PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane.

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11-positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245-dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97- vs. Golgin-245-dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin-mediated cell polarity and cell-cell junctions.

A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility.

BACKGROUND: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. METHODS: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. RESULTS: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. CONCLUSIONS: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.

JNK2 controls fragmentation of the Golgi complex and the G2/M transition through phosphorylation of GRASP65.

In mammalian cells, the Golgi complex is composed of stacks that are connected by membranous tubules. During G2, the Golgi complex is disassembled into isolated stacks. This process is required for entry into mitosis, indicating that the correct inheritance of the organelle is monitored by a 'Golgi mitotic checkpoint'. However, the regulation and the molecular mechanisms underlying this Golgi disassembly are still poorly understood. Here, we show that JNK2 has a crucial role in the G2-specific separation of the Golgi stacks through phosphorylation of Ser277 of the Golgi-stacking protein GRASP65 (also known as GORASP1). Inhibition of JNK2 by RNA interference or by treatment with three unrelated JNK inhibitors causes a potent and persistent cell cycle block in G2. JNK activity becomes dispensable for mitotic entry if the Golgi complex is disassembled by brefeldin A treatment or by GRASP65 depletion. Finally, measurement of the Golgi fluorescence recovery after photobleaching demonstrates that JNK is required for the cleavage of the tubules connecting Golgi stacks. Our findings reveal that a JNK2-GRASP65 signalling axis has a crucial role in coupling Golgi inheritance and G2/M transition.

Early targets of miR-34a in neuroblastoma.

Several genes encoding for proteins involved in proliferation, invasion, and apoptosis are known to be direct miR-34a targets. Here, we used proteomics to screen for targets of miR-34a in neuroblastoma (NBL), a childhood cancer that originates from precursor cells of the sympathetic nervous system. We examined the effect of miR-34a overexpression using a tetracycline inducible system in two NBL cell lines (SHEP and SH-SY5Y) at early time points of expression (6, 12, and 24 h). Proteome analysis using post-metabolic labeling led to the identification of 2,082 proteins, and among these 186 were regulated (112 proteins down-regulated and 74 up-regulated). Prediction of miR-34a targets via bioinformatics showed that 32 transcripts held miR-34a seed sequences in their 3'-UTR. By combining the proteomics data with Kaplan Meier gene-expression studies, we identified seven new gene products (ALG13, TIMM13, TGM2, ABCF2, CTCF, Ki67, and LYAR) that were correlated with worse clinical outcomes. These were further validated in vitro by 3'-UTR seed sequence regulation. In addition, Michigan Molecular Interactions searches indicated that together these proteins affect signaling pathways that regulate cell cycle and proliferation, focal adhesions, and other cellular properties that overall enhance tumor progression (including signaling pathways such as TGF-ß, WNT, MAPK, and FAK). In conclusion, proteome analysis has here identified early targets of miR-34a with relevance to NBL tumorigenesis. Along with the results of previous studies, our data strongly suggest miR-34a as a useful tool for improving the chance of therapeutic success with NBL.

Molecular networks that regulate cancer metastasis.

Tumor metastases are responsible for approximately 90% of all cancer-related deaths. Although many patients can be cured, in the US and UK, cancer still causes 730,000 deaths every year, and it is second only to cardiovascular disease as a cause of death. The functional roles of many critical players involved in metastasis have been delineated in great detail in recent years, due to the draft of the human genome and to many associated discoveries. Here, we address several genetic events and critical factors that define the metastatic phenotype acquired during tumorigenesis. This involves molecular networks that promote local cancer-cell invasion, single-cell invasion, formation of the metastatic microenvironment of primary tumors, intravasation, lymphogenic metastasis, extravasation, and metastatic outgrowth. Altogether, these functional networks of molecules contribute to the development of a selective environment that promotes the seeding and malignant progression of tumorigenic cells in distant organs. We include here candidate target proteins and signaling pathways that are now under clinical investigation. Although many of these trials are still ongoing, they provide the basis for the development of new aspects in the treatment of metastatic cancers, which involves inhibition of these proteins and their molecular networks.

Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models.  CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Targeting the Ubiquitin-Proteasome System and Recent Advances in Cancer Therapy.

Ubiquitination is a reversible post-translational modification based on the chemical addition of ubiquitin to proteins with regulatory effects on various signaling pathways. Ubiquitination can alter the molecular functions of tagged substrates with respect to protein turnover, biological activity, subcellular localization or protein-protein interaction. As a result, a wide variety of cellular processes are under ubiquitination-mediated control, contributing to the maintenance of cellular homeostasis. It follows that the dysregulation of ubiquitination reactions plays a relevant role in the pathogenic states of human diseases such as neurodegenerative diseases, immune-related pathologies and cancer. In recent decades, the enzymes of the ubiquitin-proteasome system (UPS), including E3 ubiquitin ligases and deubiquitinases (DUBs), have attracted attention as novel druggable targets for the development of new anticancer therapeutic approaches. This perspective article summarizes the peculiarities shared by the enzymes involved in the ubiquitination reaction which, when deregulated, can lead to tumorigenesis. Accordingly, an overview of the main pharmacological interventions based on targeting the UPS that are in clinical use or still in clinical trials is provided, also highlighting the limitations of the therapeutic efficacy of these approaches. Therefore, various attempts to circumvent drug resistance and side effects as well as UPS-related emerging technologies in anticancer therapeutics are discussed.

Golgi Complex: A Signaling Hub in Cancer.

The Golgi Complex is the central hub in the endomembrane system and serves not only as a biosynthetic and processing center but also as a trafficking and sorting station for glycoproteins and lipids. In addition, it is an active signaling hub involved in the regulation of multiple cellular processes, including cell polarity, motility, growth, autophagy, apoptosis, inflammation, DNA repair and stress responses. As such, the dysregulation of the Golgi Complex-centered signaling cascades contributes to the onset of several pathological conditions, including cancer. This review summarizes the current knowledge on the signaling pathways regulated by the Golgi Complex and implicated in promoting cancer hallmarks and tumor progression.

Combinatorial Strategies to Target Molecular and Signaling Pathways to Disarm Cancer Stem Cells.

Cancer is an urgent public health issue with a very huge number of cases all over the world expected to increase by 2040. Despite improved diagnosis and therapeutic protocols, it remains the main leading cause of death in the world. Cancer stem cells (CSCs) constitute a tumor subpopulation defined by ability to self-renewal and to generate the heterogeneous and differentiated cell lineages that form the tumor bulk. These cells represent a major concern in cancer treatment due to resistance to conventional protocols of radiotherapy, chemotherapy and molecular targeted therapy. In fact, although partial or complete tumor regression can be achieved in patients, these responses are often followed by cancer relapse due to the expansion of CSCs population. The aberrant activation of developmental and oncogenic signaling pathways plays a relevant role in promoting CSCs therapy resistance. Although several targeted approaches relying on monotherapy have been developed to affect these pathways, they have shown limited efficacy. Therefore, an urgent need to design alternative combinatorial strategies to replace conventional regimens exists. This review summarizes the preclinical studies which provide a proof of concept of therapeutic efficacy of combinatorial approaches targeting the CSCs.

Corrigendum: Combinatorial Strategies to Target Molecularand Signaling Pathways to Disarm Cancer Stem Cells.

[This corrects the article DOI: 10.3389/fonc.2021.689131.].

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer.

M2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC). Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-ß enhancement, IL-17F secretion, and extracellular vesicle protein content modulation. We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Galectin-1 and its involvement in hepatocellular carcinoma aggressiveness.

Hepatocellular carcinoma is one of the most common cancers worldwide. Despite several efforts to elucidate hepatocellular carcinoma molecular pathogenesis, it is still not fully understood. To acquire further insights into the molecular mechanisms of hepatocellular carcinoma, we performed a systematic functional genomic approach on human HuH-7 and JHH-6 cells. The subsequent analysis of the differentially expressed genes in human specimens revealed a molecular signature of 11 genes from which we selected the LGALS1 gene, which was overexpressed in hepatocellular carcinoma. The expression analysis in humans of Galectin-1 (Gal-1), the protein encoded by LGALS1, showed a Gal-1 preferential accumulation in the stromal tissue around hepatocellular carcinoma tumors. Moreover, a significant association between increased expression of Gal-1 in hepatocellular carcinoma and the presence of metastasis was observed. Interestingly, Gal-1 overexpression resulted in an increase of cell migration and invasion. In conclusion, this study provides a portfolio of targets useful for future investigations into molecular marker-discovery studies on a large number of patients and functional assays. In addition, our data provide evidence that Gal-1 plays a role in hepatocellular carcinoma cell migration and invasion, and we suggest that further studies should be conducted to fully establish the role of Gal-1 in hepatocellular carcinoma pathogenesis and evaluate Gal-1 as a potential molecular therapeutic target.

Shp1 in Solid Cancers and Their Therapy.

Shp1 is a cytosolic tyrosine phosphatase that regulates a broad range of cellular functions and targets, modulating the flow of information from the cell membrane to the nucleus. While initially studied in the hematopoietic system, research conducted over the past years has expanded our understanding of the biological role of Shp1 to other tissues, proposing it as a novel tumor suppressor gene functionally involved in different hallmarks of cancer. The main mechanism by which Shp1 curbs cancer development and progression is the ability to attenuate and/or terminate signaling pathways controlling cell proliferation, survival, migration, and invasion. Thus, alterations in Shp1 function or expression can contribute to several human diseases, particularly cancer. In cancer cells, Shp1 activity can indeed be affected by mutations or epigenetic silencing that cause failure of Shp1-mediated homeostatic maintenance. This review will discuss the current knowledge of the cellular functions controlled by Shp1 in non-hematopoietic tissues and solid tumors, the mechanisms that regulate Shp1 expression, the role of its mutation/expression status in cancer and its value as potential target for cancer treatment. In addition, we report information gathered from the public available data from The Cancer Genome Atlas (TCGA) database on Shp1 genomic alterations and correlation with survival in solid cancers patients.

SOCS3 and IRS-1 gene expression differs between genotype 1 and genotype 2 hepatitis C virus-infected HepG2 cells.

BACKGROUND: The poor response to antiviral treatment of hepatitis C virus (HCV)-infected patients with genotype 1b has been associated with a higher prevalence of metabolic syndrome. However, the molecular link between these clinical entities is not clear. The goal of this study was to clarify the role of genotype 1b and 2 in the genetic expression of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate 1 (IRS-1). METHODS: We infected human hepatocellular carcinoma cell line (HepG2) cells with human HCV genotype 1b or 2 and measured the gene and protein expression of SOCS3 at various times. We also evaluated impairment in the insulin pathway by analysis of IRS-1 and phospho-AKT. For the control, we used HepG2 cell cultures treated with non-infectious serum. We also demonstrated the occurrence of HCV infection by the detection of both positive and negative strands in the cells and culture medium. To test infection of the HepG2 cells, we performed quantitative real-time polymerase chain reaction (qRT-PCR) of viral load at different time points. We analyzed the viral genotype in the pellet and supernatant. RESULTS: At each time point, we found positive and negative strands in the infected cells, while in the medium we found positive, but no negative strands. We also detected the presence of the correct genotype in the medium. Two weeks following infection when the viral load was higher, we tested genotype 1b and 2 infected cells. SOCS3 gene expression was significantly higher in genotype 1b-infected cells (median 2.56; mean 2.82+/-0.59) compared with genotype 2 (median 1.34; mean 1.46+/-0.31) (p=0.04) and control cells (median 1.09; mean 1.02+/-0.11, p=0.02). There was no difference between cells exposed to genotype 2 and control cells. Conversely, IRS-1 was significantly lower in genotype 1b-infected cells (median 15.97; mean 15.45+/-0.67) compared with genotype 2-infected cells (median 16.45; mean 16.44+/-0.01, p=0.04). Statistically significant differences were seen when comparing the pAKT/AKT ratio in genotype 1b-infected cells (0.19+/-0.034) and not genotype 1b-infected (genotype 2-infected and non-infected) cells (0.253+/-0.004, p=0.03). This inverse regulation is compatible with interactions between the molecular expression of SOCS3, IRS-1 and phospho-AKT mediated by the genotype 1b virus. CONCLUSIONS: Up-regulation of the SOCS3 gene might be one of the mechanisms governing non-response to therapy and expression of insulin resistance mediated via a direct mechanism at this level of genotype 1b HCV.

Rhes is involved in striatal function.

The development and the function of central nervous system depend on thyroid hormones. In humans, the lack of thyroid hormones causes cretinism, a syndrome of severe mental deficiency. It is assumed that thyroid hormones affect the normal development and function of the brain by activating or suppressing target gene expression because several genes expressed in the brain have been shown to be under thyroid hormone control. Among these, the Rhes gene, encoding a small GTP-binding protein, is predominantly expressed in the striatal region of the brain. To clarify the role of Rhes in vivo, we disrupted the Rhes gene by homologous recombination in embryonic stem cells and generated mice homozygous for the Rhes null mutation (Rhes(-/-)). Rhes(-/-) mice were viable but weighed less than wild-type mice. Furthermore, they showed behavioral abnormalities, displaying a gender-dependent increase in anxiety levels and a clear motor coordination deficit but no learning or memory impairment. These results suggest that Rhes disruption affects selected behavioral competencies.

PARP1-produced poly-ADP-ribose causes the PARP12 translocation to stress granules and impairment of Golgi complex functions.

Poly-ADP-ribose-polymerases (PARPs) 1 and 2 are nuclear enzymes that catalyze the poly-ADP-ribosylation of nuclear proteins transferring poly-ADP-ribose (PAR) polymers to specific residues. PARPs and PAR intervene in diverse functions, including DNA repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Our study is focused on one of these PARPs, PARP12, a Golgi-localized mono-ADP-ribosyltransferase that under stress challenge reversibly translocates from the Golgi complex to stress granules. PARP1 activation and release of nuclear PAR drive this translocation by direct PAR binding to the PARP12-WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and the disassembly of the Golgi membranes, followed by a block in anterograde-membrane traffic. Notably, these functions can be rescued by reverting the stress condition (by drug wash-out). Altogether these data point at a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular-membrane traffic.

Aurora-A recruitment and centrosomal maturation are regulated by a Golgi-activated pool of Src during G2.

The Golgi apparatus is composed of stacks of cisternae laterally connected by tubules to form a ribbon-like structure. At the onset of mitosis, the Golgi ribbon is broken down into discrete stacks, which then undergo further fragmentation. This ribbon cleavage is required for G2/M transition, which thus indicates that a 'Golgi mitotic checkpoint' couples Golgi inheritance with cell cycle transition. We previously showed that the Golgi-checkpoint regulates the centrosomal recruitment of the mitotic kinase Aurora-A; however, how the Golgi unlinking regulates this recruitment was unknown. Here we show that, in G2, Aurora-A recruitment is promoted by activated Src at the Golgi. Our data provide evidence that Src and Aurora-A interact upon Golgi ribbon fragmentation; Src phosphorylates Aurora-A at tyrosine 148 and this specific phosphorylation is required for Aurora-A localization at the centrosomes. This process, pivotal for centrosome maturation, is a fundamental prerequisite for proper spindle formation and chromosome segregation.

Attenuation of Rhes activity significantly delays the appearance of behavioral symptoms in a mouse model of Huntington's disease.

Huntington's disease (HD) is a neuropsychiatric disorder characterized by choreiform movement of the limbs, cognitive disability, psychosis and dementia. It is invariably associated with an abnormally long CAG expansion within the IT15 gene on human chromosome 4. Although the mutant huntingtin protein is ubiquitously expressed in HD patients, cellular degeneration occurs predominantly in neurons within the corpus striatum and cerebral cortex. The Ras homolog Rhes is expressed very selectively in the precise brain areas affected by HD. Recent in vitro work suggests that Rhes may be a co-factor with mutant huntingtin in cell death. The objective of the present study was to examine whether the inhibition of Rhes would attenuate or delay the symptoms of HD in vivo. We used a transgenic mouse model of HD crossed with Rhes knockout mice to show that the behavioral symptoms of HD are regulated by Rhes. HD(+)/Rhes(-/-) mice showed significantly delayed expression of HD-like symptoms in this in vivo model. Drugs that block or inhibit the actions of Rhes may be useful as the first treatments for HD.

Dipyridamole prevents triple-negative breast-cancer progression.

Dipyridamole is a widely prescribed drug in ischemic disorders, and it is here investigated for potential clinical use as a new treatment for breast cancer. Xenograft mice bearing triple-negative breast cancer 4T1-Luc or MDA-MB-231T cells were generated. In these in vivo models, dipyridamole effects were investigated for primary tumor growth, metastasis formation, cell cycle, apoptosis, signaling pathways, immune cell infiltration, and serum inflammatory cytokines levels. Dipyridamole significantly reduced primary tumor growth and metastasis formation by intraperitoneal administration. Treatment with 15 mg/kg/day dipyridamole reduced mean primary tumor size by 67.5 % (p = 0.0433), while treatment with 30 mg/kg/day dipyridamole resulted in an almost a total reduction in primary tumors (p = 0.0182). Experimental metastasis assays show dipyridamole reduces metastasis formation by 47.5 % in the MDA-MB-231T xenograft model (p = 0.0122), and by 50.26 % in the 4T1-Luc xenograft model (p = 0.0292). In vivo dipyridamole decreased activated ß-catenin by 38.64 % (p < 0.0001), phospho-ERK1/2 by 25.05 % (p = 0.0129), phospho-p65 by 67.82 % (p < 0.0001) and doubled the expression of IkBα (p = 0.0019), thus revealing significant effects on Wnt, ERK1/2-MAPK and NF-kB pathways in both animal models. Moreover dipyridamole significantly decreased the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in primary tumors (p < 0.005), and the inflammatory cytokines levels in the sera of the treated mice. We suggest that when used at appropriate doses and with the correct mode of administration, dipyridamole is a promising agent for breast-cancer treatment, thus also implying its potential use in other cancers that show those highly activated pathways.