Targeting Of Epidermal Growth Factor Receptor By RNAi Improves The Cytotoxic Effects Of Paclitaxel On Androgen-independent Prostate Cancer In Vitro And In Vivo

Background and objective:Prostate cancer is one of the most common type of cancer in men; however, therapeutic options are limited in advantage stage. In particular, for patients at high risk of progression to more advanced PCs or those diagnosed in the late stages with metastatic and hormone-refractory prostate cancers (HRPCs), The current chemotherapeutic treatments against the metastatic HRPCs generally show no significant, or in case of docetaxel-based regimens, modest beneficial effects for improving the overall survival rate and outcome of patients in the clinics, and ultimately result in the death of patients. The median patient age in the recent multi-center trials of first-line chemotherapy for androgen-independent prostate cancer (AIPC) trials was >65 years, Older patients may not tolerate standard or investigational cytotoxic chemotherapies as well as younger patients.The epidermal growth factor receptor (EGFR) , as one of the members of the EGFR/ErbB receptor family of receptor tyrosine kinases (RTKs), is a 170 kDa type I transmembrane glycoprotein containing a ligand-binding ectodomain, a single hydrophobic transmembrane region, and a cytoplasmic tail which includes a tyrosine kinase domain and docking sites for a variety of signaling effectors, At least seven ligands can bind to the extracellular part of EGFR including EGF and transforming growth factor alpha (TGFα), Ligand binding induces homo- and/or heterodimerization, thereby resulting in kinase activation and phosphorylation of tyrosines in the ErbB tail. The downstream signaling network generated by EGF-R activation is able to control cell growth, survival and migration. 50–90% of HRPC cells show an overexpression of EGFR. The human EGFR tyrosine kinases are part of a network of pathways which are implicated in the development and progression of prostate cancer, in particular as part of the progression to androgen-independent growth, EGFR plays a critical role in tumor growth, and the prostate tissue becomes more susceptible to the growth-promoting action of EGF family growth factors during androgen withdrawal, The general inhibition of tyrosine kinase signaling pathways provides therapeutic advantage against prostate cancer metastasis, Therefore, inhibiting the activation of growth factor receptors, especially EGFR, may be a promising strategy for the treatment of prostate cancer.Blockade of EGF receptor signaling pathway represents a new perspective on the development of novel and selective anticancer therapies. Although considerable progress has been made in the application of EGF receptor-targeted antibodies and small molecule tyrosinekinase inhibitors, none of these agents is curative. Phase II study was undertaken to evaluate the efficacy of tyrosinekinase inhibitors in patients with HRPC, the results were disappointing .Current therapies of its expression are still need to be improved.In RNA interference (RNAi), duplexes of 21-23nt RNAs (small interfering RNA, siRNA) corresponding to mRNA sequences of particular genes are used to efficiently inhibit the expression of the target proteins in mammalian cells. It is approved that vectors containing shRNA expressing cassette could induce gene silencing of the target gene. Prostate cancer is located in the prostate only, fit to puncture according to it's anatomy, and has sepecific tumor MAPKs which are prone to follow-up, so prostate cancer is fit to inject the vectors containing shRNA expressing cassette for local gene targeted therapies.This study is to probe the effect of pshRNA-GFP LVEGFRRNAi on the PC-3 cell's growth and the expression of EGFR protein, and further confirm the effect of inhibiting AIPC cells, thirdly to to investigate the efficacy and safety of pshRNA-GFP LVEGFRRNAi combined with paclitaxel and Gefitinib for the treatment of AIPC cells in vitro and in vivo.Methods: 1.The target sequences were selected according to homological region of three EGFR transcript variants, and the siRNA expression vectors were constructed. 2. HT-1080(a human fibrosarcoma cell line) cells were transfected with the three recombinant vectors, and interference effect was detected by RT-PCR. 3. the most effective siRNA expression vector was co-transfected with lentiviral package plasmids into 293T cells to product lentiviral particles. 4. PC-3 cells were infected by the recombinant lentivirus, and the RNA interference effect was detected. 5. The pshRNA-GFP LVEGFRRNAi was used to treat the non-hormone-dependent prostate cancer in nude mice, the inhibition effect was investigated, and the expression levels of EGFR,MAPK,Akt,PKC,p-MAPK,p-Akt were measured. 6. Nude mice burdened with prostate cancer were injected intravenously with virus supernant which contained pshRNA-GFP LVEGFRRNAi, then treated with paclitaxel or Gefitinib, the cooperative antitumor effect was investigated.Result: 1.The siRNA sequences targeted to EGFR gene were designed and the expression vectors were cloned and verified by sequencing. 2. HT1080 cells were transfected with siRNA expression vectors. 24 hours after transfection, the transfect efficiency was about 70%. Real-time PCR analysis revealed that siRNA expression vectors can inhibit the expression of EGFR. The second vector had a better interference effect, and the interference effect reach about 90%.3. The lentiviral package vectors and siRNA expression vector were co-transfected to 293T cells, and high quality pshRNA-GFP LVEGFRRNAi was obtained. 4. pshRNA-GFP LVEGFRRNAi was used to infect the PC-3 cells. Compared with control group , real-time PCR results showed that the expression of EGFR decreased 90%(1-0.00851/0.08818), and results of western blot revealed the inhibition effect was 88.9% (1-0.05396/0.48455).After three passages, the inhibition effect remained 90%. MTT measurement showed that EGFR-RNAi stable PC-3 cell line which expresses EGFR in a low level has a lower proliferation rate. 5. The inhibition effect on PC-3 cells of RNAi is similar as Gefitinib (5mg/L), and the combination of these two treatments, can inhibit the growth of PC-3 better. 6. The expression levels of EGFR,MAPK,Akt,PKC,p-MAPK,p-Akt in EGFR-RNAi stable cell line were down-regulated significantly compared with the cells treated with Gefitinib (5mg/L). 7. Intravenous injection of the pshRNA-GFP LVEGFRRNAi encoding the siRNA targeting EGFR can inhibit the growth of tumor in nude mice. 8. PC-3 were transplanted into nude mice, and treated with pshRNA-GFP LVEGFRRNAi and paclitaxel or Gefitinib. The results showed that RNAi plus paclitaxel treatment can inhibit tumor growth with 56.8% tumor suppression. The tumor growth inhibition rate of the combination of Gefitinib and RNAi was as high as 54.0%. Compared to the control group, the group of RNAi plus Gefitinib and paclitaxel showed the highest effect against tumor with tumor growth inhibition rate as much as 63.5% (P<0.01) . The EGFR and Akt expression levels of the RNAi group or the RNAi plus Gefitinib group were decreased, but the MAPK levels were not affected.Conclusion: SiRNA-mediated inhibition of EGFR gene induced by shRNA expressing pshRNA-GFP LVEGFRRNAi is capable of suppressing EGF receptor expression with significantly inhibiting cellular proliferation and tumor growth due to cell cycle arrest and apoptosis induced. There was cooperation effect between pshRNA-GFP LVEGFRRNAi, paclitaxel and Gefitinib. RNAi targeting EGFR may constitute a useful approach in the treatment of human androgen-independent prostate cancer (AIPC).