REGgamma Expression Pattern In Breast Cancer Patients And Its Regulation Function On Estrogen Receptor Alpha In Breast Cancer Cells

REGgamma is a proteasome coactivator which regulates proteolytic activity ineukaryotic cells. Recently, the elevated expression of REGgamma was reported in a numberof human carcinomas. However, its precise role in the pathogenesis of breast cancer is stillunclear.1. High expression of REGgamma is associated with metastasis and poorprognosis of patients with breast cancerObjectives: To investigate the expression pattern of REGgamma in breast cancerpatients and to explore the possible relationship between REGgamma expression and breastcancer patient clinicopathological features as well as clinical outcomes.Methods: Immunohistochemical (IHC) staining and Western blot analysis wereperformed to investigate the expression of REGgamma in136human breast cancer tissueswith the paired peritumoural normal breast tissues and140breast benign disease tissuesamples, and the normal breast tissues and nonmetastatic axillary lymph nodes (ALNs)were employed as well.Results:1. REGgamma is highly expressed in human breast cancer tissues. Our datashowed that111out of136(81.6%) breast cancer tissue samples were REGgamma positive,which was significantly higher than breast benign disease tissues (9/140,6.4%, p <0.001)and lower than metastatic ALNs (116/116,100%, p <0.001).2. REGgamma expression ispositively correlated with poor clinicopathological features in patient with breast cancer.REGgamma expression level did not correlate with breast cancer patient age (p=0.702),patient menopausal state (p=0.171), progesterone receptor (PR) status (p=0.211) orhuman epidermal growth factor receptor-2(HER-2) status (p=0.557). However,REGgamma expression was positively correlated with breast tumour size (p <0.001), ALN metastasis state (p <0.001), tumour TNM stage (p=0.038), histological differentiation (p=0.001) and estrogen receptor alpha (ERα) status (p <0.001).3. REGgamma expression iscorrelated with poor clinical prognosis in patient with breast cancer. The5-year disease-freeand overall survival rates of patients with negative/low level of REGgamma weresignificantly higher than those of patients with high level of REGgamma expression (p <0.05). Cox regression analyses further indicated that REGgamma could serve as a novelindependent prognostic factor for breast cancer (OR=4.369, p=0.008).Conclusions: REGgamma was highly expressed in breast cancer and metastatic ALNtissues compared to the control normal tissues. The high expression of REGgamma mightpredict metastasis and poor prognosis in breast cancer.2. MMTV-PyMT mouse is an ideal mouse model to investigate the expressionpattern of REGgamma in vivoObjectives: To investigate the expression pattern of REGgamma in MMTV-PyMTmouse model (human breast cancer mouse model) and to explore the relationship betweenREGgamma expression and MMTV-PyMT tumour-bearing mouse outcomes.Methods: IHC staining and Western blot analysis were performed to investigate theexpression difference of REGgamma between MMTV-PyMT mice (FVB/N-Tg MMTV-PyVT634Mul/J mice,#002374) and control FVB ones (FVB/NJ, nontransgenic,#001800).Mouse breast cancer tissues, normal breast tissues, metastatic lung tissues and normal lungtissues samples were employed.Results:1. REGgamma was highly expressed in MMTV-PyMT mouse mammarycarcinomas and metastatic lung tumours. In MMTV-PyMT mice,14out of20(70%) mousemammary carcinomas were REGgamma positive, which was significantly higher thancontrol (0/20,0%, p <0.001) and lower than metastatic lung tumour (20/20,100%, p=0.027).2. No REGgamma protein expression in FVB mouse normal breast tissue andnormal lung tissue, but a significant high level of REGgamma expression was detected inMMTV-PyMT mouse breast carcinoma tissue (p=0.0051) and metastatic lung tumour (p=0.0190).3. In a set of MMTV-PyMT mice overall survival (OS) analysis (10mice for eachgroup), mice with the high expression level of REGgamma showed lower percent ofsurvival than that of mice with the low expression of REGgamma. Conclusions: MMTV-PyMT was an ideal mouse model to further investigate thebiological functions of REGgamma in breast cancer research. Moreover, these resultsfurther indicated a prognostic role of REGgamma in MMTV-PyMT mouse mammary glandcarcinoma progression.3. REGgamma in vitro function on human breast cancer cell proliferation,motility and invasion abilityObjectives: To investigate the in vitro function of REGgamma in ERα positive breastcancer cell phenotype such as proliferation, motility and invasion ability in MCF7andBT474cells.Methods: Small interfering RNA (siRNA), Western blot analysis, cell proliferationMTS analysis, cell motility analysis and transwell cell invasion assay were performed toinvestigate the possible function of REGgamma on breast cancer cells MCF7and BT474between REGgamma-siRNA treated and the control cells.Results:1. After REGgamma down regulation by siRNA treatment, breast cancer cellgrowth declined significantly in both MCF7(p=0.0033) and BT474cells (p=0.0114).2.As showed by cell motility assay, a significant decrease of cell moveability was found inboth MCF7(p <0.0001) and BT474cells (p <0.0001) after REGgamma down regulation.3.Transwell assay data showed that cell invasion ability decreased significantly in both MCF7(invasion index=0.2864) and BT474cells (invasion index=0.3928) after REGgamma-siRNA treatment.Conclusions: Cell growth and moveability decreased significantly after REGγ downregulation by siRNA in ERα positive breast cancer cell MCF7and BT474, suggesting thatREGgamma had potential to promote breast cancer cell malignant progression.4. REGγ regulates ERα protein expression via ubiquitin-proteasome pathwayObjectives: To investigate the possible in vitro function of REGγ on regulating ERαexpression in ERα positive breast cancer cells.Methods: Small interfering RNA, Western blot analysis, Real-time PCR analysis, ERαprotein half-life CHX chase and ERα-ubiquitin co-immunoprecipitation analysis wereperformed to investigate regulation function of REGgamma on ERα in breast cancer cells MCF7and BT474.Results:1. After REGgamma-siRNA treatment, ERα protein expression decreasedsignificantly in both MCF7(p=0.0108) and BT474(p=0.0065) cells. However, ERαmRNA expression had no obvious change in both MCF7(p=0.3011) and BT474(p=0.2178) cells.2. ERα protein half-life time decreased significantly in REGgamma-siRNAtreated cells (~2hours) compared with the control cells (~4hours). ERα protein degradedmore quickly in REGgamma-siRNA treated MCF7(p=0.0093) and BT474(p=0.0155)cells.3. ERα degradation effect which caused by REGgamma-siRNA can be totallyeliminated by proteasome inhibitor MG132.4. ERα-ubiquitin co-IP data showedREGgamma had function on regulating ERα ubiquitin-proteasome mediated degradationprocess. With the presence of REGgamma, ERα protein was less ubiquitinylation degradedcompared with the REGgamma-siRNA treated MCF7and BT474cells.Conclusions: The regulation function of REGgamma on regulating ERα only tookplace after ERα transcriptional stage. The stability and proteasome-mediated degradation ofERα can be regulated by REGgamma. REGgamma had function to stabilize ERα proteinand slow down the ubiquitin-proteasome mediated degradation process of ERα.