Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression

Microarrays of human breast carcinomas were provided by the Yale Cancer Center Critical Technologies group. Two microarrays were used in this study: Yale Tissue Microarray (YTMA) -23, containing 246 breast cancer cases with complete clinical records and more than 30 years of follow-up (Additional file 1), and YTMA-89, consisting of 54 recurrent breast cancer cases. Paraffin-embedded, formalin-fixed specimens of breast carcinoma tissue were identified from the archives of the Yale University Department of Pathology as available from 1961 to 1983. Complete treatment information was unavailable for the entire cohort of 246 primary breast cancer cases, but most patients were treated with postsurgical local radiation. None of the node-negative patients were given adjuvant systemic therapy. Among the node-positive patients, approximately 15% were given chemotherapy primarily consisting of adriamycin, cytoxan and 5-fluorouracil, and some were given tamoxifen (post-1978) [31, 32]. Another two adjacent arrays composed of 50 breast cancers with matching normal adjacent tissues were obtained from Cybrdi, Inc. (CC08-1-07; Cybrdi, Inc., Rockville, MD, USA). Ethical approval for this study was obtained from the human research ethical advisory committee of Tsinghua University.

The human breast cancer cells MCF-7 and MDA-MB-231 (American Type Culture Collection, Manassas, VA, USA) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C and 5% CO₂.

After dewaxing and hydration, tissue microarray slides were immersed into preheated citric acid buffer (pH 6.5) at 90°C to 95°C for 20 minutes with microwaving. After being blocked with 5% horse serum for 30 minutes, slides were incubated with anti-Pokemon or anti-survivin antibodies (1:50; Abcam, Cambridge, MA, USA) at 4°C in a humid box overnight. Thereafter slides were washed three times and then incubated with horseradish peroxidase-conjugated secondary antibody (1:800; Pierce Biotechnology, Rockford, IL, USA) at room temperature for 1 hour. Enhanced 3,3'-diaminobenzidine staining buffer (Pierce Biotechnology) was used to visualize signals. Staining intensity was evaluated blindly by at least one researcher and one pathologist and was scored from 0 to 3, representing negative and low, intermediate and high staining, respectively.

Pokemon-expressing plasmid was generated by inserting the encoding region into pcDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA) at the site of the HindIII restriction enzyme. Pokemon primer pairs were 5'-CTT AAG CTT GCC ACC ATG GCC GGC GGC GTG G-3' and 5'-GTC AAG CTT TTA GGC GAG TCC GGC TGT GAA GTT AC-3'. Survivin promoter was subcloned into pGL4.10 basic plasmid (Promega, Madison, WI, USA) at the BamHI and EcoRV restriction enzyme sites to drive luciferase expression. The amplification primer pairs were 5'-GTC AGA TCT AGT GAA AAG GAG TTG TTC CTT TCC TCC CTC-3' and 5'-GTC AAG CTT GCC GCC GCC GCC ACC TC-3' for a 2,082-bp fragment, 5'-GTC AAG CTT GCC GCC GCC GCC GCC ACC TC-3' and 5'-GTC AGA TCT AAA GAC AGT GGA GGC ACC AGG C-3' for a 1,054-bp fragment, and 5'-GTC AAG CTT GCC GCC GCC GCC ACC TC-3' and 5'-GTC AGA TCT TTG GGA TTA CAG GCA TGC ACC AC-3' for a 441-bp fragment.

Mutants (pLuc-95 m and pLuc-231 m) of survivin promoter were generated using an in vitro site-directed mutagenesis system (Promega). The GGGTG sequence in the binding site of Pokemon was replaced by AAAAA and confirmed by sequencing.

Pokemon expression in MDA-MD-231 breast cancer cells was knocked down using chemically synthesized small interfering RNA (siRNA) siRNA#1 (targeting 426 to 444 bp, sense 5'-GCU GGA CCU UGU AGA UCA Att-3', and antisense 5'-UUG AUC UAC AAG GUC CAG Ctt-3'), siRNA#2 (targeting 476 to 494 bp, sense 5'-AGU ACC UCG AGU UCU UCC Att-3', and antisense 5'-UGG AAG AAC UCG AGG UAC Utt-3') [13] and siRNA#3 (targeting 624 to 642 bp, sense 5'-GGA GUA CCU CGA GUU CUU Ctt-3', and antisense 5'-GAA GAA CUC GAG GUA CUC Ctt-3'). The siRNA were introduced as described previously [33], and protein knockdown was examined using Western blot analysis [34]. Soluble proteins (30 μg) were probed with anti-Pokemon (1:500), anti-survivin, anti-p14ARF and anti-Bcl-2 antibodies (1:500) (Abcam). Loading variations were normalized against β-actin, which was identified by anti-β-actin monoclonal antibody (Sigma, St. Louis, MO, USA).

Transient gene delivery was carried out to assess the effect of Pokemon on survivin promoter activity in MDA-MB-231 and MCF-7 cells as described previously [35, 36]. Briefly, 1 × 10⁵ cells were mixed with survivin promoter constructs and Pokemon expression vector or Pokemon siRNA#1. At 48 hours after transfection, cell extracts were prepared with 1 × lysis buffer, and a 10-μl aliquot of the supernatant was mixed with 50 μl of luciferase assay reagent (Promega) and analyzed with a Microplate Luminometer (Beckman Coulter, Fullerton, CA, USA). Luciferase activity was normalized by using a Renilla luciferase internal control.

MCF-7 and MDA-MB-231 cells were fixed by the addition of 1% formaldehyde to the medium for 10 minutes. Formaldehyde was quenched by the addition of 1 × glycine for 5 minutes at room temperature. A chromatin immunoprecipitation (ChIP) assay was performed as described previously [37] with 3 μl of immunoprecipitated DNA and primers (forward 5'-GTC AAG CTT GCC GCC GCC GCC ACC TC-3' and reverse 5'-GTC AGA TCT TTG GGA TTA CAG GCA TGC ACC AC-3') located at -441 to -418 bp and -17 to +1 bp of the survivin promoter.

Spearman's rank-correlation coefficients were used to assess the relationship between Pokemon and survivin expression, the Wilcoxon rank-sum test or the Kruskal-Wallis test were utilized with categorical variables and Kaplan-Meier survival curves were produced to examine the relationship between Pokemon expression and mortality. Results were considered statistically significant at P < 0.05.

The YTMA-23 tissue microarray, provided by the Yale Cancer Center Critical Technology Group, was evaluated for Pokemon expression. This array consists of 246 breast cancer cases and normal controls, among which 182 malignant tissues were histologically interpretable. The results showed that Pokemon was undetectable in normal breast lobules, but overexpressed in 158 (86.8%) of 182 cancerous tissues. The level of Pokemon expression was assessed according to the intensity of immunostaining and assigned the score of 0 to 3 (negative to most intensive) (Figure 1A). This YTMA-23 microarray came with complete clinical records and follow-up, and therefore the correlation between Pokemon expression and clinical pathological parameters was analyzed. As summarized in Table 1, Pokemon expression correlated positively with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014), but not with patient age, tumor type and nuclear grade. Pokemon was also found to be overexpressed in 28 (80%) of 35 interpretable recurrent breast tumors in another YTMA-89 microarray.

Tumor size and lymph node metastasis are well-known prognostic factors for breast cancer, and therefore the effect of Pokemon expression on patient survival was further analyzed using Kaplan-Meier plots. As shown in Figure 1B, Pokemon expression was negatively correlated with overall survival (n = 175, P = 0.01) and in particular disease-related survival (n = 79, P = 0.0134) of breast cancer patients, indicating that Pokemon is a negative prognostic indicator.

The estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) genes are well-established biomarkers and therapeutic targets for breast cancer. The correlation of Pokemon expression with these three molecular markers was assessed using Kruskal-Wallis tests, and the results showed that Pokemon expression did not correlate with ER, PR or HER2 alone or with any combination of the three genes.

The positive correlation of Pokemon expression with disease progression suggests that Pokemon may promote cancer development. To determine the underlying mechanisms, we attempted to identify genes regulated by Pokemon in MCF-7 breast cancer cells. A total of six MCF-7 cell clones with stable Pokemon overexpression were isolated (Additional file 2), and three of them were subjected to cDNA microarray analysis. Figure 2 shows representative data of microarray analysis, in which a total of 121 genes were found to have more than a twofold increased or decreased expression in Pokemon-overexpressing cells compared to the vector control. On the basis of the Kyoto Encyclopedia of Genes and Genomes and the Gene Ontology databases, these genes are classified into seven signaling and/or metabolic pathways (Figure 2C). Notably, these Pokemon target genes are mainly involved in apoptosis, cell cycle, differentiation and biosynthesis, suggesting the importance of Pokemon in cell growth, proliferation and carcinogenesis.

Interestingly, BIRC5 (survivin), a negative prognostic factor for breast cancer [38], was found to be one of the downstream targets of Pokemon. Therefore, transfection experiments were performed to determine the role of Pokemon in inducing the expression of survivin. MCF-7 cells were transfected with Pokemon expression vector to increase the level of Pokemon, while MDA-MD-231 cells were transfected with Pokemon siRNA to knock down its expression. As shown in Figure 2D(I), three different siRNA targeting 426 to 444 bp, 476 to 494 bp and 624 to 642 bp of Pokemon mRNA successfully knocked down its expression and in turn led to a decrease of survivin, proving the cDNA microarray data. A time-course study showed that significant silencing of Pokemon by siRNA was observed at 48 hours after transfection. Interestingly, Pokemon silencing in MDA-MD-231 led to upregulation of p14ARF, but had no effect on Bcl-2 expression. In addition, transient expression of Pokemon in MCF-7 cells induced survivin upregulation in a parallel manner to Pokemon levels (Figure 2D(II)), supporting the cDNA microarray data from stable clones. Taken together, these results suggest that Pokemon indeed induces survivin expression.

To confirm that the induction of survivin by Pokemon indeed occurs in breast cancer, adjacent tissue microarrays containing 50 breast cancer tissue samples and matching adjacent normal breast tissue samples were subjected to immunohistological analysis of Pokemon and survivin expression. As shown in Figure 3, the levels of survivin and Pokemon expression were found to be highly correlated (r = 0.6799, P < 0.0001). In normal breast tissues, the expression levels of both Pokemon and survivin were low (data not shown).

We further explored the mechanism by which Pokemon regulates the expression of survivin. The promoter region of survivin, containing 2,080 bp upstream of the transcription start site, was cloned and used to drive the expression of luciferase reporter in MCF-7 and MDA-MB-231 breast cancer cells. Progressive deletions of the 5' end of the survivin promoter revealed that the -441-bp fragment possesses full promoter activity (Figure 4A). Promoter motif analyses recognized two GT boxes (GGGTG), located at -231 to -227 bp and -95 to -91 bp, which are potential binding sites of Pokemon. Therefore, this promoter fragment was used to investigate the regulatory role of Pokemon on its activity. As shown in Figure 4B, cotransfection of PCDNA3.1(+)/Pokemon greatly increased the activity of -pluc-441 in both MCF-7 and MDA-MB-231 cells, while the delivery of Pokemon siRNA significantly reduced its basal activity, suggesting Pokemon as a positive regulator of survivin promoter. Furthermore, replacing GGGTG with AAAAA in these two GT boxes of pluc-441 (luciferase plasmid at -441 bp) abrogated the promoter's responsiveness to Pokemon in both MCF-7 and MDA-MB-231 cells (Figure 4B), and interaction between Pokemon and survivin promoter was further confirmed by ChIP assay. As shown in Figure 4C, survivin promoter fragment was amplified by polymerase chain reaction assay from chromatin precipitated by an anti-Pokemon antibody, but not by nonspecific immunoglobulin G. These data suggest that Pokemon upregulates survivin expression through direct binding to the GT boxes in its promoter.