Hypoxia regulates SOX2 expression to promote prostate cancer cell invasion and sphere formation

Abstract

SOX2 is an embryonic stem cell marker that in prostate cancer has been associated not only with tumorigenesis but also metastasis. Furthermore hypoxia in primary tumors has been linked to poor prognosis and outcomes in this disease. The goal of the present study was to investigate the impact of hypoxia on SOX2 expression and metastasis-associated functions in prostate cancer cells. A tissue microarray of 80 samples from prostate cancer patients or healthy controls was employed to examine the expression of HIF-1α and its correlation with SOX2. The role of SOX2 and HIF-1/2α in the regulation of cell invasion and sphere formation capacity under hypoxic conditions was investigated in vitro using short hairpin RNA (shRNA)-mediated knockdown in three human prostate cancer cell lines. HIF-1α expression was significantly elevated in malignant prostate tissue compared to benign or normal tissue, and in tumor samples its expression was highly correlated with SOX2. In prostate cancer cells, acute and chronic exposures to hypoxia that resulted in elevated expression levels of HIF-1α and HIF-2α, respectively, also induced SOX2. Genetic depletion of SOX2 attenuated hypoxia-induced cell functions. Knockdown of HIF-1α, but not HIF-2α, decreased acute hypoxia-mediated cell invasion and SOX2 up-regulation, whereas only HIF-2α gene silencing reduced sphere formation capacity and chronic hypoxia-mediated SOX2 up-regulation. Enhanced SOX2 expression and HIF-1α or HIF-2α associated phenotypes are dependent on the time duration of exposure to hypoxia. The present results indicate that SOX2 may be a key mediator of hypoxia-induced metastasis-associated functions and hence may serve as a potential target for therapeutic interventions for metastatic prostate cancer.

Keywords: Hypoxia; SOX2; hypoxia inducible factor; invasion; stemness.

Figure 1

HIF-1α expression and its correlation with SOX2 in prostate tissues. A. Immunostaining for HIF-1α was performed using prostate tissue arrays. Representative images from normal, BPH, and PCa staining were shown. Brown color indicates positive nuclear staining. The magnification of top and bottom lanes is ×5 and ×20, respectively. B. Semiquantitative analysis of IHC staining was performed for all samples that assessed both the percentage of cells stained and the intensity of the staining, and the analysis was reported as the H score of these two parameters. P value was calculated by Wilcoxon nonparametric rank sum test. C. Representative images of HIF-1α and SOX2 IHC staining on sample samples of TMA #54 and #67. High magnification images of positive-stained regions are presented in the upper right coner. D. Correlation analysis of HIF-1α and SOX2 by the H score using Spearsman test.

Figure 2

Impact of hypoxia on an embryonic stem cell marker expression. DU145, PC-3 and LNCaP cells were exposed to 1% oxygen for times ranging from 2 to 24 h (A), 24 to 120 h (B) or 72 and 120 h (C and D). Whole cell lysates were analyzed by western blot using actin as a loading control.

Figure 4

Hypoxia enhanced sphere formation in prostate cancer cells. PC-3 and LNCaP cells were exposed with 1% oxygen for 6 h (A) or 72 h (B). PC-3 cells were stably transfected with shRNA of HIF-1α or scramble shRNA (Control) and exposed to 1% oxygen for 72 h and protein expression was detected by western blot using actin as a loading control (C). Following exposure to hypoxia, cells were cultured under aerobic conditions in a low attachment plate at a density of 500 cells/ml. After 7 days of treatment, representative phase contrast images were taken at ×10 and ×20 magnification (A-C). The percentage of spheres per well was quantified as the ratio of the number of spheres divided by the seeding number (A, B, D). P value was analyzed by t test.

Figure 3

Acute hypoxia-induced, SOX2-mediated cell invasion is dependent on HIF-1α. Prostate cancer cells were stably transfected with shRNA of SOX2 (A, B), HIF-1α (C, D) or HIF-2α (E, F) or scramble shRNA (Control). Transfected cells were exposed to 1% oxygen for 6 h and protein expression was detected by western blot using actin as a loading control (A, C, E). To test cell invasion, transfected cells were seeded into invasion chambers (5×10⁴/chamber) and exposed to 1% O₂ for 6 h then re-cultured under aerobic conditions for 18 h. Invaded cells were scored 24 h after cell seeding. Column, mean; bars, SD (n=4). ns = no significance (t-test) (B, D, F). P value was analyzed by t test.

Figure 5

Chronic hypoxia-induced, SOX2-mediated sphere formation is dependent on HIF-2α. Prostate cancer cells were stably transfected with shRNA of HIF-2α (A, B) SOX2 (C, D), or scramble shRNA (Control). Transfected cells were exposed to 1% oxygen for 72 h and protein expression was detected by western blot using actin as a loading control (A, C). To test sphere formation, transfected cells were exposed to 1% oxygen for 72 h and cells were cultured under aerobic conditions in a low attachment plate at a density of 500 cells/ml. After 7 days of treatment, representative phase contrast images were taken at ×10 and ×20 magnification (B, D). The percentage of spheres per well was quantified as the ratio of the number of spheres divided by the seeding number (B, D). P value was analyzed by t test.