Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors.
BACKGROUND
Breast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.


METHODS
We utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.


RESULTS
We found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of alpha-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active beta-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive beta-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.


CONCLUSIONS
We found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.
Lebret
Stephanie C
SC
Deakin University, Burwood Highway, Burwood, Melbourne, 3125, Australia.
Newgreen
Donald F
DF
Thompson
Erik W
EW
Ackland
M Leigh
ML
D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D002277:Carcinoma / Q000378:metabolism* / Q000473:pathology*; D002454:Cell Differentiation; D003470:Culture Media; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D010641:Phenotype; D014407:Tumor Cells, Cultured
D003470:Culture Media