Silencing of Human Phosphatidylethanolamine-Binding Protein 4 Sensitizes Breast Cancer Cells to Tumor Necrosis Factor-α–Induced Apoptosis and Cell Growth Arrest

Although significant progress has been made (1, 2), the current therapeutic approach is not very effective in breast cancer patients (2–4). Therefore, alternative treatment protocols aimed at different targets need to be explored. Inducers of apoptosis and growth inhibition have been applied in cancer treatment; however, chemotherapy or radiation is not invariably cytotoxic to all tumor cells. It has been observed that defects in the apoptotic and growth inhibitory pathway in cancer cells confer insensitivity to the cytotoxic effects of chemotherapy and may therefore represent an important mechanism for cancer cell drug resistance (5–7). In the past few years, several studies have attempted to induce cancer cell apoptosis by targeting or silencing the antiapoptotic proteins and have shown promising results (8, 9). Understanding how to unleash the apoptotic program and growth arrest in cancer cells could aid the design of effective therapeutic interventions against resistant cancers. Enhancing the chemosensitivity of cancer cells by the transfer or interference of genes that influence death and growth of the cell is one of the most important strategies in cancer therapeutics.

Human phosphatidylethanolamine-binding protein 4 (hPEBP4) is a novel member of phosphatidylethanolamine-binding protein family identified from human bone marrow stromal cells by us (10). Overexpression of hPEBP4 has been shown to inhibit tumor necrosis factor-α (TNF-α)–induced apoptosis by inhibiting mitogen-activated protein kinase (MAPK) pathway activation and phosphatidylethanolamine externalization (10). TNF-α is a major mediator of apoptosis, antiapoptosis, and cell growth arrest and kills various tumor cell lines in vitro or mediates antitumor effect in vivo (11–13). TNF-α exerts its biological effects by binding to two types of cell surface receptors, ultimately up-regulating proapoptotic and cell cycle inhibitory proteins. The p53 tumor suppressor protein is a transcription factor that is activated in response to TNF stimuli (14). Activation of p53 affects genes associated with cell cycle arrest, DNA repair, and apoptosis including p21^waf/cip1 (15, 16), Bax (17), and Bcl-2 (18). She et al. (19) report that extracellular signal-regulated kinase 1/2 (ERK1/2) mediate UVB-induced phosphorylation of mouse p53 at serine 15. In addition, Fuchs et al. (20) show that c-jun NH₂-terminal kinase (JNK) activation via MAPK/ERK kinase (MEK) kinase 1 results in p53 phosphorylation, thus leading to the reduced inhibition of murine double minute-2 association with p53 and the increase in p53 protein half-life. Thus, there is active interaction between activation of MAPK pathway and activation of p53, both of which jointly contribute to cancer cell apoptosis and cell cycle arrest.

Considering our preliminary evidence showing high expression of hPEBP4 in MCF-7 breast cancer cells, in the present study, we first showed the preferential expression pattern of hPEBP4 in human breast cancer tissues. Based on the fact that hPEBP4 exhibits antiapoptotic function, our investigation was thus undertaken to further illustrate the antiproliferation and cell cycle arrest effects and the underlying molecular mechanisms of hPEBP4 expression silencing on breast cancer cells. Our results show that hPEBP4 is a candidate target molecule for breast cancer treatment and that silencing of hPEBP4 might represent a promising approach for the treatment of cancers that express high levels of hPEBP4.

Reagents and cell culture. MEK1 inhibitor PD98059 and JNK inhibitor SP600125 were obtained from New England Biolabs (Beverly, MA) and Calbiochem (San Diego, CA), respectively. All cells were grown in RPMI 1640 or DMEM supplemented with 10% (v/v) FCS, 4.5 g/L d-glucose, nonessential amino acids (100 μmol/L each), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L glutamine at 37°C in a 5% CO₂ atmosphere.

Reverse transcription-PCR and immunohistochemistry analysis of human phosphatidylethanolamine-binding protein 4 expression in breast cancer. Reverse transcription-PCR (RT-PCR) with primers specific for hPEBP4 was done as previously described (10). The human Tissue Microarrays of normal breast tissue and breast cancer tissue were obtained from Cybrdi (Xi'an, Shanxi, China). The arrays contain 123 dots in total and each dot represents one normal or diseased tissue spot from one individual specimen that was selected and pathologically confirmed. The arrays were fixed with formalin, embedded in paraffin, and immunostained with anti-hPEBP4 antibody (1:40 dilution) using avidin-biotin peroxidase complex method.

Generation of small interfering RNA plasmid vector. For the vector expressing a hairpin small interfering RNA (siRNA) against hPEBP4, the single-stranded oligonucleotides specific to hPEBP4, 5′-TCGAGGGAAAAGTCATCTCTCTCCTTgagtactgAAGGAGAGAGATGACTTTTCCCTTTTT-3′(sense) and 5′-CTAGAAAAAGGGAAAAGTCATCTCTCTCCTTcagtactcAAGGAGAGAGATGACTTTTCCC-3′ (antisense), were synthesized, annealed, and cloned into the SalI and XbaI cloning sites of pSuppressorNeo (Imgenex, San Diego, CA). The plasmid construct (hPEBP4-RNAi) was then confirmed by sequencing. The control plasmid, Neo, contains a scrambled sequence that does not show significant homology to rat, mouse, or human gene sequences (Imgenex).

Human phosphatidylethanolamine-binding protein 4 RNA interference assay. hPEBP4 transient siRNA assay with chemically synthesized siRNA duplex and mutated control was done as previously described (10). For stable silencing of hPEBP4 expression in MCF-7 human breast cancer cells, MCF-7 cells were transfected with the hPEBP4-RNAi or Neo plasmid using LipofectAMINE reagent (Invitrogen, Carlsbad, CA). Forty-eight hours after transfection, cells were screened under 0.8 mg/mL G418 (Merck, Darmstadt, Germany) for 25 days. Individual G418-resistant colonies were subcloned as MCF-7/Neo and MCF-7/hPEBP4-RNAi and the hPEBP4 expression was determined by reverse transcription-PCR and Western blot analysis.

Cell growth analysis. To determine the rate of cell growth, MCF-7/Neo and MCF-7/hPEBP4-RNAi cells were seeded into 96-well plates at 3 × 10³ cells per well and treated with 5 ng/mL TNF-α for various incubation periods. On the day of harvest, 100 μL of spent medium were replaced with an equal volume of fresh medium containing 10% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL stock. Plates were incubated at 37°C for 4 or 16 hours, then 100 μL of DMSO (Sigma, St. Louis, MO) were added to each well, and plates were shaken at room temperature for 10 minutes. The cell growth was determined by measuring the absorbance of the converted dye at a wavelength of 570 nm. For [³H]thymidine incorporation assay, each well was supplemented with 0.5 μCi of [³H]thymidine (Amersham, Buckinghamshire, England) and incubated for a further 16 hours. Then, cells were harvested onto glass fibers and the proliferation of cells was detected using a liquid Scintillation Counter (Wallac, Turku, Finland).

Cell cycle and apoptosis assay. The cell cycle was analyzed by flow cytometry as described (21). Briefly, 1 × 10⁶ cells were harvested and washed in PBS, then fixed in 75% alcohol for 30 minutes at 4°C. After washing in cold PBS thrice, cells were resuspended in 1 mL of PBS solution with 40 μg of propidium iodide (Sigma) and 100 μg of RNase A (Sigma) for 30 minutes at 37°C. Samples were then analyzed for their DNA content by FACSCalibur (Becton Dickinson, Mountain View, CA). For apoptosis assay, cells were washed, resuspended in the staining buffer, and examined with ApoAlert Annexin V Apoptosis kit (Becton Dickinson) and propidium iodide according to the instruction of the manufacturer. FL-SA-Ro staining was done as described (22). Stained cells were analyzed by fluorescence-activated cell sorting (FACScalibur, Becton Dickinson).

Coimmunoprecipitation and in vitro protein binding assay. MCF-7 cell lysates were precleared with protein A-Sepharose beads (Sigma) and immunoprecipitation was done using anti-hPEBP4 polyclonal antibody cross-linked to protein-A Sepharose beads. Samples were either subjected to Western blot analysis directly or incubated with extracts of HEK293 cells transfected with either Raf-1-FLAG or MEK1-FLAG [full-length Raf-1 and MEK-1 with a COOH-terminal FLAG-tag subcloned into pcDNA3.1/Myc-His(−)B expression vector] as previously described (10).

Western blot analysis. A BCA Protein Assay Reagent Kit (Pierce, Rockford, IL) was used to measure protein concentration. Samples containing equal amounts of protein were separated by 12% SDS-PAGE and transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Blots were probed with antibodies specific for phospho-ERK1/2, phospho-MEK-1, phospho-Raf-1, phospho-JNK1/2, ERK1/2, p53, phospho-p53^ser15, Bcl-2, Bax, Bcl-xL (Cell Signaling, Beverly, MA), p21^waf/cip1, p27, cyclin D1, cyclin A, cyclin E, Raf-1, and MEK-1 (Santa Cruz, Santa Cruz, CA) with appropriate horse radish peroxidase–conjugated antibodies as secondary antibodies (Cell Signaling). SuperSignal West Femto Maximum Sensitivity substrate (Pierce) was used for the chemiluminescent visualization of proteins.

Statistical analysis. Statistical analysis (Fisher's exact test) was done using the computer program SPSS version 6.1.

Human phosphatidylethanolamine-binding protein 4 is highly expressed in human breast carcinoma tissues. hPEBP4 was found to promote cellular resistance to TNF-α-induced apoptosis (10). We also observed that MCF-7 breast cancer cells express high level of hPEBP4. Therefore, we propose that hPEBP4 may be one candidate target molecule for breast cancer treatment. Thus, hPEBP4 expression pattern in normal and tumorous breast tissues was first examined. We evaluated its expression in clinical specimens of normal and breast cancer tissues from Chinese females by RT-PCR and immunohistochemical analysis. As shown in Fig. 1, there was no detectable expression of hPEBP4 in all the six samples of human normal breast tissue as determined by RT-PCR (Fig. 1A, lanes 1 and 2 represent two different samples, with a further four not shown). However, all six of the assayed breast cancer tissue samples displayed strong hPEBP4 expression (Fig. 1A, lanes 3-8). The expression pattern was further confirmed by immunohistochemistry with anti-hPEBP4 polyclonal antibody in the same samples, which revealed significant staining in breast cancer cells (Fig. 1B, a and b represent one sample, others not shown) but an absence of hPEBP4 staining in normal breast tissues (Fig. 1B, c and d represent one of six normal samples). Using human Tissue Microarrays, an additional 123 female breast tissue samples, including 80 cases of infiltrating duct carcinoma, 10 cases of benign lesions, and 33 cases of normal breast tissue, were tested for the expression of hPEBP4 protein by immunohistochemistry. In the tissue arrays, we used the standard immunohistochemical protocol and criteria for the judgment of positive or negative signals. As shown in Fig. 1B, the yellow reaction product indicates the expression of hPEBP4. More importantly, the expression of hPEBP4 was shown to be present in a very high percentage of breast cancer tissue (64%) but in very low percentages of benign lesion (10%) and normal breast tissue (3.3%) as shown in Table 1. The difference in prevalence of hPEBP4 between breast cancer and benign lesion or normal breast tissue was found to be highly significant (P = 0.0000) but there is no significant difference between normal breast tissue and benign lesions (P = 0.226).

These data show the preferential expression pattern of hPEBP4 in human breast cancer tissues, taken together with its antiapoptotic function, suggesting that silencing of hPEBP4 expression may be promising for breast cancer treatment.

Endogenous human phosphatidylethanolamine-binding protein 4 associates with Raf 1 or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 in MCF-7 cells. When overexpressed in L929 cells, hPEBP4 was found colocalized with lysosomes, and TNF-α stimulates its transfer to the cell membrane where it binds to Raf-1 and MEK1, thus inhibiting downstream MEK1/ERK activation (10). How about the physiologic interaction of endogenous hPEBP4 with Raf-1 and MEK1 in human breast cancer cells? Thus, possibility was tested in MCF-7 cells with or without TNF-α treatment. Anti-hPEBP4 polyclonal antibody, which specifically recognizes the first 99 amino acids of hPEBP4 (the region sharing the lowest homology with other phosphatidylethanolamine-binding proteins), was used to immunoprecipitate endogenous hPEBP4 from TNF-α-stimulated MCF-7 cell lysates. As shown in Fig. 2A, Raf-1 and MEK1 were detected in the immunoprecipitates of MCF-7 cells treated with TNF-α but not in those of unstimulated cells. This interaction was further confirmed by in vitro protein binding assay. MCF-7 cell lysates were immobilized on protein A beads complexed with anti-hPEBP4 antibody or an irrelevant anti–green fluorescent protein antibody, then incubated with extracts of HEK293 cells transfected with either Raf-1-FLAG or MEK1-FLAG. Endogenous hPEBP4 was shown to specifically bind to Raf-1 and MEK1 in vitro (Fig. 2B). No binding was detected when anti–green fluorescent protein antibody complexed beads were used. Our data provide definite evidence that hPEBP4 binds to Raf-1 or MEK-1 on TNF-α treatment, dissociates the Raf-1-MEK complex, and thus functions as a competitive inhibitor of MEK phosphorylation, which can explain why Ras/Raf-1/MEK/ERK signaling pathway activated by TNF-α was enhanced in hPEBP4-silenced cells.

Promotion of mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization contributes to the increased tumor necrosis factor-α–induced apoptosis in MCF-7 cells by human phosphatidylethanolamine-binding protein 4 silencing. Our previous study showed that silencing of hPEBP4 expression significantly sensitizes target cells to TNF-α-induced apoptosis. Next, we want to confirm the phenomenon in MCF-7 cells and further investigate the mechanisms by which silencing of hPEBP4 expression significantly sensitizes MCF-7 cells to TNF-α-induced apoptosis. As shown in Fig. 3A and B, interference with hPEBP4 siRNA robustly enhanced the activation of ERK1/2, MEK1, and JNK by TNF-α in MCF-7 cells but had no effect on Raf-1 activation, which further confirmed the negative role of hPEBP4 in TNF-α-induced activation of the Ras/Raf/MEK1/ERK pathway and JNK observed in L929 cells (10). TNF-α treatment also elicited greater phosphatidylethanolamine exposure in hPEBP4 siRNA versus control transfectants (Fig. 3C). To determine whether the shown activation of ERK and JNK was relevant to the apoptosis sensitization induced by hPEBP4 interference, we preincubated hPEBP4-silenced MCF-7 cells with MEK1 inhibitor (PD98059) or JNK inhibitor (SP600125; ref. 23), and then cells were stimulated with TNF-α. As shown in Fig. 3D, pretreatment with PD98059 or SP600125 significantly decreased the apoptotic percentage of hPEBP4-silenced MCF-7 cells [from 65.1% to 36.7% (PD98059) or 38.2% (SP600125)]. However, neither of these inhibitors could completely reverse the potentiating effect of hPEBP4 silencing on TNF-α-induced apoptosis, indicating that hPEBP4-mediated inhibition of ERK and JNK activation is only partially responsible for its suppression of TNF-α-induced apoptosis. Taken together, it seems that the sensitization of hPEBP4-silenced MCF-7 cells to TNF-α-induced apoptosis is due to the potentiated activation of the Raf-1/MEK/ERK pathway, JNK, and phosphatidylethanolamine externalization following TNF-α treatment.

Because cross talk among MAPK family members has been shown to regulate their activity (24, 25), we investigated the possibility that increased ERK1/2 activation by hPEBP4 interference might be responsible for the observed increase in JNK activation. To inhibit ERK1/2 activation, hPEBP4-silenced MCF-7 cells were pretreated with PD98059. PD98059 completely inhibited TNF-α-mediated ERK1/2 activation (phospho-ERK in Fig. 3E, compare lanes 4, 8, and 12 to lanes 3, 7, and 11). However, PD98059 had no inhibitory effect on TNF-α-induced JNK activation (phospho-JNK, compare lanes 8 and 12 to lanes 7 and 11). The effect of PD98059 on hPEBP4 silencing-enhanced JNK activation as above was then studied. In the presence of PD98059, the increased activation of JNK by hPEBP4 interference was prevented (compare lanes 4 and 2). Therefore, activation of ERK1/2 is required for TNF-α-induced greater activation of JNK by hPEBP4 silencing in MCF-7 cells.

Up-regulation of p53 and apoptosis-related proteins in human phosphatidylethanolamine-binding protein 4–silenced MCF-7 cells and its requirement for extracellular signal-regulated kinase 1/2 and c-jun NH₂-terminal kinase activation. p53 is well known as a tumor suppression gene involved in both cell growth arrest and apoptosis (26). Members of the MAPK family have also been shown to phosphorylate and stabilize p53, thus regulating apoptosis-related protein expression (27–31). Therefore, the effect of hPEBP4 silencing on p53 and apoptotic proteins expression was examined. As shown in Fig. 4, p53 was apparently stabilized by TNF-α and down-regulation of hPEBP4 by siRNA enhanced this effect in MCF-7 cells. Simultaneously, silencing of hPEBP4 expression further potentiated TNF-α-induced up-regulation of phospho-p53^ser15, p21^waf/cip1, and Bax and down-regulation of Bcl-2 and Bcl-xL. However, the protein level of Bad, the other apoptosis-related protein, remained unchanged.

We also observed the relationship of MAPK pathway activation with the up-regulation of apoptosis-inducing proteins in hPEBP4-silenced MCF-7 cells. We found that MEK1 inhibitor (PD98059) could partially inhibit the hPEBP4 silencing-induced potentiation of apoptosis-related proteins expression by TNF-α, consistent with the result of apoptosis induction. JNK inhibitor (SP600125) had no effect on the level of phospho-p53^ser15 but enhanced p53 stability, suggesting that activated JNK phosphorylated p53 at other sites such as serine 34 (25, 26, 32). These results suggest that hPEBP4 silencing promoted TNF-α-induced up-regulation of p53 and apoptosis-related proteins regulated by p53, and that the promotion by hPEBP4 silencing requires ERK1/2 and JNK activation.

Silencing of human phosphatidylethanolamine-binding protein 4 expression increases senstitivity of MCF-7 cells to tumor necrosis factor-α–induced growth inhibition and cell cycle arrest. Considering that silencing of hPEBP4 expression enhanced TNF-α-induced stability of p53 and up-regulation of p21^waf/cip1, the cell cycle inhibitors, we then investigated the effect of hPEBP4 interference on MCF-7 cell growth. MCF-7 human breast cancer cells were stably transfected with hPEBP4-RNAi or Neo plasmids. The expression levels of hPEBP4 in MCF-7 cells stably transfected with hPEBP4-RNAi plasmid was almost completely interfered (Fig. 5A), similar to that in the transiently transfected cells (10). As shown in Fig. 5B and C, MCF-7/hPEBP4-RNAi cells had growth characteristics which were similar to MCF-7/Neo cells; however, they were more sensitive to TNF-α-induced cell growth arrest, suggesting that silencing of hPEBP4 itself does not affect MCF-7 cell proliferation but increases sensitivity of MCF-7 cells to TNF-α-induced cell arrest. We went further to analyze the cell cycle kinetics of hPEBP4-silenced MCF-7 cells. Representative cell cycle profiles of transfected MCF-7 cells are shown as histograms in Fig. 5D with data expressed as mean percentage of cells in each cell cycle phase 24 hours after 10 ng/mL TNF-α treatment or not, derived from three independent experiments. MCF-7/hPEBP4-RNAi cells did not differ in their baseline cycle kinetics from MCF-7/Neo control. After TNF-α treatment, however, MCF-7/hPEBP4-RNAi cells showed a higher proportion of cells in G₀-G₁ phase (93.4%) compared with MCF-7/Neo control (65.6%) and a decrease in the proportion of cells in S phase and G₂-M phase (3.3% and 3.4%, respectively) relative to that observed in controls (19.9% and 15.4%, respectively). Pretreatment of cells with PD98059 or SP600125 also significantly decreased hPEBP4-RNAi–induced TNF-α sensitivity, indicating that enhancement of ERK or JNK activation by hPEBP4 silencing contributes to the p53/p21^waf/cip1–mediated G₀-G₁ check point. This is consistent with the observations that enforced ERK activation by overexpression of a constitutively activated Raf1 led to G₀-G₁ arrest (33–35).

Because the appropriate temporal activation of cyclin E/cyclin-dependent kinase 2, cyclin D1/cyclin-dependent kinase 2, and cyclin A/cyclin-dependent kinase 2 is required for progression through the G₁ and S entry, we simultaneously examined expression of cyclins A, E, and D1 in MCF-7/hPEBP4-RNAi cells following TNF-α stimulation. Interestingly, cyclin A and cyclin E expressions were significantly decreased in MCF-7/hPEBP4-RNAi when compared with MCF-7/Neo after TNF-α treatment for 24 hours whereas cyclin D1 expression was not (Fig. 5E).

Insights into the mechanisms underlying chemotherapeutic drug resistance have been gained from a better understanding of the pathways involved in apoptosis and cell growth. Defects in the pathway of apoptosis and growth arrest have been observed in cancer cells to confer insensitivity to the cytotoxic effects of chemotherapy and may therefore represent an important mechanism for development of drug resistance (7, 15). The direct antitumor properties of proinflammatory cytokines are generally considered to reside in their ability to inhibit tumor growth (36) or cause cell death (37). In our previous study (10), we showed that hPEBP4, a new member of the phosphatidylethanolamine-binding protein family, can inhibit TNF-α-induced apoptosis. Here we found that silencing of hPEBP4 expression in MCF-7 cells promoted TNF-α-induced apoptosis, as well as TNF-α-induced ERK and JNK activation. In addition, the increased TNF-α-induced cell death by hPEBP4 silencing was partially inhibited by PD98059 or SP600125, inhibitors of ERK and JNK activation, respectively. Thus, our results suggest that ERK and JNK might act under certain circumstances in a proapoptotic fashion. Similar data have been reported for cisplatin-induced apoptosis and Apo2 ligand/TNF-related apoptosis-inducing ligand–induced apoptosis (28, 29, 31). Simultaneously, the endogenous hPEBP4 association with both MEK1 and Raf-1 after TNF-α stimulation was observed using coimmunoprecipitation and in vitro protein binding assays confirmed our assumption that TNF-α stimulates hPEBP4 travels to a membrane proximal position where it binds to Raf-1 or MEK-1, dissociating the Raf-1-MEK complex and inhibiting MEK/ERK phosphorylation on TNF-α treatment.

In addition to apoptosis induction, growth arrest also plays a pivotal role in the cytotoxic activity of most chemotherapeutic drugs, and defects in this pathway provide a basis for drug resistance in many cancers (38, 39). We thereby investigated the effect of hPEBP4 silencing on cancer cell growth. Interestingly, silencing of hPEBP4 itself did not affect MCF-7 cell growth but sensitized MCF-7 cells to TNF-α-induced growth inhibition. Consistently, enhancement of TNF-α-induced G₀-G₁ phase arrest by hPEBP4 silencing was abolished by the inhibitors of ERK1/2 or JNK, indicating that activation of ERK and JNK is required for the increased cell arrest by TNF-α in hPEBP4-silenced MCF-7 cells.

Many chemotherapeutic drugs exert their anticancer activities by inducing apoptosis and cell growth arrest, and in many cases the p53 pathway has been identified as the effector of such signals. Under normal physiologic conditions, p53 is a short-lived protein. In response to cellular stresses, p53 protein is stabilized and activated via posttranscriptional modifications, such as phosphorylation. Activated p53 affects its downstream genes including genes involved in growth arrest (such as p21^waf/cip1) and apoptosis (such as Bcl-2 and Bax), which direct target cells primarily to growth arrest and apoptosis (40–43). Kinases that have been reported to phosphorylate p53 include JNK⁴³ and ERK1/2^28-31. In our study, we showed that silencing of hPEBP4 in MCF-7 cells promotes stability of p53, up-regulation p21^waf/cip, phospho-p53^ser15, and Bax, and down-regulation of Bcl-2 and Bcl-xL. Interestingly, PD98059 and SP600125 could reverse the promotion. Although the specific inhibitor of JNK, SP600125, partially restored the enhanced p53 accumulation, it did not affect phospho-p53^ser15 level, suggesting that JNK phosphorylates p53 at other sites, which is consistent with the previous report that ERK1/2 mediated DNA damage-induced phosphorylation of mouse p53 at serine 15 (20, 28, 29) and JNK at serine 34 (25–27, 31). Simultaneously, the expressions of cyclin E and cyclin A, which control the progression of the cell cycle from the G₀-G₁ phase to the S phase, decreased more dramatically in hPEBP4-silenced MCF-7 cells compared with control MCF-7 cells whereas expression of cyclin D1 not.

The evidence for the role of gene alterations associated with cancer in general, and in breast adenocarcinomas in particular, is still accumulating. Most of the known genetic mechanisms involved in the tumor origination, progression, and regression involve the up-regulation of antiapoptotic molecules. Of particular interest is the preferential expression pattern of hPEBP4 in cancer tissues and its antiapoptotic effect (10), which suggests that hPEBP4 may play a role in tumor origination, progression, and regression. Besides abundant expression in breast carcinoma cells, hPEBP4 is also highly expressed in ovarian and prostate cancer cells. hPEBP4 could be particularly important if the point at which it exerts an effect is common to apoptosis and growth arrest induced by other anticancer agents. Actually, we also evaluated the effect of hPEBP4 on the sensitivity of MCF-7 cells to two other relevant tumor apoptosis–inducing ligands, Fas ligand and TNF-related apoptosis-inducing ligand. The results showed that MCF-7 cells were not sensitive to Fas ligand; however, silencing of hPEBP4 expression could potentiate sensitivity of MCF-7 cells to TNF-related apoptosis-inducing ligand–induced apoptosis (Supplementary Figure). Therefore, by decreasing hPEBP4 expression in breast cancer cells and ovarian cancer cells, the threshold at which chemotherapeutic agents trigger cancerous cells to undergo apoptosis and growth inhibition may be lowered, leading to a more favorable response of cancer cells to chemotherapeutic agents. Moreover, hPEBP4 high expression in some cancers, such as breast, prostate, and ovary cancer, and its ability to function as a antiapoptotic molecule outline a promising approach for the treatment of cancers by silencing hPEBP4 expression in cancer cells, thus potentiating their sensitivity to both endogenous and chemotherapeutic agent–mediated apoptosis and growth arrest induction. Our findings warrant further studies to explore the clinical ramifications of therapeutic targeting of hPEBP4.

We thank Y. Li, Y. Zheng, X. Zuo, W. Ni, and M. Jin for their expert technical assistance and Drs. T. Wan, L. Zhang, and J. Wang for their helpful discussion.