Aberrant hypermethylation of the HOXD10 gene in papillary thyroid cancer with BRAFV600E mutation
- Authors:
- Published online on: October 25, 2017 https://doi.org/10.3892/or.2017.6058
- Pages: 338-348
Abstract
Introduction
Thyroid cancer is the most prevalent endocrine malignancy in humans. The incidence of thyroid cancer has increased in recent years. Papillary thyroid cancer (PTC) is a major type (80–85%) of thyroid cancer (1). Along with the improvement of diagnostic approaches for thyroid cancer, more and more PTC cases have been diagnosed (2). Although the prognosis of most PTC patients is optimistic, the recurrence rate was found to be relatively high after a 15-year follow-up (3–5). A small group of PTC patients appear to have a higher risk of recurrence and metastasis (6). To distinguish the population of patients with higher risk is an important task in the clinic. Appropriate biomarkers could be used to help evaluate the recurrence and metastasis risk of PTC. However, there are no effective biomarkers currently used in the clinic. New molecular biomarkers are urgently needed for the identification of the PTC patients with higher recurrence and metastasis risk.
Genetic research, which has been used as a molecular method for genetic discovery, has played a vital role in understanding the process of tumorigenesis. BRAFV600E mutation, by far, is the most common genetic event found in PTC, occurring in 20–50% of cases (7–11). Yet, PTC tumorigenesis may be regulated by epigenetic events as well. DNA methylation is the most common epigenetic regulatory mechanism in tumorigenesis. Evaluating the methylation status of DNA could be useful for the diagnosis, prognostic evaluation and predicting the risk for recurrence and metastasis of PTC (12–15).
In previous studies, we performed methylated DNA immunoprecipitation sequencing (MethylCap-seq) assay and established a database of the genome-wide DNA methylation profile of PTC. HOXD10 was one of the candidate genes that were aberrantly hypermethylated in PTC (Fig. 1). In the present study, we aimed to analyze the function and methylation status of the HOXD10 gene in PTC and to elucidate the relationship between HOXD10 methylation, HOXD10 expression, BRAF mutation and clinicopathological characteristics.
Materials and methods
Clinical samples
Human primary PTCs and adjacent non-tumor tissues (2 cm away from the tumor edge) were collected from patients who were initially surgically treated at the Department of Head and Neck Surgery, Fudan University Shanghai Cancer Center (Shanghai, China). All the patients had received lobectomy and isthmectomy plus ipsilateral central lymph node dissection. Additional modified lateral lymph node dissection was performed in patients with clinically suspicious lateral lymph node metastasis. All of the samples were pathologically confirmed. Totally 152 PTC patients were enrolled from April 2014 to December 2014. All the samples were stored at −80°C. Informed consent for the use of the tissues for clinical research was obtained before surgery, and the study protocol and consent form were approved by the Ethics Committee of Fudan University Shanghai Cancer Center. The tumor-node-metastasis (TNM) stages were determined according to the American Joint Cancer Committee (AJCC) TNM grading system, 7th edition. The clinicopathological data of the patients enrolled are summarized in Table I.
Cell culture and 5-Aza-2-deoxycytidine treatment
Human PTC cell lines TPC-1, BCPAP, K1, W3 were used for the present study (16). The cell lines TPC-1 and BCPAP were routinely cultured at 37°C in RPMI-1640 medium with 10% fetal bovine serum (FBS). K1 and W3 cells were cultured in DMEM/Hams F-12 medium (Invitrogen Life Technologies, Inc., Carlsbad, CA, USA). All the media were supplemented with penicillin/streptomycin. In some experiments, tumor cells were treated with 5 µm/ml 5-Aza-2′-deoxycytidine (5-Aza) for 72 h as a demethylation treatment. Media and 5-Aza were replenished every 24 h.
Plasmid construction and cell transfection
The HOXD10-overexpressing plasmid was constructed by cloning of the full-length HOXD10 open reading frame into the mammalian expression vector pcDNA3.1 with BamHI and XhoI restriction enzyme sites. The sequences were confirmed by DNA sequencing. PTC TPC-1 cells were cultured into a 6-well plate for 24 h and transfected with pcDNA3.1-HOXD10 or empty vector pcDNA3.1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After confirming the transfection efficiency by RT-PCR and western blotting in the surviving colonies, cells were transferred into a 6-well plate, and cultivated for further use.
Cell migration assay
PTC cells transfected with pcDNA3.1 vector or pcDNA3.1-HOXD10 were used for cell migration assays. Cell migration was assessed by modified Boyden Transwell chambers assay (Corning, Corning, NY, USA). Briefly, 2×104 cells/well were plated into 100 µl of no FBS medium in the upper chamber, and 500 µl of medium containing 10% FBS was added to the lower chamber. The cells were incubated for 12 h. The nonmigratory cells in the upper chamber were removed with a cotton swab. The cells on the bottom of the membrane were fixed and stained with polyfluoroalkoxy (PFA) and crystal violet stain solution (0.5%). The number of visible cells was counted by fluorescence microscope in 5 random high power fields. All the experiments were repeated 3 times.
Cell apoptosis and cell cycle
Analysis of cell apoptosis was performed using the PE Annexin V apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) by flow cytometric analysis (FCA). Briefly, stably transfected PTC cell line TPC-1 was suspended in annexin binding buffer, Alexa Fluor 488 Annexin V and propidium iodide (PI) working solution were added in sequence. The stained cells were finally analyzed by FACScan flow cytometry (BD Biosciences). Cell cycle distribution was detected by the Cycletest™ Plus DNA Reagent kit (BD Biosciences). Briefly, transfected cells were harvested and washed in PBS. Cellular DNA was stained with 125 µg/ml PI for 20 min at 4°C in dark. The cells were then sorted by FACSCalibur, and cell cycle distribution was determined using the ModFit LT software (Verity Software House, Topsham, ME, USA).
Western blot analysis
Total proteins were extracted from the stably transfected cells using RIPA lysis buffer. Lysates were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Primary antibodies were used as follows: HOXD10 (1:1,000; Abcam, Cambridge, MA, USA) and Tubulin (1:1,000; Proteintech Group, Chicago, IL, USA). The blots were developed using chemiluminescence with Las 4000 imaging system (Fujifilm, Tokyo, Japan).
DNA extraction and bisulphite conversion
Fresh-frozen tissue specimens and PTC cell lines were homogenized using a bead homogenizer and genomic DNA was extracted using the Genomic DNA Extraction kit (Tiangen, Beijing, China) according to the manufacturer. The DNA concentration was determined by NanoDrop 1000. The bisulfite conversion is described by Yu et al (17). The DNA sample was then stored at-20°C until further use.
Quantitative methylation-specific PCR (Q-MSP)
Q-MSP assay was performed to analyze the methylation level of the HOXD10 gene in PTC. We used a plasmid vector to construct a standard curve for absolute quantification PCR. The repetitive DNA element ALU was used as an internal reference. Briefly, quantitative PCR was carried out in a final reaction mixture of 20 µl containing 4 µl bisulfite-treated DNA, 500 nM of each primer, 250 nM TaqMan probe, 1.875 mM MgCl2, 200 µM deoxyguanosine triphosphate and 0.5 U platinum Taq polymerase in the King Hot Start Taq polymerase reaction system (Ruian Biotech, China). The reaction involved an initial pre-denaturation for 3 min at 94°C, followed by 40 cycles with denaturation for 15 sec at 94°C, annealing and extension for 60 sec at 60°C in an ABI 7500 Fast Real-Time instrument. The final results are presented as methylated gene copies (HOXD10/ALU*100).
The specific primers and TaqMan probes for the target gene HOXD10 and the internal reference gene ALU are presented in Table II. The HOXD10 gene and the promoter CpG island were searched for using the UCSC Human Genome Browser and PubMed (Fig. 1). The primers and TaqMan probes of HOXD10 for Q-MSP assay were designed by JIELI Biotechnology (Shanghai, China). The specific primers and TaqMan probes for the ALU gene were previously described by Weisenberger et al (18).
BRAF mutational screening
BRAF gene mutational status was analyzed in both clinical samples and PTC cell lines. BRAF mutational screening was analyzed by PCR followed by DNA sequencing at Boshang Biotechnology (Shanghai, China). The specific PCR primers for the BRAFV600E mutation region are presented in Table II.
RNA extraction and quantitative real-time PCR
Fresh-frozen tissue specimens were homogenized using a bead homogenizer. Genomic RNA from cell lines and tissues was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's protocol.
The expression of the HOXD10 gene was analyzed in both clinical samples and PTC cell lines by RT-PCR. Reverse transcription reaction was performed using 1 µg of total RNA with PrimeScript RT reagent kit with gDNA Eraser (Perfect Real-Time; RR047A; Takara, Dalian, Japan). The expression level of HOXD10 was determined by RT-PCR using SYBR Premix Ex Taq (Tli RNaseH Plus; RR420A; Takara). The PCR reaction was performed in a 20-µl volume containing up to 100 ng of template cDNA in a Cyclelight 480 PCR system. The reaction involved an initial denaturation for 3 min at 94°C, followed by 40 cycles with denaturation for 5 sec at 94°C, annealing for 60 sec at 60°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The expression level of HOXD10 was calculated using the 2−ΔCt method. The primers for RT-PCR were designed using the website http://www.embnet.sk/cgi-bin/primer3_www.cgi. The RT-PCR primer sequences are shown in Table II.
Statistical analysis
Statistical analyses were performed by Student's and paired t-tests, and Chi-square test. The odds ratios (ORs) for relationships between each variable and HOXD10 methylation were calculated by univariate logistic regression analysis. Multivariate logistic regression analysis was used to analyze the relationship between invasion and other clinicopathological characteristics including the methylation status of HOXD10. All confidence intervals (CIs) were stated at the 95% confidence level. A value of P<0.05 was considered to be statistically significant. SPSS 19.0 was used for data analysis (SPSS, Inc., Chicago, IL, USA). Figures were constructed using GraphPad Prism 5, Adobe Illustrator CS4 and Stata/SE 12.0.
Results
HOXD10 promoter is hypermethylated in PTC tissues
Q-MSP was designed to detect the methylation level of HOXD10 in PTC tissues. PTC and adjacent normal thyroid tissues (152 pairs) were tested by Q-MSP assay. The results of Q-MSP are shown in Fig. 2A. The overall methylation levels of the HOXD10 promoter were significantly higher in PTC tissues than levels in the adjacent normal thyroid tissues (P=0.02). Our findings showed that the promoter region of the HOXD10 gene was hypermethylated in 17.76% (27/152) of PTC tissues and in 10.53% (16/152) of adjacent normal thyroid tissues, using a cut-off value of 88 (HOXD10/ALU*100) (Fig. 2B).
HOXD10 mRNA expression is decreased in PTC tissues
To determine the relationship between the methylation status of the HOXD10 gene and its expression level, we evaluated the HOXD10 mRNA levels in 152 pairs of PTC tissue samples by RT-PCR. The result showed that the expression level of the HOXD10 gene was significantly decreased in the PTC tissues when compared with the level in the adjacent normal thyroid tissues (P=0.008) (Fig. 3A). Low expression of HOXD10 was found in 46.7% (71/152) of the PTC tissues and in only 13.8% (21/152) of the adjacent normal thyroid tissues (Table V), using a cut-off value of 0.06 (relative value).
Hypermethylation and low expresssion of HOXD10 is associated with BRAFV600E mutation in PTC tissues
To analyze the relationship between DNA methylation and BRAF mutation, we tested the BRAFV600E mutation in 4 PTC cell lines (TPC-1, BCPAP, K1 and W3) and 152 PTC clinical samples. Our results showed that BRAFV600E mutation occurred in 64 (42.1%) PTC patients and in 2 PTC cell lines (BCPAP and K1). In accordance with previous studies, no BRAF mutation was found in TPC-1 and W3 cell lines. Moreover, we observed that the hypermethylation and low-expresssion of HOXD10 was related to BRAFV600E mutation in PTC tissues. The expression of HOXD10 was significantly lower in PTC tissues with BRAFV600E mutation than in those without the mutation (P=0.022) (Table V). However, the methylation status of HOXD10 did not show statistical difference between PTC tissues with and without BRAFV600E mutation (P=0.669) (Table III). However, further analysis in PTC tissues with BRAFV600E mutation showed that the methylation levels of HOXD10 were significantly higher in tumor tissues than levels in the adjacent normal thyroid tissues (P=0.01). While in PTC tissues without BRAFV600E mutation such a significant difference was not found (P=0.50) (Fig. 2C).
5-Aza-2-deoxycytidine treatment reverts the expression of the HOXD10 gene in PTC cell lines
To further verify the relationship between the methylation status of the HOXD10 gene and its expression level, we detected the expression level of HOXD10 and performed 5-Aza treatment in 4 PTC cell lines: TPC-1, W3 (BRAF wild-type) and BCPAP, K1 (BRAFV600E mutation) to ascertain whether the changes in the methylation level influence the expression of HOXD10. The results are shown in Fig. 3B. HOXD10 mRNA was found to be weakly expressed in the TPC-1 and W3 cell lines, while the expression of HOXD10 in the BCPAP and K1 cell lines showed no significant decrease compared with the normal thyroid tissues. After a 72 h treatment of 5-Aza, the expression of HOXD10 in the TPC-1 and W3 cell lines was significantly increased (57 and 396 times, respectively), while there were no significant changes in the expression of HOXD10 in the BCPAP and K1 cell lines.
HOXD10 suppresses the migration and induces the apoptosis of PTC cells
To understand the potential functions of HOXD10 in PTC, we overexpressed HOXD10 in the TPC-1 cell line. TPC-1 cells were transfected with pcDNA3.1-HOXD10 or pcDNA3.1 vector. The transfection efficiency was confirmed by RT-PCR and western blotting (Fig. 4A). We observed that the overexpression of HOXD10 suppressed TPC-1 cell migration significantly compared to the control vector transfectants through a Transwell assay (P<0.01; Fig. 4B). To explore the mechanisms underlying the inhibition of cell proliferation by the overexpression of HOXD10, we assessed cell apoptosis and cell cycle by flow cytometry. The overexpression of HOXD10 induced the apoptosis of TPC-1 cells when compared to the empty vector-transfected cells. Additionally, we observed that the HOXD10-overexpressing TPC-1 cells showed higher S and G2 phase populations in comparison to the empty vector transfectants (Fig. 4C). In summary, the present study showed that the overexpression of HOXD10 inhibited the migration of TPC-1 cells and also induced the apoptosis in vitro. The results implied that HOXD10 may act as a tumor-suppressor in PTC.
The aberrant hypermethylation of the HOXD10 gene is associated with clinicopathological characteristics
The relationship between the HOXD10 methylation status and clinicopathological characteristics was analyzed to evaluate the prognostic value of the HOXD10 gene as a biomarker of PTC. Chi-square analysis and univariate logistic regression analysis revealed that age >45 (OR 3.881, 95% CI 1.930–9.847; P=0.003) and invasion (OR 2.972, 95% CI 1.157–7.633; P=0.027) were associated with the hypermethylation status of HOXD10 (Table III and Fig. 5), while, no relationship was found between HOXD10 hypermethylation status and sex, tumor size, multifocality, bilaterality, lymph node metastasis or Hashimoto's thyroiditis. The multivariate regression analysis was also performed to find the correlationship between invasion and other clinicopathological characteristics including methylation status of HOXD10. The result indicated that the hypermethylation of HOXD10 (OR 3.779, 95% CI 1.283–11.128; P=0.016) as well as tumor size >1 cm (OR 7.456, 95% CI 1.484–37.459; P=0.015) were independent risk factors of invasion in PTC (Table IV). In a word, our results showed the potential clinical value of HOXD10 methylation in PTC as a biomarker.
The relationship between HOXD10 expression and other clinicopathological characteristics including BRAFV600E mutation was also analyzed. Age >45 (P=0.022) and BRAFV600E mutation (P=0.022) were found to be associated with low expression of HOXD10 (Table V). However, no relationship was found between HOXD10 expression status and sex, tumor size, invasion, multifocality, bilaterality, lymph node metastasis or Hashimoto's thyroiditis. In addition, no significant correlation was found between BRAFV600E mutation and other clinicopathological characteristics.
Discussion
DNA methylation is one of the most common molecular events in cancers, along with genetic alterations, leading to carcinogenesis. Evaluating the status of DNA methylation could be useful for the diagnosis and prognostic evaluation of cancers and may be helpful in clarifying the process of tumorigenesis (12,19–22). We have paid close attention to DNA methylation in thyroid cancer in recent years. Our previous studies established a genome-wide DNA methylome database of PTC by MethylCap-seq, demonstrating that the HOXD10 gene was aberrantly hypermethylated in PTC.
Previous studies recognized HOXD10 as a sequence-specific transcription factor, mainly involved in cell differentiation and limb development (23,24). In recent years, its function in tumorigenesis has been gradually recognized (25,26). The homebox superfamily plays an important role in cell differentiation and morphogenesis. The dysregulation of the HOX gene may affect various pathways and play roles in tumorigenesis and metastasis (25,27). Several HOX genes (such as HOXB13, HOXA5 and HOXC6) have been found aberrantly expressed through promoter methylation in malignancies including lung, breast and gastrointestinal cancer (25,26,28–33). Emerging studies have found that the expression of HOXD10 is decreased in various tumors (such as breast and gastric cancer), and have considered HOXD10 as a candidate tumor-suppressor gene (34–39). However, the methylation and expression status of the HOXD10 gene, and its biological significance in PTC have not been identified.
In the present study, 152 pairs of PTC samples were collected for relative research, including Q-MSP, RT-PCR and BRAF mutation sequencing. Cytology experiments with 4 PTC cell lines were carried out to explore the relationship between the methylation and expression status of HOXD10. Overexpression transfection of HOXD10 in TPC-1 cells was designed to research the function of HOXD10 in PTC. The results showed that the methylation level of the HOXD10 gene was significantly higher in PTC tissues, compared with that noted in the adjacent normal thyroid tissues (P=0.02). RT-PCR assay showed that the expression of HOXD10 was significantly decreased in PTC cell lines and tumor tissues than that observed in the adjacent normal thyroid tissues (P=0.008), which was in accordance with the Q-MSP results. 5-Aza treatment reverted the expression of the HOXD10 gene in PTC cell lines, which demonstrated that the decreased expression of HOXD10 was caused by aberrant promoter hypermethylation. Moreover, the overexpression of HOXD10 suppressed the migration of TPC-1 cells, and promoted the cell apoptosis, implying that HOXD10 may act as a tumor suppressor in PTC. In addition, statistical analysis showed the potential clinical value of HOXD10 methylation as a biomarker. Besides, further stratified analysis showed that low expression of HOXD10 was related to BRAFV600E mutation (P=0.022). Also, the HOXD10 methylation level of PTC was significantly higher than that of adjacent normal thyroid tissues in patients with BRAFV600E mutation (P=0.01). In conclusion, we found that the HOXD10 gene was downregulated through promoter hypermethylation in PTC and HOXD10 may act as a tumor suppressor. Moreover, the aberrant hypermethylation and low expession of HOXD10 were associated with BRAFV600E mutation in PTC.
It is widely accepted that PTC with BRAFV600E mutation is a flag of high-risk. Detection of BRAFV600E mutation is an important clinical application for the diagnosis and prognostic prediction of PTC. In addition, the present study suggests that HOXD10 may be a candidate tumor suppressor and it also has interactions with the BRAF gene. The results showed that the low expression of HOXD10 was associated with BRAFV600E mutation (P=0.022). In addition, in PTCs with BRAFV600E mutation, the methylation levels of HOXD10 were significantly higher in PTC tissues than in adjacent normal thyroid tissues. While no significant difference was observed in PTCs without BRAFV600E mutation. In addition, the PTC cell lines with BRAFV600E mutation (K1, BCPAP) showed relatively high expression of HOXD10 and low sensitivity to 5-Aza treatment. On the contrary, the PTC cell lines without BRAFV600E mutation (TPC-1, W3) showed relatively low expression of HOXD10 and high sensitivity to 5-Aza treatment. The results indicated the possible interaction between HOXD10 gene and BRAFV600E mutation. One possible hypothesis to explain the results was that the hypermethylation of HOXD10 may be an accompanied event in BRAFV600E-mutated PTC, where BRAFV600E mutation plays the major role in tumorigenesis. While in wild-type PTC, HOXD10 hypermethylation may play an important role in tumorigenesis. Combining the detections of HOXD10 methylation and BRAF mutation may be a good choice to improve clinical diagnostic and prognostic accuracy.
Our results showed that the HOXD10 gene may be involved in PTC tumorigenesis and it may act with BRAFV600E mutation. However, the underlying mechanism of HOXD10 is still not clear. Wang et al (39) reported that HOXD10 regulates multiple downstream genes including IGFBP3 in gastric cancer. Reintroduction of HOXD10 upregulated IGFBP3, activated caspase-3 and caspase-8, and subsequently induced cell apoptosis. Yang et al (40) claimed that HOXD10 acted as a tumor suppressor via the inhibition of RHOC/AKT/MAPK pathway in cholangiocellular carcinoma. The upregulation of HOXD10 led to the dephosphorylation of AKT and ERK, implying that the PI3K/AKT and MAPK pathways were significantly inactivated. According to the above studies, MAPK pathways may be a key point for the interaction between BRAF and HOXD10, since BRAF is one of the most important regulatory gene in the MAPK pathways. However, further research is needed to clarify the mechanisms of the interaction between BRAF and the HOXD10 gene.
Combined with clinical data, the hypermethylation status of the HOXD10 promoter was significantly correlated with age >45 (OR 3.881, 95% CI 1.930–9.847; P=0.003) and invasion (OR 2.972, 95% CI 1.157–7.633; P=0.027). These 2 clinical characteristics usually predict a worse prognosis of PTC. Patients with an age >45 years and patients with primary tumor invasion may have a higher chance of recurrence and metastasis, leading to worse survive (41–43). Our analysis indicated that the hypermethylation of the HOXD10 gene was an independent risk factor for invasion in PTC. This indicates that the HOXD10 gene may play a role in PTC tumorigenesis and its methylation status could be used to predict the prognosis of PTC as a biomarker.
Recently, the incidence of thyroid cancer particularly PTC has significantly increased (2,44). It is well known that patients with PTC usually have good prognosis, but a small population of patients have a relatively higher recurrence risk (5,42). New biomarkers are urgently needed for the diagnosis and prognostic prediction of PTC. Currently, several biomarkers have been used to improve the diagnostic accuracy in PTC, such as the detection of BRAFV600E mutation (45–49). DNA methylation of tumor suppressors (such as Rassf1A and RARβ2) and thyroid-specific genes (such as TSHR) have been determined to be associated with BRAFV600E mutation, which may also play a role in PTC tumorigenesis (50–52). However, apart from genetic biomarkers, research must provided new ideas to search for viable epigenetic biomarkers to improve the diagnostic and prognostic accuracy in PTC. According to the present study, the hypermethylation of HOXD10 may be a promising biomarker for the diagnosis and prognostic prediction of PTC.
In summary, the present study firstly studied the methylation profile of HOXD10 and explored its functions in PTC. HOXD10 may act as a tumor suppressor in PTC. The decreased expression of the HOXD10 gene caused by aberrant hypermethylation was shown in PTCs particularly in those with BRAFV600E mutation. The epigenetic suppression of the HOXD10 gene may play a role in the tumorigenesis of PTC, and it may be a prospective biomarker for the diagnosis and prognostic prediction of PTC.
Acknowledgements
The present study was sponsored by the National Natural Science Foundation of China (81372368) and the Natural Science Foundation of Shanghai (12ZR1406800). The authors are grateful to Q.-H.J. for kindly providing the PTC cell lines TPC-1, K1, BCPAP and W3.
References
|
Kilfoy BA, Zheng T, Holford TR, Han X, Ward MH, Sjodin A, Zhang Y, Bai Y, Zhu C, Guo GL, et al: International patterns and trends in thyroid cancer incidence, 1973–2002. Cancer Causes Control. 20:525–531. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Xiang J, Wu Y, Li DS, Shen Q, Wang ZY, Sun TQ, An Y and Guan Q: New clinical features of thyroid cancer in eastern China. J Visc Surg. 147:e53–e56. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Noguchi S, Noguchi A and Murakami N: Papillary carcinoma of the thyroid. I. Developing pattern of metastasis. Cancer. 26:1053–1060. 1970. View Article : Google Scholar : PubMed/NCBI | |
|
Mazzaferri EL and Jhiang SM: Long-term impact of initial surgical and medical therapy on papillary and follicular thyroid cancer. Am J Med. 97:418–428. 1994. View Article : Google Scholar : PubMed/NCBI | |
|
Hundahl SA, Cady B, Cunningham MP, Mazzaferri E, McKee RF, Rosai J, Shah JP, Fremgen AM, Stewart AK and Hölzer S: Initial results from a prospective cohort study of 5583 cases of thyroid carcinoma treated in the united states during 1996. U.S. and German Thyroid Cancer Study Group. An American College of Surgeons Commission on Cancer Patient Care Evaluation study. Cancer. 89:202–217. 2000. View Article : Google Scholar : PubMed/NCBI | |
|
Patron V, Bedfert C, Le Clech G, Aubry K and Jegoux F: Pattern of lateral neck metastases in N0 papillary thyroid carcinoma. BMC Cancer. 11:82011. View Article : Google Scholar : PubMed/NCBI | |
|
Xing M: BRAF mutation in thyroid cancer. Endocr Relat Cancer. 12:245–262. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
da Silva RC, de Paula HS, Leal CB, Cunha BC, de Paula EC, Alencar RC, Meneghini AJ, Silva AM, Gontijo AP, Wastowski IJ, et al: BRAF overexpression is associated with BRAFV600E mutation in papillary thyroid carcinomas. Genet Mol Res. 14:5065–5075. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Schulten HJ, Alotibi R, Al-Ahmadi A, Ata M, Karim S, Huwait E, Gari M, Al-Ghamdi K, Al-Mashat F, Al-Hamour O, et al: Effect of BRAF mutational status on expression profiles in conventional papillary thyroid carcinomas. BMC Genomics. 16 Suppl 1:S62015. View Article : Google Scholar : PubMed/NCBI | |
|
Xing M, Alzahrani AS, Carson KA, Shong YK, Kim TY, Viola D, Elisei R, Bendlová B, Yip L, Mian C, et al: Association between BRAFV600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol. 33:42–50. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Yarchoan M, LiVolsi VA and Brose MS: BRAF mutation and thyroid cancer recurrence. J Clin Oncol. 33:7–8. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Jones PA and Baylin SB: The epigenomics of cancer. Cell. 128:683–692. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Kondo Y and Issa JP: DNA methylation profiling in cancer. Expert Rev Mol Med. 12:e232010. View Article : Google Scholar : PubMed/NCBI | |
|
Baylin SB and Ohm JE: Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat Rev Cancer. 6:107–116. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Jaenisch R and Bird A: Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nat Genet. 33 Suppl:S245–S254. 2003. View Article : Google Scholar | |
|
Schweppe RE, Klopper JP, Korch C, Pugazhenthi U, Benezra M, Knauf JA, Fagin JA, Marlow LA, Copland JA, Smallridge RC, et al: Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J Clin Endocrinol Metab. 93:4331–4341. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Yu J, Zhang HY, Ma ZZ, Lu W, Wang YF and Zhu JD: Methylation profiling of twenty four genes and the concordant methylation behaviours of nineteen genes that may contribute to hepatocellular carcinogenesis. Cell Res. 13:319–333. 2003. View Article : Google Scholar : PubMed/NCBI | |
|
Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, Ehrlich M and Laird PW: Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res. 33:6823–6836. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Xing M: Gene methylation in thyroid tumorigenesis. Endocrinology. 148:948–953. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Samowitz WS, Albertsen H, Sweeney C, Herrick J, Caan BJ, Anderson KE, Wolff RK and Slattery ML: Association of smoking, CpG island methylator phenotype, and V600E BRAF mutations in colon cancer. J Natl Cancer Inst. 98:1731–1738. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Hou P, Ji M and Xing M: Association of PTEN gene methylation with genetic alterations in the phosphatidylinositol 3-kinase/AKT signaling pathway in thyroid tumors. Cancer. 113:2440–2447. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Porra V, Ferraro-Peyret C, Durand C, Selmi-Ruby S, Giroud H, Berger-Dutrieux N, Decaussin M, Peix JL, Bournaud C, Orgiazzi J, et al: Silencing of the tumor suppressor gene SLC5A8 is associated with BRAF mutations in classical papillary thyroid carcinomas. J Clin Endocrinol Metab. 90:3028–3035. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Lance-Jones C, Omelchenko N, Bailis A, Lynch S and Sharma K: Hoxd10 induction and regionalization in the developing lumbosacral spinal cord. Development. 128:2255–2268. 2001.PubMed/NCBI | |
|
Gurnett CA, Keppel C, Bick J, Bowcock AM and Dobbs MB: Absence of HOXD10 mutations in idiopathic clubfoot and sporadic vertical talus. Clin Orthop Relat Res. 462:27–31. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Samuel S and Naora H: Homeobox gene expression in cancer: Insights from developmental regulation and deregulation. Eur J Cancer. 41:2428–2437. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Shah N and Sukumar S: The Hox genes and their roles in oncogenesis. Nat Rev Cancer. 10:361–371. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Botas J: Control of morphogenesis and differentiation by HOM/Hox genes. Curr Opin Cell Biol. 5:1015–1022. 1993. View Article : Google Scholar : PubMed/NCBI | |
|
Jung C, Kim RS, Zhang H, Lee SJ, Sheng H, Loehrer PJ, Gardner TA, Jeng MH and Kao C: HOXB13 is downregulated in colorectal cancer to confer TCF4-mediated transactivation. Br J Cancer. 92:2233–2239. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Raman V, Martensen SA, Reisman D, Evron E, Odenwald WF, Jaffee E, Marks J and Sukumar S: Compromised HOXA5 function can limit p53 expression in human breast tumours. Nature. 405:974–978. 2000. View Article : Google Scholar : PubMed/NCBI | |
|
Friedmann Y, Daniel CA, Strickland P and Daniel CW: Hox genes in normal and neoplastic mouse mammary gland. Cancer Res. 54:5981–5985. 1994.PubMed/NCBI | |
|
Rauch T, Wang Z, Zhang X, Zhong X, Wu X, Lau SK, Kernstine KH, Riggs AD and Pfeifer GP: Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay. Proc Natl Acad Sci USA. 104:pp. 5527–5532. 2007; View Article : Google Scholar : PubMed/NCBI | |
|
Shiraishi M, Sekiguchi A, Terry MJ, Oates AJ, Miyamoto Y, Chuu YH, Munakata M and Sekiya T: A comprehensive catalog of CpG islands methylated in human lung adenocarcinomas for the identification of tumor suppressor genes. Oncogene. 21:3804–3813. 2002. View Article : Google Scholar : View Article : Google Scholar : PubMed/NCBI | |
|
Shiraishi M, Sekiguchi A, Oates AJ, Terry MJ and Miyamoto Y: HOX gene clusters are hotspots of de novo methylation in CpG islands of human lung adenocarcinomas. Oncogene. 21:3659–3662. 2002. View Article : Google Scholar : View Article : Google Scholar : PubMed/NCBI | |
|
Carrio M, Arderiu G, Myers C and Boudreau NJ: Homeobox D10 induces phenotypic reversion of breast tumor cells in a three-dimensional culture model. Cancer Res. 65:7177–7185. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Vardhini NV, Rao PJ, Murthy PB and Sudhakar G: HOXD10 expression in human breast cancer. Tumour Biol. 35:10855–10860. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Sekar P, Bharti JN, Nigam JS, Sharma A and Soni PB: Evaluation of p53, HoxD10, and E-Cadherin status in breast cancer and correlation with histological grade and other prognostic factors. J Oncol. 702527:20142014. | |
|
Hakami F, Darda L, Stafford P, Woll P, Lambert DW and Hunter KD: The roles of HOXD10 in the development and progression of head and neck squamous cell carcinoma (HNSCC). Br J Cancer. 111:807–816. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Myers C, Charboneau A, Cheung I, Hanks D and Boudreau N: Sustained expression of homeobox D10 inhibits angiogenesis. Am J Pathol. 161:2099–2109. 2002. View Article : Google Scholar : PubMed/NCBI | |
|
Wang L, Chen S, Xue M, Zhong J, Wang X, Gan L, Lam EK, Liu X, Zhang J, Zhou T, et al: Homeobox D10 gene, a candidate tumor suppressor, is downregulated through promoter hypermethylation and associated with gastric carcinogenesis. Mol Med. 18:389–400. 2012. View Article : Google Scholar : View Article : Google Scholar : PubMed/NCBI | |
|
Yang H, Zhou J, Mi J, Ma K, Fan Y, Ning J, Wang C, Wei X, Zhao H and Li E: HOXD10 acts as a tumor-suppressive factor via inhibition of the RHOC/AKT/MAPK pathway in human cholangiocellular carcinoma. Oncol Rep. 34:1681–1691. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Baek SK, Jung KY, Kang SM, Kwon SY, Woo JS, Cho SH and Chung EJ: Clinical risk factors associated with cervical lymph node recurrence in papillary thyroid carcinoma. Thyroid. 20:147–152. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Wada N, Masudo K, Nakayama H, Suganuma N, Matsuzu K, Hirakawa S, Rino Y, Masuda M and Imada T: Clinical outcomes in older or younger patients with papillary thyroid carcinoma: Impact of lymphadenopathy and patient age. Eur J Surg Oncol. 34:202–207. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Ito Y, Hirokawa M, Jikuzono T, Higashiyama T, Takamura Y, Miya A, Kobayashi K, Matsuzuka F, Kuma K and Miyauchi A: Extranodal tumor extension to adjacent organs predicts a worse cause-specific survival in patients with papillary thyroid carcinoma. World J Surg. 31:1194–1201. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Oh CM, Jung KW, Won YJ, Shin A, Kong HJ and Lee JS: Age-period-cohort analysis of thyroid cancer incidence in Korea. Cancer Res Treat. 47:362–369. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Park KS, Oh YL, Ki CS and Kim JW: Evaluation of the Real-Q BRAFV600E detection assay in fine-needle aspiration samples of thyroid nodules. J Mol Diagn. 17:431–437. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Zou M, Baitei EY, Alzahrani AS, BinHumaid FS, Alkhafaji D, Al-Rijjal RA, Meyer BF and Shi Y: Concomitant RAS RET/PTC, or BRAF mutations in advanced stage of papillary thyroid carcinoma. Thyroid. 24:1256–1266. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Hwang TS, Kim WY, Han HS, Lim SD, Kim WS, Yoo YB, Park KS, Oh SY, Kim SK and Yang JH: Preoperative RAS mutational analysis is of great value in predicting follicular variant of papillary thyroid carcinoma. Biomed Res Int. 2015:6970682015. View Article : Google Scholar : PubMed/NCBI | |
|
Armstrong MJ, Yang H, Yip L, Ohori NP, McCoy KL, Stang MT, Hodak SP, Nikiforova MN, Carty SE and Nikiforov YE: PAX8/PPARγ rearrangement in thyroid nodules predicts follicular-pattern carcinomas, in particular the encapsulated follicular variant of papillary carcinoma. Thyroid. 24:1369–1374. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Gómez Sáez JM: Diagnostic and prognostic markers in differentiated thyroid cancer. Curr Genomics. 12:597–608. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Hoque MO, Rosenbaum E, Westra WH, Xing M, Ladenson P, Zeiger MA, Sidransky D and Umbricht CB: Quantitative assessment of promoter methylation profiles in thyroid neoplasms. J Clin Endocrinol Metab. 90:4011–4018. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Hu S, Ewertz M, Tufano RP, Brait M, Carvalho AL, Liu D, Tufaro AP, Basaria S, Cooper DS, Sidransky D, et al: Detection of serum deoxyribonucleic acid methylation markers: A novel diagnostic tool for thyroid cancer. J Clin Endocrinol Metab. 91:98–104. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Smith JA, Fan CY, Zou C, Bodenner D and Kokoska MS: Methylation status of genes in papillary thyroid carcinoma. Arch Otolaryngol Head Neck Surg. 133:1006–1011. 2007. View Article : Google Scholar : PubMed/NCBI |
