Modulation of Gene Expression in Precancerous Rat Esophagus by Dietary Zinc Deficit and Replenishment Chang-Gong Liu, 2 Liang Zhang, 1 Yubao Jiang, 1 Devjani Chatterjee, 1 Carlo M. Croce, 2 Kay Huebner, 2 and Louise Y.Y. Fong 1 1 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania and 2 Comprehensive Cancer Center, Ohio State University, Columbus, Ohio Abstract Zinc deficiency in rats enhances esophageal cell proliferation, causes alteration in gene expression, and promotes esopha- geal 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 analy- ses 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 z2-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 metallothio- nein-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. Immu- nohistochemistry 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. (Cancer Res 2005; 65(17): 7790-9) Introduction Esophageal cancer is an important cause of morbidity and mortality worldwide, with a 5-year survival of only f10% (1). In 2003 in the United States, 13,900 new cases were diagnosed and 13,000 deaths were attributed to this malignancy (2). Whereas alcohol and tobacco consumption are major risk factors in industrialized countries, other factors, such as nutritional defi- ciencies, including that of zinc (3–5), and exposure to environ- mental carcinogens, including N -nitrosomethylbenzylamine (NMBA; ref. 6, 7), play an important role in the pathogenesis of esophageal squamous cell cancer (ESCC) in high incidence areas in northern China and Iran. We have developed zinc-deficient rat and mouse esophageal and oral cancer models (8–16) and found that zinc deficiency creates a precancerous condition in the tongue, esophagus, and forestomach by causing unrestrained cell proliferation and altering gene expression (9–11, 16). In particular, cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in a variety of human premalignant and malignant lesions (17–19), is up-regulated by zinc deficiency (16). Thus, zinc deficiency accelerates carcinogenesis in the esophagus and forestomach caused by a single exposure to the carcinogen NMBA in rats (10), in p53 -deficient mice (12), and in mice overexpressing cyclin D1 (13). In addition, zinc deficiency, by inducing cell proliferation in the entire upper aerodigestive tract, facilitates the development of tumors at multiple sites (tongue, esophagus, and forestomach) in rats exposed to the tongue carcinogen 4-nitroquinoline 1-oxide (NQO; ref. 16). On the contrary, zinc replenishment in zinc-deficient rats rapidly reduces COX-2 expression (16) and stimulates apoptosis by inducing Bax expression thereby inhibiting NMBA-induced esophageal carcino- genesis (14). In a 2005 report, Abnet et al. (5) established a connection between zinc deficiency and ESCC in humans by using X-ray fluorescence spectroscopy to measure zinc, copper, iron, nickel, and sulfur in esophageal biopsy samples obtained from residents in a high ESCC incidence area in China. Subjects were matched on baseline histology and followed for 16 years. Ninety percent of subjects in the highest zinc quartile versus 65% of the subjects in the lowest quartile were alive and cancer free after 16 years. There were no associations with cancer risk for any of the other elements studied. These findings in humans are consistent with our conclusion from rodent model studies that zinc deficiency promotes esophageal cancer. The rapid tumor initiation and reversal in zinc-deficient rat esophagus offers a unique opportunity to identify molecular markers in the precancerous esophagus that are responsible for the divergent tumorigenic potential caused by zinc deficit and replacement. To date, only a few studies have used DNA array and differential display techniques to identify genes in rodent tissues in vivo that are sensitive to dietary zinc supply (20–23). Blanchard et al. (20) first reported that dietary zinc deficiency modulates expression of genes of rat intestine in vivo ; 32 genes were modulated, representing genes that influence signaling pathways, growth, redox, and energy use. Differential display screening of the murine thymic transcriptome during dietary zinc deprivation and Note: C-G. Liu and L. Zhang contributed equally to this work. Requests for reprints: Louise Y.Y. Fong, Comprehensive Cancer Center, Ohio State University, Room 388A, Tzagournis Medical Research Facility, 420 W. 12th Avenue, Columbus, OH 43210. E-mail: Louise.Fong@osumu.edu. I2005 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-05-1345 Cancer Res 2005; 65: (17). September 1, 2005 7790 www.aacrjournals.org Research Article Research. on February 26, 2016. © 2005 American Association for Cancer cancerres.aacrjournals.org Downloaded from