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Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells
D Califano1, C Rizzo, A D'Alessio
1Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, c/o Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli "Federico II," via S. Pansini 5, 80131 Naples, Italy.
Abstract:
Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.
Insights
Specific Ret mutations driving endocrine cancers activate the Ras pathway, leading to cell differentiation. Inhibiting Ras reverses this, but Ret-2B shows faster reversion than Ret-2A, implicating SNT signaling.
Area of Science:
- Oncology
- Cell Biology
- Molecular Biology
Background:
- Germline mutations in the Ret receptor tyrosine kinase gene predispose to multiple endocrine neoplasia (MEN) types 2A and 2B, and familial medullary thyroid carcinoma.
- The precise mechanisms by which distinct Ret isoforms (Ret-2A and Ret-2B) lead to different neoplastic diseases are not fully understood.
- Expression of mutated Ret isoforms in PC12 cells induces cellular differentiation.
Purpose of the Study:
- To investigate the role of the Ras pathway in mediating the phenotypic changes induced by Ret-2A and Ret-2B mutants in PC12 cells.
- To elucidate the downstream signaling events triggered by constitutively active Ret mutants.
Main Methods:
- Utilized an inducible vector expressing a dominant-negative Ras (Ras p21(N17)) to inhibit Ras activity.
- Analyzed molecular and morphological changes in PC12 cells upon expression of Ret-2A or Ret-2B mutants.
- Assessed tyrosine phosphorylation of SNT (suc1-associated neurotrophic factor-induced tyrosine phosphorylation target) and its association with Sos substrate.
Main Results:
- Ret-induced molecular and morphological changes in PC12 cells are mediated by Ras-dependent pathways.
- Inhibition of Ras activity reversed Ret-induced differentiation, with faster morphological reversion observed in Ret-2B-transfected cells compared to Ret-2A-transfected cells.
- SNT was found to be chronically tyrosine phosphorylated and associated with the Sos substrate in Ret-transfected cells, indicating its role as a Ret substrate.
Conclusions:
- Activation of the Ras cascade is an essential pathway triggered by chronically active Ret mutants in PC12 cells.
- SNT acts as a substrate for both Ret-2A and Ret-2B mutants, potentially mediating the activation of the Ras cascade.
- Differential kinetics of differentiation reversion suggest distinct downstream effects of Ret-2A and Ret-2B mutants despite Ras pathway involvement.