RESUMEN
Disturbance of protein kinase activity may result in dramatic consequences that often lead to cancer development and progression. In tumors of blood origin, both tyrosine kinases and serine/threonine kinases are altered by different types of mutations, critically regulating cancer hallmarks. CK1α and CK2 are highly conserved, ubiquitously expressed and constitutively active pleiotropic kinases, which participate in multiple biological processes. The involvement of these kinases in solid and blood cancers is well documented. CK1α and CK2 are overactive in multiple myeloma, leukemias and lymphomas. Intriguingly, they are not required to the same degree for the viability of normal cells, corroborating the idea of "druggable" kinases. Different to other kinases, mutations on the gene encoding CK1α and CK2 are rare or not reported. Actually, these two kinases are outside the paradigm of oncogene addiction, since cancer cells' dependency on these proteins resembles the phenomenon of "non-oncogene" addiction. In this review, we will summarize the general features of CK1α and CK2 and the most relevant oncogenic and stress-related signaling nodes, regulated by kinase phosphorylation, that may lead to tumor progression. Finally, we will report the current data, which support the positioning of these two kinases in the therapeutic scene of hematological cancers.
Asunto(s)
Quinasa de la Caseína II/metabolismo , Caseína Quinasa Ialfa/metabolismo , Neoplasias Hematológicas/metabolismo , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Caseína Quinasa Ialfa/antagonistas & inhibidores , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Terapia Molecular Dirigida , Estrés FisiológicoRESUMEN
Mesenchymal stem cells (MSCs) are well known for their beneficial effects, differentiation capacity and regenerative potential. Dental-derived MSCs (DSCs) are more easily accessible and have a non-invasive isolation method rather than MSCs isolated from other sources (umbilical cord, bone marrow, and adipose tissue). In addition, DSCs appear to have a relevant neuro-regenerative potential due to their neural crest origin. However, it is now known that the beneficial effects of MSCs depend, at least in part, on their secretome, referring to all the bioactive molecules (neurotrophic factors) released in the conditioned medium (CM) or in the extracellular vesicles (EVs) in particular exosomes (Exos). In this review, we described the similarities and differences between various DSCs. Our focus was on the secretome of DSCs and their applications in cell therapy for neurological disorders. For neuro-regenerative purposes, the secretome of different DSCs has been tested. Among these, the secretome of dental pulp stem cells and stem cells from human exfoliated deciduous teeth have been the most widely studied. Both CM and Exos obtained from DSCs have been shown to promote neurite outgrowth and neuroprotective effects as well as their combination with scaffold materials (to improve their functional integration in the tissue). For these reasons, the secretome obtained from DSCs in combination with scaffold materials may represent a promising tissue engineering approach for neuroprotective and neuro-regenerative treatments.
Asunto(s)
Células Madre Mesenquimatosas , Enfermedades del Sistema Nervioso , Humanos , Secretoma , Ingeniería de Tejidos , Tratamiento Basado en Trasplante de Células y Tejidos , Enfermedades del Sistema Nervioso/terapiaRESUMEN
PURPOSE: Breast cancer (BC) is the most common malignancy that affects women, and it is, to date, their leading cause of death. Luminal A molecular subtype accounts for 40% of BC and is characterized by hormone receptors positive/human epidermal growth factor 2 expression and current treatment consists of surgery plus aromatase inhibitor therapy. Interestingly, several studies demonstrated that the heavy metal cadmium (Cd), classified as a group 1 human carcinogen and widely spread in the environment, exerts estrogen-like activities in several tissues and suggested an intriguing relationship between increased Cd exposure and BC incidence. Thus, aim of this study was to evaluate effects of Cd on Luminal A BC estrogen receptor (ER) positive/progesterone receptor positive cell models in vitro to characterize the mechanism(s) involved in breast cell homeostasis disruption. METHODS: T47D and MCF7 were exposed to Cd (0.5-1 µM) for 6-24 h to evaluate potential alterations in: cells viability, steroid receptors and intracellular signaling by western blot. Moreover, we evaluated the expression of inflammatory cytokines interleukin by RT-PCR. RESULTS: Our results showed a significant induction of androgen receptor (AR) and an increased AR/ER ratio. Further, Cd exposure increased pro-inflammatory cytokines interleukin (IL)6, IL8 and tumor necrosis factor α levels. Finally, as previously demonstrated by our group, Cd alters pathways such as mitogen-activated protein kinase family and protein kinase B. CONCLUSION: In conclusion, our study demonstrates that Cd modifies the expression and pattern of ERs and AR in BC cell lines, suggesting an alteration of BC cells homeostasis, likely predisposing to a carcinogenetic microenvironment.
Asunto(s)
Neoplasias de la Mama , Disruptores Endocrinos , Femenino , Humanos , Neoplasias de la Mama/patología , Cadmio/toxicidad , Disruptores Endocrinos/farmacología , Andrógenos/farmacología , Receptores Androgénicos/metabolismo , Receptores de Estrógenos/metabolismo , Citocinas , Estrógenos , Interleucina-6 , Línea Celular Tumoral , Microambiente TumoralRESUMEN
Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the KD value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death.
Asunto(s)
Adhesinas Bacterianas , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Adhesión Bacteriana , Interacciones Huésped-Patógeno , Lectinas , Humanos , Adhesinas Bacterianas/metabolismo , Adhesinas Bacterianas/genética , Antígenos CD/metabolismo , Antígenos CD/genética , Lectinas/metabolismo , Lectinas/genética , Lectinas/inmunología , Animales , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Unión Proteica , Ratones , Células CHO , Cricetulus , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Neisseria meningitidis/inmunología , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismo , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Células Epiteliales/inmunología , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/inmunología , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Neisseria meningitidis Serogrupo B/genética , Neisseria meningitidis Serogrupo B/inmunología , Neisseria meningitidis Serogrupo B/metabolismoRESUMEN
Primary malignant brain tumors are the most common solid neoplasm in childhood. Despite recent advances, many children affected by aggressive or metastatic brain tumors still present poor prognosis, therefore the development of more effective therapies is urgent. Cancer stem cells (CSCs) have been discovered and isolated in both pediatric and adult patients with brain tumors (e.g., medulloblastoma, gliomas and ependymoma). CSCs are a small clonal population of cancer cells responsible for brain tumor initiation, maintenance and progression, displaying resistance to conventional anticancer therapies. CSCs are characterized by a specific repertoire of surface markers and intracellular specific pathways. These unique features of CSCs biology offer the opportunity to build therapeutic approaches to specifically target these cells in the complex tumor bulk. Treatment of pediatric brain tumors with classical chemotherapeutic regimen poses challenges both for tumor location and for the presence of the blood-brain barrier (BBB). Lastly, the application of chemotherapy to a developing brain is followed by long-term sequelae, especially on cognitive abilities. Novel avenues are emerging in the therapeutic panorama taking advantage of nanomedicine. In this review we will summarize nanoparticle-based approaches and the efficacy that NPs have intrinsically demonstrated and how they are also decorated by biomolecules. Furthermore, we propose novel cargoes together with recent advances in nanoparticle design/synthesis with the final aim to specifically target the insidious CSCs population in the tumor bulk.
RESUMEN
The TAR-DNA binding protein (TDP43) is a nuclear protein whose cytoplasmic inclusions are hallmarks of Amyotrophic Lateral Sclerosis (ALS). Acute stress in cells causes TDP43 mobilization to the cytoplasm and its aggregation through different routes. Although acute stress elicits a strong phenotype, is far from recapitulating the years-long aggregation process. We applied different chronic stress protocols and described TDP43 aggregation in a human neuroblastoma cell line by combining solubility assays, thioflavin-based microscopy and flow cytometry. This approach allowed us to detect, for the first time to our knowledge in vitro, the formation of 25 kDa C-terminal fragment of TDP43, a pathogenic hallmark of ALS. Our results indicate that chronic stress, compared to the more common acute stress paradigm, better recapitulates the cell biology of TDP43 proteinopathies. Moreover, we optimized a protocol for the detection of bona fide prions in living cells, suggesting that TDP43 may form amyloids as a stress response.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Humanos , Esclerosis Amiotrófica Lateral/genética , Línea Celular , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Neuroblastoma/metabolismo , Proteinopatías TDP-43/metabolismoRESUMEN
Despite decades of research, no therapies are available to halt or slow down the course of neuro-degenerative disorders. Most of the drugs developed to fight neurodegeneration are aimed to alleviate symptoms, but none has proven adequate in altering the course of the pathologies. Cell therapy has emerged as an intriguing alternative to the classical pharmacological approach. Cell therapy consists of the transplantation of stem cells that can be obtained from various embryonal and adult tissues. Whereas the former holds notable ethical issue, adult somatic stem cells can be obtained without major concerns. However, most adult stem cells, such as those derived from the bone marrow, are committed toward the mesodermal lineage, and hence need to be reprogrammed to induce the differentiation into the neurons. The discovery of neural crest stem cells in the dental pulp, both in adults' molar and in baby teeth (dental pulp stem cells and stem cells from human exfoliated deciduous teeth, respectively) prompted researchers to investigate their utility as therapy in nervous system disorders. In this review, we recapitulate the advancements on the application of these stem cells in preclinical models of neurodegenerative diseases, highlighting differences and analogies in their maintenance, differentiation, and potential clinical application.
Asunto(s)
Células Madre Mesenquimatosas , Adulto , Humanos , Pulpa Dental , Células Madre , Sistema Nervioso , Diente PrimarioRESUMEN
Medullary thyroid carcinoma (MTC) is a malignant tumor with challenging management. Multi-targeted kinase inhibitors (MKI) and tyrosine-kinase inhibitors (TKI) with high specificity for RET protein are approved for advanced MTC treatment. However, their efficacy is hindered by evasion mechanisms of tumor cells. Thus, the aim of this study was the identification of an escape mechanism in MTC cells exposed to a highly selective RET TKI. TT cells were treated with TKI, MKI, and/or the HH-Gli inhibitors, GANT61 and Arsenic Trioxide (ATO), in the presence or absence of hypoxia. RET modifications, oncogenic signaling activation, proliferation and apoptosis were assessed. Additionally, cell modifications and HH-Gli activation were also evaluated in pralsetinib-resistant TT cells. Pralsetinib inhibited RET autophosphorylation and RET downstream pathways activation in normoxic and hypoxic conditions. Additionally, pralsetinib impaired proliferation, induced the activation of apoptosis and, in hypoxic cells, downregulated HIF-1α. Focusing on escape molecular mechanisms associated with therapy, we observed increased Gli1 levels in a subset of cells. Indeed, pralsetinib stimulated the re-localization of Gli1 into the cell nuclei. Treatment of TT cells with both pralsetinib and ATO resulted in Gli1 down-regulation and impaired cell viability. Moreover, pralsetinib-resistant cells confirmed Gli1 activation and up-regulation of its transcriptionally regulated target genes. Altogether, we showed that pralsetinib impairs MTC cell growth and induces cell death, also in hypoxic conditions. The HH-Gli pathway is a new molecular mechanism of escape to pralsetinib therapy that can be overcome through combined therapy.
Asunto(s)
Neoplasias de la Tiroides , Humanos , Proteína con Dedos de Zinc GLI1/metabolismo , Transducción de Señal , Trióxido de Arsénico , Neoplasias de la Tiroides/genéticaRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer-related mortality and chemoresistance is a major medical issue. The epithelial-to-mesenchymal transition (EMT) is the primary step in the emergence of the invasive phenotype and the Hedgehog-GLI (HH-GLI) and NOTCH signaling pathways are associated with poor prognosis and EMT in CRC. CRC cell lines harboring KRAS or BRAF mutations, grown as monolayers and organoids, were treated with the chemotherapeutic agent 5-Fluorouracil (5-FU) alone or combined with HH-GLI and NOTCH pathway inhibitors GANT61 and DAPT, or arsenic trioxide (ATO) to inhibit both pathways. Treatment with 5-FU led to the activation of HH-GLI and NOTCH pathways in both models. In KRAS mutant CRC, HH-GLI and NOTCH signaling activation co-operate to enhance chemoresistance and cell motility, while in BRAF mutant CRC, the HH-GLI pathway drives the chemoresistant and motile phenotype. We then showed that 5-FU promotes the mesenchymal and thus invasive phenotype in KRAS and BRAF mutant organoids and that chemosensitivity could be restored by targeting the HH-GLI pathway in BRAF mutant CRC or both HH-GLI and NOTCH pathways in KRAS mutant CRC. We suggest that in KRAS-driven CRC, the FDA-approved ATO acts as a chemotherapeutic sensitizer, whereas GANT61 is a promising chemotherapeutic sensitizer in BRAF-driven CRC.
RESUMEN
Medullary Thyroid Carcinoma (MTC) is a rare neuroendocrine tumour whose diagnosis includes evaluating calcitonin serum levels, which can present fluctuations unrelated to MTC. Here, we investigated circulating DNA fragmentation and methylation changes as potential biomarkers using ddPCR on cell-free DNA (cfDNA) isolated from the plasma of MTC patients. For cfDNA fragmentation analysis, we investigated the fragment size distribution of a gene family and calculated short fragment fraction (SFF). Methylation analyses evaluated the methylation levels of CG_16698623, a CG dinucleotide in the MGMT gene that we found hypermethylated in MTC tissues by analyzing public databases. The SFF ratio and methylation of CG_16698623 were significantly increased in plasma from MTC patients at diagnosis, and patients with clinical remission or stable disease at follow-up showed no significant SFF difference compared with healthy subjects. Our data support the diagnostic value of cfDNA traits that could enable better management of MTC patients.
RESUMEN
The Ser-Thr kinase CK2 plays important roles in sustaining cell survival and resistance to stress and these functions are exploited by different types of blood tumors. Yet, the physiological involvement of CK2 in normal blood cell development is poorly known. Here, we discovered that the ß regulatory subunit of CK2 is critical for normal hematopoiesis in the mouse. Fetal livers of conditional CK2ß knockout embryos showed increased numbers of hematopoietic stem cells associated to a higher proliferation rate compared to control animals. Both hematopoietic stem and progenitor cells (HSPCs) displayed alterations in the expression of transcription factors involved in cell quiescence, self-renewal, and lineage commitment. HSPCs lacking CK2ß were functionally impaired in supporting both in vitro and in vivo hematopoiesis as demonstrated by transplantation assays. Furthermore, KO mice developed anemia due to a reduced number of mature erythroid cells. This compartment was characterized by dysplasia, proliferative defects at early precursor stage, and apoptosis at late-stage erythroblasts. Erythroid cells exhibited a marked compromise of signaling cascades downstream of the cKit and erythropoietin receptor, with a defective activation of ERK/JNK, JAK/STAT5, and PI3K/AKT pathways and perturbations of several transcriptional programs as demonstrated by RNA-Seq analysis. Moreover, we unraveled an unforeseen molecular mechanism whereby CK2 sustains GATA1 stability and transcriptional proficiency. Thus, our work demonstrates new and crucial functions of CK2 in HSPC biology and in erythropoiesis.
RESUMEN
Mantle cell lymphoma (MCL) is an incurable B cell non-Hodgkin lymphoma, characterized by frequent relapses. In the last decade, the pro-survival pathways related to BCR signaling and Bcl-2 have been considered rational therapeutic targets in B cell derived lymphomas. The BTK inhibitor Ibrutinib and the Bcl-2 inhibitor Venetoclax are emerging as effective drugs for MCL. However, primary and acquired resistance also to these agents may occur. Protein Kinase CK2 is a S/T kinase overexpressed in many solid and blood-derived tumours. CK2 promotes cancer cell growth and clonal expansion, sustaining pivotal survival signaling cascades, such as the ones dependent on AKT, NF-κB, STAT3 and others, counteracting apoptosis through a "non-oncogene" addiction mechanism. We previously showed that CK2 is overexpressed in MCL and regulates the levels of activating phosphorylation on S529 of the NF-κB family member p65/RelA. In the present study, we investigated the effects of CK2 inactivation on MCL cell proliferation, survival and apoptosis and this kinase's involvement in the BCR and Bcl-2 related signaling. By employing CK2 loss of function MCL cell models, we demonstrated that CK2 sustains BCR signaling (such as BTK, NF-κB and AKT) and the Bcl-2-related Mcl-1 expression. CK2 inactivation enhanced Ibrutinib and Venetoclax-induced cytotoxicity. The demonstration of a CK2-dependent upregulation of pathways that may antagonize the effect of these drugs may offer a novel strategy to overcome primary and secondary resistance.
RESUMEN
Mantle Cell Lymphoma (MCL) is still an incurable B-cell malignancy characterized by poor prognosis and frequent relapses. B Cell Receptor (BCR) signaling inhibitors, in particular of the kinases BTK and PI3Kγ/δ, have demonstrated clinically meaningful anti-proliferative effects in B cell tumors. However, refractoriness to these drugs may develop, portending a dismal prognosis. Protein kinase CK1α is an emerging pro-growth enzyme in B cell malignancies. In multiple myeloma, this kinase sustains ß-catenin and AKT-dependent survival and is involved in the activation of NF-κB in B cells. In this study, we analyzed the role of CK1α on MCL cell survival and proliferation, on the regulation of BCR-related BTK, NF-κB, PI3K/AKT signaling cascades and the effects of CK1α chemical inhibition or gene silencing in association with the BTK inhibitor Ibrutinib or the PI3Kγ/δ inhibitor Duvelisib. CK1α was found highly expressed in MCL cells as compared to normal B cells. The inactivation/loss of CK1α caused MCL cell apoptosis and proliferation arrest. CK1α sustained BCR signaling, in particular the NF-κB, AKT and BTK pathways by modulating the phosphorylation of Ser 652 on CARD11, Ser 536 p65 on NF-κB, Ser 473 on AKT, Tyr 223 on BTK, as well as the protein levels. We also provided evidence that CK1α-mediated regulation of CARD11 and BTK likely implicates a physical interaction. The combination of CK1α inhibition with Ibrutinib or Duvelisib synergistically increased cytotoxicity, leading to a further decrease of the activation of BCR signaling pathways. Therefore, CK1α sustains MCL growth through the regulation of BCR-linked survival signaling cascades and protects from Ibrutinib/Duvelisib-induced apoptosis. Thus, CK1α could be considered as a rational molecular target for the treatment of MCL, in association with novel agents.
RESUMEN
Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.
Asunto(s)
Ciclo Celular/fisiología , Proliferación Celular/fisiología , Glioblastoma/patología , Receptor Muscarínico M2/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Neoplasias Encefálicas/genética , División Celular/fisiología , Línea Celular Tumoral , Receptores ErbB/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Células Madre Neoplásicas/patología , Receptor Muscarínico M2/genética , Transducción de Señal/efectos de los fármacosRESUMEN
In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100⯵M) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper action appear to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation.