Identification of targets of Twist1 transcription factor in thyroid cancer cells.
BACKGROUND
Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors. Twist1 is a basic helix-loop-helix transcription factor involved in cancer development and progression. We showed that Twist1 affects thyroid cancer cell survival and motility.


OBJECTIVE
We aimed to identify Twist1 targets in thyroid cancer cells.


METHODS
Transcriptional targets of Twist1 were identified by gene expression profiling the TPC-Twist1 cells in comparison with control cells. Functional studies were performed by silencing in TPC-Twist1 and in CAL62 cells the top 10 upregulated genes and by evaluating cell proliferation, survival, migration, and invasion. Chromatin immunoprecipitation was performed to verify direct binding of Twist1 to target genes. Quantitative RT-PCR was applied to study the expression level of Twist1 target genes in human thyroid carcinoma samples.


RESULTS
According to the gene expression profile, the top functions enriched in TPC-Twist1 cells were cellular movement, cellular growth and proliferation, and cell death and survival. Silencing of the top 10 upregulated genes reduced viability of TPC-Twist1 and of CAL62 cells. Silencing of COL1A1, KRT7, and PDZK1 also induced cell death. Silencing of HS6ST2, THRB, ID4, RHOB, and PDZK1IP also impaired migration and invasion of TPC-Twist1 and of CAL62 cells. Chromatin immunoprecipitation showed that Twist1 directly binds the promoter of the top 10 upregulated genes. Quantitative RT-PCR showed that HS6ST2, COL1A1, F2RL1, LEPREL1, PDZK1, and PDZK1IP1 are overexpressed in thyroid carcinoma samples compared with normal thyroids.


CONCLUSIONS
We identified a set of genes that mediates Twist1 biological effects in thyroid cancer cells.
Dipartimento di Medicina Molecolare e Biotecnologie Mediche (G.D.M., F.M.O., T.C.B., P.S.), Università di Napoli "Federico II," Italy 80131; Dipartimento di Area Medica (C.U.), Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy 56126; Dipartimento di Patologia Chirugica, Medica (F.B.), Molecolare e dell'Area Critica dell' Università di Pisa, Italy 56124; Experimental Oncology 1 (R.M.), Centro di Riferimento Oncologico, Aviano, Italy 33081; and Dipartimento di Scienze Motorie e del Benessere (G.S.), Universita' "Parthenope," 80133 Napoli, Italy 80133.
Gennaro
G
Di Maro
Francesca Maria
FM
Orlandella
Tammaro Claudio
TC
Bencivenga
Paolo
P
Salerno
Clara
C
Ugolini
Fulvio
F
Basolo
Roberta
R
Maestro
Giuliana
G
Salvatore
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D002277:Carcinoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002291:Carcinoma, Papillary / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051796:Inhibitor of Differentiation Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D015238:Sulfotransferases / Q000235:genetics; D000077273:Thyroid Cancer, Papillary; D037021:Thyroid Hormone Receptors alpha / Q000235:genetics; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051879:Twist-Related Protein 1 / Q000235:genetics* / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000235:genetics
D051792:Basic Helix-Loop-Helix Proteins; D051796:Inhibitor of Differentiation Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D015238:Sulfotransferases; D037021:Thyroid Hormone Receptors alpha; D051879:Twist-Related Protein 1; D020743:rhoB GTP-Binding Protein; C490128:ID4 protein, human; C509768:PDZK1IP1 protein, human; C103173:TWIST1 protein, human; C485757:HS6ST2 protein, human