Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

Abstract

Background: Roles of cancer stem cells and early growth response gene 1 (Egr1) in carcinogenesis have been extensively studied in lung cancer. However, the role of Egr1 in the metastasis of lung cancer remains undetermined, especially in regard to stem cell-related pathways.

Methods: Egr1, osteopontin (OPN) and Oct4 expression in human lung cancer was determined by performing immunohistochemistry. Immunoblotting, ELISA, luciferase reporter assay, chromatin immunoprecipitation assay and RT-PCR were performed to validate the regulation of Oct4-Egr1-OPN axis. Moreover, the effect of Oct4-Egr1-OPN axis on lung cancer progression was evaluated by cell migration assay and mice study.

Results: We detected Oct4, Egr1, and OPN expression in clinical specimens from 79 lung cancer patients, including 72 adenocarcinomas and 7 squamous cell carcinomas. High expression of Oct4, Egr1, and OPN accounted for 53, 51, and 57% of the patients, respectively. All of the three biomarkers were positively correlated in clinical human lung cancer. Patients with high expression of OPN were significantly associated with shorter disease-free survivals than those with low expression of OPN (p < 0.05). In lung cancer cells, Oct4 transactivated the Egr1 promoter and upregulated Egr1 expression. In a human lung cancer xenograft model, Oct4-overexpressing tumors expressed elevated levels of Egr1. Furthermore, overexpression of Oct4 in lung cancer cells increased the metastatic potential.

Conclusions: Egr1 exerts a promoting effect on cancer metastasis in Oct4-overexpressing lung cancer. Thus, therapeutic strategies targeting the Oct4/Egr1/OPN axis may be further explored for the treatment of lung cancer, especially when lung cancer is refractory to conventional treatment due to cancer stem cells.

Keywords: Egr1; Lung cancer; Metastasis; Oct4; Osteopontin.

Fig. 1

Oct4, Egr1, and OPN are overexpressed in lung cancer and Egr1 expression is correlated with shorter survival. a, Representative staining for Oct4, Egr1, and OPN in human lung tumors graded as low and high expression. b, Numbers of tumors expressing high or low levels of Egr1 in tumors with high or low Oct4 expression. c. Numbers of tumors expressing high or low levels of OPN in tumors with high or low Egr1 expression. d, Tumor recurrence rates between high- and low- expression of Oct4, Egr1, and OPN in lung cancer patients, none of them reached statistical significance (Oct4: p = 0.273; Egr1: 0.261; OPN: 0.253). e-g, Kaplan-Meier curves for disease-free survivals in patients with high (red line) and low (black line) Oct4 (e), Egr1 (f), and OPN (g) expression

Fig. 2

Oct4 binds to the Egr1 promoter to transactivate Egr1 in lung cancer cells. a, H1299 cells that had been cotransfected with pGL2-Egr1p and pTCY-Renilla for 24 h were transduced with LV.Oct4 or LV.Null (left panel). A549 cells stably overexpressing Oct4 and vector control cells were cotransfected with pGL2-Egr1p and pTCY-Renilla (right panel). After 48 h, cell lysates were harvested, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity of vector control cells was set to 100, and the values are presented as relative promoter activities. b, Schematic representation of two deletion constructions containing different lengths of the Egr1 promoter (left panel). Numbering is relative to the translational start site at + 1. H1299 cells were cotransfected with full-length or different deletion constructs of the Egr1 promoter and pTCY-Renilla. After 24 h, the cells were transfected with pCMV-tag2B-Oct4 or the control vector pCMV-tag2B. Cell lysates were assessed for firefly and Renilla luciferase activities 48 h later. RLU, relative luciferase unit. c, ChIP analysis showing direct binding of Oct4 to the Oct4 response element (ORE)-containing region in the Egr1 promoter. Cross-linked chromatin of A549 cells was immunoprecipitated with anti-Oct4 or anti-IgG antibody combined with protein G agarose beads, followed by PCR amplification of the Egr1 promoter region encompassing the ORE (between − 309 to − 413 bp) region. Data are mean ± SEM. *, p < 0.05; ***, p < 0.001

Fig. 3

Overexpression of Oct4 enhances Egr1 expression, OPN production, and migratory capability of human lung cancer cells. a and b, H1299 cells (left panel) were transiently transfected with 6 μg of pSin-EF2-Oct4-Pur or the control plasmid pSin-EF2-Pur, and their RNA (a) and protein (b) levels of Oct4 and Egr1 were analyzed at 48 post-transfection by RT-PCR (a) and immunoblot (b) analysis. A549 cells (right panel) stably overexpressing Oct4 and vector control cells were examined for Oct4 and Egr1 expression by RT-PCR (a) and immunoblot (b) analysis. Expression of GAPDH (a) and β-actin (b) served as the loading control. c, The levels of OPN in the supernatants of Oct4-overexpressing and control A549 cells were quantified by ELISA. d, Migratory capabilities of A549-Oct4 and A549-vector cells were detected by a Boyden chamber assay. Cells that migrated through the membrane of the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy and photographed (left panel), and three fields in each membrane were counted (right panel). Data are mean ± SEM. **, p < 0.01

Fig. 4

Oct4-overexpressing lung tumors express higher levels of Egr1 in a mouse tumor model, and knockdown of Oct4 reduces the migratory capability of lung cancer cells. a, Immunohistochemical staining for Oct4, Egr1 and OPN in the tumors excised at day 60 from NOD/SCID mice that had been inoculated with 5 × 10⁶ of A549-Oct4 cells via the tail vein. b, Lung tissues of tumor-bearing mice were harvested, and the number of metastatic nodules was counted. Data are mean ± SEM. *, p < 0.05; **, p < 0.01

Fig. 5

Knockdown of Egr1 reduced the levels of OPN secreted into the culture medium and decreased the migratory capability. a, Levels of OPN in the supernatants of Egr1 knockdown and control A549-Oct4 cells were quantified by ELISA. b, Migratory capabilities of Egr1 knockdown or control A549-Oct4 cells via lentivirus-mediated delivery of shRNA for 48 h. Cells that migrated through the membrane to the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy and photographed (left panel), and three fields in each membrane were counted (right panel). Data are mean ± SEM. *, p < 0.05; **, p < 0.01