Oncogenic CUL4A determines the response to thalidomide treatment in prostate cancer

Abstract

Thalidomide is experimentally used to treat various human cancers; however, clinical responses to thalidomide are sporadic. Here we demonstrate that CUL4A plays an oncogenic role in prostate cancer development and prostate cancer cells with higher level of CUL4A are particularly sensitive to thalidomide treatment. We show that CUL4A is frequently overexpressed in human primary prostate cancer and cell lines. Notably, subjects with tumors that highly expressed CUL4A had poor overall survival. CUL4A downregulation inhibited cell proliferation and induced apoptosis in vitro and in vivo, whereas CUL4A overexpression transformed human normal prostate epithelial cells and promoted invasion, which was attenuated by the extracellular signal-regulated kinase (ERK) inhibitor. We further show that the sensitivity to thalidomide is positively correlated with CUL4A expression in a panel of prostate cell lines. Ectopic CUL4A expression greatly enhanced sensitivity to thalidomide, while its downregulation conferred resistance to this drug. Mechanistically, thalidomide decreased CUL4A in a time- and dose-dependent manner, consequently leading to inaction of ERK pathway. Finally, we show that cereblon level is correlated with CUL4A expression and downregulated in thalidomide-resistant prostate cancer cell. Our results offer the first evidence that CUL4A is a potential therapeutic target for prostate cancer and may serve as a biomarker for assessing prognosis of human prostate cancer and response to thalidomide treatment.

Introduction

Prostate cancer is the leading noncutaneous malignancy in American men. In 2010 alone, it was estimated that prostate cancer would be diagnosed in 217,730 men, and 27,350 would die of the disease [1]. Although progress has been made in understanding molecular genetics of prostate cancer [2], the clinical treatment of metastatic prostate cancer still remains a challenge. Docetaxel-based chemotherapy is the current standard management that could prolong patient overall survival; however, the responses are not durable and eventual progression of disease is inevitable [3, 4]. Alternative treatment strategies and novel approaches are urgently demanded for this disease.

Thalidomide was originally developed as a sedative but lately withdrawn from market due to potent teratogenic effects in humans. It is emerging as a treatment for inflammatory disease and cancers because of its antiangiogenic, immunomodulatory, and anti-inflammatory effects [5]. Although the precise mechanism of anti-tumor effects remains elusive, recent data have indicated that thalidomide inhibits angiogenesis and modulates inflammatory cytokines [6, 7]. While thalidomide and its analog have had much success in the treatment of hematological diseases, such as multiple myeloma, investigations in several solid tumors achieved variable degrees of success. For example, thalidomide monotherapy has been fairly ineffective in breast, ovarian, renal, and head and neck squamous cell carcinoma [8–11]. In addition, some clinical studies failed to show survival advantage of combination therapy of thalidomide and cytotoxic agents compared with cytotoxic agents alone in lung and prostate cancer [12–14]. The possible reason for lack of efficiency in general population of patients is the inherent heterogeneity of tumor [15–17]. Therefore, a greater understanding of mechanisms of thalidomide is essential to define the subgroups of patients who might benefit most from thalidomide-based regimens.

CUL4A controls a wide range of cellular processes, including proliferation, differentiation, and apoptosis. CUL4A has been shown to be essential for the ubiquitination of several well-defined tumor suppressor genes, such as p21, p27, DDB2, and p53, which provides the rationale for evaluating CUL4A as a potential oncogene [18–21]. CUL4A amplification or overexpression has been reported in some of human cancers, including breast cancer, squamous cell carcinomas, adrenocortical carcinomas, childhood medulloblastoma, and hepatocellular [22–27] and is associated with poor prognosis in node-negative breast cancer [28]. Recently, we have shown that CUL4A is overexpressed and amplified in 64 % primary malignant pleural mesothelioma, and downregulation of CUL4A with shRNA causes cell cycle arrest and growth inhibition through upregulation of p21 and p27 proteins [29]. Because CUL4A resides in a locus that is frequently amplified in prostate cancer [30] and given the fact that dysregulation of CUL4A is implicated in multiple tumor types, we sought to investigate the role of CUL4A in prostate cancer. In addition, recent findings suggest that thalidomide exerts teratogenic effects, at least in part by direct binding to CRBN [31], which forms an E3 ubiquitin ligase complex with CUL4A. This finding promotes us to investigate whether thalidomide exerts anti-tumor effects through inhibiting CUL4A pathway.

In this study, we showed that CUL4A is frequently found overexpressed in primary prostate cancer and cell lines, specifically in patients with high Gleason score, prostate specific antigen (PSA), and lymph node and seminal vesicle invasion. Moreover, CUL4A expression level predicted poor overall patient survival. We further demonstrated that CUL4A contributed to prostate cancer development through the regulation of extracellular signal-regulated kinase (ERK) pathway. Finally, we showed that thalidomide suppressed cancer cells survival by inhibiting CUL4A, and prostate cancer cells with high CUL4A level were particularly sensitive to thalidomide treatment. These data indicated that CUL4A is a potential novel therapeutic target for prostate cancer, and clinically implicated patients with high CUL4A levels might benefit most from thalidomide-based regimens.

Materials and methods

Human tissue samples

Clinical tissue specimens from patients were obtained from the archives of Shanghai Changhai Hospital with the approval of the medical ethics committee and with informed consent.

Immunohistochemistry

A tissue microarray was constructed using formalin-fixed, paraffin-embedded tissue samples derived from 121 prostate cancers, 8 normal prostate, and 13 benign prostatic hyperplasia (BPH). Immunohistochemistry stains were visualized using the EnVision+ system (Dako). Primary antibodies for CUL4A (#ab84382; Abcam) and ki-67 (#ZM-0166) were used. All of staining was assessed by pathologists blinded to the origination of the samples and subject outcome. The widely accepted German semi-quantitative scoring system in considering the staining intensity and area extent was used. Each specimen was assigned a score according to the intensity of the nucleic, cytoplasmic, and/or membrane staining (no staining=0; weak staining=1, moderate staining=2, strong staining=3) and the extent of stained cells (0 %=0, 1–24 %=1, 25–49 %=2, 50–74 %=3, 75–100 %=4). The final immunoreactive score was determined by multiplying the intensity score with the extent of score of stained cells, ranging from 0 (the minimum score) to 12 (the maximum score). Overall, final immunoreac-tive score ≤6 was defined as low expression, while >6 was defined as high expression.

Xenograft

All animal experiments were done in accordance with institutional guideline for animal welfare. 22RV1-Ctrol and 22Rv1-shCUL4A cells were injected s.c. into 5–6-week-old male athymic nude mice (nu/nu) with eight tumors per cell line. When solid tumors became visible after 1 week, tumor growth was monitored every 3 days indicated in the figure, and tumor sizes were calculated using the formula π/6×ab², where a is the longest dimension of the tumor and b is the width. After 4 weeks, mice were sacrificed and tumors were removed. Half of the harvested tumors were snap frozen in OCT (Miles Inc., Elkhart, IN, USA) in liquid nitrogen and the other half fixed in 10 % paraformaldehyde for paraffin embedding and subsequently processed for TUNEL assay and immunohistochemistry separately.

Statistical analyses

The data shown represent mean values ± standard deviation (SD). Data were analyzed by chi-square test or t test which is indicated in the figure legends. Survival analysis was performed using the Kaplan–Meier method. A p value <0.05 was considered statistically significant.

Results

Overexpression of CUL4A in prostate cancer cell lines and patient-derived tumor samples

We first examined the expression of CUL4A in a panel of prostate cancer cell lines, including LNCaP, C4-2, 22RV1, CWR22R, DU145, and PC3, as well as two immortalized nontumorigenic human prostate epithelial cell line, RWPE-1 and PWR-1E. Both mRNA and protein levels of CUL4A were greatly elevated in all human prostate cancer cell lines in comparison to nontumorigenic human prostate epithelial cell line, but expression levels of CUL4A varied among them (Fig. 1a, b). CUL4A protein levels were correlated with its mRNA levels (Fig. 1a, b).

Fig. 1.

Overexpression of CUL4A in prostate cancer cell lines and tissue samples. a Western blot analysis of CUL4A protein level in PWR-1E, RWPE-1, LNCAP, C4-2, CWR22, 22RV1, PC-3, and DU-145 showed that all prostate cancer cell lines examined elevated expression of CUL4A. b Comparison of mRNA expression levels of CUL4A in same panel of cell lines using qRT-PCR analysis. The average of expression levels in prostate cancer cells is high. c Comparison of mRNA expression levels of CUL4A in 17 pairs of tumor and adjacent normal samples using qRT-PCR. d Comparison of mRNA expression levels of CUL4A in 13 cancer tissue samples versus 13 benign prostatic hyperplasia (BPH). Patients with prostate cancer had higher CUL4A mRNA expression than normal (P=0.0044 obtained by t test)

We next investigated CUL4A expression in primary prostate cancer by performing quantitative RT-PCR. The first study was carried out in tumor and adjacent normal prostate tissues from 17 clinically and pathologically annotated cases of prostate cancer and demonstrated CUL4A overexpression in 58.8 % (10 of 17) of prostate cancer (Fig. 1c). To confirm this observation, CUL4A mRNA levels were examined in another independent set of samples that contain 13 BPH and 13 prostate cancers. As showed in Fig. 1d, mRNA levels of CUL4A in prostate cancer was much higher than in BPH (p=0.0044). More significantly we measured CUL4A protein levels by immunohistochemistry in tissue microarrays containing specimen cores of normal prostate (n=8), BPH (n=13), and prostate carcinoma (n=121). Prostate cancer showed strong CUL4A staining, whereas weak and no staining was observed in BPH and normal prostate (Fig. 2a, Supplementary Fig. 1). These data suggest that CUL4A is frequently overexpressed in prostate cancer.

Fig. 2.

High expression level of CUL4A is associated with poor prognosis in prostate cancer. a Representative immunohistochemical staining of CUL4A expression in normal, benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), and prostate cancer with varied Gleason score (G4, G6, G8, and G10) from tissue microarray. b High level of CUL4A is significantly associated with high PSA (p=0.019), high Gleason score (p=0.033), lymph node positive status (p=0.05), and seminal vesicle positive status (p=0.029). All p values are obtained from chi-square test. c Kaplan–Meier overall survival curves for prostate cancer patients according to tumor expression of CUL4A. The patients were divided into a group with high and low level of CUL4A expression (p=0.034, long-rank test)

Protein level of CUL4A is a prognostic biomarker for poor clinic outcome in patients with prostate cancer

We assessed the association between CUL4A levels and clinical factors and outcomes using a cohort of prostate cancer patients in the tissue microarray study. High CUL4A levels were significantly associated with high PSA (p=0.019), high Gleason score (p=0.033), lymph node positive (p=0.05), and seminal vesicle positive (p=0.029) (Fig. 2a, b and Supplementary Table 1). Patients whose tumor with high CUL4A expression had significantly shorter overall survival period using Kaplan–Meier log-rank analysis (P=0.034) (Fig. 2c). Collectively, these data indicate that CUL4A expression is a prognostic predictor of poor clinical outcome in prostate cancer patients.

Knockdown of CUL4A inhibits prostate cancer cell growth in vitro and in vivo

To explore the functional role of CUL4A in prostate cancer, we designed a short hairpin RNA (shRNA) against CUL4A (shCUL4A) and generated stable transfectants in 22RV1 cells. A scrambled shRNA was served as a control (shCtrl). The shRNA effectively reduced CUL4A protein expression in 22RV1 cell line (Fig. 3a, inset). 22RV1 with shCUL4A exhibited a consistent decrease in cell proliferation over the time course examined, with almost 35 % decrease in proliferation compared to cells with shCtrl at day 6 (Fig. 3a). Previous studies have shown that CUL4A deficiency leads to apoptosis in hematopoietic cell. We raised the question whether the inhibition of 22RV1 cell proliferation by CUL4A knockdown was due to apoptosis. Indeed, as shown in Fig. 3b, the incidences of inducible cell death were significantly exhibited in 22RV1 with shCUL4A as evidenced by TUNEL assay (Fig. 3b). Furthermore, CUL4A knockdown significantly reduced soft agar clonogenicity of 22RV1 both in number and in size (Fig. 3c).

Fig. 3.

CUL4A knockdown inhibits prostate cell growth and induces apoptosis in vitro and in vivo. All p values were obtained from t test. a Downregulation of CUL4A protein levels by shRNA. 22RV1 cells with shCUL4A significantly decreased proliferation compared to 22RV1 cells with shCtrl. Means and SDs were calculated from individual experiments with three replicate assays. b Knockdown of CUL4A induces apoptosis in 22RV1 cells. The apoptosis is detected by TUNEL assay. c Soft agar colony formation assay of 22RV1 cell with shCUL4A and shCtrl, showing knockdown of CUL4A decreased the number and size of colonies. The average number and size of colonies per 1,000 cells plated and SDs were calculated from three biological replicates. Representative phase contrast micrographs of colonies are shown in top panel. d Knockdown of CUL4A inhibits 22RV1 cells growth in vivo. The mean tumor volume ± SD for each group is shown (n=7 mice per group). e The size and weight of tumor formed from 22RV1-shCUL4A are significantly smaller than these of tumors from 22RV1-shCtrl. f TUNEL assay of frozen samples derived from 22RV1-shCUL4A and 22RV1-shCtrl showed that knockdown CUL4A induce apoptosis in vivo

We next investigated the effects of CUL4A knockdown on tumor growth in vivo. 22RV1 cells with shCUL4A grew substantially slower than control cells as evaluated by tumor volumes (Fig. 3d). Furthermore, CUL4A knockdown resulted in a significant decrease in both tumor weight and size compared with the cells with shCtrl (Fig. 3e). To confirm CUL4A is knocked down in vivo by shRNA, we did CUL4A immunohistochemical staining in these xenograft tumors. It was indeed that CUL4A protein is significantly reduced (Supplementary Fig. 2a). Consistent with in vitro data, it was found that the proliferation index Ki67 in xenograft tumors with shCUL4A remarkably decreased in comparison to the tumors with shCtrl (Supplementary Fig. 2b), indicating CUL4A knockdown inhibits prostate cancer cell proliferation in vivo. Finally, we showed that CUL4A knockdown also induced apoptosis in vivo (Fig. 3f). Taken together, these results demonstrate that CUL4A plays a crucial role in prostate cancer tumorigenesis and that knockdown of CUL4A is sufficient to inhibit tumor growth in vitro and in vivo.

Overexpression of CUL4A induces anchorage-independent growth and invasion

Anchorage-independent growth and invasion are two crucial events in tumor initiation and progression. To further study the function of CUL4A overexpression in prostate epithelial cells, immortalized nontumorigenic human prostate epithelial cells RWPE-1 were engineered to stably express CUL4A. Western blot analysis showed that the CUL4A-transfected clones (RWPE-1-CUL4A) had higher level of CUL4A protein compared to the pBabe-empty transfected controls (RWPE-1-Ctrl) (Fig. 4a, inset). We observed that RWPE-1-CUL4A cells grew robustly and formed large round colonies within 10 days in soft agar. In contrast, RWPE-1-Ctrl cells were unable to form macroscopic colonies under the soft agar conditions tested (Fig. 4a). This suggested overexpression of CUL4A transformed RWPE-1 cells into malignancy. Furthermore, overexpression of CUL4A significantly increased RWPE-1 cells migration using Boyden chamber assay (Fig. 4b and Supplementary Fig. 3), indicating that overexpression of CUL4A might also contribute to prostate cancer progression.

Fig. 4.

Ectopic CUL4A expression transforms normal prostate epithelial cells and induces invasion via ERK pathway. a Stably transfected cells RWPE-1 colony formation in soft agar presented as the average number of spheres per 1,000 cells plated ±SD; n=3 biological replicates. b CUL4A significantly promoted RWPE-1 invasion, which was abrogated by U0126. Matrigel invasion assays of RWPE-1 cells stably infected with virus expressing CUL4A or control. CUL4A-mediated invasion was abrogated by U0126. c Knockdown CUL4A significantly decreased total ERK level in 22RV1, while ectopic CUL4A expression increased total ERK expression in RWPE-1. d Knockdown CUL4A significantly decreased mRNA level of ERK1 and 2 in 22RV1, while ectopic CUL4A expression increased mRNA level of ERK1 and 2 in RWPE-1. e Soluble chromatin was prepared and precipitated with anti-triMeK4H3 or a control non-immune IgG from PC-3-Ctrl and PC-3-CUL4A cells. Fold enrichment relative to input was calculated. The results are expressed as the mean ± SD of triplicate values for each sample

CUL4A activates ERK signaling pathway via transcriptional regulation

The Ras/ERK signaling pathway is frequently activated in many types of human cancer including prostate cancer. It has been reported that RWPE-1 cells undergo malignant transformation by overexpression of v-Ki-Ras [32]. These evidences promote us to investigate whether CUL4A contributes to prostate cancer development via ERK signaling pathway. As showed in Fig. 4c, the total levels of both ERK proteins strongly increased in RWPE-1 cells with overexpression of CUL4A, while knockdown CUL4A by shRNA in 22RV1 strongly decreased the total levels of both ERKs protein (Fig. 4c). We further found that the transcriptional level of both ERK1 and 2 changed according to CUL4A level (Fig. 4d), indicating that CUL4A activates ERK signaling pathway via transcriptional regulation. Previous study showed that CUL4A regulates histone methylation at H3K4 [33, 35]. Thus, we proposed that CUL4A transcriptional activates ERK through enrichment of H3K4 trimethylation at ERK promoter. We showed that RWPE-1-CUL4A expressed higher H3K4 trimethylation compared with RWPE-1-Ctr. ChIP assays indicate that H3K4 trimethylation at the ERK1 and ERK2 promoter is significantly higher in RWPE-1-CUL4A compared with RWPE-1-Ctr (Fig. 4e). Next we monitored CUL4A-mediated malignant phenotype change after ERK inhibitor U0126 treatment in RWPE-1-CUL4A and RWPE-1-Ctrl cells. As expected, CUL4A-mediated invasion was attenuated by ERK inhibitor U0126 (Fig. 4b and Supplementary Fig. 3). Collectively, these results indicate that ERK is a downstream effector of CUL4A-mediated malignant transformation.

CUL4A determines response to thalidomide in prostate cancer

To investigate potential association between CUL4A expression and thalidomide treatment response, we first assessed the growth inhibitory effect of thalidomide in a panel of prostate cell lines with varied endogenous CUL4A expression as showed in Fig. 1a, b. Cell viability was determined by MTT assay after 72 h of treatment by thalidomide. Interestingly, we found that the cells (LNCAP, C4-2, CWR22R, 22RV1) with high level of CUL4A were particularly sensitive to thalidomide, while the cells (PC-3, DU-145, RWPE-1, and PWR-1E) with low level of CUL4A showed resistant (Fig. 5a). To establish a direct link between CUL4A and thalidomide sensitivity, we ectopically overexpressed CUL4A in the thalidomide-resistant PC-3 and RWPE-1 cells, resulting in increased sensitivity to this drug (Fig. 5b and supplementary Fig. 4). In the revise experiment, we down-regulated expression levels of CUL4A using shRNA in the thalidomide-sensitive 22RV1 cells, leading to resistance to this drug (Fig. 5c).

Fig. 5.

Expression levels of CUL4A determine the sensitivity to thalid-omide in prostate cancer cells. a Prostate cells (LNCAP, C4-2, CWR22, 22RV1) with high level of CUL4A are sensitive to thalidomide, whereas cells (RWPE-1, PWR-1E, PC-3, DU-145) with low level of CUL4A are resistant to this drug. Points present the mean (n=3) and bars presents SD. b Ectopic CUL4A expression in thalidomide-resistant PC-3 cells increases sensitivity to this treatment. c Downregulation of CUL4A with specific shRNA in thalidomide-sensitive 22RV1 cell confers the resistance to this treatment. d Effects of thalidomide on CUL4A and p-ERK. Thalidomide decreased CUL4A and p-ERK in a dose- and time-dependent manner. e Thalidomide induces cell death in sensitive LNCAP cells, but not in resistant PC-3 cell. LNCAP and PC-3 cells were exposed to thalidomide (0, 5, 20, 200 μg/ml) for 48 h; cell lysates were prepared and assayed for CUL4A and PARP by western blotting. f Downregulation of CUL4Awith specific shRNA in thalidomide-sensitive 22RV1 cell reduces cell death to this treatment. g Ectopic CUL4A in thalidomide-resistant PC-3 cells induces cell death to this treatment

Next, we sought to explore the mechanism underling the association of CUL4A and thalidomide treatment response. We first investigate whether thalidomide affects the expression of CUL4A. Indeed thalidomide induced downregulation of CUL4A and its downstream ERK in 22RV1 cells in a time and dose-dependent manner (Fig. 5d). Previous studies indicated that the growth inhibition of thalidomide was mediated by apoptosis induction. We found that thalidomide induced the apoptosis only in sensitive cell lines LNCAP in a dose-dependent manner (Fig. 5e). However, no apoptotic evidence could be found in resistant cells PC-3 at all tested concentrations (Fig. 5e). Furthermore, knockdown of CUL4A blocked apoptosis induction by thalidomide in sensitive 22RV1 cells (Fig. 5f). Reversely, ectopic overexpressions of CUL4A in resistant PC-3 promoted thalidomide-induced apoptosis (Fig. 5g). These results suggest that CUL4A was the key molecular determinant of thalidomide-induced apoptosis in prostate cancer cells.

We established secondary thalidomide-resistant C4-2 cells (C4-2-R) by treating them with thalidomide for 3 months (Fig. 6a). Immunoblotting analysis showed that C4-2-R expressed very low CUL4A compared with its parental cells (Fig. 6b), which suggested that CUL4A is lost during acquired thalidomide resistance. Collectively, downregulation of CUL4A was the major molecular determinant of thalidomide-induced apoptosis in prostate cancer cells, and basal CUL4A level is a biomarker for the response to thalidomide treatment.

Fig. 6.

Loss of CUL4A expression confers secondary thalidomide resistance. a C4-2 thalidomide-resistant cells (C4-2-R) cells were established after 3 months treatment of C4-2 cells by thalidomide. b Protein levels of CUL4A in C4-2-R cells decreased in comparison to C4-2 cells

In 2010, researchers discovered the teratogenic effects of thalidomide stem from the drug's ability to bind to cereblon, a subunit of a Cul4A-containing E3 ubiquitin ligase complex. However, whether cereblon enables the drug's therapeutic effects is unknown. Zhu et al. further revealed that cereblon is required for the antimyeloma activity of thalidomide derivatives (IMiDs). Their important findings indicated that cereblon is also a therapeutic target of thalidomide and IMiDs and a possible biomarker for the clinical assessment of antimyeloma efficacy [34]. To interpret whether this is the similar in prostate cancer, we detected cereblon mRNA level by qPCR in prostate cell lines. The results showed that cereblon is overexpressed in most cancer cell lines and those with high Cul4A levels (i.e., LNCAP, C4-2, CWR22, 22RV1) also have high cereblon expression (Fig. 7a). To study the relationship between Cul4A and cereblon, we first decreased Cul4A expression by siRNA in LNCAP and found that cereblon expression is significantly decreased (Fig. 7b). Similar experiments showed that Cul4A expression was downregu-lated by cereblon-siRNA (Fig. 7c). Collectively, these two genes may form a positive feedback loop. To study if these gene expression is correlated in clinical samples, we detected Cul4A and cereblon mRNA in 27 prostate cancer tissues. Cereblon levels were plotted against the Cul4A levels of mRNA. Statistical analysis of Cul4A and cereblon expression found a Pearson's correlation coefficient of 0.7929 (Fig. 7d, P<0.0001). Lastly, we found that cereblon expression is significantly decreased in thalidomide-resistant C4-2-R (Fig. 7e).

Fig. 7.

CRBN is correlated with CUl4A expression and downregulated in thalidomide-resistant prostate cancer cell. a mRNA expression levels of CRBN in same panel of prostate cell lines as used in Fig 1a using qRT-PCR analysis. b LNCAP was transfected with CUL4A-siRNA and control; CUL4A and CRBN expression was measured by qRT-PCR. c LNCAP was transfected with CRBN-siRNA and control; CUL4A and CRBN expression was measured by qRT-PCR. d The level of CRBN mRNA was positively correlated with that of CUL4A mRNA in prostate cancer samples. e mRNA levels of CRBN in C4-2-R cells decreased in comparison to C4-2 cells

Discussion

In this study, we provided both in vitro and in vivo evidences that CUL4A possesses oncogenic role in the development of prostate cancer. We found that CUL4A was frequently overexpressed in prostate cancer cell lines and primary prostate tumors and showed that overexpression of CUL4A was significantly associated with high PSA and Gleason score, local invasion, and poor overall survival of patients with prostate cancer. Consistent with previous report in breast cancer study, high level of Cul4A is associated with poor prognosis in node-negative breast cancer [28]. Furthermore, we showed that knockdown of CUL4A by shRNA could suppress cell growth, decrease colony formation and induce apoptosis, and inhibit tumor formation in vivo. Reversely, overexpression of CUL4A resulted in transforming normal prostate epithelial cell RWP-1E and enhancing its invasion.

It has been reported that CUL4A is essential for ubiquitination of several well-defined tumor suppressors, including p21, p27, DDB2, and p53 [18–21]. Our study suggests that CUL4A activates ERK pathway. Overexpression of CUL4A dramatically increased ERK protein while knockdown of CUL4A remarkably decreased ERK. Surprisingly our results also suggested that the regulation of ERK by CUL4A is through transcription rather than ubiquitination. Some studies have showed that CUL4A regulates histone methylation [35]. We further showed that CUL4A upregu-lates ERK through enrichment of trimethylated H3K4 at ERK1 and ERK2 promoters. Importantly, CUL4A-induced malignant phenotype can be partially reversed by ERK inhibitor U0126, which suggests that CUL4A exert the oncogenic effect in prostate cancer through upregulation of ERK.

Although thalidomide has clinically proven activity in hematologic malignancies such as multiple myeloma and mantle cell lymphoma, development toward clinical use of thalidomide in solid tumor has been slow, and the results have not been promising. In this study, we found that there is a variation in response to thalidomide treatment in different prostate cancer cell lines, which is significantly linked to the level of CUL4A. Prostate cancer cells with high level of CUL4A are particularly sensitive to thalidomide. Along this line, we also demonstrated that the apoptotic effects of thalidomide in prostate cancer cells also depend on high level of CUL4A. To further delineate the mechanism underlying this phenomenon, we use overexpression and knockdown strategy to manipulate the expression of CUL4A in prostate cancer cells. The results showed that overexpression of CUL4A in thalidomide-resistant cells increase its sensitivity and apoptosis to this drug, while knockdown of CUL4A in thalidomide-sensitive cells results in resistance and decreased apoptosis to this drug. Moreover, we found the decrease in protein level of CUL4A in secondary thalidomide-resistant cells that were generated from long-term treatment of sensitive cells. Taking all together, we suggested that CUL4A determines thalidomide-induced apoptosis and CUL4A expression is a predictive factor for the response to thalidomide treatment in prostate cancer. Interestingly, we also found that CRBN level is correlated with CUL4A expression in both prostate cell lines and samples. Notably, CRBN mRNA is significantly decreased in thalidomide-resistant prostate cancer cell lines, which is similar to the observation in myeloma [34].

Personalized medicine for molecular targeted therapy has been a common approach to improve responsiveness in cancer therapy. In addition, evidence indicates that the major hindrance of molecular targeted therapy in solid tumors, especially prostate cancer, is very low response rate and high costs. Hence, the ability to select patients with a high response rate by detecting drug-related biomarkers is important to improve clinical efficacy. In this regard, this study has shown that CUL4A level is a good biomarker for predicting the response of prostate cancer to thalidomide; this may provide a promising strategy for designing clinical trials of thalidomide in patients with prostate cancer.

In summary, our data demonstrated that CUL4A is an oncogene and is a novel prognostic biomarker for prostate cancer. Furthermore, CUL4A level is also a good potential biomarker for predicting the response of prostate cancer to thalidomide.