RAC2 promotes abnormal proliferation of quiescent cells by enhanced JUNB expression via the MAL-SRF pathway

Abstract

Radiation-induced lung injury (RILI) occurs most often in radiotherapy of lung cancer, esophageal cancer, and other thoracic cancers. The occurrence of RILI is a complex process that includes a variety of cellular and molecular interactions, which ultimately result in carcinogenesis. However, the underlying mechanism is unknown. Here we show that Ras-related C3 botulinum toxin substrate 2 (RAC2) and transcription factor jun-B (JUNB) were upregulated in non-small cell carcinoma (NSCLC) tissues and were associated with poor prognoses for NSCLC patients. Ionizing radiation also caused increased expression of RAC2 in quiescent stage cells, and the reentry of quiescent cells into a new cell cycle. The activity of the serum response factor (SRF) was activated by RAC2 and other Rho family genes (RhoA, ROCK, and LIM kinase). Consequently, JUNB acted as an oncogene and induced abnormal proliferation of quiescent cells. Together, the results showed that RAC2 can be used as a target gene for radiation protection. A better understanding of the RAC2 and JUNB mechanisms in the molecular etiology of lung cancer will be helpful in reducing cancer risks and side effects during treatment of this disorder. Our study therefore provides a new perspective on the involvement of RAC2 and JUNB as oncogenes in the tumorigenesis of NSCLC.

Keywords: RAC2; Radiation-induced lung injury; Tumorigenesis.

Figure 1.

RAC2 and JUNB are upregulated in non-small cell carcinoma (NSCLC) tissues and were correlated with poor prognoses. (a, b) The relative expressions of RAC2 and JUNB (b) in NSCLC tissues were analyzed using the Cancer Genome Atlas data set. Kaplan-Meier progression-free survival and overall survival curves were used to analyze RAC2 and JUNB expression levels. X = 1095. (c, d) The absolute expression levels of RAC2 (c) and JUNB (d) were determined in 23 clinical lung cancer tissues and four normal lung tissues using quantitative RT-PCR. The data are expressed as the mean±SEM.

Figure 2.

The effects of RAC2 on in vitro quiescent cell proliferation after ionizing radiation. (a) The expression of RAC2 and JUNB were measured by western blotting. (b, c) Grayscale analyses of RAC2 and JUNB in quiescent cells and quiescent shRAC2 cells after treatment with 2 Gy of X-ray irradiation. (d–g) Flow cytometry showing significant increases in the percentages of cells in the S or G₂/M phases when the cells were treated with 2 Gy X-ray irradiation. (h) The abnormal proliferation of quiescent cells after X-ray irradiation. (i) The CCK-8 assay was used to determine the viability of quiescent cells and quiescent shRAC2 cells after exposure to X-ray irradiation. (j-k) An immunofluorescent assay was used to determine the percentages of Ki67 positive quiescent cells and quiescent shRAC2 cells after exposure to X-ray irradiation. The error bars denote the mean±SE derived from three independent experiments.

Figure 3.

The in vivo and in vitro expressions of RAC2 and Rho GTPase pathway proteins after exposure to X-ray irradiation. (a) The expression of RAC2, RhoA, ROCK, LIM kinase, and JUNB were measured by western blotting. (b) The grayscale analyses of RAC2, RhoA, ROCK, LIM kinase, and JUNB. (c) The target area of X-ray exposure using computed tomography as guidance. (d) The expression of RAC2 and JUNB were measured by western blotting in mice (n = 4). (e) The grayscale analyses of RAC2 and JUNB in mice (n = 4). (f) The expressions of RAC2, RhoA, ROCK, LIM kinase, and JUNB were measured by western blotting in mice (n = 7). (g) The grayscale analyses of RAC2, RhoA, ROCK, LIM kinase, and JUNB in mice (n = 7). The error bars denote the mean±SE from three independent experiments.

Figure 4.

Ionizing radiation increased the activity of the serum response factor (SRF) by promoting the nuclear accumulation of megakaryocytic acute leukemia (MAL) factor. (a) A graphical representation of the plasmid construction to determine the SRF activity. (b) The intracellular protein hybridization of MAL and SRF. (c) The percentage of MAL in the nucleus. (d) Results of a Co-IP experiment using MAL antibody as the immunoprecipitating antibody to confirm interactions between MAL and its binding partners SRF. Normal rabbit IgG was used for Co-IP and served as a negative control, whereas normal cells lysate without Co-IP served as a positive control. The heavy chain of IgG served as a loading control. (e) The SRF activity was determined using the luciferase reporter assay. The error bars denote the mean± SE derived from three independent experiments.

Figure 5.

Graphical representation of the roles of the RAC2 and Rho GTPase pathways in quiescent cells involved in recycling and abnormal proliferation.