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Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin.

ABSTRACT: We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes for alpha-tropomyosin. Therefore, we evaluated the mechanism by which Ras causes the downregulation of tropomyosin expression. By using RIE-1 cells that harbor inducible expression of activated H-Ras(12V), we determined that the downregulation of tropomyosin expression correlated with the onset of morphological transformation. We found that the reversal of Ras transformation caused by inhibition of extracellular signal-regulated kinase activation corresponded to a restoration of tropomyosin expression. Inhibition of p38 activity in Raf-expressing RIE-1 cells caused both morphological transformation and loss of tropomyosin expression. Thus, a reduction in tropomyosin expression correlated strictly with morphological transformation of RIE-1 cells. However, forced overexpression of tropomyosin in Ras-transformed cells did not reverse morphological or growth transformation, a finding consistent with the possibility that multiple changes in gene expression contribute to Ras transformation. We also determined that tropomyosin expression was low in two human tumor cell lines, DLD-1 and HT1080, that harbor endogenous mutated alleles of ras, but high in transformation-impaired, derivative cell lines in which the mutant ras allele has been genetically deleted. Finally, treatment with azadeoxycytidine restored tropomyosin expression in Ras-transformed RIE-1, HT1080, and DLD-1 cells, suggesting a role for DNA methylation in downregulating tropomyosin expression.

SUBMITTER: Shields JM

PROVIDER: S-EPMC133695 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin.

Shields Janiel M JM Mehta Heena H Pruitt Kevin K Der Channing J CJ

Molecular and cellular biology 20020401 7

We showed previously that activated Ras, but not Raf, causes transformation of RIE-1 epithelial cells, demonstrating the importance of Raf-independent pathways in mediating Ras transformation. To assess the mechanism by which Raf-independent effector signaling pathways contribute to Ras-mediated transformation, we recently utilized representational difference analysis to identify genes expressed in a deregulated fashion by activated Ras but not Raf. One gene identified in these analyses encodes ...[more]

PMID: 11884615

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Project description:Ectopic programmed cell death ligand 1 (PD-L1) expression in non-small cell lung cancers (NSCLCs) is related to immune evasion by cancer, and it is a molecular target of immune checkpoint therapies. Although some altered signals in NSCLCs are responsible for ectopic PD-L1 expression, the precise mechanisms remain obscure. Because we found a higher frequency of EGFR/KRAS mutations in NSCLC cell lines with high PD-L1 expression (p < 0.001), we evaluated the relationships between downstream signals and PD-L1 expression, particularly in three KRAS-mutant adenocarcinoma cell lines. The MEK inhibitor U0126 (20 μM) significantly decreased the surface PD-L1 levels by 50-60% compared with dimethyl sulfoxide (p < 0.0001). Phorbol 12-myristate 13-acetate stimulation (100 nM, 15 min) increased (p < 0.05) and two ERK2 siRNAs as well as KRAS siRNAs decreased (p < 0.05) PD-L1 expression. The transcriptional activity of the potential AP-1 site (+4785 to +5056 from the transcription start site) in the PD-L1 gene was demonstrated by luciferase assays, which was inhibited by U0126. The chromatin immunoprecipitation assay demonstrated the binding of cJUN to the AP-1 site. Two STAT3 siRNAs decreased PD-L1 expression by 10-32% in two of the three KRAS-mutant lung adenocarcinoma cell lines (p < 0.05), while the PI3K inhibitor LY294002 (40 μM) did not change the expression level. Supervised cluster analysis and gene set enrichment analysis between the PD-L1-high and -low NSCLCs revealed a correlation between PD-L1 expression and genes/pathways related to cell motility/adhesion. These results indicate that MAPK signaling is the dominant downstream signal responsible for ectopic PD-L1 expression, in which STAT3 is also involved to some extent. Furthermore, MAPK signaling may control the expression of PD-L1 and several genes related to enhanced cell motility. Our findings suggest that MAPK, along with STAT3, is important for determining PD-L1 expression, which could be useful for targeted therapies against lung cancers.

Dataset Information

Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin.

Publications

Opposing roles of the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades in Ras-mediated downregulation of tropomyosin.

Similar Datasets

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