Nm23-H1-stabilized hnRNPA2/B1 promotes internal ribosomal entry site (IRES)-mediated translation of Sp1 in the lung cancer progression

Abstract

Our recent studies have indicated that specificity protein-1 (Sp1) accumulates substantially in the early stage of lung cancer but is partially decreased in the late stages, which is an important factor in the progression of the cancer. In this study, we found that Nm23-H1 and hnRNPA2/B1 could be recruited to the 5'UTR of Sp1 mRNA. In investigating the clinical relevance of Nm23-H1/Sp1 levels, we found a positive correlation between lung cancer patients with poor prognosis and low levels of Sp1 and Nm23-H1, suggesting an association between Nm23-H1/Sp1 levels and survival rate. Knockdown of Nm23-H1 inhibits lung cancer growth but increases lung cancer cell malignancy, which could be rescued by overexpression of Sp1, indicating that Nm23-H1-induced Sp1 expression is critical for lung cancer progression. We also found that Nm23-H1 increases the protein stability of hnRNPA2/B1and is thereby co-recruited to the 5'UTR of Sp1 mRNA to regulate cap-independent translational activity. Since the Sp1 level is tightly regulated during lung cancer progression, understanding the molecular mechanisms underlying the regulation by Nm23-H1/hnRNPA2B1 of Sp1 expression in the various stages of lung cancer will be beneficial for lung cancer therapy in the future.

Figure 1

Nm23-H1 and hnRNPA2/B1 overexpressed in lung cancer are recruited to the 5′-UTR of Sp1 mRNA. (A) In vitro transcribed pGEM-Sp1-5′-UTR RNA was used as a probe and was incubated with the total cell lysates of HeLa cells. The pulldown mixtures were analyzed using sliver staining and LC MS/MS. (B) Samples were harvested from cells with GFP-Nm23-H1 or myc-hnRNPA2/B1 overexpression for IP with antibodies against GFP or myc. RNA fragments were isolated from IP samples for RT-PCR. (C) Samples were collected from cells with GFP or GFP-Nm23-H1 expression for IP with anti-Nm23-H1 (a) or anti-GFP (b). IP samples were analyzed by Western blotting with antibodies against the indicated proteins. (D) The levels of Sp1, Nm23-H1and hnRNPA2/B1 in the various cancer cell lines were studied by Western blotting with antibodies against indicated proteins. (E,F) The levels of Sp1, Nm23-H1and hnRNPA2/B1 in mice with Kras^G12D- and EGFR^L858R-induced lung cancer were studied using IHC staining (E) and Western blotting (F). After three independent experiments, the levels were quantified and statistically analyzed with t-test, *p < 0.05; **p < 0.01; ***p < 0.005.

Figure 2

The relevance between Sp1/Nm23-H1 levels and the prognosis of lung cancer patients. (A) Samples from lung cancer patients were prepared for IHC staining with antibodies against Sp1 and Nm23-H1. (B) After IHC staining samples from 179 patients with anti-Sp1 antibodies and 185 patients with anti-Nm23-H1 antibodies, the relationship between Sp1/Nm23-H1 levels and the stage of lung cancer of the patients was analyzed. (C) The survival rate was analyzed between patients with higher Sp1 or lower Sp1 expression (a), patients with higher Nm23-H1 or lower Nm23-H1 expression (b), and between patients with higher Sp1/Nm23-H1 or lower Sp1/Nm23-H1 expression (c) by Kaplan–Meier analysis. The P-value was determined by a two-sided log-rank test. (D). The level of hnRNPA2/B1 in the clinical samples with high levels of Sp1 and Nm23-H1 or with low levels of Sp1 and Nm23-H1 was studied by Western blotting with anti-hnRNPA2/B1 antibodies.

Figure 3

Nm23-H1 regulates Sp1 and hnRNPA2/B1. (A–C) Nm23-H1 (A,B) or hnRNPA2/B1 (C) was knocked down in CL1-5 cells; thus, the mRNA and protein samples were harvested for real time-PCR and Western blotting with antibodies against indicated proteins. After three independent experiments, the levels of the proteins and mRNA were quantified and statistically analyzed with a t-test, *p < 0.05; **p < 0.01. (D) The pRF and pRSp1F plasmids were transferred into lung cancer cells with Nm23-H1 knockdown (a) or GFP-Nm23-H1 overexpression (b). After 24 h of incubation, cell extracts were collected for a luciferase activity assay and analyzed with t-test, *p < 0.05; ***p < 0.005. (E) Cells with or without knockdown of Nm23-H1 were treated with cyclohexmide. Samples harvested at the indicated time points were analyzed by Western blotting with antibodies against hnRNPA2/B1, Nm23-H1 and tubulin (a). After three independent experiments, the protein level of hnRNPA2/B1 was quantified (b). Samples were harvested from H1299 cells with or without GFP-Nm23-H1 expression treated MG132 (10 μM) for IP with anti-hnRNPA2/B1 antibodies, and then samples were analyzed by Immunoblotting assay with indicated antibodies (c).

Figure 4

Nm23-H1 and hnRNPA2/B1 increase cell proliferation. (A,B) Nm23-H1 and hnRNPA2/B1 were silenced in CL1-5 and H1299 cell lines, respectively, for cell counting (A) and a colony-formation assay (B). After three independent experiments, the levels were quantified and statistically analysis with a t-test, *p < 0.05; **p < 0.01; ***p < 0.005.

Figure 5

The role of Nm23-H1-mediated Sp1 expression in the epithelial and mesenchymal transition of lung cancer cells. (A) Morphology of CL1-5 cells with Nm23-H1 knockdown (a) or overexpression (b) was observed with a microscope (20x), and the samples harvested from GFP-Nm23-H1-over expressed cells were analyzed by Western blotting with antibodies against the indicated proteins (c). (B,C) The distribution of F-actin in the knockdown Nm23-H1 (B) or hnRNPA2/B1 (C) in CL1-5 and H1299 cell lines was studied by immunostaining with anti-F-actin antibodies (a). The EMT-related proteins were studied by Western blotting with antibodies against the indicated proteins (b). (D) Cell morphology was observed in Nm23-H1 knockdown cells with GFP or GFP-Sp1 overexpression (a), and various EMT-related proteins were analyzed by Western blotting with antibodies against the indicated proteins (b).

Figure 6

Nm23-H1-mediated Sp1 expression is involved in lung cancer malignancy. (A,B) The cell migration ability was assayed with a wound healing assay (a) and transwell assay (b) in Nm23-H1 knockdown (A) or GFP-Nm23-H1-overexpressed cells (B). After three independent experiments, the migratory area and cell number were quantified and statistical analysis was done with a t-test, *p < 0.05; ***p < 0.005. (C) CL1-5 cells with Nm23-H1 knockdown were injected into the lateral tail vein of SCID mice. Six weeks later, all mice were sacrificed and the number of tumor nodules was calculated (a). Tumor formation on the lung was studied by H&E staining (b). (D) Cell mobility was assayed in the Nm23-H1-silenced cells with or without GFP-Sp1 expression by transwell assay (a). After three independent experiments were finished, the cell number in the chamber was quantified, and the statistical analysis was performed with a t-test, ***p < 0.005 (b).

Figure 7

The effect of hnRNPA2/B1 on Sp1-mediated lung cancer migration. (A,B) Cell mobility was assayed with a wound healing assay (a) and transwell assay (b) in hnRNPA2/B1 knockdown (A) or myc-hnRNPA2/B1-overexpressed cells (B). After three independent experiments, the migratory area and cell number were quantified, and statistical analysis was done with a t-test, *p < 0.05; ***p < 0.005. (C) The protein levels and cell mobility were assayed on the hnRNPA2/B1-silenced cells with or without GFP-Sp1 expression by Western blotting with antibodies against the indicated proteins (a) and by transwell assay (b). After three independent experiments were finished, the cell number on the chamber was quantified and the statistically analysis was performed with t-test, ***p < 0.005.

Figure 8

Akt activation might be involved in the Nm23-H1-mediated IRES-translational activity of Sp1 mRNA. (A) Samples were harvested from H1299 and CL1-5 cell lines treated with Akt inhibitors, Ly294002 and MK2206, and were analyzed by Western blotting with antibodies against the indicated proteins (a). After three independent experiments, the proteins levels were quantified and statistical analysis was done with a t-test, *p < 0.05; **p < 0.01; ***p < 0.005 (b). (B–F) Samples harvested from H1299 cells with overexpression of GFP, GFP-Nm23-H1, GFP-Nm23-H1(S120A) or GFP-Nm23-H1(S122A) were analyzed by Western blotting with antibodies against the indicated proteins and real time PCR (B), 5′UTR-driven luciferase activity (C), RNA-IP with anti-GFP antibodies (D), cell counting (E) and a colony-formation assay (F). After three independent experiments, the proteins levels were quantified and statistical analysis was done with a t-test, *p < 0.05; **p < 0.01; ***p < 0.005. (G) The proposed model shows that Nm23-H1 positively regulates Sp1 expression through an increase in its translational activity in a cap-independent manner.