CCDC6

Gene Summary

Gene:CCDC6; coiled-coil domain containing 6
Aliases: H4, PTC, TPC, TST1, D10S170
Location:10q21
Summary:This gene encodes a coiled-coil domain-containing protein. The encoded protein is ubiquitously expressed and may function as a tumor suppressor. A chromosomal rearrangement resulting in the expression of a fusion gene containing a portion of this gene and the intracellular kinase-encoding domain of the ret proto-oncogene is the cause of thyroid papillary carcinoma.[provided by RefSeq, Sep 2010]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:coiled-coil domain-containing protein 6
HPRD
Source:NCBIAccessed: 27 February, 2015

Ontology:

What does this gene/protein do?
Show (5)

Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 27 February 2015 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Chromosome 10
  • RTPCR
  • Neoplastic Cell Transformation
  • Translocation
  • FISH
  • Adenocarcinoma
  • Papillary Carcinoma
  • Non-Small Cell Lung Cancer
  • Cytoskeletal Proteins
  • Carcinoma
  • Receptor Protein-Tyrosine Kinases
  • Thyroid Cancer
  • Signal Transduction
  • Gene Silencing
  • Molecular Sequence Data
  • Drosophila Proteins
  • Mutation
  • Radiation-Induced Cancer
  • Thyroid Gland
  • Gene Rearrangement
  • Chromosome Inversion
  • Quinazolines
  • Cell Proliferation
  • Base Sequence
  • Protein-Tyrosine Kinases
  • DNA Damage
  • Transfection
  • Gene Fusion
  • DNA Sequence Analysis
  • Proteins
  • Cancer Gene Expression Regulation
  • Lung Cancer
  • Proto-Oncogene Proteins
  • Messenger RNA
  • Piperidines
  • Chromosome Aberrations
  • Proto-Oncogene Proteins c-ret
  • Phosphorylation
  • HeLa Cells
  • Oncogene Fusion Proteins
Tag cloud generated 27 February, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (5)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Entity Topic PubMed Papers
Thyroid CancerRET-PTC1 Rearangements in Papillary Thyroid Cancer
The PTC1 fusion gene is present in approximately 30% of papillary thyroid carcinomas. It involves an inversion of 10q which fuses the RET protooncogene to the D10S170 (H4) gene. High incidence of RET/PTC1 rearrangements have been reported in thyroid cancers in people living in areas adjacent to Chernobyl following the nuclear accident. SCID mice transplanted with normal human thyroid tissues and then exposed to radiation preferentially developed RET/PTC1 compared to other RET rearrangements (Mizuno,2000). A FISH study (Nikiforova, 2000) suggests that RET and PTC1 are more frequently juxtaposed in the nucleus of thyroid cells compared to other cells. This close proximity of the genes in thyroid cells may promote the PTC1 fusion since a single radiation track may be sufficient to produce a double-strand break in each gene at the same site in the nucleus.
View Publications216
Thyroid CancerCCDC6 and Thyroid Cancer View Publications13
Lung CancerCCDC6 and Lung Cancer View Publications8
Lung Cancer, Non-Small CellCCDC6 and Non-Small Cell Lung Cancer View Publications4
-CCDC6 and Papillary Carcinoma View Publications4

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CCDC6 (cancer-related)

Rao PJ, Vardhini NV, Parvathi MV, et al.
Prevalence of RET/PTC1 and RET/PTC3 gene rearrangements in Chennai population and its correlation with clinical parameters.
Tumour Biol. 2014; 35(10):9539-48 [PubMed] Related Publications
Thyroid cancer is one of the most common endocrine disorders in the world. In India, about 42 million people suffer from various thyroid disorders. However, based on population-based cancer registry (PBCR) and Chennai cancer registry, thyroid cancer is emerging as a common cancer particularly in Chennai. Papillary thyroid carcinoma is considered as the most prevalent cancer constituting about 80-85 % of thyroid malignancies. Rearranged during transfection (RET)/papillary thyroid carcinoma (PTC) gene rearrangements are one of the major genetic alterations found in papillary thyroid carcinoma. This present study aims at estimating the frequency of RET/PTC1 and RET/PTC3 gene rearrangements in Chennai population and investigating the correlation between RET/PTC gene expressions with clinical parameters. Formalin-fixed paraffin-embedded (FFPE) tumor tissues obtained from 30 patients with papillary thyroid carcinoma were analyzed. Initially, to differentiate classic and follicular variants of papillary thyroid carcinoma samples, immunohistochemistry was performed. Thereafter, total RNA was isolated, and quantitative evaluation of RET/PTC1 and RET/PTC3 gene rearrangements by real-time PCR was performed. Chi-square test was performed to understand the correlation between positive and negative mutations of RET/PTC messenger RNA (mRNA) expression with clinical parameters. RET/PTC3 gene rearrangements were identified in 26/30 (86.67 %) cases, and none of the patient in our series had RET/PTC1 gene rearrangements. There was no statistically significant difference observed between positive and negative mutations of RET/PTC3 mRNA expression with clinic pathological parameters. Our results indicate that RET/PTC3 gene rearrangements are the most prevalent form of rearrangements in PTCs of Chennai population.

Minna E, Romeo P, De Cecco L, et al.
miR-199a-3p displays tumor suppressor functions in papillary thyroid carcinoma.
Oncotarget. 2014; 5(9):2513-28 [PubMed] Free Access to Full Article Related Publications
Thyroid cancer incidence is rapidly increasing. Papillary Thyroid Carcinoma (PTC), the most frequent hystotype, usually displays good prognosis, but no effective therapeutic options are available for the fraction of progressive PTC patients. BRAF and RET/PTC are the most frequent driving genetic lesions identified in PTC. We developed two complementary in vitro models based on RET/PTC1 oncogene, starting from the hypothesis that miRNAs modulated by a driving PTC-oncogene are likely to have a role in thyroid neoplastic processes. Through this strategy, we identified a panel of deregulated miRNAs. Among these we focused on miR-199a-3p and showed its under-expression in PTC specimens and cell lines. We demonstrated that miR-199a-3p restoration in PTC cells reduces MET and mTOR protein levels, impairs migration and proliferation and, more interesting, induces lethality through an unusual form of cell death similar to methuosis, caused by macropinocytosis dysregulation. Silencing MET or mTOR, both involved in survival pathways, does not recapitulate miR-199a-3p-induced cell lethality, thus suggesting that the cooperative regulation of multiple gene targets is necessary. Integrated analysis of miR-199a-3p targets unveils interesting networks including HGF and macropinocytosis pathways. Overall our results indicate miR-199a-3p as a tumor suppressor miRNA in PTC.

Mizukami T, Shiraishi K, Shimada Y, et al.
Molecular mechanisms underlying oncogenic RET fusion in lung adenocarcinoma.
J Thorac Oncol. 2014; 9(5):622-30 [PubMed] Related Publications
BACKGROUND: Oncogenic RET fusion, caused by an inversion in chromosome 10, was recently identified as a driver mutation for the development of lung adenocarcinoma (LADC). Nevertheless, the molecular mechanism(s) underlying the rearrangement of the RET locus during lung carcinogenesis are unknown.
METHODS: Genomic segments containing breakpoint junctions for RET fusions were cloned and analyzed by genomic polymerase chain reaction and genome capture sequencing using a next-generation sequencer to identify the mechanisms involved in DNA strand breaks and illegitimate joining of DNA ends. Of the 18 cases studied, 16 were identified by screening 671 LADC cases and two were previously published.
RESULTS: Almost all (17 of 18, 94%) of the breakpoints in RET were located within a 2.0-kb region spanning exon 11 to intron 11 and no breakpoint occurred within 4 bp of any other. This suggested that as in papillary thyroid carcinoma, DNA strand breaks formed at nonspecific sites within this region trigger RET fusion. Just over half of the RET fusions in LADC (10 of 18, 56%) were caused by simple reciprocal inversion, and two DNA-repair mechanisms, namely nonhomologous end joining and break-induced replication, were deduced to have contributed to the illegitimate joining of the DNA ends.
CONCLUSIONS: Oncogenic RET fusion in LADC occurs through multiple pathways and involves the illegitimate repair of DNA strand breaks through mechanisms different from those identified in papillary thyroid carcinoma, where RET fusion also functions as a driver mutation.

Tsuta K, Kohno T, Yoshida A, et al.
RET-rearranged non-small-cell lung carcinoma: a clinicopathological and molecular analysis.
Br J Cancer. 2014; 110(6):1571-8 [PubMed] Article available free on PMC after 18/03/2015 Related Publications
BACKGROUND: To elucidate clinicopathological characteristics of non-small-cell lung carcinoma (NSCLC) cases carrying RET rearrangements causing oncogenic fusions to identify responders to therapy with RET tyrosine kinase inhibitors.
METHODS: We investigated 1874 patients with carcinomas, including 1620 adenocarcinomas (ADCs), 203 squamous cell carcinomas (SCCs), 8 large cell carcinomas, and 43 sarcomatoid carcinomas (SACs). Fluorescence in situ hybridisation (FISH) and/or reverse transcription-PCR (RT-PCR) were performed to detect RET gene rearrangement.
RESULTS: In all, 22 cases (1.2%) showed RET rearrangements; all cases were of ADC histology. Of the 22 patients, 19 possessed KIF5B-RET fusion genes, whereas 3 possessed CCDC6-RET fusion genes. The RET-rearranged tumours were significantly more common in younger patients (P=0.038) and tended to occur in patients with no history of smoking (P=0.051). In addition, RET rearrangements were not associated with gender, occupational history (particularly radioactive exposure), tumour size, lymph node status, tumour stage, or patient survival. The predominant growth pattern in RET-rearranged ADCs was lepidic in 6 cases, papillary in 9 cases, acinar in 2 cases, micropapillary in 1 case, and solid in 4 cases. Cells with cytoplasmic mucin production were at least focally present in 12 of the 22 (54.5%) RET-rearranged ADC cases. Among the 21 analysed RET-rearranged tumours, RET immunopositivity was observed in 15 cases (71.4%), and was significantly associated with RET rearrangement (P<0.001).
CONCLUSIONS: The RET rearrangements were observed in 1.2% of NSCLCs. All cases of RET rearrangement were ADCs. The RET rearrangements were more likely to be observed in younger patients. Although cytoplasmic mucin production was at least focally present in 54.5% of RET-rearranged ADCs, specific histological features were not detected.

Lubitz CC, Economopoulos KP, Pawlak AC, et al.
Hobnail variant of papillary thyroid carcinoma: an institutional case series and molecular profile.
Thyroid. 2014; 24(6):958-65 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
BACKGROUND: Papillary thyroid carcinoma (PTC) is increasing in incidence while mortality is unchanged. Identifying patients with higher risk of recurrence and death is essential. Case series identify the hobnail variant of PTC (HVPTC), which is characterized by micropapillary architecture, apocrine features, and loss of cellular polarity. Herein, we describe the clinical course, pathologic features, and mutational profile of patients at our institution with HVPTC.
METHODS: A query into the surgical pathologic database (2009-2012) was performed, and clinicopathologic data were collected on all patients carrying the diagnosis of HVPTC. BRAF(V600E) testing was performed on paraffin-embedded blocks using SNaPshot mutational analysis.
RESULTS: Twelve patients with HVPTC were identified, with an average age of 54.1±18.8 years. Seven patients (63.6%) were AJCC Stage III or IV at presentation. Tumors were large (3.7±2.0 cm), some were multifocal (33.3%), and frequently with extrathyroidal extension (58.3%), lymphovascular invasion (41.7%), and lymph node metastasis (75%). Forty percent of the patients had concomitant tall cell features (TCF), and two had small foci of undifferentiated (anaplastic) thyroid carcinoma (ATC). Eighty percent of tumors undergoing mutational analysis had the BRAF(V600E) mutation, and the remaining 20% harbored a RET/PTC1 gene rearrangement. No other known thyroid cancer mutations were identified on SNaPshot analysis. At median follow-up of 26 months, four patients had recurrent or persistent disease, one of whom died from the disease one year after surgery.
CONCLUSIONS: The hobnail variant of PTC has an aggressive behavior, with a high incidence of infiltrative tumors and metastatic disease. Strikingly, all tumors in our series harbored a PTC-associated genetic abnormality, either a BRAF(V600E) mutation (80%) or a RET/PTC1 rearrangement (20%). This histologic variant warrants further study, and patients with this diagnosis should be observed closely for recurrence.

Dacic S, Luvison A, Evdokimova V, et al.
RET rearrangements in lung adenocarcinoma and radiation.
J Thorac Oncol. 2014; 9(1):118-20 [PubMed] Related Publications
BACKGROUND: RET rearrangement, a hallmark of radiation-induced thyroid cancer, has been reported to occur in 1% of lung adenocarcinoma patients. Patients with this rearrangement tend to be younger and never smokers, raising a possibility of other causes, such as radiation. We hypothesized that RET chromosomal rearrangement may represent a genetic mechanism of radiation-induced lung cancer.
METHODS: Two hundred forty-five consecutive primary lung adenocarcinomas without history of radiation and 38 lung adenocarcinoma patients with a history of therapeutic radiation for breast carcinoma or mediastinal Hodkgin lymphoma were tested for RET rearrangement by fluorescence in situ hybridization. Human lung adenocarcinoma cells (201T) were subjected to γ radiation and tested for RET gene fusions by reverse transcriptase-polymerase chain reaction and Southern blot hybridization.
RESULTS: We identified one case with RET rearrangement in the group without history of radiation (1 of 240; 0.4%) and two cases in the group with history of radiation (2 of 37; 5.4%; P=0.0436). Both these patients were women, who were former smokers with a history of breast carcinoma treated with surgery and radiation. Furthermore, we found that RET fusions could be directly induced in 201T human lung cells by exposure to 1 Gy of γ radiation. All fusions identified were between RET and KIF5B genes, and no RET fusions to CCDC6 or NCOA4 genes, characteristic for thyroid cancer, were identified in the irradiated lung cells.
CONCLUSION: RET fusions may represent a genetic mechanism of radiation-induced lung adenocarcinoma.

Yoo SS, Jin G, Jung HJ, et al.
RET fusion genes in Korean non-small cell lung cancer.
J Korean Med Sci. 2013; 28(10):1555-8 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
Recently, rearranged during transfection (RET) fusions have been identified in approximately 1% of non-small cell lung cancer (NSCLC). To know the prevalence of RET fusion genes in Korean NSCLCs, we examined the RET fusion genes in 156 surgically resected NSCLCs using a reverse transcriptase polymerase chain reaction. Two KIF5B-RET fusions and one CCDC6-RET fusion were identified. All three patients were females and never smokers with adenocarcinomas. RET fusion genes were mutually exclusive from EGFR, KRAS mutations and EML4-ALK fusion. RET fusion genes occur 1.9% (3 of 156) of surgically treated NSCLC patients in Koreans.

Staibano S, Ilardi G, Leone V, et al.
Critical role of CCDC6 in the neoplastic growth of testicular germ cell tumors.
BMC Cancer. 2013; 13:433 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
BACKGROUND: DNA damage response has been clearly described as an anti-cancer barrier in early human tumorigenesis. Moreover, interestingly, testicular germ cell tumors (TGCTs) have been reported to lack the DNA Damage Response (DDR) pathway activation. CCDC6 is a pro-apoptotic phosphoprotein substrate of the kinase ataxia telangectasia mutated (ATM) able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners. In our study we sought to determine whether CCDC6 could have a role in the patho-genesis of testicular germ cell tumors.
METHODS: To achieve this aim, analysis for CCDC6 expression has been evaluated on serial sections of the mouse testis by immunohistochemistry and on separate populations of murine testicular cells by western blot. Next, the resistance to DNA damage-induced apoptosis and the production of reactive oxygen species has been investigated in GC1 cells, derived from immortalized type B murine germ cells, following CCDC6 silencing. Finally, the CCDC6 expression in normal human testicular cells, in Intratubular Germ Cell Neoplasia Unclassified (IGCNU), in a large series of male germ cell tumours and in the unique human seminoma TCam2 cell line has been evaluated by immunohistochemistry and by Western Blot analyses.
RESULTS: The analysis of the CCDC6 expression revealed its presence in Sertoli cells and in spermatogonial cells. CCDC6 loss was the most consistent feature among the primary tumours and TCam2 cells. Interestingly, following treatment with low doses of H₂O₂, the silencing of CCDC6 in GC1 cells caused a decrease in the oxidized form of cytochrome c and low detection of Bad, PARP-1 and Caspase 3 proteins. Moreover, in the silenced cells, upon oxidative damage, the cell viability was protected, the γH2AX activation was impaired and the Reactive Oxygen Species (ROS) release was decreased.
CONCLUSIONS: Therefore, our results suggest that the loss of CCDC6 could aid the spermatogonial cells to be part of a pro-survival pathway that helps to evade the toxic effects of endogenous oxidants and contributes to testicular neoplastic growth.

Endo T, Kobayashi T
Concurrent overexpression of RET/PTC1 and TTF1 confers tumorigenicity to thyrocytes.
Endocr Relat Cancer. 2013; 20(6):767-76 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
A variant located on 14q13.3 nearest to thyroid transcription factor-1 (TTF1) predisposes individuals to thyroid cancer, but whether this variant is related to the RET/PTC rearrangement associated with human papillary thyroid carcinomas (PTCs) is unknown. The aims of this study were to investigate the effects of RET/PTC1 on the expression of thyroid-specific genes in thyrocytes and their relationship with malignant transformation of the thyrocytes. In the absence or presence of TSH, an extracellular signal-regulated kinase was phosphorylated in FRTL5 cells that stably expressed RET/PTC1, and these cells grew independently of TSH. FRTL (RET/PTC1) cells produced 566% more thyroglobulin mRNA and 474% more Na+/I- symporter mRNA than did the control FRTL (pcDNA) cells. FRTL (RET/PTC1) cells expressed 468% more Ttf1 mRNA than did FRTL (pcDNA) cells, but these two cell types did not differ significantly with respect to Pax8 or Ttf2 mRNA levels. When FRTL (RET/PTC1) cells and FRTL (pcDNA), cells were injected into each of nine nude mice, each mouse developed a single tumor at the site of FRTL (RET/PTC1) cell injection; in contrast, tumor formation never occurred at sites of FRTL (cDNA) cells injection. Tumors resulting from FRTL (RET/PTC1) cells retained (125)I-uptake activity; moreover, the cells invaded into surrounding skeletal muscle. When overexpression of Ttf1 in FRTL (RET/PTC1) cells was silenced, the cells completely lost their tumorigenic potential. Exogenous TTF1 cDNA enhanced the tumorigenicity of BHP18-21v cells, human PTC cells that express RET/PTC1, in nude mice. These results indicated that concurrent overexpression of RET/PTC1 and TTF1 confers tumorigenicity to FRTL5 and BHP18-21v cells in nude mice.

Boaventura P, Pereira D, Celestino R, et al.
Genetic alterations in thyroid tumors from patients irradiated in childhood for tinea capitis treatment.
Eur J Endocrinol. 2013; 169(5):673-9 [PubMed] Related Publications
OBJECTIVE: Exposure to ionizing radiation at young age is the strongest risk factor for the occurrence of papillary thyroid carcinoma (PTC). RET/PTC rearrangements are the most frequent genetic alterations associated with radiation-induced PTC, whereas BRAF and RAS mutations and PAX8-PPARG rearrangement have been associated with sporadic PTC. We decided to search for such genetic alterations in PTCs of patients subjected in childhood to scalp irradiation.
DESIGN: We studied 67 thyroid tumors from 49 individuals irradiated in childhood for tinea capitis scalp epilation: 36 malignant (12 cases of conventional PTC (cPTC), two cPTC metastases, 20 cases of follicular variant PTC (FVPTC), one oncocytic variant of PTC and one follicular carcinoma) and 31 follicular thyroid adenomas.
METHODS: The lesions were screened for the BRAF(V600E) and NRAS mutations and for RET/PTC and PAX8-PPARG rearrangements.
RESULTS: BRAF(V600E) mutation was detected in seven of 14 (50%) cPTC and two of 20 FVPTC (10%) (P=0.019). NRAS mutation was present in one case of FVPTC (5%). RET/PTC1 rearrangement was found, by RT-PCR, in one of 17 cases (5.9%) and by fluorescence in situ hybridization in two of six cases (33%). PAX8-PPARG rearrangement was not detected in any carcinoma. None of the follicular adenomas presented any of the aforementioned genetic alterations.
CONCLUSIONS: The prevalence of BRAF(V600E) mutation in our series is the highest reported in series of PTCs arising in radiation-exposed individuals. The prevalence of RET/PTC1 rearrangement fits with the values recently described in a similar setting.

Spina R, Filocamo G, Iaccino E, et al.
Critical role of zinc finger protein 521 in the control of growth, clonogenicity and tumorigenic potential of medulloblastoma cells.
Oncotarget. 2013; 4(8):1280-92 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells. The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1-/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression. Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1-/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential. Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

Proietti A, Sartori C, Borrelli N, et al.
Follicular-derived neoplasms: morphometric and genetic differences.
J Endocrinol Invest. 2013; 36(11):1055-61 [PubMed] Related Publications
BACKGROUND: The distinction between follicular adenomas (FAs) and well differentiated follicular and papillary carcinomas is often a demanding task and sometimes only intuitive.
AIM: We report an histomorphological evaluation of follicular neoplasms [FAs, follicular carcinomas (FCs), and follicular variant of papillary carcinomas (FVPTCs)], supported by a qualitative and quantitative image analysis and by a molecular characterization.
MATERIAL AND METHODS: Tumor fibrosis and haemorrhage, neoplastic capsule thickness, follicle diameter, number of neoplastic cells, nuclear diameter of neoplastic cells, vessels density, vessels area and intratumoral distribution were evaluated. Ras and BRAF mutations, RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements were analyzed. Correlations with clinico-pathological features have been studied.
RESULTS: We found that FAs had a more extensive intratumoral haemorrhage, while malignant neoplasms were characterized by an evident fibrosis, higher cellularity and larger size. FVPTCs had higher nuclear diameter; cells count was higher in the minimally invasive follicular thyroid carcinomas, as well as a thickener neoplastic capsule. The CD34 stain showed a higher microvessel density in the FVPTCs group. A higher peripheral vessels distribution was observed only in malignant neoplasms. We observed overall Ras mutations in 2.4% of adenomas, in 41.5% of FVPTCs, and in 44.8% of FCs. It is outstanding that there is a marked difference in the Ras mutation distribution between the benign and malignant tumors in our series.
CONCLUSIONS: We found that genotyping of Ras gene family together with an accurate analysis of selected morphological features could help in the differential diagnosis of follicular-derived thyroid neoplasms.

Ali HM, Maksimenko A, Urbinati G, et al.
Effects of silencing the RET/PTC1 oncogene in papillary thyroid carcinoma by siRNA-squalene nanoparticles with and without fusogenic companion GALA-cholesterol.
Thyroid. 2014; 24(2):327-38 [PubMed] Related Publications
BACKGROUND: RET/PTC1 is the most prevalent type of gene rearrangement found in papillary thyroid carcinoma (PTC). Previously, we introduced a new noncationic nanosystem for targeted RET/PTC1 silencing by efficient delivery of small interfering RNA (siRNA) using the "squalenoylation" approach. With the aim of improving these results further, we designed new squalenoyl nanostructures consisting of the fusogenic peptide GALA-cholesterol (GALA-Chol) and squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC1.
METHODS: The siRNA RET/PTC1-SQ bioconjugate was synthesized. The corresponding NPs were prepared with or without GALA-Chol by nanoprecipitation and then characterized for their size and zeta potential. The effects of NPs on BHP 10-3 SCmice and TPC-1 cell viability (MTT assay), gene and protein silencing (reverse transcription-quantitative polymerase chain reaction [rt-qPCR], Western blot), and cellular uptake (fluorescent microscopy) were studied. In vivo gene silencing efficiency of siRNA RET/PTC1-SQ NPs was assessed by administration in nude mice via either intratumoral (i.t.) or intravenous (i.v.) routes. Tumor growth was followed for 19 days. Tumors were then collected, and RET/PTC1 gene and protein inhibitions were assessed by RT-qPCR and Western blot.
RESULTS: The combination of siRNA RET/PTC1-SQ bioconjugate and GALA-Chol leads to stable NPs of ∼200 nm diameter. In vitro, the results revealed that combining GALA-Chol with siRNA RET/PTC1-SQ NPs decreased cell viability, enhanced cellular internalization, and induced gene silencing efficiency in both human PTC (BHP 10-3 SCmice and TPC-1) cell lines. On the contrary, in vivo, the siRNA RET/PTC1-SQ GALA-Chol NPs were not found to be efficient either in gene silencing or in tumor growth inhibition, compared to siRNA RET/PTC1-SQ NPs both via i.t. and i.v. routes (p<0.001).
CONCLUSIONS: Conversely to siRNA RET/PTC1-SQ NPs, the siRNA RET/PTC1-SQ GALA-Chol NPs are efficient in vitro but not in vivo. Finally, NPs of siRNA RET/PTC1-SQ were found to be efficient silencers of the RET/PTC1 fusion oncogene in in vivo applications even at a concentration lower than used in a previously published study.

Okamoto K, Kodama K, Takase K, et al.
Antitumor activities of the targeted multi-tyrosine kinase inhibitor lenvatinib (E7080) against RET gene fusion-driven tumor models.
Cancer Lett. 2013; 340(1):97-103 [PubMed] Related Publications
RET gene fusions are recurrent oncogenes identified in thyroid and lung carcinomas. Lenvatinib is a multi-tyrosine kinase inhibitor currently under evaluation in several clinical trials. Here we evaluated lenvatinib in RET gene fusion-driven preclinical models. In cellular assays, lenvatinib inhibited auto-phosphorylation of KIF5B-RET, CCDC6-RET, and NcoA4-RET. Lenvatinib suppressed the growth of CCDC6-RET human thyroid and lung cancer cell lines, and as well, suppressed anchorage-independent growth and tumorigenicity of RET gene fusion-transformed NIH3T3 cells. These results demonstrate that lenvatinib can exert antitumor activity against RET gene fusion-driven tumor models by inhibiting oncogenic RET gene fusion signaling.

Bansal M, Gandhi M, Ferris RL, et al.
Molecular and histopathologic characteristics of multifocal papillary thyroid carcinoma.
Am J Surg Pathol. 2013; 37(10):1586-91 [PubMed] Related Publications
Papillary thyroid carcinoma (PTC) is frequently multifocal, which can represent either intraglandular spread from a single primary tumor or multiple synchronous primary tumors (MSPTs). To distinguish and characterize these entities, we investigated whether multifocal PTCs contain genetically similar or different mutations and have particular histopathologic characteristics. In 60 cases of PTC with 2 to 4 discrete tumor foci, each focus was tested for BRAF, NRAS, HRAS, and KRAS point mutations and RET/PTC1 and RET/PTC3 rearrangements and analyzed for various histopathologic features. Overall, BRAF mutations were found in 43% of tumors, RAS in 27%, and RET/PTC in 2%. Four different patterns of mutation occurrence were identified: (i) 2 foci containing different mutations (30%); (ii) 1 tumor containing a mutation and another carrying no mutations (32%); (iii) both/all tumors containing the same mutation (25%); (iv) all tumors having no mutations (13%). The 30% of cases with 2 different mutations represent a group of tumors that are unequivocally MSPT. These tumors more commonly occurred in different lobes, although they could be located as close as 0.6 cm from each other. Moreover, MSPTs typically demonstrated distinct histologic variants/microscopic features, were encapsulated or had a smooth border, and showed no microscopic peritumoral dissemination. In conclusion, we demonstrate that at least 30% of multifocal PTCs represent unequivocal MSPTs that develop through distinct molecular alterations and that as many as 60% of multifocal PTCs are likely MSPTs. Histopathologically, MSPTs are typically located in different lobes, have distinct growth patterns, and do not show microscopic peritumoral dissemination.

Li ZJ, Mack SC, Mak TH, et al.
Evasion of p53 and G2/M checkpoints are characteristic of Hh-driven basal cell carcinoma.
Oncogene. 2014; 33(20):2674-80 [PubMed] Related Publications
Basal cell carcinoma (BCC), the most common type of cancer, is characterized by aberrant Hedgehog (Hh) pathway activity. Mutations in pathway components, such as PATCHED1 (PTCH1), are commonly found in BCC. While the tumor suppressor role of PTCH1 in BCC is well established, how Hh pathway activation disrupts normal skin homeostasis to promote BCC formationremains poorly understood. Like Ptc1, Sufu is a major negative regulator of the Hh pathway. Previously, we showed that inactivation of Sufu in the skin does not result in BCC formation. Why loss of Ptc1, but not Sufu, in the epidermis induces BCC formation is unclear. In this report, we utilized gene expression profiling to identify biological pathways and processes that distinguish Sufu from Ptc1 mutants, and discovered a novel role for Sufu in cell cycle regulation. We demonstrated that the Hh pathway activation inSufu and Ptc1 mutant skin is associated with abnormal cell cycle entry, ectopic expression of D-type cyclins and increasedDNA damage. However, despite the presence of DNA damage, p53 stabilization was impaired in the mutant skin. Alternative mechanism to halt genomic instability is the activation of G2/M cell cycle checkpoint, which can occur independent of p53. We found that while Ptc1 mutant cells continue to cycle, which would favor genomic instability, loss of Sufu results in G2/M cell cycle arrest.This finding may explain why inactivation of Sufu is not sufficient to drive BCC formation. Taken together, these studies revealed a unique role for Sufu in G2/M phase progression, and uncovered the molecular and cellular features associated with Hh-driven BCC.

Suzuki M, Makinoshima H, Matsumoto S, et al.
Identification of a lung adenocarcinoma cell line with CCDC6-RET fusion gene and the effect of RET inhibitors in vitro and in vivo.
Cancer Sci. 2013; 104(7):896-903 [PubMed] Related Publications
Rearrangements of the proto-oncogene RET are newly identified potential driver mutations in lung adenocarcinoma (LAD). However, the absence of cell lines harboring RET fusion genes has hampered the investigation of the biological relevance of RET and the development of RET-targeted therapy. Thus, we aimed to identify a RET fusion positive LAD cell line. Eleven LAD cell lines were screened for RET fusion transcripts by reverse transcription-polymerase chain reaction. The biological relevance of the CCDC6-RET gene products was assessed by cell growth, survival and phosphorylation of ERK1/2 and AKT with or without the suppression of RET expression using RNA interference. The efficacy of RET inhibitors was evaluated in vitro using a culture system and in an in vivo xenograft model. Expression of the CCDC6-RET fusion gene in LC-2/ad cells was demonstrated by the mRNA and protein levels, and the genomic break-point was confirmed by genomic DNA sequencing. Mutations in KRAS and EGFR were not observed in the LC-2/ad cells. CCDC6-RET was constitutively active, and the introduction of a siRNA targeting the RET 3' region decreased cell proliferation by downregulating RET and ERK1/2 phosphorylation. Moreover, treatment with RET-inhibitors, including vandetanib, reduced cell viability, which was accompanied by the downregulation of the AKT and ERK1/2 signaling pathways. Vandetanib exhibited anti-tumor effects in the xenograft model. Endogenously expressing CCDC6-RET contributed to cell growth. The inhibition of kinase activity could be an effective treatment strategy for LAD. LC-2/ad is a useful model for developing fusion RET-targeted therapy.

Heidenreich WF, Saran A, Atkinson M, Pazzaglia S
A mechanistic model for medulloblastoma induction in mice.
Radiat Res. 2013; 179(5):610-4 [PubMed] Related Publications
Medulloblastomas in Patched heterozygous mice (Ptc1(+/-) mice) are induced with high probability by ionizing radiation applied in the immediate post-natal period. A mathematical model is described here that accommodates the dependence of the medulloblastoma incidence on dose, age at exposure and age. The model assumes that the first step in the development of the cancer is already present in all cells of the patched mouse due to germ-line inactivation of one allele of the patched tumor suppressor gene. The subsequent rate-limiting step is dependent linearly on dose at least up to 3 Gy. The observed strong decrease in carcinogenic effect of radiation between exposure on day 1 and day 10 is described by a physiological elimination of target cells during post-natal maturation of the brain. A single malignant cell develops into a tumor following a gamma-distribution with mean of about 160 days. The multiplicity of medulloblastomas is predicted.

Frasca F, Vella V, Nicolosi ML, et al.
Thyroid cancer cell resistance to gefitinib depends on the constitutive oncogenic activation of the ERK pathway.
J Clin Endocrinol Metab. 2013; 98(6):2502-12 [PubMed] Related Publications
CONTEXT: Poorly differentiated thyroid carcinomas are refractory to common anticancer therapies, and novel inhibitors are being tested in these deadly malignancies. The epidermal growth factor receptor (EGFR) tyrosine kinase represents an attractive target for treatment because it is up-regulated in thyroid cancer and plays a role in cancer progression. However, EGFR inhibitors have provided poor results in thyroid carcinomas.
OBJECTIVE: We evaluated the possible mechanism underlying the resistance of thyroid cancer cells to EGFR inhibitors.
DESIGN: We tested the effect of the EGFR tyrosine kinase inhibitor gefitinib in a panel of thyroid cancer cell lines.
RESULTS: We found that in most of the cell lines, although gefitinib inhibited EGFR phosphorylation, it was poorly effective in reducing cell viability. gefitinib, however, was able to inhibit epidermal growth factor-induced cell migration and matrix invasion. In most thyroid cancer cell lines, gefitinib significantly inhibited Akt phosphorylation by inhibiting EGFR activation, but it had limited or no effect on ERK phosphorylation. The poor cell response to gefitinib was associated with genetic alterations, leading to constitutive activation of the ERK pathway, including BRAF(V600E) and HRAS(G12A/Q61R) mutations and RET/PTC1 rearrangement. When BRAF(V600E)-positive thyroid cancer cells were incubated with the specific BRAF inhibitor PLX4032, sensitivity to gefitinib was restored. Similar results were obtained with rat sarcoma and RET/papillary thyroid cancer inhibitors.
CONCLUSIONS: These results indicate that thyroid cancer resistance to gefitinib is due to the constitutive activation of the mitogenic pathway by either signals downstream of EGFR or other tyrosine kinase receptors. This resistance can be overcome by the combined use of selective inhibitors.

Wang P, Wang YG, Zhao WJ, et al.
[The correlation between BRAF mutations, RET/PTC rearrangements and platelet-derived growth factor B expression in papillary thyroid carcinomas].
Zhonghua Nei Ke Za Zhi. 2012; 51(12):987-91 [PubMed] Related Publications
OBJECTIVE: To investigate the prevalence of BRAF T1799A mutation and RET/PTC rearrangement in Qingdao and detect the expression of platelet-derived growth factor B (PDGF-B) in order to investigate the correlation between gene mutation and PDGF-B.
METHODS: Fresh tissue from 48 papillary thyroid carcinomas (PTC) patients was examined for BRAF mutation RET rearrangements (RET/PTC1 and RET/PTC3) by PCR, followed by direct-sequence analysis. The expression of PDGF was analyzed by immunohistochemistry.
RESULTS: Among the 48 patients, 14 (29.2%) were micro PTC; 18 (37.5%) had BRAF T1799A mutations and 23(47.9%) had RET/PTC rearrangement. There were 17 (35.4%) cases of RET/PTC1 and 6 (12.5%) of RET/PTC3, with no multiple rearrangements. Both BRAF T1799A mutation and RET/PTC rearrangement were present in 6 (12.5%) cases of non-micro PTC. The level of PDGF-B expression in BRAF T1799A positive was higher than that in the negative, and the level of PDGF-B expression in RET/PTC3 was higher than that in RET/PTC1 (P < 0.05). The more advanced neoplasm stage was, the stranger PDGF-B expression was.
CONCLUSIONS: The incidence of BRAF T1799A mutation and RET/PTC rearrangement is higher in Qingdao. BRAF T1799A mutation and RET/PTC3 rearrangement in patients suggests a poorer prognosis than the negative one. The BRAF T1799A mutation and RET/PTC3 rearrangement may strengthen the expression of PDGF-B. Both variations suggest a poor prognosis.

Wang R, Hu H, Pan Y, et al.
RET fusions define a unique molecular and clinicopathologic subtype of non-small-cell lung cancer.
J Clin Oncol. 2012; 30(35):4352-9 [PubMed] Related Publications
PURPOSE: The RET fusion gene has been recently described in a subset of non-small-cell lung cancers (NSCLCs). Because we have limited knowledge about these tumors, this study was aimed at determining the clinicopathologic characteristics of patients with NSCLC harboring the RET fusion gene.
PATIENTS AND METHODS: We examined the RET fusion gene in 936 patients with surgically resected NSCLC using a reverse transcriptase polymerase chain reaction (PCR) plus quantitative real-time PCR strategy, with validation using immunohistochemical and fluorescent in situ hybridization assays. A subset of 633 lung adenocarcinomas was also studied for EGFR, KRAS, HER2, and BRAF mutations, as well as ALK rearrangements. Patient characteristics, including age, sex, smoking history, stage, grade, International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification of subtypes of lung adenocarcinoma, and relapse-free survival, were collected.
RESULTS: Of 936 patients with NSCLC, the RET fusion gene was exclusively detected in 13 patients (11 of 633 patients with adenocarcinomas and two of 24 patients with adenosquamous cell carcinomas). Of the 13 patients, nine patients had KIF5B-RET, three patients had CCDC6-RET, and one patient had a novel NCOA4-RET fusion. Patients with lung adenocarcinomas with RET fusion gene had more poorly differentiated tumors (63.6%; P = .029 for RET v ALK, P = .007 for RET v EGFR), with a tendency to be younger (≤ 60 years; 72.7%) and never-smokers (81.8%) and to have solid subtype (63.6%) and a smaller tumor (≤ 3 cm) with N2 disease (54.4%). The median relapse-free survival was 20.9 months.
CONCLUSION: RET fusion occurs in 1.4% of NSCLCs and 1.7% of lung adenocarcinomas and has identifiable clinicopathologic characteristics, warranting further clinical consideration and targeted therapy investigation.

Luise C, Merolla F, Leone V, et al.
Identification of sumoylation sites in CCDC6, the first identified RET partner gene in papillary thyroid carcinoma, uncovers a mode of regulating CCDC6 function on CREB1 transcriptional activity.
PLoS One. 2012; 7(11):e49298 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424) which are highly conserved in vertebrates. We demonstrate that the post-translational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.

Celestino R, Sigstad E, Løvf M, et al.
Survey of 548 oncogenic fusion transcripts in thyroid tumors supports the importance of the already established thyroid fusions genes.
Genes Chromosomes Cancer. 2012; 51(12):1154-64 [PubMed] Related Publications
Neoplasms frequently present structural chromosomal aberrations that can alter the level of expression of a protein or to the expression of an aberrant chimeric protein. In the thyroid, the PAX8-PPARG fusion is present in the neoplastic lesions that have a follicular architecture-follicular thyroid carcinoma (FTC) and follicular variant of papillary thyroid carcinoma (FVPTC), and less frequently in follicular thyroid adenoma (FTA), while the presence of RET/PTC fusions are largely restricted to papillary thyroid carcinoma (PTC). The ability to detect fusion genes is relevant for a correct diagnosis and for therapy. We have developed a new fusion gene microarray-based approach for simultaneous analysis of all known and predicted fusion gene variants. We did a comprehensive screen for 548 known and putative fusion genes in 27 samples of thyroid tumors and three positive controls-one thyroid cancer cell line (TPC-1) and two PTCs with known CCDC6-RET (alias RET/PTC1) fusion gene, using this microarray. Within the thyroid tumors tested, only well known, previously reported fusion genes in thyroid oncology were identified. Our results reinforce the pathogenic role played by RET/PTC1, RET/PTC3, and PAX8-PPARG fusion genes in thyroid tumorigenesis.

Cantara S, D'Angeli F, Toti P, et al.
Expression of the ring ligase PRAJA2 in thyroid cancer.
J Clin Endocrinol Metab. 2012; 97(11):4253-9 [PubMed] Related Publications
INTRODUCTION: In thyroid cells, binding of TSH to its receptor increases cAMP levels, sustaining thyrocytes growth and hormone production. The main cAMP effector enzyme is protein kinase A (PKA). Praja2 is a widely expressed RING (Really Interesting New Gene) ligase, which degrades the regulatory subunits of PKA, thus controlling the strength and duration of PKA signaling in response to cAMP. Differentiated thyroid cancer expresses a functional TSH receptor, and its growth and progression are positively regulated by TSH and cAMP signaling.
AIM: We aimed to analyze the expression of praja2 in a group of 36 papillary thyroid cancer (PTC), 14 benign nodules, and six anaplastic thyroid cancers (ATC).
METHODS: We measured praja2 mRNA levels by quantitative RT-PCR and praja2 expression by Western blot and immunohistochemistry. Possible association between praja2 mRNA and the presence of known mutations was evaluated.
RESULTS: We found a statistical significant increase of mRNA levels in PTC tissue samples, compared with benign nodules and ATC. In particular, mRNA levels were maximal in differentiated thyroid cancer (PTC), progressively decreasing in more aggressive tumors, ATC having the lowest amount of praja2 mRNA. Accordingly, higher levels of praja2 protein were detected in lysates from PTC, compared with ATC. By immunohistochemistry, in PTC sections we observed a marked increase of cytoplasmic praja2 signal, which significantly decreased in less differentiated thyroid tumors, completely disappearing in ATC. Studies in cultured cells stably expressing RET/PTC1 oncogene or mutant BRAF revealed a direct correlation between praja2 mRNA levels and malignant phenotype of transformed cells. Similar results were obtained using thyroid cancer tissues carrying the same mutations.
CONCLUSIONS: praja2 is markedly overexpressed in differentiated thyroid cancer, and its levels inversely correlate with the malignant phenotype of the tumor. Thus, praja2 is a novel cancer-related gene whose expression is linked to the histotype and mutational status of the thyroid tumor.

Gandhi M, Evdokimova V, Nikiforov YE
Frequency of close positioning of chromosomal loci detected by FRET correlates with their participation in carcinogenic rearrangements in human cells.
Genes Chromosomes Cancer. 2012; 51(11):1037-44 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
It has been well established that genes participating in oncogenic rearrangements are non-randomly positioned and frequently close to each other in human cell nuclei. However, the actual distance between these fusion partners has never been determined. The phenomenon of fluorescence resonance energy transfer (FRET) is observed when a donor fluorophore is close (<10 nm) to transfer some of it energy to an acceptor fluorophore. The aim of this study was to validate the use of FRET on directly labeled DNA molecules to assess the frequency of positioning at <10 nm distances between genes known to be involved in rearrangement and to correlate it with their probability to undergo rearrangement. In the validation experiments, the frequency of FRET-sensitized emission (SE) was found to be 93-96% between probes for the immediately adjacent chromosomal regions as compared to 0.1-0.2% between probes for the random loci located on large linear separation. Further, we found that the frequency of FRET-SE between four pairs of genes that form rearrangements in thyroid cancer was 5% for RET and CCDC6, 4% for RET and NCOA4, 2% for BRAF and AKAP9, and 2% for NTRK1 and TPR. Moreover, the frequency with which FRET was observed showed strong correlation (r = 0.9871) with the prevalence of respective rearrangements in thyroid cancer. Our findings demonstrate that FRET can be used as a technique to analyze proximity between specific DNA regions and that the frequency of gene positioning at distances allowing FRET correlates with their probability to undergo chromosomal rearrangements.

Merolla F, Luise C, Muller MT, et al.
Loss of CCDC6, the first identified RET partner gene, affects pH2AX S139 levels and accelerates mitotic entry upon DNA damage.
PLoS One. 2012; 7(5):e36177 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
CCDC6 was originally identified in chimeric genes caused by chromosomal translocation involving the RET proto-oncogene in some thryoid tumors mostly upon ionizing radiation exposure. Recognised as a pro-apoptotic phosphoprotein that negatively regulates CREB1-dependent transcription, CCDC6 is an ATM substrate that is responsive to genotoxic stress. Here we report that following genotoxic stress, loss or inactivation of CCDC6 in cancers that carry the CCDC6 fusion, accelerates the dephosphorylation of pH2AX S139, resulting in defective G2 arrest and premature mitotic entry. Moreover, we show that CCDC6 depleted cells appear to repair DNA damaged in a shorter time compared to controls, based on reporter assays in cells. High-troughput proteomic screening predicted the interaction between the CCDC6 gene product and the catalytic subunit of Serin-Threonin Protein Phosphatase 4 (PP4c) recently identified as the evolutionarily conserved pH2AX S139 phosphatase that is activated upon DNA Damage. We describe the interaction between CCDC6 and PP4c and we report the modulation of PP4c enzymatic activity in CCDC6 depleted cells. We discuss the functional significance of CCDC6-PP4c interactions and hypothesize that CCDC6 may act in the DNA Damage Response by negatively modulating PP4c activity. Overall, our data suggest that in primary tumours the loss of CCDC6 function could influence genome stability and thereby contribute to carcinogenesis.

Zunich SM, Valdovinos M, Douglas T, et al.
Osteoblast-secreted collagen upregulates paracrine Sonic hedgehog signaling by prostate cancer cells and enhances osteoblast differentiation.
Mol Cancer. 2012; 11:30 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
BACKGROUND: Induction of osteoblast differentiation by paracrine Sonic hedgehog (Shh) signaling may be a mechanism through which Shh-expressing prostate cancer cells initiate changes in the bone microenvironment and promote metastases. A hallmark of osteoblast differentiation is the formation of matrix whose predominant protein is type 1 collagen. We investigated the formation of a collagen matrix by osteoblasts cultured with prostate cancer cells, and its effects on interactions between prostate cancer cells and osteoblasts.
RESULTS: In the presence of exogenous ascorbic acid (AA), a co-factor in collagen synthesis, mouse MC3T3 pre-osteoblasts in mixed cultures with human LNCaP prostate cancer cells or LNCaP cells modified to overexpress Shh (LNShh cells) formed collagen matrix with distinct fibril ultrastructural characteristics. AA increased the activity of alkaline phosphatase and the expression of the alkaline phosphatase gene Akp2, markers of osteoblast differentiation, in MC3T3 pre-osteoblasts cultured with LNCaP or LNShh cells. However, the AA-stimulated increase in Akp2 expression in MC3T3 pre-osteoblasts cultured with LNShh cells far exceeded the levels observed in MC3T3 cells cultured with either LNCaP cells with AA or LNShh cells without AA. Therefore, AA and Shh exert a synergistic effect on osteoblast differentiation. We determined whether the effect of AA on LNShh cell-induced osteoblast differentiation was mediated by Shh signaling. AA increased the expression of Gli1 and Ptc1, target genes of the Shh pathway, in MC3T3 pre-osteoblasts cultured with LNShh cells to at least twice their levels without AA. The ability of AA to upregulate Shh signaling and enhance alkaline phosphatase activity was blocked in MC3T3 cells that expressed a dominant negative form of the transcription factor GLI1. The AA-stimulated increase in Shh signaling and Shh-induced osteoblast differentiation was also inhibited by the specific collagen synthesis inhibitor 3,4-dehydro-L-proline.
CONCLUSIONS: Matrix collagen, formed by osteoblasts in the presence of AA, potentiates Shh signaling between Shh-expressing prostate cancer cells and osteoblasts. Collagen and Shh signaling exert a synergistic effect on osteoblast differentiation, a defining event in prostate carcinoma bone metastasis. Investigations into paracrine interactions among prostate cancer cells, osteoblasts, and osteoblast-synthesized matrix proteins advance our understanding of mechanisms contributing to prostate cancer bone metastasis.

Dinets A, Hulchiy M, Sofiadis A, et al.
Clinical, genetic, and immunohistochemical characterization of 70 Ukrainian adult cases with post-Chornobyl papillary thyroid carcinoma.
Eur J Endocrinol. 2012; 166(6):1049-60 [PubMed] Article available free on PMC after 01/06/2015 Related Publications
BACKGROUND: Increased incidence of papillary thyroid carcinoma (PTC) is observed as a consequence of radiation exposure in connection to the Chornobyl nuclear plant accident in 1986. In this study, we report a cohort of adult Ukrainian patients diagnosed with PTC from 2004 to 2008 following exposure at the age of 18 years or younger.
METHODS: In total, 70 patients were identified and clinically characterized. The common BRAF 1799T>A mutation was assessed by pyrosequencing, the RET/PTC1 and RET/PTC3 (NCOA4) rearrangements by RT-PCR, and the expression of Ki-67 (MIB-1 index), BCL2, cyclin A, and cyclin D1 by immunohistochemistry.
RESULTS: In total, 46/70 (66%) cases carried a BRAF mutation and/or a RET/PTC rearrangement. A BRAF mutation was detected in 26 tumors, RET/PTC1 in 20 cases, and RET/PTC3 in four cases. In four of these cases, BRAF mutation and RET/PTC rearrangement were coexisting. The BRAF mutation was underrepresented among PTCs with accompanying chronic lymphocytic thyroiditis (CLT) compared with PTCs without this feature (12 vs 44%). MIB-1 proliferation index determined by double staining with leukocyte common antigen was low (mean 0.8%; range 0.05-4.5%). Moreover, increased expression of cyclin A was observed in PTCs with a tumor size >2 cm compared with PTCs ≤2 cm (1.2 vs 0.6%). BCL2 and cyclin D1 showed frequent expression but without associations to clinical characteristics or amplification of the CCND1 locus.
CONCLUSIONS: Our results suggest that this cohort has frequent BRAF mutation, RET/PTC1 rearrangement, and low proliferation index. Furthermore, BRAF 1799T>A was underrepresented in PTCs with CLT, and cyclin A expression was associated with increased PTC tumor size.

Piscazzi A, Costantino E, Maddalena F, et al.
Activation of the RAS/RAF/ERK signaling pathway contributes to resistance to sunitinib in thyroid carcinoma cell lines.
J Clin Endocrinol Metab. 2012; 97(6):E898-906 [PubMed] Related Publications
CONTEXT: Sunitinib is currently being evaluated in advanced human thyroid carcinomas, based on the rationale that the vascular endothelial growth factor and platelet-derived growth factor receptors and the RET/PTC rearrangement are valuable targets for the treatment of this malignancy. However, criteria for selecting thyroid tumors that may benefit from sunitinib are lacking.
DESIGN: The effect of activating somatic mutations in the KRAS and BRAF genes on the responsiveness to sunitinib was evaluated in a panel of thyroid cancer cell lines harboring wild-type KRAS and BRAF genes, the RET/PTC1 rearrangement, the G12R KRAS, or the V600E BRAF mutation.
RESULTS: Sunitinib was found to selectively inhibit cell proliferation, induce cell accumulation in the G0-G1 phase, and inhibit the phosphorylation of ERK1/2 in both KRAS/BRAF wild-type thyroid cancer cells and in tumor cells harboring the RET/PTC rearrangement, whereas it was completely ineffective in KRAS- or BRAF-mutated thyroid carcinoma cells. This differential antitumor activity of sunitinib did not correlate with the expression profile of the vascular endothelial growth factor receptors 1, 2, and 3, platelet-derived growth factor receptor-α and cKIT genes. Of note, the constitutive activation of RAS/RAF/ERK signaling in KRAS/BRAF wild-type cells by transfection of the R12 HRAS or V600E BRAF mutants or stimulation with epithelial growth factor resulted in the loss of responsiveness to sunitinib, whereas pharmacological inhibition of MAPK kinase activity resulted in the resensitization of KRAS- or BRAF-mutated cells to the multikinase inhibitor.
CONCLUSIONS: The constitutive activation of the RAS/RAF/ERK pathway may favor resistance to sunitinib in thyroid carcinoma cells.

Gupta S, Ajise O, Dultz L, et al.
Follicular variant of papillary thyroid cancer: encapsulated, nonencapsulated, and diffuse: distinct biologic and clinical entities.
Arch Otolaryngol Head Neck Surg. 2012; 138(3):227-33 [PubMed] Related Publications
OBJECTIVE: To examine genotypic and clinical differences between encapsulated, nonencapsulated, and diffuse follicular variant of papillary thyroid carcinoma (EFVPTC, NFVPTC, and diffuse FVPTC, respectively), to characterize the entities and identify predictors of their behavior.
DESIGN: Retrospective medical chart review and molecular analysis.
SETTING: Referral center of a university hospital.
PATIENTS: The pathologic characteristics of 484 consecutive patients with differentiated thyroid cancer who underwent surgery by the 3 members of the New York University Endocrine Surgery Associates from January 1, 2007, to August 1, 2010, were reviewed. Forty-five patients with FVPTC and in whom at least 1 central compartment lymph node was removed were included.
MAIN OUTCOME MEASURES: Patients with FVPTC were compared in terms of age, sex, tumor size, encapsulation, extrathyroid extension, vascular invasion, central nodal metastases, and the presence or absence of mutations in BRAF, H-RAS 12/13, K-RAS 12/13, N-RAS 12/13, H-RAS 61, K-RAS 61, N-RAS 61, and RET/PTC1.
RESULTS: No patient with EFVPTC had central lymph node metastasis, and in this group, 1 patient (4.5%) had a BRAF V600E mutation and 2 patients (9%) had RAS mutations. Of the patients with NFVPTC, none had central lymph node metastasis (P > .99) and 2 (11%) had a BRAF V600E mutation (P = .59). Of the patients with diffuse FVPTC, all had central lymph node metastasis (P < .001), and 2 (50%) had a BRAF V600E mutation (P = .06).
CONCLUSIONS: FVPTC consists of several distinct subtypes. Diffuse FVPTC seems to present and behave in a more aggressive fashion. It has a higher rate of central nodal metastasis and BRAF V600E mutation in comparison with EFVPTC and NFVPTC. Both EFVPTC and NFVPTC behave in a similar fashion. The diffuse infiltrative pattern and not just presence or absence of encapsulation seems to determine the tumor phenotype. Understanding the different subtypes of FVPTC will help guide appropriate treatment strategies.

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