Involvement Of RACK1 In Regulation Of Proliferation, Migration And Invasion/Metastasis In Human Breast Carcinoma And The Implicated Mechanisms

IntroductionThe incidence and mortality of breast carcinoma are increasing each year, which is one of the major causes of death in women. Among various prognostic indicators of breast carcinoma, disease recurrence and metastasis are key factors affecting the outcome of primary breast carcinoma therapy. Identification of sensitive and specific biomarkers for metastatic potential and their regulatory genes in breast carcinoma cells, therefore, is important for both early clinical detection and effective prophylactic therapy.Human RACK1 (receptor of activated C kinase 1) is located in 5q35.3, with a cDNA length of 951 bp that codes for a 35 kDa protein. Several published reports have focused on the effects of RACK1 expression on melanoma and oral squamous cell carcinoma metastasis. The exact role RACK1 plays in breast carcinoma proliferation, migration and invasion/metastasis is, however, still largely unclear. Specifically, there has been no report on the relation and mechanisms underlying RACK1 expression and the biological behavior of breast carcinoma in either Chinese literature or elsewhere.Using three experimental models - respected clinical specimens of human breast carcinoma, established breast carcinoma cell lines stably transfected with RACK1, and xenograft in nude mouse mammary pad - this study investigated the impact of RACK1 on the metastatic potential of breast carcinoma in an effort to identify RACK1 as a biomarker predicting early metastasis and a potential target for therapy. Part I Screening a Human Mammary cDNA Library with PI3K p110αas Bait and the Interaction ConfirmationPurpose To identify novel PI3K p110α-interacting proteinsMethods A yeast two hybrid system was utilized (pGBKT7-PIK3CA as a bait) to screen a human mammary cDNA librasy. After verification in yeast cells, we performed IP and IF to confirm the interation between PI3K p110αand RACK1Results We screened a human mammary cDNA library with PI3K p110αas bait. Of the several newly implicated proteins, RACK1 was chosen for successive detailed analysis. The immunoprecipitation and immunofluorescence analyses sufficiently identified RACK1 as a novel binding partner of PI3K p110α.Conclusions RACK1 is a novel binding partner of PI3K p110α.Part II Promotion of Proliferation, Migration and Invasion/Metastasis by RACK1 in Human Breast CarcinomaPurpose To investigate the effect of RACK1 on proliferation, migration and invasion/metastasis of human breast carcinomaMethods We established breast carcinoma cell lines stably-transfected with RACK1. MTT assay and flow cytometry was used to evaluate the effects of RACK1 on proliferation. Migration assaywas used to analyze the effects of RACK1 on migration in vitro. Transwell invasion assay was utilized to evaluate the effects of RACK1 on invasion. Furthermore, an orthotopic nude mouse model was established to investigate the effects of RACK1 on tumor growth and metastasis in vivo.Results Experiments in breast carcinoma cell lines stably-transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration and invasion. Conclusions RACK1 promotes proliferation, migration and invasion/metastasis in human breast carcinoma in vitro and in vivo.PartⅢThe Mechanisms Underlying the Promotion of Proliferation, Migration and Invasion/Metastasis Induced by RACK1 in Human Breast CarcinomaPurpose To clarify possible mechanisms underlying the promotion of proliferation, migration and invasion/metastasis induced by RACK1 in human breast CarcinomaMethods We used Western blot to evaluate the cell cycle related protein expression level and activations of PI3K/Akt and MAPK pathway in breast carcinoma cells stably transfected with RACK1 treated with Rho kinase inhibitor and siRNA specifically targeting RACK1, compared with control cells. We used IP and IF to investigate the interaction of RACK1 and Rho A. Trans well migration assay was also applied to investigate the changes in migration of breast carcinoma cell treated with Rho kinase inhibitor. Besides, gelatin zymographic analysis, GTP-Rho pull-down assay and immnohistochemistry were utilized in this section as well.Results Experiments in breast carcinoma cell lines stably-transfected with RACK1, as well as nude mouse models, showed that RACK1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Conversely, knockdown of RACK1 by siRNA in vitro inhibited proliferation, migration and invasion. In cell lines stably-transfected with RACK1, p-Akt, cyclin D1, cyclin D3 and CD 147 expression, as well as MMP2 activity, were elevated. RACK1-induced migration could be inhibited by the addition of Rho-kinase inhibitor.Conclusions RACK1 upregulates proliferation via PI3K/Akt pathway, targeting p21, cyclin D1 and cyclin D3; migration via interaction with RhoA and activation of RhoA/Rho kinase pathway; invasion via CD 147 and MMPs.PartⅣRACK1 Expression and its Correlation with Clinicopathological Indicators in 160 Breast Carcinoma Patients Purpose To clarify RACK1 Expression and its correlation with clinicopathological indicators in 160 breast carcinoma patientsMethods IHC was performed in 160 breast carcinoma samples to investigate RACK1 Expression and its correlation with clinicopathological indicators.Results RACK1 immunoreactivity was readily detected in the cytoplasm and occasionally in the nucleus. Cases scoring 0,1 and 2 were 66,54 and 40, respectively. We did observe significant correlations of RACK1 with tumor size (P= 0.046), lymph node metastasis (P = 0.027), clinical stage (P = 0.007) and histological grading (P = 0.004). Pearson's correlation coefficient between RACK1 and Ki67 was 0.930, P<0.001. Kaplan-Meier survival analysis of 160 cases revealed a correlation between higher RACK1 expression levels and shorter disease-specific survival times (P<0.001). Factors that affected survival by univariate analyses included tumor size, lymph node metastasis, RACK1, Ki67 and HER-2 (all P<0.05). The ROC areas for RACK1, Ki67, ER, PR and HER-2 were 0.833,0.766,0.446,0.387 and 0.689, respectively.Conclusions RACK1 is a superior independent biomarker for diagnosis and prognosis comparing with currently widely-used diagnostic index in breast carcinoma.