Follicular thyroid cancers demonstrate dual activation of PKA and mTOR as modeled by thyroid-specific deletion of Prkar1a and Pten in mice.

CONTEXT: Thyroid cancer is the most common form of endocrine cancer, and it is a disease whose incidence is rapidly rising. Well-differentiated epithelial thyroid cancer can be divided into papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although FTC is less common, patients with this condition have more frequent metastasis and a poorer prognosis than those with PTC. OBJECTIVE: The objective of this study was to characterize the molecular mechanisms contributing to the development and metastasis of FTC. DESIGN: We developed and characterized mice carrying thyroid-specific double knockout of the Prkar1a and Pten tumor suppressor genes and compared signaling alterations observed in the mouse FTC to the corresponding human tumors. SETTING: The study was conducted at an academic research laboratory. Human samples were obtained from academic hospitals. PATIENTS: Deidentified, formalin-fixed, paraffin-embedded (FFPE) samples were analyzed from 10 control thyroids, 30 PTC cases, five follicular variant PTC cases, and 10 FTC cases. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Mouse and patient samples were analyzed for expression of activated cAMP response element binding protein, AKT, ERK, and mammalian target of rapamycin (mTOR). Murine FTCs were analyzed for differential gene expression to identify genes associated with metastatic progression. RESULTS: Double Prkar1a-Pten thyroid knockout mice develop FTC and recapitulate the histology and metastatic phenotype of the human disease. Analysis of signaling pathways in FTC showed that both human and mouse tumors exhibited strong activation of protein kinase A and mTOR. The development of metastatic disease was associated with the overexpression of genes required for cell movement. CONCLUSIONS: These data imply that the protein kinase A and mTOR signaling cascades are important for the development of follicular thyroid carcinogenesis and may suggest new targets for therapeutic intervention. Mouse models paralleling the development of the stages of human FTC should provide important new tools for understanding the mechanisms of FTC development and progression and for evaluating new therapeutics.

Multi-institutional phase II study of selumetinib in patients with metastatic biliary cancers.

PURPOSE: Biliary cancers (BCs) carry a poor prognosis, but targeting the RAS/RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) pathway is of significance. Selumetinib is an inhibitor of MEK1/2, so this trial was designed to determine the safety and efficacy of selumetinib in BC. PATIENTS AND METHODS: This was a multi-institutional phase II study of selumetinib at 100 mg given orally twice per day to patients with advanced BC. The primary end point was response rate. All patients were required to provide tissue before enrolling. The levels of phosphorylated ERK (pERK) and AKT (pAKT) were assessed by immunohistochemistry. Tumors were genotyped for the presence of BRAF- and/or RAS-activating mutations. RESULTS: Twenty-eight eligible patients with a median age of 55.6 years were enrolled. Thirty-nine percent of patients had received one prior systemic therapy. Three patients (12%) had a confirmed objective response. Another 17 patients (68%) experienced stable disease (SD), 14 of whom (56%) experienced prolonged SD (> 16 weeks). Patients gained an average nonfluid weight of 8.6 pounds. Median progression-free survival was 3.7 months (95% CI, 3.5 to 4.9) and median overall survival was 9.8 months (95% CI, 5.97 to not available). Toxicities were mild, with rash (90%) and xerostomia (54%) being most frequent. Only one patient experienced grade 4 toxicity (fatigue). All patients had tissue available for analysis. No BRAF V600E mutations were found. Two patients with short-lived SD had KRAS mutations. Absence of pERK staining was associated with lack of response. CONCLUSION: Selumetinib displays interesting activity and acceptable tolerability in patients with metastatic BC. Our results warrant further evaluation of selumetinib in patients with metastatic BC.

KiSS-1/G protein-coupled receptor 54 metastasis suppressor pathway increases myocyte-enriched calcineurin interacting protein 1 expression and chronically inhibits calcineurin activity.

OBJECTIVE: Tumor metastasis is a critical determinant of death from cancer. Metastin, a product of the KiSS-1 gene, is an endogenously expressed metastasis suppressor that is the ligand for G protein-coupled receptor 54 (GPR54), a Gq/11-coupled receptor. In the present study, our goal was to define the basis of GPR54 action using thyroid cancer cells as a model. DESIGN AND RESULTS: We used GPR54-null thyroid cancer cells to create a stable GPR54 overexpression model. Cell growth and cell migration of the GPR54-expressing lines were inhibited by recombinant metastin, and metastin stimulated the protein kinase C, ERK, and phosphatidylinositol-3-kinase pathways. To identify metastin-regulated genes, we performed microarray analyses using RNA isolated from GPR54 stable transfectants before and after 1 and 24 h of metastin stimulation. Consistent increases in expression of the gene encoding myocyte-enriched calcineurin interacting protein 1 (MCIP-1), an inhibitor of calcineurin, were identified and confirmed using real-time RT-PCR and Western blot. Functionally, metastin treatment of GPR54-expressing cells initially increased calcineurin activity, followed by a prolonged reduction in calcineurin activity for 24 and 48 h, consistent with the pattern of MCIP-1 expression. In addition, treatment with cyclosporin A, a calcineurin inhibitor, blocked cell migration. Lymph node metastasis in papillary thyroid cancers demonstrated loss of MCIP-1 expression in comparison with primary tumors. CONCLUSIONS: These data suggest a role for MCIP-1 and calcineurin inhibition in GPR54-mediated metastasis suppression in human cancers.

AKT activation promotes metastasis in a mouse model of follicular thyroid carcinoma.

The phosphatidylinositol 3-kinase/AKT pathway is crucial to many cell functions, and its dysregulation in tumors is a common finding. The molecular basis of follicular thyroid cancer metastasis is not well understood but may also be influenced by AKT activation. We previously created a knockin mutant mouse that expresses a mutant thyroid hormone receptor-beta gene (TRbetaPV mouse) that spontaneously develops thyroid cancer and distant metastasis similar to human follicular thyroid cancer. In this study, we investigated whether our mouse model exhibits similar AKT activation as human follicular thyroid cancer. Western blot analysis on thyroids from both wild-type and TRbeta(PV/PV) mice revealed elevation of activated AKT in TRbeta(PV/PV) mice. Immunohistochemistry and confocal microscopy reveal activated AKT in both the thyroid and metastatic lesions of TRbeta(PV/PV) mice. Whereas all three AKT isoforms were overexpressed in primary tumors from TRbeta(PV/PV) mice in the cytoplasm of thyroid cancer cells, only AKT1 was also found in the nucleus, matching the localization of activated AKT in a pattern similar to human follicular thyroid cancer. In the metastases, all AKT isoforms correlated with phosphorylated AKT nuclear localization. We created primary thyroid cell lines derived from TRbeta(PV/PV) mice and found reduction of phosphorylated AKT levels or AKT downstream targets diminishes cell motility. Activated AKT is common to both human and mouse follicular thyroid cancer and is correlated with increased cell motility in vitro and metastasis in vivo. Thus, TRbeta(PV/PV) mice could be used to further dissect the detailed pathways underlying the progression and metastasis of follicular thyroid carcinoma.

Thyroid follicular adenomas may display features of follicular carcinoma and follicular variant of papillary carcinoma.

Thyroid follicular adenomas (FA) are encapsulated tumors lacking vascular, capsular or lymphatic invasion and the typical nuclear features of papillary carcinoma (PC). However, some FA demonstrate nuclear atypia reminiscent of either follicular carcinomas (FC) or follicular variant of papillary carcinomas (FVPC), suggesting they may represent precursors of malignant transformation. We hypothesized that an objective evaluation of nuclear chromatin patterns could be used to define atypical follicular tumors (AFT) that are likely to be premalignant. To test this hypothesis, we used a computer-aided image analysis system to define the chromatin pattern of nuclei from thyroid tumors. To validate the system, we analyzed 3000 nuclei from 10 FA, 10 FC, and 10 FVPC samples and accurately distinguished between these classes of tumors. Then, we analyzed nine AFT and, in parallel, we analyzed the tumors for activating mutations of N2-RAS and over-expression of RET. The predominant chromatin pattern of AFT was of FA type in two cases, FC type in two cases, and PC type in three cases. One case contained similar numbers of FC and PC nuclei and one was comprised of a mixture of the three nuclear types. Neither RAS mutation nor RET overexpression were detected in FA. N2-RAS mutations were found in 33% of AFT, 20% of FC and 20% of FVPC without correlation with chromatin pattern. Over-expression of RET was detected in 45% of AFT, 20% of FC and 50% of FVPC and was correlated with PC nuclei. These results show that AFT are a heterogeneous group of tumors, containing genuine benign tumors and tumors that share morphological and molecular features with follicular and papillary carcinomas that might be precursors of both types of thyroid carcinomas.