Repression of YAP by NCTD disrupts NSCLC progression

Abstract

The efficacy of available lung cancer therapeutic interference is significantly limited by various resistance mechanisms to those drugs. Activation of the oncogene YAP underlying the initiation, progression, and metastasis of lung cancer associates with poor prognosis and confers drug resistance against targeted therapy. In this study, we evaluated the specificity of norcantharidin (NCTD) in repressing YAP to inhibit non-small cell lung carcinoma (NSCLC) progression. Our study revealed that YAP signal pathways were aberrantly activated in lung cancer tissues and cells which rendered more proliferative and invasive phenotypes to human lung cancer cells. We confirmed that NCTD specifically repressed YAP signaling pathway to interfere the YAP-mediated non-small cell lung carcinoma progression and metastasis via arresting cell cycle, enhancing apoptosis and inducing senescence. We also found NCTD-mediated repression of YAP decreased epithelial-to-mesenchymal transition (EMT) and reduced the motile and invasive cellular phenotype in vitro via enhancing E-cadherin and decreasing fibronectin/vimentin. Mechanistic investigations revealed that NCTD transcriptionally downregulated YAP and post-translationally modulated the subcellular redistribution of YAP between nucleus and cytoplasm. Collectively, our results indicated that NCTD is a novel therapeutic drug candidate for NSCLC which specifically and sensitively target YAP signal pathway.

Keywords: EMT; NCTD; YAP; cell cycle arrest; lung cancer.

Figure 1. Upregulation of YAP in lung cancer cells

(A) Gel-based RT-PCR with densitometric quatitation demonstrating elevated expression of YAP in human NSCLC tissues compared with their normal adjacent lung tissues. (B) Immunoblotting with densitometric quantitation demonstrating increased protein level of Yap and decreased p-YAP in human NSCLC tissues compared with their normal adjacent lung tissues. (C, D) Immunofluorescent staining of Yap proteins showing increased YAP accumulated in nuclear in NSCLC samples compared with their normal adjacent lung tissues while more YAP was localized in cytoplasm of those normal adjacent lung tissues. (E) Immunoblotting with densitometric quantitation demonstrating increased protein level of total Yap as well as nuclear Yap and decreased p-YAP in Calu-6, H1299, and A549 cells than normal cell line. **P < 0.001 ***P < 0.0001 by Student's t-test

Figure 2. Activation of YAP drives NSCLC progression

(A) Knockdown of YAP decreased transcription activity of YAP in A549 and H1299 cells as shown in Gel-based RT-PCR with densitometric quantitation. (B) Immunoblotting with densitometric quantitation demonstrating decreased YAP and Vimentin protein expressions and increased E-cadherin protein level in A549 cells with siRNA-YAP treatment. (C) Knockdown of Yap arrested clonal formation to delay the clonogenecity of A549 cells. (D) Scratch test showing that knockdown of Yap significantly decreased scar healing rates at 36 hours after siRNA-YAP treatment in A549 cells. **P < 0.001 ***P < 0.0001 by Student's t-test.

Figure 3. NCTD represses YAP signaling pathway

(A) Immunofluorescent staining of Yap proteins demonstrating that NCYD treatment at 8 μM for 36 hours enhanced the translocation of Yap from nuclear to cytoplasm in A549 cells. (B) Immunoblotting showing decreased YAP in nuclear and relatively enriched inside cytoplasm in A549 cells treated with NCTD at 8 μM for 36 h. (C, D) NCTD dose-dependently reduced Yap mRNA(C) and protein level (D, G), but increased phosphorylated YAP (D) in A549 cells for 72 hours. (E, F) NCTD time-dependently reduced Yap mRNA(E) and protein level (F) in A549 cells at 15 μΜ. (G, H) NCTD dose-dependently decreased CYR61 and CTGF at both mRNA(G) and protein (H) level from 2 μM to 16 μM in A549 cells for 72 hours. **P < 0.001 ***P < 0.0001 by Student's t-test.

Figure 4. NCTD interferes the YAP-mediated NSCLC cell proliferation

(A) Treatment of NCTD at15 μM for 72 h induced significant G2 arrests and Yap blocks the effect of NCTD in A549 cells as showing in both representative histograms of cell cycle distribution and their quantitation analysis. (B) In vitro proliferation assay demonstrating that NCTD at 15 μM significantly arrested cellular proliferation of A549 and H1299 cells with or without stably expressing YAP, YAPS127A, YAP and TEAD for 72 hours. (C) Cell growth assay showing that NCTD at 15 μM time-dependently and significantly arrested cellular proliferation of A549 cells with or without stably expressing YAP, YAPS127A, YAP and TEAD. (D) Soft agar colony formation assay demonstrating that ectopic expression of Yap significantly enhanced colony formation density which was blocked by NCTD at 15 μM for 72 hours in H1299 cells. ***P < 0.0001 by Student's t-test.

Figure 5. Yap blocks the NCTD-mediated NSCLC cell senescence and apoptosis

(A) SA-β-Gal assay showing that NCTD at 15 μM significantly increased cell senescence phenotype in A549 cells for 72 h, in which ectopic expression of YAP, YAPS127A, YAP and TEAD completely blocked NCTD-induced senescence enhancement. (B) Flow cytometric assay demonstrating that treatment of NCTD at 15 μM significantly induced apoptosis in A549 cells for 72 h, in which ectopic expression of YAP, YAPS127A, YAP and TEAD partially blocked NCTD-induced apoptosis. (C) Immunoblotting showing that NCTD dose-dependently increased pro-apoptosis protein caspase-3 in A549 and H1299 cells for 72 hours. ***P < 0.0001 by Student's t-test.

Figure 6. NCTD interferes the Yap-mediated NSCLC cell invasiveness and EMT

(A) Scratch assay showing treatment of NCTD at 15 μM for 12–24 hours dramatically decreased Yap-induced enhancement in A549 cell migration. (B) Matrigel invasion assay identified that treatment of NCTD at 15 μM for 72 hours dramatically decreased cell invasive growth and overexpressing YAP partially blocked NCTD-induced inhibition in cell invasive growth. (C, D) NCTD dose-dependently increased E-cadherin and increased fibronectin and vimentin in mRNA(C) and protein (D) level in A549 cells for 72 hours. ***P < 0.0001 by Student's t-test. **P < 0.001 ***P < 0.0001 by Student's t-test.