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. 2022 Jan 24;8(1):36.
doi: 10.1038/s41420-022-00818-8.

Targeting ADRB2 enhances sensitivity of non-small cell lung cancer to VEGFR2 tyrosine kinase inhibitors

Yingzhuo Xu #  1 , Jian Wang #  1 , Xu Wang #  1 , Xiaoshu Zhou  1 , Jing Tang  1 , Xiaohua Jie  1 , Xijie Yang  1 , Xinrui Rao  1 , Yunhong Xu  1 , Biyuan Xing  1 , Zhenyu Li  2 , Gang Wu  3
Affiliations

Targeting ADRB2 enhances sensitivity of non-small cell lung cancer to VEGFR2 tyrosine kinase inhibitors

Yingzhuo Xu et al. Cell Death Discov. .

Abstract

Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) tyrosine kinase inhibitors (TKIs) have achieved remarkable clinical progress in the treatment of non-small-cell lung cancer; however, resistance has limited their therapeutic efficacy. Therefore, understanding the mechanisms of VEGF-TKI and ICI resistance will help to develop effective treatment strategies for patients with advanced NSCLC. Our results suggested that treatment with VEGFR2-TKIs upregulated ADRB2 expression in NSCLC cells. Propranolol, a common ADRB2 antagonist, significantly enhanced the therapeutic effect of VEGFR2-TKIs by inhibiting the ADRB2 signaling pathway in NSCLC cells in vitro and in vivo. Mechanically, the treatment-induced ADRB2 upregulation and the enhancement of ADRB2/VEGFR2 interaction caused resistance to VEGFR2-TKIs in NSCLC. And the inhibition of the ADRB2/CREB/PSAT1 signaling pathway sensitized cells to VEGFR2-TKIs. We demonstrated that ADRB2 signaling is crucial in mediating resistance to VEGFR2-TKIs and provided a novel promising combinatory approach to enhance the antitumor effect of VEGFR2-TKIs in NSCLC combining with propranolol.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. ADRB2 expression is upregulated by VEGFR2-TKIs treatment in NSCLC.
A The overlapping analysis of apatinib upregulated genes and neuroactive ligand associated genes. B The results of the overlapped genes in RNA-seq data. C H522, H460, and A549 cells were treated with apatinib (20 μM) for 48 h. Western blotting was used to analyze the ADRB2 protein level. D RT-PCR was used to analyze the expression of ADRB2 mRNA. E The expression of ADRB2 was demonstrated by flow cytometry. F Statistical analysis of flow cytometry. G Representative images of immunofluorescent staining for ADRB2. H H522 and H460 cells were exposed to vehicle or anlotinib. The expression of ADRB2 was demonstrated by flow cytometry. I Statistical analysis of flow cytometry. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, compared with control).
Fig. 2
Fig. 2. ADRB2 agonist terbutaline (TERB) promoted apatinib resistance in NSCLC.
A H522 and H460 cells were exposed to vehicle or TERB (25 μM). IC50 of apatinib was measured with CCK8 assay. B Pictures of colony formation in H522 and H460 cells treated with vehicle, TERB, apatinib, or both modalities. C The clone numbers in four groups. D Representative images of EdU (EdU labeled with red, nuclei with blue). E The percentages of positive cells in four groups. (**P < 0.01, ***P < 0.001, NS not significant, compared with apatinib).
Fig. 3
Fig. 3. ADRB2 antagonist propranolol combined with apatinib inhibits cell proliferation.
A H522 and H460 cells were exposed to various concentrations of apatinib and propranolol for 48 h, and CCK8 assay was used to detect the cell viability. B Combination index (CI) was determined by CompuSyn software. C the CCK-8 assay detected the viability of H522 and H460 cells after anlotinib and propranolol treatment. D Representative images of H522 and H460 clones treated with DMSO, propranolol, apatinib, or both modalities. E The clone numbers in four groups. F Representative images of EDU (Red, EdU marked proliferative cells, blue, Hoechst 33342 marked nuclei). G The percentages of positive cells in four groups. (***P < 0.001, ****P < 0.0001, compared with apatinib).
Fig. 4
Fig. 4. ADRB2 antagonist propranolol combined with apatinib can promote apoptosis in NSCLC.
A H522 and H460 cells were treated with/without apatinib (20 μM) and propranolol (50 μM) for 48 h. The apoptosis index was detected by flow cytometry. B The percentage of apoptotic cells in H522 and H460 cells. C Western blot revealed expression level of caspase3 and cleaved-caspase3 protein. D H522 and H460 cells were treated with/without anlotinib (2.5 μM) and propranolol (50 μM) for 48 h. The apoptosis index was detected by flow cytometry. E The percentage of apoptotic cells in H522 and H460 cells. F Flow cytometry showed the proportion of cells at different stages of cell cycle. The percentages of cell cycle phases in four groups. G Flow cytometry was used to analyze ROS levels in H522 and H460 cells. (*P < 0.05; **P < 0.01; **P < 0.001; ****P < 0.0001, compared with apatinib).
Fig. 5
Fig. 5. The mechanism of apatinib resistance in NSCLC cells.
A H522 and H460 cells were treated with or without apatinib (20 μM) and propranolol (50 μM) for 48 h. The relative protein level of CREB, p-CREB, PSAT1 was detected through Western blot. B A positive correlation between CREB and PSAT1 expression in lung cancer according to TCGA database. C qRT-PCR analysis of PSAT1 mRNA levels in H522 and H460 cells after KG-501 treatment. D Western blot analysis of CREB, p-CREB, PSAT1 expression in H522 and H460 cells after ADRB2 knockdown. E H522 and H460 cells were transfected with indicated siRNAs for 48 h, and the samples were exposed to different concentrations of apatinib. IC50 of apatinib was measured with CCK8 assay. F H460 cells were treated with/without apatinib (20 μM) and KG-501 (25 μM) for 48 h. The apoptosis index was detected by flow cytometry. G The percentage of apoptotic cells in H522 and H460 cells. H Representative co-localization images stained with ADRB2 (green) and VEGFR2 (red) in H522 and H460 cells. I Anti-ADRB2 antibodies immunoprecipitated endogenous ADRB2 and Co-IP VEGFR2 by Western blot analysis. (**P < 0.01, compared with vehicle).
Fig. 6
Fig. 6. Combined apatinib and propranolol treatment significantly decreases H460-xenograft growth in vivo.
A Mice were grouped for treatment with 0.9% normal saline group, apatinib (50 mg/kg/day), propranolol (40 mg/kg/day), or both modalities by intragastric injection during the study. Tumors were dissected from nude mice. The inset picture shows the tumor in the respective group. B The growth curve of tumor volume was shown. The data represent mean tumor volumes ± SEMs. C Tumor weight of four groups was compared on the last day. The tumor volumes and weights were presented as means ± SDs (n = 6 for each group). D Body weight changes of mice during drug administration. The data were presented as the mean of tumor volumes ± SEMs. E Immunohistochemistry staining of Ki67 of tumor sections in each group. F IHC score of Ki67 staining. Pictures were captured at a light microscope with ×20 magnifications. (compared with apatinib) G ADRB2 immunohistochemical staining of lung tumors in each group. H IHC score of ADRB2 staining. Pictures were captured at a light microscope with ×20 magnifications. (*P < 0.05, **P < 0.01, compared with control).
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