Function Study Of RACK1Protein In Prostate Cancer

Prostate cancer is one of the most common male urogenital carcinomas. In Europeand United States, especially in the latter, the incidence of prostate cancer is the leadingcancer and higher than lung cancer. At present, the incidence of prostate cancer, althoughfar lower than western countries, but showed a significant growth trend in china withchanging lifestyles, longer life expectancy and the improvement of diagnosis. Tumorinvasion and metastasis was the maximal barrier influencing therapeutic effect and was theleading cause of death in Patients with Prostate cancer. It’s unclear that how Prostatecancer cells obtain the potential of invasion and metastasis. To explore molecularmechanism about invasion and metastasis of prostate cancer and try to find newtherapeutic targets exhibited important theoretical significance and clinically Practicalvalue.RACK1is a highly conserved intracellular adaptor protein with significant homologyto Gβ. However, it is now well established that RACK1interacts with a large number ofproteins either directly or as part of a larger complex. As a scaffold protein, RACK1integrates inputs from distinct signaling pathways and is critical for fundamentalcellularactivities such as cell proliferation, transcription and protein synthesis, as well as variousneuronal functions. In cancer, RACK1becomes a focal point for transformation signaling.RACK1recruits ribosomes and PKC (discussed above) and scaffolds central componentsof the MAPK pathway and the PI3K pathway. As well as this, RACK1scaffolds a host ofother different kinases and phosphatases, and the activity of several of these proteins isaltered in cancer. All of these proteins, signaling pathways and protein complexes requiretight regulation. Subtle changes in the protein expression (both up and down) of afundamental protein such as RACK1can be expected to have dramatic consequences onthe regulation of these key pathways and therefore on the development and progression ofneoplastic disease.Using three experimental models-respected clinical specimens of human prostatcarcinoma, established prostate cancer cell lines stably transfected with RACK1, andxenograft in nude mouse. This study investigated the impact of RACK1on the metastatic potential of prostate cancer in an effort to identify RACK1as a biomarker predicting earlymetastasis and apotential target for therapy.Part E xpression ofR A C K1、Ⅰ PTEN and Ki67in prostate cancerObjective: To investigate expression of RACK1、PTEN and Ki67in prostate cancer, andrelationship between their expressions and clinical stage and pathological grade in prostatecancer.Methods:42cases of prostate cancer(57～87years old, averagely66.7years old)Paraffin-embeded tissues were collected by department of urology, The First AffiliatedHospital of Soochow University during2003～2011. Prostatic cancer tissues werecategorized according to Gleason scoring. No prostate cancer Patients received endocrinetherapy or other treatment. Consecutive rescetion spceimens of4m were performed.Immunohistochemistry was carried out to detect RACK1、PTEN and Ki67expression. Therelationship between RACK1、PTEN and Ki67expession and related clinicopathologicfeatures was statistically analyzed.Results: RACK1was expressed in42prostate canser tissue spedimen with differentdegree. Cases scoring0,1,2, and3were12,14,11, and5respectively. PTEN deletionexpressin of (32/42，76.19%) and high positive expressing of Ki67in (29/42，69.04%).Pearson’s correlation coefficient between RACK1and Ki67was0.870, p<0.001andnegatively correlation with PTEN respectively. Kaplan-Meier survival analysis revealed acorrelation between higher RACK1expression levels and shorter disease-specific survivaltimes (p<0.001). Factors that affected survival by univariate analyses include clinical stage,Pathological grade, RACK, Ki67and PTEN.Conclusions:(1) RACK1was expressed inprostate cancer and correlated strikingly withclinical stage and pathological grade.(2) Deletion expressing of PTEN and positive expressing of Ki67and RACK1correlatedsignificantly with clinical stage and pathological grade in prostate cancer. However,RACK1is a superior independent biomarker for diagnosis and prognosis comparing withcurrently widely-used diagnostic index in prostate cancer. Part II Effects of Proliferation, Invasion by RACK1on prostate cancercclls DU145and LNcapObjective:To investigate the effects of RACK1interference on proliferation and invasionof prostate cancer cells.Methods: The DU145and Lncap cells were transfected with siRNA sense plasmid byLiposome-mediated transfection and gained thestable interference model. MTT assay andflow cytometry was used to evaluate the effects of RACK I on proliferation. Migrationassay wasused to analyze the effects of RACK I on migration in vitro. Trans well invasionassay was utilized to evaluate the effects of RACK1oninvasion. Furthermore, the nudemouse model was established toinvestigate the effects of RACK1interference on tumorgrowth invivo.Results: experiments in prostate cancer cell lines stably-transfected with RACK1siRNA.The effects of RACK1silencing on tumor cells growth in vitro and observed a significantdecrease in the growth rate of cells and RACK1silencing compared with control cells inboth the proliferation and colony formation Assays. Conversely, RACK1silencing had noeffect on the apoptosis rate of these cells，as determined using a TUNEL assay. As well asnude mouse models,showed that the RACK1by siRNA in vivo inhibited proliferationmigration and invasion.Conclusions:(1) The tumor cells transfected RACK1by siRNA and stably expressedinterference effects in DU145and Lncap cells.(2) RACK1interference inhibit prostatetumor cclls proliferation,migration and invasion/metastasis in vitro and in vivo.Part Ⅲ The Mechanisms Underlying the Proliferation, and InvasionInduced by RACK1in Human Prostate Cancer cellsObjective: To explore the molecular mechanisms of proliferation/invasion and tumorAngiogenesis induced by RACK1in prostate cance cells.Methods: We applyed Western blot to detect the protein expressionlevel and interferencedgroup of Akt/MAPK both in transfected with RACK1siRNA and control cells’Immunohistochemistry were utilized to determine the marker of vascular endothelial cell-CD31in transplanted tumor tissue induced by RACK1siRNA. Calculate the new vascular quantity by MVD analysis. We used real-time PCR to investigate the angiogenicfactors such as VEGF-A,VEGF-B, VEGF-C, Ang1, Ang2, HGF, FGF2, and PIGF. Toidentify a correlation between the suppression of angiogenic factors and thephosphorylation of Akt and MAPK after RACK1knockdown.Results: Compared with controls, the stably-transfected siRNA RACK1tumor cells wereexpressed significantly lower level of p-Akt,p-MAPK, but not the PLCAfter RACK1interferenced, the marker of vascular endothelial cell–CD31strikingly reduced intransplant tumor also affected MVD. Detected by real-time PCR, VEGF-B and FGF2mRNA were showed dramaticaly decreased inhbited to40-50%in transplant tumor tissue.These results suggest that suppression of p-Akt and p-MAPK occurs at a later stage, and isnot involved in the upstream signaling of the suppression of VEGF-B and FGF2transcription.Conclusion:(1) The proliferation and invasion of prostate cancer cells were affected byRACK1siRNA through inhibited Akt/MAPK signaling pathway.(2) Angiogenic factorssuch as VEGF-B, FGF2may involved in prostate cancer tumor angiogensis induced byRACK1.(3) RACK1interference inhibited p-Akt and p-MAPK activation as well asinhibited VEGF-B and FGF2may through different pathway.(4) We identified RACK1interference from xenograft and comfirmed affected p-Akt/p-MAPK as well asVEGF-B/FGF2expression.