Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction.

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.

Tigecycline Opposes Bortezomib Effect on Myeloma Cells Decreasing Mitochondrial Reactive Oxygen Species Production.

Multiple myeloma is an incurable plasma cell malignancy. Most patients end up relapsing and developing resistance to antineoplastic drugs, like bortezomib. Antibiotic tigecycline has activity against myeloma. This study analyzed tigecycline and bortezomib combination on cell lines and plasma cells from myeloma patients. Apoptosis, autophagic vesicles, mitochondrial mass, mitochondrial superoxide, cell cycle, and hydrogen peroxide were studied by flow cytometry. In addition, mitochondrial antioxidants and electron transport chain complexes were quantified by reverse transcription real-time PCR (RT-qPCR) or western blot. Cell metabolism and mitochondrial activity were characterized by Seahorse and RT-qPCR. We found that the addition of tigecycline to bortezomib reduces apoptosis in proportion to tigecycline concentration. Supporting this, the combination of both drugs counteracts bortezomib in vitro individual effects on the cell cycle, reduces autophagy and mitophagy markers, and reverts bortezomib-induced increase in mitochondrial superoxide. Changes in mitochondrial homeostasis and MYC upregulation may account for some of these findings. These data not only advise to avoid considering tigecycline and bortezomib combination for treating myeloma, but caution on the potential adverse impact of treating infections with this antibiotic in myeloma patients under bortezomib treatment.

Germline gain-of-function MMP11 variant results in an aggressive form of colorectal cancer.

Matrix metalloproteinase-11 (MMP11) is an enzyme with proteolytic activity against matrix and nonmatrix proteins. Although most MMPs are secreted as inactive proenzymes and are later activated extracellularly, MMP11 is activated intracellularly by furin within the constitutive secretory pathway. It is a key factor in physiological tissue remodeling and its alteration may play an important role in the progression of epithelial malignancies and other diseases. TCGA colon and colorectal adenocarcinoma data showed that upregulation of MMP11 expression correlates with tumorigenesis and malignancy. Here, we provide evidence that a germline variant in the MMP11 gene (NM_005940: c.232C>T; p.(Pro78Ser)), identified by whole exome sequencing, can increase the tumorigenic properties of colorectal cancer (CRC) cells. P78S is located in the prodomain region, which is responsible for blocking MMP11's protease activity. This variant was detected in the proband and all the cancer-affected family members analyzed, while it was not detected in healthy relatives. In silico analyses predict that P78S could have an impact on the activation of the enzyme. Furthermore, our in vitro analyses show that the expression of P78S in HCT116 cells increases tumor cell invasion and proliferation. In summary, our results show that this variant could modify the structure of the MMP11 prodomain, producing a premature or uncontrolled activation of the enzyme that may contribute to an early CRC onset in these patients. The study of this gene in other CRC cases will provide further information about its role in CRC development, which might improve patient treatment in the future.

New and Old Key Players in Liver Cancer.

Liver cancer represents a major health problem worldwide with growing incidence and high mortality, hepatocellular carcinoma (HCC) being the most frequent. Hepatocytes are likely the cellular origin of most HCCs through the accumulation of genetic alterations, although hepatic progenitor cells (HPCs) might also be candidates in specific cases, as discussed here. HCC usually develops in a context of chronic inflammation, fibrosis, and cirrhosis, although the role of fibrosis is controversial. The interplay between hepatocytes, immune cells and hepatic stellate cells is a key issue. This review summarizes critical aspects of the liver tumor microenvironment paying special attention to platelets as new key players, which exert both pro- and anti-tumor effects, determined by specific contexts and a tight regulation of platelet signaling. Additionally, the relevance of specific signaling pathways, mainly HGF/MET, EGFR and TGF-ß is discussed. HGF and TGF-ß are produced by different liver cells and platelets and regulate not only tumor cell fate but also HPCs, inflammation and fibrosis, these being key players in these processes. The role of C3G/RAPGEF1, required for the proper function of HGF/MET signaling in HCC and HPCs, is highlighted, due to its ability to promote HCC growth and, regulate HPC fate and platelet-mediated actions on liver cancer.

Lymphocyte antigen 6G6D-mediated modulation through p38α MAPK and DNA methylation in colorectal cancer.

In addition to being novel biomarkers for poor cancer prognosis, members of Lymphocyte antigen-6 (Ly6) gene family also play a crucial role in avoiding immune responses to tumors. However, it has not been possible to identify the underlying mechanism of how Ly6 gene regulation operates in human cancers. Transcriptome, epigenome and proteomic data from independent cancer databases were analyzed in silico and validated independently in 334 colorectal cancer tissues (CRC). RNA mediated gene silencing of regulatory genes, and treatment with MEK and p38 MAPK inhibitors were also tested in vitro. We report here that the Lymphocyte antigen 6G6D is universally downregulated in mucinous CRC, while its activation progresses through the classical adenoma-carcinoma sequence. The DNA methylation changes in LY6G6D promoter are intimately related to its transcript regulation, epigenomic and histological subtypes. Depletion of DNA methyltransferase 1 (DNMT1), which maintains DNA methylation, results in the derepression of LY6G6D expression. RNA-mediated gene silencing of p38α MAPK or its selective chemical inhibition, however, reduces LY6G6D expression, reducing trametinib's anti-inflammatory effects. Patients treated with FOLFOX-based first-line therapy experienced decreased survival due to hypermethylation of the LY6G6D promoter and decreased p38α MAPK signaling. We found that cancer-specific immunodominant epitopes are controlled by p38α MAPKs signaling and suppressed by DNA methylation in histological variants with Mucinous differentiation. This work provides a promising prospective for clinical application in diagnosis and personalized therapeutic strategies of colorectal cancer.

C3G Protein, a New Player in Glioblastoma.

C3G (RAPGEF1) is a guanine nucleotide exchange factor (GEF) for GTPases from the Ras superfamily, mainly Rap1, although it also acts through GEF-independent mechanisms. C3G regulates several cellular functions. It is expressed at relatively high levels in specific brain areas, playing important roles during embryonic development. Recent studies have uncovered different roles for C3G in cancer that are likely to depend on cell context, tumour type, and stage. However, its role in brain tumours remained unknown until very recently. We found that C3G expression is downregulated in GBM, which promotes the acquisition of a more mesenchymal phenotype, enhancing migration and invasion, but not proliferation. ERKs hyperactivation, likely induced by FGFR1, is responsible for this pro-invasive effect detected in C3G silenced cells. Other RTKs (Receptor Tyrosine Kinases) are also dysregulated and could also contribute to C3G effects. However, it remains undetermined whether Rap1 is a mediator of C3G actions in GBM. Various Rap1 isoforms can promote proliferation and invasion in GBM cells, while C3G inhibits migration/invasion. Therefore, other RapGEFs could play a major role regulating Rap1 activity in these tumours. Based on the information available, C3G could represent a new biomarker for GBM diagnosis, prognosis, and personalised treatment of patients in combination with other GBM molecular markers. The quantification of C3G levels in circulating tumour cells (CTCs) in the cerebrospinal liquid and/or circulating fluids might be a useful tool to improve GBM patient treatment and survival.

TWEAK promotes migration and invasion in MEFs through a mechanism dependent on ERKs activation and Fibulin 3 down-regulation.

TWEAK regulates multiple physio-pathological processes in fibroblasts such as fibrosis. It also induces migration and invasion in tumors and it can activate p38 MAPK in various cell types. Moreover, p38α MAPK promotes migration and invasion in several cancer cells types and in mouse embryonic fibroblasts (MEFs). However, it remains unknown if TWEAK could promote migration in fibroblasts and whether p38α MAPK might play a role. Our results reveal that TWEAK activates ERKs, Akt, and p38α/ß MAPKs and reduces secreted Fibulin 3 in MEFs. TWEAK also increases migration and invasion in wt and p38α deficient MEFs, which indicates that p38α MAPK is not required to mediate these effects. In contrast, ERKs inhibition significantly decreases TWEAK-induced migration and Fibulin 3 knock-down mimics TWEAK effect. These results indicate that both ERKs activation and Fibulin 3 down-regulation would contribute to mediate TWEAK pro-migratory effect. In fact, the additional regulation of ERKs and/or p38ß as a consequence of Fibulin 3 decrease might be also involved in the pro-migratory effect of TWEAK in MEFs. In conclusion, our studies uncover novel mechanisms by which TWEAK would favor tissue repair by promoting fibroblasts migration.

C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.

BACKGROUND: Megakaryopoiesis allows platelet formation, which is necessary for coagulation, also playing an important role in different pathologies. However, this process remains to be fully characterized. C3G, an activator of Rap1 GTPases, is involved in platelet activation and regulates several differentiation processes. METHODS: We evaluated C3G function in megakaryopoiesis using transgenic mouse models where C3G and C3GΔCat (mutant lacking the GEF domain) transgenes are expressed exclusively in megakaryocytes and platelets. In addition, we used different clones of K562, HEL and DAMI cell lines with overexpression or silencing of C3G or GATA-1. RESULTS: We found that C3G participates in the differentiation of immature hematopoietic cells to megakaryocytes. Accordingly, bone marrow cells from transgenic C3G, but not those from transgenic C3GΔCat mice, showed increased expression of the differentiation markers CD41 and CD61, upon thrombopoietin treatment. Furthermore, C3G overexpression increased the number of CD41+ megakaryocytes with high DNA content. These results are supported by data obtained in the different models of megakaryocytic cell lines. In addition, it was uncovered GATA-1 as a positive regulator of C3G expression. Moreover, C3G transgenic megakaryocytes from fresh bone marrow explants showed increased migration from the osteoblastic to the vascular niche and an enhanced ability to form proplatelets. Although the transgenic expression of C3G in platelets did not alter basal platelet counts, it did increase slightly those induced by TPO injection in vivo. Moreover, platelet C3G induced adipogenesis in the bone marrow under pathological conditions. CONCLUSIONS: All these data indicate that C3G plays a significant role in different steps of megakaryopoiesis, acting through a mechanism dependent on its GEF activity.

p38 MAPK down-regulates fibulin 3 expression through methylation of gene regulatory sequences: role in migration and invasion.

p38 MAPKs regulate migration and invasion. However, the mechanisms involved are only partially known. We had previously identified fibulin 3, which plays a role in migration, invasion, and tumorigenesis, as a gene regulated by p38α. We have characterized in detail how p38 MAPK regulates fibulin 3 expression and its role. We describe here for the first time that p38α, p38γ, and p38δ down-regulate fibulin 3 expression. p38α has a stronger effect, and it does so through hypermethylation of CpG sites in the regulatory sequences of the gene. This would be mediated by the DNA methylase, DNMT3A, which is down-regulated in cells lacking p38α, but once re-introduced represses Fibulin 3 expression. p38α through HuR stabilizes dnmt3a mRNA leading to an increase in DNMT3A protein levels. Moreover, by knocking-down fibulin 3, we have found that Fibulin 3 inhibits migration and invasion in MEFs by mechanisms involving p38α/ß inhibition. Hence, p38α pro-migratory/invasive effect might be, at least in part, mediated by fibulin 3 down-regulation in MEFs. In contrast, in HCT116 cells, Fibulin 3 promotes migration and invasion through a mechanism dependent on p38α and/or p38ß activation. Furthermore, Fibulin 3 promotes in vitro and in vivo tumor growth of HCT116 cells through a mechanism dependent on p38α, which surprisingly acts as a potent inducer of tumor growth. At the same time, p38α limits fibulin 3 expression, which might represent a negative feed-back loop.

Met signaling in cardiomyocytes is required for normal cardiac function in adult mice.

Hepatocyte growth factor (HGF) and its receptor, Met, are key determinants of distinct developmental processes. Although HGF exerts cardio-protective effects in a number of cardiac pathologies, it remains unknown whether HGF/Met signaling is essential for myocardial development and/or physiological function in adulthood. We therefore investigated the requirement of HGF/Met signaling in cardiomyocyte for embryonic and postnatal heart development and function by conditional inactivation of the Met receptor in cardiomyocytes using the Cre-α-MHC mouse line (referred to as α-MHCMet-KO). Although α-MHCMet-KO mice showed normal heart development and were viable and fertile, by 6 months of age, males developed cardiomyocyte hypertrophy, associated with interstitial fibrosis. A significant upregulation in markers of myocardial damage, such as ß-MHC and ANF, was also observed. By the age of 9 months, α-MHCMet-KO males displayed systolic cardiac dysfunction. Mechanistically, we provide evidence of a severe imbalance in the antioxidant defenses in α-MHCMet-KO hearts involving a reduced expression and activity of catalase and superoxide dismutase, with consequent reactive oxygen species accumulation. Similar anomalies were observed in females, although with a slower kinetics. We also found that Met signaling down-regulation leads to an increase in TGF-ß production and a decrease in p38MAPK activation, which may contribute to phenotypic alterations displayed in α-MHCMet-KO mice. Consistently, we show that HGF acts through p38α to upregulate antioxidant enzymes in cardiomyocytes. Our results highlight that HGF/Met signaling in cardiomyocytes plays a physiological cardio-protective role in adult mice by acting as an endogenous regulator of heart function through oxidative stress control.

CRISPR/Cas9 screenings unearth protein arginine methyltransferase 7 as a novel essential gene in prostate cancer metastasis.

Due to the limited effectiveness of current treatments, the survival rate of patients with metastatic castration-resistant prostate cancer (mCRPC) is significantly reduced. Consequently, it is imperative to identify novel therapeutic targets for managing these patients. Since the invasive ability of cells is crucial for establishing and maintaining metastasis, the aim of this study was to identify the essential regulators of invasive abilities of mCRPC cells by conducting two independent high-throughput CRISPR/Cas9 screenings. Furthermore, some of the top hits were validated using siRNA technology, with protein arginine methyltransferase 7 (PRMT7) emerging as the most promising candidate. We demonstrated that its inhibition or depletion via genetic or pharmacological approaches significantly reduces invasive, migratory and proliferative abilities of mCRPC cells in vitro. Moreover, we confirmed that PRMT7 ablation reduces cell dissemination in chicken chorioallantoic membrane and mouse xenograft assays. Molecularly, PRMT7 reprograms the expression of several adhesion molecules by methylating various transcription factors, such as FoxK1, resulting in the loss of adhesion from the primary tumor and increased motility of mCRPC cells. Furthermore, PRMT7 higher expression correlates with tumor aggressivity and poor overall survival in prostate cancer patients. Thus, this study demonstrates that PRMT7 is a potential therapeutic target and potential biomarker for mPCa.

p38α mediates cell survival in response to oxidative stress via induction of antioxidant genes: effect on the p70S6K pathway.

We reveal a novel pro-survival role for mammalian p38α in response to H(2)O(2), which involves an up-regulation of antioxidant defenses. The presence of p38α increases basal and H(2)O(2)-induced expression of the antioxidant enzymes: superoxide-dismutase 1 (SOD-1), SOD-2, and catalase through different mechanisms, which protects from reactive oxygen species (ROS) accumulation and prevents cell death. p38α was found to regulate (i) H(2)O(2)-induced SOD-2 expression through a direct regulation of transcription mediated by activating transcription factor 2 (ATF-2) and (ii) H(2)O(2)-induced catalase expression through regulation of protein stability and mRNA expression and/or stabilization. As a consequence, SOD and catalase activities are higher in WT MEFs. We also found that this p38α-dependent antioxidant response allows WT cells to maintain an efficient activation of the mTOR/p70S6K pathway. Accordingly, the loss of p38α leads to ROS accumulation in response to H(2)O(2), which causes cell death and inactivation of mTOR/p70S6K signaling. This can be rescued by either p38α re-expression or treatment with the antioxidants, N-acetyl cysteine, or exogenously added catalase. Therefore, our results reveal a novel homeostatic role for p38α in response to oxidative stress, where ROS removal is favored by antioxidant enzymes up-regulation, allowing cell survival and mTOR/p70S6K activation.

C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity.

We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.

TWEAK prevents TNF-α-induced insulin resistance through PP2A activation in human adipocytes.

Visceral fat is strongly associated with insulin resistance. Obesity-associated adipose tissue inflammation and inflammatory cytokine production are considered key mediators of insulin signaling inhibition. TWEAK is a relatively new member of the TNF cytokine superfamily, which can exist as full length membrane-associated (mTWEAK) and soluble (sTWEAK) isoforms. Although TWEAK has been shown to have important functions in chronic inflammatory diseases its physiological role in adipose tissue remains unresolved. In this study, we explore the molecular mechanisms involved in the modulation of TNF-α-induced effects on insulin sensitivity by sTWEAK in a human visceral adipose cell line and also in primary human adipocytes obtained from visceral fat depots. Our data reveal that sTWEAK ameliorates TNF-α-induced insulin resistance on glucose uptake, GLUT4 translocation and insulin signaling without affecting other metabolic effects of TNF-α such as lipolysis or apoptotis. Co-immunoprecipitation experiments in adipose cells revealed that pretreatment with sTWEAK specifically inhibits TRAF2 association with TNFR1, but not with TNFR2, which mediates insulin resistance. However, sTWEAK does not affect other downstream molecules activated by TNF-α, such as TAK1. Rather, sTWEAK abolishes the stimulatory effect of TNF-α on JNK1/2, which is directly involved in the development of insulin resistance. This is associated with an increase in PP2A activity upon sTWEAK treatment. Silencing of the PP2A catalytic subunit gene overcomes the dephosphorylation effect of sTWEAK on JNK1/2, pointing to PP2A as a relevant mediator of sTWEAK-induced JNK inactivation. Overall, our data reveal a protective role of TWEAK in glucose homeostasis and identify PP2A as a new driver in the modulation of TNF-α signaling by sTWEAK.

C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion.

BACKGROUND: Previous studies by our group and others have shown that C3G interacts with Bcr-Abl through its SH3-b domain. RESULTS: In this work we show that C3G and Bcr-Abl form complexes with the focal adhesion (FA) proteins CrkL, p130Cas, Cbl and Abi1 through SH3/SH3-b interactions. The association between C3G and Bcr-Abl decreased upon Abi1 or p130Cas knock-down in K562 cells, which suggests that Abi1 and p130Cas are essential partners in this interaction. On the other hand, C3G, Abi1 or Cbl knock-down impaired adhesion to fibronectin, while p130Cas silencing enhanced it. C3G, Cbl and p130Cas-SH3-b domains interact directly with common proteins involved in the regulation of cell adhesion and migration. Immunoprecipitation and immunofluorescence studies revealed that C3G form complexes with the FA proteins paxillin and FAK and their phosphorylated forms. Additionally, C3G, Abi1, Cbl and p130Cas regulate the expression and phosphorylation of paxillin and FAK. p38α MAPK also participates in the regulation of adhesion in chronic myeloid leukemia cells. It interacts with C3G, CrkL, FAK and paxillin and regulates the expression of paxillin, CrkL and α5 integrin, as well as paxillin phosphorylation. Moreover, double knock-down of C3G/p38α decreased adhesion to fibronectin, similarly to the single silencing of one of these genes, either C3G or p38α. These suggest that C3G and p38α MAPK are acting through a common pathway to regulate cell adhesion in K562 cells, as previously described for the regulation of apoptosis. CONCLUSIONS: Our results indicate that C3G-p38αMAPK pathway regulates K562 cell adhesion through the interaction with FA proteins and Bcr-Abl, modulating the formation of different protein complexes at FA.

C3G down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process.

Previous data indicate that C3G (RapGEF1) main isoform is highly expressed in liver progenitor cells (or oval cells) compared to adult mature hepatocytes, suggesting it may play an important role in oval cell biology. Hence, we have explored C3G function in the regulation of oval cell properties by permanent gene silencing using shRNAs. We found that C3G knock-down enhanced migratory and invasive ability of oval cells by promoting a partial epithelial to mesenchymal transition (EMT). This is likely mediated by upregulation of mRNA expression of the EMT-inducing transcription factors, Snail1, Zeb1 and Zeb2, induced in C3G-silenced oval cells. This EMT is associated to a higher expression of the stemness markers, CD133 and CD44. Moreover, C3G down-regulation increased oval cells clonogenic capacity by enhancing cell scattering. However, C3G knock-down did not impair oval cell differentiation into hepatocyte lineage. Mechanistic studies revealed that HGF/MET signaling and its pro-invasive activity was impaired in oval cells with low levels of C3G, while TGF-ß signaling was increased. Altogether, these data suggest that C3G might be tightly regulated to ensure liver repair in chronic liver diseases such as non-alcoholic steatohepatitis. Hence, reduced C3G levels could facilitate oval cell expansion, after the proliferation peak, by enhancing migration.

New functions of C3G in platelet biology: Contribution to ischemia-induced angiogenesis, tumor metastasis and TPO clearance.

C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli.

In vivo production of fluorine-18 in a chicken egg tumor model of breast cancer for proton therapy range verification.

Range verification of clinical protontherapy systems via positron-emission tomography (PET) is not a mature technology, suffering from two major issues: insufficient signal from low-energy protons in the Bragg peak area and biological washout of PET emitters. The use of contrast agents including 18O, 68Zn or 63Cu, isotopes with a high cross section for low-energy protons in nuclear reactions producing PET emitters, has been proposed to enhance the PET signal in the last millimeters of the proton path. However, it remains a challenge to achieve sufficient concentrations of these isotopes in the target volume. Here we investigate the possibilities of 18O-enriched water (18-W), a potential contrast agent that could be incorporated in large proportions in live tissues by replacing regular water. We hypothesize that 18-W could also mitigate the problem of biological washout, as PET (18F) isotopes created inside live cells would remain trapped in the form of fluoride anions (F-), allowing its signal to be detected even hours after irradiation. To test our hypothesis, we designed an experiment with two main goals: first, prove that 18-W can incorporate enough 18O into a living organism to produce a detectable signal from 18F after proton irradiation, and second, determine the amount of activity that remains trapped inside the cells. The experiment was performed on a chicken embryo chorioallantoic membrane tumor model of head and neck cancer. Seven eggs with visible tumors were infused with 18-W and irradiated with 8-MeV protons (range in water: 0.74 mm), equivalent to clinical protons at the end of particle range. The activity produced after irradiation was detected and quantified in a small-animal PET-CT scanner, and further studied by placing ex-vivo tumours in a gamma radiation detector. In the acquired images, specific activity of 18F (originating from 18-W) could be detected in the tumour area of the alive chicken embryo up to 9 h after irradiation, which confirms that low-energy protons can indeed produce a detectable PET signal if a suitable contrast agent is employed. Moreover, dynamic PET studies in two of the eggs evidenced a minimal effect of biological washout, with 68% retained specific 18F activity at 8 h after irradiation. Furthermore, ex-vivo analysis of 4 irradiated tumours showed that up to 3% of oxygen atoms in the targets were replaced by 18O from infused 18-W, and evidenced an entrapment of 59% for specific activity of 18F after washing, supporting our hypothesis that F- ions remain trapped within the cells. An infusion of 18-W can incorporate 18O in animal tissues by replacing regular water inside cells, producing a PET signal when irradiated with low-energy protons that could be used for range verification in protontherapy. 18F produced inside cells remains entrapped and suffers from minimal biological washout, allowing for a sharper localization with longer PET acquisitions. Further studies must evaluate the feasibility of this technique in dosimetric conditions closer to clinical practice, in order to define potential protocols for its use in patients.

Centrosome Dynamics and Its Role in Inflammatory Response and Metastatic Process.

Metastasis is a process by which cancer cells escape from the location of the primary tumor invading normal tissues at distant organs. Chromosomal instability (CIN) is a hallmark of human cancer, associated with metastasis and therapeutic resistance. The centrosome plays a major role in organizing the microtubule cytoskeleton in animal cells regulating cellular architecture and cell division. Loss of centrosome integrity activates the p38-p53-p21 pathway, which results in cell-cycle arrest or senescence and acts as a cell-cycle checkpoint pathway. Structural and numerical centrosome abnormalities can lead to aneuploidy and CIN. New findings derived from studies on cancer and rare genetic disorders suggest that centrosome dysfunction alters the cellular microenvironment through Rho GTPases, p38, and JNK (c-Jun N-terminal Kinase)-dependent signaling in a way that is favorable for pro-invasive secretory phenotypes and aneuploidy tolerance. We here review recent data on how centrosomes act as complex molecular platforms for Rho GTPases and p38 MAPK (Mitogen activated kinase) signaling at the crossroads of CIN, cytoskeleton remodeling, and immune evasion via both cell-autonomous and non-autonomous mechanisms.

HGK promotes metastatic dissemination in prostate cancer.

Metastasis is the process of cancer cell dissemination from primary tumors to different organs being the bone the preferred site for metastatic homing of prostate cancer (PCa) cells. Prostate tumorigenesis is a multi-stage process that ultimately tends to advance to become metastatic PCa. Once PCa patients develop skeletal metastases, they eventually succumb to the disease. Therefore, it is imperative to identify essential molecular drivers of this process to develop new therapeutic alternatives for the treatment of this devastating disease. Here, we have identified MAP4K4 as a relevant gene for metastasis in PCa. Our work shows that genetic deletion of MAP4K4 or pharmacological inhibition of its encoded kinase, HGK, inhibits metastatic PCa cells migration and clonogenic properties. Hence, MAP4K4 might promote metastasis and tumor growth. Mechanistically, our results indicate that HGK depleted cells exhibit profound differences in F-actin organization, increasing cell spreading and focal adhesion stability. Additionally, HGK depleted cells fails to respond to TNF-α stimulation and chemoattractant action. Moreover, here we show that HGK upregulation in PCa samples from TCGA and other databases correlates with a poor prognosis of the disease. Hence, we suggest that it could be used as prognostic biomarker to predict the appearance of an aggressive phenotype of PCa tumors and ultimately, the appearance of metastasis. In summary, our results highlight an essential role for HGK in the dissemination of PCa cells and its potential use as prognostic biomarker.