Zinc treatment reverses and anti-Zn-regulated miRs suppress esophageal carcinomas in vivo.

Esophageal squamous cell carcinoma (ESCC) is a deadly disease with few prevention or treatment options. ESCC development in humans and rodents is associated with Zn deficiency (ZD), inflammation, and overexpression of oncogenic microRNAs: miR-31 and miR-21. In a ZD-promoted ESCC rat model with upregulation of these miRs, systemic antimiR-31 suppresses the miR-31-EGLN3/STK40-NF-κB-controlled inflammatory pathway and ESCC. In this model, systemic delivery of Zn-regulated antimiR-31, followed by antimiR-21, restored expression of tumor-suppressor proteins targeted by these specific miRs: STK40/EGLN3 (miR-31), PDCD4 (miR-21), suppressing inflammation, promoting apoptosis, and inhibiting ESCC development. Moreover, ESCC-bearing Zn-deficient (ZD) rats receiving Zn medication showed a 47% decrease in ESCC incidence vs. Zn-untreated controls. Zn treatment eliminated ESCCs by affecting a spectrum of biological processes that included downregulation of expression of the two miRs and miR-31-controlled inflammatory pathway, stimulation of miR-21-PDCD4 axis apoptosis, and reversal of the ESCC metabolome: with decrease in putrescine, increase in glucose, accompanied by downregulation of metabolite enzymes ODC and HK2. Thus, Zn treatment or miR-31/21 silencing are effective therapeutic strategies for ESCC in this rodent model and should be examined in the human counterpart exhibiting the same biological processes.

Abrogation of esophageal carcinoma development in miR-31 knockout rats.

MicroRNA-31 (miR-31) is overexpressed in esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary Zn deficiency and inflammation. In a Zn deficiency-promoted rat ESCC model with miR-31 up-regulation, cancer-associated inflammation, and a high ESCC burden following N-nitrosomethylbenzylamine (NMBA) exposure, systemic antimiR-31 delivery reduced ESCC incidence from 85 to 45% (P = 0.038) and miR-31 gene knockout abrogated development of ESCC (P = 1 × 10-6). Transcriptomics, genome sequencing, and metabolomics analyses in these Zn-deficient rats revealed the molecular basis of ESCC abrogation by miR-31 knockout. Our identification of EGLN3, a known negative regulator of nuclear factor κB (NF-κB), as a direct target of miR-31 establishes a functional link between oncomiR-31, tumor suppressor target EGLN3, and up-regulated NF-κB-controlled inflammation signaling. Interaction among oncogenic miR-31, EGLN3 down-regulation, and inflammation was also documented in human ESCCs. miR-31 deletion resulted in suppression of miR-31-associated EGLN3/NF-κB-controlled inflammatory pathways. ESCC-free, Zn-deficient miR-31-/- rat esophagus displayed no genome instability and limited metabolic activity changes vs. the pronounced mutational burden and ESCC-associated metabolic changes of Zn-deficient wild-type rats. These results provide conclusive evidence that miR-31 expression is necessary for ESCC development.

Human-like hyperplastic prostate with low ZIP1 induced solely by Zn deficiency in rats.

Prostate cancer is a leading cause of cancer death in men over 50 years of age, and there is a characteristic marked decrease in Zn content in the malignant prostate cells. The cause and consequences of this loss have thus far been unknown. We found that in middle-aged rats a Zn-deficient diet reduces prostatic Zn levels (P = 0.025), increases cellular proliferation, and induces an inflammatory phenotype with COX-2 overexpression. This hyperplastic/inflammatory prostate has a human prostate cancer-like microRNA profile, with up-regulation of the Zn-homeostasis-regulating miR-183-96-182 cluster (fold change = 1.41-2.38; P = 0.029-0.0003) and down-regulation of the Zn importer ZIP1 (target of miR-182), leading to a reduction of prostatic Zn. This inverse relationship between miR-182 and ZIP1 also occurs in human prostate cancer tissue, which is known for Zn loss. The discovery that the Zn-depleted middle-aged rat prostate has a metabolic phenotype resembling that of human prostate cancer, with a 10-fold down-regulation of citric acid (P = 0.0003), links citrate reduction directly to prostatic Zn loss, providing the underlying mechanism linking dietary Zn deficiency with miR-183-96-182 overexpression, ZIP1 down-regulation, prostatic Zn loss, and the resultant citrate down-regulation, changes mimicking features of human prostate cancer. Thus, dietary Zn deficiency during rat middle age produces changes that mimic those of human prostate carcinoma and may increase the risk for prostate cancer, supporting the need for assessment of Zn supplementation in its prevention.

Integration of metabolomics, transcriptomics, and microRNA expression profiling reveals a miR-143-HK2-glucose network underlying zinc-deficiency-associated esophageal neoplasia.

Esophageal squamous cell carcinoma (ESCC) in humans is a deadly disease associated with dietary zinc (Zn)-deficiency. In the rat esophagus, Zn-deficiency induces cell proliferation, alters mRNA and microRNA gene expression, and promotes ESCC. We investigated whether Zn-deficiency alters cell metabolism by evaluating metabolomic profiles of esophageal epithelia from Zn-deficient and replenished rats vs sufficient rats, using untargeted gas chromatography time-of-flight mass spectrometry (n = 8/group). The Zn-deficient proliferative esophagus exhibits a distinct metabolic profile with glucose down 153-fold and lactic acid up 1.7-fold (P < 0.0001), indicating aerobic glycolysis (the "Warburg effect"), a hallmark of cancer cells. Zn-replenishment rapidly increases glucose content, restores deregulated metabolites to control levels, and reverses the hyperplastic phenotype. Integration of metabolomics and our reported transcriptomic data for this tissue unveils a link between glucose down-regulation and overexpression of HK2, an enzyme that catalyzes the first step of glycolysis and is overexpressed in cancer cells. Searching our published microRNA profile, we find that the tumor-suppressor miR-143, a negative regulator of HK2, is down-regulated in Zn-deficient esophagus. Using in situ hybridization and immunohistochemical analysis, the inverse correlation between miR-143 down-regulation and HK2 overexpression is documented in hyperplastic Zn-deficient esophagus, archived ESCC-bearing Zn-deficient esophagus, and human ESCC tissues. Thus, to sustain uncontrolled cell proliferation, Zn-deficiency reprograms glucose metabolism by modulating expression of miR-143 and its target HK2. Our work provides new insight into critical roles of Zn in ESCC development and prevention.

Reduction in squamous cell carcinomas in mouse skin by dietary zinc supplementation.

Inadequate dietary Zn consumption increases susceptibility to esophageal and other cancers in humans and model organisms. Since Zn supplementation can prevent cancers in rodent squamous cell carcinoma (SCC) models, we were interested in determining if it could have a preventive effect in a rodent skin cancer model, as a preclinical basis for considering a role for Zn in prevention of human nonmelanoma skin cancers, the most frequent cancers in humans. We used the 7,12-dimethyl benzanthracene carcinogen/phorbol myristate acetate tumor promoter treatment method to induce skin tumors in Zn-sufficient wild-type and Fhit (human or mouse protein) knockout mice. Fhit protein expression is lost in >50% of human cancers, including skin SCCs, and Fhit-deficient mice show increased sensitivity to carcinogen induction of tumors. We hypothesized that: (1) the skin cancer burdens would be reduced by Zn supplementation; (2) Fhit(-/-) (Fhit, murine fragile histidine triad gene) mice would show increased susceptibility to skin tumor induction versus wild-type mice. 30 weeks after initiating treatment, the tumor burden was increased ~2-fold in Fhit(-/-) versus wild-type mice (16.2 versus 7.6 tumors, P < 0.001); Zn supplementation significantly reduced tumor burdens in Fhit(-/-) mice (males and females combined, 16.2 unsupplemented versus 10.3 supplemented, P = 0.001). Most importantly, the SCC burden was reduced after Zn supplementation in both strains and genders of mice, most significantly in the wild-type males (P = 0.035). Although the mechanism(s) of action of Zn supplementation in skin tumor prevention is not known in detail, the Zn-supplemented tumors showed evidence of reduced DNA damage and some cohorts showed reduced inflammation scores. The results suggest that mild Zn supplementation should be tested for prevention of skin cancer in high-risk human cohorts.

MicroRNA dysregulation and esophageal cancer development depend on the extent of zinc dietary deficiency.

Zinc deficiency (ZD) increases the risk of esophageal squamous cell carcinoma (ESCC), and marginal ZD is prevalent in humans. In rats, marked-ZD (3 mg Zn/kg diet) induces a proliferative esophagus with a 5-microRNA signature (miR-31, -223, -21, -146b, -146a) and promotes ESCC. Here we report that moderate and mild-ZD (6 and 12 mg Zn/kg diet) also induced esophageal hyperplasia, albeit less pronounced than induced by marked-ZD, with a 2-microRNA signature (miR-31, -146a). On exposure to an environmental carcinogen, ~16% of moderate/mild-ZD rats developed ESCC, a cancer incidence significantly greater than for Zn-sufficient rats (0%) (P ≤ 0.05), but lower than marked-ZD rats (68%) (P < 0.001). Importantly, the high ESCC, marked-ZD esophagus had a 15-microRNA signature, resembling the human ESCC miRNAome, with miR-223, miR-21, and miR-31 as the top-up-regulated species. This signature discriminated it from the low ESCC, moderate/mild-ZD esophagus, with a 2-microRNA signature (miR-31, miR-223). Additionally, Fbxw7, Pdcd4, and Stk40 (tumor-suppressor targets of miR-223, -21, and -31) were downregulated in marked-ZD cohort. Bioinformatics analysis predicted functional relationships of the 3 tumor-suppressors with other cancer-related genes. Thus, microRNA dysregulation and ESCC progression depend on the extent of dietary Zn deficiency. Our findings suggest that even moderate ZD may promote esophageal cancer and dietary Zn has preventive properties against ESCC. Additionally, the deficiency-associated miR-223, miR-21, and miR-31 may be useful therapeutic targets in ESCC.

Repression of Esophageal Neoplasia and Inflammatory Signaling by Anti-miR-31 Delivery In Vivo.

BACKGROUND: Overexpression of microRNA-31 (miR-31) is implicated in the pathogenesis of esophageal squamous cell carcinoma (ESCC), a deadly disease associated with dietary zinc deficiency. Using a rat model that recapitulates features of human ESCC, the mechanism whereby Zn regulates miR-31 expression to promote ESCC is examined. METHODS: To inhibit in vivo esophageal miR-31 overexpression in Zn-deficient rats (n = 12-20 per group), locked nucleic acid-modified anti-miR-31 oligonucleotides were administered over five weeks. miR-31 expression was determined by northern blotting, quantitative polymerase chain reaction, and in situ hybridization. Physiological miR-31 targets were identified by microarray analysis and verified by luciferase reporter assay. Cellular proliferation, apoptosis, and expression of inflammation genes were determined by immunoblotting, caspase assays, and immunohistochemistry. The miR-31 promoter in Zn-deficient esophagus was identified by ChIP-seq using an antibody for histone mark H3K4me3. Data were analyzed with t test and analysis of variance. All statistical tests were two-sided. RESULTS: In vivo, anti-miR-31 reduced miR-31 overexpression (P = .002) and suppressed the esophageal preneoplasia in Zn-deficient rats. At the same time, the miR-31 target Stk40 was derepressed, thereby inhibiting the STK40-NF-κΒ-controlled inflammatory pathway, with resultant decreased cellular proliferation and activated apoptosis (caspase 3/7 activities, fold change = 10.7, P = .005). This same connection between miR-31 overexpression and STK40/NF-κΒ expression was also documented in human ESCC cell lines. In Zn-deficient esophagus, the miR-31 promoter region and NF-κΒ binding site were activated. Zn replenishment restored the regulation of this genomic region and a normal esophageal phenotype. CONCLUSIONS: The data define the in vivo signaling pathway underlying interaction of Zn deficiency and miR-31 overexpression in esophageal neoplasia and provide a mechanistic rationale for miR-31 as a therapeutic target for ESCC.

Targeted expression of ornithine decarboxylase antizyme prevents upper aerodigestive tract carcinogenesis in p53-deficient mice.

Upper aerodigestive tract (UADT) cancers of the oral cavity and esophagus are a significant global health burden, and there is an urgent need to develop relevant animal models to identify chemopreventive and therapeutic strategies to combat these diseases. Antizyme (AZ) is a multifunctional negative regulator of cellular polyamine levels, and here, we evaluate the susceptibility of keratin 5 (K5)-AZ transgenic mice to tumor models that combine chemical carcinogenesis with dietary and genetic risk factors known to influence human susceptibility to UADT cancer and promote UADT carcinogenesis in mice. First, p53(+/-) and K5-AZ/p53(+/-) (AZ/p53(+/-)) mice were placed on a zinc-deficient (ZD) or zinc-sufficient (ZS) diet and chronically exposed to 4-nitroquinoline 1-oxide. Tongue tumor incidence, multiplicity and size were substantially reduced in both ZD and ZS AZ/p53(+/-) mice compared with p53(+/-). AZ expression also reduced progression to carcinoma in situ or invasive carcinoma and decreased expression of the squamous cell carcinoma biomarkers K14, cyclooxygenase-2 and metallothionein. Next, AZ-expressing p53(+/-) and p53 null mice were placed on the ZD diet and treated with a single dose of N-nitrosomethylbenzylamine. Regardless of p53 status, forestomach (FST) tumor incidence, multiplicity and size were greatly reduced with AZ expression, which was also associated with a significant decrease in FST epithelial thickness along with reduced proliferation marker K6 and increased differentiation marker loricrin. These studies demonstrate the powerful tumor suppressive effects of targeted AZ expression in two distinct and unique mouse models and validate the polyamine metabolic pathway as a target for chemoprevention of UADT cancers.

Dysregulation of miR-31 and miR-21 induced by zinc deficiency promotes esophageal cancer.

Zinc deficiency (ZD) increases the risk of esophageal squamous cell carcinoma (ESCC). In a rat model, chronic ZD induces an inflammatory gene signature that fuels ESCC development. microRNAs regulate gene expression and are aberrantly expressed in cancers. Here we investigated whether chronic ZD (23 weeks) also induces a protumorigenic microRNA signature. Using the nanoString technology, we evaluated microRNA profiles in ZD esophagus and six additional tissues (skin, lung, pancreas, liver, prostate and peripheral blood mononuclear cells [PBMC]). ZD caused overexpression of inflammation genes and altered microRNA expression across all tissues analyzed, predictive of disease development. Importantly, the inflammatory ZD esophagus had a distinct microRNA signature resembling human ESCC or tongue SCC miRNAomes with miR-31 and miR-21 as the top-up-regulated species. Circulating miR-31 was also the top-up-regulated species in PBMCs. In ZD esophagus and tongue, oncogenic miR-31 and miR-21 overexpression was accompanied by down-regulation of their respective tumor-suppressor targets PPP2R2A and PDCD4. Importantly, esophageal miR-31 and miR-21 levels were directly associated with the appearance of ESCC in ZD rats, as compared with their cancer-free Zn-sufficient or Zn-replenished counterparts. In situ hybridization analysis in rat and human tongue SCCs localized miR-31 to tumor cells and miR-21 to stromal cells. In regressing tongue SCCs from Zn-supplemented rats, miR-31 and miR-21 expression was concomitantly reduced, establishing their responsiveness to Zn therapy. A search for putative microRNA targets revealed a bias toward genes in inflammatory pathways. Our finding that ZD causes miR-31 and miR-21 dysregulation associated with inflammation provides insight into mechanisms whereby ZD promotes ESCC.

Zinc supplementation suppresses 4-nitroquinoline 1-oxide-induced rat oral carcinogenesis.

Dietary zinc (Zn) deficiency is implicated in the pathogenesis of human oral-esophageal cancers. In rats, Zn deficiency causes increased cell proliferation and cyclooxygenase-2 (COX-2) overexpression and enhances oral carcinogenesis by 4-nitroquinoline 1-oxide (NQO). Zn replenishment reverses all these effects. We questioned whether Zn has antitumor efficacy in a Zn-sufficient animal by investigating in Zn-sufficient rats (i) the efficacy of Zn supplementation on the progression of tongue squamous cell carcinogenesis induced by drinking water exposure to high (20-30 p.p.m.) and low (10 p.p.m.) doses of NQO and (ii) the modulating effects of Zn supplementation on biomarker expression in tongue lesions by immunohistochemistry. In rats exposed to high doses of NQO, Zn supplementation significantly reduced the incidence of papillomas from 100 to 64.7% (P=0.018) and invasive carcinomas from 93.8 to 52.9% (P=0.017). In rats exposed to low doses of NQO, where only minimally invasive carcinomas developed, Zn supplementation significantly reduced tumor multiplicity, incidence of tumors (1-2 mm), hyperplasia, dysplasia, papillomas and progression to carcinoma. Immunohistochemical analysis of carcinomas showed that Zn supplementation caused a shift to a less proliferative/aggressive cancer phenotype by reducing cell proliferation, stimulating apoptosis and decreasing expression of the key tumor markers cyclin D1, p53 and COX-2. Additionally, Zn supplementation significantly reduced cell proliferation in non-lesional tongue squamous epithelia, thereby suppressing tumor development. Together, the results demonstrate that Zn supplementation has chemopreventive efficacy against oral carcinogenesis in nutritionally complete animals. Our data suggest that Zn supplementation may be efficacious in the chemoprevention of human oral cancer.

Effect of zinc supplementation on N-nitrosomethylbenzylamine-induced forestomach tumor development and progression in tumor suppressor-deficient mouse strains.

Zinc deficiency is associated with high incidences of esophageal and other cancers in humans and leads to a highly proliferative hyperplastic condition in the upper gastrointestinal tract in laboratory rodents. Zn replenishment reduces the incidence of lingual, esophageal and forestomach tumors in Zn-deficient rats and mice. While previous animal studies focused on Zn deficiency, we have investigated the effect of Zn supplementation on carcinogenesis in Zn-sufficient mice of wild-type and tumor suppressor-deficient mouse strains. All mice received N-nitrosomethylbenzylamine and half the mice of each strain then received Zn supplementation. At killing, mice without Zn supplementation had developed more tumors than Zn-supplemented mice: wild-type C57BL/6 mice developed an average of 7.0 versus 5.0 tumors for Zn supplemented (P < 0.05); Zn-supplemented Fhit-/- mice averaged 5.7 versus 8.0 for control mice (P < 0.01); Zn-supplemented Fhit-/-Nit1-/- mice averaged 5.4 versus 9.2 for control mice (P < 0.01) and Zn-supplemented Fhit-/-Rassf1a-/- (the murine gene) mice averaged 5.9 versus 9.1 for control mice (P < 0.01). Zn supplementation reduced tumor burdens by 28% (wild-type) to 42% (Fhit-/-Nit1-/-). Histological analysis of forestomach tissues also showed significant decreases in severity of preneoplastic and neoplastic lesions in Zn-supplemented cohorts of each mouse strain. Thus, Zn supplementation significantly reduced tumor burdens in mice with multiple tumor suppressor deficiencies. When Zn supplementation was begun at 7 weeks after the final carcinogen dose, the reduction in tumor burden was the same as observed when supplementation began immediately after carcinogen dosing, suggesting that Zn supplementation may affect tumor progression rather than tumor initiation.

Zinc deficiency activates S100A8 inflammation in the absence of COX-2 and promotes murine oral-esophageal tumor progression.

Zinc (Zn)-deficiency (ZD) is implicated in the pathogenesis of human oral-esophageal cancers. Previously, we showed that in ZD mice genetic deletion of cyclooxygenase-2 (Cox-2) enhances N-nitrosomethylbenzylamine-induced forestomach carcinogenesis. By contrast, Cox-2 deletion offers protection in Zn-sufficient (ZS) mice. We hypothesize that ZD activates pathways insensitive to COX-2 inhibition, thereby promoting carcinogenesis. This hypothesis is tested in a Cox-2(-/-) mouse tongue cancer model that mimics pharmacologic blockade of COX-2 by firstly examining transcriptome profiles of forestomach mucosa from Cox-2(-/-) and wild-type mice on a ZD vs. ZS diet, and secondly investigating the roles of identified markers in mouse forestomach/tongue preneoplasia and carcinomas. In Cox-2(-/-) mice exposed to the tongue carcinogen 4-nitroquinoline 1-oxide, dietary ZD elicited tongue/esophagus/forestomach carcinomas that were prevented by ZS. The precancerous ZD:Cox-2(-/-) vs. ZS:Cox-2(-/-) forestomach had an inflammatory signature with upregulation of the proinflammation genes S100a8 and S100a9. Bioinformatics analysis revealed overrepresentation of inflammation processes comprising S100a8/a9 and an nuclear factor (NF)-κB network with connectivity to S100A8. Immunohistochemistry revealed co-overexpression of S100A8, its heterodimeric partner S100A9, the receptor for advanced glycation end-products (RAGE), NF-κB p65, and cyclin D1, in ZD:Cox-2(-/-) forestomach/tongue preneoplasia and carcinomas, evidence for the activation of a RAGE-S100A8/A9 inflammatory pathway. Accumulation of p53 in these carcinomas indicated activation of additional inflammatory pathways. Zn-replenishment in ZD:Cox-2(-/-) mice reversed the inflammation and inhibited carcinogenesis. Thus, ZD activates alternative inflammation-associated cancer pathways that fuel tumor progression and bypass the antitumor effect of Cox-2 ablation. These findings have important clinical implications, as combination cancer therapy that includes Zn may improve efficacy.

Nit1 and Fhit tumor suppressor activities are additive.

The fragile histidine triad gene (human FHIT, mouse Fhit) has been shown to act as a tumor suppressor gene. Nit1 and Fhit form a fusion protein, encoded by the NitFhit gene in flies and worms, suggesting that mammalian Nit1 and Fhit proteins, which are encoded by genes on different chromosomes in mammals, may function in the same signal pathway(s). A previous study showed that Nit1 deficiency in knockout mice confers a cancer prone phenotype, as does Fhit deficiency. We have now assessed the tumor susceptibility of Fhit(-/-)Nit1(-/-) mice and observed that double knockout mice develop more spontaneous and carcinogen-induced tumors than Fhit(-/-) mice, suggesting that the extent of tumor susceptibility due to Nit1 and Fhit deficiency is additive, and that Nit1 and Fhit affect distinct signal pathways in mammals. Nit1, like Fhit, is present in cytoplasm and mitochondria but not nuclei. Because Fhit deficiency affects responses to replicative and oxidative stress, we sought evidence for Nit1 function in response to such stresses in tissues and cultured cells: when treated with hydroxyurea, the normal kidney-derived double-deficient cells appear not to activate the pChk2 pathway and when treated with H(2)O(2), show little evidence of DNA damage, compared with wild type and Fhit(-/-) cells. The relevance of Nit1 deficiency to human cancers was examined in human esophageal cancer tissues, and loss of Nit1 expression was observed in 48% of esophageal adenocarcinomas.

UCbase & miRfunc: a database of ultraconserved sequences and microRNA function.

Four hundred and eighty-one ultraconserved sequences (UCRs) longer than 200 bases were discovered in the genomes of human, mouse and rat. These are DNA sequences showing 100% identity among the three species. UCRs are frequently located at genomic regions involved in cancer, differentially expressed in human leukemias and carcinomas and in some instances regulated by microRNAs (miRNAs). Here we present UCbase & miRfunc, the first database which provides ultraconserved sequences data and shows miRNA function. Also, it links UCRs and miRNAs with the related human disorders and genomic properties. The current release contains over 2000 sequences from three species (human, mouse and rat). As a web application, UCbase & miRfunc is platform independent and it is accessible at http://microrna.osu.edu/.UCbase4.

Zinc replenishment reverses overexpression of the proinflammatory mediator S100A8 and esophageal preneoplasia in the rat.

BACKGROUND & AIMS: Zinc deficiency is implicated in the pathogenesis of human esophageal cancer. In the rat esophagus, it induces cell proliferation, modulates genetic expression, and enhances carcinogenesis. Zinc-replenishment reverses proliferation and inhibits carcinogenesis. The zinc-deficient rat model allows the identification of biological differences affected by zinc during early esophageal carcinogenesis. METHODS: We evaluated gene expression profiles of esophageal epithelia from zinc-deficient and replenished rats vs zinc-sufficient rats using microarray analysis. We characterized the role of the top-up-regulated gene S100A8 in esophageal hyperplasia/reversal and in chemically induced esophageal carcinogenesis in zinc-modulated animals by immunohistochemistry and real-time quantitative polymerase chain reaction. RESULTS: The hyperplastic-deficient esophagus has a distinct expression signature with the proinflammation genes S100 calcium binding protein A8 (S100A8) and A9 (S100A9) up-regulated 57-fold and 5-fold, respectively. Zinc replenishment rapidly restored to control levels the expression of S100A8/A9 and 27 other genes and reversed the hyperplastic phenotype. With its receptor for advanced glycation end products (RAGE), colocalization and overexpression of S100A8 protein occurred in the deficient esophagus that overexpressed nuclear factor kappaBeta p65 and cyclooxygenase-2 (COX-2) protein. Zinc replenishment, but not a COX-2 inhibitor, reduced the overexpression of these 4 proteins. Additionally, esophageal S100A8/A9 messenger RNA levels were associated directly with the diverse tumorigenic outcome in zinc-deficient and zinc-replenished rats. CONCLUSIONS: In vivo zinc regulates S100A8 expression and modulates the link between S100A8-RAGE interaction and downstream nuclear factor kappaBeta/COX-2 signaling. The finding that zinc regulates an inflammatory pathway in esophageal carcinogenesis may lead to prevention and therapy for this cancer.

Prevention of upper aerodigestive tract cancer in zinc-deficient rodents: inefficacy of genetic or pharmacological disruption of COX-2.

Zinc deficiency in humans is associated with an increased risk of upper aerodigestive tract (UADT) cancer. In rodents, zinc deficiency predisposes to carcinogenesis by causing proliferation and alterations in gene expression. We examined whether in zinc-deficient rodents, targeted disruption of the cyclooxygenase (COX)-2 pathway by the COX-2 selective inhibitor celecoxib or by genetic deletion prevent UADT carcinogenesis. Tongue cancer prevention studies were conducted in zinc-deficient rats previously exposed to a tongue carcinogen by celecoxib treatment with or without zinc replenishment, or by zinc replenishment alone. The ability of genetic COX-2 deletion to protect against chemically-induced forestomach tumorigenesis was examined in mice on zinc-deficient versus zinc-sufficient diet. The expression of 3 predictive biomarkers COX-2, nuclear factor (NF)-kappa B p65 and leukotriene A(4) hydrolase (LTA(4)H) was examined by immunohistochemistry. In zinc-deficient rats, celecoxib without zinc replenishment reduced lingual tumor multiplicity but not progression to malignancy. Celecoxib with zinc replenishment or zinc replenishment alone significantly lowered lingual squamous cell carcinoma incidence, as well as tumor multiplicity. Celecoxib alone reduced overexpression of the 3 biomarkers in tumors slightly, compared with intervention with zinc replenishment. Instead of being protected, zinc-deficient COX-2 null mice developed significantly greater tumor multiplicity and forestomach carcinoma incidence than wild-type controls. Additionally, zinc-deficient COX-2-/- forestomachs displayed strong LTA(4)H immunostaining, indicating activation of an alternative pathway under zinc deficiency when the COX-2 pathway is blocked. Thus, targeting only the COX-2 pathway in zinc-deficient animals did not prevent UADT carcinogenesis. Our data suggest zinc supplementation should be more thoroughly explored in human prevention clinical trials for UADT cancer.

Inactivation of the Wwox gene accelerates forestomach tumor progression in vivo.

The WWOX gene encodes a tumor suppressor spanning the second most common human fragile site, FRA16D. Targeted deletion of the Wwox gene in mice led to an increased incidence of spontaneous and ethyl nitrosourea-induced tumors. In humans, loss of heterozygosity and reduced or loss of WWOX expression has been reported in esophageal squamous cell cancers (SCC). In the present study, we examined whether inactivation of the Wwox gene might lead to enhanced esophageal/forestomach tumorigenesis induced by N-nitrosomethylbenzylamine. Wwox+/- and Wwox+/+ mice were treated with six intragastric doses of N-nitrosomethylbenzylamine and observed for 15 subsequent weeks. Ninety-six percent (25 of 26) of Wwox+/- mice versus 29% (10 of 34) of Wwox+/+ mice developed forestomach tumors (P = 1.3 x 10(-7)). The number of tumors per forestomach was significantly greater in Wwox+/- than in Wwox+/+ mice (3.2 +/- 0.34 versus 0.47 +/- 0.17; P < 0.0001). In addition, 27% of Wwox+/- mice had invasive SCC in the forestomach, as compared with 0% of wild-type controls (P = 0.002). Intriguingly, forestomachs from Wwox+/- mice displayed moderately strong Wwox protein staining in the near-normal epithelium, but weak and diffuse staining in SCC in the same tissue section, a result suggesting that Wwox was haploinsufficient for the initiation of tumor development. Our findings provide the first in vivo evidence of the tumor suppressor function of WWOX in forestomach/esophageal carcinogenesis and suggest that inactivation of one allele of WWOX accelerates the predisposition of normal cells to malignant transformation.

Fez1/Lzts1 absence impairs Cdk1/Cdc25C interaction during mitosis and predisposes mice to cancer development.

The FEZ1/LZTS1 (LZTS1) protein is frequently downregulated in human cancers of different histotypes. LZTS1 is expressed in normal tissues, and its introduction in cancer cells inhibits cell growth and suppresses tumorigenicity, owing to an accumulation of cells in G2/M. Here, we define its role in cell cycle regulation and tumor progression by generating Lzts1 knockout mice. In Lzts1(-/-) mouse embryo fibroblasts (MEFs), Cdc25C degradation was increased during M phase, resulting in decreased Cdk1 activity. As a consequence, Lzts1(-/-) MEFs showed accelerated mitotic progression, resistance to taxol- and nocodazole-induced M phase arrest, and improper chromosome segregation. Accordingly, Lzts1 deficiency was associated with an increased incidence of both spontaneous and carcinogen-induced cancers in mice.

Zinc deficiency potentiates induction and progression of lingual and esophageal tumors in p53-deficient mice.

Upper aerodigestive tract (UADT) cancer, including oral and esophageal cancer, is an important cause of cancer deaths worldwide. Patients with UADT cancer are frequently zinc deficient (ZD) and show a loss of function of the pivotal tumor suppressor gene p53. The present study examined whether zinc deficiency in collaboration with p53 insufficiency (p53+/-) promotes lingual and esophageal tumorigenesis in mice exposed to low doses of the carcinogen 4-nitroquinoline 1-oxide. In wild-type mice, ZD significantly increased the incidence of lingual and esophageal tumors from 0% in zinc sufficient (ZS) ZS:p53+/+ mice to approximately 40%. On the p53+/- background, ZD:p53+/- mice had significantly greater tumor incidence and multiplicity than ZS:p53+/- and ZD:p53+/+ mice, with a high frequency of progression to malignancy. Sixty-nine and 31% of ZD:p53+/- lingual and esophageal tumors, respectively, were squamous cell carcinoma versus 19 and 0% of ZS:p53+/- tumors (tongue, P = 0.003; esophagus, P = 0.005). Immunohistochemical analysis revealed that the increased cellular proliferation observed in preneoplastic lingual and esophageal lesions, as well as invasive carcinomas, was accompanied by overexpression of cytokeratin 14, cyclooxygenase-2 and metallothionein. In summary, a new UADT cancer model is developed in ZD:p53+/- mouse that recapitulates aspects of the human cancer and provides opportunities to probe the genetic changes intrinsic to UADT carcinogenesis and to test strategies for prevention and reversal of this deadly cancer.