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. 2016 Mar 15;7(11):11756-69.
doi: 10.18632/oncotarget.7598.

Functional interactions of the cystine/glutamate antiporter, CD44v and MUC1-C oncoprotein in triple-negative breast cancer cells

Masanori Hasegawa  1 , Hidekazu Takahashi  1   2 , Hasan Rajabi  1 , Maroof Alam  1 , Yozo Suzuki  1   3 , Li Yin  1 , Ashujit Tagde  1 , Takahiro Maeda  1 , Masayuki Hiraki  1 , Vikas P Sukhatme  4 , Donald Kufe  1
Affiliations

Functional interactions of the cystine/glutamate antiporter, CD44v and MUC1-C oncoprotein in triple-negative breast cancer cells

Masanori Hasegawa et al. Oncotarget. .

Abstract

The xCT light chain of the cystine/glutamate transporter (system XC-) is of importance for the survival of triple-negative breast cancer (TNBC) cells. The MUC1-C transmembrane oncoprotein is aberrantly overexpressed in TNBC and, like xCT, has been linked to maintaining glutathione (GSH) levels and redox balance. However, there is no known interaction between MUC1-C and xCT. Here we show that silencing MUC1-C is associated with decreases in xCT expression in TNBC cells. The results demonstrate that MUC1-C forms a complex with xCT and the CD44 variant (CD44v), which interacts with xCT and thereby controls GSH levels. MUC1-C binds directly with CD44v and in turn promotes stability of xCT in the cell membrane. The interaction between MUC1-C and xCT is further supported by the demonstration that targeting xCT with silencing or the inhibitor sulfasalazine suppresses MUC1 gene transcription by increasing histone and DNA methylation on the MUC1 promoter. In terms of the functional significance of the MUC1-C/xCT interaction, we show that MUC1-C protects against treatment with erastin, an inhibitor of XC- and inducer of ferroptosis, a form of non-apoptotic cell death. These findings indicate that targeting this novel MUC1-C/xCT pathway could represent a potential therapeutic approach for promoting TNBC cell death.

Keywords: CD44v; MUC1-C; epigenetics; ferroptosis; xCT.

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Conflict of interest statement

CONFLICTS OF INTEREST

D.K. holds equity in Genus Oncology and is a consultant to the company. The other authors disclosed no potential conflicts of interest.

Figures

Figure 1
Figure 1. Downregulation of MUC1-C decreases xCT levels
A. and B. Lysates from MDA-MB-468 (A) or BT-20 (B) cells were precipitated with anti-MUC1-C, anti-xCT or a control IgG. The precipitates were immunoblotted with the indicated antibodies. C. and D. MDA-MB-468/tet-MUC1shRNA (C) and BT-20/tet-MUC1shRNA (D) cells were treated with 200 ng/ml DOX for the indicated times. Membrane fractions were immunoblotted with the indicated antibodies. E. and F. Intracellular glutamate levels were determined in MDA-MB-468/tet-MUC1shRNA (E) and BT-20/tet-MUC1shRNA (F) cells cultured in the presence of 200 ng/ml DOX for 72 h. The results (mean±SD of 3 determinations) are expressed as relative glutamate levels as compared with that obtained from cells without DOX treatment (assigned a value of 1).
Figure 2
Figure 2. MUC1-C interacts with CD44v
A. Lysates from MDA-MB-468 cells were precipitated with anti-MUC1-C or a control IgG. The precipitates were immunoblotted with the indicated antibodies. B. Amino acid sequence of the MUC1 cytoplasmic domain (MUC1-CD). GST, GST-MUC1-CD or the indicated GST-MUC1-CD fragments were incubated with His-CD44-ICD. The adsorbates were immunoblotted with anti-CD44. Input of the GST proteins was assessed by Coomassie blue staining. C. Amino acid sequence of the CD44-ICD. GST, GST-CD44-ICD or the indicated GST-CD44-ICD fragments were incubated with MUC1-CD. The adsorbates were immunoblotted with anti-MUC1-C. Input of the GST proteins was assessed by Coomassie blue staining. D. Lysates (15 and 45 μg) from MDA-MB-468, BT-20, ZR-75-1 and MCF-7 cells were immunoblotted with the indicated antibodies (left). CD44v9 mRNA levels in the indicated cells were determined by qRT-PCR (right). The results (mean±SD of 4 determinations) are expressed as relative CD44v9 mRNA levels as compared with that obtained for MCF-7 cells (assigned a value of 1). E. Lysates from 293T/MUC1-C cells transfected with an empty vector or one expressing CD44v8-10 were precipitated with anti-MUC1-C or a control IgG. The precipitates were immunoblotted with the indicated antibodies.
Figure 3
Figure 3. MUC1 increases xCT stability
A. 293T cells were transfected to stably express MUC1-C or a control vector. Lysates from the transduced cells were immunoblotted with the indicated antibodies (left). Lysates from 293T/MUC1-C cells were precipitated with anti-MUC1-C or a control IgG. The precipitates were immunoblotted with the indicated antibodies (right). B. Membrane fractions (left) and total cell lysates (right) from 293T/vector or 293T/MUC1-C cells were immunoblotted with the indicated antibodies. C. 293T/vector and 293T/MUC1-C cells were exposed to 50 μg/ml CHX for the indicated times. Total cell lysates were immunoblotted with the indicated antibodies. D. Intensities of the xCT signals as compared to those obtained for β-actin (xCT/β-actin ratio) for the CHX-treated 293T/vector and 293T/MUC1-C cells are plotted relative to the control (time 0; assigned a value of 1). E. MDA-MB-468/CshRNA and MDA-MB-468/MUC1shRNA cells were exposed to CHX (50 μg/ml) for the indicated times. Total cell lysates were immunoblotted with the indicated antibodies. F. Intensities of the xCT signals as compared to those obtained for β-actin (xCT/β-actin ratio) for the CHX-treated MDA-MB-468/CshRNA and MDA-MB-468/MUC1shRNA cells are plotted relative to the control (time 0; assigned a value of 1).
Figure 4
Figure 4. Targeting xCT suppresses MUC1 expression
A. and B. MDA-MB-468 (A) or BT-20 (B) cells were treated with 1.0 μM or 1.5 μM SASP for 72 h, respectively. MUC1 mRNA levels were determined by qRT-PCR (left). The results (mean±SD of 4 determinations) are expressed as relative MUC1 mRNA levels as compared with that obtained for the untreated control cells (assigned a value of 1). Lysates from control and SASP-treated cells were immunoblotted with the indicated antibodies (right). C. and D. MDA-MB-468 (C) and BT-20 (D) cells were transfected to stably express a control shRNA (CshRNA) or an xCT shRNA (xCTshRNA). MUC1 mRNA levels were determined by qRT-PCR (left). The results (mean±SD of 4 determinations) are expressed as relative MUC1 mRNA levels as compared with that obtained for the CshRNA cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right).
Figure 5
Figure 5. Glutamate suppresses MUC1 expression
A. and B.. MDA-MB-468 (A) or BT-20 (B) cells were treated with 20 mM glutamate for 72 h. MUC1 mRNA levels in the control and glutamate treated cells were determined by qRT-PCR (left). The results (mean±SD of 4 determinations) are expressed as relative MUC1 mRNA levels as compared with that obtained for the untreated control cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). C. and D. MDA-MB-468 (C) or BT-20 (D) cells were treated with 10 μM or 20 μM compound 968 for 72 h, respectively. MUC1 mRNA levels were determined by qRT-PCR (left). The results (mean±SD of 4 determinations) are expressed as relative MUC1 mRNA levels as compared with that obtained for the untreated control cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right).
Figure 6
Figure 6. Targeting xCT induces histone and DNA methylation of the MUC1 promoter
A. and B. MDA-MB-468 (A) or BT-20 (B) cells were treated with 1.0 mM or 1.5 mM SASP for 72 h, respectively. Soluble chromatin from control and SASP treated cells was precipitated with anti-H3K9me2, anti-H3K9me3 or a control IgG. The final DNA samples were amplified by qPCR with pairs of primers for the MUC1 promoter region or control region from the GAPDH promoter. The results (mean±SD of 3 determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (untreated cells, assigned a value of 1). C. and D. Soluble chromatin from MDA-MB-468/CshRNA, MDA-MB-468/xCTshRNA (C), BT-20/CshRNA and BT-20/xCTshRNA (D) cells was precipitated with anti-H3K9me2, anti-H3K9me3 or a control IgG. The final DNA samples were amplified by qPCR with pairs of primers for the MUC1 promoter region or control GAPDH promoter. The results (mean±SD of 3 determinations) are expressed as the relative fold enrichment compared with that obtained with the IgG control (CshRNA cells, assigned a value of 1). E. Genomic DNA from the indicated MDA-MB-468 (left) and BT-20 (right) cells was subjected to immunoprecipitation of methylated DNA (MeDIP) and the precipitates were analyzed by qPCR of the MUC1 gene promoter. The results (mean±SD of 3 determinations) are expressed as relative fold enrichment compared to that obtained from CshRNA expressing cells (assigned a value of 1).
Figure 7
Figure 7. MUC1-C protects against erastin-induced ferroptosis
A. and B. MDA-MB-468/tet-CshRNA (open bars) and MDA-MB-468/tet-MUC1shRNA (solid bars) cells were treated with 200 ng/ml DOX for 7 d. A. Cells were then plated in a 96 well plate, and exposed to 1 μM erastin (labeled E) with or without 2 μM Fer-1 (labeled F) for 24 h. The results (mean±SD of 8 determinations) are expressed as percentage cell death as determined by Alamar blue analysis. B. The indicated cells were analyzed for relative GSH levels (mean±SD of 3 determinations) as compared with that obtained for erastin-treated, DOX- cells (assigned a value of 1)(right). C. and D. BT-20/tet-CshRNA (open bars) and BT-20/tet-MUC1shRNA (solid bars) cells were treated with 200 ng/ml DOX for 7 d. C. Cells were then plated in a 96 well plate, and exposed to 0.5 μM erastin (labeled E) with or without 2 μM Fer-1 (labeled F) for 12 h. The results (mean±SD of 8 determinations) are expressed as percentage cell death as determined by Alamar blue analysis. D. The indicated cells were analyzed for relative GSH levels (mean±SD of 3 determinations) as compared with that obtained for erastin-treated, DOX- cells (assigned a value of 1)(right). E. Schema of the proposed MUC1-C/xCT regulatory loop. MUC1-C forms a complex with xCT and CD44v in the TNBC cell membrane through a direct interaction between the MUC1-C cytoplasmic domain (MUC1-CD) and the CD44v intracellular domain (CD44v-ICD). CD44v interacts with xCT through their extracellular domains [7]. MUC1-C stabilizes xCT; thus targeting MUC1-C decreases xCT in the cell membrane. In addition, targeting xCT with silencing or SASP increases intracellular glutamate and thereby suppresses MUC1 transcription by increasing H3K9 and DNA methylation on the MUC1 promoter. The MUC1-C/xCT loop suppresses ferroptosis and promotes TNBC cell survival.
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