IntroductionThe majority of deaths from breast cancer are a result of metastases; however, little is understood about the genetic alterations underlying their onset. Genetic profiling has identified the adhesion molecule plakoglobin as being three-fold reduced in expression in primary breast tumors that have metastasized compared with nonmetastatic tumors. In this study, we demonstrate a functional role for plakoglobin in the shedding of tumor cells from the primary site into the circulation.MethodsWe investigated the effects of plakoglobin knockdown on breast cancer cell proliferation, migration, adhesion, and invasion in vitro and on tumor growth and intravasation in vivo. MCF7 and T47D cells were stably transfected with miRNA sequences targeting the plakoglobin gene, or scramble vector. Gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Cell proliferation, adhesion, migration, and invasion were measured by cell counting, flow cytometry, and scratch and Boyden Chamber assays. For in vivo experiments, plakoglobin knockdown and control cells were inoculated into mammary fat pads of mice, and tumor growth, shedding of tumor cells into the bloodstream, and evidence of metastatic bone lesions were monitored with caliper measurement, flow cytometry, and microcomputed tomography (μCT), respectively.ResultsPlakoglobin and γ-catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered after transfection with the scrambled sequence. Reduced plakoglobin resulted in significantly increased in MCF7 and T47D cell proliferation in vitro and in vivo, compared with control, with significantly more tumor cells being shed into the bloodstream of mice bearing plakoglobin knockdown tumors. In addition, plakoglobin knockdown cells showed a >250% increase in invasion through basement membrane and exhibited reduced cell-to-cell adhesion compared with control cells.ConclusionDecreased plakoglobin expression increases the invasive behavior of breast cancer cells. This is the first demonstration of a functional role for plakoglobin/γ-catenin in the metastatic process, indicating that this molecule may represent a target for antimetastatic therapies.

No abstract

The majority of deaths from breast cancer are a result of metastases, however, little is understood about the genetic alterations underlying their onset. A recent study of human breast cancer biopsies showed alterations of expression of 9 genes in primary tumours that had metastasized compared to benign tumours. Of these, plakoglobin was the most altered, with metastatic tumours showing a 3x decrease in expression compared to benign. Furthermore, expression of plakoglobin is reduced upon autocrine production of human growth hormone, which is associated with increased breast tumour cell invasion in vivo. Plakoglobin codes for the adhesion protein gamma catenin, an integral part of the cadherin-catenin complex involved in cell-to-cell adhesion. The aim of the current study is to investigate the effects of plakoglobin knockdown on cell proliferation, migration, adhesion and invasion in vitro and in vivo. The non-metastatic human breast cancer cell lines MCF7 and T47D that express high levels of plakoglobin and gamma-catenin were stably transfected with miRNA to two different regions of the plakoglobin gene, or scramble vector, to produce plakoglobin knockdown and controls for each cell line. Plakoglobin gene expression was monitored by qPCR and gamma-catenin expression by Western blot. Cell proliferation was assessed 24, 48, 72 and 96 hours post seeding; cell migration and invasion were measured over 24 and 48 hours using scratch and modified Boyden Chamber assays. Cell-cell adhesion was assessed by spheroid formation in agar matrix. For in vivo experiments, MCF7 plakoglobin knockdown and control cells were inoculated into the 5th and 10th mammary fat pads of 12-week old female balb/c mice (n=10 per group). Tumour growth was monitored by caliper measurements and local invasion was investigated 6 weeks after tumour inoculation on histological sections. Shedding of tumour cells into the blood stream and evidence of metastatic bone lesions was monitored by flow cytometry and μCT, respectively. Plakoglobin and gamma catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered following transfection with the scrambled vector. Furthermore, knockdown of plakoglobin did not effect expression of its binding partner, e-cadherin. Reduced expression of plakoglobin resulted in a more than 3-fold increase in MCF7 and T47D cell proliferation in vitro (p<0.005) and a 2-fold increase in MCF-7 proliferation in vivo (P<0.05), compared with control. In addition plakoglobin knockdown cells showed a 6 - 15 fold increase in invasion through basement membrane (p<0.005), a 2-fold increase in migration (p<0.005) and exhibited reduced cell-to-cell adhesion compared with control cells. Data show that decreased expression of plakoglobin increases the early pro-metastatic behaviour of cells including increased cell proliferation, migration and invasion. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-20.