Identification of a prognostic LncRNA signature for ER-positive, ER-negative and triple-negative breast cancers.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Epigenetic genes regulated by the BRAFV600E signaling are associated with alterations in the methylation and expression of tumor suppressor genes and patient survival in melanoma.

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.

Expression of NOTCH1, NOTCH4, HLA-DMA and HLA-DRA is synergistically associated with T cell exclusion, immune checkpoint blockade efficacy and recurrence risk in ER-negative breast cancer.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Mutations in KMT2C, BCOR and KDM5C Predict Response to Immune Checkpoint Blockade Therapy in Non-Small Cell Lung Cancer.

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.

A Novel Metabolic Reprogramming Strategy for the Treatment of Diabetes-Associated Breast Cancer.

Diabetes is directly related to the risk of breast cancer (BC) occurrence and worsened BC prognosis. Currently, there are no specific treatments for diabetes-associated BC. This paper aims to understand the fundamental mechanisms of diabetes-induced BC progression and to develop personalized treatments. It reports a metabolic reprogramming strategy (MRS) that pharmaceutical induction of glucose import and glycolysis with metformin and NF-κB inhibitor (NF-κBi) while blocking the export of excessive lactate via inhibiting monocarboxylate transporter 4 (MCT4) leads to a metabolic crisis within the cancer cells. It demonstrates that the MRS shifts the metabolism of BC cells toward higher production of lactate, blocks lactate secretion, prompts intracellular acidification and induces significant cytotoxicity. Moreover, a novel MCT4 inhibitor CB-2 has been identified by structure-based virtual screening. A triple combination of metformin, CB-2, and trabectedin, a drug that impedes NF-κB signaling, strongly inhibits BC cells. Compared to normal glucose condition, MRS elicits more potent cancer cell-killing effects under high glucose condition. Animal model studies show that diabetic conditions promote the proliferation and progression of BC xenografts in nude mice and that MRS treatment significantly inhibits HG-induced BC progression. Therefore, inhibition of MCT4 combined with metformin/NF-κBi is a promising cancer therapy, especially for diabetes-associated BC.

Basal-like breast cancer with low TGFß and high TNFα pathway activity is rich in activated memory CD4 T cells and has a good prognosis.

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, TGFß 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 TGFß 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 TGFß and STAT3 pathways, respectively. TNFα/TGFß 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 TGFß 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 TGFß and high TNFα pathway activities. These results suggested that cooperation of TNFα and TGFß signaling may be involved in the regulation of memory T cells and anti-cancer immunity in BLBCs. Our data also demonstrate that TNFα/TGFß pathway combination may represent a better biomarker for BLBC prognosis and clinical management.

BRAF mutation-selective inhibition of thyroid cancer cells by the novel MEK inhibitor RDEA119 and genetic-potentiated synergism with the mTOR inhibitor temsirolimus.

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 4 cell lines that harbored BRAF mutation but had no or modest effects on the other 4 cells that harbored wild-type BRAF (IC(50) 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 2 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 2 drugs were also seen on the growth of FTC133 xenograft tumors in nude mice. Overall, the effects of the 2 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.

AR pathway activity correlates with AR expression in a HER2-dependent manner and serves as a better prognostic factor in breast cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

BRAF/MEK Pathway is Associated With Breast Cancer in ER-dependent Mode and Improves ER Status-based Cancer Recurrence Prediction.

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.

Association of an anaplastic lymphoma kinase pathway signature with cell de-differentiation, neoadjuvant chemotherapy response, and recurrence risk in breast cancer.

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 < 0.01), and this association was more significant in HER2-negative and grade 1&2 tumors than in HER2-positive and grade 3 tumors. ALK pathway activity was also positively associated with recurrence risk in breast cancer patients from 30 datasets annotated with survival information (overall hazard ratio = 1.21, P < 0.01), particularly in patients with age > 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.

Concomitant dysregulation of the estrogen receptor and BRAF/MEK signaling pathways is common in colorectal cancer and predicts a worse prognosis.

PURPOSE: 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. METHODS: 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. RESULTS: 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 < 0.001) and stage II CRC (n = 535; overall HR: 1.976, 95% CI: 1.306-2.989, p = 0.001). CONCLUSIONS: Combined activity of the ER and BRAF/MEK pathways may represent a novel biomarker for CRC prognosis and clinical management.

Association of the T1799A BRAF mutation with tumor extrathyroidal invasion, higher peripheral platelet counts, and over-expression of platelet-derived growth factor-B in papillary thyroid cancer.

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.

Potent inhibition of thyroid cancer cells by the MEK inhibitor PD0325901 and its potentiation by suppression of the PI3K and NF-kappaB pathways.

BACKGROUND: 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. METHODS: We examined the effects of PD0325901 on a variety of cellular and molecular activities of thyroid cancer cell lines with distinct genotypes. RESULTS: 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. CONCLUSIONS: 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.

Suppression of BRAF/MEK/MAP kinase pathway restores expression of iodide-metabolizing genes in thyroid cells expressing the V600E BRAF mutant.

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.

Genetic alterations and their relationship in the phosphatidylinositol 3-kinase/Akt pathway in thyroid cancer.

PURPOSE: To investigate the overall occurrence and relationship of genetic alterations in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in thyroid tumors and explore the scope of this pathway as a therapeutic target for thyroid cancer. EXPERIMENTAL DESIGN: We examined collectively the major genetic alterations and their relationship in this pathway, including PIK3CA copy number gain and mutation, Ras mutation, and PTEN mutation, in a large series of primary thyroid tumors. RESULTS: Occurrence of any of these genetic alterations was found in 25 of 81 (31%) benign thyroid adenoma (BTA), 47 of 86 (55%) follicular thyroid cancer (FTC), 21 of 86 (24%) papillary thyroid cancer (PTC), and 29 of 50 (58%) anaplastic thyroid cancer (ATC), with FTC and ATC most frequently harboring these genetic alterations. PIK3CA copy gain was associated with increased PIK3CA protein expression. A mutual exclusivity among these genetic alterations was seen in BTA, FTC, and PTC, suggesting an independent role of each of them through the PI3K/Akt pathway in the tumorigenesis of the differentiated thyroid tumors. However, coexistence of these genetic alterations was increasingly seen with progression from differentiated tumor to undifferentiated ATC. Their coexistence with BRAF mutation was also frequent in PTC and ATC. CONCLUSIONS: The data provide strong genetic implication that aberrant activation of PI3K/Akt pathway plays an extensive role in thyroid tumorigenesis, particularly in FTC and ATC, and promotes progression of BTA to FTC and to ATC as the genetic alterations of this pathway accumulate. Progression of PTC to ATC may be facilitated by coexistence of PI3K/Akt pathway-related genetic alterations and BRAF mutation. The PI3K/Akt pathway may thus be a major therapeutic target in thyroid cancers.

BRAF V600E maintains proliferation, transformation, and tumorigenicity of BRAF-mutant papillary thyroid cancer cells.

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.

Lack of mutations in the thyroid hormone receptor (TR) alpha and beta genes but frequent hypermethylation of the TRbeta gene in differentiated thyroid tumors.

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 TRalpha and TRbeta 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 TRalpha and TRbeta 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 TRbeta was also analyzed. RESULTS: We found no TRalpha gene mutation in 17 papillary thyroid cancers (PTCs) and 11 follicular thyroid cancers (FTCs), and no TRbeta gene mutation in 16 PTCs and 12 FTCs. We also found no methylation of the TRalpha gene in 33 PTCs, 31 FTCs, 20 follicular thyroid adenomas (FTAs), and 10 thyroid tumor cell lines. In contrast, we found hypermethylation of the TRbeta 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 TRbeta 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 TRbeta 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.

Inhibitory effects of the mitogen-activated protein kinase kinase inhibitor CI-1040 on the proliferation and tumor growth of thyroid cancer cells with BRAF or RAS mutations.

CONTEXT: Targeting MAPK kinase (MEK) in the MAPK pathway is a potentially effective therapeutic strategy for thyroid cancer. OBJECTIVE: The objective of the study was to investigate genotype-dependent therapeutic potential of the MEK inhibitor CI-1040 for thyroid cancer. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

High prevalence and mutual exclusivity of genetic alterations in the phosphatidylinositol-3-kinase/akt pathway in thyroid tumors.

CONTEXT: Genetic alterations in the phosphatidylinositol-3-kinase (PI3K)/Akt pathway and their role in thyroid tumor pathogenesis in Chinese people remain undefined. OBJECTIVE: 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. DESIGN: 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). RESULTS: 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%) (P < 0.01) harbored one of the mutations, and when PIK3CA copy gain was included, 16 of 31 FTC (52%) vs. five of 62 FTA (8%) (P < 0.01) 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. CONCLUSION: 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.