br various types of cancer including
various types of cancer, including prostate, breast, colorectal, liver and CeCa [41,42]. In the particular case of CeCa, several studies have suggested that TGF-β1 expression directly correlates with the degree of disease progression [8,43–45]. In addition, a dual role of TGF-β1 has been suggested during the development of CeCa, either as an anti-on-cogenic factor in precancerous cervical lesions or as a tumor promoter in advanced stages [6,46]. In contrast, the expression of TGF-β1 in CeCa has been positively correlated with the expression of HR-HPV onco-genes . In fact, the E6 and E7 oncogenes of HPV-16 induce the activation of the human TGF-β1 promoter through the Sp1 recognition sequence . However, the production of high concentrations of TGF-
β in different tumors has been associated with hypoxia conditions that occur in the TME , suggesting that other factors can influence the production of TGF-β1 to promote tumor growth. Recent studies have shown that in pathological states, the activation of A2AR or A2BR in different cell types produces TGF-β to exert immunosuppressive and anti-inflammatory effects [34–36]. We previously reported that CeCa 946414-94-4 exposed to AMP generate large amounts of Ado to suppress the effector functions of CTL . The present study provided the first evidence that CeCa cells are induced to produce TGF-β1 through the adenosinergic pathway. CeCa cells cultured in the presence of AMP or Ado were induced to express and secrete TGF-β1, and blockade of A2AR and A2BR with specific antagonists reversed this effect. In addition, the produced TGF-β1 induced or maintained CD73 expression in CeCa tumor cells. It is known that Ado exerts inhibitory effects on the effector
cells of the immune system through its interaction with A2AR and A2BR, which are coupled to G proteins and increase the levels of cAMP, thereby decreasing the production of proinflammatory cytokines and increasing the synthesis of immunosuppressive cytokines, such as IL-10 and TGF-β [49–51]. In the present study, concentrations of Ado > 10 μM were necessary to induce an increase greater than 50% in the production of TGF-β1 in CeCa tumor cells with respect to the basal production, and the addition of ZM241385 and MRS1754 strongly re-versed the TGF-β1 production induced by Ado. These results suggested that signaling pathways similar to the effector cells of the immune system may occur in CeCa tumor cells to induce the production of TGF-β1 via A2AR and A2BR. Moreover, the present study also demonstrated that the TGF-β1 produced by tumor cells is important for maintaining or inducing CD73 expression in CeCa tumor cells. Addition of neu-tralizing anti-TGF-β or the inhibition of TGF-βRI strongly reversed CD73 expression in the CeCa cells, suggesting a feedback loop between the adenosinergic pathway and TGF-β1 production to maintain the immunosuppressive status of CeCa cells through generating Ado and TGF-β1, which promotes tumor progression. It is known that TGF-βRI is phosphorylated once TGF-β binds to TGF-βRII to constitute a functional receptor . Recently, a comprehensive molecular and integrative study of CeCa revealed that the TGFBR2 gene, present exclusively in cervical squamous tumors, is significantly mutated in more than 70% of analyzed tumors , thus illustrating the clinical importance of this pathway as a therapeutic target. According to the present study, the alterations reported in the signaling pathway of TGF-β in CeCa  may significantly contribute to maintain an active adenosinergic pathway and therefore the immunosuppressive capacity in the TME of CeCa.
The present study provided evidence for the first time that the adenosinergic pathway plays an important role in the induction of the secretion and expression of TGF-β1 in CeCa tumor cells through gen-eration of Ado and activation of A2AR and A2BR. Moreover, the present study demonstrated that TGF-β1 produced through this pathway maintains CD73 expression in tumor cells, suggesting a feedback loop to maintain the immunosuppressive status in CeCa through the production of Ado and TGF-β1. These results implied that this route may have clinical importance as a therapeutic target.
Conflict of interests
The authors declare that they have no conflicts of interest.
The present study was performed with the following funding: DGAPA-PAPIIT (Grant IN226516) to MLMG; CONACYT (Grant 240635) to AMG; and Instituto Mexicano del Seguro Social (IMSS) for supporting AMG (Grants FIS-1161, FIS-1014, FIS-1383 and FIS-1613). We also appreciate the support given to Dr. Rosario García Rocha for the Postdoctorate Scholarship Program of UNAM. We also thank Dr. Lorudes A. Arriaga Pizano and MSc. Jessica Lakshmi Prieto Chávez from the Flow Cytometry Instrument Center of IMSS for their support in the flow cytometry analysis as well as to Dr. Patricia Piña Sánchez of the Molecular Oncology Laboratory of UIMEO (XXI Century CMN, IMSS) for the allowing the use of necessary equipment for the analysis of the cell samples.