Tumor necrosis factor alpha and interleukin 11 secreted by malignant breast epithelial cells inhibit adipocyte differentiation by selectively down-regulating CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma: Mechanism of desmoplastic reaction

The dense layer of fibroblasts that accumulate around malignant breast epit helial cells (i.e.. desmoplastic reaction) arises from the breast adipose t issue and provides structural and biochemical support for breast cancer. We report herein a number of epithelial-stromal interactions responsible for desmoplastic reaction in breast cancer using cultured 3T3-L1 murine fibrobl asts and human adipose fibroblasts. which can be activated with a mixture o f hormones to differentiate to mature adipocytes. Adipocyte differentiation was inhibited by coculturing fibroblasts with various breast cancer cell l ines (T47D, MCF-7. SSC202. SSC78. and SSC30) completely or by breast cancer cell conditioned media in a dose-dependent manner: on the other hand, adip ocyte differentiation was not inhibited by coculturing with normal human pr imary mammary epithelial cell conditioned medium. This tumor effect was eli minated using neutralizing antibodies against tumor necrosis factor (TNF)-a lpha or interleukin (IL)-Il. TNF-alpha and IL-II levels were 2.5-3 times hi gher in T47D conditioned medium compared with control medium, and TNF-alpha transcripts were detectable in T47D hut not in 3T3-L1 cells in culture, in dicating that the malignant epithelial cell is the major site of cytokine p roduction. This was confirmed in vivo in mastectomy specimens, where immuno reactive TNF-alpha and IL-II were readily detectable in malignant epithelia l tells but not in the majority of the surrounding fibroblasts. Adipocyte d ifferentiation is mediated by the expression of a cascade of adipogenic tra nscription factors, including CCAAT/enhancer binding protein (C/EBP)beta, C /EBP delta. peroxisome proliferator-activated receptor (PPAR)gamma and C/EB P alpha. C/EBP alpha and PPAR gamma are essential for this process. We demo nstrated by Northern analysis that exposure of activated 3T3-L1 cells to T4 7D cell conditioned medium strikingly decreased the levels of PPAR gamma an d C/EBP alpha transcripts and increased the levels of C/EBP beta and C/EBP delta transcripts. In these 3T3-L1 cells, inhibition of differentiation was also confirmed by markedly suppressed le, els of aP2 mRNA. H lich is an ad ipocyte-specific gene. These in vitro observations were confirmed in sectio ns of human malignant breast tumors, where immunoreactive C/ERP alpha was r eadily detectable in adipose fibroblasts distant to the tumor hut not in in tratumoral fibroblasts. Treatment of 3T3-L1 cells with T47D cell conditione d medium or TNF-a changed neither the numbers of cells in G(o)-G(i), S, and G(2) phases nor the rate of [H-3]thymidine incorporation, thus ruling out a proliferative effect of malignant cells on the surrounding fibroblasts. I n summary, desmoplastic reaction primarily occurs via the action of cytokin es (TNF-alpha and IL-I1) secreted by the malignant epithelial cells to inhi bit differentiation of adipose fibroblasts to mature adipocytes. This tumor -induced block in adipocyte differentiation is mediated by the selective in hibition of expression of the essential adipogenic transcription factors. i .e., PPAR gamma and C/EBP alpha.