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Supplementary Figures S1-S2; Supplementary Table S1.

Purpose: This study was designed to explore the role of IQGAP1 in the invasiveness of thyroid cancer and its potential as a novel prognostic marker and therapeutic target in this cancer.Experimental Design: We examined IQGAP1 copy gain and its relationship with clinicopathologic outcomes of thyroid cancer and investigated its role in cell invasion and molecules involved in the process.Results: We found IQGAP1 copy number (CN) gain ≥3 in 1 of 30 (3%), 24 of 74 (32%), 44 of 107 (41%), 8 of 16 (50%), and 27 of 41 (66%) of benign thyroid tumor, follicular variant papillary thyroid cancer (FVPTC), follicular thyroid cancer (FTC), tall cell papillary thyroid cancer (PTC), and anaplastic thyroid cancer, respectively, in the increasing order of invasiveness of these tumors. A similar tumor distribution trend of CN ≥4 was also seen. IQGAP1 copy gain was positively correlated with IQGAP1 protein expression. It was significantly associated with extrathyroidal and vascular invasion of FVPTC and FTC and, remarkably, a 50%–60% rate of multifocality and recurrence of BRAF mutation–positive PTC (P = 0.01 and 0.02, respectively). The siRNA knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion and colony formation. Coimmunoprecipitation assay showed direct interaction of IQGAP1 with E-cadherin, a known invasion-suppressing molecule, which was upregulated when IQGAP1 was knocked down. This provided a mechanism for the invasive role of IQGAP1 in thyroid cancer. In contrast, IQGAP3 lacked all these functions.Conclusions: IQGAP1, through genetic copy gain, plays an important role in the invasiveness of thyroid cancer and may represent a novel prognostic marker and therapeutic target for this cancer. Clin Cancer Res; 16(24); 6009–18. ©2010 AACR.

Supplementary Figure 1 from Genetic Alterations in the Phosphoinositide 3-Kinase/Akt Signaling Pathway Confer Sensitivity of Thyroid Cancer Cells to Therapeutic Targeting of Akt and Mammalian Target of Rapamycin

Supplementary Figure Legend from Genetic Alterations in the Phosphoinositide 3-Kinase/Akt Signaling Pathway Confer Sensitivity of Thyroid Cancer Cells to Therapeutic Targeting of Akt and Mammalian Target of Rapamycin

Reliable biomarkers to predict the outcome and treatment response of estrogen receptor (ER)-negative breast cancer (BC) are urgently needed. Since immune-related signaling plays an important role in the tumorigenesis of ER-negative BC, we asked whether Notch genes, alone or in combination with other immune genes, can be used to predict the clinical outcome and immune checkpoint blockade (ICB) for this type of cancer.We analyzed transcriptome data of 6918 BC samples from five independent cohorts, 81 xenograft triple-negative BC tumors that respond differently to ICB treatment and 754 samples of different cancer types from patients treated with ICB agents.We found that among four Notch genes, the expression levels of NOTCH1 and NOTCH4 were positively associated with recurrence of ER-negative BC, and that combined expression of these two genes (named Notch14) further enhanced this association, which was comparable with that of the Notch pathway signature. Analysis of 1182 immune-related genes revealed that the expression levels of most HLA genes, particularly HLA-DMA and -DRA, were reversely associated with recurrence in ER-negative BC with low, but not high Notch14 expression. A combined expression signature of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA was more prognostic for ER-negative and triple-negative BCs than previously reported immune-related signatures. Furthermore, we found that the expression levels of these four genes were also synergistically associated with T cell exclusion score, infiltration of specific T cells and ICB efficacy in ER-negative BC, thereby providing a potential molecular mechanism for the synergistic effect of these genes on BC.Our data indicate that a gene signature composed of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA may serve as a potential promising biomarker for predicting ICB therapy efficacy and recurrence in ER-negative/triple-negative BCs.

Efficient predictive biomarkers are urgently needed to identify non-small cell lung cancer (NSCLC) patients who could benefit from immune checkpoint blockade (ICB) therapy. Since chromatin remodeling is required for DNA repair process, we asked whether mutations in chromatin remodeling genes could increase tumor mutational burden (TMB) and predict response to ICB therapy in NSCLC. Analysis of seven ICB-treated NSCLC cohorts revealed that mutations of three chromatin remodeling-related genes, including

Background Aberrant activation of anaplastic lymphoma kinase (ALK) signaling has been found to be involved in the tumorigenesis of multiple types of cancer. The aim of this study was to determine the role of this pathway in the pathogenesis of breast cancer. Methods An ALK pathway signature that we generated previously was used to compute the ALK pathway activity in 6381 breast cancer samples from 42 microarray datasets, and the associations between ALK pathway signature score and clinical variables were examined using logistic regression and survival analyses. Results Our results indicated that high ALK pathway activity was a significant risk factor for hormone receptor‐negative, high‐grade breast cancer in the 42 datasets. ALK pathway activity was positively associated with pathological complete response (pCR) in 15 datasets annotated with patient's neoadjuvant chemotherapy response information (overall odds ratio = 1.67, P 50 years old, with positive lymph nodes, or with residual disease after neoadjuvant chemotherapy. Conclusions ALK may be involved in breast cancer tumorigenesis, and ALK pathway signature score may serve as a prognostic biomarker for breast cancer.

The development of multi-gene signatures has led to improvements in identification of breast cancer patients at high risk of recurrence. The prognostic power of commercially available gene signatures is mostly restricted to estrogen receptor (ER)-positive breast cancer. On the contrary, immune-related gene signatures predict prognosis only in ER-negative breast cancer. This study aimed to develop a better prognostic signature for breast cancer. The expressions of long non-coding RNA (lncRNA) genes from 30 independent microarray datasets with a total of 4813 samples were analyzed. A prognostic lncRNA signature was developed based on likelihood-ratio Cox regression analysis. Survival analysis was used to compare the prognostic efficiencies of our signature and 10 previously reported prognostic gene signatures. Cox regression analysis on 30 independent datasets showed that the 6-lncRNA signature identified in this study performed as well as five commercially available signatures in recurrence prediction for ER-positive breast cancer. In ER-negative breast cancer, this lncRNA signature was as prognostic as three immune-related gene signatures. Moreover, our lncRNA signature also demonstrated a good capacity to predict recurrence risk for triple-negative breast cancer. Function analysis showed that several lncRNAs in this signature were probably involved in cell proliferation and immune processes. A six-LncRNA signature was identified that is prognostic for ER-positive, ER-negative, and triple-negative breast cancers and thus deserves further validation in prospective studies.

Recurrence is a major cause of colorectal cancer (CRC)-related death. As yet, the accurate identification of CRC patients at high risk of recurrence is still a major clinical challenge. Previously, we found that an estrogen receptor (ER) pathway gene signature may predict disease recurrence in CRC patients. The aim of this study is to evaluate the potential application of additional pathway-specific gene signatures in the prediction of CRC recurrence. The activities of 26 cancer-related pathways in CRC were semi-quantified using gene signature-based Bayesian binary regression analysis, and putative associations of the pathways with cancer recurrence risk were assessed using survival analysis. Among the 26 pathways tested, inactivation of the estrogen receptor (ER) pathway was found to be one of the most common events in CRC. Inactivation of this pathway was found to be frequently accompanied by over-activation of the BRAF/MEK pathway, and these two pathways were found to be associated with opposite effects on several clinicopathological CRC features, including microsatellite instability, subsite location, advanced stage and recurrence. Survival analysis of four independent CRC patient cohorts revealed that while the BRAF/MEK pathway was more strongly associated with recurrence than the ER pathway in mixed-stage CRCs, the ER pathway was a better predictor of recurrence than the BRAF/MEK pathway in stage II CRC. A combined use of these two pathways improved the prediction of CRC recurrence in both mixed stage CRC (n = 1122; overall HR: 2.518, 95% CI: 1.570–4.038, p

Efficient predictive biomarkers are urgently needed to identify non-small cell lung cancer (NSCLC) patients who could benefit from immune checkpoint blockade (ICB) therapy. Since chromatin remodeling is required for DNA repair process, we asked whether mutations in chromatin remodeling genes could increase tumor mutational burden (TMB) and predict response to ICB therapy in NSCLC. Analysis of seven ICB-treated NSCLC cohorts revealed that mutations of three chromatin remodeling-related genes, including KMT2C, BCOR and KDM5C, were significantly associated with ICB response, and combined mutations of these three genes further enhance this association. NSCLC patients with KMT2C/BCOR/KDM5C mutations had comparable clinical outcomes to TMB-high patients in terms of objective response rate, durable clinical benefit and overall survival. Although KMT2C/BCOR/KDM5C mutations were positively correlated with TMB levels in NSCLC, the association of this mutation with better ICB response was independent of tumor TMB and programmed death-ligand 1 (PD-L1) level, and combination of KMT2C/BCOR/KDM5C mutations with TMB or PD-L1 further improve the prediction of ICB response in NSCLC patients. Cancer Genome Atlas (TCGA) pan-cancer analysis suggested that the association of KMT2C/BCOR/KDM5C mutations with ICB response observed here might not result from DNA repair defects. In conclusion, our data indicate that KMT2C/BCOR/KDM5C mutation has the potential to serve as a predictive biomarker, alone or combined with PD-L1 expression or TMB, for ICB therapy in NSCLC.

Basal-like breast cancer (BLBC) is a type of high-grade invasive breast cancer with high risk of recurrence, metastases, and poor survival. Immune activation in BLBC is a key factor that influences both cancer progression and therapeutic response, although its molecular mechanisms are not well clarified. In this study, we examined five cancer immunity-related pathways (IFNα, IFNγ, STAT3, TGFβ and TNFα) in four large independent breast cancer cohorts (n = 6,381) and their associations with the prognosis of breast cancer subtypes. Activities of the 5 pathways were calculated based on corresponding pathway signatures and associations between pathways and clinical outcomes were examined by survival analysis. Among the five PAM50-based subtypes, BLBC had the highest IFNα, IFNγ, TNFα pathway activities, and the lowest TGFβ activity. The IFNα, IFNγ, TNFα pathway activities were negatively correlated with BLBC recurrence. In contrast, positive association and no association with BLBC recurrence were observed for TGFβ and STAT3 pathways, respectively. TNFα/TGFβ pathway combination improved the prediction of recurrence and chemotherapy response of BLBCs. Immune cell subset analysis in BLBC showed that M0, M1 and M2 macrophage levels were associated with either TNFα or TGFβ pathways, whereas the level of activated memory CD4 T cells were associated with both pathways. Moreover, this T cell subset was most abundant in BLBCs with low TGFβ and high TNFα pathway activities. These results suggested that cooperation of TNFα and TGFβ signaling may be involved in the regulation of memory T cells and anti-cancer immunity in BLBCs. Our data also demonstrate that TNFα/TGFβ pathway combination may represent a better biomarker for BLBC prognosis and clinical management.

Androgen receptor (AR) antagonists are currently tested in multiple clinical trials for different breast cancer (BC) subtypes, which emphasizes the need for clarifying the role of AR in this type of cancer. Previous studies showed that AR expression was associated with a favorable prognosis in ER-positive BC. However, the true biological effect of AR signaling in BC is not clear. An AR pathway signature was generated to compute AR pathway activity in BCs (n = 6439) from 46 microarray datasets. Associations of AR pathway activity and AR expression with BC prognosis were compared by survival analysis. AR pathway activity showed moderate positive and negative correlations with AR expression in HER2-positive and HER2-negative BCs, respectively. AR pathway activity increased while AR expression decreased in ER-negative BCs. Like ER and progesterone receptor (PR) expression, AR expression was also negatively associated with tumor grade, neoadjuvant response, and recurrence risk in BC. By contrast, AR pathway activity was positively, and more significantly, associated with these clinical features. Moreover, the AR pathway, but not AR expression, was significantly associated with recurrence risk in BC patients treated with endocrine therapy. These data suggest that, although AR expression probably reflects well-differentiated states of BC and is thus associated with favorable prognosis in BC, the biological effects of AR signaling confers worse outcomes in BC. Our findings encourage the continued evaluation of AR antagonists for BC treatment and support that AR pathway activity serves as a better prognostic factor than AR expression in BC.

The role of ALK signaling in the pathogenesis of breast cancer (BC) is not clear. Previously we generated a gene signature for ALK pathway based on the difference of gene expression profiles between tumor cells with activated and inactive ALK pathway. Here, this signature was used to compute ALK pathway activity in BC samples from 42 microarray datasets, and the associations between ALK pathway score and the clinical outcome were examined by logistic regression and survival analysis. Our results indicated that high ALK pathway activity was a significant risk factor for the presence of higher-grade breast cancer with loss of ER and PR expression in the 42 datasets (n= 6381). ALK pathway activity was also positively associated with pathological complete response (pCR) in 15 datasets annotated with patient’s neoadjuvant response information (n= 2093, overall OR 1.67, p=2.00E-12), and with recurrence risk in 30 datasets annotated with patient’s survival information (n= 4678, overall HR 1.21, p=3.31E-08). The associations of ALK pathway activity with pCR and recurrence were more significant in HER2 negative and grade 1&2 tumors than in HER2 positive and grade 3 tumors. Notably, the association between ALK and tumor recurrence was statistically significant in patient with age>50 but not ≤50 years old, in patient with positive but not negative lymph node and in patients with residual disease but not pCR following neoadjuvant chemotherapy. These data indicate that ALK may be involved in BC tumorigenesis and ALK pathway signature represents a novel biomarker for BC clinical management.

The associations of estrogen receptor (ER) and progesterone receptor (PR) pathways with the prognosis of colorectal cancer (CRC) are still controversial. The aim of this study was to readdress these issues by introducing a gene signature-based approach to semiquantitate pathway activity. In this approach, the ER and PR pathway activities in CRC were computed based on the expression profiles of the signature genes of ER and PR pathways, respectively. The results showed that the ER pathway activity was progressively significantly decreased from normal colorectal mucosa, colorectal adenoma to CRC. ER pathway signaling was a favorable factor for the presence of microsatellite stability (MSS) in CRC in seven cohorts tested, while was an unfavorable factor for cancer recurrence in all four CRC cohorts tested (n = 1122; overall HR: 0.311, 95% CI: 0.199-0.488, P < 0.001). Subset stratification in stage II patients showed that ER pathway remained significantly inversely associated with recurrence. PR pathway was also suppressed in colorectal tumors and inversely associated with recurrence of CRC, but to a much lesser extent than ER pathway. Moreover, the inverse association of PR pathway with cancer recurrence was more likely observed in CRC with high ER pathway activity, suggesting the interactions between the two pathways. PR pathway was not associated with MSS in CRC, but it was more significant than ER pathway associated with advance cancer stages and cancer response to adjuvant chemotherapy. These results suggested the potential application of the gene signatures of ER and PR pathways, especially the former, as novel markers for prognosis and management of CRC.

Background Aberrant BRAF/MEK signaling was found in nearly 50% of human malignancies and proved to play a critical role in the tumorigenesis of multiple cancers. However, this pathway was relatively seldom studied in breast cancer, and the role of this pathway in the pathogenesis of breast cancer is still controversial. Materials and Methods Breast cancer gene expression data from The Cancer Genome Atlas (TCGA) and 43 Affymetrix microarray datasets were analyzed. The BRAF/MEK pathway activity was presented with phosphorylated ERK level (for the TCGA dataset) or computed by a gene signature-based algorithm (for Affymetrix datasets). Aberrant activation of BRAF/MEK pathway in breast cancer was assessed in matched normal/tumor tissues. The associations of the BRAF/MEK pathway with clinical outcome in patients with breast cancer were analyzed by logistic regression, Cox regression, and Kaplan-Meier methods. Results Down-regulation of the BRAF/MEK pathway was observed in atypical ductal hyperplasia, ductal carcinoma in situ, and invasive breast cancers, with the exception of human epidermal growth factor receptor 2-positive and triple-negative breast cancers. Higher BRAF/MEK pathway activities were associated with better survival in estrogen receptor (ER)-positive (overall hazard ratio [HR], 0.85; P = 5.47E−5; n = 3128) or progesterone receptor-positive (overall HR, 0.85; P = 4.19E−3; n = 1537) breast cancers, but with worse survival in ER-negative (overall HR, 1.13; P = .01; n = 1107) or progesterone receptor-negative (overall HR, 1.13; P = .01; n = 1219) breast cancers. Combination with BRAF/MEK pathway activities could improve ER status-based recurrence prediction for breast cancer. Conclusion BRAF/MEK pathway was associated with the recurrence risk of breast cancer in an ER status-dependent mode. Combination with BRAF/MEK pathway activities could improve the ER status-based recurrence prediction in breast cancer.

Cancer stem cells (CSCs) have been recently identified in thyroid neoplasm. Anaplastic thyroid cancer (ATC) contains a higher percentage of CSCs than well-differentiated thyroid cancer. The signaling pathways and the transcription factors that regulate thyroid CSC self-renewal remain poorly understood.The objective of this study is to use two ATC cell lines (KAT-18 and SW1736) as a model to study the role of the sonic hedgehog (Shh) pathway in maintaining thyroid CSC self-renewal and to understand its underlying molecular mechanisms.The expression and activity of aldehyde dehydrogenase (ALDH), a marker for thyroid CSCs, was analyzed by Western blot and ALDEFLUOR assay, respectively. The effect of three Shh pathway inhibitors (cyclopamine, HhAntag, GANT61), Shh, Gli1, Snail knockdown, and Gli1 overexpression on thyroid CSC self-renewal was analyzed by ALDEFLUOR assay and thyrosphere formation. The sensitivity of transfected KAT-18 cells to radiation was evaluated by a colony survival assay.Western blot analysis revealed that ALDH protein levels in five thyroid cancer cell lines (WRO82, a follicular thyroid cancer cell line; BCPAP and TPC1, two papillary thyroid cancer cell lines; KAT-18 and SW1736, two ATC cell lines) correlated with the percentage of the ALDH(High) cells as well as Gli1 and Snail expression. The Shh pathway inhibitors, Shh and Gli1 knockdown, in KAT-18 cells decreased thyroid CSC self-renewal and increased radiation sensitivity. In contrast, Gli1 overexpression led to increased thyrosphere formation, an increased percentage of ALDH(High) cells, and increased radiation resistance in KAT-18 cells. Inhibition of the Shh pathway by three specific inhibitors led to decreased Snail expression and a decreased number of ALDH(High) cells in KAT-18 and SW1736. Snail gene knockdown decreased the number of ALDH(High) cells in KAT-18 and SW1736 cells.The Shh pathway promotes the CSC self-renewal in ATC cell lines by Gli1-induced Snail expression.

The BRAF V600E mutation causes impaired expression of sodium iodide symporter (NIS) and radioiodine refractoriness of thyroid cancer, but the underlying mechanism remains undefined. In this study, we hypothesized that histone deacetylation at the NIS (SLC5A5) promoter was the mechanism. Using the chromatin immunoprecipitation approach, we examined histone acetylation status on the lysine residues H3K9/14, H3K18, total H4, and H4K16 at the NIS promoter under the influence of BRAF V600E. We found that expression of stably or transiently transfected BRAF V600E inhibited NIS expression while the deacetylase inhibitor SAHA stimulated NIS expression in PCCL3 rat thyroid cells. Although BRAF V600E enhanced global histone acetylation, it caused histone deacetylation at the NIS promoter while SAHA caused acetylation in the cells. In human thyroid cancer BCPAP cells harboring homozygous BRAF V600E mutation, BRAF V600E inhibitor, PLX4032, and MEK inhibitor, AZD6244, increased histone acetylation of the NIS promoter, suggesting that BRAF V600E normally maintained histone in a deacetylated state at the NIS promoter. The regions most commonly affected with deacetylation by BRAF V600E were the transcriptionally active areas upstream of the translation start that contained important transcription factor binding sites, including nucleotides −297/−107 in the rat NIS promoter and −692/−370 in the human NIS promoter. Our findings not only reveal an epigenetic mechanism for BRAF V600E-promoted NIS silencing involving histone deacetylation at critical regulatory regions of the NIS promoter but also provide further support for our previously proposed combination therapy targeting major signaling pathways and histone deacetylase to restore thyroid gene expression for radioiodine treatment of thyroid cancer.

RAS proteins control many cellular processes, including cell migration, proliferation, differentiation, and survival (1). Extracellular signals are transduced intracellularly through the RAS pathway, and the amplitude and duration of this signaling are critical to appropriate biological responses of the cell. The normal signaling of this pathway is delicately regulated by various modulators that function as enhancers or inhibitors of RAS and its downstream pathways. The main pathways downstream of RAS in human tumorigenesis include RAF→ MEK → ERK (MAPK) and PI3K→AKT→mTOR (PI3K) pathways. The RAS GTPase-activating protein (RasGAP) RASAL1 that catalyzes RAS inactivation (2–5) and other negative modulators of the RAS signaling system (6–17) are presumably tumor suppressor genes (TSGs), and their aberrant silencing has been observed in various cancers. Germline genetic alterations in some of these negative modulators, such as NF1, SPRED1, PTEN, LKB1, TSC1, and TSC2, are associated with inherited cancer syndromes and development of a variety of cancers (18,19). Thus, like the genetic activation of the classical components of RAS signaling–coupled pathways, such as RAS and BRAF in the MAPK pathway and RAS and PIK3CA in the PI3K pathway, genetic or epigenetic inactivation of the negative modulators of these pathways is presumably an important alternative mechanism in human tumorigenesis. Follicular cell–derived thyroid cancer is a common endocrine malignancy with a rapidly rising incidence globally (20,21). Thyroid cancer histologically consists mainly of papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), and anaplastic thyroid cancer (ATC) (22). RAS-coupled MAPK and PI3K pathways play a fundamental role in thyroid tumorigenesis (23). Genetic alterations are the fundamental driving force for the aberrant activation of these two pathways in thyroid cancer and occur in a thyroid tumor type–predilected manner. BRAF mutation that activates the MAPK pathway is most common in PTC (24,25), whereas RAS, PTEN, and PIK3CA mutations that activate the PI3K pathway are most common in FTC and ATC (26). These classical genetic alterations activating the MAPK and PI3K pathways account for about 65% to 70% of thyroid cancers, leaving 30% to 35% of thyroid cancers with no known genetic background responsible for aberrant signaling of the RAS-coupled MAPK and PI3K pathways (23). In this study, we investigated alternative RAS signaling–related genes that may play an important role in thyroid tumorigenesis through the RAS-coupled MAPK and PI3K pathways. We focused on negative modulators of RAS signaling that are potential TSGs and, for the first time, specifically identified RASAL1 as a major TSG with common epigenetic and genetic alterations in thyroid cancer.

We have previously reported that the BRAFV600E signaling causes genome-wide aberrations in gene methylation in melanoma cells. To explore the potential molecular mechanisms for this epigenetic effect of BRAFV600E, in this in silico study we analyzed 11 microarray datasets retrieved from NCBI GEO database and examined the relationship of the expression of the epigenetic genes (genes involved in epigenetic regulation) with BRAFV600E signaling, methylation and expression of tumor-suppressor genes (TSGs) in melanoma, and patient survival with this cancer. Among 273 epigenetic genes examined, 12 genes were down-regulated (named DD genes) and 16 were up-regulated (UU genes) by suppression of the BRAFV600E signaling using inhibitors. While the expression of 245 non-DD/UU genes overall had no correlation with the expression and methylation of a set of potential TSGs, the expression of DD genes was significantly correlated negatively with the TSG expression and positively with TSG methylation. Expression of UU genes was positively, albeit weakly, associated with the TSG expression. Overall, no correlation was found between UU gene expression and TSG methylation. Importantly, the expression of DD genes, but not UU genes, was significantly associated with decreased survival of patients with melanoma. Interestingly, the promoters of DD genes contain more binding motifs of c-fos and myc, two BRAFV600E signaling-related transcription factors, than those of UU and non-DD/UU genes. Thus, these results link epigenetic genes to methylation and suppression of tumor suppressor genes as a mechanism involved in BRAFV600E-promoted melanoma tumorigenesis and uncover a novel molecular signature that predicts a poor prognosis of melanoma.

Purpose: This study was designed to explore the role of IQGAP1 in the invasiveness of thyroid cancer and its potential as a novel prognostic marker and therapeutic target in this cancer. Experimental Design: We examined IQGAP1 copy gain and its relationship with clinicopathologic outcomes of thyroid cancer and investigated its role in cell invasion and molecules involved in the process. Results: We found IQGAP1 copy number (CN) gain ≥3 in 1 of 30 (3%), 24 of 74 (32%), 44 of 107 (41%), 8 of 16 (50%), and 27 of 41 (66%) of benign thyroid tumor, follicular variant papillary thyroid cancer (FVPTC), follicular thyroid cancer (FTC), tall cell papillary thyroid cancer (PTC), and anaplastic thyroid cancer, respectively, in the increasing order of invasiveness of these tumors. A similar tumor distribution trend of CN ≥4 was also seen. IQGAP1 copy gain was positively correlated with IQGAP1 protein expression. It was significantly associated with extrathyroidal and vascular invasion of FVPTC and FTC and, remarkably, a 50%–60% rate of multifocality and recurrence of BRAF mutation–positive PTC (P = 0.01 and 0.02, respectively). The siRNA knockdown of IQGAP1 dramatically inhibited thyroid cancer cell invasion and colony formation. Coimmunoprecipitation assay showed direct interaction of IQGAP1 with E-cadherin, a known invasion-suppressing molecule, which was upregulated when IQGAP1 was knocked down. This provided a mechanism for the invasive role of IQGAP1 in thyroid cancer. In contrast, IQGAP3 lacked all these functions. Conclusions: IQGAP1, through genetic copy gain, plays an important role in the invasiveness of thyroid cancer and may represent a novel prognostic marker and therapeutic target for this cancer. Clin Cancer Res; 16(24); 6009–18. ©2010 AACR.

Heparanase-1 (HPR1), an endoglycosidase that specifically degrades heparan sulfate (HS) proteoglycans, is overexpressed in a variety of malignancies. Our present study sought to determine whether oncogene BRAF and RAS mutations lead to increased HPR1 expression. Reverse transcription-polymerase chain reaction analysis revealed that HPR1 gene expression was increased in HEK293 cells transiently transfected with a mutant BRAF or RAS gene. Flow cytometric analysis revealed that B-Raf activation led to loss of the cell surface HS, which could be blocked by two HPR1 inhibitors: heparin and PI-88. Cotransfection of a BRAF or RAS mutant gene with HPR1 promoter-driven luciferase reporters increased luciferase reporter gene expression in HEK293 cells. Knockdown of BRAF expression in a BRAF-mutated KAT-10 tumor cell line led to the suppression of HPR1 gene expression, subsequently leading to increased cell surface HS levels. Truncational and mutational analyses of the HPR1 promoter revealed that the Ets-relevant elements in the HPR1 promoter were critical for BRAF activation-induced HPR1 expression. Luciferase reporter gene expression driven by a four-copy GA binding protein (GABP) binding site was significantly lower in BRAF siRNA-transfected KAT-10 cells than in the control siRNA-transfected cells. We further showed that BRAF knockdown led to suppression of the expression of the GABPβ, an Ets family transcription factor involved in regulating HPR1 promoter activity. Taken together, our study suggests that B-Raf kinase activation plays an important role in regulating HPR1 expression. Increased HPR1 expression may contribute to the aggressive behavior of BRAF-mutated cancer.

We examined the therapeutic potential of a novel MEK inhibitor, RDEA119, and its synergism with the mTOR inhibitor temsirolimus in thyroid cancer cell lines. RDEA119 potently inhibited the proliferation of the four cell lines that harbored BRAF mutation, but had no or modest effects on the other four cells that harbored wild-type BRAF (IC50 of 0.034 – 0.217 μM vs. 1.413 – 34.120 μM). This inhibitory effect of RDEA119 in selected cell lines OCUT1 (BRAF V600E+, PIK3CA H1047R+) and SW1376 (BRAF V600E+) was enhanced by combination with the mTOR inhibitor temsirolimus. The PTEN-deficient cell FTC133 was highly sensitive to temsirolimus but insensitive to RDEA119 and simultaneous treatment with the latter enhanced the sensitivity of the cell to the former. The KAT18 (wild-type) cell was not sensitive to either drug alone, but became sensitive to the combination of the two drugs. The drug synergy was confirmed by combination index and isobologram analyses. RDEA119 and temsirolimus also showed synergistic effects on autophagic death of OCUT1 and KAT18 cells selectively tested. Dramatic synergistic effects of the two drugs were also seen on the growth of FTC133 xenograft tumors in nude mice. Overall, the effects of the two drugs on cell proliferation or autophagic death, either alone or in combination, were more pronounced in cells that harbored genetic alterations in the MAP kinase and PI3K/Akt pathways. Thus, these results demonstrated the important therapeutic potential of the novel MEK inhibitor RDEA119 and its synergism with temsirolimus in thyroid cancer.

We investigated the genotype-dependent therapeutic potential of targeting the phosphoinositide 3-kinase (PI3K)/Akt pathway for thyroid cancer. Proliferation of TPC1, Hth7, FTC133, OCUT1, K1, and BCPAP cells that harbored PI3K/Akt-activating genetic alterations was potently inhibited by the Akt inhibitor perifosine, whereas SW1736, Hth74, WRO, KAT18, and TAD2 cells that harbored no genetic alterations had no or only modest responses. Inhibition of Akt phosphorylation by perifosine was seen in these cells. Genetic-dependent apoptosis was induced by perifosine in cells selectively tested. Similarly, potent inhibition of cell proliferation by the mammalian target of rapamycin (mTOR) inhibitor temsirolimus occurred in virtually all the cells harboring genetic alterations, whereas modest inhibition was seen in some of the cells not harboring genetic alterations. Temsirolimus inhibited the phosphorylation of p70S6K, a substrate of mTOR. Knockdown of Akt1/2 or mTOR by shRNA approach inhibited the proliferation and colony formation of FTC133 and OCUT1 cells that harbored genetic alterations in the PI3K/Akt pathway but had no effect on SW1736 and KAT18 cells that did not. Transfection with PIK3CA mutants greatly sensitized SW1736 cells to perifosine and temsirolimus. Growth of xenograft tumors derived from FTC133 cells but not SW1736 cells in nude mice was dramatically inhibited by perifosine. Thus, this work for the first time shows that genetic alterations in the PI3K/Akt pathway confer thyroid cancer cells addiction to this pathway and their sensitivity to inhibition by targeting Akt and mTOR. This genotype-based targeting of the PI3K/Akt pathway using Akt and mTOR inhibitors may offer an effective therapeutic strategy for thyroid cancer and warrants further studies. [Cancer Res 2009;69(18):7311–9]

Targeting MAPK kinase (MEK) in the MAPK pathway is a potentially effective therapeutic strategy for thyroid cancer.The objective of the study was to investigate genotype-dependent therapeutic potential of the MEK inhibitor CI-1040 for thyroid cancer.We examined the effects of CI-1040 on proliferation, apoptosis, transformation, thyroid gene reexpression, and xenograft tumor growth with respect to genotypes in 10 thyroid tumor cell lines.Cell proliferation was potently inhibited by CI-1040 in cells harboring BRAF or RAS mutations but not in cells harboring RET/PTC rearrangement or wild-type alleles. For example, the IC50 values for BRAF mutation-harboring KAT10 cells and DRO cells and H-RAS mutation-harboring C643 cells were 0.365, 0.031, and 0.429 microm, respectively, whereas the IC50 values for RET/PTC1-harboring TPC1 cells and the wild-type MRO and WRO cells were 44, 46, and 278 microm, respectively. Proapoptotic effect of CI-1040 was seen in DRO cells, and cytostatic effect was seen in other cells. Down-regulation of cyclin D1 and reexpression of some thyroid genes were induced by CI-1040 in some BRAF mutation-harboring cells, and transformation was inhibited in all cells. CI-1040 also inhibited the growth of xenograft tumors in nude mice derived from KAT10 or C643 cells but not that derived from MRO cells.We for the first time demonstrated potent inhibitory effects of a MEK inhibitor, CI-1040, on thyroid cancer cells, some of which, particularly cell proliferation and tumor growth, seemed to be BRAF mutation or RAS mutation selective. Our data encourage a clinical trial on CI-1040 in thyroid cancer patients.

Purpose: The V600E BRAF mutant plays an important role in the pathogenesis of papillary thyroid cancer (PTC) and is associated with loss of expression of thyroid iodide-metabolizing genes. This study was done to investigate the restorability of expression of these genes by suppressing the BRAF/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway in V600E BRAF–harboring thyroid cells and to explore the mechanisms involved. Experimental Design: We used inducible expression of V600E BRAF, small interfering RNA transfection, and MEK-specific inhibitor to alter the MAP kinase pathway activities and subsequently examined the changes in expression, promoter activities, and methylation status of thyroid genes. Results: MEK inhibitor U0126 or cessation of V600E BRAF expression in PCCL3 cells restored expression of thyroid genes silenced by induced expression of V600E BRAF. U0126 also restored the expression of these genes in V600E BRAF–harboring PTC-derived NPA cells. Knockdown of BRAF by specific small interfering RNA restored expression of some of these genes in NPA cells. Luciferase reporter assay using thyroid-stimulating hormone receptor gene as a model showed that the promoter activity was modulated by the MAP kinase pathway. Promoter methylation in association with DNA methyltransferase expression played a role in gene silencing by MAP kinase pathway in NPA cells. Conclusions: We showed the restorability of expression of thyroid iodide-metabolizing genes silenced by V600E BRAF, and linked this process to gene methylation in PTC cells. The results provide clinical implications that therapeutic targeting at the BRAF/MEK/MAP kinase pathway may be a good approach in restoring thyroid gene expression for effective radioiodine therapy for BRAF mutation-harboring PTC.

TTV is a DNA virus with high genetic heterogeneity. To investigate the novel isolates of the virus, blood samples were collected from subjects who lived in various parts of China and suffered from hepatitis or were asymptomatic carriers. Nested PCR was carried out to amplify a 3.2-kb fragment using primers deduced from the prototype TTV (TA278). The ten entire 3.2-kb nt sequences were aligned with isolate TA278, SANBAN, TUS01, and SENV retrieved from GenBank, and a phylogenetic tree was constructed by Neighbor-Joining method. The analysis indicated that five novel variants of the present study have not been described before, and all TTV-related isolates could be classified into three groups. The isolate TCHN-A, B and TUS01 were included in a group, and the remaining novel isolates together with SANBAN and TA278 clustered into another group, while SEN virus formed a distinct group. The genetic distances of the five novel variants were 0.5507-0.8476 to TA278, 0.4635-0.7877 to SANBAN, 0.6064-0.7834 to TUS01 and 0.6936-0.8236 to SENV. Of these novel variants, the ORF1 consisted of 426-772 aa and ORF2 of 141-156 aa. The nt identities of ORF1 and ORF2 between those variants and TA278, SANBAN, and TUS01 were 46.1-60.8 and 48.7-63.6%, and those of aa sequences were only 27.1-52.4 and 28.9-45.5%, respectively. The first 65 aa of ORF1 were rich in arginine and most conserved with homology of 56.5-70.0%. There was a hypervariable region from aa 286 to 403 with merely 17.7-27.0% of identity. Despite a low aa identity between TA278 and the variants, they have similar hydrophilicity profiles of ORF1. There were 2-10 N-glycosylation motifs found in these variants. In conclusion, despite the high divergence, sequences of all these isolates shared common genome organisation, ORF structure, hydrophilicity patterns, and some potential motifs with TTV prototype. It is suggested that various TTV and TTV-related isolates belong to a very large and complex family, which remains to be studied.

Virus fragments homologous to TTV were detected previously from an enterically transmitted outbreak of non-A-E hepatitis [Luo et al., 1999]. To test the susceptibility of the Rhesus monkey to this virus and to establish its transmission routes, 6 Rhesus monkeys were inoculated, 3 orally and another 3 intravenously. The inoculum was prepared by extracting and filtering feces collected from a patient during the incubation period identified in the described outbreak. A second group of 3 monkeys was used for the passage study. The feces and blood samples were collected for detection of the virus by polymerase chain reaction (PCR). Four animals were subjected to liver biopsies and bile aspiration by open surgery for in situ virus detection. Viremia occurred in 4-7 days after intravenous and 7-10 days after oral inoculation. The virus was excreted in feces a few days after oral infection and simultaneously with viremia after intravenous inoculation. The virus was also detected in bile during the viremic phase. There was a prolonged carrier state with persistent viremia and virus excretion in feces for more than 6 months. Serum transaminase levels were not raised during the infection. The virus was present in both the cytoplasm and nuclei of hepatocytes, but no significant pathology was found. Therefore, the Rhesus monkey is susceptible to TT virus infection, but the virus seems nonpathogenic. Infection of the liver may be established either by oral or parenteral inoculation. The virus may be released from liver into the blood or via bile into feces, so it may be transmitted by both blood and fecal routes.

The T1790A BRAF mutation (L597Q) in childhood acute lymphoblastic leukemia is a functional oncogene

This study was supported by NIH RO-1 grant CA113507-01. The RAS → RAF → MEK → MAP kinase/ERK signaling pathway (MAP kinase pathway) is a conserved protein kinase cascade that regulates cell growth, proliferation, and differentiation in response to growth factors, cytokines, and hormones.1 Constitutive activation of this pathway is common in human cancers. Activating mutations in the gene for the B-type RAF or BRAF were identified in many human cancers.2 Over 40 missense mutations have been identified in the BRAF gene, the majority of which clusters in exons 11 and 15 and affects residues located within the kinase domain of the protein.3 The T1799A point BRAF mutation in exon 15, resulting in a V600E substitution (i.e., valine replaced by glutamic acid) in BRAF protein accounts for about 90% of all the oncogenic BRAF mutations.3 Numerous studies have shown frequent occurrence of this mutation in papillary thyroid cancer (PTC), with a prevalence of 44% on average.4 PTC is the most common thyroid cancer, accounting for 80% of thyroid malignancies.5 A few other BRAF mutations were also observed in PTC, including BRAFK601E (formerly named BRAFK600E) and BRAF599ins mutations, and their oncogenic function were welldocumented.6,7 By direct genomic DNA sequencing, we identified two cases of PTC samples from a BRAF mutation study published previously,8 whose DNA sequencing chromatogram showed an overlapping pattern of nucleotide sequence starting at base position T1799 in the BRAF gene (Fig. 1). These could represent either deletion or insertion of nucleotides in the affected alleles mixed with wild alleles. To distinguish the two possibilities, we subcloned the PCR products from tissue samples into pGEM-T vector (Promega Corporation, Madison, WI, USA) and amplified and sequenced the subcloned DNA to delineate single alleles of the BRAF gene. As illustrated in Figure 1, we confirmed two different new mutations, T1799-1801del and A1799-1816ins, in exon 15 of the BRAF gene (Fig. 1). The T1799-1801del mutation, originating from a deletion of three nucleotides (TGA) at the base position 1799-1801, resulted in a deletion of amino acid K601and a V600E amino acid substitution in the resultant BRAF protein while preserving the translation frame of the coding region of the gene (Fig. 1A). This BRAF mutation was previously found in 3/8 lymph node-metastasized PTC.9 We did not have matched lymph node tissues to analyze this mutation in the present study. The A1799-1816ins mutation originated from an insertion of eighteen nucleotides at the base position 1799-1816, resulting in the insertion of six amino acids in the BRAF protein (Fig. 1B). The inserted sequence was identical to the sequence of T1781-1798 in the BRAF gene. This is a novel and previously unknown BRAF mutation, representing an interesting and unusual genetic mutation with a repeat of a relatively long DNA sequence in a functionally critical region of the gene. This mutation resulted in a V600D amino acid substitution (i.e., valine replaced by aspartic acid), followed by addition of five amino acids (Phe-Gly-Leu-AlaThr) in the resultant BRAF protein. We blasted this inserted sequence in GenBank but found no additional gene or DNA sequence that had homology to this insertion. Although the T1799-1801del BRAF mutation was reported previously in PTC,8 its function consequence has not been characterized. It would be particularly interesting to know whether the V600E substitution associated with the deletion of its neighbor K601 in the amino acid sequence of BRAF protein still confers the BRAF kinase constitutive activation and oncogenic function. It would be similarly interesting to know whether the D600 followed by five new amino acids in the BRAF mutant from the A1799-1816ins mutation was functional. To examine their functions, we generated the two BRAF mutations by in vitro mutagenesis using wild BRAF in pEFm6 (which, together with the BRAFV600E construct, was a gift from Dr. Richard Marais) as the template and appropriately designed primers and the Quick-Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). For T1799-1801del BRAF mutation, the mutagenesis primers used were 5'-GGT GAT TTT GGT CTA GCT ACA GAA TCT CGA TGG AGT GGG TCC

Drug resistance is a major obstacle in the targeted therapy of melanoma using BRAF/MEK inhibitors. This study was to identify BRAF V600E-associated oncogenic pathways that predict resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors. We took in silico approaches to analyze the activities of 24 cancer-related pathways in melanoma cells and identify those whose activation was associated with BRAF V600E and used the support vector machine (SVM) algorithm to predict the resistance of BRAF-mutated melanoma cells to BRAF/MEK inhibitors. We then experimentally confirmed the in silico findings. In a microarray gene expression dataset of 63 melanoma cell lines, we found that activation of multiple oncogenic pathways preferentially occurred in BRAF-mutated melanoma cells. This finding was reproduced in 5 additional independent melanoma datasets. Further analysis of 46 melanoma cell lines that harbored BRAF mutation showed that 7 pathways, including TNFα, EGFR, IFNα, hypoxia, IFNγ, STAT3, and MYC, were significantly differently expressed in AZD6244-resistant compared with responsive melanoma cells. A SVM classifier built on this 7-pathway activation pattern correctly predicted the response of 10 BRAF-mutated melanoma cell lines to the MEK inhibitor AZD6244 in our experiments. We experimentally showed that TNFα, EGFR, IFNα, and IFNγ pathway activities were also upregulated in melanoma cell A375 compared with its sub-line DRO, while DRO was much more sensitive to AZD6244 than A375. In conclusion, we have identified specific oncogenic pathways preferentially activated in BRAF-mutated melanoma cells and a pathway pattern that predicts resistance of BRAF-mutated melanoma to BRAF/MEK inhibitors, providing novel clinical implications for melanoma therapy.

Mutations 1 295 228 C>T and 1 295 250 C>T (termed C228T and C250T respectively), corresponding to −124 C>T and −146 C>T from the translation start site in the promoter of the telomerase reverse transcriptase (TERT) gene, have recently been reported in human cancers, but not in thyroid cancers yet. We explored these mutations in thyroid cancers by genomic sequencing of a large number of primary tumor samples. We found the C228T mutation in 0 of 85 (0.0%) benign thyroid tumors, 30 of 257 (11.7%) papillary thyroid cancers (PTC), 9 of 79 (11.4%) follicular thyroid cancers (FTC), 3 of 8 (37.5%) poorly differentiated thyroid cancers (PDTC), 23 of 54 (42.6%) anaplastic thyroid cancers (ATC), and 8 of 12 (66.7%) thyroid cancer cell lines. The C250T mutation was uncommon, but mutually exclusive with the C228T mutation, and the two mutations were collectively found in 11 of 79 (13.9%) FTC, 25 of 54 (46.3%) ATC, and 11 of 12 (91.7%) thyroid cancer cell lines. Among PTC variants, the C228T mutation was found in 4 of 13 (30.8%) tall-cell PTC (TCPTC), 23 of 187 (12.3%) conventional PTC, and 2 of 56 (3.6%) follicular variant PTC samples. No TERT mutation was found in 16 medullary thyroid cancer samples. The C228T mutation was associated with the BRAF V600E mutation in PTC, being present in 19 of 104 (18.3%) BRAF mutation-positive PTC vs 11 of 153 (7.2%) the BRAF mutation-negative PTC samples (P=0.0094). Conversely, BRAF mutation was found in 19 of 30 (63.3%) C228T mutation-positive PTC vs 85 of 227 (37.4%) C228T mutation-negative PTC samples (P=0.0094). We thus for the first time, to our knowledge, demonstrate TERT promoter mutations in thyroid cancers, that are particularly prevalent in the aggressive thyroid cancers TCPTC, PDTC, ATC and BRAF mutation-positive PTC, revealing a novel genetic background for thyroid cancers.

The proto-oncogene PIK3CA has been well studied for its activating mutations and genomic amplifications but not single nucleotide polymorphism (SNP) in thyroid cancer. We investigated SNP rs17849071 (minor allele G and major allele T) in PIK3CA in thyroid tumors in 503 subjects by PCR and sequencing of a region of intron 9 carrying this SNP. This SNP was found in both normal and thyroid tumor tissues as well as in different generations of a studied family, confirming it to be a germline genetic event in thyroid tumor patients. In comparison with normal subjects, a dramatically lower prevalence of the heterozygous genotype G/T at rs17849071 was found in patients with follicular thyroid cancer (FTC). Specifically, rs17849071G/T was found in 15% (18/117) normal subjects vs. 1.3% (1/77) FTC patients, with an odds ratio of 0.07 (95% CI 0.01-0.55; P = 0.001). This represents a 93% risk reduction for FTC with this SNP. In contrast, no difference was seen with benign thyroid neoplasms in which the prevalence of rs17849071G/T was 13.1% (17/130), with an odds ratio of 0.83 (95% CI 0.40-1.69; P = 0.72). There was a trend of lower prevalences of rs17849071G/T and odds ratio in other types of thyroid cancer without statistical significance. We also found an interesting inverse relationship of rs17849071G/T with PIK3CA amplification. With copy number ≥4 defined as copy gain, 2.9% (1/34) rs17849071G/T vs. 19.0% (67/352) rs17849071T/T cases displayed PIK3CA amplification (P = 0.01). Conversely, 1.5% (1/68) cases with PIK3CA amplification vs. 10.4% (33/318) cases without PIK3CA amplification harbored rs17849071G/T (P = 0.01). This provides an explanation for the reciprocal relationship of rs17849071G/T with FTC, since PIK3CA amplification is an important oncogenic mechanism in thyroid cancer, particularly FTC. Thus, the present study uncovers an interesting phenomenon that rs17849071G/T is protective against FTC possibly through preventing PIK3CA amplifications.

Although BRAF(V600E) is well known to play an important role in the tumorigenesis of melanoma, its molecular mechanism, particularly the epigenetic aspect, has been incompletely understood. Here, we investigated the role of BRAF(V600E) signaling in altering gene methylation in the genome of melanoma cells using a methylated CpG island amplification/CpG island microarray system and searched for genes coupled to the BRAF(V600E) signaling through methylation aberrations. The results indicated that a wide range of genes with broad functions were linked to BRAF(V600E) signaling through their hyper- or hypomethylation. Expression of 59 genes hypermethylated upon BRAF knockdown was selectively tested and found to be largely correspondingly underexpressed, suggesting that these genes were naturally hypomethylated, and overexpressed with BRAF(V600E) in melanoma. This BRAF(V600E)-promoted hypomethylation was confirmed on genes selectively examined in primary melanoma tumors. Some of these genes were functionally tested and demonstrated to play a role in melanoma cell proliferation and invasion. As a mechanism of aberrant gene methylation driven by BRAF(V600E), expression of the DNA methyltransferase 1 and histone methyltransferase EZH2 was profoundly affected by BRAF(V600E). We have thus uncovered a previously unrecognized prominent epigenetic mechanism in the tumorigenesis of melanoma driven by BRAF(V600E). Many of the functionally important genes controlled by the BRAF(V600E) signaling through aberrant methylation may prove to be novel therapeutic targets for melanoma.

The purpose of the study was to explore optimal combinations of currently actively developed drugs for dually targeting the Ras → Raf → MAPK kinase (MEK) → MAPK/ERK (MAPK) and the phosphatidylinositol 3-kinase/Akt pathways as effective treatments for thyroid cancer.We tested the combinations of the Akt inhibitors MK2206 or perifosine with the BRAF(V600E) inhibitor PLX4032 or the MEK1/2 inhibitor AZD6244 in thyroid cancer cells harboring both the BRAF(V600E) and PIK3CA mutations.We found that MK2206 could potently, when used alone, and synergistically, when combined with either PLX4032 or AZD6244, inhibit thyroid cancer cell growth with all the combination index values lower than 1. Perifosine could potently inhibit thyroid cancer cell growth when used alone, but a strong antagonism occurred between this drug and PLX4032 or AZD6244 in the inhibition of thyroid cancer cell growth with all combination index values higher than 1. Combinations of MK2206 with PLX4032 or AZD6244 dramatically enhanced G1 cell cycle arrest induced by each drug alone. However, G2 cell cycle arrest uniquely induced by perifosine alone and G1 cell cycle arrest induced by PLX4032 or AZD6244 were both reversed by combination treatments, providing a mechanism for their antagonism. All these drugs could correspondingly inhibit the MAPK and phosphatidylinositol 3-kinase/Akt signalings, confirming their expected target effects.We demonstrated, unexpectedly, opposite outcomes of MK2206 and perifosine in their combinational treatments with BRAF(V600E)/MEK inhibitors in thyroid cancer cells. The data may help appropriate selection of these prominent drugs for clinical trials of combination therapies for thyroid cancer.

The BRAF V600E mutation plays an important role in the tumorigenesis of papillary thyroid cancer (PTC). To explore an epigenetic mechanism involved in this process, we performed a genome-wide DNA methylation analysis using a methylated CpG island amplification (MCA)/CpG island microarray system to examine gene methylation alterations after shRNA knockdown of BRAF V600E in thyroid cancer cells. Our results revealed numerous methylation targets of BRAF V600E mutation with a large cohort of hyper- or hypo-methylated genes in thyroid cancer cells, which are known to have important metabolic and cellular functions. As hypomethylation of numerous genes by BRAF V600E was particularly a striking finding, we took a further step to examine the selected 59 genes that became hypermethylated in both cell lines upon BRAF V600E knockdown and found them to be mostly correspondingly under-expressed (i.e. they were normally maintained hypomethylated and over-expressed by BRAF V600E in thyroid cancer cells). We confirmed the methylation status of selected genes revealed on MCA/CpG microarray analysis by performing methylation-specific PCR. To provide proof of concept that some of the genes uncovered here may play a direct oncogenic role, we selected six of them to perform shRNA knockdown and examined its effect on cellular functions. Our results demonstrated that the HMGB2 gene played a role in PTC cell proliferation and the FDG1 gene in cell invasion. Thus, this study uncovered a prominent epigenetic mechanism through which BRAF V600E can promote PTC tumorigenesis by altering the methylation and hence the expression of numerous important genes.

We recently demonstrated inhibition of thyroid cancer cells by the MEK inhibitor CI-1040. The objective of this study was to use a potent new-generation MEK inhibitor PD0325901 to further investigate the therapeutic potential of specifically targeting MEK in the MAP kinase pathway for thyroid cancer.We examined the effects of PD0325901 on a variety of cellular and molecular activities of thyroid cancer cell lines with distinct genotypes.PD0325901 remarkably inhibited MAP kinase pathway signaling in the thyroid cancer cells tested. It potently inhibited cell proliferation (IC(50) = 0.059-0.783 microM) and arrested cell cycle at the G0/G1 phase of cells harboring BRAF or RAS mutations but not cells harboring wild-type alleles or the RET/PTC1 rearrangement. Synergistic inhibitory effects were observed when PD0325901 was combined with phosphatidylinositol 3-kinase (PI3K) or NF-kappaB pathway inhibitors in most cells, including the RET/PTC1-harboring cells. PD0325901 could inhibit invasion and anchorage-independent growth of thyroid cancer cells independently of the type of genetic alterations. This compound did not seem to have significant proapoptotic effects, however.The MEK inhibitor PD0325901 has a wide range of potent inhibitory effects on thyroid cancer cells, some of which seemed to be genotype-selective, consistent with the results previously observed with an early-generation MEK inhibitor, CI-1040. The data provide further evidence that targeted inhibition of MEK may be therapeutically effective for thyroid cancer, particularly if the PI3K and NF-kappaB pathways are concurrently inhibited.

The relationship among BRAF mutation, platelet counts, and platelet-derived growth factor (PDGF) with respect to clinicopathological outcomes of papillary thyroid cancer (PTC) may play a role in PTC pathogenesis but remains undefined. We examined the T1799A BRAF mutation by direct genomic DNA sequencing in 108 primary PTC samples from a Chinese cohort and analyzed its relationship with clinicopathological, hematological, and other laboratory results as well as the levels of expression of PDGF in tumors. We found that the BRAF mutation was significantly associated with extrathyroidal invasion and advanced tumor stages III and IV. Specifically, extrathyroidal invasion was seen in 30/54 (56%) PTC with BRAF mutation versus 18/54 (33%) PTC without the mutation (P=0.02). Tumor stages III and IV were seen in 16/54 (30%) PTC with BRAF mutation versus 7/54 (13%) PTC without the mutation (P=0.04). The BRAF mutation was also significantly associated with a higher platelet count, with 249.28+/-53.76 x 10(9)/l in the group of patients with BRAF mutation versus 207.79+/-58.98 x 10(9)/l in the group without the mutation (P=0.001). An association of higher platelet accounts with extrathyroidal invasion was also seen, with 242.66+/-51.85 x 10(9)/l in patients with extrathyroidal invasion versus 218.49+/-59.10 x 10(9)/l in patients without extrathyroidal invasion (P=0.03). The BRAF T1799A-positive PTC tissues harbored a significantly higher level of PDGF-B than BRAF T1799A-negative PTC tissues. The data suggest that the BRAF T1799A mutation is associated with aggressive pathological outcomes of PTC in which high platelet counts and increased PDGF production may play a role.

Background: Identification of tumor suppressor genes (TSGs) silenced by CpG methylation uncovers the molecular mechanism of tumorigenesis and potential tumor biomarkers. Loss of heterozygosity at 11q25 is common multiple tumors including nasopharyngeal carcinoma (NPC). OPCML, located at 11q25, is one of the downregulated genes we identified through digital expression subtraction. Methodology/Principal Findings: Semi-quantitative RT-PCR showed frequent OPCML silencing in NPC and other common tumors, with no homozygous deletion detected by multiplex differential DNA-PCR. Instead, promoter methylation of OPCML was frequently detected in multiple carcinoma cell lines (nasopharyngeal, esophageal, lung, gastric, colon, liver, breast, cervix, prostate), lymphoma cell lines (non-Hodgkin and Hodgkin lymphoma, nasal NK/T-cell lymphoma) and primary tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues. Pharmacological and genetic demethylation restored OPCML expression, indicating a direct epigenetic silencing. We further found that OPCML is stress-responsive, but this response is epigenetically impaired when its promoter becomes methylated. Ecotopic expression of OPCML led to significant inhibition of both anchorage-dependent and -indendent growth of carcinoma cells with endogenous silencing. Conlusions/Significance: Thus, through functional epigenetics, we identified OPCML as a broad tumor suppressor, which is frequently inactivated by methylation in multiple malignancies. © 2008 Cui et al., published_or_final_version

Context: It remains inconclusive whether mutations in thyroid hormone receptor (TR) genes naturally occur in thyroid cancer and whether these genes could be suppressors of this cancer. Objectives: Our objectives were to examine further mutations of TRα and TRβ genes in thyroid cancer and also to examine their methylation as an epigenetic silencing mechanism in thyroid cancer. Experimental Design: Instead of using a cDNA sequencing approach used in previous studies, we used genomic DNA to sequence directly the coding regions of the TRα and TRβ genes to search mutations in various differentiated thyroid tumors and used methylation-specific PCR to analyze promoter methylation of these genes. Allelic zygosity status at TRβ was also analyzed. Results: We found no TRα gene mutation in 17 papillary thyroid cancers (PTCs) and 11 follicular thyroid cancers (FTCs), and no TRβ gene mutation in 16 PTCs and 12 FTCs. We also found no methylation of the TRα gene in 33 PTCs, 31 FTCs, 20 follicular thyroid adenomas (FTAs), and 10 thyroid tumor cell lines. In contrast, we found hypermethylation of the TRβ gene in 10 of 29 (34%) PTCs, 22 of 27 (81%) FTCs, five of 20 (25%) follicular thyroid adenomas, and three of 10 (30%) thyroid tumor cell lines, with the highest prevalence in FTC. We additionally examined loss of heterozygosity at TRβ and found it in three of nine (33%) PTCs and three of nine (33%) FTCs. Conclusions: Mutation is not common in TR genes, whereas hypermethylation of the TRβ gene as an alternative gene silencing mechanism is highly prevalent in thyroid cancer, particularly FTC, consistent with a possible tumor suppressor role of this gene for FTC.

Genetic alterations in the phosphatidylinositol-3-kinase (PI3K)/Akt pathway and their role in thyroid tumor pathogenesis in Chinese people remain undefined.The objective of the study was to examine the major genetic alterations and their relationship in the PI3K/Akt pathway in differentiated thyroid tumors in a Chinese cohort.We used real-time quantitative PCR for the analysis of PIK3CA copy gain and direct DNA sequencing for the detection of PIK3CA, RAS, and PTEN mutations on genomic DNA isolated from 234 thyroid tumors, including 31 follicular thyroid cancer (FTC), 141 papillary thyroid cancer (PTC), and 62 follicular thyroid adenoma (FTA).We found PIK3CA copy gain (defined as four or more copies) in nine of 31 FTC (29%), 20 of 141 PTC (14%), and five of 62 FTA (8%); PIK3CA gene mutations in four of 31 FTC (13%), one of 141 PTC (1%), and none of 62 FTA (0%); Ras mutations in three of 31 FTC (10%) and none of the 141 PTC and 62 FTA; and PTEN mutations in two of 31 FTC (6%) and none of 62 FTA (0%). Collectively, nine of 31 FTC (29%) vs. none of 62 FTA (0%) (P0.01) harbored one of the mutations, and when PIK3CA copy gain was included, 16 of 31 FTC (52%) vs. five of 62 FTA (8%) (P0.01) harbored any genetic alteration in the PI3K/Akt pathway. Mutual exclusivity was seen among all these PI3K/Akt pathway-related genetic alterations in all thyroid tumors except for two cases that harbored two genetic alterations.These data from a Chinese cohort provide further genetic evidence suggesting that dysregulated PI3K/Akt pathway plays a significant role in the pathogenesis of thyroid tumors, particularly FTC.

Context: Although the BRAF V600E mutant can initiate the formation of papillary thyroid cancer (PTC), it is unclear whether it is required to maintain cell proliferation, transformation, and tumor growth of BRAF mutation-harboring PTC. Objective: The aim of the study was to investigate whether BRAF V600E is required for the proliferation, transformation, and tumorigenicity of BRAF mutation-harboring PTC cells. Design: We addressed this issue using BRAF small interference RNA (siRNA) to transfect stably several BRAF mutation-harboring PTC cell lines, isolated clones with stable suppression of BRAF, and assessed their ability to proliferate, transform, and grow xenograft tumors in nude mice. Results: PTC cell proliferation and transformation were suppressed in specific BRAF siRNA clones, but not in control scrambled siRNA clones. Specifically, taking the advantage of stable BRAF knockdown, we were able to show continued suppression of PTC cell proliferation and transformation, or anchorage-independent colony formation in soft agar, after long-term culture. Moreover, we also demonstrated that in vivo tumorigenicity and growth of tumors from the specific BRAF siRNA cell clones in nude mice were suppressed compared with control clones. Conclusions: BRAF V600E is not only an initiator of PTC as demonstrated previously but is also a maintainer of proliferation, transformation, and tumorigenicity of PTC cells harboring BRAF mutation, and growth of tumors derived from such cells continues to depend on BRAF V600E. These results provide further support for potentially effective therapy targeted at BRAF for BRAF mutation-harboring PTC.