RET/PTC (rearranged in transformation/papillary thyroid carcinomas) tyrosine kinase phosphorylates and activates phosphoinositide-dependent kinase 1 (PDK1): an alternative phosphatidylinositol 3-kinase-independent pathway to activate PDK1

Thyroid cancers are a leading cause of death due to endocrine malignancies. RET/PTC (rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although the oncogenic potential of RET/PTC is related to intrinsic tyrosine kinase activity, the substrates for this enzyme are yet to be identified. In this report, we show that phosphoinositide-dependent kinase 1 (PDK1), a pivotal serine/threonine kinase in growth factor-signaling pathways, is a target of RET/PTC. RET/PTC and PDK1 colocalize in the cytoplasm. RET/PTC phosphorylates a specific tyrosine (Y9) residue located in the N-terminal region of PDK1. Y9 phosphorylation of PDK1 by RET/PTC requires an intact catalytic kinase domain. The short (iso 9) and long forms (iso 51) of the RET/PTC kinases (RET/PTC1 and RET/PTC3) induce Y9 phosphorylation of PDK1. Moreover, Y9 phosphorylation of PDK1 by RET/PTC does not require phosphatidylinositol 3-kinase or Src activity. RET/PTC-induced phosphorylation of the Y9 residue results in increased PDK1 activity, decrease of cellular p53 levels, and repression of p53-dependent transactivation. In conclusion, RET/PTC-induced tyrosine phosphorylation of PDK1 may be one of the mechanisms by which it acts as an oncogenic tyrosine kinase in thyroid carcinogenesis.     

RET/PTC Rearrangements Are Associated with Elevated Postoperative TSH Levels and Multifocal Lesions in Papillary Thyroid Cancer without Concomitant Thyroid Benign Disease

RET/PTC rearrangements, resulting in aberrant activity of the RET protein tyrosine kinase receptor, occur exclusively in papillary thyroid cancer (PTC). In this study, we examined the association between RET/PTC rearrangements and thyroid hormone homeostasis, and explored whether concomitant diseases such as nodular goiter and Hashimoto''s thyroiditis influenced this association. A total of 114 patients diagnosed with PTC were enrolled in this study. Thyroid hormone levels, clinicopathological parameters and lifestyle were obtained through medical records and surgical pathology reports. RET/PTC rearrangements were detected using TaqMan RT-PCR and validated by direct sequencing. No RET/PTC rearrangements were detected in benign thyroid tissues. RET/PTC rearrangements were detected in 23.68% (27/114) of PTC tissues. No association between thyroid function, clinicopathological parameters and lifestyle was observed either in total thyroid cancer patients or the subgroup of patients with concomitant disease. In the subgroup of PTC patients without concomitant disease, RET/PTC rearrangement was associated with multifocal cancer (P = 0.018). RET/PTC rearrangement was also correlated with higher TSH levels at one month post-surgery (P = 0.037). Based on likelihood-ratio regression analysis, the RET/PTC-positive PTC cases showed an increased risk of multifocal cancers in the thyroid gland (OR = 5.57, 95% CI, 1.39–22.33). Our findings suggest that concomitant diseases such as nodular goiter and Hashimoto''s thyroiditis in PTC may be a confounding factor when examining the effects of RET/PTC rearrangements. Excluding the potential effect of this confounding factor showed that RET/PTC may confer an increased risk for the development of multifocal cancers in the thyroid gland. Aberrantly increased post-operative levels of TSH were also associated with RET/PTC rearrangement. Together, our data provides useful information for the treatment of papillary thyroid cancer.     

A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.     

Inhibition of heat shock protein 90, a novel RET/PTC1-associated protein, increases radioiodide accumulation in thyroid cells

RET/PTC1 is a rearranged form of the RET tyrosine kinase commonly seen in papillary thyroid carcinomas. It has been shown that RET/PTC1 decreases expression of the sodium/iodide symporter (NIS), the molecule that mediates radioiodide therapy for thyroid cancer. Using proteomic analysis, we identify hsp90 and its co-chaperone p50cdc37 as novel proteins associated with RET/PTC1. Inhibition of hsp90 function with 17-allylamino-17-demothoxygeldanamycin (17-AAG) reduces RET/PTC1 protein levels. Furthermore, 17-AAG increases radioiodide accumulation in thyroid cells, mediated in part through a protein kinase A-independent mechanism. We show that 17-AAG does not increase the total amount of NIS protein or cell surface NIS localization. Instead, 17-AAG increases radioiodide accumulation by decreasing iodide efflux. Finally, the ability of 17-AAG to increase radioiodide accumulation is not restricted to thyroid cells expressing RET/PTC1. These findings suggest that 17-AAG may be useful as a chemotherapeutic agent, not only to inhibit proliferation but also to increase the efficacy of radioiodide therapy in patients with thyroid cancer.     

RET tyrosine kinase signaling in development and cancer

The variety of diseases caused by mutations in RET receptor tyrosine kinase provides a classic example of phenotypic heterogeneity. Gain-of-function mutations of RET are associated with human cancer. Gene rearrangements juxtaposing the tyrosine kinase domain to heterologous gene partners have been found in sporadic papillary carcinomas of the thyroid (PTC). These rearrangements generate chimeric RET/PTC oncogenes. In the germline, point mutations of RET are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Both MEN 2 mutations and PTC gene rearrangements potentiate the intrinsic tyrosine kinase activity of RET and, ultimately, activate the RET downstream targets. Loss-of-function mutations of RET cause Hirschsprung's disease (HSCR) or colonic aganglionosis. A deeper understanding of the molecular signaling of normal versus abnormal RET activity in cancer will enable the development of potential new treatments for patients with sporadic and inherited thyroid cancer or MEN 2 syndrome. We now review the role and mechanisms of RET signaling in development and carcinogenesis.     

Analysis of the expression of RET/PTC oncogenes in post-chernobyl papillary thyroid carcinomas of patients from different age groups

A comparative analysis of the expression of both, RET/PTC1 and RET/PTC3 oncogenes in papillary thyroid carcinomas (PTC) of patients from different age groups was carried out. Those were the following groups: children (mean age - 13 years, mean latency period - 13 years), young adults (mean age - 24 years, mean latency period - 14 years), adults (mean age - 38 years, mean latency period - 22 years). The presence of RET/PTC oncogenes was detected using polymerase chain reaction. In all cases the samples of both tumor and normal thyroid tissue were studied. It was established that induction of both, RET/PTC1 and RET/PTC3 rearrangements was present only in carcinoma samples. In PTCs the percentage of RET/PTC-positive tumors with increasing the age of patients has been decreasing. It should be noted that the part of carcinomas with induction of RET/PTC1 did not change with increasing the age of patients. At the same time the frequency of RET/PTC3 rearrangements with the increasing both the latency period and age of patients, significantly decreased. In conclusion, our data can evidence for the presence of correlation between the age of patients, latency period and induction of RET/PTC3 oncogenes.     

Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells

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.     

Mycobacterium avium infection of mouse macrophages inhibits IFN-gamma Janus kinase-STAT signaling and gene induction by down-regulation of the IFN-gamma receptor.

Macrophage activation is required to control the growth of intracellular pathogens. Recent data indicate that macrophages become functionally deactivated during mycobacterial infection. We studied macrophage deactivation by examining the expression of a panel of IFN-gamma-inducible genes and activation of Janus Kinase (JAK)-STAT pathway in Mycobacterium avium-infected macrophages. Reduced expression of IFN-gamma-inducible genes-MHC class II gene E beta; MHC class II transactivator; IFN regulatory factor-1; and Mg21, a gene coding for a GTP-binding protein-was observed in M. avium-infected macrophages. Decreased tyrosine phosphorylation and DNA binding activity of STAT1 in M. avium-infected macrophages stimulated with IFN-gamma was observed. Tyrosine phosphorylation of JAK1, JAK2, and IFN-gamma R alpha was also reduced in infected cells. Northern and Western blot analyses showed that a down-regulation of IFN-gamma R alpha- and beta-chain mRNA and protein occurred in M. avium-infected macrophages. The down-regulation of IFN-gamma R and inhibition of STAT1 activation were time dependent and required 4 h of infection for down-regulation of the IFN-gamma R and 8 h for STAT1 inhibition. These findings suggest that M. avium infection inhibits induction of IFN-gamma-inducible genes in mouse macrophages by down-regulating IFN-gamma R, resulting in reduced phosphorylation of IFN-gamma R alpha, JAK1, JAK2, and STAT1.