Experimental Study Of New Anti-apoptotic Protein HPEBP4 As A Potential Therapeutic Target For Human Prostate Cancer

Prostate cancer is the most common malignancy and the second leading cause of cancer mortality in men. However treatment of advanced and metastatic prostate cancer still represents a crucial problem. Recently, inducers of apoptosis have been used in cancer therapy, disruption of apoptotic function may result in cancer cell resistance. Therefore, development of more selective and effective apoptosis inducers is needed to minimise sideeffects and maximise efficacy.We detailed here a novel member of phosphatidylethanolamine-binding protein (PEBP) family, designated as human phosphatidylethanolamine-binding protein 4 (hPEBP4). As with all PEBP family members, hPEBP4 was predicted to contain a phosphatidylethanolamine (PE) binding domain. RT-PCR analysis revealed that hPEBP4 mRNA is expressed in a variety of tumor cells, including Daudi (Burkitt's lymphoma), NAMALWA (Burkitt's lymphoma), MCF-7 (breast carcinoma), LNCaP (prostate carcinoma) and CaoV-3 (ovarian carcinoma) cells. Our previous study showed that hPEBP4 inhibits TNFa-induced apoptosis by interfering with Ras/Raf/MEK1/ERK1/2 signaling, JNK activation and PE externalization via its phosphatidylethanolamine-binding domain. Silencing of hPEBP4 sensitizes breast cancer cells to TNFa-induced apoptosis and cell growth arrest. These results suggest that hPEBP4 may be a promising target in the treatment of human breast cancer. The preferential expression pattern of hPEBP4 in cancer tissues and its anti-apoptotic effect prompt us to further investigate the function and the molecular mechanisms of hPEBP4 in other malignancies highly expressing hPEBP4. Here the anti-apoptotic effect of hPEBP4 was further confirmed and its underlying mechanisms were investigated in human prostate cancer.Part I. Overexpression of hPEBP4 in human prostate cancer PC-3 cells inhibits TRAIL-induced apoptosisWe went first to confirm the expression pattern of hPEBP4 in prostate cancer tissues and prostate cancer cell lines by tissue array and RT-PCR. Tissue array analysis showed that hPEBP4 is selectively expressed in human prostate cancer tissue and the expression levels were positively correlated with Gleason grading. RT-PCR showed that hPEBP4 is highly expressed in TRAIL-resistant LNCaP cells, whereas not in TRAIL-sensitive PC-3 and DU145 cells. This expression pattern of hPEBP4 indicates that hPEBP4 may play a role in TRAIL resistance in prostate cancer.PC-3 prostate cancer cells were transfected with hPEBP4-B, p75PEBP4-B or control vector. Western blot and RT-PCR confirmed the overexpression of hPEBP4 in PC-3 stable transfectants. In the apoptosis assay, hPEBP4-B transfection inhibits TRAIL-induced apoptosis, when compared to mock transfection or transfection with p75PEBP4-B which is deficient of PE-binding domain. PI3K/Akt and MAPK pathways are important signaling pathways which account for the apoptosis resistance in prostate cancer cells especially in TRAIL resistance. We then examined the effect of hPEBP4 on these pathways. When compared to p75PEBP4-B or mock transfectants, hPEBP4-B transfectants showed higher Akt activation not only in their baseline level but also following TRAIL stimulation. Simultaneously, hPEBP4-B transfectants displayed lower ERKl/2 activation upon TRAIL stimulation. But the baseline ERKl/2 activation of transfectans was almost equal. hPEBP4 transfection did not affect TRAIL-induced JNK and p38 activation. PI3-K inhibitor, Wortmannin or LY-294002 completely inhibited TRAIL-induced Akt activation and dramatically increased the effects of TRAIL on apoptosis. And the blocking effect of hPEBP4 on TRAIL-induced apoptosis was almost reversed. These results indicate that hPEBP4 confers TRAIL resistance by Akt activation and that the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.We then examined the effect of hPEBP4 on the regulation of apoptosis-related proteins. We showed that overexpression of hPEBP4 in PC-3 cells inhibitesTRAIL-induced BID truncation and caspase 3 cleavage, down-regulates BAD, p27kipl and up-regulates Bcl-2 and Bcl-xL expression. Overexpression of hPEBP4 had neither effect on Caspase 8 and Caspase 9 activation nor effect on DR4 and DR5 expression after TRAIL stimulalation. LY294002 could partially reverse the above effects. These data suggest that hPEBP4 inhibits TRAIL-induced apoptosis via Akt activation. The activated Akt then affects its downstream targets which direct target cells to apoptosis.Part II. Silencing hPEBP4 in human prostate cancer LNCaP cells potentiates TRAIL-induced apoptosisIn Part One, we found that hPEBP4 is highly expressed in LNCaP cells and hPEBP4 plays a negative role in TRAIL-induced apoptosis. Given the high expression of hPEBP4 in LNCaP cells, the possibility that silencing hPEBP4 expression could sensitize LNCaP prostate cancer cells to apoptosis induction was then investigated. hPEBP4-RNAi or Neo plasmid were used to stably silence the expression of hPEBP4 protein in LNCaP cells. Western blot and RT-PCR confirmed the complete stable silencing of hPEBP4 expression in LNCaP cells. Compared to controls, hPEBP4-silenced LNCaP cells were more sensitive to TRAIL-induced apoptosis. Simultaneously, silencing hPEBP4 in LNCaP cells inhibited Akt activation and promoted TRAIL-induced ERK1/2 activation. U0126, the inhibitor of MEK1 partially reversed the enhanced effect of hPEBP4-silencing on TRAIL-induced apoptosis. Taken together with results of Part One, hPEBP4 inhibits TRAIL-induced apoptosis throuth Akt activation and ERK1/2 inhibition.We next examined the potential mechanisms by which hPEBP4 regulated Akt and ERK1/2 activation. In our previous report we found that hPEBP4 translocates from lysosomes to cell membrane and binds to Raf-1 or MEK1 following TNF-a treatment, thus inhibiting Raf/MEKl/ERKl/2 activation. Here immunoprecipitaion showed that hPEBP4 associated with Raf-1 and MEK1 after TRAIL stimulation whereas neither PI3K nor Akt. These data suggest that hPEBP4 may interact with Raf-1 and MEK1 after TRAIL stimulation in the same way as it did upon TNF-a stimulation, but hPEBP4-mediated Aktregulation still remains unknown.Several reports have addressed the crosstalk between PI3-K/Akt and ERKl/2 pathways, however the relationship between these two pathways still remains mystery. Here we found that PI3-K inhibitor had no effect on TRAIL-induced ERKl/2 activation in PC-3 cells whereas MEK1 inhibitor could partially reverse TRAIL-induced Akt inhibition in hPEBP4-silenced LNCaP cells, suggesting that hPEBP4 may transfer to cell membrane and binds to Raf-1 or MEK1, inhibit Raf-l/MEKl/ERKl/2 activation after TRAIL stimulation. Then the ERKl/2 inhibition may subsequently facilitate to activate Akt which is consistent with the report that ERKl/2 activation can inhibit Akt activation directly. Simultaneously, hPEBP4 may promote Akt activation directly in an unknown way. Further investigation will be required to clarify the mechanism of hPEBP4-mediated Akt regulation in TRAIL-induced apoptosis.The effect of hPEBP4 silence on apoptosis-related proteins was also examined. Silencing of hPEBP4 in LNCaP cells promotes TRAIL-induced BID truncation and caspase 3 cleavage, up-regulates p53, p27kipl and down-regulates Bcl-2, Bcl-xL expression. U0126 could partially reverse the above effects. Taken together with results of Part One, these data indicate that hPEBP4 upregulates anti-apoptotic proteins and downregulates pro-apoptotic proteins by Akt activation and ERKl/2 inhibition.Several reports have addressed that high p-Akt and low p-ERKl/2 activity predict poor clinical outcome in prostate cancer. Furthermore, hPEBP4 inhibited TRAIL-induced apoptosis via promoting Akt activation and inhibiting ERKl/2 pathway. More importantly, tissue array assay showed the higher Gleason grade prostate cancer, the more increased level of hPEBP4 expression, consistent with the expression pattern of active Akt in prostate cancer but inversely correlated with that of active ERKl/2. These data suggest the abundant expression of hPEBP4 in poorly differentiated prostate cancer may upregulate Akt activation and inhibit ERKl/2 activation, thus result in failure in treatment of prostate cancer. Therefore, silencing hPEBP4 outlines a promising approach in the treatment of prostate cancer.In summary, on the basis of the important role of hPBPP4 in cellular resistance to apoptotic stimuli, it appears that the hPEBP4 would be a potential target for drug development, which could have a profound effect on the treatment of prostate cancer. Our findings warrant further studies to explore the clinical ramifications of therapeutic targeting of hPEBP4. Altogether, silencing hPEBP4 may be a novel strategy for the development of innovative therapeutic modalities targeting apoptosis-resistant forms of malignancy that highly express hPEBP4, such as ovarian carcinoma, prostate carcinoma and breast carcinoma.