Retinoid X Receptor γ Expression Correlated with Epithelial Mesenchymal Transition, Invasion and Metastasis of Papillary Thyroid Carcinoma
|Course||Pathology and Pathophysiology|
|Keywords||thyroid epithelial mesenchymal transition loss of cellular polarity/cohesiveness tumor progression RXRγ|
[Back ground]Papillary thyroid carcinoma (PTC) is the most common malignancy of the endocrine organs in human. It is well known that PTC has good prognosis but still about 10% of patients develop recurrence in lymph node and/ or distant organs. In spite of the high frequency of nodal metastasis in PTC, the prognostic indicators obtainable from tumor are largely unknown. Previous studies have focused on the vascular endothelial growth factor (VEGF) and its effect on angiogenesis and lymphangiogenesis via VEGF receptors. Recently, epithelial mesenchymal transition (EMT), which was developed in the field of embryology, has been postulated as an essential event for tumor invasion and metastasis.EMT was observed in the embryonic development. Gary Greenburg proposed the definition of EMT firstly in Epithelia suspended in collagen gels in year 1982. Morphologically, this includes the loss of apical-basal polarity and cell-cell adhesion. At the molecular level, EMT involves the loss of expression of structural adhesion proteins, such as E-cadherin, and gain of mesenchymal markers, such as vimentin and fibronectin. Then cells undergoing EMT gain the ability to invasive into the surrounding tissue, and play a key role in the development of malignancy. Abnormal EMT is a common event in the invasion and metastasis of epithelial malignancies, which features by a loss of epithelial properties and the acquisition of mesenchymal properties. EMT has been reported to be associated with high metastatic rates in several cancers, including prostate, breast, non-small-cell lung carcinoma and colorectal cancers. In thyroid neoplasia, our group first reported the loss of cellular polarity/ cohesiveness (LOP/ C) in the invasive front of the PTC, where cancer cells were loosely or individually arranged without forming well developed papilla or follicles. A subsequent study from our group suggested that PTC cases with LOP/ C have more extrathyroid invasion and lymph node metastasis, and PTC with LOP/ C involving more than 20% of tumor area was elucidated as high-risk group of PTC with higher recurrence rates. The LOP/ C described in PTC by our group should be a morphological expression of EMT, according to the features described above. As supported by Vasko et al., reduced levels of mRNAs encoding proteins involve in cell-cell adhesion and communication and over-expression of vimentin was found in EMT in the invasive front of PTC. A recent study reported eight cases with similar hobnail appearance to have aggressive behavior and significant higher mortality rate.Nuclear receptors (NRs) form a class of transcription factors that are regulated by small lipophilic ligands includes steroids, thyroid hormone, retinoids and vitamin D3. NRs mediate their effects by ligand binding, gene activation and posttranslational events. These may be brought about by interactions with a diversity of signaling transduction pathways including MAPK, phosphatidylinositol-3-kinase (PI3K/Akt), and Wnt signal. Retinoids exhibit potent inhibitory effect on tumor cell growth and differentiation through retinoid receptors, retinoic acid receptor (RAR) and RXRs. RXRy is one of the RXRs that has two isoforms,αandβ. Variable expressions of the different receptors in thyroid cancer tissue and cell lines have been reported recently. Our previous study showed up-regulation of RXRy in tumor tissue compared with matched thyroid tissue in seven of nine PTCs. This up-regulation was later confirmed by some other researches. In addition, well differentiated pancreatic endocrine neoplasms with aberrant expression of RXRy were found to have higher metastatic potential. However, whether RXRy expression has any biological role in tumor metastasis in PTC remains unknown.We hope to solve several questions in this study:(1) The expression of RXRy mRNA and protein in PTC tumor tissue and matched non-neoplastic tissue; (2) The correlation between RXRy expression and the clinicopathological parameters; (3) The morphological and immunohistochemical features of LOP/C in the invasive front of PTC, (4) The proposal of EMT in PTC on basis of LOP/C.[Methods]1. Thyroid carcinoma tissues and matched non-neoplastic tissues were obtained from 69 patients with primary papillary thyroid carcinoma, which were fixed in 4% formalin and embedded in paraffin for HE (hematoxylin and eosin) section preparation and histological examination. The presence of the LOP/C in the invasive front was examined, and was correlated with the other clinicopathological parameters by using the Chi-square test or Fisher’s exact test as appropriate.2. Extract the total RNA from the 69 cases of paired carcinoma tissue and non-neoplastic tissue described above, synthesize the first strand cDNA, and examine the expression of RXRy by RT-PCR method and quantitative real-time PCR method.β-actin served as an endogenous control. Differences between the matched tumor and non-neoplastic thyroid tissue were calculated using the formula 2expΔCt tumor-ΔCt normal) and expressed as a fold change in expression: T/N ratio (T:tumor tissue; N:non-neoplatic tissue). The correlations between periostin expression and the LOP/C and other clinicopathological parameters were calculated.3. Extract protein from four cases with RXRy up-regulation, and compare the expression between tumor tissue and non-neoplastic tissue using western blotting.4. Immunohistochemical staining of RXRy was done to thirteen PTCs with RXRy up-regulation, and TTF-1, E-cadherin,β-catenin and vimentin were done to sixteen cases with LOP/C.5. To evaluate the immunohistochemical features of LOP/C and examine its correlation with the clinicopathological parameters.6. To examine the risk factor of LNM of PTC using stepwise logistic regression analysis. Results1. All hematoxylin-eosin (H & E) sections were reviewed by two pathologists. Histological stage grouping was confirmed by histological examination referred to UICC TNM classification and WHO classification. Poorly differentiated carcinoma (PDC) and solid variant PTCs were excluded and only common type PTC were included in this study.2. LOP/C are located in the invasive front of the papillary thyroid carcinoma. Forty two out of 69 cases (60.9%) showed LOP/C in no less than 20% tumor area, and were classified as PTC with LOP/C, while the other 27 cases (39.1%) failed to show these features in significant proportion, and were classified as PTC without LOP/C.3. The relative expression (RE) ratio between the tumor and paired non-neoplastic thyroid tissue (T/N) was calculated using the formula 2exp (ΔCt tumor-ΔCt normal) and expressed as a fold change in expression. Using a cut-off value of 1.5,59 cases (RE≥1.5) were up-regulated, and 10 cases (RE<1.5) were non-up-regulated. The difference of RXRy mRNA expression and up-regulation in tumor tissue comparing with normal tissue is statistically significant using Student’s t-test (P= 0.011). RXRγup-regulation was found in 40 cases (95.2%) of PTC with LOP/C, but in only 19 cases (70.3%) without LOP/C. The proportion of RXRy up-regulation in PTC with LOP/C was significantly higher than that without LOP/C using univariate analysis (r=0.345, P=0.004), which suggests RXRy up-regulation may have significant role in LOP/C morphology. PTCs with RXRy up-regulation showed extrathyroid invasion (r=0.293, P=0.019), advanced tumor stage (pT3 or pT4, r=0.318, P=0.016) and LNM (r=0.338, P=0.005) more frequently than those cases without. These results suggest that RXRy up-regulation mainly occurred in PTCs with extrathyroid invasion, LNM or in advanced tumor stage. In contrast, there was no statistically significance between RXRy expression and patients’ age, gender or tumor size, which indicates that RXRy up-regulation is independent of these clinical parameters. Immunohistochemically, no or less expression of RXRy was demonstrated in non-neoplastic thyroid tissue, however, strong and diffuse expression was found in carcinoma cells. Interestingly, those follicular cells with marked Hashimoto thyroiditis also showed diffuse and strong immunoreaction too. Moreover,4. The immunohistochemical features of tumor cells with LOP/C and without LOP/C in the same case were compared. Immunohistochemically, decrease or loss of TTF-1 expression in the nuclei was demonstrated in tumor cells with LOP/C in all the 16 cases, compared with those intense reaction in PTC tumor cells without LOP/C. The expression of E-cadherin was decreased in the cell membrane of the tumor cells with LOP/C, and the expression ofβ-catenin was decreased in cell membrane but increased in the cytoplasm and nuclei in PTC tumor cells with LOP/C. Although the majority of PTC expressed vimentin predominantly in the subnucleus and basal part of the cytoplasm, it was expressed extensively and strongly in the cytoplasm of the tumor cells with LOP/C.5. Multivariate analysis further indicated that LOP/C, as well as extrathyroid invasion, was one of the two independent variables indicating LNM at surgery. The risk for an individual with extrathyroid invasion to have LNM increased more than 7 times (Odds ratio,7.556) than those without. Moreover, the risk for an individual with LOP/C to have LNM increased more than 6 times (Odds ratio,6.346) than those without. Both extrathyroid invasion (Odds ratio,8.759) and advanced tumor stage (Odds ratio,6.143) are independent indicator of LNM in papillary thyroid carcinoma.Conclusions1. Both RXRγand LOP/C have correlated with the extrathyroid invasion and LNM of PTC, which suggest that LOP/C could be regarded as a morphological indicator of the prognosis of PTC. Moreover, the significant correlation between RXRy and tumor invasion indicate there should be a way to cure aggressive PTC through regulating RXRy. 2. Overall, PTC with LOP/C was demonstrated to have less immunoreaction with TTF-1, E-cadherin and aberrant immunoreaction withβ-catenin and vimentin which indicate that they have less differentiated or immature follicular cells characteristics. LOP/C may be a useful morphological feature of EMT under H&E observation, and it should be an important indicator of lymph node metastasis and aggressive clinical behavior of papillary thyroid carcinoma.3. RXRγup-regulation correlate with EMT and tumor progression in PTC.