Interleukin-7 up-regulates cyclin D1 via activator protein-1 to promote proliferation of cell in lung cancer

Abstract

Interleukin-7 is a potent regulator of lymphocyte proliferation, but it inducing growth of solid tumors is few known. We study the relationship between Interleukin-7 and the regulator of the cell cycle, cyclin D1 and the mechanism of Interleukin-7 regulating cell growth in human lung cancer. We detected expression of cyclin D1 and its impact on the prognosis of lung cancer patients. Using Western blot, reverse transcriptase-PCR, Co-Immunoprecipitation, and Chromatin Immunoprecipitation, we investigated how Interleukin-7 regulated cyclin D1 in vitro and in nude mice. We found that, in lung cancer cell lines and in nude mice, Interleukin-7/Interleukin-7 receptor increased the expression of cyclin D1 and phosphorylation of c-Fos/c-Jun, induce c-Fos and c-Jun heterodimer formation, and enhanced c-Fos/c-Jun DNA-binding activity to regulate cyclin D1. In addition, lymph node metastasis, tumor stage, and cyclin D1 were the strongest predictors of survival in 100 human non-small cell lung cancer specimens analyzed. Taken together, our results provided evidence that Interleukin-7/Interleukin-7 receptor induced cyclin D1 up-regulation via c-Fos/c-Jun pathway to promote proliferation of cells in lung cancer.

Fig. 1

IL-7 promoting the growth of cell in lung cancer cell lines. MTT analysis showing IL-7 inducing cells proliferation of lung cancer A549 (a), LH7 (b) cells, and blocking IL-7R with siRNA IL-7R inhibiting these cells proliferation

Fig. 2

IL-7 accelerates G1/S-phase progression in lung cancer cell lines. The FACS analysis indicated that IL-7 could promote G1/S-phase transition in A549 (a) and LH7 (b) cells. However, siRNA against IL-7R inhibited G1/S-phase progression in A549 and LH7 cells

Fig. 3

IL-7 up-regulates cyclin D1 in lung cancer cell lines. The RT-PCR and Western blot analysis showing induction expression of cyclin D1 mRNA and protein after Il-7 stimulation and reduction expression of cyclin D1 mRNA and protein after blocking IL-7R with siRNA IL-7R in A549 (a, b) and LH7 (c, d) cells. However, after Il-7 stimulation or blocking IL-7R with siRNA, IL-7R did not affect the expression of cyclin C and cyclin E protein and mRNA in A549 (a, b) and LH7 (c, d) cells

Fig. 4

Down-regulating cyclin D1 inhibit G1/S-phase progression in lung cancer cell lines. The FACS analysis indicated that siRNA against cyclin D1 inhibited G1/S-phase progression in A549 (a) and LH7 (b) cells

Fig. 5

IL-7 up-regulates the expression and phosphorylation of c-Fos, c-Jun in lung cancer cell lines. Western blot analysis showing up-regulation of c-Fos, c-Jun, and p-c-Jun proteins in A549 (a) and LH7 (b) cells treated with IL-7, and blocking IL-7R with siRNA IL-7R down-regulating the expression of c-Fos, c-Jun, and p-c-Jun proteins

Fig. 6

Inhibiting AP-1 down-regulating the level of cyclin D1 expression in lung cancer cell lines. The RT-PCR and Western blot analysis showing reduction in cyclin D1 mRNA and protein after inhibiting AP-1 with AP-1-specific inhibitor SP600125 in A549 (a) and LH7 (b) cells

Fig. 7

IL-7 enhances the DNA-binding activity of AP-1 to the Promoter of Cyclin D1. The CHIP analysis showing that AP-1 (c-Fos/c-Jun) could bind with cyclin D1 promoter, and the activity of binding was enhanced by IL-7 in A549 cell line. However, inhibiting IL-7R with siRNA IL-7R decreased the activity of binding AP-1 with cyclin D1 promoter

Fig. 8

IL-7 promoting the growth of xenograft tumors of nude mice. The growth curve of the tumor analysis showing IL-7 inducing xenograft tumors proliferation, and blocking IL-7R or inhibiting AP-1 inhibiting xenograft tumors proliferation in nude mice

Fig. 9

The RT-PCR and Western blot analysis showing induction of cyclin D1 mRNA and protein after Il-7 stimulation and reduction in cyclin D1 mRNA and protein after blocking IL-7R or inhibiting AP-1 in nude mice (a, b). Western blot analysis showing up-regulation of c-Fos, c-Jun, and p-c-Jun proteins in nude mice treated with IL-7, and blocking IL-7R with IL-7R-specific antibody (sc-662) or inactivating AP-1 with AP-1 inhibitor (SP600125) down-regulating the expression of c-Fos, c-Jun, and p-c-Jun proteins (c). IL-7 could induce the formation of AP-1 (c-Fos/c-Jun) heterodimmer via IL-7R with CoIP method in vivo (d). The CHIP analysis showing that AP-1 (c-Fos/c-Jun) could bind with cyclin D1 promoter, and the activity of binding were enhanced by IL-7 in xenograft tumors (e)

Fig. 10

The expressions of cyclin D1 in NSCLC correlates with the level of IL-7 and IL-7R. Immunohistochemical straining of consecutive serial sections of NSCLC tissues. Brown-yellow particles of IL-7 (a), IL-7R (b), and cyclin D1 (c) were observed in cancer cells (relationship between cyclin D1 expression with IL-7 (P = 0.001, R = 11.063) and with IL-7R (P = 0.025, R = 5.365); 400 × magnification)

Fig. 11

Survival of NSCLC patients correlates with the expression of cyclin D1. Kaplan–Meier survival plots for patients with NSCLC, grouped according to cyclin D1 protein expression. Correlation between overall survival of patients with cyclin D1 expression was found to be statistically significant (P = 0.000). All patients alive at their last follow-up are indicated by tick marks on the plot