Post-Transcriptional Up-Regulation of PDGF-C by HuR in Advanced and Stressed Breast Cancer

Breast cancer is a heterogeneous disease characterized by multiple genetic alterations leading to the activation of growth factor signaling pathways that promote cell proliferation. Platelet-derived growth factor-C (PDGF-C) is overexpressed in various malignancies; however, the involvement of PDGF-C in breast cancers and the mechanisms underlying PDGF-C deregulation remain unclear. Here, we show that PDGF-C is overexpressed in clinical breast cancers and correlates with poor prognosis. PDGF-C up-regulation was mediated by the human embryonic lethal abnormal vision-like protein HuR, which stabilizes the PDGF-C transcript by binding to two predicted AU-rich elements (AREs) in the 3'-untranslated region (3'-UTR). HuR is up-regulated in hydrogen peroxide-treated or ultraviolet-irradiated breast cancer cells. Clinically, HuR levels are correlated with PDGF-C expression and histological grade or pathological tumor-node-metastasis (pTNM) stage. Our findings reveal a novel mechanism underlying HuR-mediated breast cancer progression, and suggest that HuR and PDGF-C are potential molecular candidates for targeted therapy of breast cancers.


Correlation of Platelet-Derived Growth Factor-C (PDGF-C) Expression with Poor Prognosis of Breast Cancers
The expression of PDGF-C was examined in clinical breast cancer specimens of different pathological tumor-node-metastasis (pTNM) stages (Table 1). PDGF-C was detected in 49 of 81 (60.5%) breast cancer cases, and significantly increased PDGF-C levels were observed in high stage breast cancers ( Figure 1A). The expression of PDGF-C was significantly correlated with histological grade (p = 0.021) and pTNM stage (p = 0.023) ( Table 2). Patients were grouped according to the level of PDGF-C expression, as determined by immunohistochemical staining, which showed that PDGF-C-high breast carcinomas were associated with short disease-free survival compared with PDGF-C-low or -negative breast cancers ( Figure 1B). PDGF-C expression was higher in the tumorigenic and invasive breast cancer cell line MDA-MB-231 than in the non-invasive cell line MCF-7 ( Figure 1C). Knockdown of PDGF-C inhibited the proliferation and invasiveness of MDA-MB-231 cells ( Figure 1D-F). Taken together, these results suggest that PDGF-C expression is correlated with the malignant phenotype and poor prognosis of breast cancers.  and (F) Relative invasion activities compared with untransfected (ctrl) cells were determined by counting the numbers of invaded cells in a Transwell matrigel assay. Data are represented as the mean ± standard deviation (SD) of three independent assays for E and F. * p < 0.05, ** p < 0.01, compared with the control group.

Coordinated Expression of HuR and PDGF-C in Breast Cancers
To elucidate the mechanism underlying the up-regulation of PDGF-C in advanced breast cancers, a luciferase reporter construct containing the PDGF-C promoter was generated and introduced into breast cancer cells with a distinct invasive potential. No significant differences in the activity of the PDGF-C promoter were detected between MCF-7 and MDA-MB-231 cells (Figure 2A), which was in contrast to a significantly higher stability of luciferase transcripts flanked by the PDGF-C 3'-UTR in MDA-MB-231 cells than in MCF-7 cells ( Figure 2B). These data indicate that PDGF-C may be regulated at the post-transcriptional level in breast cancer cells. The RNA-binding protein HuR binds to and stabilizes specific mRNAs and is thus implicated in diverse pathophysiological processes [12]. To determine whether PDGF-C is post-transcriptionally regulated by HuR, we first predicted the HuR-binding sites on the PDGF-C mRNA, and found multiple putative HuR-binding sites on the transcript ( Figure 2C). Consistently, HuR was expressed at high level in advanced breast cancers ( Figure 2D). The expression of PDGF-C was significantly correlated with histological grade (p = 0.021), pTNM stage (p = 0.023), and HuR (p = 0.029). Other clinicopathological factors examined showed no significant correlations with PDGF-C expression (Table 2). Therefore, high PDGF-C levels were associated with the up-regulation of HuR in advanced breast carcinomas.

Direct Targeting and Stabilization of PDGF-C Transcripts by HuR in Breast Cancers
We next examined whether PDGF-C is directly up-regulated by HuR in neoplastic mammary cells. We found that PDGF-C levels were correlated with the expression of HuR in breast cancer cell lines with varied invasion potentials ( Figure 3A) Knockdown of HuR downregulated PDGF-C in MDA-MB-231 cells ( Figure 3B,C). An RNA-immunoprecipitation (IP) confirmed the direct association of HuR with the PDGF-C transcript ( Figure 3D). Reporter plasmids consisting of the luciferase coding sequence flanked by intact or truncated 3'-UTR of PDGF-C were co-transfected with control or HuR siRNAs into MDA-MB-231 cells ( Figure 3E). The results showed that the absence of either the second or fourth proximal putative HuR-binding site significantly decreased luciferase activity, suggesting decreased mRNA stability due to attenuated HuR binding ( Figure 3E). Knockdown of HuR further inhibited luciferase activity in the 3'-UTR constructs encompassing at least one of the two putative HuR-binding sites, supporting the protective role of these sites possibly mediated by HuR binding and stabilization of the transcripts ( Figure 3E). Taken together, these findings indicated that the PDGF-C transcript was stabilized by direct interaction with HuR in breast cancer cells.

Stress-Induced HuR Regulation of PDGF-C in Breast Cancers
HuR expression plays a role in maintaining the malignant state of advanced carcinomas; however, induced expression of HuR, which has been reported frequently, may represent an adaptive response to diverse stress situations [17]. Consistent with the well-defined role of HuR in oxidative stress [18,19], treatment of MCF-7 cells with hydrogen peroxide (H2O2) up-regulated HuR, leading to the up-regulation of PDGF-C ( Figure 4A). Similarly, a concomitant increase in HuR and PDGF-C was observed upon exposure of MCF-7 cells to ultraviolet (UV) irradiation ( Figure 4B). Knockdown of HuR or PDGF-C sensitized MCF-7 cells to H2O2-or UV-triggered cell death ( Figure 4C). Therefore, HuR-mediated up-regulation of PDGF-C is involved in the cellular protective responses to stress.

Discussion
Breast cancers are heterogeneous malignancies driven by germline or somatically accumulated genetic mutations [16]. Excessive signaling by steriod hormone and epidermal growth factor receptors is a characteristic of most breast cancers [2]. However, alternative inherent alterations also contribute to the occurrence and progression of breast cancers [2,16]. Here, we showed that platelet-derived growth factor-C (PDGF-C) is redundantly expressed in malignant cells, and its overexpression is correlated with advanced stage and poor prognosis of clinical breast cancers. Our findings are in agreement with previous reports that PDGF-C is overexpressed in various malignancies and plays essential regulatory roles in the tumor microenvironment [4,7,20,21]. Consistent with a pan-epithelial expression pattern of PDGF receptors [4], we found that PDGF-C plays a role in the accelerated proliferation of cultured breast cancer cells and confers resistance against stress-induced neoplastic cell death. However, our results do not exclude the effect of PDGF-C on tumor-associated macrophages, fibroblasts, and vascular endothelial cells, which expedite the progression of breast carcinomas [3,8,20,22].
The past decades have witnessed breakthroughs towards understanding of the post-transcriptional gene regulation machinery [10,11]. Unlike the degradation and translational suppression of messenger RNAs by microRNAs, HuR is among a cohort of RNA-binding proteins that regulate gene expression by stabilizing gene transcripts [10][11][12]. HuR is implicated in cell cycle and apoptosis regulation, angiogenesis, inflammation, and tumorigenesis by targeting numerous genes including cytokines and cyclins [23][24][25].
In the present study, we show that HuR binds to and stabilizes the PDGF-C transcript in advanced breast cancers, resulting in increased PDGF-C levels in neoplastic cells. Consistently, HuR expression was correlated with high levels of PDGF-C in late-stage breast cancers, suggesting that PDGF-C is among the key mediators of HuR-induced malignant transformation.
As a master regulator of gene transcript stability and splicing, HuR itself is tightly regulated at multiple levels including by transcriptional activation and post-translational modifications [17,26,27]. Nucleo-cytoplasmic shuttling plays a pivotal role in fine-tuning HuR activity under physiological or stress conditions [17,28,29]. The exact mechanisms underlying the regulation of HuR expression and cytoplasmic translocation remain unclear; however, various stress stimuli such as UV light, DNA damaging agents, or T cell activation promote the translocation of HuR to the cytoplasm, while AMP-activated kinase inhibits it [17,26,30,31]. Stress promotes the p38 MAPK-induced cytoplasmic translocation of HuR and the stability of ARE-containing mRNAs [32]. In the present study, we observed that H2O2 treatment or UV irradiation up-regulated HuR and PDGF-C expression in MCF-7 cells. Given that the effectiveness of standard cancer treatment is largely based on the induction of oxidative or DNA damage stresses [33][34][35], the role of HuR up-regulation in cancer resistance to clinical chemotherapy or radiotherapy merits further investigation. Collectively, our results shed light on the roles of HuR and PDGF-C in mammary carcinogenesis and indicate that these oncoproteins may serve as potential targets for the molecular classification and treatment of breast cancers.

Cell Invasion Assay
Cell invasion assay was performed in a 24-well Transwell (Corning, New York, NY, USA) on a polycarbonate filter pre-coated with 30 μg of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). 2 × 10 4 cells suspended in 0.2 mL of serum-free medium were added to the upper well of the chamber, and 600 μL of medium supplemented with 10% FBS was added to the lower well. After incubation at 37 °C in 5% CO2 for 24 h, the cells on the upper chamber were removed by a cotton swab. Invaded cells on the bottom of the membranes were fixed with methanol and stained with 0.1% crystal violet solution. Cells were photographed under the microscope, and the cell numbers were counted by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Springs, MD, USA) in five randomly selected fields.3.6. RNA Immunoprecipitation.
Cells were lysed in buffer containing 0.5% NP40, 0.5% Na Deoxycholate, 300 U/mL Superase Inhibitor (Ambion, Austin, TX, USA), and protease inhibitor (pH 7.9). Samples were treated with 30 U of Turbo DNase (Ambion) and incubated 15 min at 37 °C. After centrifuging for 5 min at 1350× g at 4 °C, 10% of the supernatant was saved as input, and the rest was subjected to immunoprecipitation at 4 °C overnight using 100 μL of Dynabeads Protein G conjugated with anti-HuR antibody (Proteintech) or IgG. Beads were washed three times at 4 °C with PBS supplemented with 1% NP40, 0.5% Na Deoxycholate, additional 300 mM NaCl, and 1:200 superase inhibitor. The immunocomplexes were eluted from the beads by the addition of 100 μL extraction buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1.3% SDS) plus Superase inhibitor and incubated for 30 min at 65 °C. RNA was extracted from input samples and immunoprecipitates using Trizol reagent (Invitrogen). PCR was performed to amplify the cDNA of PDGF-C and GAPDH (ctrl) as described above in "Reverse transcriptase polymerase chain reaction (PCR)". The results were normalized relative to the input control.

Enzyme Linked Immunosorbent Assay (ELISA) for PDGF-C
The total proteins of cultured MCF-7 cells were extracted using NE-PER Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) according to the manufacturer's protocol, and protein concentration was determined using a Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). PDGF-C levels were measured using an ELISA kit (Yanxin Biological Technology, Shanghai, China) according to the manufacturer's instructions. The protein concentrations of PDGF-C were normalized and expressed as pictograms per milligram of total cellular protein.

Clinical Sample Collection
A total of 81 primary breast cancer patients were enrolled between January 2012 and February 2013 in Tangdu Hospital of the Fourth Military Medical University (Xi'an) in China. The clinical characteristics of patients were obtained from hospital records. Sample collection was approved by the Ethics Committee of the Fourth Military Medical University.

Immunohistochemistry
Formalin-fixed breast cancer samples were embedded in paraffin. Serial 4 μm sections were obtained using a Leica microtome. The sections were then deparaffinized in xylene and rehydrated in a descending ethanol series. Tissue antigen retrieval was performed using 0.01 mol/L Na-citrate buffer (pH 6.0) in a steamer at 100 °C for 15 min. The sections were then incubated in 0.3% H2O2 for 10 min to remove endogenous tissue peroxidase activity. After washing with PBS, nonspecific tissue binding sites were blocked for 20 min at room temperature with 10% horse serum. The tissue sections were then incubated overnight with a rabbit anti-HuR antibody (1:100; Proteintech) or a rabbit anti-PDGF-C antibody (1:200, GeneTex). The slides were washed with PBS and incubated for 1 h with biotin-labeled anti-rabbit secondary antibodies (1:200 in phosphate-buffered saline containing 1% normal horse serum) for 45 min. The tissue sections were incubated with the avidin-biotinylated reagent (Gene Tech, Shanghai, China) for 1 h. Antibody binding was visualized using III Detection System/Mo & Rb (Gene Tech) at 37 °C and then lightly counterstained with Mayer's hematoxylin. As a positive control for PDGF-C expression, we used a human breast carcinoma cell line known to overexpress PDGF-C. When more than 10% of the cancer cells showed cytoplasmic staining for PDGF-C, the tumor was judged as positive for PDGF-C expression. We observed normal colon glands as a positive control for HuR staining.
The percentage of positive cells and intensity of staining were measured as indexes of HuR staining status. The percentage of positive cells was scored as follows: Zero (0% positive cells), 1 (<25% positive cells), 2 (25% to <50% positive cells), 3 (50% to <75% positive cells), and 4 (>75% positive cells). The intensity of cytoplasmic and nuclear staining was scored as follows: Zero (negative staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). For the immunoreactivity score, values from 0 to 12 were multiplied by the positive cell score and intensity of staining score. Cases were classified as having negative or weak expression when the immunoreactivity score was 0 to 6; a score from 7 to 12 was regarded as strong expression.

Statistical Analysis
All statistical analyses were performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). The association between the staining index and other clinicopathological variables was evaluated by the chi-squared test. A probability value below 0.05 was considered as statistically significant.

Conclusions
In the present study, we showed that PDGF-C expression was positively correlated with late-stage clinical breast cancers. PDGF-C was up-regulated at the post-transcriptional level by the human embryonic lethal abnormal vision (ELAV)-like protein HuR, which stabilizes mRNA transcripts via association with the 3'-UTR of target genes. HuR was induced by H2O2 treatment or UV irradiation of breast cancer cells. Clinically, HuR levels were correlated with PDGF-C expression and histological grade or pTNM stage. These findings reveal a novel mechanism underlying the role of HuR in breast cancer progression and suggest that HuR and PDGF-C could serve as molecular targets for the treatment of breast cancers.