Aberrant expression of zinc transporter ZIP4 (SLC39A4) significantly contributes to human pancreatic cancer pathogenesis and progression

Abstract

Zinc is an essential trace element and catalytic/structural component used by many metalloenzymes and transcription factors. Recent studies indicate a possible correlation of zinc levels with the cancer risk; however, the exact role of zinc and zinc transporters in cancer progression is unknown. We have observed that a zinc transporter, ZIP4 (SLC39A4), was substantially overexpressed in 16 of 17 (94%) clinical pancreatic adenocarcinoma specimens compared with the surrounding normal tissues, and ZIP4 mRNA expression was significantly higher in human pancreatic cancer cells than human pancreatic ductal epithelium (HPDE) cells. This indicates that aberrant ZIP4 up-regulation may contribute to the pancreatic cancer pathogenesis and progression. We studied the effects of ZIP4 overexpression in pancreatic cancer cell proliferation in vitro and pancreatic cancer progression in vivo. We found that forced expression of ZIP4 increased intracellular zinc levels, increased cell proliferation by 2-fold in vitro, and significantly increased tumor volume by 13-fold in the nude mice model with s.c. xenograft compared with the control cells. In the orthotopic nude mice model, overexpression of ZIP4 not only increased the primary tumor weight (7.2-fold), it also increased the peritoneal dissemination and ascites incidence. Moreover, increased cell proliferation and higher zinc content were also observed in the tumor tissues that overexpressed ZIP4. These data reveal an important outcome of aberrant ZIP4 expression in contributing to pancreatic cancer pathogenesis and progression. It may suggest a therapeutic strategy whereby ZIP4 is targeted to control pancreatic cancer growth.

Fig. 1.

ZIP4 expression in human pancreatic cancer tissue specimens and cell lines. (A) cDNA microarray analysis was done by using Affymetrix chips containing 6,800 genes. Microdissected samples from pancreatic adenocarcinoma (10), chronic pancreatitis (5), and normal pancreas (5) were analyzed. *, P < 0.05. (B) Validation of microarray results of ZIP4 mRNA levels in 17 patients. Total RNA was extracted from tissues, and the mRNA levels for ZIP4 were analyzed by real-time PCR and normalized to that of the house keeping gene, β-actin. Relative mRNA level is presented as fold increase for both tumor and normal samples. All data shown are the means ± SD of three separate experiments. *, P < 0.05. (C) Immunohistochemical staining of ZIP4 expression in human pancreatic cancer tissue and its normal surrounding tissue. Dark brown color represents positive staining of ZIP4. Arrowheads, normal ductal epithelial cells; arrows, tumor epithelial cells. (D) Expression of ZIP4 in human pancreatic cancer cell lines by real-time RT-PCR analysis. The mRNA levels of ZIP4 in eight human pancreatic cancer cell lines were examined by real-time RT-PCR as above. All data shown are the means ± SD of three separate experiments.

Fig. 2.

Effect of ZIP4 on MIA CaPa-2 cell proliferation. (A) Overexpression of ZIP4 mRNA in MIA PaCa-2 cells. mRNA levels in MIA-V and MIA-ZIP4 cells were examined with real-time RT-PCR. Human ZIP4 mRNA levels were normalized to that of human β-actin. ZIP4 mRNA levels in MIA-ZIP4 cells were significantly higher than that in MIA-V cells. *, P < 0.01. (B) Overexpression of ZIP4 protein in MIA PaCa-2 cells detected by Western blot. Specific anti-ZIP4 Ab (1:500) was used to probe the protein bands. (C) Zinc concentration in MIA PaCa-2 cells. MIA-V and MIA-ZIP4 cells were treated with 4 μM TPEN for 1 h at 37°C and then incubated with DMEM in the presence of 10 μM ZnCl₂ for 5 min before collection. The zinc concentration was examined by ICPMS. *, P < 0.05. (D) MTS assay. MIA-V and MIA-ZIP4 cells were seeded in 96-well plates (2 × 10³ cells per well) and serum-starved for 24 h before examining the cell proliferation. Absorbance at 490 nm was recorded daily until day 5 after starvation. Data were expressed as the means ± SD of triplicate values. *, P < 0.05. (E) Zinc-dependent assay. MIA-V and MIA-ZIP4 cells in 96-well plates (2 × 10³ cells per well) were treated with 4 μM TPEN for 1 h at 37°C and then incubated with DMEM in the presence of 0, 1, 5, 25, 50, and 125 μM ZnCl₂. Absorbance at 490 nm was recorded at day 1 after treatment. Data were expressed as the means ± SD of triplicate values. *, P < 0.05.

Fig. 3.

Effects of ZIP4 on pancreatic cancer growth in the nude mouse model of s.c. xenograft. (A) MIA-ZIP4 or MIA-V cells (3 × 10⁶) were s.c. inoculated into the right flank of nude mice (n = 10 per treatment group). Tumor size was measured weekly for 6 weeks. Tumor volume was calculated by the formula: tumor volume [mm³] = (length [mm]) × (width [mm])² × 0.52. *, P < 0.01. A representative s.c. tumor mass from each group was shown in the Insets. (B) The s.c. tumors were removed and processed for immunohistochemistry analysis. A monoclonal Ab against Ki67 was used to stain the tissue slides from MIA-V and MIA-ZIP4 groups. s.c. tumors from MIA-ZIP4 group showed much increased cell proliferation by Ki67 staining compared with that of the MIA-V mice. (C) Zinc concentration in nude mouse s.c. tumors. The s.c. tumors were removed and homogenized for zinc detection with ICPMS. The average zinc concentration in the MIA-V group and MIA-ZIP4 group was presented. *, P < 0.05.

Fig. 4.

Effects of ZIP4 on pancreatic tumor progression in the nude mouse model of orthotopic xenograft. (A) Tumor weight. MIA-ZIP4 or MIA-V cells (3 × 10⁶) were orthotopically inoculated into the pancreas of nude mice (n = 5 per treatment group). Tumor weight was measured after the mice were euthanized at 7 weeks. *, P < 0.01. A representative orthotopic tumor mass from each group was shown in the Insets. (B) Primary tumors. The orthotopic primary tumors were removed, and a representative picture from each group was shown. (C) Picture of gross appearance. Pictures of the gross appearance and the primary tumors of each group were shown. (D) Zinc concentration in nude mouse primary pancreas tumors. The orthotopic pancreas tumors were removed and homogenized for zinc detection with ICPMS. The average zinc concentration in the MIA-V group and MIA-ZIP4 group was presented. *, P < 0.05.