Targeting PRMT5/Akt signalling axis prevents human lung cancer cell growth

Abstract

The emerging evidence reveals that protein arginine methyltransferase 5 (PRMT5) is involved in regulation of tumour cell proliferation and cancer development. Nevertheless, the exact role of PRMT5 in human lung cancer cell proliferation and the underlying molecular mechanism remains largely obscure. Here, we showed that PRMT5 was highly expressed in human lung cancer cells and lung cancer tissues. Furthermore, we generated PRMT5 stable knockdown cell lines (A549 and H1299 cells) and explored the functions of PRMT5 in lung cancer cell proliferation. We found that the down-regulation of PRMT5 by shRNA or the inhibition of PRMT5 by specific inhibitor GSK591 dramatically suppressed cyclin E1 and cyclin D1 expression and cell proliferation. Moreover, we uncovered that PRMT5 promoted lung cancer cell proliferation via regulation of Akt activation. PRMT5 was directly co-localized and interacted with Akt, but not PTEN and mTOR. Down-regulation or inhibition of PRMT5 markedly reduced Akt phosphorylation at Thr308 and Ser473, whereas the expression of PTEN and mTOR phosphorylation was unchanged, indicating that PRMT5 was an important upstream regulator of Akt and induced lung cancer cell proliferation. Altogether, our results indicate that PRMT5 promotes human lung cancer cell proliferation through direct interaction with Akt and regulation of Akt activity. Our findings also suggest that targeting PRMT5 may have therapeutic potential for treatment of human lung cancer.

Keywords: Akt; PRMT5; PTEN; lung cancer; mTOR; proliferation.

Figure 1

PRMT5 is overexpressed in human lung cancer cells and tissues. (A) PRMT5 protein expression level was detected by Western blotting in different human lung cancer cell lines compared with normal human foetal lung fibroblast cells (IMR90). (B) Quantitative analysis of PRMT5 protein expression level in different human lung cancer cell lines compared with IMR90. *P < 0.05 vs IMR90. (C) PRMT5 mRNA expression level was detected by qRT‐PCR in normal tissues and lung cancer tissues. *P < 0.05 vs normal tissues. (D) PRMT5 protein expression level was determined by Western blotting in normal tissues and lung cancer tissues. (E) Quantitative analysis of PRMT5 protein expression level in normal tissues and lung cancer tissues. *P < 0.05 vs normal tissues

Figure 2

Knockdown of PRMT5 suppresses proliferation of lung cancer cells. (A, B) A549 and H1299 cells were infected with lentivirus containing PRMT5 and scramble (scr) shRNA and PRMT5 mRNA expression level was measured by qRT‐PCR. *P < 0.05 vs scr. (C, D) A549 and H1299 cells were infected with lentivirus containing PRMT5 and scramble (scr) shRNA and the PRMT5 protein expression level was detected by Western blotting. (E, F) Quantitative analysis of PRMT5 protein expression level in A549 and H1299 cells. *P < 0.05 vs scr. (G, H) A549 and H1299 cells were infected with lentivirus containing PRMT5 and scramble (scr) shRNA and the cell proliferation were measured by CCK‐8 assay at the indicated time points. *P < 0.05 vs scr. (I) The cyclin E1 and cyclin D1 expression level was detected by Western blotting when PRMT5 was down‐regulated in A549 and H1299 cells. (J, K) Quantitative analysis of cyclin E1 and cyclin D1 protein expression level in A549 and H1299 cells. *P < 0.05 vs scr

Figure 3

Blocking PRMT5 by GSK591 represses proliferation of lung cancer cell. (A) IMR90 and A549 cells were treated with PRMT5 inhibitor GSK591 or vehicle at the indicated concentrations for 4 days and cell proliferation was measured by CCK‐8 assay. *P < 0.05 IMR90 vs A549 cells. (B) IMR90 and H1299 cells were treated with PRMT5 inhibitor GSK591 or vehicle at the indicated concentrations for 4 days and cell proliferation was measured by CCK‐8 assay. *P < 0.05 IMR90 vs H1299 cells. (C) A549 and H1299 cells were treated with PRMT5 inhibitor GSK591 (1 μmol/L) or vehicle and the cyclin E1 and cyclin D1 protein expression level was detected by Western blotting. (D, E) Quantitative analysis of cyclin E1 and cyclin D1 protein expression level in A549 and H1299 cells. *P < 0.05 vs vehicle

Figure 4

PRMT5 directly interacts with Akt in lung cancer cells. (A) The co‐localization of endogenous PRMT5 and p‐Akt‐Ser473 (activated Akt) were shown in A549 cells using immunofluorescence. (B) A549 cells were infected with lentivirus containing Akt and scramble shRNA and cell extracts from A549 cells were immunoprecipitated with an antibody against Akt and immunoblotted with an antibody against PRMT5. (C) A549 cells were infected with lentivirus containing PRMT5 and scramble shRNA and cell extracts from A549 cells were immunoprecipitated with an antibody against PRMT5 and immunoblotted with an antibody against Akt. (D) A549 cells were infected with lentivirus containing PRMT5 and scramble shRNA and cell extracts from A549 cells were immunoprecipitated with an antibody against PRMT5 and immunoblotted with an antibody against PTEN. (E) A549 cells were infected with lentivirus containing PRMT5 and scramble shRNA and cell extracts from A549 cells were immunoprecipitated with an antibody against PRMT5 and immunoblotted with an antibody against mTOR. (F) The co‐localization of endogenous PRMT5 and p‐Akt‐Ser473 (activated Akt) were shown in H1299 cells using immunofluorescence. (G) H1299 cells were infected with lentivirus containing Akt and scramble shRNA and cell extracts from H1299 cells were immunoprecipitated with an antibody against Akt and immunoblotted with an antibody against PRMT5. (H) H1299 cells were infected with lentivirus containing PRMT5 and scramble shRNA and cell extracts from H1299 cells were immunoprecipitated with an antibody against PRMT5 and immunoblotted with an antibody against Akt. (I) H1299 cells were infected with lentivirus containing PRMT5 and scramble shRNA and cell extracts from H1299 cells were immunoprecipitated with an antibody against PRMT5 and immunoblotted with an antibody against PTEN. (J) H1299 cells were infected with lentivirus containing PRMT5 and scramble shRNA and cell extracts from H1299 cells were immunoprecipitated with an antibody against PRMT5 and immunoblotted with an antibody against mTOR

Figure 5

PRMT5 regulates Akt activation in lung cancer cell. (A) A549 and H1299 cells were infected with lentivirus containing PRMT5 and scramble shRNA and the indicated proteins were detected by Western blotting. (B, C) Quantitative analysis of indicated proteins expression level in A549 and H1299 cells. *P < 0.05 vs scr. (D) A549 and H1299 were treated with PRMT5 inhibitor GSK591 (1 μmol/L) or vehicle for 4 days and indicated proteins were detected by Western blotting. (E, F) Quantitative analysis of indicated proteins expression level in A549 and H1299 cells upon PRMT5 inhibitor stimulation. *P < 0.05 vs vehicle. (G, H) A549 and H1299 cells were infected with lentivirus containing PRMT5 and scramble shRNA and the kinase activity of Akt was evaluated by in vitro kinase assay kit. (I) Western blotting analysis of indicated proteins expression level from paired human lung cancer tissues and adjacent normal tissues. (J) Quantitative analysis of indicated proteins expression level in human lung cancer tissues and normal tissues. *P < 0.05 vs normal tissues