apyrase and Breast-Neoplasms

Breast cancer (BC) is a public health problem worldwide, causing suffering and premature death among women. As a heterogeneous disease, BC-specific diagnosis and treatment are challenging. Ectonucleotidases are related to tumor development and their expression may vary among BC. miRNAs may participate in epigenetic events and may regulate ectonucleotidases in BC. This study aimed to evaluate the expression of ectonucleotidases according to BC subtypes and to predict if there is post-transcriptional regulation of them by miRNAs. MCF 10A (non-tumorigenic), MCF7 (luminal BC), and MDA-MB-231 (triple-negative BC - TNBC) breast cell lines were used and ENTPD1 (the gene encoding for NTPDase1) and NT5E (the gene encoding for ecto-5'-nucleotidase) gene expression was determined. Interestingly, the expression of ENTPD1 was only observed in MCF7 and NT5E was lower in MCF7 compared to MDA-MB-231 cell line. ATP, ADP, and AMP hydrolysis were observed on the surface of all cell lines, being higher in MDA-MB-231. Like qPCR, the activity of AMP hydrolysis was also lower in the MCF7 cells, which may represent a striking feature of this BC subtype. In silico analyses confirmed that the miRNAs miR-101-3p, miR-141-3p, and miR-340-5p were higher expressed in MCF7 cells and targeted NT5E mRNA. Altogether, data suggest that the regulation of NT5E by miRNAs in MCF7 lineage may direct the molecular profile of luminal BC. Thus, we suggest that the roles of ecto-5'-nucleotidase and the aforementioned miRNAs must be unraveled in TNBC to be possibly defined as diagnostic and therapeutic targets.

Topics: 5'-Nucleotidase; Antigens, CD; Apyrase; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Triple Negative Breast Neoplasms

Luminal-like breast cancer (BC) constitutes the majority of BC subtypes, but, differently from highly aggressive triple negative BC, is poorly infiltrated by the immune system. The quality of the immune infiltrate in luminal-like BCs has been poorly studied, thereby limiting further investigation of immunotherapeutic strategies. By using high-dimensional single-cell technologies, we identify heterogeneous behavior within the tissue-resident memory CD8+ T (Trm) cells infiltrating luminal-like tumors. A subset of CD127- CD39

Topics: Apyrase; Breast Neoplasms; CD8-Positive T-Lymphocytes; Humans; Prognosis; Single-Cell Analysis

Some breast tumors metastasize aggressively whereas others remain dormant for years. The mechanism governing metastatic dormancy remains largely unknown. Through high-parametric single-cell mapping in mice, we identify a discrete population of CD39

Topics: Animals; Antigens, CD; Apyrase; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Humans; Immunotherapy; Lung; Mammary Neoplasms, Experimental; Mice, Inbred BALB C; Mice, Inbred NOD; Mice, Knockout; Neoplasm Metastasis; Programmed Cell Death 1 Receptor

B cells can promote or inhibit immune responses against breast cancer. We investigated changes in the frequency of B cells with stimulatory or regulatory capacity in breast tumor draining lymph nodes during cancer progression.. We isolated mononuclear cells from fresh axillary lymph nodes (LNs) of 44 patients with breast cancer and stained lymphocytes with antibodies against CD19, CD80, CD86, CD39 and CD73. To assess programmed death-1 (PD-1) and PD-ligand 1 (PD-L1) expression, lymphocytes were briefly stimulated, stained for CD19, PD-1 and PD-L1, and examined with flow cytometry.. The frequency of CD80. CD86

Topics: Adult; Aged; Apyrase; Axilla; B-Lymphocyte Subsets; B-Lymphocytes, Regulatory; B7-2 Antigen; B7-H1 Antigen; Breast Neoplasms; Female; Flow Cytometry; Humans; Lymph Nodes; Middle Aged; Prognosis; Programmed Cell Death 1 Receptor

The ability of CD8

Topics: Adenosine Triphosphate; Animals; Antigens, CD; Apyrase; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cytokines; Female; Humans; Lymphatic Metastasis; Lymphocytes, Tumor-Infiltrating; Male; Melanoma; Mice, Inbred BALB C; Mice, Knockout; Tumor Microenvironment; Xenograft Model Antitumor Assays

Our previous work has demonstrated that extracellular ATP is an important pro-invasive factor, and in this study, we tapped into a possible mechanism involved. We discovered that ATP could upregulate both the intracellular expression and secretion of S100A4 in breast cancer cells and fibroblasts. Apart from stimulating breast cancer cell motility via intracellular S100A4, ATP enhanced the ability of breast cancer cells to transform fibroblasts into cancer-associated fibroblast (CAF)-like cells, which in turn secreted S100A4 to further promote cancer cell motility. Both apyrase and niclosamide treatments could inhibit metastasis of inoculated tumors to lung, liver and kidney in mice model, and CAFs from these treated tumors exhibited weakened migration-stimulating capacity for breast cancer cells. Collectively, our data indicate that extracellular ATP promotes the interactions between breast cancer cells and fibroblasts, which work collaboratively via production of S100A4 to exacerbate breast cancer metastasis.

Topics: Adenosine Triphosphate; Animals; Apyrase; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Movement; Female; Gene Expression Profiling; Gene Knockdown Techniques; Humans; MCF-7 Cells; Mice; Mice, Nude; Niclosamide; Oligonucleotide Array Sequence Analysis; Primary Cell Culture; RNA, Messenger; RNA, Small Interfering; S100 Calcium-Binding Protein A4; Up-Regulation

Regulatory T cells (Tregs) play a major role in tumor escape from immunosurveillance by suppressing effector cells. The number of Tregs is increased in tumor sites and peripheral blood of breast cancer patients. However, the data regarding phenotypic and functional heterogeneity of Treg subpopulations in breast cancer are limited. The present study aimed to investigate the number and suppressive potential of Tregs that possess natural naïve-(N nTregs), effector/memory-like (EM nTregs), and Tr1-like phenotypes in breast cancer patients and healthy women.. The study included 10 HW and 17 primary breast cancer patients. Numbers of CD4. Herein, we demonstrate that the percentages of N nTregs, EM nTregs, CD25. Collectively, the current study shows phenotypic alterations in suppressive receptors of Treg subsets, suggesting that breast cancer patients have increased activity of N nTregs, EM nTregs and Tr1 cells; and EM nTregs and CD25

Topics: Adult; Aged; Apyrase; Breast Neoplasms; CTLA-4 Antigen; Female; Forkhead Transcription Factors; Humans; Interleukin-2 Receptor alpha Subunit; Interleukins; Middle Aged; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory

Extracellular ATP has been shown to either inhibit or promote cancer growth and migration; however, the mechanism underlying this discrepancy remained elusive. Here we demonstrate the divergent roles of ATP and adenosine released by bone osteocytes on breast cancers. We showed that conditioned media (CM) collected from osteocytes treated with alendronate (AD), a bisphosphonate drug, inhibited the migration of human breast cancer MDA-MB-231 cells. Removal of the extracellular ATP by apyrase in CM abolished this effect, suggesting the involvement of ATP. ATP exerted its inhibitory effect through the activation of purinergic P2X receptor signaling in breast cancer cells evidenced by the attenuation of the inhibition by an antagonist, oxidized ATP, as well as knocking down P2X7 with small interfering RNA (siRNA), and the inhibition of migration by an agonist, BzATP. Intriguingly, ATP had a biphasic effect on breast cancer cells-lower dosage inhibited but higher dosage promoted its migration. The stimulatory effect on migration was blocked by an adenosine receptor antagonist, MRS1754, ARL67156, an ecto-ATPase inhibitor, and A2A receptor siRNA, suggesting that in contrast to ATP, adenosine, a metabolic product of ATP, promoted migration of breast cancer cells. Consistently, non-hydrolyzable ATP, ATPγS, only inhibited but did not promote cancer cell migration. ATP also had a similar inhibitory effect on the Py8119 mouse mammary carcinoma cells; however, adenosine had no effect owing to the absence of the A2A receptor. Consistently, ATPγS inhibited, whereas adenosine promoted anchorage-independent growth of MDA-MB-231 cells. Our in vivo xenograft study showed a significant delay of tumor growth with the treatment of ATPγS. Moreover, the extent of bone metastasis in a mouse intratibial model was significantly reduced with the treatment of ATPγS. Together, our results suggest the distinct roles of ATP and adenosine released by osteocytes and the activation of corresponding receptors P2X7 and A2A signaling on breast cancer cell growth, migration and bone metastasis.

Topics: Adenosine; Adenosine Triphosphate; Alendronate; Animals; Apyrase; Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Humans; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Transplantation; Osteocytes; Receptor, Adenosine A2A; Receptors, Purinergic P2X; RNA Interference; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells.. Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards γ-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry.. Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to γ-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39.. Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches.

Topics: Antigens, CD; Apoptosis; Apyrase; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Separation; DNA Mutational Analysis; Female; Flow Cytometry; Gamma Rays; Gene Expression Regulation, Neoplastic; Humans; Monocytes; Mutation; Necrosis; Nucleotides; Tumor Suppressor Protein p53; X-Rays

The activities of NTPDase (EC 3.6.1.5, apyrase, CD39) and 5'-nucleotidase (EC 3.1.3.5, CD73) enzymes were analyzed in platelets from breast cancer patients. Initially, patients were compared in terms of length (years) of tamoxifen use. The following groups were studied: breast cancer patients who did not use tamoxifen, patients using tamoxifen for 1-48 months, patients using tamoxifen for 49-84 months, and controls (healthy subjects). Results demonstrated that adenosine triphosphate (ATP) hydrolysis was enhanced (F(3,114)=8.53; P<0.001) and adenosine diphosphate (ADP) hydrolysis was reduced (F(3,106)=5.09, P=0.002) as a function of tamoxifen use, while adenosine monophosphate (AMP) hydrolysis was unchanged. Next, patients were compared statistically according to disease stage, determined by the tumor-node-metastasis (TNM) staging system for classifying breast tumor. ATP hydrolysis was significantly elevated in patients with stage I and II breast cancer (F(4,113)=4.35; P=0.003), but was normal in patients with stage III and IV cancer. ADP hydrolysis was reduced in stages II to IV (F(4,105)=3.88, P=0.006) and AMP hydrolysis was elevated in stage II (F(4,105)=3.45 P=0.01), but was normal in stages III and IV. Platelet aggregation time was similar in all patients regardless of tamoxifen use or disease stage. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were also within the normal range and similar among all groups. Similarly, fibrinogen and fibrin degradation product (FDP) were unchanged in all groups. In conclusion, our study demonstrated for the first time that hydrolysis of adenine nucleotides is modified in platelets from breast cancer patients taking tamoxifen.

Topics: 5'-Nucleotidase; Adenine Nucleotides; Adult; Aged; Analysis of Variance; Apyrase; Blood Platelets; Breast Neoplasms; Carcinoma, Ductal, Breast; Female; Fibrinogen; Humans; Hydrolysis; Immunohistochemistry; Middle Aged; Neoplasm Staging; Platelet Aggregation; Prothrombin Time; Tamoxifen

MCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP). MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml). This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma. This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine. MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma. Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tissue factor activity expression. Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50, 0.1 microM) and rhodostomin (IC50, 0.03 microM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC50, 0.54 mM).

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Apyrase; Breast Neoplasms; Crotalid Venoms; Cysteine Proteinase Inhibitors; Female; Hirudins; Humans; Intercellular Signaling Peptides and Proteins; Oligopeptides; Peptides; Phospholipases A; Phospholipases A2; Platelet Aggregation; Platelet Aggregation Inhibitors; Sphingosine; Tumor Cells, Cultured

We studied the effects on platelet function of cells isolated from freshly dissociated human tumor tissues (11 breast carcinomas, 9 colon carcinomas and 1 lymph node metastasis from melanoma) obtained at surgery as compared with cultured human tumor cells: namely, human melanoma 1402 cell line derived from a primary tumor and two lines derived from lymph node metastases (ME 7110/2 and Me 665/1) as well as a human hepatoma cell line (Hep G2). The three melanoma cell lines activated platelets by producing ADP, as evidenced by the inhibitory effect of apyrase and by the direct measurement of the agonist in the supernatants of tumor cell suspensions; this production was much greater by the cells derived from metastases than by the cells derived from the primary tumor. On the other hand, aggregation induced by Hep G2 hepatoma cells was unaffected by apyrase and was inhibited by hirudin or concanavalin A, suggesting that the cells aggregate platelets by producing thrombin, probably through tissue factor activity of the cells themselves. Cells isolated from 16 of the 21 human tumor tissues possessed a potent platelet-aggregating effect, which was not inhibited by apyrase, hirudin or concanavalin A, but was virtually abolished by the cysteine protease inhibitors iodoacetic acid or p-hydroxymercuri-phenylsulfonate. Collectively, our data demonstrate that cells isolated from freshly dissociated tumor tissues activate platelets through tumor-associated cysteine proteinases rather than by the ADP- or thrombin-dependent mechanisms characteristic of cultured human tumor cell lines.

Topics: Adenosine Diphosphate; Adult; Apyrase; Blood Platelets; Breast Neoplasms; Cell Communication; Cell Separation; Colonic Neoplasms; Concanavalin A; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Hirudins; Humans; Melanoma; Platelet Aggregation; Tumor Cells, Cultured

Platelets may promote the development of metastasis, and tumor cells that aggregate platelets are believed to be more malignant. We studied three different human mammary carcinoma cell lines, which had different interactions with human platelet-rich plasma (PRP). The MCF-7 and the T47-D cell lines induced an adenosine diphosphate (ADP)-mediated platelet aggregation. The third cell line, MDA-MB 231 did not induce any platelet aggregation. On the contrary, this cell line inhibited ADP- and arachidonic acid-induced platelet aggregation. This inhibiting activity is mainly adenosine-mediated. The mechanism by which platelets may contribute to the dissemination of cancer could be related to platelet growth factors. MCF-7 and T47-D cell lines induced a release of platelet-derived growth factor (PDGF). On the contrary, the MDA-MB 231 cell line did not induce any platelet release. The role of these platelet growth factors in tumor cell growth is discussed.

Topics: 6-Ketoprostaglandin F1 alpha; Adenosine Diphosphate; Alprostadil; Apyrase; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Breast Neoplasms; Carcinoma; Cell Line; Creatine Kinase; Humans; Neoplasm Metastasis; Platelet Aggregation; Thromboxane B2