Gene regulation by phorbol 12-myristate 13-acetate in MCF-7 and MDA-MB-231, two breast cancer cell lines exhibiting highly different phenotypes.

Lacroix M, Haibe-Kains B, Hennuy B, Laes JF, Lallemand F, Gonze I, Cardoso F, Piccart M, Leclercq G and Sotiriou C

We have examined the effects of the protein kinase C (PKC)-activator phorbol 12-myristate 13-acetate (PMA) on gene expression in two breast cancer cell (BCC) lines exhibiting highly different phenotypes. These are the estrogen receptor ·(ER·)-positive, weakly invasive, luminal epithelial-like MCF-7 and the ER·-negative, highly invasive, fibroblast-like MDA-MB-231. They express constitutively low and high PKC activities, respectively. After a 24-h exposition to 100 nM PMA, the number of genes showing an altered expression at the 2-fold change level was much higher in MCF-7 (n=435) than in MDA-MB-231 (n=18) BCC. Four of these genes, namely CDC2, CENPA, NR4A1and MMP10, were altered in the same way in both cell lines. Two genes were regulated in an opposite way: ID1and EVA1. Many of the genes down-regulated in MCF-7 BCC appeared to be preferentially expressed in the G1, S, and/or G2 phases of the cell cycle. The ER·gene, ESR1, and other genes associated to the ER·-positive, luminal epithelial-like BCC phenotype were down-regulated, while a series of genes related to a more aggressive, fibroblast-like BCC phenotype were up-regulated. Other altered genes were notably linked to cell architecture, supporting profound effects of PMA on cell morphology and motility, as well as on the interactions between BCC and their neighboring proteins. Of note, all the modulated genes involved in proteolysis and its control were up-regulated. In summary, PMA effects suggest that PKC activation may induce, to some extent, a more fibroblast-like phenotype in the ER·-positive, luminal epithelial-like MCF-7 BCC, and significantly modulate the interactions of these cells with their environment. Introduction based on the initial observation that it was able to promote skin tumor formation, phorbol 12-myristate 13-acetate (PMA) has been widely used in experimental oncology. PMA is an activator of various isoforms of protein kinase C (PKC). Total PKC activity is elevated in breast cancer tissues, suggesting an association with tumorigenicity (1). In breast cancer cell (BCC) lines, a relation has been found between a high PKC activity and the absence of estrogen receptor ·(ER·), and PKC activity is generally higher in aggressive and invasive BCC lines (2). Treatment with PMA has been shown to drastically increase the invasiveness of low-PKC-expressing, ER·-positive MCF-7 BCC. A PKC inhibitor (H7) reversed the PMA effects on these cells. Similar effects of PMA were observed for cell chemo-taxis (3). A prolonged (hours) exposure of MCF-7 to PMA led to a significant growth reduction, an effect seen to a much lesser extent in MDA-MB-231 cells (4). In MCF-7 BCC stably transfected with the PMA-responsive protein kinase C ·isoform, various changes were observed: enhanced proliferation rate, anchorage- independent growth, dramatic morphologic alterations including loss of an ‘epithelioid’ appearance, significant increase in the level of the mesenchymal/fibroblastic marker vimentin, increased tumorigenicity in nude mice, a significant reduction in ER·expression, and a decrease in estrogen-dependent gene expression (5). These observations suggest that PKC activation by PMA may produce considerable changes in gene expression, at least in MCF-7 BCC.