Central Role of the Threonine Residue within the p+1 Loop of Receptor Tyrosine Kinase in STAT3 Constitutive Phosphorylation in Metastatic Cancer Cells

Abstract

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met^p+1loop→Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr^p+1loop residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr^p+1loop→Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK^Metp+1loop with 2B mutation and wild-type EPH members the Thr^p+1loop residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET^M918T, nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr^p+1loop residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.

We thank S. M. Jhiang for wild-type and mutant RET constructs, P. Accornero and K. Furge for mouse Met constructs, A. Danilkovitch-Miagkova for human RON construct, R. Arceci for mouse KIT construct, and E. B. Pasquale, B. Wang, and R. Zhou for various EPH and ephrin constructs. Patient samples were obtained from Rhode Island Hospital and Mayo Clinic Foundation.

This study was partially supported by NIH RO1 grant (CA82549) to Y.E.C. and NIH COBRE grant (RR-15578) to Brown University.