BACKGROUND: Somatic mutation of the BRAF gene is one to be the most commonly known genetic change in thyroid tumors especially papillary thyroid cancers. The T1799A activating point mutation is detected in >98% of the thyroid tumors, and result in substitution of amino acid valine at position 600 to glutamic acid. METHOD: In this study, we evaluated BRAF mutation in 95 Indian thyroid tumors by pyrosequencing assay. RESULTS: Overall, 36 cases (38%) showed presence of BRAF V600E mutation, while none of the cases showed V600K mutation. BRAF mutation was found predominantly in female patients in comparison to males (38.4% vs. 36.4%, p=.86). Likewise, smaller sized tumors (

Melanoma is significantly associated with mutant BRAF gene, a suitable target for siRNA-based anti-melanoma therapy. However, a tumor-specific delivery system is a major hurdle for clinical applications. Here, we developed a novel nano-carrier, FA-GNR-siBRAF for safe topical application, which consists of folic acid (FA) as the tumor-targeting moiety, golden nanorods (GNR) providing photothermal capability to kill tumor cells under laser irradiation, and siRNA specifically silencing BRAF (siBRAF). The in vitro and in vivo results revealed that FA-GNR-siBRAF displayed high transfection rates, and subsequently induced remarkable gene knockdown of BRAF, resulting in suppression of melanoma growth due to the interruption of the MEK/ERK pathway. Combinatorial photothermal effects and BRAF knockdown by FA-GNR-siBRAF effectively killed tumor cells through apoptosis, with enhanced efficiency than individual treatments. Therefore, the FA-GNR-siBRAF simultaneously induced BRAF gene silencing and photothermal effects which achieved synergistic efficacy in the treatment of melanoma, paving a new path for developing clinical treatment methods for melanoma.

MNS16A, a functional polymorphic tandem repeat minisatellite, is located in the promoter region of an antisense transcript of the human telomerase reverse transcriptase gene. MNS16A promoter activity depends on the variable number of tandem repeats (VNTR) presenting varying numbers of transcription factor binding sites for GATA binding protein 1. Although MNS16A has been investigated in multiple cancer epidemiology studies with incongruent findings, functional data of only two VNTRs (VNTR-243 and VNTR-302) were available thus far, linking the shorter VNTR to higher promoter activity.For the first time, we investigated promoter activity of all six VNTRs of MNS16A in cell lines of colorectal, lung and prostate cancer using Luciferase reporter assay. In all investigated cell lines shorter VNTRs showed higher promoter activity. While this anticipated indirect linear relationship was affirmed for colorectal cancer SW480 (P = 0.006), a piecewise linear regression model provided significantly better model fit in lung cancer A-427 (P = 6.9 x 10-9) and prostate cancer LNCaP (P = 0.039). In silico search for transcription factor binding sites in MNS16A core repeat element suggested a higher degree of complexity involving X-box binding protein 1, general transcription factor II-I, and glucocorticoid receptor alpha in addition to GATA binding protein 1.Further functional studies in additional cancers are requested to extend our knowledge of MNS16A functionality uncovering potential cancer type-specific differences. Risk alleles may vary in different malignancies and their determination in vitro could be relevant for interpretation of genotype data.

Emergence of resistance, unavoidable systemic toxicity and unsatisfactory efficacy arethe main obstacles for traditional cancer therapy. Combination with phosphorothioate modified antisense oligonucleotides (PSASODN) against human telomerase reverse transcriptase (hTERT) may enhance the therapeutic effect of irradiation. However, the effect of PSASODN against hTERT on the antitumor effects of irradiation in liver cancer remain unclear. In the current study, Walker 256 cells were transfected with hTERT PSASODN. Cell proliferation and cell viability were measured using the MTT assay and cell senescence was examined by SAbetagal staining. Telomerase activity was determined by telomeric repeat amplification protocolpolymerase chain reactionELISA. Cell apoptosis was assayed by flow cytometry and DNA damage was determined by the comet assay.The PSASODN was demonstrated to have an inhibitory effect on cell proliferation and accelerated effect on cell senescence by inhibiting telomerase activity. PSASODN promoted the irradiationinduced inhibition of cell viability and telomerase activity, and irradiationinduced DNA damage and cell apoptosis via the activation of apoptosisassociated proteins. Taken together, these results indicated that combined treatment of PSASODN with irradiation significantly enhanced tumor inhibition. Therefore, PSASODN provides an experimental foundation for gene therapy and is proposed for application in clinical treatment of liver cancer combined with radiotherapy.

High rates of telomerase reverse transcriptase (TERT) promoter mutations have recently been described in urothelial carcinoma (UC). Unlike UC in the bladder, adenocarcinomas account for the majority of urachal cancer (UrC) cases. As data in UrC is unclear, we analyzed TERT promoter mutations in a large cohort of UrC for its differential diagnostic, clinicopathological and prognostic significance. UrC cases from six academic centers were analyzed for c.-146C>T (C250T) and c.-124C>T (C228T) TERT promoter mutations by PCR and Sanger sequencing. Clinicopathological and survival data were collected. The cohort consisted of 15 men (56%) and 12 women (44%) with a median age of 50 years including 23 adenocarcinomas, two squamous cell carcinomas (SCC), one UC and one undifferentiated carcinoma. In one case of (mucinous) urachal adenocarcinoma a C228T mutation was detected (1/23; 4%), like in a case of SCC in addition to one C250T mutation in the UC case. TERT promoter mutations are very rare in urachal adenocarcinomas (unlike in UC) with differential diagnostic implications. Additionally, the low TERT promoter mutation rate in urachal adenocarcinomas is more comparable to colorectal adenocarcinomas than to UC, giving further support to recent genetic findings and therapeutic considerations.

Importance: BRAF V600E and TERT promoter mutations can coexist in papillary thyroid cancer (PTC). This genetic duet was indicated to be involved in the aggressiveness of PTC, but its prognostic value in PTC-related mortality remains to be specifically established. Objective: To establish the prognostic power of this genetic duet in PTC-specific mortality. Design, Setting, and Participants: This genetic-clinical correlation study examined BRAF V600E and TERT promoter mutations (chr5:1,295,228C>T and chr5:1,295,250C>T) and PTC-specific mortality in 1051 patients (764 women and 287 men) with a median (interquartile range [IQR]) age of 46 (36-57) years, with a median (IQR) follow-up time of 89 (48-142) months (7.4 years). Main Outcomes and Measures: BRAF V600E and TERT promoter mutation patterns and associated patient deaths caused by PTC. Results: Papillary thyroid cancer-specific mortality occurred in 4 of 629 patients (0.6%) with neither mutation; 7 of 292 (2.4%) with BRAF V600E alone; 4 of 64 (6.3%) with TERT promoter mutation alone; and 15 of 66 (22.7%) with the genetic duet; and deaths per 1000-person years in patients harboring neither mutation, BRAF V600E alone, TERT mutation alone, or both mutations were 0.80 (95% CI, 0.30-2.13), 3.08 (95% CI, 1.47-6.46), 6.62 (95% CI, 2.48-17.64), and 29.86 (95% CI, 18.00-49.52), respectively. Compared with patients harboring neither mutation, HRs (95% CIs) for PTC-specific mortality were 3.08 (0.87-10.84) for BRAF V600E alone; 8.18 (2.04-32.75) with TERT mutation alone; and 37.77 (12.50-114.09) with both mutations. Papillary thyroid cancer-specific mortality for cases with both mutations remained significant (HR, 9.34; 95% CI, 2.53-34.48) after adjustment for clinicopathological factors, and the genetic duet showed a strong incremental and synergistic impact over either mutation alone. Kaplan-Meier analyses revealed a flat PTC-specific patient survival curve with neither mutation, a modest decline in the curve with either mutation alone, and a sharp decline in the curve with coexisting mutations. Even more robust mortality associations of the genetic duet were seen when only conventional-variant PTC (CPTC) was analyzed (HR, 54.46; 95% CI, 12.26-241.82), which remained strongly significant (HR, 18.56; 95% CI, 2.97-116.18) after adjustment for clinicopathological factors. Conclusions and Relevance: These results demonstrate a simple 4-genotype classification of PTC, particularly CPTC, with a disease-specific mortality risk order of the genetic duet>>>>BRAF V600E alone = TERT promoter mutation alone > wild-type for both genes, representing a powerful molecular prognostic system that can help pinpoint patients with the highest mortality risk.

Context: Little is known about the frequency of key mutations in thyroid cancer metastases and its relationship with the primary tumor genotype. Objectives: To evaluate the frequency of TERT promoter (TERTp), BRAF, and NRAS mutations in metastatic thyroid carcinomas, analyzing primary thyroid tumors, lymph node metastases (LNMs), and distant metastases. Design and Patients: Mutation analysis was performed in 437 tissue samples from 204 patients, mainly with papillary thyroid carcinomas (PTCs; n = 180), including 196 LNMs and 56 distant metastases. All the distant metastases included corresponded to radioiodine-refractory metastatic tissue. Results: We found the following mutation frequency in primary PTCs, LNMs, and distant metastases, respectively: TERTp: 12.9%, 10.5%, and 52.4%; BRAF: 44.6%, 41.7%, and 23.8%; and NRAS: 1.2%, 1.3%, and 14.3%. There was a significant concordance between the primary tumor genotype and the corresponding LNM for all the genes, in particular BRAF-mutated PTC. The overall concordance between primary tumors and respective distant metastases was low. In the group of patients with PTCs, we found a high frequency of TERTp mutations and a low frequency of BRAF mutations in distant metastases, in comparison with the paired primary tumors. When present in distant metastases, BRAF mutations frequently coexisted with TERTp mutations. Conclusions: When the genotype of primary tumors is compared with the genotype of LNMs, the concordance is high for all the genes studied. On the other hand, distant metastases show an enrichment in TERTp mutations and a decrease in BRAF mutations. TERTp mutations may play a role in distant metastases.

Synergistic effects of BRAFV600E and TERT promoter mutations on the poor clinical outcomes in papillary thyroid cancer (PTC) have been demonstrated. The potential mechanism of this phenomenon has been proposed: MAPK pathway activation by the BRAFV600E mutation may upregulate E-twenty six (ETS) transcription factors, increasing TERT expression by binding to the ETS-binding site generated by the TERT promoter mutation; however, it has not yet been fully proven. This article provides transcriptomic insights into the interaction between BRAFV600E and TERT promoter mutations mediated by ETS factors in PTC. RNA sequencing data on 266 PTCs from The Cancer Genome Atlas and 65 PTCs from our institute were analyzed for gene expression changes and related molecular pathways, and the results of transcriptomic analyses were validated by in vitro experiments. TERT mRNA expression was increased by the coexistence of BRAFV600E and TERT promoter mutations (fold change, 16.17; q-value = 7.35 x 10-12 vs no mutation). In the ETS family of transcription factors, ETV1, ETV4 and ETV5 were upregulated by the BRAFV600E/MAPK pathway activation. These BRAFV600E-induced ETS factors selectively bound to the mutant TERT promoter. The molecular pathways activated by BRAFV600E were further augmented by adding the TERT promoter mutation, and the pathways related to immune responses or adhesion molecules were upregulated by TERT expression. The mechanism of the synergistic effect between BRAFV600E and TERT promoter mutations on cancer invasiveness and progression in PTC may be explained by increased TERT expression, which may result from the BRAF-induced upregulation of several ETS transcription factors.

BACKGROUND/AIMS: Ovarian cancer is often diagnosed at later stages with poor prognosis. Recent studies have associated the expression of deubiquitylase USP7 with the survival of ovarian cancers. Being a cysteine protease, USP7 could become a target for pharmacological intervention. Therefore, in this study, we assessed the influence of its inhibitor P5091 on ovarian cancer cells. METHODS: Ovarian cancer cells were treated with P5091, and cell proliferation was measured with MTT assay; cell morphology was inspected under a phase-contrast microscope; cell cycle and cell death were examined by flow cytometry. To gain mechanistic insights into its effects, immunoblotting was performed to detect USP7, HDM2, p53, p21, apoptosis and autophagy related proteins. RESULTS: P5091 effectively suppressed the growth of ovarian cancer cells, caused cell cycle blockage, and induced necrosis and apoptosis with more severe phenotypes observed in HeyA8 cells with wild-type p53 than in OVCAR-8 cells with mutant p53. P5091 also prompted autophagy, with more efficient p62 degradation in HeyA8. CONCLUSION: P5091 shows efficacy in suppressing ovarian cancers harbouring wild-type and mutant p53. Its effects seemed to be enhanced by wild-type p53. The potency of this USP7 inhibitor also correlated with autophagy to some extent. Therefore, the pharmacological targeting of USP7 may serve as a potential therapeutic strategy and warrants further investigation.

The Hippo pathway is an evolutionarily conserved signaling pathway that plays important roles in stem cell biology, tissue homeostasis, and cancer development. Vestigial-like 4 (Vgll4) functions as a transcriptional co-repressor in the Hippo-Yes-associated protein (YAP) pathway. Vgll4 inhibits cell proliferation and tumor growth by competing with YAP for binding to TEA-domain proteins (TEADs). However, the mechanisms by which Vgll4 itself is regulated are unclear. Here we identified a mechanism that regulates Vgll4's tumor-suppressing function. We found that Vgll4 is phosphorylated in vitro and in vivo by cyclin-dependent kinase 1 (CDK1) during antimitotic drug-induced mitotic arrest and also in normal mitosis. We further identified Ser-58, Ser-155, Thr-159, and Ser-280 as the main mitotic phosphorylation sites in Vgll4. We also noted that the nonphosphorylatable mutant Vgll4-4A (S58A/S155A/T159A/S280A) suppressed tumorigenesis in pancreatic cancer cells in vitro and in vivo to a greater extent than did wild-type Vgll4, suggesting that mitotic phosphorylation inhibits Vgll4's tumor-suppressive activity. Consistent with these observations, the Vgll4-4A mutant possessed higher-binding affinity to TEAD1 than wild-type Vgll4. Interestingly, Vgll4 and Vgll4-4A markedly suppressed YAP and beta-catenin signaling activity. Together, these findings reveal a previously unrecognized mechanism for Vgll4 regulation in mitosis and its role in tumorigenesis.

The receptor tyrosine kinase rearranged during transfection (RET) is an oncogenic driver activated in multiple cancers, including non-small cell lung cancer (NSCLC), medullary thyroid cancer (MTC), and papillary thyroid cancer. No approved therapies have been designed to target RET; treatment has been limited to multikinase inhibitors (MKI), which can have significant off-target toxicities and limited efficacy. BLU-667 is a highly potent and selective RET inhibitor designed to overcome these limitations. In vitro, BLU-667 demonstrated >/=10-fold increased potency over approved MKIs against oncogenic RET variants and resistance mutants. In vivo, BLU-667 potently inhibited growth of NSCLC and thyroid cancer xenografts driven by various RET mutations and fusions without inhibiting VEGFR2. In first-in-human testing, BLU-667 significantly inhibited RET signaling and induced durable clinical responses in patients with RET-altered NSCLC and MTC without notable off-target toxicity, providing clinical validation for selective RET targeting.Significance: Patients with RET-driven cancers derive limited benefit from available MKIs. BLU-667 is a potent and selective RET inhibitor that induces tumor regression in cancer models with RET mutations and fusions. BLU-667 attenuated RET signaling and produced durable clinical responses in patients with RET-altered tumors, clinically validating selective RET targeting. Cancer Discov; 8(7); 836-49. (c)2018 AACR.See related commentary by Iams and Lovly, p. 797This article is highlighted in the In This Issue feature, p. 781.

KRAS is one of the most mutated genes in human cancer. It is controlled by a G4 motif located upstream of the transcription start site. In this paper, we demonstrate that 8-oxoguanine (8-oxoG), being more abundant in G4 than in non-G4 regions, is a new player in the regulation of this oncogene. We designed oligonucleotides mimicking the KRAS G4-motif and found that 8-oxoG impacts folding and stability of the G-quadruplex. Dimethylsulphate-footprinting showed that the G-run carrying 8-oxoG is excluded from the G-tetrads and replaced by a redundant G-run in the KRAS G4-motif. Chromatin immunoprecipitation revealed that the base-excision repair protein OGG1 is recruited to the KRAS promoter when the level of 8-oxoG in the G4 region is raised by H2O2. Polyacrylamide gel electrophoresis evidenced that OGG1 removes 8-oxoG from the G4-motif in duplex, but when folded it binds to the G-quadruplex in a non-productive way. We also found that 8-oxoG enhances the recruitment to the KRAS promoter of MAZ and hnRNP A1, two nuclear factors essential for transcription. All this suggests that 8-oxoG in the promoter G4 region could have an epigenetic potential for the control of gene expression.

Metastasis represents an end stage in the evolution of cancer progression and has been related to specific genetic pathways. Overexpression of mutant RAS in particular appears to promote invasion and metastasis, although exactly how this occurs has not been well characterized. It was previously showed that activation of the WASF3 protein regulates actin cytoskeleton dynamics that promote invasion. In this report, how WASF3 overexpression interacts with mutant RAS to increase invasion and metastasis was investigated. The ability of RAS to promote invasion and metastasis was shown to be dependent on WASF3 activation in a PI3K and AKT dependent manner. Proteomics analysis demonstrates the presence of AKT in the WASF3 immunocomplex which is enhanced by overexpression of mutant RAS. During these processes activation of ERK1/2 is not affected by loss of WASF3 expression. Analysis of the relative involvement of p85 and p110 in the WASF3 complex demonstrates that mutant RAS promotes dissociation of p85 promoting activation of p110. These studies provide a deeper understanding of the critical role for WASF3 in facilitating increased invasion potential in cancer cells expressing mutant RAS and supports the idea that targeting WASF3 in metastatic cells overexpressing RAS may be used to suppress invasion and metastasis.

Purpose Sorafenib and lenvatinib are oral multikinase inhibitors targeting vascular endothelial growth factor receptor (VEGFR) and approved for radioiodine (RAI)-refractory differentiated thyroid cancer (DTC). However, there are no approved second- or third-line therapies. MET is implicated in resistance to VEGFR inhibitors. Cabozantinib is an oral multikinase inhibitor targeting MET in addition to VEGFR and is approved for medullary thyroid cancer. In a phase I study of cabozantinib, five of eight patients with DTC previously treated with a VEGFR-targeted therapy had an objective response to cabozantinib. Patients and Methods Patients with RAI-refractory disease with Response Evaluation Criteria in Solid Tumor (RECIST) measurable disease and evidence of progression on prior VEGFR-targeted therapy were enrolled in this single-arm phase II study. The cabozantinib starting dose was 60 mg/day orally but could be escalated to 80 mg if the patient did not experience a response. Patients underwent tumor assessment according to RECIST v1.1 every 8 weeks. In this study, if at least five of 25 response-evaluable patients had an objective response, cabozantinib would be considered a promising agent in this patient population. Results Twenty-five patients were enrolled. The median age was 64 years, and 64% of patients were men. Twenty-one patients had received only one prior VEGFR-targeted therapy (sorafenib, pazopanib, or cediranib), and four patients had received two such therapies. The most common treatment-related adverse events were fatigue, weight loss, diarrhea, palmar-plantar erythrodysesthesia, and hypertension. One drug-related death was noted. Of the 25 patients, 10 (40%) had a partial response, 13 (52%) had stable disease, and two (8%) had nonevaluable disease. The median progression-free survival and overall survival were 12.7 months and 34.7 months, respectively. Conclusion Cabozantinib demonstrated clinically significant, durable objective response activity in patients with RAI-refractory DTC who experienced disease progression while taking prior VEGFR-targeted therapy.

BACKGROUND: Receptor tyrosine kinases (RTKs) play crucial roles in numerous cancer cell processes including cell survival, proliferation, and migration. MEK1/2 MAPK kinases are very important for cancer survival and development. Anaplastic thyroid carcinoma (ATC) is a deadly type of thyroid cancer and there are no very effective systemic treatment strategies for ATC so far. Also, ATC can easily become resistant to therapy of traditional therapeutic drugs for ATC, such as doxorubicin. Drug combination treatment could be a promising therapeutic strategy for ATC, especially for drug resistant ATC. METHODS: We explored the combination effect between a MEK1/2 inhibitor SL327 and a multi-targeted RTK inhibitor Sunitinib Malate in doxorubicin resistant ATC cells using cell viability assay, cell migration assay, nuclei morphology and caspase-3 activity analysis, as well as in vivo tumor growth assay. RESULTS: There is a significant additive effect between SL327 and Sunitinib Malate in reducing viability, increasing apoptosis, and suppressing migration of doxorubicin-resistant ATC cells. Importantly, combination of SL327 and Sunitinib Malate induced significant additive suppression of in vivo doxorubicin-resistant ATC tumor growth. CONCLUSIONS: Our results suggest that the combination of MEK1/2 inhibitor and RTK inhibitor is promising for treatment of ATC especially doxorubicin-resistant ATC. The combination might not only enhance the anti-cancer efficacy, but also reduce the side effects and overcome drug resistance developed in ATC treatment. All these might provide useful information for clinical therapeutics of ATC.

Metastasis to lymph nodes and distant organs is the main challenge in the treatment of papillary thyroid cancer. In the current investigation, we aimed to evaluate the synergistic effects of celecoxib (CX) and sodium valproate (VPA) against cell survival, invasiveness properties, and expression of metalloproteinase-2 and -9 (MMP-2 and MMP-9) in papillary thyroid cancer cell line, B-CPAP cells. The effect of CX and VPA on B-CPAP cells viability and apoptosis were investigated using MTT assay and annexin V/7-AAD flowcytometry, respectively. The effects of the drugs on invasiveness properties of B-CPAP cells and expression of MMP-2 and MMP-9 were evaluated using transwell assay and real time PCR, respectively. MTT assay showed that CX and VPA decreased viability of B-CPAP cells dose dependently (IC50 32.4microM and 6.8 mM, respectively). Combination of CX (5 muM) and VPA (2.5 and 5 mM) increased apoptosis, and reduced cell migration and invasion of B-CPAP cell, synergistically. Real time PCR results showed that both CX (5 microM) and VPA (2.5 and 5 mM) reduced MMP-2 expression (P < 0.05="" but="" had="" no="" significant="" effects="" the="" expression="" of="" mmp-9.="" our="" findings="" suggest="" that="" cx="" and="" vpa="" synergistically="" increase="" apoptosis="" and="" suppress="" migration="" and="" invasion="" of="" b-cpap="" cells="" through="" inhibition="" of="" mmp-2="" expression.="">

Lung cancer is one of most common types of cancer worldwide. Lung cancer results in a death higher rate each year compared to colon, breast and prostate cancer combined. Celecoxib is a selective inhibitor of cyclooxygenase-2 (COX2), an enzyme of which the expression is induced by various stimuli, such as inflammation. In addition, celecoxib triggers COX-2 loading on exosomes. Exosomes are small vesicles composed of a lipid bilayer membrane and are found in most biological fluids, such as blood breast milk and urine. In this study, we focused on exosomes containing COX-2 proteins from lung cancer cells to determine their involvement in the interaction with neighbor cells following treatment with celecoxib. We found that celecoxib induced COX-2 expression in both the cytosol and exosomes in lung cancer cells. Exosomes from celecoxib-treated lung cancer cell culture supernatant were isolated and incubated with several types of cells. The THP-1, monocytic leukemia cell line effectively absorbed COX-2 by lung cancer cell-derived exosomes. Following incubation with exosomes, the COX-2 protein level was increased in the THP-1 cells; however, COX-2 mRNA expression was not affected. Moreover, prostaglandin E2 (PGE2) and vascular endothelial growth factor (VEGF) production by THP-1 cells was increased following incubation with exosomes from celecoxib-treated lung cancer cells. Conditioned medium from THP-1 following incubation with exosomes promoted formation in EA.hy926 cells. Taken together, our findings suggest that celecoxib induces COX-2 expression in lung cancer cells, and that highly expressed COX-2 in exosomes can be transferred to other cells.

Anaplastic thyroid carcinoma (ATC) although rare is the most deadly form of thyroid cancer. The fatality rate for ATC is high-pitched, the survival rate at 1 year after diagnosis is <20%. control="" of="" atc="" is="" severely="" hard="" and="" widespread="" with="" unpredictability.="" we="" previous="" proved="" that="" histone="" gene="" reviser="" and="" epigenetic="" changes="" role="" significant="" parts="" in="" papillary="" and="" anaplastic="" thyroid="" cancer="" tumorigenesis.="" herein="" the="" goal="" of="" this="" study="" was="" to="" investigate="" the="" anti-tumor="" activities="" of="" a="" hdac="" inhibitor="" hnha="" alone="" and="" in="" combination="" with="" sorafenib="" in="" atc="" cells="" in="" vitro="" and="" in="" vivo="" and="" to="" explore="" its="" effects="" apoptotic="" cell="" death="" pathways.="" three="" atc="" cell="" lines="" were="" exposed="" to="" sorafenib="" in="" the="" presence="" or="" absence="" of="" hnha="" and="" cell="" viability="" was="" determined="" by="" mtt="" assay.="" effects="" of="" combined="" treatment="" cell="" cycle="" and="" intracellular="" signaling="" pathways="" were="" assessed="" by="" flow="" cytometry="" and="" western="" blot="" analysis.="" the="" atc="" cell="" lines="" xenograft="" model="" was="" used="" to="" examine="" the="" anti-tumor="" activity="" in="" vivo.="" our="" data="" showed="" that="" hnha="" and="" sorafenib="" synergistically="" decreased="" cell="" viability="" in="" atc="" cells="" and="" also="" significantly="" increased="" apoptotic="" cell="" death="" in="" these="" cells="" as="" proved="" by="" the="" cleavage="" of="" caspase-3="" and="" dna="" fragmentation.="" hnha="" and="" sorafenib="" combination="" was="" reduced="" anti-apoptotic="" factor="" in="" atc.="" thus="" combination="" therapy="" with="" hnha="" and="" sorafenib="" significantly="" decreased="" vessel="" density="" and="" most="" significantly="" reduced="" tumor="" volume="" and="" increased="" survival="" in="" atc="" xenografts.="" these="" results="" propose="" that="" hnha="" in="" combination="" with="" sorafenib="" has="" significant="" anti-cancer="" activity="" in="" preclinical="" models="" potentially="" suggesting="" a="" new="" clinical="" approach="" for="" patients="" of="" advanced="" thyroid="" cancer="" type.="">