|Year : 2013 | Volume
| Issue : 7 | Page : 173-177
RASSF7 and RASSF8 proteins are predictive factors for development and metastasis in malignant thyroid neoplasms
Xi Li1, Ge Zhao2, Yinghou Wang1, Jian Zhang1, Zhiquan Duan1, Shijie Xin1
1 Department of Vascular and Thyroid Surgery, The First Affiliated Hospital of China Medical University, Shenyang 110001, China
2 Department of Obstetrics, The First Affiliated Hospital of China Medical University, Shenyang 110001, China
|Date of Web Publication||30-Nov-2013|
Department of Vascular and Thyroid Surgery, The First Affiliated Hospital of China Medical University, Shenyang 110001
Source of Support: None, Conflict of Interest: None
Objectives: To study the expression status of Ras-association domain family 7 (RASSF7) and Ras-association domain family 8 (RASSF8) in nodular thyroid goiter (NTG), medullary thyroid carcinoma (MTC), and papillary thyroid carcinoma (PTC) and establish whether a correlation exists between the presence of RASSF7 and RASSF8 proteins and clinicopathological parameters of the disease.
Materials and Methods: RASSF7 and RASSF8 protein expression was examined immunohistochemically on paraffin-embedded thyroid tissues from 112 cases with PTC, 20 cases with MTC, and 38 cases with NTG.
Results: The immunohistochemical expression of RASSF7 and RASSF8 was higher in PTC and MTC than in NTG (P < 0.001). In thyroid carcinomas, RASSF7 and RASSF8 expression was significantly correlated with a more advanced TNM stage (P = 0.035, P = 0.037), age, sex, lymph node metastasis (P = 0.006, P = 0.002), and tumor size (P = 0.012, P = 0.015).
Conclusions: RASSF7 and RASSF8 expression was increased in PTC and MTC compared to NTG, and this may be linked to the development and progression of thyroid carcinoma. In addition, these proteins were correlated with more advanced tumor stages, tumor size, and metastasis.
Keywords: Immunohistochemistry, RASSF7, RASSF8, thyroid carcinoma
|How to cite this article:|
Li X, Zhao G, Wang Y, Zhang J, Duan Z, Xin S. RASSF7 and RASSF8 proteins are predictive factors for development and metastasis in malignant thyroid neoplasms. J Can Res Ther 2013;9, Suppl S2:173-7
|How to cite this URL:|
Li X, Zhao G, Wang Y, Zhang J, Duan Z, Xin S. RASSF7 and RASSF8 proteins are predictive factors for development and metastasis in malignant thyroid neoplasms. J Can Res Ther [serial online] 2013 [cited 2021 May 19];9:173-7. Available from: https://www.cancerjournal.net/text.asp?2013/9/7/171/122519
| > Introduction|| |
Thyroid carcinomas represent the majority of endocrine neoplasms worldwide, and their incidence rates have steadily increased over the past few decades.  According to studies, the increase in the incidence of thyroid carcinomas can be explained by a higher frequency of early tumor detection due to the use of more sophisticated diagnostic techniques.  Thyroid carcinomas can be divided into three types, including well-differentiated carcinomas (papillary and follicular carcinomas), poorly differentiated carcinomas (medullary carcinoma), and undifferentiated carcinomas, due to well-established clinical findings and histological criteria. , The most common type of thyroid carcinoma is papillary thyroid carcinoma (PTC), which has a rapidly growing incidence worldwide.  Medullary thyroid carcinoma (MTC) is a rare malignant tumor originating from parafollicular C cells of the thyroid, first described by Hazard et al.,  and annually accounts for more deaths than most endocrine carcinomas combined.
The Ras proto-oncogenes belong to a super-family of GTPases that participate in a number of cellular processes.  So, Ras proteins are the key in many signaling pathways that transfer information from the extracellular environment to the internal cellular compartments.  Thus, mutation-induced deregulation of Ras activity may result in important alterations of cell growth control and differentiation. Mutation-induced deregulation of the Ras superfamily of proteins, or of upstream and downstream signaling components, do not only play an important role in mechanisms of tumor formation but also result in important alterations of normal cell growth control and differentiation. 
However, in mammals, the Ras signal transduction pathway is mediated by many effectors, including proteins with a Ras-associated (RalGDS/AF-6) (RA) domain, and the RA-domain is a common feature of the genes in the Ras-association domain family (RASSF). ,
The RASSF proteins carry several characteristic domains and function as adaptor proteins in many important biological processes, such as pro-apoptotic pathways, cell cycle and cytoskeleton regulation. The family can be split into two groups, the classical RASSF proteins (RASSF1-6) and the four recently added N-terminal RASSF proteins (RASSF7-10), due to the difference in domain architecture and sequence of the RA-domains. The classical RASSF family members have been reported to be involved in many biological processes such as microtubule stability, cell cycle control, and apoptosis, and are generally considered as tumor suppressors.  However, the importance of the RA-domain in many of the classical RASSF protein functions remains to be clarified. To date, the Ras-binding status of N-terminal RASSF family members remains largely unknown, especially RASSF7 and RASSF8.
In the present study, we use immunohistochemical method to determine the expression of the RASSF7 and RASSF8 proteins in benign and malignant thyroid tissues. We also investigate the correlation between RASSF7 and RASSF8 protein expression and clinicopathological variables in cases of primary thyroid carcinoma. To our knowledge, this is the first study to analyze RASSF7 and RASSF8 protein expression in benign and malignant thyroid tissues.
| > Materials and Methods|| |
Paraffin blocks from 170 patients, including 38 cases of nodular thyroid goiter (NTG), 112 cases of PTC, and 20 cases of MTC, were selected from the Department of Pathology in First Affiliated Hospital of China Medical University between 2008 and 2011. Patient records were obtained from the Medical Records Department at our hospital and also from pathology reports. None of these patients had a history of familial thyroid carcinoma or neck external irradiation. The diagnosis of malignant tumor was based on characteristic cytologic features, which include nuclear irregularity (nuclear grooves, clearing, and increased size) and pseudoinclusions.  All resected specimens were fixed in 10% neutral buffered formalin (pH 7.4), embedded in paraffin, cut into 5-μm sections, and stained with hematoxylin and eosin (H and E). Informed consent was obtained from all patients who donated their specimens, and all experiments were approved by the hospital's ethics committee.
Immunohistochemical staining was performed for both RASSF7 and RASSF8 to evaluate immunoreactivity in the above-mentioned tissue samples. Paraffin-embedded tissue sections fixed in formalin were used. Slides were deparaffinized, rehydrated, and subjected to microwave heat antigen retrieval in 10-mm citrate buffer (pH 6.0) for 20-25 min. After blocking endogenous peroxidase activity, the sections were incubated with primary antibodies against RASSF7 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and RASSF8 (1:200 dilution; Santa Cruz Biotechnology) overnight at 4°C. After washing with phosphate-buffered saline (PBS), staining was performed by the Elivision™ plus two-step system (Maixin Bio, Fuzhou, China). Immunoreactivity was visualized using the chromogen, 3,3'diamino-benzidine (DAB) (Maixin Bio, China). Slides were then counterstained with hematoxylin, washed, dehydrated with alcohol and xylene, and mounted onto coverslips. Appropriate positive and negative controls were run simultaneously with the patient specimen.
Evaluation of immunohistochemistry
Stained tissue sections were reviewed and scored according to the procedure of Sung et al.  Briefly, the staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The percentage of staining was scored as 0 (0%), 1 (1-25%), 2 (26-50%), 3 (51-75%), or 4 (76-100%). The staining intensity and percentage of staining were then multiplied to generate the immunoreactivity score for each case, ranging from 0 to 12. Tumor tissues with a final staining score ≥4 were considered to be high expression, while tissues with a score <4 were considered low expression.
Descriptive statistics were used according to the distribution of variables. The Mann-Whitney U test was used for comparison of the immunohistochemistry scores. The Chi-squared test or Fisher's exact test was used for the comparison of the frequency or proportions of single variables. Analyses were performed with SPSS software, version 16.0 (SPSS, Chicago, IL, USA). P < 0.05 were considered statistically significant.
| > Results|| |
The RASSF7 and RASSF8 protein expression of immunohistochemistry was observed in epithelial cells, with no staining in stromal or endothelial cells. The positive results revealed moderate-to-strong cytoplasmic RASSF7 and RASSF8 immunoreactivity, mainly to the carcinoma cells. In contrast, it found little immunostaining in NTG. Moreover, the expression of these proteins was significantly (P < 0.05) upregulated in PTC (82.61%, 91.07%) and MTC (75%, 80%) compared with NTG (8.57%, 5.56%) [Figure 1].
|Figure 1: Immunohistochemical analysis of RASSF7 and RASSF8 protein expression in NTG and thyroid carcinoma tissues (×200). (a, d, g) The HE of NTG, PTC, and MTC. (b and c) Negative RASSF7 and RASSF8 expression in NTG. (e and h) Strong RASSF7 expression in PTC and MTC. (f and i) Strong RASSF8 expression in PTC and MTC|
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The association between the immunohistochemical expression of RASSF7 and RASSF8 and the clinicopathological data was analyzed in cases of primary thyroid carcinoma (PTC and MTC). The expression of RASSF7 and RASSF8 was significantly higher at an advanced TNM stage (stages III and IV), in tumor size, and lymph node metastasis (P < 0.05) [Table 1]. However, the expression of these proteins was not significantly associated with sex or age.
|Table 1: Associations of RASSF7 and RASSF8 expression with clinicopathological characteristics in patients with malignant thyroid lesions|
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| > Discussion|| |
To our knowledge, there are limited data available in the literature on the expression status of RASSF proteins in thyroid carcinomas. This is the first time that the immunohistochemical expression of the RASSF7 and RASSF8 proteins was analyzed in a number of benign and malignant thyroid lesions. Recently, several additional RA-domain - containing family members have been identified and designated RASSF7 and RASSF8. RASSF7, previously known as HRC1 (HRAS1 cluster 1) and C11orf13, was first identified during the characterization of a 55-kb region of DNA surrounding HRAS1, located within a cluster of genes situated about 32-kb upstream of HRAS1 on human chromosome 11p15.  RASSF8's sequence was first deposited into the National Center for Biotechnology Information (NCBI) database by Hoon and Yuzuki in 1996 (accession number Q8NHQ8) and was found to be located on chromosome 12 and referred to as C12orf2 (chromosome 12 open reading frame 2), also named human carcinoma-associated HoJ-1. 
The present study demonstrated that RASSF7 and RASSF8 protein expression was significantly upregulated in thyroid carcinoma tissues compared with NTGs. Unlike other members of RASSF which were considered as tumor suppressors, RASSF7 had increased expression in various carcinomas. Brandt et al. found the expression of RASSF7 was increased in pancreatic ductal adenocarcinoma relative to normal tissue by DNA microarray analysis. ,, Meanwhile, Lowe et al. reported in another pancreatic carcinoma, islet cell tumors, RASSF7 was upregulated through the same methods, and the study also concluded that RASSF7 could identify islet cell tumors.  In addition, with tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis in 12 ovarian clear cell carcinoma cell lines, David et al. observed RASSF7 had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines.  Similarly, in the endometrioid endometrial adenocarcinoma, George et al. detected the elevated RASSF7 expression among approximately 6000 unique genes that distinguish malignant from normal endometrium.  Additionally, RASSF7 expression was increased in the MCF7 breast carcinoma cell line by hypoxia  and in human umbilical vein endothelial cells.  Consequently, our results about RASSF7, but not about RASSF8, were consistent with the results of the studies performed previously.
Though RASSF8 was found to be generally expressed in human adult tissues (liver, lung, pancreas, heart, kidney, brain, skeletal muscle, placenta, and colon),  there are several evidences to suggest that it might be a tumor suppressor, especially in lung carcinoma. In 2006, Falvella et al. reported reduced RASSF8 transcript levels in lung adenocarcinoma compared to normal lung tissue.  RASSF8 expression was downregulated in male germ cell tumors, in addition to lung adenocarcinoma.  Moreover, RASSF8 could be identified as a candidate gene related to leukemia and lymphoma formation in a study on retroviral-induced blood carcinomas in mice.  These discrepancies might be due to the different antibodies used for immunohistochemistry or the different tissue processing methods used.
Our present study also showed that increased immunohistochemical data of RASSF7 and RASSF8 were associated with a more advanced TNM stage (stages III and IV), tumor size, and lymph node metastasis in patients with thyroid carcinoma (PTC and MTC). We firstly investigated an association between increased RASSF7 and RASSF8 expression and the clinicopathological data. Nonetheless, currently there is not enough evidence to suggest that increased expression of RASSF7 and RASSF8 promotes carcinoma formation. Recino et al. addressed, for the first time, knocking down RASSF7 in human cell lines inhibited cell growth, resulted in cell death, and induced defects in mitosis, including aberrant spindle formation and a failure in chromosomal congression.  With regard to RASSF8, overexpression of this isoform suppressed anchorage-independent growth of the lung carcinoma cell lines A549 and H520.  Furthermore, transient and stable depletion of RASSF8 in H1792 and A549 lung carcinoma cells had an important role in stabilizing the actin cytoskeleton and thus repressing cell migration, providing further evidence for the role of RASSF8 as a tumor suppressor gene,  which may be distinct from what we observed. The reason of the distinction is that RASSF8 has been described as an important tumor suppressor in lung carcinogenesis initially by virtue of its locality within the lung tumor-associated D12S1034 minisatellite. 
| > Conclusions|| |
In our study, we demonstrated the upregulation of RASSF7 and RASSF8 in thyroid carcinomas by immunohistochemistry, proving to be a useful diagnostic marker for discriminating malignant from benign thyroid. On the other hand, the association of RASSF7 and RASSF8 protein expression with clinicopathological parameters provided evidence for its possible implication in the development and progression of thyroid carcinoma. However, these findings are provisional and need to be further confirmed in cohort studies including a higher proportion of patients with advanced nodal and metastatic disease. There is clearly a long way to go, but further analysis of RASSF7 and RASSF8 in thyroid will show if these also represent potential therapeutic targets.
| > Acknowledgments|| |
This work was supported by the National Nature Science Foundation of China (grant number: 81170295). The authors are grateful to Center of Experimental Medicine and Laboratory Technology and Central Laboratory of China Medical University for support.
| > References|| |
|1.||Chen AY, Jemal A, Ward EM. Increasing incidence of differentiated thyroid cancer in the United States, 1988-2005. Cancer 2009;15:3801-7. |
|2.||Davies L, Welch HG. Increasing incidence of thyroid cancer in the United States, 1973-2002. JAMA 2006;295:2164-7. |
|3.||DeLellis RA, Williams ED. Thyroid and parathyroid tumors: Introduction. In: DeLellis RA, Lloyd RV, Heitz PU, Eng C, editors. World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of Endocrine Organs. Lyon: IARC Press; 2004. p. 51-6. |
|4.||Hsin MKY, Wan IYP. Robotic thymectomy: The Hong Kong experience. Thorac Cancer 2011;2:84-9. |
|5.||Wartofsky L. Increasing world incidence of thyroid cancer: Increased detection or higher radiation exposure? Hormones 2010;9:103-8. |
|6.||Hazard JB, Hawk WA, Crile G Jr. Medullary (solid) carcinoma of the thyroid; a clinicopathologic entity. J Clin Endocrinol Metab 1959;19:152-61. |
|7.||Cully M, Downward J. SnapShot: Ras Signaling. Cell 2008;133:1292-1292.e. 1. |
|8.||Malumbres M, Barbacid M. RAS oncogenes: The first 30 years. Nat Rev Cancer 2003;3:459-65. |
|9.||Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer 2003;3:11-22. |
|10.||Wohlgemuth B, Kliel C, Kramer A, Serrano L, Wittinghofer F, Herrmann C. Recognizing and defining true Ras binding domains I: Biochemical analysis. J Mol Biol 2005;348:741-58. |
|11.||Sherwood V, Manbodh R, Sheppard C, Chalmers AD. RASSF7 is a member of a new family of Ras association domain-containing proteins and is required for completing mitosis. Mol Biol Cell 2008;19:772-82. |
|12.||Barut F, Onak Kandemir N, Bektas S, Bahadir B, Keser S, Ozdamar SO. Universal markers of thyroid malignancies: Galectin-3, HBME-1, and cytokeratin-19. Endocr Pathol 2010;21:80-9. |
|13.||Sung CO, Han SY, Kim SH. Low expression of claudin-4 is associated with poor prognosis in esophageal squamous cell carcinoma. Ann Surg Oncol 2011;18:273-81. |
|14.||Sobin LH, Fleming ID. TNM Classification of Malignant Tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997;80:1803-4. |
|15.||Weitzel JN, Kasperczyk A, Mohan C, Krontiris TG. The HRAS1 gene cluster: Two upstream regions recognizing transcripts and a third encoding a gene with a leucine zipper domain. Genomics 1992;14:209-19. |
|16.||Debeer P, Schoenmakers EF, Thoelen R, Holvoet M, Kuittinen T, Fabry G, et al. Physical map of a 1.5 mb region on 12p11.2 harbouring a synpolydactyly associated chromosomal breakpoint. Eur J Hum Genet 2000;8:861-70. |
|17.||Brandt R, Grützmann R, Bauer A, Jesnowski R, Ringel J, Löhr M, et al. DNA microarray analysis of pancreatic malignancies. Pancreatology 2004;4:587-97. |
|18.||Friess H, Ding J, Kleeff J, Fenkell L, Rosinski JA, Guweidhi A, et al. Microarray-based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer. Cell Mol Life Sci 2003;60:1180-99. |
|19.||Logsdon CD, Simeone DM, Binkley C, Arumugam T, Greenson JK, Giordano TJ, et al. Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer. Cancer Res 2003;63:2649-57. |
|20.||Lowe AW, Olsen M, Hao Y, Lee SP, Taek Lee K, et al. Gene expression patterns in pancreatic tumors, cells and tissues. PLoS One 2007;2:e323. |
|21.||Tan DS, Lambros MB, Rayter S, Natrajan R, Vatcheva R, Gao Q, et al. PPM1D is a potential therapeutic target in ovarian clear cell carcinomas. Clin Cancer Res 2009;15:2269-80. |
|22.||Mutter GL, Baak JP, Fitzgerald JT, Gray R, Neuberg D, Kust GA, et al. Global expression changes of constitutive and hormonally regulated genes during endometrial neoplastic transformation. Gynecol Oncol 2001;83:177-85. |
|23.||Camps C, Buffa FM, Colella S, Moore J, Sotiriou C, Sheldon H, et al. hsa-miR-210 is induced by hypoxia and is an independent prognostic factor in breast cancer. Clin Cancer Res 2008;14:1340-8. |
|24.||Liang GP, Su YY, Chen J, Yang ZC, Liu YS, Luo XD. Analysis of the early adaptive response of endothelial cells to hypoxia via a long serial analysis of gene expression. Biochem Biophys Res Commun 2009;384:415-9. |
|25.||Lock FE, Underhill-Day N, Dunwell T, Matallanas D, Cooper W, Hesson L, et al. The RASSF8 candidate tumor suppressor inhibits cell growth and regulates the Wnt and NFkappaB signaling pathways. Oncogene 2010;29:4307-16. |
|26.||Falvella FS, Manenti G, Spinola M, Pignatiello C, Conti B, Pastorino U, et al. Identification of RASSF8 as a candidate lung tumor suppressor gene. Oncogene 2006;25:3934-8. |
|27.||Korkola JE, Houldsworth J, Chadalavada RS, Olshen AB, Dobrzynski D, Reuter VE, et al. Down-regulation of stem cell genes, including those in a 200-kb gene cluster at 12p13.31, is associated with in vivo differentiation of human male germ cell tumors. Cancer Res 2006;66:820-7. |
|28.||Weiser KC, Liu B, Hansen GM, Skapura D, Hentges KE, Yarlagadda S, et al. Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma. Mamm Genome 2007;18:709-22. |
|29.||Recino A, Sherwood V, Flaxman A, Cooper WN, Latif F, Ward A, et al. Human RASSF7 regulates the microtubule cytoskeleton and is required for spindle formation, Aurora B activation and chromosomal congression during mitosis. Biochem J 2010;430:207-13. |
|30.||Yanagitani N, Kohno T, Sunaga N, Kunitoh H, Tamura T, Tsuchiya S, et al. Localization of a human lung adenocarcinoma susceptibility locus, possibly syntenic to the mouse Pas1 locus, in the vicinity of the D12S1034 locus on chromosome 12p11.2-p12.1. Carcinogenesis 2002;23:1177-83. |