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. 2017 Jan 10;8(2):3528-3541.
doi: 10.18632/oncotarget.13869.

Loss of Expression of the Recycling Receptor, FcRn, Promotes Tumor Cell Growth by Increasing Albumin Consumption

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Free PMC article

Loss of Expression of the Recycling Receptor, FcRn, Promotes Tumor Cell Growth by Increasing Albumin Consumption

Rafal Swiercz et al. Oncotarget. .
Free PMC article

Abstract

Tumor cells rely on high concentrations of amino acids to support their growth and proliferation. Although increased macropinocytic uptake and lysosomal degradation of the most abundant serum protein, albumin, in Ras-transformed cells can meet these demands, it is not understood how the majority of tumor cells that express wild type Ras achieve this. In the current study we reveal that the neonatal Fc receptor, FcRn, regulates tumor cell proliferation through the ability to recycle its ligand, albumin. By contrast with normal epithelial cells, we show that human FcRn is present at very low or undetectable levels in the majority of tumor cell lines analyzed. Remarkably, shRNA-mediated ablation of FcRn expression in an FcRn-positive tumor cell line results in a substantial growth increase of tumor xenografts, whereas enforced expression of this receptor by lentiviral transduction has the reverse effect. Moreover, intracellular albumin and glutamate levels are increased by the loss of FcRn-mediated recycling of albumin, combined with hypoalbuminemia in tumor-bearing mice. These studies identify a novel role for FcRn as a suppressor of tumor growth and have implications for the use of this receptor as a prognostic indicator and therapeutic target.

Keywords: albumin; hypoalbuminemia; neonatal Fc receptor (FcRn); recycling receptor; tumor suppressor.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. The majority of breast and prostate tumor cell lines express very low or undetectable levels of FcRn
A. Immunoblotting of cell lysates using antibodies specific for FcRn α-chain, β2-microglobulin (β2m) and β-actin. Cropped images are shown with molecular weights (kDa) on the right. B. Cells were pulsed with 10 μg/ml Alexa 647-labeled MST-HN or Alexa 647-labeled H435A for 40 minutes at 37°C in medium (pH 6.0). Alexa 647 levels (mean fluorescence intensity, MFI) were determined using flow cytometry and mean values for triplicate samples are shown. Control indicates autofluorescence levels of cells. C. Cell lines were pulsed with 1 μg/ml Alexa 647-labeled FcRn-specific (bivalent) nanobody for 40 minutes at 37°C in medium (pH 6.0) and analyzed using flow cytometry as in panel (B). Control indicates autofluorescence levels of cells. For panels (B) and (C), error bars represent S.D. Significant differences between MST-HN or nanobody levels are indicated by * (one-way ANOVA, p < 0.05). Data are representative of at least two independent experiments.
Figure 2
Figure 2. The level of FcRn expression regulates the intracellular levels of albumin in tumor cells
A. HCC1419 cells transduced with empty vector (EV), scrambled shRNA (scramble) or shRNAs targeting FcRn (sh-5, sh-6) were pulsed with 1 μg/ml Alexa 647-labeled FcRn-specific nanobody for 40 minutes at 37°C in medium (pH 6.0). Alexa 647 levels (MFI) were determined using flow cytometry and mean values for triplicate samples are shown. Control indicates autofluorescence levels of cells. The right hand panel shows immunoblotting of cell lysates using antibodies specific for FcRn α-chain and β-actin. B. DU145 cell lines transduced with empty vector (EV) and FcRn expression constructs (WT-FcRn or H166A-FcRn) were analyzed as in panel (A). C, D. HCC1419 cell lines (same as in panel A) were pulsed with 1.5 μM (100 μg/ml) MSA (C) or 1.5 μM (100 μg/ml) HSA (D) for 0 h or 2 hrs at 37°C at pH 7.4. Cell lysates were used in immunoblotting with antibodies specific for albumin (MSA/HSA), FcRn α-chain and β-actin. Albumin levels were quantitated and normalized relative to HCC1419 cells. E, F. DU145 cell lines (same as in panel B) were treated with MSA (E) or HSA (F) as in panels (C) and (D), and albumin levels quantitated and normalized relative to DU145 cells. G. DU145 cell lines were pulsed with 1.5 μM Alexa 647-labeled MSA for 2 hrs. Cells were chased for the indicated times. Alexa 647 levels (MFI) were determined using flow cytometry and mean values for triplicate samples are shown (left panel). The right hand panel shows the MFI values normalized to the no chase (0 time point) MFI value for each cell line. H. HCC1419 cell lines transduced with scrambled shRNA (scramble), sh-5 or sh-5 plus shRNA resistant FcRn (Res-FcRn) were treated and albumin levels relative to ‘scramble’ analyzed as in panel (C). For panels (C-F) and (H), mean normalized signal levels derived from three independent immunoblotting experiments are shown. For the immunoblots, cropped images are shown with molecular weights (kDa) on the right. For panels (A-H), error bars represent S.D. Significant differences are indicated by * (one-way ANOVA, p < 0.05). For panels (A, B) and (G), data are representative of at least two independent experiments.
Figure 3
Figure 3. Decreased pH affects fluid phase uptake by the cell lines
A, B. HCC1419 and DU145 cell lines were treated with MSA as in Figure 2C, except that the incubation medium was adjusted to the indicated pH. Immunoblotting of cell lysates using antibodies specific for FcRn α-chain and β-actin was performed. Cropped images are shown with molecular weights (kDa) on the right. C, D. HCC1419 and DU145 cell lines were pulsed with 1.5 μM Alexa 647-labeled H435A for 2 hrs in medium adjusted to the indicated pH. Alexa 647 levels (MFI) were determined using flow cytometry and mean values for triplicate samples are shown. Error bars represent S.D. Significant differences are indicated by * (one-way ANOVA, p < 0.05). For panels (A-D), data are representative of two independent experiments.
Figure 4
Figure 4. Pinocytic uptake and recycling rates of tumor cells are not affected by lentiviral transduction or the level of FcRn expression
A. HCC1419 cell lines transduced with empty vector (EV), scrambled shRNA (scramble) or shRNAs (sh-5, sh-6) were pulsed for 0-60 minutes with 1.5 μM tetramethylrhodamine-labeled 70 kDa dextran, or pulsed for 5 minutes with 20 μg/ml Alexa 647-labeled transferrin followed by chase periods of 0-60 minutes. B. DU145 cell lines transduced with empty vector (EV) or FcRn expression constructs (WT-FcRn or H166A-FcRn) were treated with Alexa 647-labeled dextran or Alexa 647-labeled transferrin as in panel (A). Flow cytometry was used to determine cell-associated fluorescence. The MFI values represent means derived from triplicate samples. Error bars represent S.D. Data are representative of two independent experiments.
Figure 5
Figure 5. The expression level of FcRn controls the growth of tumor xenografts
A. Mice (n = 6-8 mice/group) were implanted with HCC1419 cells transduced with shRNAs targeting FcRn (sh-5, sh-6) or empty vector (EV) and tumor size monitored (upper panel). Statistically significant differences between the EV group and knockdown (sh-5, sh-6) groups from days 18-31 are indicated by * (two-way ANOVA with Tukey post-hoc comparison; p < 0.05). Immunoblotting analysis of lysates of tumors (HCC1419 cell lines) extracted at the end of the experiment using antibodies specific for human FcRn α-chain and β-actin is shown in the lower panel. Cropped images are shown with molecular weights (kDa) on the right. Data are representative of two independent assays. B. Mice (n = 9-10 mice/group) were implanted with DU145 cells transduced with FcRn expression constructs (WT-FcRn or H166A-FcRn) or empty vector (EV) and tumor size monitored (upper panel). Statistically significant differences from days 21-28 (EV vs. WT-FcRn) and days 21-24 (H166A-FcRn vs. WT-FcRn) are indicated by * (two-way ANOVA with Tukey post-hoc comparison; p < 0.05). Immunoblotting analysis of lysates of tumors (DU145 cell lines) was carried out and displayed as described in panel (A). C. Serum albumin levels at day 21 for mice within different groups for experiment shown in panel (B). D. Plot of tumor size vs. serum albumin levels (day 21) for mice implanted with DU145 cells transduced with EV or H166A-FcRn. Statistically significant differences between groups in panel (C) are indicated by * (one-way ANOVA, p < 0.05). Data are representative of at least two independent experiments.
Figure 6
Figure 6. The expression level of FcRn regulates intracellular glutamate levels and cell proliferation
A. HCC1419 cells transduced with empty vector (EV), scrambled shRNA (scramble) or shRNAs targeting FcRn (sh-5, sh-6) were cultured in base medium containing 100 μM glucose and 1.5 μM MSA or 2 mM glutamine for 24 hrs. Cells were lysed and glutamate levels determined. B. HCC1419 cell lines were cultured in the same medium as for panel A for 16 hours. BrdU levels in cells were determined following 4 hrs incubation with BrdU. C, D. DU145 cells transduced with empty vector (EV) or FcRn expression constructs (WT-FcRn or H166A-FcRn) were treated as in panels A and B to assess intracellular glutamate levels and proliferation, respectively. For panels A-D, mean values for triplicate samples are shown. Error bars represent S.D. Significant differences are indicated by * (one-way ANOVA, p < 0.05). Data are representative of at least two independent experiments.

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References

    1. Cantor JR, Sabatini DM. Cancer cell metabolism: one hallmark, many faces. Cancer Discov. 2012;2:881–898. - PMC - PubMed
    1. Commisso C, Davidson SM, Soydaner-Azeloglu RG, Parker SJ, Kamphorst JJ, Hackett S, Grabocka E, Nofal M, Drebin JA, Thompson CB, Rabinowitz JD, et al. MG Vander Heiden, et al. Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature. 2013;497:633–637. - PMC - PubMed
    1. Kamphorst JJ, Nofal M, Commisso C, Hackett SR, Lu W, Grabocka E, MG Vander Heiden, Miller G, Drebin JA, Bar-Sagi D, Thompson CB, Rabinowitz JD. Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein. Cancer Res. 2015;75:544–553. - PMC - PubMed
    1. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012;72:2457–2467. - PMC - PubMed
    1. Gupta D, Lis CG. Pretreatment serum albumin as a predictor of cancer survival: a systematic review of the epidemiological literature. Nutr J. 2010;9:69. - PMC - PubMed

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