Pathway specific gene expression profiling reveals oxidative stress genes potentially regulated by transcription co-activator LEDGF/p75 in prostate cancer cells

Abstract

Background: Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator and autoantigen that is overexpressed in tumors, including prostate cancer (PCa). This oncoprotein promotes resistance to cell death induced by oxidative stress and chemotherapy by mechanisms that remain unclear. To get insights into these mechanisms we identified candidate target stress genes of LEDGF/p75 using pathway-specific gene expression profiling in PCa cells.

Methods: A "Human oxidative stress and antioxidant defense" qPCR array was used to identify genes exhibiting significant expression changes in response to knockdown or overexpression of LEDGF/p75 in PC-3 cells. Validation of array results was performed by additional qPCR and immunoblotting.

Results: Cytoglobin (CYGB), Phosphoinositide-binding protein PIP3-E/IPCEF-1, superoxidase dismutase 3 (SOD3), thyroid peroxidase (TPO), and albumin (ALB) exhibited significant transcript down- and up-regulation in response to LEDGF/p75 knockdown and overexpression, respectively. CYGB gene was selected for further validation based on its emerging role as a stress oncoprotein in human malignancies. In light of previous reports indicating that LEDGF/p75 regulates peroxiredoxin 6 (PRDX6), and that PRDXs exhibit differential expression in PCa, we also examined the relationship between these proteins in PCa cells. Our validation data revealed that changes in LEDGF/p75 transcript and protein expression in PCa cells closely paralleled those of CYGB, but not those of the PRDXs.

Conclusions: Our study identifies CYGB and other genes as stress genes potentially regulated by LEDGF/p75 in PCa cells, and provides a rationale for investigating their role in PCa and in promoting resistance to chemotherapy- and oxidative stress-induced cell death.

Fig. 1

Verification of LEDGF/p75 depletion and overexpression in PC-3 cells. Real-time PCR (qPCR) and immunoblotting were used to determine the levels of LEDGF/p75 transcript (upper panels) and protein (lower panels), respectively, in PC-3 cells with transient knockdown (A), stable knockdown (B), and stable overexpression (C) of LEDGF/p75. Cells were transfected with siLEDGF/p75, shLEDGF/p75, and retroviral vector harboring full-length LEDGF/p75 to induce transient knockdown, stable knockdown, and stable overexpression, respectively. PC-3 cells with mock transfection (Nuc) or transfection with small interfering scrambled RNA duplex (siSD), short hairpin scrambled RNA (shSCR), and empty vector (Vector) served as corresponding controls. LEDGF/p75 mRNA levels were normalized to GAPDH mRNA levels and expressed relative to normalized corresponding control values respectively. *P < 0.01; **P < 0.001. P values were determinedwith two-tailed Student’s t-test.

Fig. 2

Identification of stress genes potentially regulated by LEDGF/p75. A human oxidative stress and antioxidant defense qPCR-array was used to identify genes exhibiting significant up- or down-regulation of transcript levels in PC-3 cells with transient knockdown (A), stable knockdown (B), and stable overexpression (C) of LEDGF/p75. Messenger RNA levels were normalized using the average expression of five housekeeping genes (β2M, HPRT1, RPL13A, GAPDH, β-actin) and expressed relative to normalize dmRNA values of respective controls. Gene expression displaying significant differential regulation (fold-change ≥ 2; *P < 0.05, **P < 0.01; ***P < 0.001) was represented graphically. P values were determined with two-tailed Student’s t-test.

Fig. 3

Analysis of changes in CYGB protein expression in response to knockdown or overexpression of LEDG/p75. Expression of CYGB protein was evaluated by immunoblotting in PC-3 cells with transient knockdown (A) and stable overexpression of LEDGF/p75 (B), to further validate the qPCR data. Protein loading was assessed with antibody to β-actin.

Fig. 4

Transcript and protein expression changes of PRDXs in PC-3 cells in response to knockdown or overexpression of LEDGF/p75. PRDX expression was evaluated using the qPCR gene microarray (upper panels) and immunoblotting (lower panels) in PC-3 cells with transient knockdown (A), stable knockdown (B), and stable overexpression (C) of LEDGF/p75. Levels of PRDX mRNA were normalized using the average expression of five housekeeping genes in the qPCR microarray, and expressed relative to normalized corresponding control values respectively. Protein loading inimmunoblots was assessed with antibody to β-actin. P values were determined with two-tailed Student’s t-test and were not significant for any of the PRDXs.

Fig. 5

Expression analysis of LEDGF/p75, CYGB, PRDX3 and PRDX6 in a panel of human prostate cell lines. Protein expression was evaluated by immunoblotting in a panel of prostate cell lines (A) that included non-tumor (BRF-55T, PrEc, PrSc), a nontumorigenic immortalized normal human prostate epithelial cell line (RWPE-1), a tumorigenic transformed RWPE-1 cell line (RWPE-2), androgen-independent (DU-145, PC-3), and androgen-responsive (LNCaP, MDA-PCa-2b, BRF-41T, and 22RV1) cell lines. LEDGF/p75 transcript overexpression in a docetaxel resistant PC-3 cell line (PC-3-DR) was verified using qPCR (B). The expression of these four proteins was also evaluated by immunoblotting in PC-3-DR cells (C), PC-3-DR cells with transient LEDGF/p75 knockdown (D), and PC-3 cells stably overexpressing LEDGF/75 with transient knockdown of the protein (E). Parental PC-3 cells served as control for (B) and (C), while siSD transfected-PC-3-DR cells and LEDGF/p75-overexpressing PC-3 cells served as corresponding controls in (D) and (E), respectively. Protein loading was assessed with antibody toβ-actin. *P < 0.001. P values were determined with two-tailed Student’s t-test.

Fig. 6

Expression analysis of LEDGF/p75 and CYGB in docetaxel resistant DU 145 cells. LEDGF/p75 and CYGB transcript and protein overexpression in a docetaxel resistant DU 145 cell line (DU 145-DR) were verified using qPCR (A) and immunoblotting (B), respectively. Transient knockdown of LEDGF/p75 and its effect on CYGB expression were evaluated in DU145-DR cells by qPCR (C) and immunoblotting (D). Protein loading was assessed with antibody to β-actin. *P < 0.05, **P < 0.001. P values were determined with two-tailed Student’s t-test.