Journal: Nature medicine
Article Title: Metastatic cancers promote cachexia through altered zinc homeostasis in skeletal muscle
doi: 10.1038/s41591-018-0054-2
Figure Lengend Snippet: (a) qRT-PCR analysis of Zip14 expression in purified muscle progenitor subpopulations from either non-tumor-bearing control mice (Con) or mice bearing 4T1 or C26m2 metastases (Tb) harvested five weeks after tumor-cell injection (see and ). CD45 − CD31 − CD34 + Sca1 + and CD45 − CD31 − CD34 + integrin-α7 + cells were purified from gastrocnemius muscles using a combination of magnetic and flow-cytometry-assisted sorting (see ). For 4T1 model, n=4 control mice for isolated CD34 + Sca1 + cells and n=3 mice for the other groups. For C26m2 model, n=3 mice per group. (b) ZIP14 immunofluorescence analysis using muscle sections from either non-tumor-bearing control mice or mice bearing C26m2 metastases five weeks after tumor-cell injection. ZIP14 (red), DAPI (blue). Scale bars, 50 μm. Boxed area in upper panels is magnified in the corresponding lower panels. A representative image from three independent experiments is shown. (c,d) Immunofluorescence analysis showing myosin heavy chain (MyHC) expression in C2C12 myoblasts infected with adenovirus expressing either control (Adeno-Con) or Zip14 cDNA (Adeno- Zip14 ) and differentiated for 6 days, either with 0 or 50 μM ZnCl 2 (zinc) replenished daily. Representative images and quantitation (d) are shown. Scale bars, 25 μm. Data presented as percentage of MyHC fluorescence signal in corresponding untreated myotubes (d). Data representative of three independent experiments (c,d). (e) qRT-PCR analysis of MyoD , Myf5 and Mef2c expression in untreated and zinc-treated C2C12 cells expressing either Adeno-Con or Adeno- Zip14 , represented as a heatmap. Adenovirus-infected C2C12 cells were differentiated for 2 days and then treated with either 0 or 50 μM ZnCl 2 (zinc) for 24 hours. Data is representative of four independent experiments. (f) RNA-Seq analysis of MyoD , Myf5 and Mef2c expression shown as heatmap (RNA seq shown in and ) comparing TA muscles from non-tumor-bearing control mice to mice bearing 4T1 or C26m2 metastases, collected 5 weeks post tumor-cell injection. (g) MyHC and tropomyosin (Tm) protein expression by immunoblot analysis in C2C12 cells infected with adenovirus expressing either control or Zip14 cDNA and differentiated for 3 days followed by treatment with either 0 or 50 μM ZnCl 2 for 24 hours. Data is representative of three independent experiments. Uncropped immunoblot images are shown in Supplementary Fig. 6 . (h) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from mice intramuscularly injected with adeno-associated virus expressing either shCon or sh Zip14 and subsequently injected with C26m2 cancer cells (see ). Age-matched, non-tumor-bearing mice were used as a control. Data is representative of three independent experiments. Uncropped immunoblot images are shown in . (i) Immunoblot analysis probing for MyHC and Tm in gastrocnemius muscles from the indicated groups. Muscles were isolated from Zip14 WT and KO mice with (Tb) or without (Con) 4T1 metastases. Another cohort of Zip14 KO mice were injected intramuscularly with AAV-Con ( mCherry ) or AAV- Zip14 in the gastrocnemius muscle and injected four weeks later with 4T1 tumor cells. All mice were harvested 5 weeks post tumor-cell injection. Data is representative of three independent experiments. Uncropped immunoblot images are shown in . Error bars represent SEM and all data were represented by mean ± SEM. P values in (a,d) were determined by two-tailed, unpaired Student’s t test. Con, control; Tb, tumor-bearing; Tm, Tropomyosin. (j) Working model: During cancer progression and metastasis development, cytokines such as TNF-α and TGF-β upregulate the expression of Zip14 , a metal ion transporter, in muscle progenitor and mature muscle cells. This causes an aberrant accumulation of zinc in these muscle cells. ZIP14 expression and zinc uptake in muscle progenitor cells represses key myogenic genes such as MyoD and Mef2c , and blocks muscle differentiation. ZIP14 expression in mature muscle cells causes myosin heavy chain loss, which promotes cancer-induced muscle atrophy in metastatic cancers. Tf, tumor factors; Ca, cancer cells; Nr, normal cells.
Article Snippet: Cells were then incubated for 1 hour with primary antibody: rabbit polyclonal antibodies against mouse ZIP14 (1:500 developed in our laboratory), Desmin (1:500 Sigma, D8281), mouse monoclonal antibodies against fast MyHC (1:500 Sigma, clone MY-32) or tropomyosin (1:500 DSHB, CH1) followed by 30 mins incubation with Alexa Fluor 568-labeled secondary antibody (Thermo Fisher).
Techniques: Quantitative RT-PCR, Expressing, Purification, Injection, Flow Cytometry, Isolation, Immunofluorescence, Infection, Quantitation Assay, Fluorescence, RNA Sequencing Assay, Western Blot, Two Tailed Test
