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1.
Biology (Basel) ; 10(9)2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34571806

RESUMO

Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.

2.
Viruses ; 13(6)2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067349

RESUMO

Europe is experiencing a third wave of COVID-19 due to the spread of highly transmissible SARS-CoV-2 variants. A number of positive and negative factors constantly shape the rates of COVID-19 infections, hospitalization, and mortality. Among these factors, the rise in increasingly transmissible variants on one side and the effect of vaccinations on the other side create a picture deeply different from that of the first pandemic wave. Starting from the observation that in several European countries the number of COVID-19 infections in the second and third pandemic wave increased without a proportional rise in disease severity and mortality, we hypothesize the existence of an additional factor influencing SARS-CoV-2 dynamics. This factor consists of an immune defence against severe COVID-19, provided by SARS-CoV-2-specific T cells progressively developing upon natural exposure to low virus doses present in populated environments. As suggested by recent studies, low-dose viral particles entering the respiratory and intestinal tracts may be able to induce T cell memory in the absence of inflammation, potentially resulting in different degrees of immunization. In this scenario, non-pharmaceutical interventions would play a double role, one in the short term by reducing the detrimental spreading of SARS-CoV-2 particles, and one in the long term by allowing the development of a widespread (although heterogeneous and uncontrollable) form of immune protection.


Assuntos
COVID-19/imunologia , SARS-CoV-2/imunologia , Linfócitos T/imunologia , COVID-19/prevenção & controle , Relação Dose-Resposta Imunológica , Exposição Ambiental , Humanos , Memória Imunológica
3.
Front Oncol ; 10: 592891, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33194755

RESUMO

Severe coronavirus disease 2019 (COVID-19) causes an uncontrolled activation of the innate immune response, resulting in acute respiratory distress syndrome and systemic inflammation. The effects of COVID-19-induced inflammation on cancer cells and their microenvironment are yet to be elucidated. Here, we formulate the hypothesis that COVID-19-associated inflammation may generate a microenvironment favorable to tumor cell proliferation and particularly to the reawakening of dormant cancer cells (DCCs). DCCs often survive treatment of primary tumors and populate premetastatic niches in the lungs and other organs, retaining the potential for metastatic outgrowth. DCCs reawakening may be promoted by several events associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, including activation of neutrophils and monocytes/macrophages, lymphopenia and an uncontrolled production of pro-inflammatory cytokines. Among pro-inflammatory factors produced during COVID-19, neutrophil extracellular traps (NETs) released by activated neutrophils have been specifically shown to activate premetastatic cancer cells disseminated in the lungs, suggesting they may be involved in DCCs reawakening in COVID-19 patients. If confirmed by further studies, the links between COVID-19, DCCs reactivation and tumor relapse may support the use of specific anti-inflammatory and anti-metastatic therapies in patients with COVID-19 and an active or previous cancer.

4.
Breast Cancer Res ; 22(1): 117, 2020 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-33126915

RESUMO

Severe coronavirus disease 2019 (COVID-19) causes a hyperactivation of immune cells, resulting in lung inflammation. Recent studies showed that COVID-19 induces the production of factors previously implicated in the reawakening of dormant breast cancer cells such as neutrophil extracellular traps (NETs). The presence of NETs and of a pro-inflammatory microenvironment may therefore promote breast cancer reactivation, increasing the risk of pulmonary metastasis. Further studies will be required to confirm the link between COVID-19 and cancer recurrence. However, an increased awareness on the potential risks for breast cancer patients with COVID-19 may lead to improved treatment strategies to prevent metastatic relapse.


Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/virologia , Infecções por Coronavirus/imunologia , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/virologia , Pneumonia Viral/imunologia , Betacoronavirus/imunologia , Neoplasias da Mama/patologia , COVID-19 , Infecções por Coronavirus/virologia , Armadilhas Extracelulares/imunologia , Feminino , Humanos , Pulmão/imunologia , Pulmão/patologia , Recidiva Local de Neoplasia/patologia , Neutrófilos/imunologia , Pandemias , Pneumonia/imunologia , Pneumonia/virologia , Pneumonia Viral/virologia , SARS-CoV-2 , Microambiente Tumoral/imunologia
5.
J Exp Clin Cancer Res ; 39(1): 2, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31910865

RESUMO

BACKGROUND: Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown. METHODS: A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets. RESULTS: Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes. CONCLUSIONS: These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.


Assuntos
Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos , Células-Tronco Neoplásicas/metabolismo , Regulação para Cima , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , MAP Quinase Quinase Quinase 5/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Oxaliplatina/farmacologia , Prognóstico
6.
Nutrients ; 11(12)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31816977

RESUMO

Colorectal cancer (CRC) is the third commonly diagnosed cancer and the second leading cause of cancer-related deaths worldwide. Global CRC burden is expected to increase by 60% in the next decade, with low-income countries experiencing an escalation of CRC incidence and mortality in parallel to the adoption of western lifestyles. CRC incidence is also sharply increasing in individuals younger than 50 years, often presenting at advanced stages and with aggressive features. Both genetic and environmental factors have been recognized as major contributors for the development of CRC, the latter including diet-related conditions such as chronic inflammation and obesity. In particular, a diet rich in fat and sugars (Western-style diet, WSD) has been shown to induce multiple pathophysiological changes in the intestine linked to an increased risk of CRC. In this scenario, dietary factors have been recently shown to play novel unexpected roles in the regulation of intestinal stem cells (ISCs) and of the gut microbiota, which represent the two main biological systems responsible for intestinal homeostasis. Furthermore, diet is increasingly recognized to play a key role in the neoplastic transformation of ISCs and in the metabolic regulation of colorectal cancer stem cells. This review illustrates novel discoveries on the role of dietary components in regulating intestinal homeostasis and colorectal tumorigenesis. Particular focus is dedicated to new areas of research with potential clinical relevance including the effect of food components on ISCs and cancer stem cells (CSCs), the existence of CRC-specific microbial signatures and the alterations of intestinal homeostasis potentially involved in early-onset CRC. New insights on the role of dietary factors in intestinal regulation will provide new tools not only for the prevention and early diagnosis of CRC but also for improving the effectiveness of current CRC therapies.


Assuntos
Neoplasias Colorretais , Dieta , Trato Gastrointestinal/fisiologia , Homeostase , Células-Tronco/fisiologia , Microbioma Gastrointestinal , Humanos
7.
Front Oncol ; 9: 1245, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31824842

RESUMO

Although stem cell factor (SCF)/cKIT interaction plays key functions in erythropoiesis, cKIT signaling in human erythroid cells is still poorly defined. To provide new insights into cKIT-mediated erythroid expansion in development and disease, we performed phosphoproteomic profiling of primary erythroid progenitors from adult blood (AB), cord blood (CB), and Polycythemia Vera (PV) at steady-state and upon SCF stimulation. While AB and CB, respectively, activated transient or sustained canonical cKIT-signaling, PV showed a non-canonical signaling including increased mTOR and ERK1 and decreased DEPTOR. Accordingly, screening of FDA-approved compounds showed increased PV sensitivity to JAK, cKIT, and MEK inhibitors. Moreover, differently from AB and CB, in PV the mature 145kDa-cKIT constitutively associated with the tetraspanin CD63 and was not endocytosed upon SCF stimulation, contributing to unrestrained cKIT signaling. These results identify a clinically exploitable variegation of cKIT signaling/metabolism that may contribute to the great erythroid output occurring during development and in PV.

8.
Cancers (Basel) ; 11(10)2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31619007

RESUMO

Breast cancer is the most frequent cancer among women worldwide. Therapeutic strategies to prevent or treat metastatic disease are still inadequate although great progress has been made in treating early-stage breast cancer. Cancer stem-like cells (CSCs) that are endowed with high plasticity and self-renewal properties have been shown to play a key role in breast cancer development, progression, and metastasis. A subpopulation of CSCs that combines tumor-initiating capacity and a dormant/quiescent/slow cycling status is present throughout the clinical history of breast cancer patients. Dormant/quiescent/slow cycling CSCs are a key component of tumor heterogeneity and they are responsible for chemoresistance, tumor migration, and metastatic dormancy, defined as the ability of CSCs to survive in target organs and generate metastasis up to two decades after diagnosis. Understanding the strategies that are used by CSCs to resist conventional and targeted therapies, to interact with their niche, to escape immune surveillance, and finally to awaken from dormancy is of key importance to prevent and treat metastatic cancer. This review summarizes the current understanding of mechanisms involved in CSCs chemoresistance, dissemination, and metastasis in breast cancer, with a particular focus on dormant cells. Finally, we discuss how advancements in the detection, molecular understanding, and targeting of dormant CSCs will likely open new therapeutic avenues for breast cancer treatment.

9.
J Exp Clin Cancer Res ; 38(1): 373, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439019

RESUMO

BACKGROUND: An increasing number of anticancer agents has been proposed in recent years with the attempt to overcome treatment-resistant cancer cells and particularly cancer stem cells (CSC), the major culprits for tumour resistance and recurrence. However, a huge obstacle to treatment success is the ineffective delivery of drugs within the tumour environment due to limited solubility, short circulation time or inconsistent stability of compounds that, together with concomitant dose-limiting systemic toxicity, contribute to hamper the achievement of therapeutic drug concentrations. The synthetic retinoid Fenretinide (4-hydroxy (phenyl)retinamide; 4-HPR) formerly emerged as a promising anticancer agent based on pre-clinical and clinical studies. However, a major limitation of fenretinide is traditionally represented by its poor aqueous solubility/bioavailability due to its hydrophobic nature, that undermined the clinical success of previous clinical trials. METHODS: Here, we developed a novel nano-micellar fenretinide formulation called bionanofenretinide (Bio-nFeR), based on drug encapsulation in an ion-pair stabilized lipid matrix, with the aim to raise fenretinide bioavailability and antitumour efficacy. RESULTS: Bio-nFeR displayed marked antitumour activity against lung, colon and melanoma CSC both in vitro and in tumour xenografts, in absence of mice toxicity. Bio-nFeR is suitable for oral administration, reaching therapeutic concentrations within tumours and an unprecedented therapeutic activity in vivo as single agent. CONCLUSION: Altogether, our results indicate Bio-nFeR as a novel anticancer agent with low toxicity and high activity against tumourigenic cells, potentially useful for the treatment of solid tumours of multiple origin.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Fenretinida/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Micelas , Células-Tronco Neoplásicas/efeitos dos fármacos , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Apoptose , Disponibilidade Biológica , Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Fenretinida/química , Fenretinida/farmacocinética , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cancers (Basel) ; 11(8)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31344798

RESUMO

Molecular alterations are not randomly distributed in colorectal cancer (CRC), but rather clustered on the basis of primary tumor location underlying the importance of colorectal cancer sidedness. We aimed to investigate whether circulating tumor cells (CTC) characterization might help clarify how different the patterns of dissemination might be relative to the behavior of left- (LCC) compared to right-sided (RCC) cancers. We retrospectively analyzed patients with metastatic CRC who had undergone standard baseline CTC evaluation before starting any first-line systemic treatment. Enumeration of CTC in left- and right-sided tumors were compared. The highest prognostic impact was exerted by CTC in left-sided primary cancer patients, even though the lowest median number of cells was detected in this subgroup of patients. CTC exhibit phenotypic heterogeneity, with a predominant mesenchymal phenotype found in CTC from distal compared to proximal primary tumors. Most CTC in RCC patients exhibited an apoptotic pattern. CTC in left-sided colon cancer patients exhibit a predominant mesenchymal phenotype. This might imply a substantial difference in the biology of proximal and distal cancers, associated with different patterns of tumor cells dissemination. The poor prognosis of right-sided CRC is not determined by the hematogenous dissemination of tumor cells, which appears to be predominantly a passive shedding of non-viable cells. Conversely, the subgroup of poor-prognosis left-sided CRC is reliably identified by the presence of mesenchymal CTC.

11.
Front Oncol ; 9: 626, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31355143

RESUMO

Cancer treatment with either standard chemotherapy or targeted agents often results in the emergence of drug-refractory cell populations, ultimately leading to therapy failure. The biological features of drug resistant cells are largely overlapping with those of cancer stem cells and include heterogeneity, plasticity, self-renewal ability, and tumor-initiating capacity. Moreover, drug resistance is usually characterized by a suppression of proliferation that can manifest as quiescence, dormancy, senescence, or proliferative slowdown. Alterations in key cellular pathways such as autophagy, unfolded protein response or redox signaling, as well as metabolic adaptations also contribute to the establishment of drug resistance, thus representing attractive therapeutic targets. Moreover, a complex interplay of drug resistant cells with the micro/macroenvironment and with the immune system plays a key role in dictating and maintaining the resistant phenotype. Recent studies have challenged traditional views of cancer drug resistance providing innovative perspectives, establishing new connections between drug resistant cells and their environment and indicating unexpected therapeutic strategies. In this review we discuss recent advancements in understanding the mechanisms underlying drug resistance and we report novel targeting agents able to overcome the drug resistant status, with particular focus on strategies directed against dormant cells. Research on drug resistant cancer cells will take us one step forward toward the development of novel treatment approaches and the improvement of relapse-free survival in solid and hematological cancer patients.

12.
Cell Death Dis ; 10(7): 529, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31332161

RESUMO

Fenretinide is a synthetic retinoid characterized by anticancer activity in preclinical models and favorable toxicological profile, but also by a low bioavailability that hindered its clinical efficacy in former clinical trials. We developed a new formulation of fenretinide complexed with 2-hydroxypropyl-beta-cyclodextrin (nanofenretinide) characterized by an increased bioavailability and therapeutic efficacy. Nanofenretinide was active in cell lines derived from multiple solid tumors, in primary spheroid cultures and in xenografts of lung and colorectal cancer, where it inhibited tumor growth independently from the mutational status of tumor cells. A global profiling of pathways activated by nanofenretinide was performed by reverse-phase proteomic arrays and lipid analysis, revealing widespread repression of the mTOR pathway, activation of apoptotic, autophagic and DNA damage signals and massive production of dihydroceramide, a bioactive lipid with pleiotropic effects on several biological processes. In cells that survived nanofenretinide treatment there was a decrease of factors involved in cell cycle progression and an increase in the levels of p16 and phosphorylated p38 MAPK with consequent block in G0 and early G1. The capacity of nanofenretinide to induce cancer cell death and quiescence, together with its elevated bioavailability and broad antitumor activity indicate its potential use in cancer treatment and chemoprevention.


Assuntos
Antineoplásicos/uso terapêutico , Fenretinida/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Cancer Immunol Res ; 6(6): 658-670, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29622580

RESUMO

Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1). Such mice are unresponsive to this family of cytokines. Compared with parental neu+/- mice (neuT mice), IFNAR1-/- neu+/- mice (IFNAR-neuT mice) showed earlier onset and increased tumor multiplicity with marked vascularization. IFNAR-neuT tumors exhibited deregulation of genes having adverse prognostic value in breast cancer patients, including the breast cancer stem cell (BCSC) marker aldehyde dehydrogenase-1A1 (ALDH1A1). An increased number of BCSCs were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 enzymatic activity, clonogenic assay, and tumorigenic capacity. In vitro exposure of neuT+ mammospheres and cell lines to antibodies to IFN-I resulted in increased frequency of ALDH+ cells, suggesting that IFN-I controls stemness in tumor cells. Altogether, these results reveal a role of IFN-I in neuT-driven spontaneous carcinogenesis through intrinsic control of BCSCs. Cancer Immunol Res; 6(6); 658-70. ©2018 AACR.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Interferon Tipo I/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptor ErbB-2/metabolismo , Transdução de Sinais , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Camundongos Knockout , Camundongos Transgênicos , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Receptor ErbB-2/genética , Ensaio Tumoral de Célula-Tronco
14.
Cell Biol Toxicol ; 34(6): 459-469, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29478126

RESUMO

Biobanking of molecularly characterized colorectal cancer stem cells (CSCs) generated from individual patients and growing as spheroids in defined serum-free media offer a fast, feasible, and multi-level approach for the screening of targeted therapies and drug resistance molecular studies. By combining in vitro and in vivo analyses of cetuximab efficacy with genetic data on an ongoing collection of stem cell-enriched spheroids, we describe the identification and preliminary characterization of microsatellite stable (MSS) CSCs that, despite the presence of the KRAS (G12D) mutation, display epidermal growth factor (EGF)-dependent growth and are strongly inhibited by anti-EGF-receptor (EGFR) treatment. In parallel, we detected an increased resistance to anti-EGFR therapy of microsatellite instable (MSI) CSC lines irrespective of KRAS mutational status. MSI CSC lines carried mutations in genes coding for proteins with a role in RAS and calcium signaling, highlighting the role of a genomically unstable context in determining anti-EGFR resistance. Altogether, these results argue for a multifactorial origin of anti-EGFR resistance that emerges as the effect of multiple events targeting direct and indirect regulators of the EGFR pathway. An improved understanding of key molecular determinants of sensitivity/resistance to EGFR inhibition will be instrumental to optimize the clinical efficacy of anti-EGFR agents, representing a further step towards personalized treatments.


Assuntos
Neoplasias Colorretais/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Bancos de Espécimes Biológicos/tendências , Cetuximab/farmacologia , Neoplasias Colorretais/fisiopatologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/fisiologia , Humanos , Mutação , Panitumumabe , Medicina de Precisão/métodos , Proteínas Proto-Oncogênicas p21(ras)/genética , Esferoides Celulares/fisiologia , Células Tumorais Cultivadas/fisiologia
15.
Gut ; 67(5): 903-917, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-28389531

RESUMO

OBJECTIVE: Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. DESIGN: To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. RESULTS: The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. CONCLUSIONS: LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.


Assuntos
Antineoplásicos/farmacologia , Quinase 1 do Ponto de Checagem/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Pirazinas/farmacologia , Pirazóis/farmacologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Neoplasias Colorretais/genética , Replicação do DNA/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Mutação , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Supressora de Tumor p53/genética
16.
Ecancermedicalscience ; 11: ed74, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29225696

RESUMO

Colorectal cancer (CRC) holds the woeful record of being the most preventable but least prevented type of cancer. Although healthy lifestyles and screening programs associated to early polyp removal greatly reduce CRC incidence and CRC-related death, the overall lack of information and of effective preventive policies is promoting an overwhelming global escalation of this disease. Moreover, new challenges such as the increasing occurrence of aggressive CRC in young adults highlight underlying changes not only in the incidence but also in the nature of this disease. In this scenario, CRC prevention should also undergo a significant transformation, embracing not only individual initiatives but also issues of social and international relevance. The nascent network of countries surrounding the Mediterranean basin (COLOMED, the COLOrectal cancer MEDiterranean network) serves as a paradigm of international cooperation aimed at broadening scientific collaboration and promoting effective health policies in CRC research and prevention.

17.
Int J Biol Markers ; 32(4): e415-e420, 2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-28604994

RESUMO

BACKGROUND: Several studies have raised the issue of the inadequacy of CellSearch® to detect the entire pool of circulating tumor cells (CTCs) from blood of cancer patients, suggesting that cells expressing low levels of epithelial cell adhesion molecule (EpCAM) are not recognized by the capture reagent. In this exploratory study, we aimed to evaluate the status of EpCAM in CTCs isolated from a group of metastatic colorectal cancer patients, in 40% of whom, CTC had been found to be undetected by the CellSearch® system. METHODS: CTCs were analyzed using both a microfiltration method (ScreenCell) and CellSearch® in parallel. Furthermore, since EpCAM exists in 2 different variants, we investigated the presence of both its intracellular domain (EpICD) and extracellular domain (EpEX) through immunofluorescence staining of CTCs on filters. RESULTS: Results from immunofluorescence experiments demonstrated that, overall, EpICD and/or EpEX was expressed in 176 CTCs detected by ScreenCell, while the CellSearch® system was able to capture only 10 CTCs. CONCLUSIONS: This is the first demonstration that the low sensitivity of CellSearch® to detect CTCs in colorectal cancer patients is not due to the lack of EpCAM.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Molécula de Adesão da Célula Epitelial/sangue , Células Neoplásicas Circulantes/patologia , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/sangue , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Biópsia Líquida , Masculino , Pessoa de Meia-Idade
18.
Nat Commun ; 7: 12589, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27586544

RESUMO

Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/ß-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.


Assuntos
Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Pluripotentes/fisiologia , beta Catenina/metabolismo , Animais , Reprogramação Celular/genética , Fator de Crescimento Epidérmico/genética , Camadas Germinativas/citologia , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Proteína Nodal/metabolismo , Proteína Smad2/metabolismo , Proteínas Wnt/metabolismo
19.
Exp Hematol ; 44(12): 1138-1155.e4, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27592389

RESUMO

To assess the role of abnormal transforming growth factor-beta (TGF-ß) signaling in the pathogenesis of primary myelofibrosis (PMF), the effects of the TGF-ß receptor-1 kinase inhibitor SB431542 on ex vivo expansion of hematopoietic cells in cultures from patients with JAK2V617+-polycythemia vera (PV) or PMF (JAK2V617F+, CALRpQ365f+, or unknown) and from normal sources (adult blood, AB, or cord blood, CB) were compared. In cultures of normal sources, SB431542 significantly increased by 2.5-fold the number of progenitor cells generated by days 1-2 (CD34+) and 6 (colony-forming cells) (CB) and that of precursor cells, mostly immature erythroblasts, by days 14-17 (AB and CB). In cultures of JAK2V617F+-PV, SB431542 increased by twofold the numbers of progenitor cells by day 10 and had no effect on that of precursors cells by days 12-17 (∼fourfold increase in all cases). In contrast, SB431542 had no effect on the number of either progenitor or precursor cells in cultures of JAK2V617F+ and CALR pQ365fs+ PMF. These ontogenetic- and disease-specific effects were associated with variegation in the ability of SB431542 to induce CD34+ cells from AB (increased), CB (decreased), or PV and PMF (unaffected) into cycle and erythroblasts in proliferation (increased for AB and PV and unaffected for CB and PMF). Differences in expansion of erythroblasts from AB, CB, and PV were associated with differences in activation of TGF-ß signaling (SHCY317, SMAD2S245/250/255, and SMAD1S/S/SMAD5S/S/SMAD8S/S) detectable in these cells by phosphoproteomic profiling. In conclusion, treatment with TGF-ß receptor-1 kinase inhibitors may reactivate normal hematopoiesis in PMF patients, providing a proliferative advantage over the unresponsive malignant clone.


Assuntos
Terapia de Alvo Molecular , Mielofibrose Primária/etiologia , Mielofibrose Primária/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Antígenos CD34/metabolismo , Benzamidas/farmacologia , Biomarcadores , Ciclo Celular , Células Cultivadas , Dioxóis/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Sangue Fetal , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Janus Quinase 2/metabolismo , Camundongos , Mutação , Fenótipo , Policitemia Vera/metabolismo , Mielofibrose Primária/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/sangue , Fator de Crescimento Transformador beta/metabolismo
20.
Stem Cells Transl Med ; 5(4): 511-23, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26956206

RESUMO

UNLABELLED: Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]-targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)-driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC-enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC-enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti-EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance. SIGNIFICANCE: Colorectal cancer stem cells (CSCs) have been shown to be responsible for tumor propagation, metastatic dissemination, and relapse. However, molecular pathways present in CSCs, as well as mechanisms of therapy resistance, are mostly unknown. Taking advantage of genetically characterized CSC lines derived from colorectal tumors, this study provides an extensive analysis of CSC response to EGFR-targeted therapy in vivo and an overview of factors implicated in therapy response or resistance. Furthermore, the implementation of a biobank of molecularly annotated CSC lines provides an innovative resource for future investigations in colorectal cancer.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas/patologia , Animais , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Análise em Microsséries , Modelos Biológicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Transdução de Sinais/genética
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