Adenosine increases PD-L1 expression in mesenchymal stromal cells derived from cervical cancer through its interaction with A2AR/A2BR and the production of TGF-ß1.

Mesenchymal stromal cells (MSCs) together with malignant cells present in the tumor microenvironment (TME), participate in the suppression of the antitumor immune response through the production of immunosuppressive factors, such as transforming growth factor beta 1 (TGF-ß1). In previous studies, we reported that adenosine (Ado), generated by the adenosinergic activity of cervical cancer (CeCa) cells, induces the production of TGF-ß1 by interacting with A2AR/A2BR. In the present study, we provide evidence that Ado induces the production of TGF-ß1 in MSCs derived from CeCa tumors (CeCa-MSCs) by interacting with both receptors and that TGF-ß1 acts in an autocrine manner to induce the expression of programmed death ligand 1 (PD-L1) in CeCa-MSCs, resulting in an increase in their immunosuppressive capacity on activated CD8+ T lymphocytes. The addition of the antagonists ZM241385 and MRS1754, specific for A2AR and A2BR, respectively, or SB-505124, a selective TGF-ß1 receptor inhibitor, in CeCa-MSC cultures significantly inhibited the expression of PD-L1. Compared with CeCa-MSCs, MSCs derived from normal cervical tissue (NCx-MSCs), used as a control and induced with Ado to express PD-L1, showed a lower response to TGF-ß1 to increase PD-L1 expression. Those results strongly suggest the presence of a feedback mechanism among the adenosinergic pathway, the production of TGF-ß1, and the induction of PD-L1 in CeCa-MSCs to suppress the antitumor response of CD8+ T lymphocytes. The findings of this study suggest that this pathway may have clinical importance as a therapeutic target.

STAT5 Is Necessary for the Metabolic Switch Induced by IL-2 in Cervical Cancer Cell Line SiHa.

The tumor cells reprogram their metabolism to cover their high bioenergetic demands for maintaining uncontrolled growth. This response can be mediated by cytokines such as IL-2, which binds to its receptor and activates the JAK/STAT pathway. Some reports show a correlation between the JAK/STAT pathway and cellular metabolism, since the constitutive activation of STAT proteins promotes glycolysis through the transcriptional activation of genes related to energetic metabolism. However, the role of STAT proteins in the metabolic switch induced by cytokines in cervical cancer remains poorly understood. In this study, we analyzed the effect of IL-2 on the metabolic switch and the role of STAT5 in this response. Our results show that IL-2 induces cervical cancer cell proliferation and the tyrosine phosphorylation of STAT5. Also, it induces an increase in lactate secretion and the ratio of NAD+/NADH, which suggest a metabolic reprogramming of their metabolism. When STAT5 was silenced, the lactate secretion and the NAD+/NADH ratio decreased. Also, the expression of HIF1α and GLUT1 decreased. These results indicate that STAT5 regulates IL-2-induced cell proliferation and the metabolic shift to aerobic glycolysis by regulating genes related to energy metabolism. Our results suggest that STAT proteins modulate the metabolic switch in cervical cancer cells to attend to their high demand of energy required for cell growth and proliferation.

Immune microenvironment of cervical cancer and the role of IL-2 in tumor promotion.

The tumor microenvironment (TME) is a heterogeneous mixture of resident and tumor cells that maintain close communication through their secretion products. The composition of the TME is dynamic and complex among the different types of cancer, where the immune cells play a relevant role in the elimination of tumor cells, however, under certain circumstances they contribute to tumor development. In cervical cancer (CC) the human papilloma virus (HPV) shapes the microenvironment in order to mediate persistent infections that favors transformation and tumor development. Interleukin-2 (IL-2) is an important TME cytokine that induces CD8+ effector T cells and NKs to eliminate tumor cells, however, IL-2 can also suppress the immune response through Treg cells. Recent studies have shown that CC cells express the IL-2 receptor (IL-2R), that are induced to proliferate at low concentrations of exogenous IL-2 through alterations in the JAK/STAT pathway. This review provides an overview of the main immune cells that make up the TME in CC, as well as the participation of IL-2 in the tumor promotion. Finally, it is proposed that the low density of IL-2 produced by immunocompetent cells is used by tumor cells through its IL-2R as a mechanism to proliferate simultaneously depleting this molecule in order to evade immune response.

Inhibition of CD73 expression or A2AR blockade reduces MRP1 expression and increases the sensitivity of cervical cancer cells to cisplatin.

Recently, a link between the biological activity of CD73 in solid tumors and multidrug resistance protein (MRP) has been proposed. Cisplatin (CP) is the most widely used anticancer agent to treat advanced and recurrent cervical cancer (CC). However, multidrug resistance protein-1 (MRP1) is overexpressed in approximately 85% of these tumors and has been strongly associated with cisplatin resistance (CPR). In this study, we examine the involvement of CD73 and the interaction of adenosine (ADO) with its receptors (ARs) in MRP1 expression in CC cells. We found that ADO positively modulates MRP1 expression in CC cells in a dose-dependent manner. The inhibition of CD73 expression with a CD73-targeted siRNA and A2AR blockade with the selective antagonist ZM241385 significantly decreased MRP1 expression and the extrusive capacity of CC cells, making them significantly more sensitive to CP treatment than cancer cells treated with MK-751, a specific MRP1 inhibitor. These results suggest CD73 inhibition or blocking ADO signaling through A2AR could be strategies to reverse CPR in patients with advanced or recurrent CC, which is characterized by very low response rates to CP (10%-20%).

Role of SIRT1 in Chemoresistant Leukemia.

Leukemias of the AML, CML, and CLL types are the most common blood cancers worldwide, making them a major global public health problem. Furthermore, less than 24% of patients treated with conventional chemotherapy (low-risk patients) and 10-15% of patients ineligible for conventional chemotherapy (high-risk patients) survive five years. The low levels of survival are mainly due to toxicity and resistance to chemotherapy or other medication, the latter leading to relapse of the disease, which is the main obstacle to the treatment of leukemia. Drug resistance may include different molecular mechanisms, among which epigenetic regulators are involved. Silent information regulator 2 homolog 1 (SIRT1) is an epigenetic factor belonging to the sirtuin (SIRT) family known to regulate aspects of chromatin biology, genome stability, and metabolism, both in homeostasis processes and in different diseases, including cancer. The regulatory functions of SIRT1 in different biological processes and molecular pathways are dependent on the type and stage of the neoplasia; thus, it may act as both an oncogenic and tumor suppressor factor and may also participate in drug resistance. In this review, we explore the role of SIRT1 in drug-resistant leukemia and its potential as a therapeutic target.

Inhibition of adenosine deaminase activity reverses resistance to the cytotoxic effect of high adenosine levels in cervical cancer cells.

Adenosine (ADO) generation in the tumor microenvironment (TME) plays important roles in the promotion of tumor growth, invasion, and metastasis and in suppression of the antitumor immune response. Recently, adenosine deaminase (ADA) activity in the TME has been proposed to be a compensatory mechanism against toxic accumulation of ADO in cancerous tissues. In the present study, the expression and functional activity of ADA in cervical cancer (CeCa) tumor cells were analyzed: C33A (HPV-), CaSki (HPV + ), and HeLa (HPV + ) cells. CeCa tumor cells, as well as activated T lymphocytes (ATLs), which were used as a positive control, showed different ADA contents in the membrane and intracellularly and a strong ability to convert ADO into inosine (INO). Treatment of tumor cells with EHNA, a specific ADA inhibitor, decreased the viability of CeCa tumor cells in a dose-dependent manner. In C33A (EHNA half maximal inhibitory concentration (IC50) = 374 µM), CaSki (EHNA IC50 = 273.6 µM), and HeLa (EHNA IC50 = 252.2 µM) cells, EHNA strongly reversed the resistance of tumor cells to the cytotoxic effect of high concentrations of ADO; 38.82 ± 3.1%, 47.18 ± 4.7%, and 71.63 ± 6.9% of the cells were apoptotic, and 40 ± 4.8%, 52 ± 5.3% and 70 ± 6.8% of the cells had mitochondrial membrane damage, respectively. In ATLs (EHNA IC50 = 391.8 µM) treated with EHNA, 32.4 ± 4.4% were apoptotic, and 32 ± 4.3% had mitochondrial membrane damage. These results suggest that the presence and activity of ADA in CeCa tumor cells can provide protection against the cytotoxic effect of high ADO contents in the TME. Therefore, the inhibition of ADA could be a strategy for the treatment of CeCa.

Evidence that cervical cancer cells cultured as tumorspheres maintain high CD73 expression and increase their protumor characteristics through TGF-ß production.

Recently, a link between the biological activity of CD73 and tumorigenicity in solid tumors has been proposed. We previously reported that the generation of adenosine (Ado) by the activity of CD73 in cervical cancer (CC) cells induces transforming growth factor-beta 1 (TGF-ß1) production to maintain CD73 expression. In the present study, we analyzed the participation of TGF-ß1 in CD73 expression and the development of protumoral characteristics in CaSki CC cells cultured as tumorspheres (CaSki-T) and in monolayers (CaSki-M). Compared with those in CaSki-M cells, CD73 expression and Ado generation ability were significantly increased in CaSki-T cells. CaSki-T cells exhibited enrichment in the CSC-like phenotype due to increases in the expression levels of stem cell markers (CD49f, CK17, and P63; OCT4 and SOX2), greater sphere formation efficiency (SFE), and an increase in the percentage of side population (SP) cells. Interestingly, compared with CaSki-M cells, CaSki-T cells produced a greater amount of TGF-ß1 and presented a marked protumor phenotype characterized by a significant decrease in the expression of major histocompatibility complex class-I (MHC-I) molecules, an increase in the expression of multidrug resistance protein-I (MRP-I) and vimentin, and an increase in the protein expression levels of Snail-1 and Twist, which was strongly reversed with TGF-ß1 inhibition. These results suggest that the presence of TGF-ß1-CD73-Ado feedback loop can promote protumoral characteristics in the CC tumor microenvironment.

Evidence that the viral oncoproteins E6 and E7 of HPV induce the expression of a functional IL-2R on cervical cancer cells.

HPV-positive (HPV+) cervical cancer (CC) cells have been reported to express the IL-2 receptor (IL-2R) in contrast to virus-negative CC cells. This work was carried out to evaluate whether HPV infection induces IL-2R expression in CC cells. The analysis of the IL-2R expression data collected from The Cancer Genome Atlas (TCGA) and Genotype Tissue Expression project (GTEx) using the Xena platform demonstrate a higher expression of IL-2R subunits in CC tumors in comparison with normal tissues. Moreover IL-2Rß expression is consistently higher in HPV+ tumors versus HPV- tumors. Furthermore, it was demonstrated that transfection of the HPV E6/E7 genes into the C33A (HPV-) cell line promotes IL-2R expression and regulates proliferation in response to exogenous IL-2. Additionally, we found that HPV+ cell lines enhances their proliferation in co-culture with peripheral blood lymphocytes (PBLs). To corroborate that the viral proteins E6 and E7 were related to the effects mediated by IL-2, we used cells derived from the HeLa cell line in which the expression of E6/E7 has decreased, we found that it loses the ability to respond to the exogenous IL-2 stimuli. Finally, the importance of IL-2R in CC, as an immune escape mechanism, is discussed.

Inhibitors of Chemoresistance Pathways in Combination with Ara-C to Overcome Multidrug Resistance in AML. A Mini Review.

Acute myeloid leukemia (AML), the most common type of leukemia in older adults, is a heterogeneous disease that originates from the clonal expansion of undifferentiated hematopoietic progenitor cells. These cells present a remarkable variety of genes and proteins with altered expression and function. Despite significant advances in understanding the molecular panorama of AML and the development of therapies that target mutations, survival has not improved significantly, and the therapy standard is still based on highly toxic chemotherapy, which includes cytarabine (Ara-C) and allogeneic hematopoietic cell transplantation. Approximately 60% of AML patients respond favorably to these treatments and go into complete remission; however, most eventually relapse, develop refractory disease or chemoresistance, and do not survive for more than five years. Therefore, drug resistance that initially occurs in leukemic cells (primary resistance) or that develops during or after treatment (acquired resistance) has become the main obstacle to AML treatment. In this work, the main molecules responsible for generating chemoresistance to Ara-C in AML are discussed, as well as some of the newer strategies to overcome it, such as the inclusion of molecules that can induce synergistic cytotoxicity with Ara-C (MNKI-8e, emodin, metformin and niclosamide), subtoxic concentrations of chemotherapy (PD0332991), and potently antineoplastic treatments that do not damage nonmalignant cells (heteronemin or hydroxyurea + azidothymidine).

Adenosine augments the production of IL-10 in cervical cancer cells through interaction with the A2B adenosine receptor, resulting in protection against the activity of cytotoxic T cells.

Cervical cancer (CeCa) produces large amounts of IL-10, which downregulates the major histocompatibility complex class I molecules (HLA-I) in cancer cells and inhibits the immune response mediated by cytotoxic T lymphocytes (CTLs). In this study, we analyzed the ability of CeCa cells to produce IL-10 through the CD73-adenosine pathway and its effect on the downregulation of HLA-I molecules to evade CTL-mediated immune recognition. CeCa cells cultured in the presence of ≥10 µM AMP or adenosine produced 4.5-6 times as much IL-10 as unstimulated cells. The silencing of CD73 or the blocking of A2BR with the specific antagonist MRS1754 reversed this effect. In addition, IL-10 decreased the expression of HLA-I molecules, resulting in the protection of CeCa cells against the cytotoxic activity of CTLs. The addition of MRS1754 or anti-IL-10 reversed the decrease in HLA-I molecules and favored the cytotoxic activity of CTLs. These results strongly suggest the presence of a feedback loop encompassing the adenosinergic pathway, the production of IL-10, and the downregulation of HLA-I molecules in CeCa cells that favors immune evasion and thus tumor progression. This pathway may have clinical importance as a therapeutic target.

Detection of CD39 and a Highly Glycosylated Isoform of Soluble CD73 in the Plasma of Patients with Cervical Cancer: Correlation with Disease Progression.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the main factor in the development of cervical cancer (CC). The presence of immunosuppressive factors plays an important role in the development of this type of cancer. To determine whether CD39 and CD73, which participate in the production of immunosuppressive adenosine (Ado), are involved in the progression of CC, we compared the concentrations and hydrolytic activity of these ectonucleotidases in platelet-free plasma (PFP) samples between patients with low-grade squamous intraepithelial lesions (LSILs) (n = 18), high-grade squamous intraepithelial lesions (HSILs) (n = 12), and CC (n = 19) and normal donors (NDs) (n = 15). The concentrations of CD39 and CD73 in PFP increased with disease progression (r = 0.5929, p < 0.001). The PFP of patients with HSILs or CC showed the highest concentrations of CD39 (2.3 and 2.2 times that of the NDs, respectively) and CD73 (1.7 and 2.68 times that of the NDs, respectively), which were associated with a high capacity to generate Ado from the hydrolysis of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The addition of POM-1 and APCP, specific inhibitors of CD39 and CD73, respectively, inhibited the ADPase and AMPase activity of PFP by more than 90%. A high level of the 90 kD isoform of CD73 was detected in the PFP of patients with HSILs or CC. Digestion with endoglycosidase H and N-glycanase generated CD73 with weights of approximately 90 kD, 85 kD, 80 kD, and 70 kD. In addition, the levels of transforming grow factor-ß (TGF-ß) in the PFPs of patients with LSIL, HSIL and CC positively correlated with those of CD39 (r = 0.4432, p < 0.001) and CD73 (r = 0.5786, p < 0.001). These results suggest that persistent infection by HR-HPV and the concomitant production of TGF-ß promote the expression of CD39 and CD73 to favor CC progression through Ado generation.

Cervical cancer cells produce TGF-ß1 through the CD73-adenosine pathway and maintain CD73 expression through the autocrine activity of TGF-ß1.

In cancer, the adenosinergic pathway participates in the generation of an immunosuppressive microenvironment and in the promotion of tumor growth through the generation of adenosine (Ado). The present study analyzed the participation of Ado, generated through the functional activity of the cervical cancer (CeCa) pathway in CeCa cells, to induce the expression and secretion of TGF-ß1, as well as the participation of this factor to maintain CD73 expression. Ado concentrations greater than 10 µM were necessary to induce an increase of over 50% in the production and expression of TGF-ß1 in CeCa tumor cells. Blockade of A2AR and A2BR with the specific antagonists, ZM241385 and MRS1754, respectively, strongly reversed the production of TGF-ß1. TGF-ß1 produced by CeCa cells was necessary to maintain CD73 expression because the addition of anti-TGF-ß neutralizing antibodies or the inhibition of TGF-ßRI strongly reversed the expression of CD73 in the CeCa cells. These results suggested a feedback loop in CeCa cells that favors immunosuppressive activity through the production of TGF-ß1 and Ado as well as the autocrine activity of TGF-ß1 and expression of CD73.

HPV-16 Infection Is Associated with a High Content of CD39 and CD73 Ectonucleotidases in Cervical Samples from Patients with CIN-1.

The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-ß than those of CIN-1 patients negative for HPV-16 and ND. These results suggest that persistent infection with HR-HPV, mostly HPV-16, in CIN-1 patients may promote the expression of CD39 and CD73 through the production of TGF-ß in precursor lesions to generate an immunosuppressive microenvironment and allow its progression to CeCa.

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

BACKGROUND: In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. METHODS: We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). RESULTS: We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. CONCLUSIONS: This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

Pleiotropic Effects of IL-2 on Cancer: Its Role in Cervical Cancer.

IL-2 receptor (IL-2R) signalling is critical for normal lymphocyte proliferation, but its role in cervical cancer is not fully understood. The receptor is composed of three chains: IL-2α, IL-2ß, and IL-2γ. Intracellular signalling is initiated by ligand-induced heterodimerization of the IL-2ß and IL-2γ chains, resulting in the activation of multiple intracellular kinases. Recently, IL-2R was shown to be expressed on nonhaematopoietic cells, especially on several types of tumour cells. However, the function of this receptor on malignant cells has not been clearly defined. The expression of IL-2R and the production of IL-2 in cervical cancer cells have been documented as well as expression of molecules of the JAK-STAT pathway. In the current review we have highlighted the differences in the responses of molecules downstream from the IL-2R in normal lymphocytes and tumour cells that could explain the presence of tumour cells in an environment in which cytotoxic lymphocytes also exist and compete and also the effect of different concentrations of IL-2 that could activate effector cells of the immune system cells, which favour the elimination of tumour cells, or concentrations that may promote a regulatory microenvironment in which tumour cells can easily grow.

IL-2 enhances cervical cancer cells proliferation and JAK3/STAT5 phosphorylation at low doses, while at high doses IL-2 has opposite effects.

The IL-2R signaling is critical for normal lymphocyte proliferation. However, the role of the IL-2 signaling in cervical cancer is not yet fully understood. We show that in IL-2R-expressing cervical cancer cells, JAK1 molecules are not phosphorylated. At low doses of IL-2, the constitutive phosphorylation of JAK3 and STAT5 increases in the tumor cells and decreases in lymphocytes, whereas the opposite occurs at high doses of IL-2. Using AG-490, the activation of JAK3 and the proliferation of cervical cancer cells were inhibited. We describe differences in the response of molecules downstream the IL-2R in lymphocytes and tumor cells.

Immunization with an HPV-16 L1-based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model.

HPV L1-based virus-like particles vaccines (VLPs) efficiently induce temporary prophylactic activity through the induction of neutralizing antibodies; however, VLPs that can provide prophylactic as well as therapeutic properties for longer periods of time are needed. For this purpose, we generated a novel HPV 16 L1-based chimeric virus-like particle (cVLP) produced in plants that contains a string of T-cell epitopes from HPV 16 E6 and E7 fused to its C-terminus. In the present study, we analyzed the persistence of specific IgG antibodies with neutralizing activity induced by immunization with these cVLPs, as well as their therapeutic potential in a tumor model of C57BL/6 mice. We observed that these cVLPs induced persistent IgG antibodies for over 12 months, with reactivity and neutralizing activity for VLPs composed of only the HPV-16 L1 protein. Efficient protection for long periods of time and inhibition of tumor growth induced by TC-1 tumor cells expressing HPV-16 E6/E7 oncoproteins, as well as significant tumor reduction (57 %), were observed in mice immunized with these cVLPs. Finally, we discuss the possibility that chimeric particles of the type described in this work may be the basis for developing HPV prophylactic and therapeutic vaccines with high efficacy.

Anti-cancer potential of casein and its derivatives: novel strategies for cancer treatment.

Cancer is one of the leading causes of death worldwide, with over 10 million fatalities annually. While tumors can be surgically removed and treated with chemotherapy, radiotherapy, immunotherapy, hormonal therapy, or combined therapies, current treatments often result in toxic side effects in normal tissue. Therefore, researchers are actively seeking ways to selectively eliminate cancerous cells, minimizing the toxic side effects in normal tissue. Caseins and its derivatives have shown promising anti-cancer potential, demonstrating antitumor and cytotoxic effects on cells from various tumor types without causing harm to normal cells. Collectively, these data reveals advancements in the study of caseins and their derivative peptides, particularly providing a comprehensive understanding of the molecular mechanism of action in cancer therapy. These mechanisms occur through various signaling pathways, including (i) the increase of interferon-associated STAT1 signaling, (ii) the suppression of stemness-related markers such as CD44, (iii) the attenuation of the STAT3/HIF1-α signaling, (iv) the down-expression of uPAR and PAI-1, (v) the loss of mitochondrial membrane potential and reduced intracellular ATP production, (vi) the increase of caspase-3 activity, and (vii) the suppression of TLR4/NF-кB signaling. Therefore, we conclude that casein could be an effective adjuvant for cancer treatment.