The P2RY2 Receptor Induces Carcinoma Cell Migration and EMT Through Cross‐Talk With Epidermal Growth Factor Receptor

Abstract

Extracellular nucleotides are signaling elements present in the tumor microenvironment; however, their role in tumor growth is not completely understood. In the present study, we asked whether nucleotides regulate cell migration in ovarian carcinoma-derived cells. We observed that 100 μM UTP induced migration in SKOV-3 cells (1.57 ± 0.08 fold over basal), and RT-PCR showed expression of transcripts for the P2RY2 and P2RY4 receptors. Knockdown of P2RY2 expression in SKOV-3 cells (P2RY2-KD) abolished the UTP-induced migration. The mechanism activated by UTP to induce migration involves transactivation of the epidermal growth factor receptor (EGFR) since we observed that the EGFR kinase inhibitor AG1478 and the PI3K inhibitor Wortmannin inhibit this response (to 0.76 ± 0.23 and 0.46 ± 0.14 relative to the control, respectively). In agreement with these observations, UTP was able to modify the phosphorylation state of the EGFR; likewise, the induction of ERK1/2 phosphorylation promoted by UTP was abolished by a 30-60 min treatment with AG1478. Our data also suggested that the enhanced cell migration involves the epithelium to mesenchymal transition (EMT) process, since a 12 h stimulation of SKOV-3 cells with 100 μM UTP showed an increase in vimentin and SNAIL protein levels (459.8 ± 132.4% over basal for SNAIL). Interestingly, treatment with apyrase (10 U/mL) reduces the migration of control cells and induces a considerable enrichment of E-cadherin in the cell-cell contacts, favoring an epithelial phenotype and strongly suggesting that the nucleotides released by tumor cells and acting through the P2RY2 receptor are potential regulators of invasiveness.

Cellular Migration Ability Is Modulated by Extracellular Purines in Ovarian Carcinoma SKOV‐3 Cells

“…In previous studies, we demonstrated that SKOV‐3 cells release ATP into the extracellular medium [Vázquez‐Cuevas et al, ]. In addition, we showed that the incubation of SKOV‐3 cell cultures with apyrase induced a relocation of E‐cadherin from the cytosol to the cellular junctions [Martínez‐Ramírez et al, ].…”

“…In previous studies on ovarian carcinoma‐derived SKOV‐3 cells, we observed that purinergic signaling is able to mediate the induction of epithelium to mesenchymal transition (EMT). These cells release ATP into the extracellular space, and the addition of apyrase to the culture medium favored the epithelial phenotype, as evidenced by the location of E‐cadherin in the cellular contact zone [Martínez‐Ramírez et al, ]. Our observations led us to hypothesize that there was a balance between ATP released in SKOV‐3 cells and its catabolites, produced by ectonucleotidase activity and resulting purinergic receptor responses.…”

Nucleotides and nucleoside signaling in the regulation of the epithelium to mesenchymal transition (EMT)

“…Interestingly, treatment with apyrase, an ectonucleotidase that cleaves ATP to ADP and AMP, reduced basal migration and favored the epithelial phenotype. This action promoted a relocation of E-cadherin to cellular junctions, strongly suggesting that dephosphorylated metabolites of ATP participate in the control of EMT induction [89]. The increment in invasiveness, together with the downregulation of epithelial markers and the expression of mesenchymal markers mediated by the P2Y2 receptor, was also demonstrated in a highly metastatic breast cancer cellular line named MDA-MB-231, through a pathway mediated by PKC activity [90].…”

Assessment of cellular and serum proteome from tongue squamous cell carcinoma patient lacking addictive proclivities for tobacco, betel nut, and alcohol: Case study

“…A total of 1000 ng normalized RNA samples were reverse transcribed using a RevertAid™ H Minus first strand cDNA synthesis kit (Thermo Fisher Scientific). The integrity of the cDNA was assured by PCR with GAPDH specific primers targeting 496 bp sequence in the GAPDH cDNA (Table ); the reaction constituted 1 μL cDNA, 1× Taq buffer, 1 U Taq polymerase, 1.5 mM MgCl 2 , 0.4 mM dNTPs, 0.4 μM each primers; thermal conditions (i) 94°C for 3 min (initial denaturation) (ii) 35 cycles at 94°C for 1 min, 58°C for 1 min, 72°C for 1 min (iii) final extension (72°C for 10 min) . Relative quantitative PCR was performed on Stratagene Mx3005p real‐time PCR cycler (Agilent) using SYBR green dye chemistry.…”