Zinc and its transporter ZIP10 are involved in invasive behavior of breast cancer cells

Blackwell Publishing Asia

Naofumi Kagara,1,2 Natsumi Tanaka,1 Shinzaburo Noguchi2 and Toshio Hirano1,3,4

1 Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences, and Graduate School of Medicine, and 2Department of Breast and Endocrine Surgery, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka, 565-0871; 3Laboratory for Cytokine Signaling, RIKEN Research Center for Allergy and Immunology, (RCAI), 1-7-22, Suehiro, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan

(Received November 13, 2006/Revised December 22, 2006/Accepted December 25, 2006/Online publication March 16, 2007)

Zinc is an essential element, necessary for sustaining all life. Zinc deficiency causes taste impairments, immune deficiency, skin problems, and growth and mental retardation. Recent reports suggest that zinc is associated with an increased risk of cancer, although it is still unclear whether zinc or its transporters are involved in cancer progression. Here we show that zinc and its transporter ZIP10 are involved in the invasive behavior of breast cancer cells. The screening of clinical samples for ZIP10 mRNA expression suggested that ZIP10 was significantly associated with the metastasis of breast cancer to the lymph node. In addition, the expression of ZIP10 mRNA was higher in the invasive and metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435S than in less metastatic breast cancer cell lines, such as MCF7, T47D, ZR75-1 and ZR75-30. In in vitro cell migration assays, the depletion of zinc transporter ZIP10 and intracellular zinc inhibited the migratory activity of MDA-MB-231 and MDA-MB-435S cells. These results showed that zinc and ZIP10 play an essential role in the migratory activity of highly metastatic breast cancer cells, and suggest ZIP10 as a possible marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies. (Cancer Sci 2007; 98: 692697)

Here we demonstrate that the migratory activity of metastatic breast cancer cells was inhibited by ZIP10 knockdown and by zinc chelation. Analysis of clinical samples showed that breast cancers with lymph-node metastasis had signicantly higher ZIP10 zinc transporter expression than those without lymph-node metastasis. ZIP10 was recently shown to mediate zinc uptake and to act as a membrane transporter in vivo,(17) but this is the rst report describing a biological role for the ZIP10 zinc transporter in cancer progression. We also propose elevated ZIP10 as a candidate marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies.
Materials and Methods
Clinical samples. The breast cancer samples used in the present study were obtained through a protocol approved by the Institutional Review Board, from 177 primary breast cancer patients who underwent breast-conserving surgery or mastectomy at the Department of Breast and Endocrine Surgery, Osaka University, from February 1998 to April 2005. The median age of patients was 54 years (range 28 87 years). RNA extraction and reverse transcription. Total cellular RNA was extracted from frozen tumor specimens using TRIzol reagent, according to the protocol provided by the manufacturer (Molecular Research Center, Cincinnati, OH, USA). Three micrograms of each total RNA sample was used for cDNA synthesis by Superscript II (Life Technologies, Rockville, MD, USA), with Oligo-(dT)15 as the primer, and reaction conditions of 42C for 90 min followed by heating at 70C for 10 min. Real-time polymerase chain reaction. Real-time polymerase chain reactions (PCR) were carried out using the ABI Prism 9700 Sequence Detector System (Perkin-Elmer Applied Biosystems, Foster City, CA, USA). For quantication, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts served as the quantitative control, and each sample was normalized to its GAPDH transcript level. The primer and probe mixtures for ZIP6, ZIP10 and GAPDH were purchased from Perkin-Elmer Applied Biosystems, and the PCR method was as recommended in the manufacturers protocol. Cell culture. Human breast cancer cell lines were obtained from American Type Culture Collection, Manassas, VA, USA. MDAMB-231 and MDA-MBN-435S cells were maintained in Dulbeccos modied Eagles medium (DMEM), and MCF-7, T47D, ZR-75-1 and ZR-75-30 cells were maintained in RPMI supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 g/mL streptomycin. Conventional PCR. Conventional PCR amplication was carried out at 95C for 1 min, followed by 25 cycles of 95C for 30 s, 55C for 30 s, and 72C for 1 min. The primers used for PCR

inc is an essential heavy metal and a constituent of a great number of zinc metalloproteins in the human body.(1,2) It is important in nucleic acid metabolism, cell replication, and tissue repair and growth, and it is required for tumor growth. Zinc deciency is associated with diverse disorders, including impaired immunity, retarded growth, brain development disorders and delayed wound healing. Many reports have suggested an involvement of zinc in cancer development, and shown that the levels of zinc in the serum and malignant tissues decreases in patients with various malignancies, such as carcinoma of the liver, gallbladder, digestive tracts or prostate.(37) In contrast with these observations, in breast cancer patients the zinc levels are lower in the serum and elevated in malignant tissues.(3,6,8) In fact, studies of the role of zinc in malignant diseases have a long history of reporting contradictory and ill-dened biological processes.(9) Eukaryotic cells have a remarkable ability to regulate their levels of intracellular zinc. Although cells are exposed to micromolar ranges of free zinc, the intracellular levels of free zinc that regulate the transcription of zinc inux, efux or sequestration machinery are in the femtomolar range. Several proteins, including the ZRT IRT-like protein (ZIP) family zinc transporters, zinc transporter (ZnT) family zinc transporters and zinc-sequestering metallothioneins, maintain the intracellular zinc homeostasis.(1013) There are at least 14 human ZIP transporters, and they are believed to function in zinc inux into the cytosol.(10) Some ZIP family members are reported to be involved in cancer progression: ZIP1 is reported to be a suppressor gene for prostate cancer (14) and ZIP6/LIV1 to be involved in the metastasis of clinical breast cancer to the lymph node,(15) although this issue is controversial.(16) Nevertheless, the precise effects of human ZIP on tumor development and progression remain elusive.
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To whom correspondence should be addressed. E-mail: hirano@molonc.med.osaka-u.ac.jp

doi: 10.1111/j.1349-7006.2007.00446.x 2007 Japanese Cancer Association

were: ZIP6, 5-TCTGTCACAAATCCCCTTCA-3 (sense), 5-GGAGGGCTCTTGTGAGTCTG-3 (antisense); ZIP10, 5-CCTGGTTCCTGAAGATGAGG-3 (sense), 5-CATGGCAGAGAGGAGGTTGT-3 (antisense); E-CADHERIN, 5-GTCATTGAGCCTGGCAATTT-3 (sense), 5-GCTTGAACTGCCGAAAAATC-3 (antisense); VIMENTIN, 5-CCTTGAACGCAAAGTGGAAT-3 (sense), 5-GCTTCAACGGCAAAGTTCTC-3 (antisense); GAPDH, 5-TGAAGGTCGGAGTCAACGGAT-3 (sense), 5-CATGTGGGCCATGAGGTCCAC-3 (antisense). RNA interference. The small interfering RNA (siRNA) for human ZIP6, human ZIP10 and the control (siCONTROL non-targeting siRNA #1) were obtained from Dharmacon Inc., Chicago, IL, USA. The target sequences of the siRNA for human ZIP6 were 5-GAAGUUAUCUGUAAUCUUGUU-3, 5-GAAGUGACCUCAACUGUGUUU-3, 5-UGAAGGAACUCACUUUCUAUU3 and 5-UGACUUUGCUGUUCUACUAUU-3. For human ZIP10 they were 5-GAACGUCACUCAGUUAUUAUU-3, 5GGAAGAAUAUGAUGCUGUAUU-3, 5-CCACAAACCUGAUCGUGUAUU-3 and 5-GAAAGGACUUGUUGCUCUAUU-3. Cells were treated with a mixture of all four oligonucleotides, following the manufacturers protocol, 24 h before the zinc uptake or cell migration assays. Zinc assays. To chelate intracellular Zn, Cu, Fe, or Mn, 10 M N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; Molecular Probes, Eugene, OR, USA) 10 M disodium bathocuproine disulfonate (BCDS; Sigma-Aldrich, St. Louis, MO, USA), 10 M 2,2-dipyridyl disulfonate (Sigma-Aldrich, St. Louis, MO, USA), or 10 M sodium para-aminosalicylic acid disulfonate (PAS; Sigma-Aldrich), respectively, was added to the culture medium 24 h prior to observation. To rescue the zinc level, 5 M ZnCl2 or 1 M pyrithione was added to the TPENcontaining medium. For the 65Zn uptake assays, the cells were grown to 50% conuence, incubated at 37C for the indicated times in DMEM and 0.1 M 65ZnCl2 (Oak Ridge National Laboratory, Oak Ridge, IN, USA), washed three times with phosphatebuffered saline, and then harvested. Cell-associated radioactivity was measured with a Packard Auto-Gamma 5650 counter.
In vitro transwell migration assay and time-lapse imaging.

Table 1. Relationship between the clinicopathological parameters and ZIP6 or ZIP10 mRNA expression in breast cancer tissues Parameter Tumor size 2 cm >2 cm Unknown Histological grade I, II III Unknown Lymph node metastasis Negative Positive Unknown Estrogen receptor Positive Negative Unknown Progesterone receptor Positive Negative Unknown HER2 status Negative Positive Unknown Total n = 177 66 109 2 131 38 8 92 85 0 104 68 5 84 88 5 81 17 79 ZIP6 mean SD ( 103) 6.43 9.61 6.63 8.85 ZIP10 mean SD ( 105) 4.76 5.26 6.63 5.56

7.82 9.88* 1.88 3.73

6.42 5.56 4.20 5.51

6.44 9.32 6.55 8.87

4.51 4.87*** 7.29 5.87

8.51 9.28** 3.03 6.64

6.55 5.59 4.67 5.34

7.85 8.21 4.91 9.03

6.76 5.90 4.89 5.07

7.19 9.82** 1.81 2.83

5.18 5.06 4.01 4.11

Age range: 2887 years (mean = 54 years). *P < 0.00001, **P < 0.0001, ***P < 0.001 (Students t-test).

In vitro transwell migration assays were carried out using 5.0m pore size Costar Transwell inserts (Corning Inc., Lowell, MA, USA), according to the manufacturers protocol. To each well was added 2 104 cells, which were incubated in the presence of 10% FBS for 24 h. Cells that migrated to the bottom of the transwell membrane were xed with methanol, stained with hematoxylin and eosin, and counted under a microscope. Time-lapse images were obtained every 20 min under the indicated conditions. Migration rates (mean 1 SD [m/min]) were calculated from 20 independent cells in each experiment. Statistical analysis. Differences in expression levels of ZIP6 and ZIP10 mRNA between various subgroups were evaluated using Students t-test.
Results
Correlation of high ZIP10 mRNA expression with lymph node metastasis in clinical breast cancers. We investigated whether ZIP6

Fig. 1. mRNA expression of ZIP6 and ZIP10 in clinical breast cancers. The mRNA expression of (a) ZIP6 and (b) ZIP10 was evaluated by quantitative real-time polymerase chain reaction analysis in lymph-node metastasis-negative (LN), and -positive (LN+) breast cancers. The bar indicates the mean value.

and ZIP10 expression correlated with certain clinicopathological phenotypes of clinical breast tumor samples by real-time PCR (Table 1; Fig. 1). We observed a signicantly higher level of ZIP10 transcript in the lymph node metastasis-positive group compared with the metastasis-negative group (P = 0.00080) (Table 1; Fig. 1b), whereas no signicant difference in the ZIP6 transcript was seen (P = 0.94) (Fig. 1a). High ZIP6 mRNA expression was associated with better prognostic parameters, such as better histological grade (I or II) (P < 0.00001), estrogen receptor positivity (P < 0.0001) and HER2 negativity (P < 0.0001) (Table 1), consistent with previous ndings that ZIP6 expression is estrogen regulated and not associated with malignant phenotypes in
Kagara et al.

clinical breast cancer.(16) These clinical ndings suggested that the zinc transporter ZIP10 is more likely than ZIP6 to correlate with cancer invasion and metastasis.
Correlation of high ZIP10 mRNA expression with the invasive phenotype of breast cancer cell lines. To test whether zinc transporters

are involved in the metastatic phenotype of breast cancer cells, we analyzed the mRNA expression of the ZIP (ZIP114), which mediate zinc inux into the cytosol(10) in several breast cancer cell lines. These included the invasive and metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435S, which express low E-cadherin and high vimentin, and the less metastatic
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Fig. 2. mRNA expression of ZIP10 in breast cancer cell lines. (a) mRNA expression of ZIP6, ZIP10, E-CADHERIN, and VIMENTIN was evaluated by semiquantitative reverse transcription polymerase chain reaction (PCR) in less metastatic breast cancer cell lines (MCF7, T47-D, ZR75-1, ZR75-30) and highly metastatic lines (MDA-MB-231 and MDA-MB435S), and (b) the relative mRNA expression of ZIP10 in these six breast cancer cell lines was evaluated by real-time PCR.

breast cancer cell lines MCF-7, T-47D, ZR-75-1 and ZR-75-30. Among the ZIP zinc transporters we screened, the expression of ZIP10 was relatively upregulated in the highly metastatic cell-lines MDA-MB-231 and MDA-MB-435S (Fig. 2a), which we conrmed by real-time PCR (Fig. 2b), whereas the expression of ZIP6 was not. These results also suggest that ZIP10 may be a major ZIP that regulates human breast cancer progression.
ZIP10 is essential for the invasive behavior of MDA-MB-231 and MDA-MB-435S breast cancer cells in vitro. To examine whether

Fig. 3. ZIP10 mRNA expression and 65Zn uptake of MDA-MB-231 and MDA-MB-435S cells expressing control, ZIP6, ZIP10 or ZIP6/ZIP10 small interfering RNA (siRNA). (a) Semiquantitative reverse transcription polymerase chain reaction (PCR) and (b) real-time PCR showed the knockdown effect of ZIP6 siRNA and/or ZIP10 siRNA. (c) Relative zinc uptake activity was evaluated by the counts per minute (CPM) of cells incubated with the 65Zn isotope. Each figure shows a representative result of at least three experiments.

ZIP6 or ZIP10 is involved in breast cancer progression, we generated ZIP6-, ZIP10- and ZIP6/10-knockdowns and control MDA-MB-231 and MDA-MB-435S cells using siRNA for human ZIP6, human ZIP10 and the control. Reverse transcriptionPCR and real-time PCR analyses of these MDAMB-231 and MDA-MB-435S knockdown cells showed that mRNA expression of ZIP6 and ZIP10 was specically depleted by each siRNA in both cell types (Fig. 3a,b). In a transwell migration assay, the control and ZIP6-knockdown MDA-MB-231 cells exhibited high migration activity, which was inhibited by ZIP10 or ZIP6/10 depletion (Fig. 4a). Time-lapse analysis of these cells also highlighted their requirement for ZIP10 in their migratory behavior (Fig. 4c,d). Control and ZIP6-knockdown MDA-MB-231 cells showed active migration, reecting their
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invasive property, whereas ZIP10- and ZIP6/10-knockdown MDA-MB-231 cells exhibited poor migratory activity. Similar results were obtained using MDA-MB-435S cells (Fig. 4a,c,d). These results indicated that ZIP10, but not ZIP6, is involved in the migratory activity of these breast cancer cells, and suggested that zinc transported via ZIP10 has essential regulatory functions in this activity.
Zinc is essential for the invasive behavior of MDA-MB-231 and MDA-MB-435S breast cancer cells in vitro. Because ZIP6 and ZIP10

belong to the ZIP zinc-transporter family, whose members are thought to mediate zinc inux into the cytosol, we carried out a 65Zn uptake assay using the knockdown cells. ZIP10- and ZIP6/10-knockdown MDA-MB-231 or MDA-MB-435S cells incorporated signicantly less 65Zn than their respective controls and ZIP6-knockdown cells (Fig. 3c). This indicated that ZIP10 was involved in the zinc uptake of MDA-MB-231 or MDA-MB435S breast cancer cells, whereas ZIP6-knockdown had no signicant effect on zinc uptake in these cells, and suggested that zinc transported by ZIP10 might be involved in the migratory activity of these cells. To examine whether zinc is
doi: 10.1111/j.1349-7006.2007.00446.x 2007 Japanese Cancer Association

Fig. 4. The effects of ZIP10 knockdown on cancer cell migration. (a) MDA-MB-231 and MDA-MB435S cells expressing control, ZIP6, ZIP10 or ZIP6/ ZIP10 small interfering RNA (siRNA) were incubated with or without 5 M ZnCl2 (Zn2+) or 1 M zinc pyrithione (pyr), for 24 h during in vitro transwell migration assays. (b) MDA-MB-231 and MDA-MB435S cells were incubated with dimethylsulfoxide (control), 10 M TPEN, 10 M TPEN + 5 M ZnCl2 (Zn2+), 10 M disodium bathocuproine disulfonate (BCDS), 10 M 2,2-dipyridyl, or 10 M sodium paraaminosalicylic acid (PAS) for 24 h during in vitro transwell migration assays. Error bars represent the standard deviations for three replicates. (c) The migratory tracks of individual cells were obtained by time-lapse imaging and (d) the migration rate (m/min) over a 24-h incubation period was calculated. Cells expressing control, ZIP6, ZIP10 and ZIP6/10 siRNA were incubated without TPEN, and those expressing control siRNA were incubated with TPEN. Three views of representative migratory tracks from each condition are shown in panel (c). Lines and asterisks represent the tracks and the starting points of cell movement, respectively. Error bars represent the standard deviations for 20 cells (d).

involved in breast cancer cell migration, we carried out in vitro migration assays using MDA-MB-231 and MDA-MB-435S cells treated with the membrane-permeable zinc chelator TPEN. In in vitro migration assays, TPEN inhibited the migration of MDA-MB-231 cells (Fig. 4b,c,d). This inhibition was neutralized by the addition of free zinc ion in the culture medium (Fig. 4b), indicating that the effect of TPEN was simply due to its chelation activity, not due to the other toxicities of the reagent. The chelation of intracellular Cu, Fe and Mn by 10 M disodium bathocuproine disulfonate, 10 M 2,2-dipyridyl and 10 M sodium para-aminosalicylic acid, respectively, did not inhibit migratory activity of MDA-MB-231 cells (Fig. 4b). Similar results were obtained using the MDA-MB-435S cells (Fig. 4a,c,d). Thus, intracellular zinc has essential functions in the migratory activity of these breast cancer cells. In addition, the defect in the migratory activity of the ZIP10-knockdown MDA-MB-231 and MDA-MB-435S cells could not be rescued by raising the level of intracellular free zinc ion using ZnCl2 with or without zinc ionophore pyrithione (Fig. 4a), suggesting that zinc transported by ZIP10, but not intracellular free zinc ion, was necessary for breast cancer cell migration. Together, these
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results indicate that the zinc transporter ZIP10 is signicantly involved in the migratory activity of MDA-MB-231 and MDAMB-435S cells, and that the zinc transported via ZIP10 has essential functions in the migratory behavior of these breast cancer cells.
Discussion

Previous studies have indicated that zinc is related to the progression of cancers,(18) including carcinoma of the prostate(4) and liver.(7) The involvement of some kinds of zinc transporters in cancer metastasis, such as the lymph node metastasis of breast cancers, has also been reported.(15) Our experiments established that zinc transported via ZIP10 but not ZIP6 is involved in the invasive behavior of breast cancer cells. Four lines of evidence support this idea. First, the depletion of intracellular zinc and ZIP10 in invasive breast cancer cells caused similar defects in the migratory activity of these cells. Second, the knockdown of ZIP10 in invasive breast cancer cells caused the downregulation of their zinc-uptake activity. Third, the expression level of ZIP10 mRNA in breast cancer
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cell lines was correlated with their invasiveness. Finally, the expression of ZIP10 mRNA in clinical breast tumor samples was correlated with metastasis to lymph nodes. Many previous reports have shown that zinc levels are higher in breast cancer than in normal breast tissue,(6,8,9,19) and that breast cancer tissue has a signicantly high uptake of zinc.(20) In contrast, the serum zinc level is lower in patients with advanced breast cancers, leading to the idea that measuring the serum zinc levels could be useful for estimating the extent and prognosis of malignant breast diseases.(3,21,22) In our study, highly metastatic and advanced breast cancers showed higher expression of the ZIP10 zinc transporter than non-metastatic breast cancers. ZIP10 functions as a zinc importer, so our data suggest that more advanced breast cancers, which express high levels of ZIP10, can import larger amounts of zinc into the cytosol from the serum. This is consistent with previous observations of a high zinc level in advanced breast cancer tissue and a corresponding low level in serum. It is possible that highly metastatic and advanced breast cancers take in more zinc at least in part via the ZIP10 transporter, thereby acquiring greater migratory activities. Our experiments showed that impaired migratory activity by ZIP10 suppression was not recoverable by increasing the intracellular free zinc ion level. This indicates that not free zinc ion but only the zinc transported via ZIP10 is essential for the increased migratory activity of breast cancer cells. Considering that intracellular free zinc ion is quite a little and almost all intracellular zinc exists in a form bound to zinc-containing molecules, our results suggest that there might be a specic chaperon molecule that transfers zinc imported through each zinc transporter to its functional target molecule, which requires zinc to regulate cell migration activities. This might be one of the mechanisms underlying the functional difference between ZIP10 and ZIP6 in cancer migratory activity. Our study demonstrated an involvement of ZIP10 in human breast cancer progression and invasion. However, previous reports have shown that the ZIP6 LIV1 zinc transporter is involved in breast cancer metastasis to the lymph node.(15) ZIP10 and ZIP6 are most closely related to each other in the ZIP zinc transporter family, based on amino acid sequence,(23) indicating that they may have similar functions in regulating cell migration. However, our present study clearly showed that ZIP6 has no signicant role in zinc uptake and cell migration of breast cancer cells, even though the basal expression level of ZIP6 is much higher than that of ZIP10. In addition, our clinicopathological analysis also indicated that high ZIP6 mRNA expression is associated with better prognostic parameters, which is consistent with a recent report about ZIP6.(16) Thus we think that ZIP10, but not ZIP6, plays a major role in breast cancer progression. At the moment, we do not know the reason why there is a functional difference between ZIP10 and ZIP6. This issue remains to be claried.

Our present data showed that ZIP10 expression had close correlations with epithelialmesenchymal transition (EMT) markers such as E-cadherin and vimentin, whereas ZIP6 expression did not. This suggested the involvement of ZIP10 in EMT, but the suppression of ZIP10 changed neither the morphologies nor the EMT markers of MDA-MB-231 or MDA-MB-435S cells (data not shown). Although we previously reported the involvement of zinc transporter ZIP6 in EMT during gastrulation of zebrash,(24) the similar involvement of zinc transporter ZIP10 in EMT of human breast cancer was not shown, at least in the two breast cancer cell lines we examined. This may be due to the difference between normal cells and cancer cells. Furthermore, this suggested that ZIP10 regulates cancer cell migratory activities by other mechanisms. To clarify this issue, we investigated the expression of cell adhesion molecules, but we found no signicant effects of ZIP10 knockdown on the levels of extracellular matrix proteins such as bronectin and vitronectin, their receptors, including integrin family members 15, V, and 1, 3 and 5, or integrin-binding proteins, such as -catenin, vinculin and talin (data not shown). In the meantime, zinc involvement in cytoskeleton-dependent cellular phenomena have been described, in the Fc-epsilon receptor I (RI)-induced translocation of granules in mast cell degranulation,(25) and the lipopolysaccharide (LPS)-induced upregulation of major histocompatibility complex class II and costimulatory molecules in dendritic cells. (26) These studies support our idea that zinc is probably involved in the cytoskeletal reorganization of breast cancer cells, although this issue remains to be claried. Further investigations are needed to elucidate the novel mechanisms behind zinc- and ZIP10-dependent cancer cell migration. Two recent reports demonstrate the zinc-uptake activity of ZIP10 and its metal-regulatory transcription factor 1 (MTF-1)dependent expression; however, neither report refers to the biological functions of ZIP10.(17,27) The present study is the rst to demonstrate a role for zinc and ZIP10 in cancer development, although the elucidation of the precise molecular mechanisms of their involvement will require further study. Our data suggest that ZIP10 may be a candidate marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies, in addition to providing a new conceptual basis for understanding zinc-dependent signaling.
Acknowledgments
We thank S. Yamashita for his critical comments and helpful suggestions. We thank R. Masuda for secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology in Japan, and the Japan Society for the Promotion of Science Research Fellowship Division.

References
1 Vallee BL, Falchuk KH. The biochemical basis of zinc physiology. Physiol Rev 1993; 73: 79118. 2 Vallee BL, Auld DS. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 1990; 29: 564759. 3 Chakravarty PK, Ghosh A, Chowdhury JR. Zinc in human malignancies. Neoplasma 1986; 33: 8590. 4 Costello LC, Franklin RB. The clinical relevance of the metabolism of prostate cancer; zinc and tumor suppression: connecting the dots. Mol Cancer 2006; 5: 17. 5 Gupta SK, Singh SP, Shukla VK. Copper, zinc, and Cu/Zn ratio in carcinoma of the gallbladder. J Surg Oncol 2005; 91: 2048. 6 Margalioth EJ, Schenker JG, Chevion M. Copper and zinc levels in normal and malignant tissues. Cancer 1983; 52: 86872. 7 Tashiro H, Kawamoto T, Okubo T, Koide O. Variation in the distribution of trace elements in hepatoma. Biol Trace Elem Res 2003; 95: 4963. 8 Schwartz AE, Leddicotte GW, Fink RW, Friedman EW. Trace elements in normal and malignant human breast tissue. Surgery 1974; 76: 3259.

9 Mulay IL, Roy R, Knox BE, Suhr NH, Delaney WE. Trace-metal analysis of cancerous and noncancerous human tissues. J Natl Cancer Inst 1971; 47: 113. 10 Eide DJ. The SLC39 family of metal ion transporters. Pugers Arch 2004; 447: 796800. 11 Gaither LA, Eide DJ. Eukaryotic zinc transporters and their regulation. Biometals 2001; 14: 25170. 12 Liuzzi JP, Cousins RJ. Mammalian zinc transporters. Annu Rev Nutr 2004; 24: 15172. 13 Palmiter RD, Huang L. Efux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pugers Arch 2004; 447: 74451. 14 Franklin RB, Feng P, Milon B et al. hZIP1 zinc uptake transporter down regulation and zinc depletion in prostate cancer. Mol Cancer 2005; 4: 32. 15 Manning DL, Robertson JF, Ellis IO et al. Oestrogen-regulated genes in breast cancer: association of pLIV1 with lymph node involvement. Eur J Cancer 1994; 30A: 6758. 16 Kasper G, Weiser AA, Rump A et al. Expression levels of the putative zinc

696

doi: 10.1111/j.1349-7006.2007.00446.x 2007 Japanese Cancer Association

17 18 19 20 21

transporter LIV-1 are associated with a better outcome of breast cancer patients. Int J Cancer 2005; 117: 96173. Kaler P, Prasad R. Molecular cloning and functional characterization of novel zinc transporter, rZip10 (Slc39a10), involved in zinc uptake across rat renal brush border membrane. Am J Physiol Renal Physiol 2007; 292: F21729. Prasad AS, Kucuk O. Zinc in cancer prevention. Cancer Metastasis Rev 2002; 21: 2915. Rizk SL, Sky-Peck HH. Comparison between concentrations of trace elements in normal and neoplastic human breast tissue. Cancer Res 1984; 44: 53904. Tupper R, Watts RW, Wormall A. The incorporation of 65Zn in mammary tumours and some other tissues of mice after injection of the isotope. Biochem J 1955; 59: 2648. Gupta SK, Shukla VK, Vaidya MP et al. Serum trace elements and Cu/Zn ratio in breast cancer patients. J Surg Oncol 1991; 46: 17881.

22 Yucel I, Arpaci F, Ozet A et al. Serum copper and zinc levels and copper/ zinc ratio in patients with breast cancer. Biol Trace Elem Res 1994; 40: 318. 23 Kambe T, Yamaguchi-Iwai Y, Sasaki R, Nagao M. Overview of mammalian zinc transporters. Cell Mol Life Sci 2004; 61: 4968. 24 Yamashita S, Miyagi C, Fukada T, Kagara N, Che YS, Hirano T. Zinc transporter LIVI controls epithelialmesenchymal transition in zebrash gastrula organizer. Nature 2004; 429: 298302. 25 Kabu K, Yamasaki S, Kamimura D et al. Zinc is required for Fc epsilon RI-mediated mast cell activation. J Immunol 2006; 177: 1296305. 26 Kitamura H, Morikawa H, Kamon H et al. Toll-like receptor-mediated regulation of zinc homeostasis inuences dendritic cell function. Nat Immunol 2006; 7: 9717. 27 Wimmer U, Wang Y, Georgiev O, Schaffner W. Two major branches of anticadmium defense in the mouse: MTF-1/metallothioneins and glutathione. Nucl Acids Res 2005; 33: 571527.

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