Identification of targets of Twist1 transcription factor in anaplastic thyroid cancer

Thyroid cancer is the most common malignancy of endocrine organs and accounts for about 1% of newly diagnosed cancer cases. Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors; it is characterized by local invasion, distant metastasis, chemoresistance and radioresistance. Effective systemic therapy capable of reversing the aggressive biology of this disease is currently not available. Therefore, there is an urgent need for a better understanding of the molecular mechanisms underlying anaplastic thyroid cancer pathogenesis. Through, a cDNA microarray analysis we have isolated Twist1 as a gene up-regulated in ATC. Twist1 is a basic helix-loop-helix transcription factor that has been shown to contribute to cancer development and progression but its mechanism of action is poorly defined. In this Dissertation we showed that approximately 50% of ATCs up-regulated Twist1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Silencing of Twist1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of Twist1 in thyroid cancer cells induced resistance to apoptosis and increased cell migration and invasion. To uncover the molecular mechanisms underlying Twist1 biological effects, we have performed a gene expression profile of Twist1 ectopically expressing thyroid cancer TPC cells in comparison to vector control cells. We founded 158 genes up-regulated and 221 genes down-regulated by more than 1.5 fold in TPC-Twist1 cells. We used the Ingenuity Systems Pathway Analysis (IPA) software to classify these genes. Consistent with the biological function of Twist1 in ATC, the top three molecular and cellular functions enriched in TPC-Twist1 cells were: cellular movement, cellular growth and proliferation, cell death and survival. In this dissertation we focused on the up-regulated genes by more than 4 fold. Silencing of the 11 top up-regulated genes reduced cell viability of TPC-Twist1 cells. Silencing of COL1A1, KRT7, PDZK1 induced also apoptosis. Silencing of HS6ST2, THRB, ID4, RHOB, and PDZK1IP, also impaired cell migration and invasion of TPC-Twist1 cells. Thus, our data demonstrate that Twist1 plays a key role in determining malignant features of anaplastic thyroid cancer cells. These effects are mediated by a set of genes whose expression is under Twist1 control. The identified target genes are potential novel molecular determinants of ATC

Syringicin, a new α-elicitin from an isolate of Phytophthora syringae, pathogenic to citrus fruit

Syringicin (Syr), a new acidic α-elicitin, has been isolated from culture filtrates of Phytophthora syringae, which is the causal agent of citrus fruit rot. Its molecular weight (10194.6±0.2) determined by ES-MS is very similar to that calculated using the mean isotopic composition and considering the occurrence of disulfide bridges. The primary structure was obtained by the combined use of Edmann degradation with MALDI-MS and by comparison with already known a-elicitins. Syr induces a hypersensitive response and electrolyte leakage in tobacco. These are characteristic elicitor properties of the group and in agreement with the molecular mechanism recently proposed for this kind of protein. Finally, its possible applications in biological agriculture and biomedicine are briefly discussed

TWIST1/miR-584/TUSC2 pathway induces resistance to apoptosis in thyroid cancer cells

TWIST1, a transcription factor, plays a pivotal role in cancer initiation and progression. Anaplastic thyroid carcinoma (ATC) is one of the deadliest human malignancies; TWIST1 is overexpressed in ATC and increases thyroid cancer cell survival, migration and invasion. The molecular mechanisms underlying the effects of TWIST1 are partially known. Here, using miRNome profiling of papillary thyroid cancer cells (TPC-1) ectopically expressing TWIST1, we identified miR-584. We showed that TWIST1 directly binds miR-584 using chromatin immunoprecipitation. Importantly, miR-584 was up-regulated in human ATC compared to papillary thyroid carcinoma (PTC) and normal thyroid samples. Overexpression of miR-584 in TPC cells induced resistance to apoptosis, whereas stable transfection of anti-miR-584 in TPC-TWIST1 and 8505C cells increased the sensitivity to apoptosis. Using bioinformatics programs, we identified TUSC2 (tumor suppressor candidate 2) as a novel target of miR-584. TUSC2 mRNA and protein levels were decreased in TPC miR-584 and increased in TPC-TWIST1 anti-miR-584 cells. Luciferase assays demonstrated direct targeting. Restored expression of TUSC2 rescued the inhibition of apoptosis induced by miR-584. Finally, qRT-PCR and immunohistochemical analysis showed that TUSC2 was down-regulated in ATC and PTC samples compared to normal thyroids. In conclusion, our study identified a novel TWIST1/miR-584/TUSC2 pathway that plays a role in resistance to apoptosis of thyroid cancer cells

Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin

The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v. San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation. WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pl of 5.8. The inhibition constants (Ki) for Bacillus licheniformis subtilisin and bovine pancreatic α-chymotrypsin are 3.92×10-9 M and 7.24×10-9 M, respectively. The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines. The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the CI-2 type and with maize subtilisin-chymotrypsin inhibitor MPI. Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors. The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis