Expression pattern of carbonic anhydrase IX in Medullary thyroid carcinoma supports a role for RET-mediated activation of the HIF pathway.

Medullary thyroid carcinoma is a relatively rare tumor with poor prognosis and therapy response. Its phenotype is determined by both genetic alterations (activating RET oncoprotein) and physiological stresses, namely hypoxia [activating hypoxia-inducible factor (HIF)]. Here, we investigated the cooperation between these two mechanisms. The idea emerged from the immunohistochemical analysis of carbonic anhydrases (CA) IX and XII expression in thyroid cancer. Although CAXII was present in all types of thyroid carcinomas, CAIX, a direct HIF target implicated in tumor progression, was associated with aggressive medullary and anaplastic carcinomas, and its expression pattern in medullary thyroid carcinomas suggested contribution of both hypoxic and oncogenic signaling. Therefore, we analyzed the CA9 promoter activity in transfected tumor cells expressing RET and/or the HIF-α subunit. We showed that overexpression of both wild-type and mutant RET can increase the CA9 promoter activity induced by HIF-1 (but not HIF-2) in hypoxia. Similar results were obtained with another HIF-1-regulated promoter derived from the lactate dehydrogenase A gene. Moreover, inhibition of the major kinase pathways, which transmit signals from RET and regulate HIF-1, abrogated their cooperative effect on the CA9 promoter. Thus, we brought the first experimental evidence for the crosstalk between RET and HIF-1 that can explain the increased expression of CAIX in medullary thyroid carcinoma and provide a rationale for therapy simultaneously targeting both pathways.

In vivo 5FU-exposed human medullary thyroid carcinoma cells contain a chemoresistant CD133+ tumor-initiating cell subset.

BACKGROUND: The hierarchical model of solid tumor proposes the existence of rare tumor cell subpopulations with stem-cell properties. The glycoprotein prominin-1 (CD133) represents one of the cancer stem-cell markers in several tumor types. The CD133+ cell subpopulation was shown to be enriched for tumor-initiating and highly chemoresistant cells in human cancer(s). METHODS: We investigated whether CD133+ cells derived from human medullary thyroid carcinoma (MTC) possess tumor-initiating properties in vivo and exhibit differential responses to chemotherapeutic agents. We demonstrated that separated CD133+ cells from the human MTC cell line TT are enriched for tumor-initiating cells as demonstrated by tumor formation in vivo. Nevertheless, TT CD133+ cells do not exhibit increased chemoresistance in comparison to parental cells. However, when MTC xenotransplants were treated with the chemotherapeutic drug 5-fluorouracil (5FU) in vivo, CD133 expression increased in MTC cells. RESULTS: This cell line, designated FTTiv isolated from the drug-exposed xenotransplants, exhibits a significantly different response to 5FU associated with the substantial change in the expression profile of genes involved in 5FU metabolism and drug resistance. Moreover, the CD133+ tumor-initiating subpopulation derived from these drug-exposed FTTiv cells is significantly more resistant to 5FU and retains the chemoresistant properties upon FTTiv culture propagation. CONCLUSIONS: These data suggest that the chemoresistant phenotype and the CD133+ MTC subpopulation emerged in response to chemotherapy in vivo.

Local bystander effect induces dormancy in human medullary thyroid carcinoma model in vivo.

The extent of local bystander effect induced by fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (yCD) in combination with 5-fluorocytosine (5FC) was evaluated in xenogeneic model of human medullary thyroid carcinoma (MTC). This approach to gene-directed enzyme/prodrug therapy (GDEPT) induces strong bystander cytotoxicity. Effector yCD-TT mixed with target EGFP-TT cells in a ratio 2:9 could achieve significant tumor regression and 14-fold decrease in serum marker calcitonin upon 5FC administration. Histopathological analysis unraveled that antitumor effect resulted in tumor dormancy and proliferation arrest of remaining tumor cell clusters in vivo. yCD/5FC combination represents another GDEPT approach to achieve tumor growth control in MTC.

Effect of methotrexate on inflammatory cells redistribution in experimental adjuvant arthritis.

The aim of this study was to evaluate the morphological changes in the spleen, the thymus and the knee joints of rats with experimental adjuvant arthritis induced by Mycobacterium butyricum in the incomplete Freund's adjuvant and the effect of treatment with methotrexate (MTX). Particular attention was aimed on the redistribution of granulocytes in the tissues during the inflammatory process. Clinical parameters, e.g., joint edema, body weight and of gamma glutamyl transferase (GGT) activity as an inflammatory marker, have also been determined. Induction of adjuvant arthritis caused a significant decrease in granulocyte number in the spleen and vice versa a significant increase in the knee joints, but without significant changes in the thymus. Treatment with methotrexate reversed this phenomenon by increasing the granulocyte number in the spleen and decreasing it in knee joints. MTX decreased the joint edema as well as the activity of GGT in the spleen, modified the size of the white pulp of the spleen and increased the cortex/medulla ratio in the thymus. The observed changes support the anti-inflammatory and immunomodulatory properties of MTX supporting its use as the first-line medication in patients with rheumatoid arthritis.

Bystander cytotoxicity in human medullary thyroid carcinoma cells mediated by fusion yeast cytosine deaminase and 5-fluorocytosine.

In our work, we have evaluated efficiency of gene-directed enzyme/prodrug therapy (GDEPT) based on combination of fusion yeast cytosine deaminase (yCD) and 5-fluorocytosine (5FC) on model human medullary thyroid carcinoma (MTC) cell line TT. We determined the efficiency of this GDEPT approach in suicide and bystander cytotoxicity induction. We have shown significant bystander effect in vitro and 5FC administration resulted in potent antitumor effect in vivo. Furthermore, we have unraveled high efficiency of cell-mediated GDEPT, when human mesenchymal stromal cells (MSC) were used as delivery vehicles in direct cocultures in vitro. Nevertheless, effector MSC exhibited inhibitory effect on TT cell proliferation and abrogated TT xenotransplant growth in vivo. We suggest that yCD/5FC combination represents another experimental treatment modality to be tested in MTC and our data further support the exploration of MSC antitumor potential for future use in metastatic MTC therapy.