Constitutive ERK1/2 Activation in Esophagogastric Rib Bone Marrow Micrometastatic Cells Is MEK-independent

Abstract

In this study, we examined the mitogen-activated protein kinase (MAPK) cascade in micrometastatic cell lines generated from rib bone marrow (RBM) of patients undergoing resection of esophagogastric malignancies. The molecular mechanism(s) involved in esophagogastric MAPK activation have not previously been investigated. Constitutive activation of both ERK1 and -2 isoforms was evident in each of the five RBM cell lines. Elk-1, a transcription factor activated by the ERK1/2 pathway was also found to be constitutively activated. Cell lines generated from metastases of involved lymph nodes (OC2) and ascites (OC1) of patients with esophageal cancer do not display, however, hyperphosphorylation of ERK1/2. Constitutive RBM ERK1/2 activation is protein kinase C and phosphatidylinositol 3-kinase dependent. Surprisingly, constitutive ERK1/2 activation is MEK-independent. Pharmacological inhibition of MEK with two specific inhibitors, PD 98059 and U0126, were both ineffective in blocking ERK activation. Similarly, the use of a dominant negative MEK mutant was without effect. Interestingly, experiments overexpressing two different dominant negative Pak1 mutants significantly reduced RBM ERK1/2 activation, albeit not to the same extent for all cell lines. We also examined the role of three different phosphatases, PAC1, MKP-1, and -2. While RBM ERK1/2 activation was found to be PAC1-and MKP-2-independent, surprisingly, MKP-1 was down-regulated in all five RBM cell lines. In conclusion, we provide evidence for the first time for a MEK-independent constitutive ERK1/2 activation pathway in esophagogastric RBM cell lines. These findings have important implications for drug treatment strategies which currently target MEK in other forms of cancer.Esophageal cancer is an aggressive tumor which responds poorly to treatment and has a poor prognosis (1, 2). Approximately half of patients diagnosed with localized esophageal cancer die of metastatic disease within the first 2 years following tumor resection. We have succeeded in developing a number of esophagogastric cell lines from the rib bone marrow of patients (3). We have previously shown that these cells are viable, proliferate, and grow independently in tissue culture and form malignant tumors in athymic nude mice (4). These metastatic cells are representative of the disseminated progenitors of secondary tumors and are the appropriate targets for treatment.The precise molecular events leading to the acquisition of the metastatic phenotype remain largely unknown. Members of the Ras superfamily of small GTP-binding proteins have been implicated in tumor progression and are found to be activated in 20 to 30% of tumors (5). In its active GTP-bound state, Ras activates the serine threonine kinase Raf (6). Raf upon activation in turn phosphorylates the dual specific kinase MEK 1 (also known as MAP kinase kinase or MAPKK) which in turn phosphorylates the MAP kinases ERK1 and -2 (for extracellular regulated kinases 1 and 2). Although MEK is the only known kinase directly downstream of Raf...

Dominant-Negative Notch3 Receptor Inhibits Mitogen-Activated Protein Kinase Pathway and the Growth of Human Lung Cancers

“…Phosphorylation is usually transient even in the continued presence of activating stimuli, indicating that protein phosphatases represent an important mechanism for MAPK control. Down-regulation of MKPs or resistance to dephosphorylation by MKPs is often observed in human tumors (36)(37)(38). Recent data show that Notch antagonizes EGF in developing C. elegans, and one mechanism is through up-regulation of LIP-1, a homologue of mammalian MAPK phosphatases (28).…”

ERK1/2 Achieves Sustained Activation by Stimulating MAPK Phosphatase-1 Degradation via the Ubiquitin-Proteasome Pathway

“…4B. (24,35,36). More recently, decreased expression of MKP-1 has been strongly associated with the advanced progression of human ovarian cancers (37).…”

“…This may be attributable to elevated Ras-Raf-MKK1/2 signaling (20 -22). Nonetheless, constitutive activation of ERK1/2 has been reported to be independent of MKK1/2 and Raf-1 (23,24), and the mechanism by which sustained ERK1/2 activation occurs remains elusive. We recently reported that Pb(II) persistently stimulates ERK1/2 without activating JNK, p38, and ERK5 in CL3 human lung cancer cells and normal human fibroblasts, and sustained ERK1/2 signaling is essential for cellular nucleotide excision repair synthesis, anti-cytotoxicity, and anti-mutagenesis (25).…”

Dual-Specificity Phosphatase 1 Ubiquitination in Extracellular Signal-Regulated Kinase–Mediated Control of Growth in Human Hepatocellular Carcinoma

“…This frequently depends on up-regulation of the RAS/ MEK cascade. However, constitutive ERK overexpression may also occur independently of the RAS/MEK signaling (10,11). Recent studies (12,13) indicate that prolonged activation of ERK promotes phosphorylation at the Ser 296 residue of its inhibitor, dual-specificity phosphatase 1 (DUSP1; also known as MAPK phosphatase-1).…”