Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Mar 12;9(3):e91509.
doi: 10.1371/journal.pone.0091509. eCollection 2014.

DLK1 promotes lung cancer cell invasion through upregulation of MMP9 expression depending on Notch signaling

Lin Li  1 , Jinjing Tan  1 , Ying Zhang  1 , Naijun Han  1 , Xuebing Di  1 , Ting Xiao  1 , Shujun Cheng  1 , Yanning Gao  1 , Yu Liu  1
Affiliations

DLK1 promotes lung cancer cell invasion through upregulation of MMP9 expression depending on Notch signaling

Lin Li et al. PLoS One. .

Abstract

The transmembrane and secreted protein delta-like 1 homolog (DLK1) belongs to the EGF-like family. It is widely accepted that DLK1 plays important roles in regulating cell differentiation, such as adipogenesis and osteogenesis. Aberrant expression of DLK1 has been found in various types of human cancers, including lung cancer. A previous study in this lab has revealed that DLK1 is associated with tumor invasion, although the mechanism is still unknown. To explore the potential effects that DLK1 might have on invasion, DLK1 was overexpressed or knocked down in the human lung cancer cell lines. The protein's influences on cell invasion were subsequently evaluated. A transwell assay showed that DLK1 overexpression significantly promoted cancer cell invasion. Western blotting and gelatin zymography analysis indicated that DLK1 could affect both matrix metalloproteinase-9 (MMP9) expression and its extracellular activity. An analysis of NOTCH1 and HES1 gene expression and Notch intracellular domain (NICD) nuclear translocation during DLK1 stimulation or depletion demonstrated that DLK1 could activate Notch signaling in lung cancer cells. Additionally, the elevated expression of MMP9 induced by DLK1 stimulation could be significantly decreased by inhibiting Notch signaling using γ-secretase inhibitor (GSI). The data presented in this study suggest that DLK1 can promote the invasion of lung cancer cells by upregulating MMP9 expression, which depends on Notch signaling.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effect of DLK1 overexpression on ECM invasion by the human lung cancer cells H520 and H1299 in an in vitro chemoinvasion assay.
A, representative photomicrographs of the invading cells that migrated through the Matrigel-coated membrane of the transwell, taken from three groups: H520, the parental cells, H520-pcdb, the null vector-control cells and H520-dlk1, the cells stably expressing DLK1. B, a histogram for the counts of migratory cells from the H520, H520-pcdb and H520-dlk1 groups. C, representative photomicrographs showing H1299 cells, which were transiently transfected with DLK1 (H1299-dlk1) or null vector (H1299-pcdb), that invaded through the Matrigel-coated membrane of the transwell. D, a histogram for the counts of migratory cells from the H1299-dlk1 and H1299-pcdb groups. The experiments were performed in triplicate (* t-test, p-value <0.05).
Figure 2
Figure 2. Effect of DLK1 on the expression and activity of MMP9 in human lung cancer cell lines.
A, a histogram for the relative mRNA expression of MMP9 in H520 cells transfected with DLK1 (H520-dlk1) or null vector (H520-pcdb) detected by real-time PCR analysis. The expression level of MMP9 in the blank H520 cells was used as a control and set to 1. B, the protein expression of MMP9 in H520-dlk1, H520-pcdb and H520 cells evaluated by Western blotting analysis. C, gelatin zymography showing the activity of secreted MMP9 and MMP2 in H520-dlk1, H520-pcdb and H520 cells. D, real-time PCR analysis of the relative mRNA expression of MMP9 in H1299 cells transfected with DLK1 (H1299-dlk1) or null vector (H1299-pcdb) shown in a histogram. E, Western blotting showing the protein expression of MMP9 in H1299-dlk1 and H1299-pcdb cells. F, a histogram showing the relative mRNA expression of MMP9 in DLK1 siRNA (A549-si-dlk1) or null control siRNA (A549-NC) transfected A549 cells evaluated by real-time PCR analysis. G, the protein expression of MMP9 in A549-si-dlk1, A549-NC and blank A549 cells detected by Western blotting. All of the experiments were performed in triplicate. 18S ribosomal RNA was used as an internal control in the real-time PCR analysis, whereas GAPDH was used as an internal control in the Western blotting analysis (* t-test, p-value <0.05).
Figure 3
Figure 3. Effects of DLK1 on the Notch signaling pathway in the human lung cancer cells.
A, Western blotting showing NOTCH1 expression in H520 cells after transfection with DLK1 (H520-dlk1) or null vector (H520-pcdb)examined in whole-cell lysates. GAPDH was used as an internal control. B, Western blotting evaluating nuclear NICD expression in H520-dlk1 andH520-pcdb cells. TBP was used as an internal control. C, a histogram for the relative mRNA expression of Notch target gene HES1 in the H520-dlk1 and H520-pcdb cells detected by real-time PCR analysis. H520-pcdb cells were used as a control, and its expression level of HES1 was set to 1. 18S ribosomal RNA was used as an internal control. D, Western blotting showing the expression of NOTCH1 in H1299 cells transfected with DLK1 (H1299-dlk1) or null vector (H1299-pcdb). GAPDH was used as an internal control. E, a histogram presenting the relative mRNA expression of HES1 in H1299-dlk1 and H1299-pcdb cells detected by real-time PCR analysis. F, the expression of NOTCH1 in cytoplasm were evaluated by Western blotting in A549 cells transiently transfected with DLK1 siRNA (A549-si-dlk1) or null control siRNA (A549-NC). GAPDH was used as an internal control. G, real-time PCR analysis of the relative mRNA expression of HES1 in A549-si-dlk1 and A549-NC cells, and presented in a histogram. The experiments were performed in triplicate (* t-test, p-value <0.05).
Figure 4
Figure 4. Effects of Notch signaling blockade on DLK1-regulated MMP9 expression in the human lung cancer cell line H520.
A, a bar graph of the mRNA expression of MMP9 detected by real-time PCR in H520 cells with or without DLK1 overexpression and with or without GSI treatment. The group with neither DLK1 stimulation nor GSI treatment (dlk−/GSI−) was used as a control, and the expression level of MMP9 was set to 1. 18S ribosomal RNA was used as an internal control. B, the expression of HES1 evaluated by real-time PCR in parallel to MMP9 expression is shown in a bar graph, indicating Notch signaling activities upon DLK1 overexpression or GSI treatment. The experiments were performed in triplicate (* t-test, p-value <0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2008) Cancer statistics, 2008. CA Cancer J Clin 58: 71–96. - PubMed
    1. Herbst RS, Lippman SM (2007) Molecular signatures of lung cancer—toward personalized therapy. N Engl J Med 356: 76–78. - PubMed
    1. Glittenberg M, Pitsouli C, Garvey C, Delidakis C, Bray S (2006) Role of conserved intracellular motifs in Serrate signalling, cis-inhibition and endocytosis. EMBO J 25: 4697–4706. - PMC - PubMed
    1. Bray SJ, Takada S, Harrison E, Shen SC, Ferguson-Smith AC (2008) The atypical mammalian ligand Delta-like homologue 1 (Dlk1) can regulate Notch signalling in Drosophila. BMC Dev Biol 8: 11. - PMC - PubMed
    1. Nueda ML, Baladron V, Sanchez-Solana B, Ballesteros MA, Laborda J (2007) The EGF-like protein dlk1 inhibits notch signaling and potentiates adipogenesis of mesenchymal cells. J Mol Biol 367: 1281–1293. - PubMed

Publication types

MeSH terms

Grants and funding

This work was funded by the Natural Science Foundation of China (30930042 and 81301852) and the Specialized Research Fund for the Doctoral Program of Higher Education (20111106110017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.