1,25-dihydroxyvitamin D3 and retonic acid analogues induce differentiation in breast cancer cells with function- and cell-specific additive effects.

Vitamin D3 derivatives and retinoids can induce cell cycle arrest, differentiation and cell death in many cell lines. These compounds can act cooperatively in some of their functions and may be of potential use either individually or in combination in the treatment of breast cancer. The effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), all-trans retinoic acid (ATRA) and several analogues were evaluated on malignant phenotypic traits of breast cancer cell lines MCF-7, T-47D and MDA-MB-231. Both 1,25(OH)2D3 and ATRA caused a decrease in anchorage independent colony formation in MCF-7 and T-47D cells in a dose-dependent manner. The effects of 1,25(OH)2D3 10(-10) and 10(-9) M were synergistic with ATRA 10(-8) M in T-47D cells but were antagonistic in both MCF-7 and in T-47D cells at most concentrations. Both 1,25(OH)2D3 and ATRA individually induced an accumulation of MCF-7 cells in the G1 phase of the cell cycle and an associated increase in p21WAFI/CiP1, p27KiP1 and a dephosphorylation of Rb but the effects were not additive. Both compounds inhibited the invasive capacity of MDA-MB-231 cells. 1,25(OH)2D3 but not ATRA caused an increase in E-cadherin levels in MDA-MB-231 cells. These two functions were not additive. The compounds 1,25(OH)2D3, a noncalcemic analogue 1,25(OH)2-16-ene-23-yne-D3, ATRA, AGN195183, an RARalpha-specific agonist, and AGN190168 (tazarotene), an RARbeta/gamma-selective agonist, induced differentiation as determined by measurements of lipid droplet formation. The individual effects of 1,25(OH)2-16-ene-23-yne-D3 combined with ATRA or with tazarotene at 10(-9) M each were additive in MCF-7 and MDA-MB-231 cells on lipid formation. The data demonstrate that both 1,25(OH)2D3, ATRA, and selected analogues induce a more differentiated phenotype in breast cancer cells with additive effects that are function- and cell-specific.

Intracellular FGF-2 promotes differentiation in T-47D breast cancer cells.

To test the implicated role of basic fibroblast growth factor (bFGF; FGF-2) in promoting differentiation in breast cancer, we enforced the expression of FGF-2 in T-47D breast cancer cells. Expression of FGF-2 conferred an overall less malignant phenotype to T-47D cells as revealed by their reduced proliferative response, impaired capacity for anchorage-independent growth, and invasion through Matrigel. To understand one candidate mechanism for the intracellular FGF-2-mediated anti-invasive effect, we examined the effect of FGF-2 on T-47D cell motility. Addition of recombinant FGF-2 to the growth medium markedly enhanced cell motility while constitutive expression of intracellular FGF-2 significantly inhibited the migratory potential of T-47D cells in a dominant manner. FGF-2-expressing T-47D cells also formed relatively defined branching structures in Matrigel matrices, a characteristic phenotype of differentiation in breast cancer cells. These data suggest a potential role for FGF-2 in promoting functional differentiation of breast epithelial cells.

Basic fibroblast growth factor confers a less malignant phenotype in MDA-MB-231 human breast cancer cells.

Basic fibroblast growth factor (FGF-2) expression is associated with a more differentiated phenotype, earlier stage of disease, and a better prognosis in breast cancer patients. To determine whether expression of FGF-2 can cause a less malignant phenotype, we engineered MDA-MB-231 cells, a highly dedifferentiated, invasive breast cancer cell line, to express different isoforms of FGF-2. Cells expressed either cytoplasmic, nuclear, or a combination of both FGF-2 isoforms. Western blots of 2 M NaCl washes and of conditioned medium demonstrated that these cells did not export FGF-2. Cells expressing FGF-2 had levels of fibroblast growth factor receptors equivalent with those of control cells. Transformation was assayed by anchorage-independent colony formation and tumor formation in athymic mice. All of the constructs expressing various FGF-2 isoforms had a 60-70% reduction in colony formation in soft agar, but only cells expressing the Mr 18,000 FGF-2 isoform formed fewer and smaller tumors in mice. To determine potential mechanisms responsible for a less malignant phenotype, experiments measuring invasion in Matrigel, the secretion of matrix metalloprotease activity and migration in a modified Boyden chamber and in a patch wound motility assay were carried out. Cells expressing the Mr 18,000 cytoplasmic FGF-2 moiety had a 45% decrease in invasion in Matrigel compared to vector-transfected controls. Cells expressing Mr 18,000 FGF-2 had an increase in Mr 97,000 and Mr 48,000 collagenase, demonstrating that the decreased invasive potential was not due to a down-regulation of gelatinolytic or caseinolytic matrix metalloproteinases. However, motility was decreased in both assays, primarily in cells expressing Mr 18,000 FGF-2, whereas exogenous recombinant human FGF-2 had no effect. These studies demonstrate for the first time that FGF-2 expression can cause a less malignant phenotype in breast cancer cells, possibly as a result of decreased motility and invasion.