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. 2022 Mar;13(3):6895-6907.
doi: 10.1080/21655979.2021.1977105.

Silencing of A-kinase anchor protein 4 inhibits the metastasis and growth of non-small cell lung cancer

Bo Zhang  1 , Quanteng Hu  1 , Jian Zhang  1 , Zixian Jin  1 , Yuhang Ruan  1 , Lilong Xia  2 , Chunguo Wang  1
Affiliations

Silencing of A-kinase anchor protein 4 inhibits the metastasis and growth of non-small cell lung cancer

Bo Zhang et al. Bioengineered. 2022 Mar.

Abstract

Non-small cell lung cancer (NSCLC) is one of the most malignant tumors. The treatment of advanced NSCLC can be challenging due to drug resistance. The discovery of novel cancer-testis antigens to develop new strategies for advanced metastatic NSCLC is required. AKAP4 is an oncogene discovered in some malignant tumors, and its molecular function of AKAP4 in NSCLC is unknown. This study aimed to explore the potential function of AKAP4 in the development and progression of NSCLC. AKAP-4 was found to be significantly upregulated in both clinical NSCLC tissues and NSCLC cell lines. Cell viability and migration were suppressed, apoptosis was induced, and tube formation was inhibited by the knockdown of AKAP-4, accompanied by the downregulation of VEGF, N-cadherin, EphA2, and MMP-2, and upregulation of c-AMP, PKA, and E-cadherin. In vivo xenograft experiments revealed that tumor growth was inhibited by the knockdown of AKAP4, accompanied by the activation of c-AMP/PKA signaling and inhibition of epithelial-mesenchymal transition progression. Our results show that AKAP4 might be an important target for treating NSCLC because of its function in promoting the migration and proliferation of NSCLC cells.

Keywords: AKAP4; PKA; migration; non-small cell lung cancer.

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Conflict of interest statement

The authors declare there is no conflicts of interest regarding the publication of this paper.

Figures

Figure 1.
Figure 1.
A high expression level of AKAP4 was observed in both clinical NSCLC tissues and NSCLC cell lines. (a). RT-PCR was used to determine the gene expression level of AKAP-4 in the NSCLC tissues and para-carcinoma tissues. (b). Immunohistochemical assay was used to determine the protein expression level of AKAP-4 in the NSCLC tissues and para-carcinoma tissues (**p < 0.01 vs. para-carcinoma tissues). (c). Western blotting assay was used to determine the expression level of AKAP4 in the NSCLC cell line and HEAS-2B cells (**p < 0.01 vs. HEAS-2B cells).
Figure 2.
Figure 2.
Proliferation was suppressed by the knockdown of AKAP4. (a). The gene expression level of AKAP4 was detected by RT-PCR assay. (b). The protein expression level of AKAP4 was detected by western blotting assay. (c). The cell viability was measured by CCK-8 assay. (d). The apoptosis was determined by flow cytometry (**p < 0.01 vs. siNC). The control was H460 cells incubated with blank completed medium.
Figure 3.
Figure 3.
Migration and angiogenesis were inhibited by the knockdown of AKAP4. (a). Transwell assay was used to determine the migration ability of H460 cells. (b). Tube formation assay was used to evaluate the angiogenesis of HUVECs. C. The expression level of VEGF was determined by western blotting assay (**p < 0.01 vs. siNC). The control was H460 cells incubated with blank completed medium.
Figure 4.
Figure 4.
The c-AMP/PKA signaling was activated, and EMT progression was suppressed by the knockdown of AKAP4. (a). The production of c-AMP was measured by ELISA. (b). The expression level of PKA, E-cadherin, N-cadherin, EphA2, and MMP-2 was determined by western blotting assay (**p < 0.01 vs. siNC). The control was H460 cells incubated with blank completed medium.
Figure 5.
Figure 5.
The in vivo growth of the tumor was inhibited by the knockdown of AKAP4. (a). Tumor volumes were calculated every week after the planting. (b). Tumor weights were weighed at the end of the experiments. (c). The images of the tumor tissues in each group. (d). The expression level of VEGF was determined by western blotting assay. (e). The expression level of VEGF was determined by immunohistochemical assay (**p < 0.01 vs. siNC). The controls were nude mice planted with H460 cells incubated with blank completed medium.
Figure 6.
Figure 6.
The c-AMP/PKA signaling in tumor tissues was activated by the knockdown of AKAP4. The expression level of AKAP4, c-AMP, and PKA was determined by immunohistochemical assay (**p < 0.01 vs. siNC). The controls were nude mice planted with H460 cells incubated with blank completed medium.
Figure 7.
Figure 7.
The EMT progression in tumor tissues was inhibited by the knockdown of AKAP4. The expression level of E-cadherin, N-cadherin, EphA2, and MMP-2 was determined by western blotting assay (**p < 0.01 vs. siNC). The controls were nude mice planted with H460 cells incubated with blank completed medium.
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Grants and funding

This work was supported by grants from the Taizhou Science and Technology Bureau (Grant No. 1702KY01].