RESUMO
The largest subunit of the origin recognition complex (ORC1) is essential for assembly of the prereplicative complex, firing of DNA replication origins, and faithful duplication of the genome. Here, we generated knock-in mice with LoxP sites flanking exons encoding the critical ATPase domain of ORC1. Global or tissue-specific ablation of ORC1 function in mouse embryo fibroblasts and fetal and adult diploid tissues blocked DNA replication, cell lineage expansion, and organ development. Remarkably, ORC1 ablation in extraembryonic trophoblasts and hepatocytes, two polyploid cell types in mice, failed to impede genome endoreduplication and organ development and function. Thus, ORC1 in mice is essential for mitotic cell divisions but dispensable for endoreduplication. We propose that DNA replication of mammalian polyploid genomes uses a distinct ORC1-independent mechanism.
Assuntos
Endorreduplicação/genética , Genoma/genética , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Adenosina Trifosfatases/genética , Animais , Divisão Celular/genética , Proliferação de Células/genética , Desenvolvimento Embrionário/genética , Ativação Enzimática , Feminino , Deleção de Genes , Hepatócitos/citologia , Regeneração Hepática/genética , Camundongos , Mitose/genética , Placenta/fisiologia , GravidezRESUMO
The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.
Assuntos
Carcinoma Ductal Pancreático , Metaplasia/genética , Metaplasia/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Proliferação de Células/genética , Células Cultivadas , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Fibroblastos/citologia , Fibroblastos/patologia , Deleção de Genes , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/patologia , Transdução de Sinais/genética , Fator de Crescimento Transformador alfa/metabolismo , Células Tumorais Cultivadas , Proteína Gli2 com Dedos de ZincoRESUMO
Inactivation of phosphatase and tensin homology deleted on chromosome 10 (PTEN) is linked to increased PI3K-AKT signaling, enhanced organismal growth, and cancer development. Here we generated and analyzed Pten knock-in mice harboring a C2 domain missense mutation at phenylalanine 341 (Pten(FV)), found in human cancer. Despite having reduced levels of PTEN protein, homozygous Pten(FV/FV) embryos have intact AKT signaling, develop normally, and are carried to term. Heterozygous Pten(FV/+) mice develop carcinoma in the thymus, stomach, adrenal medulla, and mammary gland but not in other organs typically sensitive to Pten deficiency, including the thyroid, prostate, and uterus. Progression to carcinoma in sensitive organs ensues in the absence of overt AKT activation. Carcinoma in the uterus, a cancer-resistant organ, requires a second clonal event associated with the spontaneous activation of AKT and downstream signaling. In summary, this PTEN noncatalytic missense mutation exposes a core tumor suppressor function distinct from inhibition of canonical AKT signaling that predisposes to organ-selective cancer development in vivo.
Assuntos
Carcinoma/genética , Mutação de Sentido Incorreto/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais , Animais , Carcinoma/enzimologia , Carcinoma/fisiopatologia , Núcleo Celular/metabolismo , Células Cultivadas , Embrião de Mamíferos , Ativação Enzimática , Feminino , Técnicas de Introdução de Genes , Camundongos , Proteína Oncogênica v-akt/genética , Proteína Oncogênica v-akt/metabolismo , Estabilidade ProteicaRESUMO
Mutated protein-coding genes drive the molecular pathogenesis of many diseases, including cancer. Specifically, mutated KRAS is a documented driver for malignant transformation, occurring early during the pathogenesis of cancers such as lung and pancreatic adenocarcinomas. Therapeutically, the indiscriminate targeting of wild-type and point-mutated transcripts represents an important limitation. Here, we leveraged on the design of miRNA-like artificial molecules (amiRNAs) to specifically target point-mutated genes, such as KRAS, without affecting their wild-type counterparts. Compared with an siRNA-like approach, the requirement of perfect complementarity of the microRNA seed region to a given target sequence in the microRNA/target model has proven to be a more efficient strategy, accomplishing the selective targeting of point-mutated KRAS in vitro and in vivo.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Interferente Pequeno/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Gefitinibe , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Polimorfismo de Nucleotídeo Único/genética , Quinazolinas/farmacologia , Interferência de RNA , Transplante HeterólogoRESUMO
BACKGROUND: A large collaborative analysis of data from 47 epidemiological studies concluded that longer duration of breastfeeding reduces the risk of developing breast cancer. Despite the strong epidemiological evidence, the molecular mechanisms linking prolonged breastfeeding to decreased risk of breast cancer remain poorly understood. METHODS: We modeled two types of breastfeeding behaviors in wild type FVB/N mice: (1) normal or gradual involution of breast tissue following prolonged breastfeeding and (2) forced or abrupt involution following short-term breastfeeding. To accomplish this, pups were gradually weaned between 28 and 31 days (gradual involution) or abruptly at 7 days postpartum (abrupt involution). Mammary glands were examined for histological changes, proliferation, and inflammatory markers by immunohistochemistry. Fluorescence-activated cell sorting was used to quantify mammary epithelial subpopulations. Gene set enrichment analysis was used to analyze gene expression data from mouse mammary luminal progenitor cells. Similar analysis was done using gene expression data generated from human breast samples obtained from parous women enrolled on a tissue collection study, OSU-2011C0094, and were undergoing reduction mammoplasty without history of breast cancer. RESULTS: Mammary glands from mice that underwent abrupt involution exhibited denser stroma, altered collagen composition, higher inflammation and proliferation, increased estrogen receptor α and progesterone receptor expression compared to those that underwent gradual involution. Importantly, when aged to 4 months postpartum, mice that were in the abrupt involution cohort developed ductal hyperplasia and squamous metaplasia. Abrupt involution also resulted in a significant expansion of the luminal progenitor cell compartment associated with enrichment of Notch and estrogen signaling pathway genes. Breast tissues obtained from healthy women who breastfed for < 6 months vs ≥ 6 months showed significant enrichment of Notch signaling pathway genes, along with a trend for enrichment for luminal progenitor gene signature similar to what is observed in BRCA1 mutation carriers and basal-like breast tumors. CONCLUSIONS: We report here for the first time that forced or abrupt involution of the mammary glands following pregnancy and lack of breastfeeding results in expansion of luminal progenitor cells, higher inflammation, proliferation, and ductal hyperplasia, a known risk factor for developing breast cancer.
Assuntos
Aleitamento Materno , Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Estrogênios/metabolismo , Inflamação/complicações , Inflamação/metabolismo , Transdução de Sinais , Animais , Biópsia , Neoplasias da Mama/patologia , Colágeno/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Epiteliais/metabolismo , Estrogênios/efeitos adversos , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Hiperplasia , Imuno-Histoquímica , Inflamação/patologia , Lactação , Camundongos , Gravidez , Receptores de Estrogênio/metabolismo , Medição de Risco , Fatores de Risco , Esteroides/metabolismoRESUMO
Group 3 innate lymphoid cells (ILC3s) are important regulators of the immune system, maintaining homeostasis in the presence of commensal bacteria, but activating immune defenses in response to microbial pathogens. ILC3s are a robust source of IL-22, a cytokine critical for stimulating the antimicrobial response. We sought to identify cytokines that can promote proliferation and induce or maintain IL-22 production by ILC3s and determine a molecular mechanism for this process. We identified IL-18 as a cytokine that cooperates with an ILC3 survival factor, IL-15, to induce proliferation of human ILC3s, as well as induce and maintain IL-22 production. To determine a mechanism of action, we examined the NF-κB pathway, which is activated by IL-18 signaling. We found that the NF-κB complex signaling component, p65, binds to the proximal region of the IL22 promoter and promotes transcriptional activity. Finally, we observed that CD11c+ dendritic cells expressing IL-18 are found in close proximity to ILC3s in human tonsils in situ. Therefore, we identify a new mechanism by which human ILC3s proliferate and produce IL-22, and identify NF-κB as a potential therapeutic target to be considered in pathologic states characterized by overproduction of IL-18 and/or IL-22.
Assuntos
Proliferação de Células , Interleucina-18/metabolismo , Interleucinas/biossíntese , Linfócitos/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais , Células Dendríticas/fisiologia , Humanos , Imunidade Inata , Interleucina-15/imunologia , Interleucinas/genética , Interleucinas/imunologia , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Regiões Promotoras Genéticas , Transdução de Sinais/imunologia , Fator de Transcrição RelA/metabolismo , Interleucina 22RESUMO
Communication between mesodermal cells and epithelial cells is fundamental to normal animal development and is frequently disrupted in cancer. However, the genes and processes that mediate this communication are incompletely understood. To identify genes that mediate this communication and alter the proliferation of cells with an oncogenic Ras genotype, we carried out a tissue-specific genome-wide RNAi screen in Caenorhabditis elegans animals bearing a let-60(n1046gf) (RasG13E) allele. The screen identifies 24 genes that, when knocked down in adjacent mesodermal tissue, suppress the increased vulval epithelial cell proliferation defect associated with let-60(n1046gf). Importantly, gene knockdown reverts the mutant animals to a wild-type phenotype. Using chimeric animals, we genetically confirm that 2 of the genes function nonautonomously to revert the let-60(n1046gf) phenotype. The effect is genotype restricted, as knockdown does not alter development in a wild type (let-60(+)) or activated EGF receptor (let-23(sa62gf)) background. Although many of the genes identified encode proteins involved in essential cellular processes, including chromatin formation, ribosome function, and mitochondrial ATP metabolism, knockdown does not alter the normal development or function of targeted mesodermal tissues, indicating that the phenotype derives from specific functions performed by these cells. We show that the genes act in a manner distinct from 2 signal ligand classes (EGF and Wnt) known to influence the development of vulval epithelial cells. Altogether, the results identify genes with a novel function in mesodermal cells required for communicating with and promoting the proliferation of adjacent epithelial cells with an activated Ras genotype.
Assuntos
Proteínas de Caenorhabditis elegans , Trifosfato de Adenosina/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Feminino , Proteínas de Helminto/genética , Ligantes , Mutação , Transdução de Sinais/genética , Vulva/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer. Unlike other types of breast cancer that can be effectively treated by targeted therapies, no such targeted therapy exists for all TNBC patients. The ADAR1 enzyme carries out A-to-I editing of RNA to prevent sensing of endogenous double-stranded RNAs. ADAR1 is highly expressed in breast cancer including TNBC. Here, we demonstrate that expression of ADAR1, specifically its p150 isoform, is required for the survival of TNBC cell lines. In TNBC cells, knockdown of ADAR1 attenuates proliferation and tumorigenesis. Moreover, ADAR1 knockdown leads to robust translational repression. ADAR1-dependent TNBC cell lines also exhibit elevated IFN stimulated gene expression. IFNAR1 reduction significantly rescued the proliferative defects of ADAR1 loss. These findings establish ADAR1 as a novel therapeutic target for TNBC tumors.
Assuntos
Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Regulação para Cima , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Transplante de Neoplasias , Isoformas de Proteínas/metabolismo , Receptor de Interferon alfa e beta/metabolismo , Análise de Sobrevida , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Platelet-derived growth factor receptor-beta (PDGFRß) is a receptor tyrosine kinase found in cells of mesenchymal origin such as fibroblasts and pericytes. Activation of this receptor is dependent on paracrine ligand induction, and its preferred ligand PDGFB is released by neighboring epithelial and endothelial cells. While expression of both PDGFRß and PDGFB has been noted in patient breast tumors for decades, how PDGFB-to-PDGFRß tumor-stroma signaling mediates breast cancer initiation, progression, and metastasis remains unclear. Here we demonstrate this paracrine signaling pathway that mediates both primary tumor growth and metastasis, specifically, metastasis to the brain. Elevated levels of PDGFB accelerated orthotopic tumor growth and intracranial growth of mammary tumor cells, while mesenchymal-specific expression of an activating mutant PDGFRß (PDGFRßD849V) exerted proproliferative signals on adjacent mammary tumor cells. Stromal expression of PDGFRßD849V also promoted brain metastases of mammary tumor cells expressing high PDGFB when injected intravenously. In the brain, expression of PDGFRßD849V was observed within a subset of astrocytes, and aged mice expressing PDGFRßD849V exhibited reactive gliosis. Importantly, the PDGFR-specific inhibitor crenolanib significantly reduced intracranial growth of mammary tumor cells. In a tissue microarray comprised of 363 primary human breast tumors, high PDGFB protein expression was prognostic for brain metastases, but not metastases to other sites. Our results advocate the use of mice expressing PDGFRßD849V in their stromal cells as a preclinical model of breast cancer-associated brain metastases and support continued investigation into the clinical prognostic and therapeutic use of PDGFB-to-PDGFRß signaling in women with breast cancer. SIGNIFICANCE: These studies reveal a previously unknown role for PDGFB-to-PDGFRß paracrine signaling in the promotion of breast cancer brain metastases and support the prognostic and therapeutic clinical utility of this pathway for patients.See related article by Wyss and colleagues, p. 594.
Assuntos
Neoplasias da Mama , MicroRNAs , Animais , Encéfalo/metabolismo , Neoplasias da Mama/genética , Células Endoteliais/metabolismo , Humanos , Camundongos , Receptor beta de Fator de Crescimento Derivado de PlaquetasRESUMO
Heterozygous mutations in the BRCA1 gene predispose women to breast and ovarian cancer, while biallelic BRCA1 mutations are a cause of Fanconi anemia (FA), a rare genetic disorder characterized by developmental abnormalities, early-onset bone marrow failure, increased risk of cancers, and hypersensitivity to DNA-crosslinking agents. BRCA1 is critical for homologous recombination of DNA double-strand breaks (DSB). Through its coiled-coil domain, BRCA1 interacts with an essential partner, PALB2, recruiting BRCA2 and RAD51 to sites of DNA damage. Missense mutations within the coiled-coil domain of BRCA1 (e.g., L1407P) that affect the interaction with PALB2 have been reported in familial breast cancer. We hypothesized that if PALB2 regulates or mediates BRCA1 tumor suppressor function, ablation of the BRCA1-PALB2 interaction may also elicit genomic instability and tumor susceptibility. We generated mice defective for the Brca1-Palb2 interaction (Brca1 L1363P in mice) and established MEF cells from these mice. Brca1 L1363P/L1363P MEF exhibited hypersensitivity to DNA-damaging agents and failed to recruit Rad51 to DSB. Brca1 L1363P/L1363P mice were viable but exhibited various FA symptoms including growth retardation, hyperpigmentation, skeletal abnormalities, and male/female infertility. Furthermore, all Brca1 L1363P/L1363P mice exhibited macrocytosis and died due to bone marrow failure or lymphoblastic lymphoma/leukemia with activating Notch1 mutations. These phenotypes closely recapitulate clinical features observed in patients with FA. Collectively, this model effectively demonstrates the significance of the BRCA1-PALB2 interaction in genome integrity and provides an FA model to investigate hematopoietic stem cells for mechanisms underlying progressive failure of hematopoiesis and associated development of leukemia/lymphoma, and other FA phenotypes. SIGNIFICANCE: A new Brca1 mouse model for Fanconi anemia (FA) complementation group S provides a system in which to study phenotypes observed in human FA patients including bone marrow failure.See related commentary by Her and Bunting, p. 4044.
Assuntos
Neoplasias da Mama , Anemia de Fanconi , Animais , Proteína BRCA1/genética , Dano ao DNA/genética , Anemia de Fanconi/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Feminino , Humanos , Masculino , Camundongos , Fenótipo , Proteínas Supressoras de Tumor/genéticaRESUMO
Alterations of the PALB2 tumor suppressor gene have been identified in familial breast, ovarian and pancreatic cancer cases. PALB2 cooperates with BRCA1/2 proteins through physical interaction in initiation of homologous recombination, in maintenance of genome integrity following DNA double-strand breaks. To determine if the role of PALB2 as a linker between BRCA1 and BRCA2 is critical for BRCA1/2-mediated tumor suppression, we generated Palb2 mouse pancreatic cancer models and compared tumor latencies, phenotypes and drug responses with previously generated Brca1/2 pancreatic cancer models. For development of Palb2 pancreatic cancer, we crossed conditional Palb2 null mouse with mice carrying the KrasG12D; p53R270H; Pdx1-Cre (KPC) constructs, and these animals were observed for pancreatic tumor development. Individual deletion of Palb2, Brca1 or Brca2 genes in pancreas per se using Pdx1-Cre was insufficient to cause tumors, but it reduced pancreata size. Concurrent expression of mutant KrasG12D and p53R270H, with tumor suppressor inactivated strains in Palb2-KPC, Brca1-KPC or Brca2-KPC, accelerated pancreatic ductal adenocarcinoma (PDAC) development. Moreover, most Brca1-KPC and some Palb2-KPC animals developed mucinous cystic neoplasms with PDAC, while Brca2-KPC and KPC animals did not. 26% of Palb2-KPC mice developed MCNs in pancreata, which resemble closely the Brca1 deficient tumors. However, the remaining 74% of Palb2-KPC animals developed PDACs without any cysts like Brca2 deficient tumors. In addition, the number of ADM lesions and immune cells infiltrations (CD3+ and F/480+) were significantly increased in Brca1-KPC tumors, but not in Brca2-KPC tumors. Interestingly, the level of ADM lesions and infiltration of CD3+ or F/480+ cells in Palb2-KPC tumors were intermediate between Brca1-KPC and Brca2-KPC tumors. As expected, disruption of Palb2 and Brca1/2 sensitized tumor cells to DNA damaging agents in vitro and in vivo. Altogether, Palb2-KPC PDAC exhibited features observed in both Brca1-KPC and Brca2-KPC tumors, which could be due to its role, as a linker between Brca1 and Brca2.
Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma/genética , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Neoplasias Pancreáticas/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Modelos Animais de Doenças , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Camundongos , Camundongos Nus , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologiaRESUMO
Orchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cell-cycle-regulated transcription has improved significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G0, G1-S, and S-G2-M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the single-cell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Diferenciação Celular , Fator de Transcrição E2F3/fisiologia , Fator de Transcrição E2F4/fisiologia , Regulação da Expressão Gênica , Proteínas Repressoras/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , TranscriptomaRESUMO
The initial molecular events that lead to malignant transformation of the fimbria of the fallopian tube (FT) through high-grade serous ovarian carcinoma (HGSC) remain poorly understood. In this study, we report that increased expression of signal transducer and activator of transcription 3 (pSTAT3 Tyr705) and suppression or loss of protein inhibitor of activated STAT3 (PIAS3) in FT likely drive HGSC. We evaluated human tissues-benign normal FT, tubal-peritoneal junction (TPJ), p53 signature FT tissue, tubal intraepithelial lesion in transition (TILT), serous tubal intraepithelial carcinoma (STIC) without ovarian cancer, and HGSC for expression of STAT3/PIAS3 (compared with their known TP53 signature) and their target proliferation genes. We observed constitutive activation of STAT3 and low levels or loss of PIAS3 in the TPJ, p53 signature, TILT, and STIC through advanced stage IV (HGSC) tissues. Elevated expression of pSTAT3 Tyr705 and decreased levels of PIAS3 appeared as early as TPJ and the trend continued until very advanced stage HGSC (compared with high PIAS3 and low pSTAT3 expression in normal benign FT). Exogenous expression of STAT3 in FT cells mediated translocation of pSTAT3 and c-Myc into the nucleus. In vivo experiments demonstrated that overexpression of STAT3 in FT secretory epithelial cells promoted tumor progression and metastasis, mimicking the clinical disease observed in patients with HGSC. Thus, we conclude that the STAT3 pathway plays a role in the development and progression of HGSC from its earliest premalignant states.Significance: Concomitant gain of pSTAT3 Tyr705 and loss of PIAS3 appear critical for initiation and development of high-grade serous carcinoma. Cancer Res; 78(7); 1739-50. ©2018 AACR.
Assuntos
Cistadenocarcinoma Seroso/genética , Neoplasias das Tubas Uterinas/genética , Chaperonas Moleculares/genética , Neoplasias Ovarianas/genética , Lesões Pré-Cancerosas/genética , Proteínas Inibidoras de STAT Ativados/genética , Fator de Transcrição STAT3/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Transformação Celular Neoplásica/genética , Cistadenocarcinoma Seroso/patologia , Neoplasias das Tubas Uterinas/patologia , Tubas Uterinas/patologia , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Lesões Pré-Cancerosas/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Quimiocina CXCL12/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Invasividade Neoplásica/patologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Neoplasias Mamárias Animais , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/patologia , Microambiente Tumoral/fisiologiaRESUMO
Engineered oncolytic viruses are used clinically to destroy cancer cells and have the ability to boost anticancer immunity. Phosphatase and tensin homolog deleted on chromosome 10 loss is common across a broad range of malignancies, and is implicated in immune escape. The N-terminally extended isoform, phosphatase and tensin homolog deleted on chromosome 10 alpha (PTENα), regulates cellular functions including protein kinase B signaling and mitochondrial adenosine triphosphate production. Here we constructed HSV-P10, a replicating, PTENα expressing oncolytic herpesvirus, and demonstrate that it inhibits PI3K/AKT signaling, increases cellular adenosine triphosphate secretion, and reduces programmed death-ligand 1 expression in infected tumor cells, thus priming an adaptive immune response and overcoming tumor immune escape. A single dose of HSV-P10 resulted in long term survivors in mice bearing intracranial tumors, priming anticancer T-cell immunity leading to tumor rejection. This implicates HSV-P10 as an oncolytic and immune stimulating therapeutic for anticancer therapy.
Assuntos
Herpesviridae/metabolismo , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Vírus Oncolíticos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Linfócitos T/metabolismo , Animais , Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/secundário , Linhagem Celular Tumoral , Feminino , Humanos , Imunidade , Cinética , Camundongos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
The importance of the tumor-associated stroma in cancer progression is clear. However, it remains uncertain whether early events in the stroma are capable of initiating breast tumorigenesis. Here, we show that in the mammary glands of non-tumor bearing mice, stromal-specific phosphatase and tensin homolog (Pten) deletion invokes radiation-induced genomic instability in neighboring epithelium. In these animals, a single dose of whole-body radiation causes focal mammary lobuloalveolar hyperplasia through paracrine epidermal growth factor receptor (EGFR) activation, and EGFR inhibition abrogates these cellular changes. By analyzing human tissue, we discover that stromal PTEN is lost in a subset of normal breast samples obtained from reduction mammoplasty, and is predictive of recurrence in breast cancer patients. Combined, these data indicate that diagnostic or therapeutic chest radiation may predispose patients with decreased stromal PTEN expression to secondary breast cancer, and that prophylactic EGFR inhibition may reduce this risk.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , PTEN Fosfo-Hidrolase/genética , Tolerância a Radiação/genética , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/radioterapia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Raios gama/efeitos adversos , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/efeitos da radiação , Humanos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/efeitos da radiação , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/efeitos da radiação , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/radioterapia , Camundongos , PTEN Fosfo-Hidrolase/deficiência , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/efeitos da radiaçãoRESUMO
The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3ß (GSKß), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.
RESUMO
The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Neoplasias Mamárias Animais/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Diferenciação Celular/genética , Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Ácido Hialurônico/administração & dosagem , Glândulas Mamárias Animais/patologia , Glândulas Mamárias Humanas/patologia , Neoplasias Mamárias Animais/patologia , Camundongos , Morfogênese/genética , Transdução de Sinais , Células Estromais/patologiaRESUMO
Despite the development of combined modality treatments against liposarcoma in recent years, a significant proportion of patients respond only modestly to such approaches, possibly contributing to local or distant recurrence. Early detection of recurrent or metastatic disease could improve patient prognosis by triggering earlier clinical intervention. However, useful biomarkers for such purposes are lacking. Using both patient plasma samples and cell lines, we demonstrate here that miR-25-3p and miR-92a-3p are secreted by liposarcoma cells through extracellular vesicles and may be useful as potential biomarkers of disease. Both miR-25-3p and miR-92a-3p stimulated secretion of proinflammatory cytokine IL6 from tumor-associated macrophages in a TLR7/8-dependent manner, which in turn promoted liposarcoma cell proliferation, invasion, and metastasis via this interaction with the surrounding microenvironment. Our findings provide novel and previously unreported insight into liposarcoma progression, identifying communication between liposarcoma cells and their microenvironment as a process critically involved in liposarcoma progression. This study establishes the possibility that the pattern of circulating miRNAs may identify recurrence prior to radiological detectability while providing insight into disease outcome and as a possible approach to monitor treatment efficacy. Cancer Res; 77(14); 3846-56. ©2017 AACR.
Assuntos
Exossomos/metabolismo , Lipossarcoma/genética , MicroRNAs/metabolismo , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Células HEK293 , Humanos , Lipossarcoma/sangue , Lipossarcoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , TransfecçãoRESUMO
Macrophages are attracted to developing tumors and can participate in immune surveillance to eliminate neoplastic cells. In response, neoplastic cells utilize NF-κB to suppress this killing activity, but the mechanisms underlying their self-protection remain unclear. Here, we report that this dynamic interaction between tumor cells and macrophages is integrally linked by a soluble factor identified as growth and differentiation factor 15 (GDF-15). In vitro, tumor-derived GDF-15 signals in macrophages to suppress their proapoptotic activity by inhibiting TNF and nitric oxide (NO) production. In vivo, depletion of GDF-15 in Ras-driven tumor xenografts and in an orthotopic model of pancreatic cancer delayed tumor development. This delay correlated with increased infiltrating antitumor macrophages. Further, production of GDF-15 is directly regulated by NF-κB, and the colocalization of activated NF-κB and GDF-15 in epithelial ducts of human pancreatic adenocarcinoma supports the importance of this observation. Mechanistically, we found that GDF-15 suppresses macrophage activity by inhibiting TGF-ß-activated kinase (TAK1) signaling to NF-κB, thereby blocking synthesis of TNF and NO. Based on these results, we propose that the NF-κB/GDF-15 regulatory axis is important for tumor cells in evading macrophage immune surveillance during the early stages of tumorigenesis.