Extracellular ATP is Differentially Metabolized on Papillary Thyroid Carcinoma Cells Surface in Comparison to Normal Cells

Abstract

The incidence of differentiated thyroid cancer has been increasing. Nevertheless, its molecular mechanisms are not well understood. In recent years, extracellular nucleotides and nucleosides have emerged as important modulators of tumor microenvironment. Extracellular ATP is mainly hydrolyzed by NTPDase1/CD39 and NTPDase2/CD39L1, generating AMP, which is hydrolyzed by ecto-5'-nucleotidase (CD73) to adenosine, a possible promoter of tumor growth and metastasis. There are no studies evaluating the expression and functionality of these ectonucleotidases on normal or tumor-derived thyroid cells. Thus, we investigated the ability of thyroid cancer cells to hydrolyze extracellular ATP generating adenosine, and the expression of ecto-enzymes, as compared to normal cells. We found that normal thyroid derived cells presented a higher ability to hydrolyze ATP and higher mRNA levels for ENTDP1-2, when compared to papillary thyroid carcinoma (PTC) derived cells, which had a higher ability to hydrolyze AMP and expressed CD73 mRNA and protein at higher levels. In addition, adenosine induced an increase in proliferation and migration in PTC derived cells, whose effect was blocked by APCP, a non-hydrolysable ADP analogue, which is an inhibitor of CD73. Taken together, these results showed that thyroid follicular cells have a functional purinergic signaling. The higher expression of CD73 in PTC derived cells might favor the accumulation of extracellular adenosine in the tumor microenvironment, which could promote tumor progression. Therefore, as already shown for other tumors, the purinergic signaling should be considered a potential target for thyroid cancer management and treatment.

Keywords: Adenosine; CD73; Extracellular ATP; NTPDases; Purinergic signaling; Thyroid papillary carcinoma.

Fig. 1

ATP, ADP and AMP hydrolysis on the surface of thyroid cells. Thyroid cells were incubated in phosphate-free buffer containing 1 mM of nucleotides at 37 °C for 10, 20, 30 and 60 min, as described in material and methods. In normal thyroid-derived cells, a Nthy-1, b FRTL-5 and c PCCL3, ATP hydrolysis was higher than AMP, while in papillary thyroid carcinoma derived cells, d TPC-1 and e K1, AMP hydrolysis was higher than ATP and ADP. Data are expressed as mean ± SD of three experiments, performed in duplicates

Fig. 2

Metabolism of extracellular ATP and expression of NTPDases by normal and thyroid cancer cells. The hydrolysis of extracellular ATP and formation of degradation product were analyzed by HPLC in comparison with reference standards. a FRTL-5, b TPC-1 and c Nthy-1 cells were incubated with 100 μM ATP and supernatant aliquots were collected after 0, 10, 20, 30, 60, 90 and 120 min. ATP, ADP, AMP and adenosine (ADO) were quantified by HPLC. Data are shown as mean ± SD of triplicates. d The expression of NTPDases in normal and thyroid cancer cell lines was analyzed by RT-qPCR. mRNA levels were normalized by ACTB gene expression. ENTPD1 and ENTPD2 mRNA were more strongly expressed in cells derived from normal thyroid, while ENTPD3 mRNA was identified only in tumor-derived thyroid cells. mRNA levels of ENTPD5 and ENTPD6 were similar in normal and tumor cells. NTPDases expression is shown as mean ± SD of at least three experiments, performed in duplicates

Fig. 3

APCP inhibits AMP hydrolysis in thyroid cancer TPC-1 cells. a TPC-1 cells were incubated with 1 mM AMP without (black bars) or with (gray bars) 10 μM APCP. AMPase activity was determined by the Malachite Green assay. Data are expressed as nmol Pi released/mg of protein and represent the mean ± SD of three experiments in triplicates. b HPLC analysis showing extracellular AMP metabolism in TPC-1cells, after incubation with 100 μM of AMP without (solid line; green) or with (dotted line; red) 1 μM APCP. APCP decreased adenosine (ADO) formation. AMP and ADO were quantified by HPLC in comparison with reference standards. c The expression of CD73 was analyzed by RT-qPCR and mRNA level was normalized with ACTB. The ectonucleotidase expression was higher (~10 fold) in tumor derived thyroid cells when compared to cells derived from normal thyroid. Data are shown as mean ± SD of three experiments, performed in duplicates. d Western blot of CD73 in normal thyroid cell line (Nthy-1) and tumoral thyroid cell (PTC-1) obtained from three independent cell cultures (n1, n2 and n3). The loading control (LC) shows representative bands of the Coomassie-stained membrane

Fig. 4

Effect of nucleotides on TPC-1 migration and proliferation in vitro. a TPC-1 cells were scratch-wounded with a micropipette tip (200 μl) and treated with serum free DMEM; or serum free DMEM plus 100 μM ATP; or 100 μM AMP; or 100 μM AMP plus 10 μM APCP; or 100 μM ADO. The wound gap was evaluated at 0, 3, 6, and 24 h post-scratching. Mean relative quantification of the fraction of the wound that remains uncovered by the migratory cells as a function of time for each treatment are shown. Bars represent means ± SEM; n = 3. ª adenosine vs. control p < 0.0001; ^b ATP vs. control p < 0.0001 and, adenosine vs. control p < 0.00001. b Determination of ATP, ADP, AMP and, adenosine effect by cell counting in a Neubauer cell chamber. After 48 h of exposure with AMP and adenosine, the percentage of TPC-1 cells was significantly elevated as compared to the control group (# AMP vs. control p=0.001; ## adenosine vs. control p=0.001). c The RT-qPCR analysis showed that Ki-67 mRNA level was increased, but not significantly, in TPC-1 cells treated with AMP or adenosine. The stimulatory effect on TPC-1 migration and proliferation caused by AMP was reverted in co-treatment with APCP (Adenosine 5′-(α,β-methylene)- diphosphate)

Fig. 5

Schematic illustration summarizing nucleotide metabolism and ectonucleotidases expression profile in normal and papillary thyroid cancer (PTC) cells. Normal thyroid cells express higher levels of ENTPD1 and 2, when compared to PTC cells, leading to increased extracellular ATP hydrolysis with consequent accumulation of ADP and AMP. In the other hand, PTC cells express ENTPD3, while ENTPD1 and 2 are absent, decreasing ATP metabolism, and its accumulation in tumor microenvironment. As CD73 is highly expressed in TPC-1 and K1 cells in comparison with normal cells, AMP is immediately metabolized to adenosine. Therefore, in the thyroid tumor microenvironment, a crosstalk between normal cells, which are more efficient to hydrolyze ATP and ADP, could provide AMP to be dephosphorylated to adenosine by tumor cells. Adenosine could be involved in several pro-tumorigenic features, as immunosuppression and angiogenesis, in the tumor microenvironment