H4(D10S170), a gene frequently rearranged with RET in papillary thyroid carcinomas: functional characterization

Abstract

Human thyroid papillary carcinomas are characterized by rearrangements of the RET protooncogene with a number of heterologous genes, which generate the RET/papillary thyroid carcinoma (PTC) oncogenes. One of the most frequent variants of these recombination events is the fusion of the intracellular kinase-encoding domain of RET to the first 101 amino acids of a gene named H4(D10S170). We have characterized the H4(D10S170) gene product, showing that it is a ubiquitously expressed 55 KDa nuclear and cytosolic protein that is phosphorylated following serum stimulation. This phosphorylation was found to depend on mitogen-activated protein kinase (MAPK) Erk1/2 activity and to be associated to the relocation of H4(D10S170) from the nucleus to the cytosol. Overexpression of the H4(D10S170) gene was able to induce apoptosis of thyroid follicular epithelial cells; conversely a carboxy-terminal truncated H4(D10S170) mutant H4(1-101), corresponding to the portion included in the RET/PTC1 oncoprotein, behaved as dominant negative on the proapoptotic function and nuclear localization of H4(D10S170). Furthermore, conditional expression of the H4(D10S170)-dominant negative truncated mutant protected cells from stress-induced apoptosis. The substitution of serine 244 with alanine abrogated the apoptotic function of H4(D10S170). These data suggest that loss of the H4(D10S170) gene function might have a role in thyroid carcinogenesis by impairing apoptosis.

CCDC6 represses CREB1 activity by recruiting histone deacetylase 1 and protein phosphatase 1

“…Earlier work has shown that CCDC6 gene product is an ubiquitously expressed 65 kDa nuclear and cytosolic protein, is phosphorylated by extracellular signalregulated protein kinase after serum stimulation (Grieco et al, 1994;Celetti et al, 2004); it has also been proposed as a proapoptotic factor (Celetti et al, 2004). More recently, it has been shown that CCDC6 is involved in ATM-mediated cellular response to DNA damage, supporting the idea that the impairment of CCDC6 gene function might have a function in thyroid carcinogenesis (Merolla et al, 2007).…”

“…The expression CCDC6 and mutant CCDC6 (1-101) plasmids and small-interfering RNAs (CCDC6i-345) have been described elsewhere (Celetti et al, 2004;Merolla et al, 2007). The expression plasmid pCMVSPORT6-CREB1 was from Invitrogen (Carlsbad, CA, USA).…”

“…The Abs used for immunoprecipitation and western blotting were anti-CCDC6 (Celetti et al, 2004), anti-CREB1, and anti-HDAC1(06720) (Upstate Biotechnology Inc., Lake Placid, NY, USA); anti-Myc(9E10), anti-ATF1, anti-ATF2, anti-ATF3, anti-CREM1, anti-CREB2, anti-PP1, and anti-a-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-pCREB Ser133 (Cell Signaling Technology, Inc., Danvers, MA, USA).…”

New therapeutic perspectives in CCDC6 deficient lung cancer cells

“…14,15 In previous work, we documented that CCDC6 (coiled-coildomain containing 6) acts as a proapoptotic protein substrate of ATM, to sustain DNA damage checkpoints in response to genotoxic events. 16,17 Moreover, CCDC6 protects genome integrity by modulating the activity of the phosphatase PP4C directed toward the dephosphorylation on S139 of the histone H2AX (gH2AX) in response to DNA damage. 18 Consistent with this, we reported that CCDC6 deficiency affects the gH2AX foci formation and the repair of the DNA DSBs.…”

“…20 In most cancers that harbour the CCDC6 gene rearrangement, the product of the normal allele is either wholly absent or functionally impaired by a dominant negative mechanism. 16 Recently, CCDC6-RET fusions and CCDC6 inactivating mutations (N394Y, T462A, S351Y, E227K; www.sanger.ac.uk/genetics/ CGP/cosmic) have been reported in NSCLC. 21,22 In this study, we examined CCDC6 protein and mRNA levels in a group of NSCLC cell lines along with DNA repair proficiency, DNA damage response and platinum-based therapy as a single agent or in combination with PARP inhibitors.…”