c-Fos over-expression promotes radioresistance and predicts poor prognosis in malignant glioma

Abstract

c-Fos is a major component of activator protein (AP)-1 complex. It has been implicated in cell differentiation, proliferation, angiogenesis, invasion, and metastasis. To investigate the role of c-Fos in glioma radiosensitivity and to understand the underlying molecular mechanisms, we downregulated c-Fos gene expression by lentivirus-mediated shRNA in glioma cell lines and subsequently analyzed the radiosensitivity, DNA damage repair capacity, and cell cycle distribution. Finally, we explored its prognostic value in 41 malignant glioma patients by immunohistochemistry. Our results showed that silencing c-Fos sensitized glioma cells to radiation by increasing radiation-induced DNA double strand breaks (DSBs), disturbing the DNA damage repair process, promoting G2/M cell cycle arrest, and enhancing apoptosis. c-Fos protein overexpression correlated with poor prognosis in malignant glioma patients treated with standard therapy. Our findings provide new insights into the mechanism of radioresistance in malignant glioma and identify c-Fos as a potentially novel therapeutic target for malignant glioma patients.

Keywords: c-Fos; malignant glioma; prognosis; radioresistance.

Figure 1. c-Fos expression and cell viability were inhibited by ShRNA

A. Western blot analysis performed on control vector and c-Fos shRNA using anti-c-Fos, and anti-α-tubulin antibodies. B. T98G glioma cell viability was inhibited by ShRNA1 and ShRNA2. *P<0.05; t-test. C. The viability of U251 glioma cells was inhibited by ShRNA1 and ShRNA2. *P<0.05; t-test.

Figure 2. c-Fos regulated radiosensitivity in T98G and U251 cell lines

A. Down-regulation of c-Fos increased the sensitivity of T98G cells to radiation. P<0.05; ANOVA test. B. Down-regulation of c-Fos increased the sensitivity of U251 cells to radiation. P<0.05; ANOVA test.

Figure 3. c-Fos depletion combined with 3 Gy radiation increased the sub-G2/M population

A. Analysis of cell cycle distribution by flow cytometry. B. Sub-population percentage analysis by flow cytometry for T98G cell line. C. Sub-population percentage analysis by flow cytometry for U251 cell line.

Figure 4. c-Fos knockdown delayed DNA damage repair in T98G and U251 cell line

A. γH2AX foci in c-Fos-depleted T98G cells vs. control at various time points following 3 Gy radiation. Original magnification, x600. B. γH2AX foci in c-Fos-depleted U251 cells vs. control at various time points following 3 Gy radiation. Original magnification, X600. C. γH2AX foci positive cells at various time points following 3 Gy radiation in both T98G-3Gy cells and T98G-ShRNA2+3Gy cells. *P<0.05; **P<0.01, t-test. D. γH2AX foci positive cells at various time points following 3 Gy radiation in both U251-3Gy cells and U251-ShRNA2+3Gy cells. *P<0.05, **P<0.01, t-test.

Figure 5. Western blot analysis of cell cycle and apoptosis-related proteins

T98G-3Gy cells, T98G-ShRNA+3Gy cells, U251-3Gy cells, and U251-ShRNA+3Gy cells were harvested 48 hours after 3 Gy radiation. Lysates were subjected to Western blot analysis with the labeled antibodies.

Figure 6. Expression of c-Fos in malignant glioma tissues detected by immunohistochemistry staining and Kaplan–Meier estimates of survival probability

Negative staining of c-Fos in glioma tissue A. 200×, B. 400×; strong staining of c-Fos in nucleus and cytoplasm C. 200×, D. 400×; High c-Fos expression levels were significantly associated with poor overall survival (P=0.015) in malignant glioma patients E.