cancer. Oncogene 2002;21:6567–72. 155:115–22. of CD44v6 as an indicator of malignant potential in 39. Kurtz A, Aigner A, Cabal-Manzano RH, et al. 45. Ma X, Zhao H, Shan J, et al. PDCD10 interacts with esophageal squamous cell carcinoma. J Cancer Res Clin Differential regulation of a fibroblast growth factor- Ste20-related kinase MST4 to promote cell growth and Oncol 2004;130:334–8. binding protein during skin carcinogenesis and wound transformation via modulation of the ERK pathway. Mol 32. Fu L, Qin YR, Xie D, et al. Identification of a-actinin 4 healing. Neoplasia 2004;6:595–602. Biol Cell 2007;18:1965–78. and 67 kDa laminin receptor as stage-specific markers 40. Chen T, Rose ME, Hwang H, Nines RG, Stoner GD. 46. Wierinckx A, Auger C, Devauchelle P, et al. A in esophageal cancer via proteomic approaches. Cancer Black raspberries inhibit N-nitrosomethylbenzylamine diagnostic marker set for invasion, proliferation, and 2007;110:2672–81. (NMBA)-induced angiogenesis in rat esophagus parallel aggressiveness of prolactin pituitary tumors. Endocr 33. Kobayashi H, Sagara J, Kurita H, et al. Clinical to the suppression of COX-2 and iNOS. Carcinogenesis Relat Cancer 2007;14:887–900. significance of cellular distribution of moesin in patients 2006;27:2301–7. 47. Fuller-Pace FV. DExD/H box RNA helicases: multi- with oral squamous cell carcinoma. Clin Cancer Res 41. Kassie F, Anderson LB, Scherber R, et al. functional proteins with important roles in transcrip- 2004;10:572–80. Indole-3-carbinol inhibits 4-(methylnitrosamino)-1-(3- tional regulation. Nucleic Acids Res 2006;34:4206–15. 34. Huang Q, Shen HM, Ong CN. Emodin inhibits tumor pyridyl)-1-butanone plus benzo(a)pyrene-induced lung 48. Yuan RH, Jeng YM, Pan HW, et al. Overexpression of cell migration through suppression of the phosphatidy- tumorigenesis in A/J mice and modulates carcinogen- KIAA0101 predicts high stage, early tumor recurrence, linositol 3-kinase-Cdc42/Rac1 pathway. Cell Mol Life Sci induced alterations in protein levels. Cancer Res 2007; and poor prognosis of hepatocellular carcinoma. Clin 2005;62:1167–75. 67:6502–11. Cancer Res 2007;13:5368–76. 35. Busserolles J, Megias J, Terencio MC, Alcaraz MJ. 42. Grigorova M, Staines JM, Ozdag H, Caldas C, 49. Hartman J, Muller P, Foster JS, Wimalasena J, Heme oxygenase-1 inhibits apoptosis in Caco-2 cells via Edwards PA. Possible causes of chromosome instability: Gustafsson JA, Strom A. HES-1 inhibits 17h-estradiol activation of Akt pathway. Int J Biochem Cell Biol 2006; comparison of chromosomal abnormalities in cancer and heregulin-h1-mediated upregulation of E2F-1. 38:1510–7. cell lines with mutations in BRCA1, BRCA2, CHK2 and Oncogene 2004;23:8826–33. 36. Dai Y, Grant S. Targeting multiple arms of the BUB1. Cytogenet Genome Res 2004;104:333–40. 50. Pujana MA, Han JD, Starlta LM, et al. Network apoptotic regulatory machinery. Cancer Res 2007;67: 43. Rodrigues SP, Fathers KE, Chan G, et al. CrkI and modeling links breast cancer susceptibility and centro- 2908–11. CrkII function as key signaling integrators for migra- some dysfunction. Nat Genet 2007;39:1338–49. 37. Halstead JR, van Rheenen J, Snel MH, et al. A role for tion and invasion of cancer cells. Mol Cancer Res 2005; 51. Knoblauch A, Will C, Goncharenko G, Ludwig S, PtdIns(4,5)P2 and PIP5Ka in regulating stress-induced 3:183–94. Wixler V. The binding of Mss4 to a-integrin subunits apoptosis. Curr Biol 2006;16:1850–6. 44. Zheng J, Rudra-Ganguly N, Powell WC, Roy-Burman regulates matrix metalloproteinase activation and 38. Chen C, Bhalala HV, Qiao H, Dong JT. A possible P. Suppression of prostate carcinoma cell invasion by fibronectin remodeling. FASEB J 2007;21:497–510.

www.aacrjournals.org 6467 Cancer Res 2008; 68: (15). August 1, 2008

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2008 American Association for Cancer Research. Carcinogen-Altered Genes in Rat Esophagus Positively Modulated to Normal Levels of Expression by Both Black Raspberries and Phenylethyl Isothiocyanate

Gary D. Stoner, Alan A. Dombkowski, Rashmeet K. Reen, et al.

Cancer Res 2008;68:6460-6467.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/68/15/6460

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2008/07/28/68.15.6460.DC1

Cited articles This article cites 51 articles, 13 of which you can access for free at: http://cancerres.aacrjournals.org/content/68/15/6460.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/68/15/6460.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/68/15/6460. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.