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1.
Nat Commun ; 15(1): 3363, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38637494

RESUMO

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.


Assuntos
Neoplasias Colorretais , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/metabolismo , Fenótipo
2.
Nat Cancer ; 5(3): 448-462, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38267628

RESUMO

Chemotherapy often generates intratumoral senescent cancer cells that strongly modify the tumor microenvironment, favoring immunosuppression and tumor growth. We discovered, through an unbiased proteomics screen, that the immune checkpoint inhibitor programmed cell death 1 ligand 2 (PD-L2) is highly upregulated upon induction of senescence in different types of cancer cells. PD-L2 is not required for cells to undergo senescence, but it is critical for senescent cells to evade the immune system and persist intratumorally. Indeed, after chemotherapy, PD-L2-deficient senescent cancer cells are rapidly eliminated and tumors do not produce the senescence-associated chemokines CXCL1 and CXCL2. Accordingly, PD-L2-deficient pancreatic tumors fail to recruit myeloid-derived suppressor cells and undergo regression driven by CD8 T cells after chemotherapy. Finally, antibody-mediated blockade of PD-L2 strongly synergizes with chemotherapy causing remission of mammary tumors in mice. The combination of chemotherapy with anti-PD-L2 provides a therapeutic strategy that exploits vulnerabilities arising from therapy-induced senescence.


Assuntos
Neoplasias Pancreáticas , Animais , Camundongos , Neoplasias Pancreáticas/metabolismo , Linfócitos T CD8-Positivos/patologia , Tolerância Imunológica , Terapia de Imunossupressão , Senescência Celular , Microambiente Tumoral
3.
Nat Metab ; 5(12): 2111-2130, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38097808

RESUMO

Fibrogenesis is part of a normal protective response to tissue injury that can become irreversible and progressive, leading to fatal diseases. Senescent cells are a main driver of fibrotic diseases through their secretome, known as senescence-associated secretory phenotype (SASP). Here, we report that cellular senescence, and multiple types of fibrotic diseases in mice and humans are characterized by the accumulation of iron. We show that vascular and hemolytic injuries are efficient in triggering iron accumulation, which in turn can cause senescence and promote fibrosis. Notably, we find that senescent cells persistently accumulate iron, even when the surge of extracellular iron has subdued. Indeed, under normal conditions of extracellular iron, cells exposed to different types of senescence-inducing insults accumulate abundant ferritin-bound iron, mostly within lysosomes, and present high levels of labile iron, which fuels the generation of reactive oxygen species and the SASP. Finally, we demonstrate that detection of iron by magnetic resonance imaging might allow non-invasive assessment of fibrotic burden in the kidneys of mice and in patients with renal fibrosis. Our findings suggest that iron accumulation plays a central role in senescence and fibrosis, even when the initiating events may be independent of iron, and identify iron metabolism as a potential therapeutic target for senescence-associated diseases.


Assuntos
Senescência Celular , Fenótipo Secretor Associado à Senescência , Humanos , Ferro , Rim , Fibrose
4.
Nat Commun ; 13(1): 6840, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36369429

RESUMO

The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.


Assuntos
Neoplasias , RNA Longo não Codificante , Sumoilação , Humanos , Neoplasias/genética , Proteínas rho de Ligação ao GTP/metabolismo , Ubiquitinas/metabolismo , RNA Longo não Codificante/genética
5.
Nat Cancer ; 3(9): 1052-1070, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35773527

RESUMO

Colorectal cancer (CRC) patient-derived organoids predict responses to chemotherapy. Here we used them to investigate relapse after treatment. Patient-derived organoids expand from highly proliferative LGR5+ tumor cells; however, we discovered that lack of optimal growth conditions specifies a latent LGR5+ cell state. This cell population expressed the gene MEX3A, is chemoresistant and regenerated the organoid culture after treatment. In CRC mouse models, Mex3a+ cells contributed marginally to metastatic outgrowth; however, after chemotherapy, Mex3a+ cells produced large cell clones that regenerated the disease. Lineage-tracing analysis showed that persister Mex3a+ cells downregulate the WNT/stem cell gene program immediately after chemotherapy and adopt a transient state reminiscent to that of YAP+ fetal intestinal progenitors. In contrast, Mex3a-deficient cells differentiated toward a goblet cell-like phenotype and were unable to resist chemotherapy. Our findings reveal that adaptation of cancer stem cells to suboptimal niche environments protects them from chemotherapy and identify a candidate cell of origin of relapse after treatment in CRC.


Assuntos
Neoplasias Colorretais , Organoides , Animais , Diferenciação Celular , Neoplasias Colorretais/tratamento farmacológico , Camundongos , Células-Tronco Neoplásicas , Recidiva
6.
Nat Cancer ; 3(4): 418-436, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35469014

RESUMO

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.


Assuntos
Anticorpos Biespecíficos , Neoplasias Epiteliais e Glandulares , Anticorpos Biespecíficos/farmacologia , Receptores ErbB/metabolismo , Humanos , Imidazóis , Neoplasias Epiteliais e Glandulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismo
7.
Cells ; 10(4)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33921436

RESUMO

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.


Assuntos
Diferenciação Celular , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Impressão Genômica , Inibidores de Proteínas Quinases/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Células Germinativas/citologia , Células Germinativas/efeitos dos fármacos , Células Germinativas/metabolismo , Humanos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacos
8.
Sci Rep ; 9(1): 14065, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575908

RESUMO

CD98 heavy chain (CD98hc) forms heteromeric amino acid (AA) transporters by interacting with different light chains. Cancer cells overexpress CD98hc-transporters in order to meet their increased nutritional and antioxidant demands, since they provide branched-chain AA (BCAA) and aromatic AA (AAA) availability while protecting cells from oxidative stress. Here we show that BCAA and AAA shortage phenocopies the inhibition of mTORC1 signalling, protein synthesis and cell proliferation caused by CD98hc ablation. Furthermore, our data indicate that CD98hc sustains glucose uptake and glycolysis, and, as a consequence, the pentose phosphate pathway (PPP). Thus, loss of CD98hc triggers a dramatic reduction in the nucleotide pool, which leads to replicative stress in these cells, as evidenced by the enhanced DNA Damage Response (DDR), S-phase delay and diminished rate of mitosis, all recovered by nucleoside supplementation. In addition, proper BCAA and AAA availability sustains the expression of the enzyme ribonucleotide reductase. In this regard, BCAA and AAA shortage results in decreased content of deoxynucleotides that triggers replicative stress, also recovered by nucleoside supplementation. On the basis of our findings, we conclude that CD98hc plays a central role in AA and glucose cellular nutrition, redox homeostasis and nucleotide availability, all key for cell proliferation.


Assuntos
Aminoácidos/metabolismo , Ciclo Celular , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Nucleotídeos/metabolismo , Aminoácidos Aromáticos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Divisão Celular , Dano ao DNA , Reparo do DNA , Cadeia Pesada da Proteína-1 Reguladora de Fusão/fisiologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Estresse Oxidativo
10.
Nat Cell Biol ; 20(2): 211-221, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29358704

RESUMO

For many patients with breast cancer, symptomatic bone metastases appear after years of latency. How micrometastatic lesions remain dormant and undetectable before initiating colonization is unclear. Here, we describe a mechanism involved in bone metastatic latency of oestrogen receptor-positive (ER+) breast cancer. Using an in vivo genome-wide short hairpin RNA screening, we identified the kinase MSK1 as an important regulator of metastatic dormancy in breast cancer. In patients with ER+ breast cancer, low MSK1 expression associates with early metastasis. We show that MSK1 downregulation impairs the differentiation of breast cancer cells, increasing their bone homing and growth capacities. MSK1 controls the expression of genes required for luminal cell differentiation, including the GATA3 and FOXA1 transcription factors, by modulating their promoter chromatin status. Our results indicate that MSK1 prevents metastatic progression of ER+ breast cancer, suggesting that stratifying patients with breast cancer as high or low risk for early relapse based on MSK1 expression could improve prognosis.


Assuntos
Neoplasias da Mama/genética , Fator de Transcrição GATA3/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Adulto , Idoso , Animais , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Diferenciação Celular/genética , Cromatina/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Prognóstico , RNA Interferente Pequeno/genética , Receptores de Estrogênio/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Stem Cell Reports ; 10(1): 257-271, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29290625

RESUMO

Mammary stem and progenitor cells are essential for mammary gland homeostasis and are also candidates for cells of origin of mammary tumors. Here, we have investigated the function of the protein kinase p38α in the mammary gland using mice that delete this protein in the luminal epithelial cells. We show that p38α regulates the fate of luminal progenitor cells through modulation of the transcription factor RUNX1, an important controller of the estrogen receptor-positive cell lineage. We also provide evidence that the regulation of RUNX1 by p38α probably involves the kinase MSK1, which phosphorylates histone H3 at the RUNX1 promoter. Moreover, using a mouse model for breast cancer initiated by luminal cells, we show that p38α downregulation in mammary epithelial cells reduces tumor burden, which correlates with decreased numbers of tumor-initiating cells. Collectively, our results define a key role for p38α in luminal progenitor cell fate that affects mammary tumor formation.


Assuntos
Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Feminino , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/patologia , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo
12.
EMBO Mol Med ; 9(7): 869-879, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28468934

RESUMO

The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient-derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP+ cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage-tracing experiments showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation toward mucosecreting- and absorptive-like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/fisiopatologia , Células-Tronco Neoplásicas/fisiologia , Organoides , Animais , Diferenciação Celular , Proliferação de Células , Feminino , Edição de Genes/métodos , Técnicas de Introdução de Genes , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Xenoenxertos , Humanos , Camundongos SCID , Receptores Acoplados a Proteínas G/genética , Coloração e Rotulagem/métodos
13.
EMBO Rep ; 15(11): 1210-8, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25296644

RESUMO

Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces "tumor-like" overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-ß acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-ß. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-ß. In addition, Irx5 expression confers a growth advantage in the presence of TGF-ß, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-ß activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-ß, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-ß pathway components.


Assuntos
Adenocarcinoma/metabolismo , Carcinogênese/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Mucosa Intestinal/metabolismo , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Regulação para Cima
14.
Nat Cell Biol ; 16(7): 695-707, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24952462

RESUMO

Aberrant activation of WNT signalling and loss of BMP signals represent the two main alterations leading to the initiation of colorectal cancer (CRC). Here we screen for genes required for maintaining the tumour stem cell phenotype and identify the zinc-finger transcription factor GATA6 as a key regulator of the WNT and BMP pathways in CRC. GATA6 directly drives the expression of LGR5 in adenoma stem cells whereas it restricts BMP signalling to differentiated tumour cells. Genetic deletion of Gata6 from mouse colon adenomas increases the levels of BMP factors, which signal to block self-renewal of tumour stem cells. In human tumours, GATA6 competes with ß-catenin/TCF4 for binding to a distal regulatory region of the BMP4 locus that has been linked to increased susceptibility to development of CRC. Hence, GATA6 creates an environment permissive for CRC initiation by lowering the threshold of BMP signalling required for tumour stem cell expansion.


Assuntos
Adenoma , Receptores de Proteínas Morfogenéticas Ósseas/genética , Neoplasias Colorretais/fisiopatologia , Fator de Transcrição GATA6/metabolismo , Regulação Neoplásica da Expressão Gênica , Células-Tronco/citologia , Células-Tronco/metabolismo , Adenoma/patologia , Animais , Antineoplásicos/farmacologia , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Feminino , Imunofluorescência , Fator de Transcrição GATA6/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Células-Tronco/efeitos dos fármacos , Proteínas Wnt/metabolismo
15.
PLoS Comput Biol ; 6(5): e1000777, 2010 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-20463876

RESUMO

Histopathological classification of human tumors relies in part on the degree of differentiation of the tumor sample. To date, there is no objective systematic method to categorize tumor subtypes by maturation. In this paper, we introduce a novel computational algorithm to rank tumor subtypes according to the dissimilarity of their gene expression from that of stem cells and fully differentiated tissue, and thereby construct a phylogenetic tree of cancer. We validate our methodology with expression data of leukemia, breast cancer and liposarcoma subtypes and then apply it to a broader group of sarcomas. This ranking of tumor subtypes resulting from the application of our methodology allows the identification of genes correlated with differentiation and may help to identify novel therapeutic targets. Our algorithm represents the first phylogeny-based tool to analyze the differentiation status of human tumors.


Assuntos
Algoritmos , Biologia Computacional/métodos , Neoplasias/genética , Adipogenia/genética , Análise de Variância , Diferenciação Celular/fisiologia , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/classificação , Neoplasias/patologia , Filogenia
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