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1.
J Clin Endocrinol Metab ; 103(9): 3169-3182, 2018 09 01.
Article in English | MEDLINE | ID: mdl-29846633

ABSTRACT

Objective: To investigate the molecular underpinnings of thyroid cancer, preclinical cell line models are crucial; however, ∼40% of these have been proven to be either duplicates of existing thyroid lines or even nonthyroid-derived lines or are not derived from humans at all. Therefore, we set out to establish procedures and guidelines that should proactively avoid these problems, which facilitated the creation of criteria to make valid preclinical models for thyroid cancer research. Design: Based on our recommendations, we systematically characterized all new cell lines that we generated by a standardized approach that included (1) determination of human origin, (2) exclusion of lymphoma, (3) DNA fingerprinting and histological comparisons to establish linkage to presumed tissue of origin, (4) examining thyroid differentiation by screening two to three thyroid markers, (5) examination of biological behavior (growth rate, tumorigenicity), and (6) presence of common thyroid cancer genetic changes (TP53, BRAF, PTEN, PIK3CA, RAS, TERT promoter, RET/PTC, PAX8/PPARγ, NF1, and EIF1AX). Results: We established seven new thyroid cell lines (LAM136, EAM306, SDAR1, SDAR2, JEM493, THJ529, and THJ560) out of 294 primary culture attempts, and 10 patient-derived tumor xenografts (PDTXs; MC-Th-95, MC-Th-374, MC-Th-467, MC-Th-491, MC-Th-493, MC-Th-504, MC-Th-524, MC-Th-529, MC-Th-560, and MC-Th-562) out of 67 attempts. All were successfully validated by our protocols. Conclusions: This standardized approach for cell line and PDTX characterization should prevent (or detect) future cross-contamination and ensure that only valid preclinical models are used for thyroid cancer research.


Subject(s)
Thyroid Neoplasms/pathology , Aged , Aged, 80 and over , Animals , Cell Differentiation , Cell Division , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , DNA Fingerprinting/methods , DNA, Neoplasm/genetics , Female , Heterografts , Humans , Male , Mice, Nude , Middle Aged , Mutation , Neoplasm Transplantation , Thyroid Neoplasms/genetics , Tumor Cells, Cultured
2.
J Cell Sci ; 125(Pt 18): 4253-63, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22718346

ABSTRACT

The Forkhead transcription factor, FoxO3a, is a known suppressor of primary tumor growth through transcriptional regulation of key genes regulating cell cycle arrest and apoptosis. In many types of cancer, in response to growth factor signaling, FoxO3a is phosphorylated by Akt, resulting in its exclusion from the nucleus. Here we show that FoxO3a remains nuclear in anaplastic thyroid carcinoma (ATC). This correlates with lack of Akt phosphorylation at serine473 in ATC cell lines and tissues of ATC patients, providing a potential explanation for nuclear FoxO3a. Mechanistically, nuclear FoxO3a promotes cell cycle progression by transcriptional upregulation of cyclin A1, promoting proliferation of human ATC cells. Silencing FoxO3a with a reverse genetics approach leads to downregulation of CCNA1 mRNA and protein. These combined data suggest an entirely novel function for FoxO3a in ATC promotion by enhancing cell cycle progression and tumor growth through transcriptional upregulation of cyclin A1. This is clinically relevant since we detected highly elevated CCNA1 mRNA and protein levels in tumor tissues of ATC patients. Our data indicate therapeutic inactivation of FoxO3a may lead to attenuation of tumor expansion in ATC. This new paradigm also suggests caution in relation to current dogma focused upon reactivation of FoxO3a as a therapeutic strategy against cancers harboring active PI3-K and Akt signaling pathways.


Subject(s)
Cyclin A1/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Regulation, Neoplastic , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Transcription, Genetic , Base Sequence , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , Cyclin A1/metabolism , Forkhead Box Protein O3 , Gene Silencing , HEK293 Cells , Humans , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins c-akt/metabolism , Thyroid Carcinoma, Anaplastic , Thyroid Neoplasms/enzymology , Thyroid Neoplasms/therapy
3.
J Clin Endocrinol Metab ; 95(12): 5338-47, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20810568

ABSTRACT

CONTEXT: Anaplastic thyroid carcinoma (ATC) is a highly aggressive carcinoma in need of therapeutic options. One critical component of drug discovery is the availability of well-characterized cell lines for identification of molecular mechanisms related to tumor biology and drug responsiveness. Up to 42% of human thyroid cancer cell lines are redundant or not of correct tissue origin, and a comprehensive analysis is currently nonexistent. Mechanistically, RhoB has been identified as a novel molecular target for ATC therapy. OBJECTIVE: The aim was to develop four ATC cell lines detailing genetic, molecular, and phenotypic characteristics and to test five classes of drugs on the cell lines to determine whether they inhibited cell proliferation in a RhoB-dependent fashion. DESIGN: Four cell lines were derived from ATC tumors. Short tandem DNA repeat and mutational status of the originating tumors and cell lines were performed along with molecular and phenotypic characterizations. Compounds were tested for growth inhibition and ability to up-regulate RhoB. RESULTS: Cell line authenticity was confirmed by DNA short tandem repeat analysis. Each proved unique regarding expression of thyroid markers, oncogene status, amplified and deleted genes, and proliferative growth rates. FTI-277, GGTI-286, lovastatin, romidepsin, and UCN-01 up-regulated RhoB and inhibited cell proliferation in a dose-responsive fashion with only romidepsin and FTI-277 being RhoB dependent. CONCLUSIONS: Molecular descriptions of thyroid lines were matched to the originating tumors, setting a new standard for cell line characterization. Furthermore, suppressed RhoB is implicated as a molecular target for therapy against ATC because five classes of drugs up-regulate RhoB and inhibit growth dose-responsively.


Subject(s)
Antineoplastic Agents/therapeutic use , Microsatellite Repeats/genetics , Mutation , rhoB GTP-Binding Protein/metabolism , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blotting, Western , Cell Division/drug effects , Cell Line, Tumor , DNA Fingerprinting , DNA, Neoplasm/genetics , DNA, Neoplasm/isolation & purification , Dimethyl Sulfoxide/pharmacology , Flow Cytometry , Genetic Markers , Humans , Thyroid Carcinoma, Anaplastic , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathology , Transfection , rhoB GTP-Binding Protein/drug effects , rhoB GTP-Binding Protein/genetics
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