The emerging role of the LIV-1 subfamily of zinc transporters in breast cancer - PubMed (original) (raw)

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The emerging role of the LIV-1 subfamily of zinc transporters in breast cancer

Kathryn M Taylor et al. Mol Med. 2007 Jul-Aug.

Abstract

Zinc transporter LIV-1 (SLC39A6) is estrogen regulated and present in increased amounts in estrogen receptor-positive breast cancer as well as in tumors that spread to the lymph nodes. The LIV-1 subfamily of ZIP zinc transporters consists of nine human sequences that share considerable homology across transmembrane domains. Many of these sequences have been shown to transport zinc and/or other ions across cell membranes. Increasingly, studies have implicated members of the LIV-1 transporter subfamily in a variety of diseases. We review these studies and report our own investigations of the role in breast cancer of the nine LIV-1 zinc transporters. We have documented the response of these transporters to estrogen and antiestrogens, and also their presence in our models of resistance to antiestrogens. Resistance to antiestrogen drugs such as tamoxifen and fulvestrant often occurs in advanced breast cancer. In these models we observed differential expression of individual LIV-1 family members, which may be related to their observed variable tissue expression. We were unable detect ZIP4, which is known to be expressed in the intestine. HKE4/SLC39A7 had elevated expression in both antiestrogen-resistant cell lines, and ZIP8 had elevated expression in fulvestrant-resistant cells. In addition, we investigated the expression of the nine LIV-1 family members in a clinical breast cancer series. Although a number of different LIV-1 family members showed some association with growth factor receptors, LIV-1 was solely associated with estrogen receptor and a variety of growth factors commonly associated with clinical breast cancer. HKE4, however, did show an association with the marker of cell proliferation Ki67 the spread of breast cancer to lymph nodes.

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Figures

Figure 1

Figure 1

Phylogenetic tree and alignment of the human members of the ZIP superfamily of zinc transporters. (A) This phylogenetic tree was drawn using ClustalW and treeview software. (B) This alignment demonstrating the highly conserved motif in TM V for the LIV-1 family was performed using ClustalW and shaded using Boxshade software. Black shading represents at least 50% identity, gray shading represents at least 50% complementary residues. (C) This alignment demonstrating the CPALLY motif directly upstream of TM I was performed using ClustalW and shaded using Boxshade software. Black shading represents at least 50% identity, gray shading represents at least 50% complementary residues.

Figure 2

Figure 2

Cellular location of three human LIV-1 family members in MCF-7 cells. MCF-7 cells expressing recombinant HKE4, LIV-1, or ZIP14 were imaged using a mouse anti-V5 antibody (Invitrogen) conjugated to Alexa Fluor 488 (green) and assembled onto slides using Vectorshield with DAPI (Vector Laboratories). All cells were incubated with Texas red phalloidin to stain F-Actin filaments red. Coverslips were viewed on a Leica RPE automatic microscope using a 63x oil immersion lens. The fluorescent superimposed images were acquired using a multiple bandpass filter set appropriate for DAPI, fluorescein, and Texas Red as well as bright field for differential interference contrast imaging.

Figure 3

Figure 3

Comparison of the effect of antihormones on the RNA expression of the nine human LIV-1 family members. MCF-7 cells were treated with 4-hydroxytamoxifen (TAM), fulvestrant (FAS), or oestradiol (E2) for seven days in serum growth factor–free DCCM medium before total RNA was extracted, reverse transcribed to cDNA, and PCR performed for individual ZIPs. All data were normalized to individual β-actin levels. The upper panel shows representative agarose gels of pS2, LIV-1, HKE4, and ZIP8 in MCF-7 treated with oestradiol (lane 1), untreated (lane 2), treated with Tamoxifen (lane 3), or treated with Fulvestrant (lane 4). M represents size markers. Lower panel shows mean ± SEM densitometric values of 8 genes, comparing their expression levels in MCF-7 cells untreated (CON), or treated with oestradiol (E2), Tamoxifen (TAM), or Fulvestrant (FAS). Statistically significant results are indicated with asterisks, and the relevant P value is given. ZIP4 was undetectable.

Figure 4

Figure 4

Comparison of RNA expression of the nine human LIV-1 family members in anti-hormone-resistant MCF-7 cells. The expression of different ZIPs in tamoxifen (TamR)- and fulvestrant (FasR)-resistant MCF-7 cells was compared with wild-type MCF-7 cells. RNA was extracted and reverse transcribed to cDNA. The upper panel shows representative gels of pS2, LIV-1, HKE4, and ZIP8 in MCF-7 (lane 1), TamR (lane 2) and FasR (lane 3) cell lines. M represents size markers. Lower panel shows mean ± SEM densitometric values of 8 genes comparing their expression levels in MCF-7, TamR, and FasR cells. Statistically significant results are indicated with asterisks, and the relevant P value is given. ZIP4 was undetectable.

Figure 5

Figure 5

Comparison of the expression of LIV-1 family members in a series of samples from breast cancer patients. (A) representative results from 10 patient tumor samples amplified with gene specific primers for ZIP5, HKE4, ZIP10, LIV-1, ZIP8, ZIP14, and β-actin. (B) distribution of the densitometric values for ZIP5, HKE4, ZIP10, LIV-1, ZIP8, and ZIP14 in the patient samples, with no correction for the number of PCR cycles.

Figure 6

Figure 6

Comparison of the expression of LIV-1 and/or ZIP5 with ER, EGFR and grade in a series of breast cancer samples. (A) shows the positive correlation of both LIV-1 and ZIP5 with estrogen receptor (ER). (B) shows the reverse relationship of LIV-1 with EGFR. (C) shows the decreasing levels of LIV-1 with worsening grade.

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