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Background: Mucinous carcinomas from different organs are morphologically similar and might share similarities at the molecular and biochemical levels that may illuminate their pathogenesis and influence management. The factors involved in the pathogenesis of mucinous carcinomas remain unknown; which is likely one contributor to the current dearth of biomarkers for detection. Because zinc changes are implicated in some cancers e.g., prostate; we assessed the possibility of a similar role in mucinous carcinomas., Methods: The goal of the current work is to study the expression of hZip1 by immunohistochemistry in mucinous carcinomas as compared with non-neoplastic epithelia and conventional carcinomas. Tissue microarray slides containing mucinous carcinomas of the ovary (n = 35), colon (n = 51), stomach (n = 32) and lung (n = 21) were used., Results: hZip1 showed persistent low expression in mucinous compared to ovarian serous carcinomas and normal tissue (P < 0.05), colonic adenocarcinoma and normal mucosa (P < 0.001), and gastric adenocarcinoma and normal epithelium (P < 0.05). hZip1 also showed low expression in pulmonary mucinous carcinomas., Conclusions: hZip1 is consistently decreased in mucinous carcinomas from a variety of organs. Despite the fact that these preliminary findings are unlikely to be of much diagnostic significance, these findings suggest that hZip1 plays a fundamental role in the carcinogenesis of mucinous tumors.

Pancreatic cancer (ductal adenocarcinoma) remains a deadly cancer with ~85% mortality, and a 5-year survival rate of ~6% or less for the past 30 years. The factors and events associated with the development of pancreatic cancer are poorly identified. As such, effective biomarkers for early detection of malignancy are lacking. Efficacious chemotherapy once the cancer is identified does not exist. Recent clinical studies have revealed that the zinc levels are consistently and markedly decreased in adenocarcinoma as compared with normal/benign pancreatic tissue. The decreased zinc is exhibited in well-differentiated malignancy and in progressing malignancy, and also exists throughout the development of PanIN. Concurrent with the decrease in zinc, RREB1 transcription factor and ZIP3 zinc uptake transporter are downregulated. Thus, a RREB1/ZIP3/Zinc transformation appears to be an early event in the development of pancreatic cancer. We propose that this transformation is necessary to prevent the accumulation of high cellular zinc levels, which result in cytotoxic effects on the developing malignant cells. This report now demonstrates that exposure of Panc1 cells to physiological concentrations of zinc that result in increased zinc uptake and accumulation also inhibits cell proliferation. The study further shows that ZIP3 is the important transporter required for the accumulation of zinc and its inhibition of proliferation. RREB1 is identified as the positive regulator of ZIP3 expression. Therefore, the pathway of RREB1/ZIP3/Zinc and its downregulation during oncogenesis exist to prevent the accumulation of cytotoxic levels of zinc during the development and progression of the malignant cells in pancreatic adenocarcinoma.

Purpose: Pancreatic adenocarcinoma is an untreatable cancer with a 5-year survival rate of about 6 % or less for the past 35 years. This lack of significant progress is largely due to the lack of elucidation and understanding of the factors involved in the development of this cancer. Recent studies identified and implicated zinc in the development and progression of pancreatic cancer. This study was conducted to establish the changes in zinc, ZIP3 zinc transporter, and Ras-responsive element-binding protein 1 (RREB-1) transcription factor as early events in the development of malignancy., Methods: In situ relative zinc determination and immunohistochemical analysis of ZIP3 and RREB-1 were performed on archived human pancreatic tissue sections and tissue microarrays. Normal/benign versus adenocarcinoma pancreas was compared. Panc1 cells were employed to determine the influence of RREB-1 on ZIP3 expression., Results: Zinc levels of normal ductal and acinar epithelium were markedly and consistently decreased in adenocarcinoma. Pancreatic intraepithelial neoplasia (PanIN) lesions also exhibited a loss of zinc. ZIP3 and RREB-1 were also markedly downregulated. Initial results indicate that RREB-1 regulates ZIP3 expression., Conclusions: These results corroborate the earlier report that zinc, ZIP3, and RREB-1 are markedly decreased in early stage adenocarcinoma. Additionally and most importantly, these changes occur in PanIN, which are thought to be precancerous lesions leading to ductal adenocarcinoma. These results support a concept that downregulation of RREB-1 causes downregulation of ZIP3, which results in decreased zinc in premalignant and carcinoma cells. The decrease in zinc is essential to remove its cytotoxic effects on malignant cells. This relationship constitutes a new concept of early genetic/metabolic events in the progressive transformation of normal cells to premalignant cells to malignant cells in the development of pancreatic cancer.

Purpose: Hepatocellular cancer (HCC) is a deadly and most rapidly increasing cancer in the USA and worldwide. The etiology and factors involved in development of HCC remain largely unknown. A marked decrease in zinc occurs in HCC. Its role and involvement in HCC has not been identified. We investigated the relationship of cellular zinc changes to the development of malignancy, and the identification of potential zinc transporters associated with the inability of hepatoma cells to accumulate zinc., Methods: The detection of relative zinc levels in situ in normal hepatic cells vs. hepatoma was performed on normal and HCC tissue sections. ZIP1, 2, 3, and 14 transporters were identified by immunohistochemistry., Results: Intracellular zinc levels are markedly decreased in HCC hepatoma cells vs. normal hepatic cells in early stage and advanced stage malignancy. ZIP14 transporter is localized at the plasma membrane in normal hepatocytes, demonstrating its functioning for uptake and accumulation of zinc. The transporter is absent in the hepatoma cells and its gene expression is downregulated. The change in ZIP14 is concurrent with the decrease in zinc. ZIP1, 2, 3 are not associated with normal hepatocyte uptake of zinc, and HCC zinc depletion. HepG2 cells exhibit ZIP14 transporter. Zinc treatment inhibits their growth., Conclusions: ZIP14 downregulation is likely involved in the depletion of zinc in the hepatoma cells in HCC. These events occur early in the development of malignancy possibly to protect the malignant cells from tumor suppressor effects of zinc. This provides new insight into important factors associated with HCC carcinogenesis.

Prostate cancer is the second leading cause of cancer deaths among men. The availability of animal models that represent the events and factors that exist in the natural history and biology of human prostate cancer is essential in dealing with prostate cancer. In recent decades and presently, emphasis has been directed at the development and employment of prostate cancer induced in transgenic mice. However, the important consistent hallmark characteristic and event of decrease in zinc and citrate and downregulation of ZIP1 zinc transporter in prostate malignancy has not been studied or identified in any animal model. We investigated the status of these parameters in TRAMP tumors as compared with human prostate cancer. The results show that citrate levels are markedly decreased in the developing and advancing stages of malignancy in TRAMP. Zinc levels are also decreased and ZIP1 transporter is lost in TRAMP tumors. In vitro studies show that zinc treatment of TRAMP C2 cells exhibits cytotoxic effects. Collectively, these results mimic the ZIP1, zinc, and citrate status and relationship that exist in human prostate cancer. This is the first report that establishes the existence of the human prostate zinc/citrate hallmark characteristic and relationship in an animal model. It now appears that the TRAMP model will be suitable for studies relating to the implications and role of zinc- and citrate-related metabolism in the development and progression of human prostate cancer.

Background: A marked decrease in the level of zinc is a consistent characteristic of prostate cancer; which results from down-regulation of ZIP1 zinc transporter. The aim of this study was to determine if RREB-1 transcription is involved in the down-regulation of ZIP1 gene expression; and to determine the expression of RREB-1 in benign and cancerous prostate in situ., Methods: Overexpression and siRNA knock down of RREB-1 were used to determine the effect of RREB-1 on hZIP1 abundance in PC-3 cells. Immunohistochemistry with tissue microarrays (TMAs) and tissue sections was used to determine the levels of RREB-1 expression in prostate in situ., Results: Overexpression of RREB-1 resulted in a decrease in the abundance of hZIP1 in the plasma membrane of PC-3 cells; whereas siRNA knock down significantly increased hZIP1 expression. Prostate TMAs and tissue sections showed an inverse relationship between RREB-1 and hZIP1 staining., Conclusions: RREB-1 overexpression results in down-regulation of hZIP1 and contributes to the loss of hZIP1 expression and zinc in prostate cancer. This is an early event in prostate carcinogenesis., (Copyright © 2011 Wiley-Liss, Inc.)

Pancreatic adenocarcinoma is an untreatable deadly cancer. The factors involved in its early development remain unknown; which contributes to the absence of biomarkers for early detection of malignancy or at-risk subjects, and the absence of efficacious therapeutic agents. Because zinc changes are implicated in some cancers, we determined if it might be involved in the development of pancreatic adenocarcinoma. With in situ Dithizone and Zinquin staining of normal pancreas and adenocarcinoma tissue sections, we show for the first time, a consistent major loss of zinc in ductal and acinar epithelium in adenocarcinoma compared to the normal epithelium. This decrease in zinc is evident in well-differentiated through poorly-differentiated stages of malignancy. Immunohistochemistry identified ZIP3 as the basilar membrane zinc uptake transporter in normal ductal/acinar epithelium; and that the transporter is absent in adenocarcinoma. In situ Rt-PCR revealed that ZIP3 gene expression is silenced in adenocarcinoma. The ZIP3 down regulation accompanied the loss of zinc in early and progressing malignancy. RREB1 transcription factor was down regulated along with ZIP3; and might be involved in the silencing of ZIP3 expression. Zinc treatment was cytotoxic to malignant Panc1 cells. The combination of concurrent zinc, ZIP3, and RREB-1 changes represent early events in the development of adenocarcinoma; and suggest that zinc might be a tumor suppressor of pancreatic cancer. This report provides the clinical foundation for further mechanistic studies that will provide important insight into pancreatic carcinogenesis, and can lead to the development of effective early biomarkers and effective therapeutic agents for pancreatic cancer.

Background: Normal prostate accumulates extremely high levels of zinc compared to other soft tissues. In contrast, the level of zinc in the prostate decreases significantly in prostate cancer. We have shown that down-regulation of the expression of the zinc transporter hZIP1 in prostate cancer is an important event that is responsible for the decrease of zinc levels. However, the mechanism of hZIP1 down-regulation is not known. We have hypothesized that hZIP1 is down-regulated through transcriptional regulation., Methods: The hZIP1 promoter was studied using luciferase reporter assays, site-directed mutagenesis, gel shift, and ChIP assay., Results: We have characterized a promoter region, downstream of the transcription start site, responsible for repression of hZIP1 transcription. We demonstrate that this region contains a binding site for the Ras-Responsive Element Binding protein 1 (RREB-1) and that the binding of RREB-1 to the hZIP1 promoter is involved in the decrease of hZIP1 transcription in PC-3 cells., Conclusion: The Ras pathway and activation of RREB-1 are involved in hZIP1 down-regulation and may play a role in the decrease of the transporter expression in prostate cancer., (Copyright 2009 Wiley-Liss, Inc.)

Background: The normal human prostate glandular epithelium has the unique function of accumulating high levels of zinc. In prostate cancer this capability is lost as an early event in the development of the malignant cells. The mechanism and factors responsible for the ability of the normal epithelial cells to accumulate zinc and the loss of this capability in the malignant cells need to be identified. We previously reported that Zip1 is an important zinc uptake transporter in prostate cells and is down regulated in the malignant cells in situ along with the depletion of zinc levels. In this report we investigated the expression of two other Zip family zinc transporters, Zip2 and Zip3 in malignant versus nonmalignant (normal and BPH) glands. Zip2 and Zip3 relative protein levels were determined by immunohistochemistry analysis of human prostate tissue sections., Results: Normal and BPH glandular epithelium consistently exhibited the strong presence of both Zip 2 and Zip3; whereas both transporters consistently were essentially non-detectable in the malignant glands. This represents the first report of the expression of Zip3 in human prostate tissue; and more importantly, reveals that ZiP2 and Zip3 are down regulated in malignant cells in situ as we also had demonstrated for Zip1. Zip2 and Zip3 transporter proteins were localized predominantly at the apical cell membrane, which is in contrast to the Zip1 localization at the basolateral membrane. Zip2 and Zip3 seemingly are associated with the re-uptake of zinc from prostatic fluid., Conclusion: These results coupled with previous reports implicate Zip2 and Zip3 along with Zip1 as important zinc uptake transporters involved in the unique ability of prostate cells to accumulate high cellular zinc levels. Zip1 is important for the extraction of zinc from circulation as the primary source of cellular zinc. Zip 2 and Zip3 appear to be important for retention of the zinc in the cellular compartment. The down regulation of all three transporters in the malignant cells is consistent with the loss of zinc accumulation in these cells. Since zinc imposes tumor suppressor effects, the silencing of the gene expression for these transporters is a required event for the manifestation of the malignant activities of the neoplastic cells. This now provides new insights into the genetic/molecular events associated with the development of prostate cancer; and supports our concept of Zip1, and now Zip2 and Zip3, as tumor suppressor genes and zinc as a tumor suppressor agent.

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H(158)WHD(161). To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.

Zinc is an essential trace element that is involved in diverse metabolic and signaling pathways. Zinc deficiency is associated with retardation of bone growth. Previous in vitro studies have suggested a direct effect of zinc on both the proliferation and differentiation of osteoblast-like cells. However, the mechanisms for uptake of zinc into osteoblasts have not been examined in detail. Several families of zinc transporters have previously been characterized in mammalian cells; such transporters function in the uptake, intracellular sequestration or efflux of zinc. In the current study, we examined zinc transport in osteoprogenitor cells and have attempted to define a functional role for a zinc transport mechanism in osteogenic differentiation. We identified at least two zinc transporters in both human mesenchymal stem cells (MSCs) and in osteoblastic cells--the ubiquitous zinc transporter, ZIP1, and LIV-1, which was previously characterized as a protein that is expressed in breast cancer cells. The subcellular localization of both these zinc transporters suggested distribution in both the plasma membrane and also diffusely in the cytoplasm. During the differentiation process of pluripotent MSCs into osteoblast-like cells, both zinc uptake and expression of the ZIP1 protein were increased. An adenoviral-mediated overexpression of ZIP1 in MSCs resulted in Alizarin-red-positive mineralization and also increased expression of specific osteoblast-associated markers, such as alkaline phosphatase, and of several osteoblast differentiation genes, including osteopontin, Cbfa1/Runx2, promyelocytic leukemia zinc finger and bone sialoprotein. An siRNA-mediated reduction of ZIP1 protein expression in MSCs caused decreased zinc uptake and inhibition of osteoblastic differentiation under osteogenic culture conditions. Finally, following overexpression of ZIP1 in MSCs, cDNA microarray analysis revealed differential regulation of several genes associated with the proliferation of osteoprogenitor cells and osteoblast differentiation. In conclusion, these studies provide important insights into the role of a plasma membrane zinc transporter in the initiation of an osteogenic lineage from MSCs.

Background: The genetic and molecular mechanisms responsible for and associated with the development and progression of prostate malignancy are largely unidentified. The peripheral zone is the major region of the human prostate gland where malignancy develops. The normal peripheral zone glandular epithelium has the unique function of accumulating high levels of zinc. In contrast, the ability to accumulate zinc is lost in the malignant cells. The lost ability of the neoplastic epithelial cells to accumulate zinc is a consistent factor in their development of malignancy. Recent studies identified ZIP1 (SLC39A1) as an important zinc transporter involved in zinc accumulation in prostate cells. Therefore, we investigated the possibility that down-regulation of hZIP1 gene expression might be involved in the inability of malignant prostate cells to accumulate zinc. To address this issue, the expression of hZIP1 and the depletion of zinc in malignant versus non-malignant prostate glands of prostate cancer tissue sections were analyzed. hZIP1 expression was also determined in malignant prostate cell lines., Results: hZIP1 gene expression, ZIP1 transporter protein, and cellular zinc were prominent in normal peripheral zone glandular epithelium and in benign hyperplastic glands (also zinc accumulating glands). In contrast, hZIP1 gene expression and transporter protein were markedly down-regulated and zinc was depleted in adenocarcinomatous glands and in prostate intra-epithelial neoplastic foci (PIN). These changes occur early in malignancy and are sustained during its progression in the peripheral zone. hZIP1 is also expressed in the malignant cell lines LNCaP, PC-3, DU-145; and in the nonmalignant cell lines HPr-1 and BPH-1., Conclusion: The studies clearly establish that hZIP1 gene expression is down regulated and zinc is depleted in adenocarcinomatous glands. The fact that all the malignant cell lines express hZIP1 indicates that the down-regulation in adenocarcinomatous glands is likely due to in situ gene silencing. These observations, coupled with the numerous and consistent reports of loss of zinc accumulation in malignant cells in prostate cancer, lead to the plausible proposal that down regulation of hZIP1 is a critical early event in the development prostate cancer.