Genetics and epigenetics of sporadic thyroid cancer.

Thyroid carcinoma is the most common endocrine malignancy, and although the disease generally has an excellent prognosis, therapeutic options are limited for patients not cured by surgery and radioiodine. Thyroid carcinomas commonly contain one of a small number of recurrent genetic mutations. The identification and study of these mutations has led to a deeper understanding of the pathophysiology of this disease and is providing new approaches to diagnosis and therapy. Papillary thyroid carcinomas usually contain an activating mutation in the RAS cascade, most commonly in BRAF and less commonly in RAS itself or through gene fusions that activate RET. A chromosomal translocation that results in production of a PAX8-PPARG fusion protein is found in follicular carcinomas. Anaplastic carcinomas may contain some of the above changes as well as additional mutations. Therapies that are targeted to these mutations are being used in patient care and clinical trials.

The thyroid cancer PAX8-PPARG fusion protein activates Wnt/TCF-responsive cells that have a transformed phenotype.

A chromosomal translocation results in the production of a paired box 8-peroxisome proliferator-activated receptor gamma (PAX8-PPARG) fusion protein (PPFP) in ∼35% of follicular thyroid carcinomas. To examine the role of PPFP in thyroid oncogenesis, the fusion protein was stably expressed in the non-transformed rat thyroid cell line PCCL3. PPFP conferred on PCCL3 cells the ability to invade through Matrigel and to form colonies in anchorage-independent conditions. PPFP also increased the fraction of cells with Wnt/TCF-responsive green fluorescent protein reporter gene expression. This Wnt/TCF-activated population was enriched for colony-forming and invading cells. These actions of PPFP required a functional PPARG DNA binding domain (DBD) within PPFP and were further stimulated by PPARG agonists. These data indicate that PPFP, through its PPARG DBD, induces Wnt/TCF pathway activation in a subpopulation of cells, and these cells have properties of cellular transformation including increased invasiveness and anchorage-independent growth.

Multiple roles for the non-coding RNA SRA in regulation of adipogenesis and insulin sensitivity.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a master transcriptional regulator of adipogenesis. Hence, the identification of PPARγ coactivators should help reveal mechanisms controlling gene expression in adipose tissue development and physiology. We show that the non-coding RNA, Steroid receptor RNA Activator (SRA), associates with PPARγ and coactivates PPARγ-dependent reporter gene expression. Overexpression of SRA in ST2 mesenchymal precursor cells promotes their differentiation into adipocytes. Conversely, knockdown of endogenous SRA inhibits 3T3-L1 preadipocyte differentiation. Microarray analysis reveals hundreds of SRA-responsive genes in adipocytes, including genes involved in the cell cycle, and insulin and TNFα signaling pathways. Some functions of SRA may involve mechanisms other than coactivation of PPARγ. SRA in adipocytes increases both glucose uptake and phosphorylation of Akt and FOXO1 in response to insulin. SRA promotes S-phase entry during mitotic clonal expansion, decreases expression of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1, and increases phosphorylation of Cdk1/Cdc2. SRA also inhibits the expression of adipocyte-related inflammatory genes and TNFα-induced phosphorylation of c-Jun NH(2)-terminal kinase. In conclusion, SRA enhances adipogenesis and adipocyte function through multiple pathways.

Invariant mRNA and mitotic protein breakdown solves the Russian Doll problem of the cell cycle.

It has been proposed that cyclical gene expression occurs at a large number of different times during the cell cycle. The existence of a large number of cycle-specific variations in mRNA and protein during the eukaryotic cell cycle raises the problem of how cell-cycle variations are regulated. This is the "infinite regression" or Russian Doll problem where postulating a cell-cycle specific control element pushes the explanation of cell-cycle variation back one step to the problem of how that control element varies during the cell cycle. PCR studies on unperturbed cells indicate Cyclin mRNA content is invariant during the cell cycle. Furthermore, calculations reveal that variations in mRNA content do not account for observed protein variations. Continuous and constant gene expression during the cell cycle, continuous protein accumulation, and protein breakdown only within the mitotic window solves the Russian Doll problem or infinite regression problem. These results, and theoretical ideas support an alternative view of the cell cycle where many of the proposed control systems do not exist.

Membrane-elution analysis of content of cyclins A, B1, and E during the unperturbed mammalian cell cycle.

BACKGROUND: Problems with whole-culture synchronization methods for the study of the cell cycle have led to the need for an analysis of protein content during the cell cycle of cells that have not been starved or inhibited. The membrane-elution method is a method that allows the study of the cell cycle by producing a culture of unperturbed, synchronized cells. RESULTS: The Helmstetter membrane-elution method for the continuous production of newborn, unperturbed, mammalian cells has been enhanced so that the collection of cells of different cell cycle ages is automated, reproducible, and relatively inexpensive. We have applied the automated membrane-elution method to the analysis of cyclin content during the cell cycle. Cyclin E protein was invariant during the cell cycle. Cyclins B1 and A accumulated continuously during the cell cycle and were degraded at mitosis. Newborn cells had ~0.5% of the cyclin B1 content of dividing cells. CONCLUSION: The expression patterns of cyclins A, B1, and E can be explained by constant mRNA levels during the cell cycle. Previously reported phase specific variations of the cyclins are not strictly necessary for cell-cycle progression. Cells produced by membrane-elution are available to other laboratories for analysis of the cell cycle.