Breakdown of the regulatory control of pyrimidine biosynthesis in human breast cancer cells

The activity of the de novo pyrimidine biosynthetic pathway in the MCF7 breast cancer cells was 4.4‐fold higher than that in normal MCF10A breast cells. Moreover, while pyrimidine biosynthesis in MCF10A was tightly regulated, increasing as the culture matured and subsequently down‐regulated in confluency, the biosynthetic rate in MCF7 cells remained elevated and invariant in all growth phases. The flux through the pathway is regulated by carbamoyl phosphate synthetase, a component of the multifunctional protein, CAD. The intracellular CAD concentration was 3.5‐ to 4‐fold higher in MCF7 cells, an observation that explains the high rate of pyrimidine biosynthesis but cannot account for the lack of growth‐dependent regulation. In MCF10A cells, up‐regulation of the pathway in the exponential growth phase resulted from MAP kinase phosphorylation of CAD Thr456. The pathway was subsequently down‐regulated by dephosphorylation of P∼Thr456 and the phosphorylation of CAD by PKA. In contrast, the CAD P∼Thr456 was persistently phosphorylated in MCF7 cells, while the PKA site remained unphosphorylated and consequently the activity of the pathway was elevated in all growth phases. In support of this interpretation, inhibition of MAP kinase in MCF7 cells decreased CAD P∼Thr456, increased PKA phosphorylation and decreased pyrimidine biosynthesis. Conversely, transfection of MCF10A with constructs that elevated MAP kinase activity increased CAD P∼Thr456 and the pyrimidine biosynthetic rate. The differences in the CAD phosphorylation state responsible for unregulated pyrimidine biosynthesis in MCF7 cells are likely to be a consequence of the elevated MAP kinase activity and the antagonism between MAP kinase‐ and PKA‐mediated phosphorylations. © 2004 Wiley‐Liss, Inc.