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, 9 (1), 15042

Loss of the Dermis Zinc Transporter ZIP13 Promotes the Mildness of Fibrosarcoma by Inhibiting Autophagy

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Loss of the Dermis Zinc Transporter ZIP13 Promotes the Mildness of Fibrosarcoma by Inhibiting Autophagy

Mi-Gi Lee et al. Sci Rep.

Abstract

Fibrosarcoma is a skin tumor that is frequently observed in humans, dogs, and cats. Despite unsightly appearance, studies on fibrosarcoma have not significantly progressed, due to a relatively mild tumor severity and a lower incidence than that of other epithelial tumors. Here, we focused on the role of a recently-found dermis zinc transporter, ZIP13, in fibrosarcoma progression. We generated two transformed cell lines from wild-type and ZIP13-KO mice-derived dermal fibroblasts by stably expressing the Simian Virus (SV) 40-T antigen. The ZIP13-/- cell line exhibited an impairment in autophagy, followed by hypersensitivity to nutrient deficiency. The autophagy impairment in the ZIP13-/- cell line was due to the low expression of LC3 gene and protein, and was restored by the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza) treatment. Moreover, the DNA methyltransferase activity was significantly increased in the ZIP13-/- cell line, indicating the disturbance of epigenetic regulations. Autophagy inhibitors effectively inhibited the growth of fibrosarcoma with relatively minor damages to normal cells in xenograft assay. Our data show that proper control over autophagy and zinc homeostasis could allow for the development of a new therapeutic strategy to treat fibrosarcoma.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
ZIP13 is downregulated in fibrosarcoma. (a) Real-time PCR analysis reveals that ZIP13 is one of the predominant zinc transporter transcripts in normal human fibroblasts. The data represent three independent experiments. (b) Real-time PCR analysis reveals that ZIP13 is downregulated in the HT1080 fibrosarcoma cell line. The data are presented as mean ± SD of three independent experiments (***P < 0.005). (c) Real-time PCR analysis reveals that ZIP13 is downregulated in different cancer cell lines compared to placental cDNA. The data represent three independent experiments. (d) Real-time PCR analysis reveals that 5 μM 5-aza-2′-deoxycytidine (5-Aza) treatment increases ZIP13 expression in the HT1080 fibrosarcoma cell line. The data are presented as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01).
Figure 2
Figure 2
ZIP13−/− cell lines reveal autophagy impairment. (a) Microscopic analysis reveals that after 3 days in culture without medium change, floating and spherical cells appear in the ZIP13−/− cultures. (b) Western blot analysis reveals the downregulation of LC3 proteins in the ZIP13−/− cell line. Uncropped blots are shown in Fig. S1b. (c) Confocal microscopic analysis reveals the decrease in LC3-positive vacuoles in the ZIP13−/− cell line after 3-day culturing without medium change (n = 5, ***P < 0.005). Scale bars = 25 μm. (d) Western blot analysis shows that bafilomycin treatment does not restore the accumulation of LC3 proteins in the ZIP13−/− cell line. The relative intensity of total LC3s was measured by the ImageJ software (http://rsbweb.nih.gov/ij/download.html). The data represent three independent experiments (***P < 0.005). Uncropped blots are shown in Fig. S2.
Figure 3
Figure 3
ZIP13−/− cell lines have a defect in autophagy induction. (a) Western blot analysis reveals that glucose depletion induces LC3 expression. –AA, amino acid depletion; -Glu, glucose depletion. Uncropped blots are shown in Fig. S5. (b) Western blot analysis reveals that glucose depletion significantly induces LC3 expression in the ZIP13+/+ cell line compared to the ZIP13−/− cell line. Uncropped blots are shown in Fig. S6. (c,d) Real-time PCR analysis reveals that the expression levels of Map1lc3a and Map1lc3b are comparable in both cell lines under normal condition (c). However, glucose depletion-induced Map1lc3a and Map1lc3b upregulation is significantly stronger in the ZIP13+/+ compared to the ZIP13−/− cell line. The relative intensity of total LC3s was measured by ImageJ software (http://rsbweb.nih.gov/ij/download.html). All data in graphs represent three independent experiments.
Figure 4
Figure 4
5-Aza treatment recovers the ZIP13 expression. (a) Real-time PCR analysis reveals that 5-Aza treatment induces Map1lc3a expression more significantly in the ZIP13−/− cell line compared to the ZIP13+/+ cell line. The data are presented as mean ± SD of three independent experiments (***P < 0.005). (b) Western blot analysis reveals that 5-aza-2′-deoxycytidine (5-Aza) treatment for 24 h increases ZIP13 expression in the ZIP13−/− cell line. Uncropped blots are shown in Fig. S7. (c) The DNA methyltransferase (DNMT) assay reveals that the DNMT activity is higher in the ZIP13−/−, compared to the ZIP13+/+ cell line. The data are presented as mean ± SD of three independent experiments (**P < 0.01). The Y-axis shows the relative intensity of the OD 450 nm value with respect to the ZIP13 + / + signal, which was set at 1.
Figure 5
Figure 5
TPEN treatment induces fibrosarcoma death in a mice model. (ad) Crystal violet assay reveals that 3-MA, chloroquine, and TPEN treatment lead to more severe cytotoxicity in the ZIP13−/− cell line and the HT1080 fibrosarcoma cell line, compared to the ZIP13+/+ cell line, as well as normal human dermal fibroblast. The data are presented as mean ± SD of three independent experiments (*P < 0.05). (e) Flowchart of HT1080 fibrosarcoma xenograft model. (f) Tumor image representative of 5 independent experiments. (g,h) Tumor volume and weight were significantly decreased by TPEN treatment compared to the control (*P < 0.05). (i) IHC analysis showed that TPEN treatment decreased LC3 and increased the level of cleaved caspase 3, compared to the control in tumor tissue. Scale bars = 500 μm.
Figure 6
Figure 6
Models for ZIP13 and zinc involvements in fibrosarcoma survival. (a) ZIP13 causes resistance to nutrient-deficiency in fibrosarcoma. (b) Zinc supports autophagy. When TPEN is applied, proper autophagy is blocked, resulting in reduced cell survival.

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