Suppressor of cytokine signaling (SOCS)-1 is expressed in human prostate cancer and exerts growth-inhibitory function through down-regulation of cyclins and cyclin-dependent kinases

Abstract

Suppressor of cytokine signaling (SOCS) proteins play a pivotal role in the development and progression of various cancers. We have previously shown that SOCS-3 is expressed in prostate cancer, and its expression is inversely correlated with activation of signal transducer and activator of transcription factor 3. We hypothesized that SOCS-1, if expressed in prostate cancer cells, has a growth-regulatory role in this malignancy. The presence of both SOCS-1 mRNA and protein was detected in all tested cell lines. To assess SOCS-1 expression levels in vivo, we analyzed tissue microarrays and found a high percentage of positive cells in both prostate intraepithelial neoplasias and cancers. SOCS-1 expression levels decreased in samples taken from patients undergoing hormonal therapy but increased in specimens from patients who failed therapy. In LNCaP-interleukin-6- prostate cancer cells, SOCS-1 was up-regulated by interleukin-6 and in PC3-AR cells by androgens; such up-regulation was also found to significantly impair cell proliferation. To corroborate these findings, we used a specific small interfering RNA against SOCS-1 and blocked expression of the protein. Down-regulation of SOCS-1 expression caused a potent growth stimulation of PC3, DU-145, and LNCaP-interleukin-6- cells that was associated with the increased expression levels of cyclins D1 and E as well as cyclin-dependent kinases 2 and 4. In summary, we show that SOCS-1 is expressed in prostate cancer both in vitro and in vivo and acts as a negative growth regulator.

Figure 1

SOCS-1 mRNA and protein levels in prostate cancer cell lines. Cells were grown under standard conditions and SOCS-1 expression levels were measured using quantitative real-time PCR and Western blot, respectively. SOCS-1 mRNA levels were normalized to levels of the housekeeping gene encoding TATA-box binding protein and those of SOCS-1 protein to the value of β-actin. Data represent mean value ± SE; n = 4 independent experiments carried out in triplicate.

Figure 2

Expression of SOCS-1 and Ki-67 in normal prostate tissue, PIN (prostate intraepithelial neoplasia) and carcinoma obtained from individuals before or after androgen-deprivation therapy (ADT). A: SOCS-1 immunoreactivity in samples from patients with prostate cancer and (B) SOCS-1 staining in human prostate cancer from a patient who failed endocrine therapy. Immunohistochemistry for SOCS-1 was performed using the ABC staining kit according to the manufacturer’s recommendations. SOCS-1 expression levels were analyzed quantitatively using a single cell resolution technique with the HistoQuest software. C: Percentage of Ki-67-positive cells in untreated, treated, and therapy-resistant prostate cancer and adjacent PIN and normal tissue. Data represent mean ± SE. D: Ki-67 staining in representative samples of human prostate cancer, adjacent PIN, and normal tissue.

Figure 3

Regulation of expression of SOCS-1 by androgen in (A) PC3-AR, (B) LNCaP-IL-6− and (C) parental LNCaP ATCC cells. The cells were treated with increasing concentrations of the synthetic androgen methyltrienolone (R1881) for 72 hours and SOCS-1 levels were determined by Western blot. The results are representative of three independent experiments.

Figure 4

IL-6 induces SOCS-1 protein but not mRNA in LNCaP-IL-6− cells and inhibits cellular growth. Cells were seeded onto six-well plates and treated with 10 ng/ml of IL-6 for the indicated time periods. RNA was isolated and transcribed into cDNA or whole cell lysates were prepared. Graphs show levels of SOCS-1 mRNA (top) and protein (middle), respectively. Inhibition of proliferation by IL-6 is also shown (bottom); n = 3 independent experiments carried out in triplicate. *P < 0.05.

Figure 5

A: Up-regulation of SOCS-1 expression by transient transfection. PC3 and DU-145 cells were seeded onto six-well plates and transfected after 24 hours with a tetracycline-responsive SOCS-1 expression vector. Protein levels of SOCS-1 were measured 48 and 72 hours later. Controls included the cells grown in vehicle (PBS) only; n = 5 independent experiments carried out in duplicate. B: SOCS-1 overexpression leads to a reduced ³H-thymidine uptake. Cells were seeded onto 96-well plates and transfected with the pBig2i plasmid. Vehicle or doxycycline (2 μg/ml) were supplemented and ³H-thymidine was added after 48 hours. The assay was analyzed 24 hours later; n = 4 independent experiments carried out in triplicate. *P < 0.05, **P < 0.01.

Figure 6

Inhibition of SOCS-1 expression causes a moderate growth advantage. Cells (DU-145 and PC3) were seeded onto six- and 96-well plates for quantitative real-time PCR, Western blot or proliferation assays, respectively. 72 hours after mock or siRNA transfection (final concentration 100 nmol/L), SOCS-1 levels (top, middle) and proliferation status (bottom) were measured; n = 3 to 5 independent experiments carried out in triplicate. *P < 0.05, **P < 0.01.

Figure 7

Long-term knock-down of SOCS-1 causes a strong stimulation of proliferation. Cells were seeded onto six- and 96-well plates for Western blot and ³H-thymidine uptake measurement, respectively. Increasing concentrations of siRNA were transfected at two consecutive time points; n = 6 independent experiments carried out in triplicate; siRNA concentration is given 1 nmoL/L. *P < 0.05, **P < 0.01.

Figure 8

Inhibition of SOCS-1 expression by siRNA leads to elevation of intracellular levels of cyclins D1 and E and cdk 2 and 4 as determined by Western blot. Experimental conditions for DU-145 (A, 3 to 4 independent experiments) and PC3 cells (B, 2 to 4 independent experiments) were the same as those described in the legend for Figure 7. Data represent mean value ± SE. *P < 0.05, **P < 0.01.

Figure 9

Inhibition of SOCS-1 expression in LNCaP-IL-6− cells leads to stimulation of proliferation but does not interfere with IL-6-induced growth inhibition. The cells were transfected with either SOCS-1 or control siRNA in the presence or absence of IL-6. Proliferation was measured by ³H-thymidine incorporation. Data represent mean values ± SE from three independent experiments. ***P < 0.001.