Imbalanced CRABP-? And FABP5 Expression And Its Relevance With Retinoic Acid Resistance Of Anaplastic Thyroid Cancers:An In Vitro And In Vivo Investigation

Background and objective:In endocrine malignancies thyroid cancer is frequent occurrence and accounts for the majority of endocrine-related cancer deaths.Most thyroid cancers(papillary and follicular types)arise from the follicular epithelium with well differentiated phenotype and,therefore,have better prognosis after thyroidectomy.Nevertheless,the anaplastic thyroid cancer(ATC),though in low incidence(about 2% in total TC cases),grow aggressively with high dissemination rates and lead to majority of ATC patients died shortly.To prevent tumor recurrence,post-operative radiotherapy,chemotherapy or their combination have been employed,of which all-trans retinoic acid/RA,a well-known cell differentiation promoter,is used to enhance radio-sensitivity of ATC patients.The data coming from clinical practice reveal that the outcomes of this therapeutic regimen are not identical.For instance,the aggressive behavior of thyroid carcinoma can be suppressed by all-trans retinoic acid induced re-differentiation;On the other hand,tretinoin therapy is not completely effective on highly aggressive thyroid cancer.Because RA-based re-differentiation therapy has been already applied in clinic,it would be necessary to cautiously evaluate the suitability of RA in ATC management.Because the re-differentiated cancer cells usually exhibit growth arrest and are sensitized to anticancer therapies,RA has been used to overcome radioiodine-resistance of thyroid cancers by promoting radioiodine uptake.However,as mentioned above,the therapeutic consequences are quite different.It has been recognized that RA signaling can be transmitted by CRABP-? as cancer suppressing and by FABP5 as cancer promoting pathway,respectively.In this context,the statuses of CRABP-? and FABP5 expression would be the critical factors in determining the fates of RA-treated cancer cells.Alternatively,the imbalanced patterns of CRABP-? and FABP5 expression may lead to different therapeutic outcomes.However,no report has been available concerning the expression patterns of these two RA-related factors in anaplastic thyroid cancers in vitro and in vivo.The current study aims to shed light on the underlying reason(s)resulting in unresponsiveness of ATC cells to RA treatment via checking and manipulating levels of CRABP-? and FABP5 expression in three RA-resistant ATC cell lines by multiple experimental approaches.Meanwhile,the CRABP-? and FABP5 expression patterns in the surgical specimens of thyroid cancers and their noncancerous counterparts are profiled by tissue microarray-based immunhistochemical staining and their potential relevance with RA sensitivities is elucidated.Materials and Method:Three human ATC cell lines,THJ-11 T,16T and 21 T cell lines,were kindly provided by Professor Professor Quentin Liu,Institute of Cancer Stem Cell,Dalian Medical University.286 achieved paraffin blocks of thyroid surgical specimens were kindly provided for tissue microarray construction by the Department of Clinical Pathology,the First Affiliated Hospital of Dalian Medical University and Department of Laboratory Medicine,the Second Hospital of South China University of Technology without impacting pathological diagnosis.In this study,cell culture,ICC,MTT assay,RT-PCR,HE staining,Western-blotting,IHC,IF and paraffin tissue microarrays and other methods carried out the following: 1)HE staining and MTT assay was used to observe;2)ICC,IF and Western Blot were used to observe the expression of CRABP-? and FABP5 in three cell lines,and the effects of CRABP-? FABP5 expression in three undifferentiated thyroid cancer cell lines;3)RT-PCR detection of retinoic acid on CRABP-? and FABP5 transcriptional level;4)using microscopic observation,HE staining and MTT detection of demethylation The changes of retinoic acid sensitivity in three cell lines after combination of agent and FABP5 inhibitor and retinoic acid,and the changes of CRABP-? and FABP5 in three cell lines before and after dosing were detected by ICC;5)Tissue microarray combined with IHC technique was used to detect the expression of CRABP-? and FABP5.Result:1.Hematoxylin and eosin(H/E)staining revealed no distinct morphological change of the three RA-treated ATC cell lines in comparison with their normally cultured or DMSO treated counterparts.MTT assays results showed that the OD values of the RA treatment group was higher than that of the normally cultured and DMSO treated cells.RA treated compared with the normally cultured cells,the difference was statistically significant(p <0.05),but small.2.The levels of CRABP-? expression were very low(faint positive)in THJ-11 T,THJ-16 T and THJ-21 T cells by ICC and IF staining,while FABP5 levels were distinctly high(strongly positive)in those ATC cells.In agreement with the above ICC and IF findings,RT-PCR and Western blot analyses show that CRABP-? is low in THJ-11 T cells and was undetectable in THJ-16 T and THJ-21 T cells.FABP5 is highly expressed in the three cell lines.3.THJ-11 T,THJ-16 T and THJ-21 T cells were treated with 5 ?M gemcitabine or 5 ?M gemcitabine and 10 ?M RA combination for 48 hours.MTT assays revealed that compared with the growth rate of non-medicated cells,the growth suppression rates of gemcitabine and gemcitabine/RA combination were 48.3% and 54.0% for THJ-11 T,41.2% and 51.3% for THJ-16 T cells,42.8% and 51.6% for THJ-21 T cells.In comparison with the growth rates of the cells treated by 10 ?M RA only,the growth suppression rates of gemcitabine and gemcitabine/RA combination were 51.9% and 57.2% for THJ-11 T,46.3% and 55.6% for THJ-16 T cells,50.0% and 54.3% for THJ-21 T cells,respectively.Significant differences of inhibitory rates can be established between the gemcitabine and the gemcitabine/RA combination treated cells4.To determine the effect of FABP5 inhibition on the RA resistant features of anaplastic thyroid cancer cells,BMS309403,a competitive inhibitor of FABP5,was used independently,prior to or concurrently with RA treatment.The results from ICC showed reduction of FABP5 nuclear translocation in BMS309403 treated cells.H/E staining showed that the cells did not undergo appearance variation after BMS309403 treatment in the three manners,in comparison with the normally cultured cells and the cells treated with RA only.The results of MTT assay showed that it failed to reverse RA resistance of ATC cells5.A total of 286 thyroid specimens were classified into 4 pathological subtypes as normal thyroid tissue/N,adenoma/A,well differentiated thyroid carcinoma/DTC and anaplastic thyroid carcinoma/ATC.The patterns of CRABP-? and FABP5 expression in the four thyroid tissue groups were analyzed using tissue microarray-based immunohistochemical staining.The results showed highly variable CRABP-? and FABP5 expression patterns not only among the four thyroid tissue groups but also the individual cases within each of the histological groups.As shown in Figure 4B,the frequencies of CRABP-?-/FABP5-,CRABP-?+/FABP5+,CRABP-?+/FABP5-and CRABP-?-/FABP5+ patterns were 50%(15),16.7%(5),3.3%(1),30.0%(9)in 30 ATC cases.And of the five CRABP-?+/FABP5+ ATC cases,one(3.3%)shows predominant CRABP-?(CRABP-??/FABP5?),three(10%)show predominant FABP5(CRABP-??/FABP5?)and one(3.3%)shows balanced CRABP-??FABP5 expression pattern.Consequently,only one out of 30 ATC cases was found with relatively higher CRABP-? expression.Conclusion:1.Different anaplastic thyroid cancer cell lines have different sensitivity to RA,and RA can promote the cell proliferation of THJ-11 T,THJ-16 T and THJ-21 T cells.2.Retinoic acid treatment can change the expression patterns of CRABP-? and FABP5 in anaplastic thyroid cancer cell lines.Examination of CRABP-? and FABP5 statuses may be the cutting edge to shed light on the reason(s)of RA-resistance of ATC cells.3.DNA demethylator and its combination therapy with RA significantly increased the retinoic acid sensitivity of THJ-11 T,THJ-16 T and THJ-21 T,and this combination therapy resulted in significant inhibition of cell proliferation and upregulation of CRABP-?.4.FABP5 inhibitor in combination with RA fail to alter RA sensitivity of THJ-16 T cells.5.The expression of CRABP-? and FABP5 in human thyroid cancer is correlated with the degree of differentiation of thyroid cancer.CRABP-? in anaplastic thyroid cancer is significantly down-regulated,which is closely related to RA resistance in anaplastic thyroid cancer.CRABP-? and FABP5 will be a pair of parameters used to predict the response of anaplastic thyroid cancer cells to RA therapy and therefore have important practical implications for the treatment of anaplastic thyroid cancer patients with individual RA.