Yubao Jiang

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.

Dietary zinc deficiency fuels esophageal cancer development by inducing a distinct inflammatory signature.

Chronic inflammation is implicated in the pathogenesis of esophageal squamous cell carcinoma (ESCC). The causes of inflammation in ESCC, however, are undefined. Dietary zinc (Zn)-deficiency (ZD) increases the risk of ESCC. We have previously shown that short-term ZD (6 weeks) in rats induces overexpression of the proinflammatory mediators S100a8 and S100a9 in the esophageal mucosa with accompanying esophageal epithelial hyperplasia. Here we report that prolonged ZD (21 weeks) in rats amplified this inflammation that when combined with non-carcinogenic low doses of the environmental carcinogen, N-nitrosomethylbenzylamine (NMBA) elicited a 66.7% (16/24) incidence of ESCC. With Zn-sufficiency, NMBA produced no cancers (0/21) (P<0.001). At tumor endpoint, the neoplastic ZD esophagus, as compared with Zn-sufficient esophagus, had an inflammatory gene signature with upregulation of numerous cancer-related inflammation genes (CXC and CC chemokines, chemokine receptors, cytokines and Cox-2) in addition to S100a8 and S100a9. This signature was already activated in the earlier dysplastic stage. Additionally, time-course bioinformatics analysis of expression profiles at tumor endpoint and before NMBA exposure revealed that this sustained inflammation was due to ZD rather than carcinogen exposure. Importantly, Zn replenishment reversed this inflammatory signature at both the dysplastic and neoplastic stages of ESCC development, and prevented cancer formation. Thus, the molecular definition of ZD-induced inflammation as a critical factor in ESCC development has important clinical implications with regard to development and prevention of this deadly disease.

Modulation of gene expression in precancerous rat esophagus by dietary zinc deficit and replenishment

Zinc deficiency in rats enhances esophageal cell proliferation, causes alteration in gene expression, and promotes esophageal carcinogenesis. Zinc replenishment rapidly induces apoptosis in the esophageal epithelium thereby reversing cell proliferation and carcinogenesis. To identify zinc-responsive genes responsible for these divergent effects, we did oligonucleotide array-based gene expression profiling analyses in the precancerous zinc-deficient esophagus and in zinc-replenished esophagi after treatment with intragastric zinc compared with zinc-sufficient esophagi. Thirty-three genes (21 up-regulated and 12 down-regulated) showed a > or = 2-fold change in expression in the hyperplastic zinc-deficient versus zinc-sufficient esophageal epithelia. Expression of genes involved in cell division, survival, adhesion, and tumorigenesis were markedly changed. The zinc-sensitive gene metallothionein-1 (MT-1 was up-regulated 7-fold, the opposite of results for small intestine and liver under zinc-deficient conditions. Keratin 14 (KRT14, a biomarker in esophageal tumorigenesis), carbonic anhydrase II (CAII, a regulator of acid-base homeostasis), and cyclin B were up-regulated >4-fold. Immunohistochemistry showed that metallothionein and keratin 14 proteins were overexpressed in zinc-deficient esophagus, as well as in lingual and esophageal squamous cell carcinoma from carcinogen-treated rats, emphasizing their roles in carcinogenesis. Calponin 1 (CNN1, an actin cross-linking regulator) was down-regulated 0.2-fold. Within hours after oral zinc treatment, the abnormal expression of 29 of 33 genes returned to near zinc-sufficient levels, accompanied by reversal of the precancerous phenotype. Thus, we have identified new molecular markers in precancerous esophagus and showed their restoration by zinc replenishment, providing insights into the interaction between zinc and gene expression in esophageal cancer development and prevention.

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.

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.

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.

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)‐κ B p65 and leukotriene A4 hydrolase (LTA4H) 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 LTA4H 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.

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.

Abstract LB-241: Silencing of microRNA-31 prevents esophageal neoplasia in zinc deficient rats.

Dietary zinc deficiency (ZD) is implicated in the pathogenesis of human esophageal squamous cell carcinoma (ESCC). microRNA-31 (miR-31) is overexpressed in human ESCC. In the rat a ZD diet promotes esophageal carcinogenesis by inducing cellular proliferation and changes in the expression of microRNA and mRNA, including overexpression of miR-31 and cancer-related proinflammation genes. Here we report that treatment of ZD rats with locked nucleic acid (LNA)-modified inhibitor of miR-31 (LNA-antimiR) prevents the development of a precancerous esophageal phenotype. Weanling rats were fed ZD or zinc-sufficient (ZS) diet for 5 weeks. Simultaneously, ZD rats were administered intravenously (twice a week) LNA-antimiR, LNA-scramble miR-31 or the vehicle saline. ZS rats received saline. Compared to ZS rats, LNA-scramble miR-31-treated or saline-treated ZD rats overexpressed miR-31 and displayed a highly proliferative and inflammatory esophageal phenotype. Treatment of ZD rats with LNA-antimiR reduced miR-31 expression in esophageal epithelia and circulating blood by ~60% and reversed the ZD-induced esophageal pathology, as evidenced by a thinned esophageal epithelium with reduced cell proliferation by PCNA immunohistochemistry and increased apoptosis by caspase-3/7 activity. Transcriptome analyses of esophageal epithelia demonstrated derepression of target mRNAs with miR-31 seed sites. In particular, Stk40 (a negative regulator of NF-κ? signaling) was demonstrated to be a bona fide miR-31 target. Using in situ hybridization and immunohistochemistry, miR-31 overexpression in ZD esophageal sections correlated with downregulation of STK40 protein, as well as with upregulation of an NF-κ? p65 - RAGE-S100A9 inflammatory pathway that in turn, was normalized by miR-31 silencing. Thus, silencing miR-31 prevents esophageal neoplasia. Overexpression of miR-31 promotes ESCC initiation by enhancing inflammation via STK40 - NF-κ? signaling. The data indicate that miR-31 may be a promising therapeutic target for improved ESCC diagnosis and prevention. Funding: National Institutes of Health grants U01 CA152758 to CMC and R01CA118560 & R21CA152505 to LYYF. Citation Format: Cristian Taccioli, Hongping Chen, Michela Garofalo, Yubao Jiang, Gianpiero Di Leva, Hansjuerg Alder, Justin Middleton, Karl J. Smalley, Arianna Bottoni, Stefan Costinean, John L. Farber, Carlo M. Croce, Louise Y Y Fong. Silencing of microRNA-31 prevents esophageal neoplasia in zinc deficient rats. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-241. doi:10.1158/1538-7445.AM2013-LB-241

Abstract 4174: Cxcl5 and Cxcl2 overexpression in esophageal carcinogenesis is associated with rapid tumor formation in zinc-deficient rats

Objectives: Zinc-deficiency (ZD) is implicated in the pathogenesis of human esophageal cancer. It induces cell proliferation, modulates genetic expression, and enhances carcinogenesis. Zinc-replenishment (ZR) reverses proliferation and inhibits carcinogenesis. Recently, we showed that a ZD diet induces overexpression of the proinflammation mediators S100a8/a9 in hyperplastic rat esophagus, providing evidence that zinc regulates an inflammatory pathway in early esophageal carcinogenesis (Gastroenterology, 136: 953-966, 2009). Here we determined the biological processes affected by zinc nutrition during rapid N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor development and its prevention. Methods: We evaluated gene expression profiles of esophageal mucosa from zinc-modulated rats at 5 weeks after an NMBA dose and at 15 weeks (endpoint) with 3 NMBA doses, using Affymetrix Rat Genome GeneChip. Here, ZD rats documented a significantly higher tumorigenic outcome than control zinc-sufficient (ZS) rats (tumor incidence, 100% vs 16.6%; multiplicity, 11 ± 3.8 vs 0.5 ± 0.3, P 25-fold); Ptgs2 (up 26-fold); and Il17f (up 2.7 fold). At endpoint, the tumor-bearing ZD esophagus displayed a neoplastic gene signature, with increased up-regulation of Cxcl5 and Cxcl2 (up 84- and 38-fold), Cxcl3 (up 9-fold), Cxcl1 (up 7-fold), Ptgs2 (up 41-fold), Il1b (up 12-fold), Il17f (up 7.5-fold), and S100a8/a9 (up 2.1-fold). Replenishing zinc led to a global reversal of abnormal gene expression, resulting in an expression profile similar to that of ZS esophagi. In parallel to mRNA expression, CXCL5, CXCL2, COX-2, and S100A8/A9 protein expression was similarly modulated by ZD and ZR, as shown by ELISA, immunoblotting, and immunohistochemistry assays. Ingenuity Pathway Analysis predicted an Il1b-centric network, with direct connections to many up-regulated genes. Conclusions: ZD fuels inflammation and drives carcinogenesis by inducing overexpression of several cancer-related inflammation genes, including Cxcl5 and Cxcl2. ZR modulates inflammatory responses and inhibits tumor growth. The data provide a new understanding of the molecular role of zinc in esophageal carcinogenesis/prevention and suggest that zinc supplementation should be more thoroughly explored in human prevention clinical trials for upper aerodigestive tract cancer. Supported by NIH grant CA118560. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4174.