Loss of plakoglobin promotes decreased cell-cell contact, increased invasion, and breast cancer cell dissemination in vivo.
BACKGROUND
The 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.


METHODS
We 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.


RESULTS
Plakoglobin 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.


CONCLUSIONS
Decreased 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.
Academic Unit of Clinical Oncology, University of Sheffield, Medical School, Beech Hill Road, Sheffield, S10 2RX, UK.
Ingunn
I
Holen
Jacob
J
Whitworth
Faith
F
Nutter
Alyson
A
Evans
Hannah K
HK
Brown
Diane V
DV
Lefley
Ivana
I
Barbaric
Mark
M
Jones
Penelope D
PD
Ottewell
D000818:Animals; D002448:Cell Adhesion; D002450:Cell Communication; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D061986:MCF-7 Cells; D015674:Mammary Neoplasms, Animal / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009361:Neoplasm Invasiveness; D009360:Neoplastic Cells, Circulating; D034622:RNA Interference; D034741:RNA, Small Interfering; D051185:gamma Catenin / Q000235:genetics / Q000378:metabolism*
C495293:Jup protein, mouse; D034741:RNA, Small Interfering; D051185:gamma Catenin