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. 2020 Feb 21;11(2):143.
doi: 10.1038/s41419-020-2344-0.

Deguelin suppresses non-small cell lung cancer by inhibiting EGFR signaling and promoting GSK3β/FBW7-mediated Mcl-1 destabilization

Feng Gao #  1   2   3 , Xinfang Yu #  4 , Ming Li #  5   6 , Li Zhou  7 , Wenbin Liu  8 , Wei Li  9 , Haidan Liu  10   11
Affiliations

Deguelin suppresses non-small cell lung cancer by inhibiting EGFR signaling and promoting GSK3β/FBW7-mediated Mcl-1 destabilization

Feng Gao et al. Cell Death Dis. .

Abstract

Activating mutations of epidermal growth factor receptor (EGFR) play crucial roles in the oncogenesis of human non-small cell lung cancer (NSCLC). By screening 79 commercially available natural products, we found that the natural compound deguelin exhibited a profound anti-tumor effect on NSCLC via directly down-regulating of EGFR-signaling pathway. Deguelin potently inhibited in vitro EGFR kinase activity of wild type (WT), exon 19 deletion, and L858R/T790M-mutated EGFR. The in silico docking study indicated that deguelin was docked into the ATP-binding pocket of EGFRs. By suppression of EGFR signaling, deguelin inhibited anchorage-dependent, and independent growth of NSCLC cell lines, and significantly delayed tumorigenesis in vivo. Further study showed that deguelin inhibited EGFR and downstream kinase Akt, which resulted in the activation of GSK3β and eventually enhanced Mcl-1 phosphorylation at S159. Moreover, deguelin promoted the interaction between Mcl-1 and E3 ligase SCFFBW7, which enhanced FBW7-mediated Mcl-1 ubiquitination and degradation. Additionally, phosphorylation of Mcl-1 by GSK3β is a prerequisite for FBW7-mediated Mcl-1 destruction. Depletion or pharmacological inactivation of GSK3β compromised deguelin-induced Mcl-1 ubiquitination and reduction. Taken together, our data indicate that enhancement of ubiquitination-dependent Mcl-1 turnover might be a promising approach for cancer treatment.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Deguelin is a candidate compound that inhibits non-small cell lung cancer (NSCLC) cells.
a The inhibitory efficacy of screened compounds on cell viability of HCC827 cells. b The structure of deguelin. c MTS assay analysis of cell viability in HBE and NL20 cells with deguelin treatment. d MTS assay analysis of cell viability in HCC827, H3255, H1975, and A549 cells with deguelin or gefitinib treatment. *p < 0.05, **p < 0.01, ***p < 0.001. e Anchorage-independent cell growth of HCC827, H3255, H1975, and A549 cells with deguelin or gefitinib treatment. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig. 2
Fig. 2. Deguelin inhibits EGFR kinase activity in vitro and ex vivo.
a Cell lysates (500 μg) from HCC827, H3255, H1975, and A549 cells were incubated with deguelin-Sepharose 4B beads (Sepharose 4B beads only as control) overnight at 4 °C. The beads were washed and boiled with loading buffer and subjected to immunoblotting (IB) analysis. b Active EGFR kinases, including EGFR Del E746-A750, EGFR L858R, EGFR L858R/T790M, and EGFR WT, were incubated with different concentrations of ATP overnight, followed by incubation with deguelin-Sepharose 4B beads for 4 h. The beads were washed and boiled with loading buffer, EGFR protein level was detected by IB analysis. c Deguelin inhibits WT and mutant EGFR kinase activities in a dose-dependent manner. Gefitinib was used as a positive control. *p < 0.05, **p < 0.01, ***p < 0.001. d Binding modes of deguelin with wildtype and mutated EGFRs predicted by molecular docking. Left, a cartoon representation of the deguelin-binding pocket in EGFR. Right, different binding modes of deguelin with four types of EGFR. The ligands were shown in yellow sticks, while proteins were depicted in cartoon representation with key residues indicated as gray sticks and labeled. Hydrogen bonds were shown as red dashed lines.
Fig. 3
Fig. 3. Deguelin suppresses EGFR signaling.
a Human NSCLC cells, including HCC827, H3255, H1975, and A549 cells, were treated with deguelin and gefitinib, whole-cell extracts (WCE) were collected and subjected to IB analysis using the primary antibodies as indicated. b Ba/F3 cells expressing EGFR WT and various EGFR mutants were pre-incubated with deguelin or gefitinib for 2 h followed by treatment with EGF or 15 min in 0.1% FBS containing cell culture medium. WCE was prepared and subjected to IB analysis.
Fig. 4
Fig. 4. Deguelin attenuates Mcl-1 expression in NSCLC cells.
a Human NSCLC cells, including HCC827, H3255, H1975, and A549 cells, were treated with deguelin for 24 h, WCE were collected and subjected to IB analysis. b HCC827 cells were transfected with siCtrl or siMcl-1, followed by treatment with deguelin for 24 h, WCE were collected and subjected to IB analysis. c HCC827 cells were transfected with Mcl-1 construct, followed by treatment with deguelin for 24 h, WCE were collected and subjected to IB analysis. d HCC827 cells were treated with DMSO, PD98059, deguelin, or LY294002 for 24 h, WCE were collected and subjected to IB analysis. e HCC827 cells were transfected with Myr-Akt1 construct, followed by treatment with deguelin for 24 h, WCE were collected and subjected to IB analysis. f HCC827 cells were treated with deguelin or SB216763 for 24 h, WCE were collected and subjected to IB analysis as indicated. g HCC827 cells stable expression sh-GFP or sh-GSK3β were treated with deguelin or DMSO for 24 h, WCE were collected and subjected to IB analysis as indicated. h HCC827 cells were transfected with GSK3β WT or GSK3β S9A construct, followed by treatment with deguelin for 24 h, WCE was collected and subjected to IB analysis.
Fig. 5
Fig. 5. Deguelin promotes Mcl-1 ubiquitination in NSCLC cells.
a HCC827 cells were transfected with various constructs as indicated and treated with deguelin for 24 h, then incubated with MG132 for another 6 h. WCE were harvested and subjected to in vivo ubiquitination assay. b HCC827 cells were transfected with various His-Ub mutant constructs as indicated. MG132 was added to the cell culture medium for 6 h before harvest. WCE was subjected to in vivo ubiquitination assay. c HCC827 (left) and H1975 (right) cells were treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was collected and subjected to endogenous Mcl-1 ubiquitination analysis. d HCC827 cells were treated with deguelin or/and SB216763 for 24 h, followed by incubation with MG132 for another 6 h. WCE was collected and subjected to endogenous Mcl-1 ubiquitination analysis. e HCC827 sh-GFP and sh-GSK3β stable cells were treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was harvested and subjected to endogenous ubiquitination analysis. f HCC827 cells were transfected with GSK3β WT or GSK3β S9A construct, treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was harvested and subjected to endogenous ubiquitination analysis.
Fig. 6
Fig. 6. FBW7 is required for deguelin-induced Mcl-1 down-regulation.
a HCC827 cells were treated with deguelin or DMSO for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to co-immunoprecipitation (co-IP) analysis. b HCC827 cells were transfected with various constructs and treated with deguelin or DMSO for 24 h, followed by incubation with MG132 for another 6 h. WCE was subjected to in vivo ubiquitination assay. c HCC827 sh-GFP and sh-FBW7 stable cells were treated with DMSO or deguelin for 24 h, followed by incubation with MG132 for another 6 h. WCE was harvested and subjected to endogenous ubiquitination analysis. d HCC827 cells were transfected with various constructs and treated with deguelin for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to co-IP analysis. e HCC827 cells were treated with deguelin or/and SB216763 for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to co-IP analysis. f HCC827 cells were transfected with various constructs and treated with deguelin for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to endogenous ubiquitination analysis. g HCC827 cells were transfected with various constructs and treated with deguelin for 24 h, followed by incubation with MG132 for another 6 h. Cell lysates were subjected to in vivo ubiquitination analysis. h HCC827 cells were transfected with various constructs and treated with cycloheximide (CHX). WCE was harvested at different time points and subjected to IB analysis. i, j Cell viability i, and colony formation j of HCC827 cells transfected with Mcl-1 WT or Mcl-1 5KR and treated with deguelin. *p < 0.05, **p < 0.01.
Fig. 7
Fig. 7. Deguelin inhibits tumor growth in vivo.
a, b Tumor volume a and tumor weight b of HCC827 xenograft tumors treated with vehicle control, deguelin, or gefitinib. c, d Tumor volume c and tumor weight d of H3255 xenograft tumors treated with vehicle control, deguelin, or gefitinib. e, f Tumor volume e, and tumor weight f of H1975 xenograft tumors treated with vehicle control, deguelin, or gefitinib. g, h Tumor volume g, and tumor weight h of A549 xenograft tumors treated with vehicle control, deguelin, or gefitinib. i Immunohistochemistry staining analysis of Ki67, p-EGFR, p-Akt, p-ERK1/2, and Mcl-1 in HCC827 xenograft tumors. *p < 0.05, **p < 0.01, ***p < 0.001.
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