Inhibition of sphingolipid biosynthesis decreases phosphorylated ERK2 in LLC-PK1 cells.

Fumonisin B(1) (FB(1)) is a fungal toxin produced by Fusarium verticillioides that inhibits ceramide synthase (CS), a key enzyme in the de novo sphingolipid biosynthesis pathway. In LLC-PK(1) cells, FB(1) inhibits cell proliferation and induces apoptosis, which can be prevented by inhibitors of serine palmitoyltransferase (SPT). Inhibition of SPT prevents the FB(1)-induced accumulation of free sphinganine, a precursor of ceramide biosynthesis. However, not all of the effects of FB(1) in LLC-PK(1) cells can be explained solely by the increase in free sphingoid bases. The downstream signaling pathways that are affected by FB(1)-induced disruption of sphingolipid biosynthesis are not well understood. This study determined, in LLC-PK(1) cells, changes in p42 MAP kinase (phosphorylated ERK2 [pERK2]) phosphorylation in response to various inhibitors of key enzymes of the de novo sphingolipid biosynthesis pathway (CS, SPT, and glucosylceramide synthase [GlcCer synthase]). The results show that inhibition of any of the three enzymes caused a similar decrease in the extent of phosphorylation of ERK2 with no reduction in total ERK2. The co-treatment of FB(1) (CS inhibitor) with SPT inhibitors or the GlcCer synthase inhibitor had no effect on the FB(1)-induced reduction in pERK2 phosphorylation, indicating that FB(1)-mediated changes in phosphorylation of pERK2 was independent of increases in free sphinganine or its metabolites or a reduction in ceramide. Nonetheless, the decrease in pERK2 phosphorylation was dependent on inhibition of de novo sphingolipid biosynthesis. Decreased pERK2 activity could contribute to the physiological effects of FB(1) in LLC-PK(1) cells that are not due to alteration in pathways modulated by free sphingoid bases and their metabolites but are sensitive to inhibition of glycosphingolipid biosynthesis.

Prolonged extracellular signal-regulated kinase 1/2 activation during fibroblast growth factor 1- or heregulin beta1-induced antiestrogen-resistant growth of breast cancer cells is resistant to mitogen-activated protein/extracellular regulated kinase kinase inhibitors.

Increased growth factor receptor signaling is implicated in antiestrogen-resistant breast tumors suggesting that abrogation of such signaling could restore or prolong sensitivity to antihormonal agents. Activation of the mitogen-activated protein/extracellular regulated kinase kinase (MEK)-extracellular regulated kinase (ERK)1/2 cascade is a common component of such pathways. We investigated the ability of the MEK activation inhibitor U0126 to block the increased growth of estrogen receptor-positive MCF-7 breast cancer cells caused by fibroblast growth factor 1 (FGF-1), heregulin beta1 (HRGbeta1), and epidermal growth factor (EGF) in the presence of the pure antiestrogen ICI 182780 (Faslodex; fulvestrant). We found that either FGF-1 or HRGbeta1 but not EGF substantially reduced the inhibitory effects of U0126 on growth and ERK1/2 activation, including the combined inhibitory effects of U0126 and ICI 182780. FGF-1 and HRGbeta1 also reduced the inhibition of ERK1/2 phosphorylation by the MEK inhibitors PD98059 and PD184161. Interestingly, a transiently transfected dominant-negative MEK1 completely abrogated activation of a coexpressed green fluorescent protein-ERK2 reporter by all three of the factors. Despite a short-lived activation of Ras and Raf-1 by all three of the growth factors, both FGF-1 and HRGbeta1, unlike EGF, induced a prolonged activation of MEK and ERK1/2 in these cells. Thus, activation of FGF-1- and HRGbeta1-specific signaling causes MEK-dependent prolonged activation of ERK1/2, which is incompletely susceptible to known MEK inhibitors. We also demonstrate that the cytosolic phospholipase A2 inhibitor arachidonyl trifluoro methyl ketone and the pan PKC inhibitor bisindolymaleimide abrogated U0126-resistant phosphorylation of ERK1/2 induced by HRGbeta1 but not by FGF-1. Phosphorylation of ERK5 by all three of the factors was also resistant to U0126 suggesting that its activation is not sufficient to overturn growth inhibition due to diminished ERK1/2 activation. Therefore, therapy combining antiestrogens and MEK inhibitors may be ineffective in some antiestrogen-resistant estrogen receptor-positive breast cancers.