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. 2014 May;15(5):633-42.
doi: 10.4161/cbt.28180. Epub 2014 Feb 20.

Targeting Met and Notch in the Lfng-deficient, Met-amplified triple-negative breast cancer

Shubing Zhang  1 , Wen-cheng Chung  1 , Lucio Miele  2 , Keli Xu  3
Affiliations

Targeting Met and Notch in the Lfng-deficient, Met-amplified triple-negative breast cancer

Shubing Zhang et al. Cancer Biol Ther. 2014 May.

Abstract

Triple negative breast cancer (TNBC) accounts for 15-20% of breast carcinomas and represents one of the most aggressive forms of this disease. Basal and claudin-low are the two main molecular subtypes among TNBCs. We previously reported that deletion of Lfng in mouse mammary gland caused deregulated Notch activation and induced basal-like and claudin-low tumors with co-selection for Met amplification. In human breast cancers, the vast majority of basal tumors and a subset of claudin-low tumors show reduced Lfng expression. Elevated Met expression and activation is associated with basal as well as claudin-low subtypes. To examine roles of Met and Notch in TNBC cells, we established two cell lines that harbor Met amplification as well as Lfng deletion, and possess features of basal and claudin-low breast cancer subtypes. Pharmacological inhibition of Met not only suppressed cell growth, tumorsphere and colony formation, but also reversed epithelial-to-mesenchymal transition and inhibited cell migration in both cell lines. In contrast, inhibition of Notch signaling using a γ-secretase inhibitor (GSI) only suppressed colony formation. Interestingly, GSI had no effect as single agent, but exerted a synergistic effect with Met inhibitor, on cell growth in 2D culture. We found that inhibition of Met resulted in downregulation of Dll ligands and upregulation of Jagged ligands, leading to differential modulation of Notch signaling. Our results suggest that combination targeting of Met and Notch may prove beneficial for TNBC patients with Met overexpression and Notch hyperactivation.

Keywords: Lfng; Met; Met inhibitor; Notch; basal-like breast cancer; claudin-low breast cancer; epithelial-to-mesenchymal transition; triple negative breast cancer; γ-secretase inhibitor.

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Figures

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Figure 1. Characterization of two TNBC cell lines B5725 and C0321 established from the Lfngflox/flox;MMTV-Cre mouse model. (A) Western blot analysis for Notch receptors in B5725 and C0321 cells. β-actin is included as loading control. (B) Copy numbers of the Met gene in B5725 and C0321 cells determined by quantitative PCR, and normalized to that of spleen cells in syngeneic FVB mouse. Shown are mean values ± standard errors derived from triplicate PCR for each sample. *P < 0.05, ***P < 0.0005. (C) Phase-contrast images of B5725 and C0321 cells in 2D culture. White arrow points to the filopodium-like protrusion. Scale bars: 50 μm.
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Figure 2. Authotopic isograft of B5725 and C0321 cell lines in FVB mice. (A and B) Representative H&E staining of mammary tumors developed from injection of B5725 or C0321 cell line (1 × 105 cells/mouse) in the mammary fat pad of FVB mice. (C and D) Anti-vimentin immunofluorescence staining of the mammary tumor sections. (E and F) Representative H&E staining of metastatic lesions (white arrows) in the lungs after injection of B5725 or C0321 cell line into the mammary fat pad. Scale bars: 50 μm.
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Figure 3. Effects of Met inhibitor SU11274 on Met expression and activation, and γ-secretase inhibitor MK-0752 on Notch receptor activation. (A) Western blot analysis of Met and phospho-Met in B5725 and C0321 cells incubated with SU11274 at a concentration of 6 μM (in DMSO) for 24 h, or incubated with same amount of DMSO as control. (B) Western blot for Notch receptors in B5725 and C0321 cells treated with 6 μM of MK-0752 for 24 h. Control was incubation with DMSO.
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Figure 4. Inhibition of Met and Notch in TNBC cells suppressed cell growth as well as tumorsphere and colony formation. (A) Cell growth curves of B5725 and C0321 cell lines under treatment of SU11274 (6 μM), MK-0752 (6 μM), or SU11274 (6 μM) plus MK-0752 (6 μM). Control is the incubation with DMSO. At each time point, relative numbers of viable cells were determined by MTS assay, and presented as mean values ± standard errors of the absorbance at 490 nm. (B) Quantification of the numbers of tumorspheres formed per 1000 cells in 2 wk. Cells were incubated with SU11274, MK-0752, or DMSO as control, at the same concentration as above. Data are presented as mean values ± standard errors from three experiments. (C) Quantification of the numbers of colonies formed in soft agar per 1000 cells, with the treatment of SU11274, MK-0752, or DMSO control at the same concentration as above. Data are presented as mean values ± standard errors from three experiments. *P < 0.05, **P < 0.005, ***P < 0.0005
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Figure 5. Inhibition of Met in TNBC cells caused downregulation of Twist, alteration in E-cadherin and vimentin expression, and reduced cell migration. (A) Western blot analysis of E-cadherin, vimentin, and Twist in B5725 and C0321 cells incubated with 6 μM SU11274 for 24, 48, and 72 h. The control cells were incubated with DMSO. (B) Representative images of the wound healing assay. Cells were incubated with SU11274 (6 μM) or DMSO as control.
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Figure 6. Increased cytokeratin expression in TNBC cells treated with Met inhibitor. Shown are representative images of the immunofluorescence staining for cytokeratin 5 (K5) and cytokeratin 8 (K8) in B5725 and C0321 cells after 48h treatment of SU11274 (6 μM) or DMSO as control. Dapi staining is for the nucleus. Merged images for anti-K5 (green) and anti-K8 (red) staining are shown at the right. Scale bar: 20 μm.
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Figure 7. Met modulates Notch signaling in TNBC cells through differential regulation of Dll and Jagged ligands. (A) Western blot analysis of Notch receptors and Dll4, Jag1 ligands in B5725 and C0321 cells incubated with SU11274 at a concentration of 6 μM (in DMSO) for 24 and 48 h, or incubated with same amount of DMSO as control. β-actin is included as the loading control. (B) Relative expression levels of Dll1, Jag1, and Jag2 in B5725 and C0321 cells treated with 6 μM of SU11274 (or DMSO as control) for 48 h. mRNA levels of each gene were determined by quantitative RT-PCR and normalized to Gapdh in each sample. Shown are mean values ± standard errors from three reactions. (C) Relative mRNA levels of Notch downstream targets Hes1, Hes5, Hey1, and Hey2 in B5725 and C0321 cells treated with 6 μM of SU11274 (or DMSO as control) for 48 h. Shown are mean values ± standard errors from three reactions. *P < 0.05, **P < 0.005, ***P < 0.0005.
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