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. 2015 Oct 27;6(33):34953-67.
doi: 10.18632/oncotarget.5547.

Skp1 in lung cancer: clinical significance and therapeutic efficacy of its small molecule inhibitors

Yong-Qiang Liu  1 , Xiao-Lu Wang  1 , Xin Cheng  1 , Yong-Zhi Lu  2 , Gui-Zhen Wang  1 , Xin-Chun Li  1 , Jian Zhang  3 , Zhe-Sheng Wen  4 , Zhi-Liang Huang  4 , Qin-Lei Gao  5 , Li-Na Yang  6 , Yong-Xian Cheng  5 , Sheng-Ce Tao  6 , Jinsong Liu  2 , Guang-Biao Zhou  1
Affiliations

Skp1 in lung cancer: clinical significance and therapeutic efficacy of its small molecule inhibitors

Yong-Qiang Liu et al. Oncotarget. .

Abstract

Skp1 is an essential adaptor protein of the Skp1-Cul1-F-box protein complex and is able to stabilize the conformation of some ubiquitin E3 ligases. However, the role Skp1 plays during tumorigenesis remains unclear and Skp1-targeting agent is lacking. Here we showed that Skp1 was overexpressed in 36/64 (56.3%) of non-small cell lung cancers, and elevated Skp1 was associated with poor prognosis. By structure-based high-throughput virtual screening, we found some Skp1-targeting molecules including a natural compound 6-O-angeloylplenolin (6-OAP). 6-OAP bound Skp1 at sites critical to Skp1-Skp2 interaction, leading to dissociation and proteolysis of oncogenic E3 ligases NIPA, Skp2, and β-TRCP, and accumulation of their substrates Cyclin B1, P27 and E-Cadherin. 6-OAP induced prometaphase arrest and exerted potent anti-lung cancer activity in two murine models and showed low adverse effect. These results indicate that Skp1 is critical to lung cancer pathogenesis, and Skp1 inhibitor inactivates crucial oncogenic E3 ligases and exhibits significant therapeutic potentials.

Keywords: 6-O-angeloylplenolin; Skp1; inhibitors; lung cancer; structure-based high-throughput virtual screening.

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Conflict of interest statement

CONFLICTS OF INTEREST

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Skp1 in lung cancer
A. Representative Western blot analyses of lysates of tumor and adjacent normal lung tissues harvested from NSCLCs (n = 64). B. The densitometry analysis of the Western blot results. C. Immunohistochemistry of Skp1 in NSCLCs using an anti-Skp1 antibody. Size bar, 50 μm. D. The immunoreactivity score was calculated. E. Overall survival of the 64 patients. F–J. A549 and H1975 cells were transfected with Skp1 specific siRNAs (F), the cell proliferation were analyzed by trypan blue exclusion analyses (G), and the clonogenic activity of cells was tested by the Flat plate clone formation assay (H, I). The cell cycle distribution of H1975 cells were analyzed (J). K, L. Effects of three Skp1-targeting compounds on lung cancer cells. The compounds were identified by structure-based high-throughput virtual screening for Skp1 inhibitors (See also Figure S1). Synchronous or asynchronous H1975 cells were treated with or without the compounds, and cell cycle distribution was determined (K). Western blot analysis of lysates of the cells treated with indicated compounds (L). Evo, Evodiamine; Lir, Liriodenine; 6-OAP, 6-O-angeloylplenolin.
Figure 2
Figure 2. 6-OAP directly binds Skp1 and interferes with SCFNIPA
A. Two potential binding pockets (P1 and P2) of Skp1 for 6-OAP, revealed by docking 6-OAP to Skp1 (PDB code: 2AST). In the left panel, Skp2 (shown as cartoon) interacts with Skp1 (shown as surface) via P1 and P2, and only Skp1 is used during the docking. In the middle and right panels, 6-OAP (shown as sticks) is predicted to interact with Skp1 (shown as cartoon) via P1 and P2, respectively. B. Chemical structure of 6-OAP and Bio-6-OAP. C. Images of Bio-6-OAP-Skp1 interaction on the slides. D. H1975 cells were treated with Biotin or Bio-6-OAP at 50 μM for 6 h, lysed, and the cell lysates were subjected to immunoprecipitation using streptavidin agarose and Western blot using indicated antibodies. E. H1975 cells were treated with Bio-6-OAP (50 μM) in the presence or absence of 6-OAP (100 μM) for 6 h, lysed, and the cell lysates were subjected to immunoprecipitation and Western blot. F. 293T cells were transfected with wild type (WT) or mutant Skp1 for 48 h, lysed, the lysates were subjected to immunoprecipitation using streptavidin (S.) agarose and Western blot using indicated antibodies. G. The cells were treated with 6-OAP, lysed, and subjected to Western blot. H. H1975 cells were treated with or without 6-OAP for 3 h, lysed, and immunoprecipitation and Western blot assays were performed (left panel). 293T cells were transfected with pcDNA3.1-flag-NIPA, treated with or without 6-OAP, and lysed for immunoprecipitation and Western blot (right panel). I. Cells were treated with 6-OAP, lysed, and Western blot was performed. J. An in vitro ubiquitination assay using SCFNIPA, Cyclin B1, and 6-OAP. K. A549 cells were synchronized to G1/S boundary and released, and treated with or without 6-OAP. Cell cycle distribution was determined (left), and the expression of NIPA and Cyclin B1 was analyzed by Western blot (right). L. A549 cells transfected with control or NIPA specific siRNA were treated with 6-OAP for 12 h, harvested for Western blot (upper) or flow cytometry analysis (lower). M. A549 cells transfected with NIPA-specific siRNA were treated with or without 6-OAP (7.5 μM) for 12 h, and analyzed by immunofluorescence labeling with anti-centromere sera, anti-α-tubulin antibody, and DAPI. Size bar, 5 μm.
Figure 3
Figure 3. Effects of 6-OAP on Skp2, β-TRCP, Fbxw7 and their substrate proteins
A. H1975 cells were treated with or without 6-OAP for 3 h, lysed, and immunoprecipitation and Western blot were performed using indicated antibodies. B. The cells were treated with 6-OAP, lysed and subjected to Western blot. C, D. 6-OAP inhibited ubiquitination of E-Cadherin, revealed by immunoprecipitation and Western blot in H1975 cells upon 6-OAP (C) and an in vitro ubiquitination assay using SCFSkp2, purified E-Cadherin and 6-OAP (D). E, F. H1975 cells were treated with 6-OAP, lysed, and analyzed by immunoprecipitation and Western blot. G. 293T cells were transfected with Flag-Skp1 and treated with 6-OAP, lysed, and the lysates were subjected to immunoprecipitation and Western blot assays.
Figure 4
Figure 4. 6-OAP induces prometaphase arrest in lung cancer cells
A. The GI50s of 6-OAP in cells is associated with the relative Skp1 expression. B. Effects of 6-OAP on clonogeinc activity of lung cancer cells. C. The synchronized or asynchronous (asyn) to G1/S boundary cells were treated with 6-OAP at indicated concentrations for 12 h. Cell cycle distribution was determined by propidium iodide (PI) staining and flow cytometry analysis. D. The cells were treated with 6-OAP, lyzed, and Western blot was performed using antibodies indicated. E. The cells were treated with 6-OAP, taxol (50 nM) or Vincristine (VCR; 50 nM) for 12 h. The polymerized (P) and soluble (S) tubulin fractions were prepared and subjected to Western blot using anti-α-tubulin antibody. T-α-tubulin, total-α-tubulin. F. A549 cells were treated with 7.5 μM 6-OAP for 12 h, and assayed by immunofluorescence labeling with anti-centromere sera (green), anti-α-tubulin antibody to visualize microtubules (red), and DAPI to counter stained DNA (blue). Size bar, 5 μm. G. A549 cells were treated with 7.5 μM 6-OAP for 12 h or 10 μM MG-132 for 3 h, and incubated in ice-cold media for 10 min and stained with anti-centromere sera, anti-α-tubulin antibody, and DAPI. H, I. The A549 (H) and 293T (I) cells were transfected with Skp1, synchronized at G1/S boundary site by thymidine treatment, and treated with 6-OAP for 12 hours. The cells were analyzed by flow cytometry to evaluate the cell cycle distribution, or lysed for Western blot analysis. Numbers under the NIPA, Skp2, and Cyclin B1 bands are the relative expression values to Actin determined by densitometry analysis. *p = 0.04.
Figure 5
Figure 5. In vivo anti-lung cancer efficacy and acute toxicity of 6-OAP
A. Nude mice subcutaneously inoculated with H1975 cells were treated with 6-OAP or vehicle for 24 days, and tumor volume was estimated every two days. B. Images of xenograft tumors obtained from the mice. C. Western blot assays using lysates of isolated tumors and indicated antibodies. D. A549-Luciferase cells were intravenously injected into SCID mice, and 1 week later the mice were randomized to receive vehicle (n = 6) or 6-OAP treatment (n = 12). The mice were detected by IVIS Spectrum. E. The relative luciferase intensity in the mice. F. Lung tissue sections of mice treated with 6-OAP or vehicle control were stained with hematoxylin-eosin and analyzed using a research microscope. Size bar, 100 μm. G. Life span of the mice. H. Western blot analysis using lysates of tumor samples isolated from the mice. I. Intravenous injection of 6-OAP at 225 to 600 mg/kg did not significantly affect the body weight of Kunming mice. J, K. The serum AST (J) and CR (K) levels were detected after intravenous injection of 6-OAP or vehicle in Kunming mice. L. A schematic representation of mechanisms of 6-OAP-induced mitosis arrest.
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