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1.
Proc Natl Acad Sci U S A ; 121(11): e2307802121, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38437557

RESUMO

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.


Assuntos
Neoplasias Ovarianas , Peptídeos , Humanos , Feminino , Interferência de RNA , Peptídeos/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/terapia , Peptídeo Hidrolases , RNA Interferente Pequeno/genética , Endopeptidases , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA
2.
Cell ; 146(4): 633-44, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21854987

RESUMO

Cancer cells within individual tumors often exist in distinct phenotypic states that differ in functional attributes. While cancer cell populations typically display distinctive equilibria in the proportion of cells in various states, the mechanisms by which this occurs are poorly understood. Here, we study the dynamics of phenotypic proportions in human breast cancer cell lines. We show that subpopulations of cells purified for a given phenotypic state return towards equilibrium proportions over time. These observations can be explained by a Markov model in which cells transition stochastically between states. A prediction of this model is that, given certain conditions, any subpopulation of cells will return to equilibrium phenotypic proportions over time. A second prediction is that breast cancer stem-like cells arise de novo from non-stem-like cells. These findings contribute to our understanding of cancer heterogeneity and reveal how stochasticity in single-cell behaviors promotes phenotypic equilibrium in populations of cancer cells.


Assuntos
Neoplasias da Mama/patologia , Cadeias de Markov , Animais , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Processos Estocásticos , Transplante Heterólogo
3.
Cell ; 138(4): 645-659, 2009 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-19682730

RESUMO

Screens for agents that specifically kill epithelial cancer stem cells (CSCs) have not been possible due to the rarity of these cells within tumor cell populations and their relative instability in culture. We describe here an approach to screening for agents with epithelial CSC-specific toxicity. We implemented this method in a chemical screen and discovered compounds showing selective toxicity for breast CSCs. One compound, salinomycin, reduces the proportion of CSCs by >100-fold relative to paclitaxel, a commonly used breast cancer chemotherapeutic drug. Treatment of mice with salinomycin inhibits mammary tumor growth in vivo and induces increased epithelial differentiation of tumor cells. In addition, global gene expression analyses show that salinomycin treatment results in the loss of expression of breast CSC genes previously identified by analyses of breast tissues isolated directly from patients. This study demonstrates the ability to identify agents with specific toxicity for epithelial CSCs.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Paclitaxel/farmacologia , Piranos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica/tratamento farmacológico , Transplante de Neoplasias , Paclitaxel/uso terapêutico , Piranos/uso terapêutico
4.
Cell ; 139(7): 1255-67, 2009 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-20064372

RESUMO

During the course of a viral infection, viral proteins interact with an array of host proteins and pathways. Here, we present a systematic strategy to elucidate the dynamic interactions between H1N1 influenza and its human host. A combination of yeast two-hybrid analysis and genome-wide expression profiling implicated hundreds of human factors in mediating viral-host interactions. These factors were then examined functionally through depletion analyses in primary lung cells. The resulting data point to potential roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of RNA-binding proteins, components of WNT signaling, and viral polymerase subunits. This multilayered approach provides a comprehensive and unbiased physical and regulatory model of influenza-host interactions and demonstrates a general strategy for uncovering complex host-pathogen relationships.


Assuntos
Interações Hospedeiro-Patógeno , Vírus da Influenza A Subtipo H1N1/metabolismo , Proteínas Virais/metabolismo , Apoptose , Células Epiteliais/virologia , Perfilação da Expressão Gênica , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Interferons/metabolismo , Pulmão/citologia , Pulmão/virologia , Proteômica , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/metabolismo , Proteínas Wnt/metabolismo
5.
Mol Cell ; 63(1): 60-71, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27320198

RESUMO

Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1 independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Homeostase , Camundongos da Linhagem 129 , Camundongos Endogâmicos CBA , Agregados Proteicos , Mapas de Interação de Proteínas , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Tempo , Fatores de Transcrição/genética , Transfecção
7.
Proc Natl Acad Sci U S A ; 114(2): 382-387, 2017 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-28028240

RESUMO

The use of proteasome inhibitors to target cancer's dependence on altered protein homeostasis has been greatly limited by intrinsic and acquired resistance. Analyzing data from thousands of cancer lines and tumors, we find that those with suppressed expression of one or more 19S proteasome subunits show intrinsic proteasome inhibitor resistance. Moreover, such proteasome subunit suppression is associated with poor outcome in myeloma patients, where proteasome inhibitors are a mainstay of treatment. Beyond conferring resistance to proteasome inhibitors, proteasome subunit suppression also serves as a sentinel of a more global remodeling of the transcriptome. This remodeling produces a distinct gene signature and new vulnerabilities to the proapoptotic drug, ABT-263. This frequent, naturally arising imbalance in 19S regulatory complex composition is achieved through a variety of mechanisms, including DNA methylation, and marks the emergence of a heritably altered and therapeutically relevant state in diverse cancers.


Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos
8.
Proc Natl Acad Sci U S A ; 114(16): 4153-4158, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28377514

RESUMO

Advances in mammography have sparked an exponential increase in the detection of early-stage breast lesions, most commonly ductal carcinoma in situ (DCIS). More than 50% of DCIS lesions are benign and will remain indolent, never progressing to invasive cancers. However, the factors that promote DCIS invasion remain poorly understood. Here, we show that SMARCE1 is required for the invasive progression of DCIS and other early-stage tumors. We show that SMARCE1 drives invasion by regulating the expression of secreted proteases that degrade basement membrane, an ECM barrier surrounding all epithelial tissues. In functional studies, SMARCE1 promotes invasion of in situ cancers growing within primary human mammary tissues and is also required for metastasis in vivo. Mechanistically, SMARCE1 drives invasion by forming a SWI/SNF-independent complex with the transcription factor ILF3. In patients diagnosed with early-stage cancers, SMARCE1 expression is a strong predictor of eventual relapse and metastasis. Collectively, these findings establish SMARCE1 as a key driver of invasive progression in early-stage tumors.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Movimento Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Recidiva Local de Neoplasia/patologia , Animais , Apoptose , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Intraductal não Infiltrante/metabolismo , Proliferação de Células , Progressão da Doença , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Recidiva Local de Neoplasia/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nature ; 483(7391): 598-602, 2012 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-22388813

RESUMO

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.


Assuntos
Reprogramação Celular , Cromatina/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Reprogramação Celular/genética , Cromatina/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste , Fibroblastos/citologia , Fibroblastos/metabolismo , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Metilação , Metiltransferases/antagonistas & inibidores , Metiltransferases/biossíntese , Metiltransferases/genética , Metiltransferases/metabolismo , Proteína Homeobox Nanog , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/antagonistas & inibidores , Fator de Transcrição YY1/metabolismo
10.
PLoS Biol ; 12(9): e1001945, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25203443

RESUMO

Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)-scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy.


Assuntos
Neoplasias da Mama/genética , Carcinoma/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , Fator 2 Relacionado a NF-E2/genética , Recidiva Local de Neoplasia/genética , eIF-2 Quinase/genética , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Desdiferenciação Celular/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , Fator 2 Relacionado a NF-E2/metabolismo , Gradação de Tumores , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Oxirredução , Fosforilação , Transdução de Sinais , Transcrição Gênica , eIF-2 Quinase/metabolismo
11.
Breast Cancer Res ; 18(1): 19, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26926363

RESUMO

BACKGROUND: Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. METHODS: Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. RESULTS: When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. CONCLUSIONS: These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.


Assuntos
Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Glândulas Mamárias Humanas/patologia , Células Estromais/efeitos dos fármacos
12.
PLoS Comput Biol ; 11(4): e1004161, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25894653

RESUMO

The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS). We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available.


Assuntos
Diferenciação Celular/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Glândulas Mamárias Humanas/citologia , Células-Tronco/citologia , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Perfilação da Expressão Gênica , Humanos , Organoides/citologia , Organoides/metabolismo , Biologia de Sistemas
13.
Nature ; 462(7269): 108-12, 2009 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-19847166

RESUMO

The proto-oncogene KRAS is mutated in a wide array of human cancers, most of which are aggressive and respond poorly to standard therapies. Although the identification of specific oncogenes has led to the development of clinically effective, molecularly targeted therapies in some cases, KRAS has remained refractory to this approach. A complementary strategy for targeting KRAS is to identify gene products that, when inhibited, result in cell death only in the presence of an oncogenic allele. Here we have used systematic RNA interference to detect synthetic lethal partners of oncogenic KRAS and found that the non-canonical IkappaB kinase TBK1 was selectively essential in cells that contain mutant KRAS. Suppression of TBK1 induced apoptosis specifically in human cancer cell lines that depend on oncogenic KRAS expression. In these cells, TBK1 activated NF-kappaB anti-apoptotic signals involving c-Rel and BCL-XL (also known as BCL2L1) that were essential for survival, providing mechanistic insights into this synthetic lethal interaction. These observations indicate that TBK1 and NF-kappaB signalling are essential in KRAS mutant tumours, and establish a general approach for the rational identification of co-dependent pathways in cancer.


Assuntos
Genes ras/genética , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Alelos , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Perfilação da Expressão Gênica , Genes Letais , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transdução de Sinais , Proteína bcl-X/metabolismo
14.
Nat Genet ; 37(10): 1047-54, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16142232

RESUMO

The aggressive clinical behavior of melanoma suggests that the developmental origins of melanocytes in the neural crest might be relevant to their metastatic propensity. Here we show that primary human melanocytes, transformed using a specific set of introduced genes, form melanomas that frequently metastasize to multiple secondary sites, whereas human fibroblasts and epithelial cells transformed using an identical set of genes generate primary tumors that rarely do so. Notably, these melanomas have a metastasis spectrum similar to that observed in humans with melanoma. These observations indicate that part of the metastatic proclivity of melanoma is attributable to lineage-specific factors expressed in melanocytes and not in other cell types analyzed. Analysis of microarray data from human nevi shows that the expression pattern of Slug, a master regulator of neural crest cell specification and migration, correlates with those of other genes that are important for neural crest cell migrations during development. Moreover, Slug is required for the metastasis of the transformed melanoma cells. These findings indicate that melanocyte-specific factors present before neoplastic transformation can have a pivotal role in governing melanoma progression.


Assuntos
Transformação Celular Neoplásica/genética , Melanócitos/metabolismo , Melanoma/patologia , Fatores de Transcrição/metabolismo , Animais , Adesão Celular , Diferenciação Celular , Movimento Celular/genética , Genes Neoplásicos/genética , Humanos , Melanócitos/citologia , Melanócitos/patologia , Melanoma/genética , Camundongos , Metástase Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Células Tumorais Cultivadas
15.
Bioorg Med Chem Lett ; 23(6): 1834-8, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23403082

RESUMO

A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations.


Assuntos
Amidas/química , Bibliotecas de Moléculas Pequenas/química , Amidas/toxicidade , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/toxicidade , Relação Estrutura-Atividade
16.
Proc Natl Acad Sci U S A ; 107(50): 21737-42, 2010 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-21098263

RESUMO

Many tumors contain heterogeneous populations of cells, only some of which exhibit increased tumorigenicity and resistance to anticancer therapies. Evidence suggests that these aggressive cancer cells, often termed "cancer stem cells" or "cancer stem-like cells" (CSCs), rely upon developmental signaling pathways that are important for survival and expansion of normal stem cells. Here we report that, in analogy to embryonic mammary epithelial biology, estrogen signaling expands the pool of functional breast CSCs through a paracrine FGF/FGFR/Tbx3 signaling pathway. Estrogen or FGF9 pretreatment induced CSC properties of breast cancer cell lines and freshly isolated breast cancer cells, whereas cotreatment of cells with tamoxifen or a small molecule inhibitor of FGFR signaling was sufficient to prevent the estrogen-induced expansion of CSCs. Furthermore, reduction of FGFR or Tbx3 gene expression was able to abrogate tumorsphere formation, whereas ectopic Tbx3 expression increased tumor seeding potential by 100-fold. These findings demonstrate that breast CSCs are stimulated by estrogen through a signaling pathway that similarly controls normal mammary epithelial stem cell biology.


Assuntos
Neoplasias da Mama/metabolismo , Estrogênios/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Comunicação Parácrina , Transdução de Sinais/efeitos dos fármacos , Proteínas com Domínio T/metabolismo , Linhagem Celular Tumoral , Feminino , Fator 9 de Crescimento de Fibroblastos/genética , Fator 9 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Humanos , Células-Tronco Neoplásicas/fisiologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas com Domínio T/genética
17.
Proc Natl Acad Sci U S A ; 107(35): 15449-54, 2010 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-20713713

RESUMO

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-beta1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-beta1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-beta1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-beta1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.


Assuntos
Neoplasias da Mama/genética , Claudinas/genética , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Mesoderma/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Análise por Conglomerados , Regulação para Baixo , Feminino , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Proteína Goosecoid/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta1/genética , Proteína 1 Relacionada a Twist/genética
18.
Diagnostics (Basel) ; 13(4)2023 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-36832208

RESUMO

The NavDx® blood test analyzes tumor tissue modified viral (TTMV)-HPV DNA to provide a reliable means of detecting and monitoring HPV-driven cancers. The test has been clinically validated in a large number of independent studies and has been integrated into clinical practice by over 1000 healthcare providers at over 400 medical sites in the US. This Clinical Laboratory Improvement Amendments (CLIA), high complexity laboratory developed test, has also been accredited by the College of American Pathologists (CAP) and the New York State Department of Health. Here, we report a detailed analytical validation of the NavDx assay, including sample stability, specificity as measured by limits of blank (LOBs), and sensitivity illustrated via limits of detection and quantitation (LODs and LOQs). LOBs were 0-0.32 copies/µL, LODs were 0-1.10 copies/µL, and LOQs were <1.20-4.11 copies/µL, demonstrating the high sensitivity and specificity of data provided by NavDx. In-depth evaluations including accuracy and intra- and inter-assay precision studies were shown to be well within acceptable ranges. Regression analysis revealed a high degree of correlation between expected and effective concentrations, demonstrating excellent linearity (R2 = 1) across a broad range of analyte concentrations. These results demonstrate that NavDx accurately and reproducibly detects circulating TTMV-HPV DNA, which has been shown to aid in the diagnosis and surveillance of HPV-driven cancers.

19.
Bioorg Med Chem Lett ; 22(10): 3571-4, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22503247

RESUMO

A high-throughput screen (HTS) with the National Institute of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) compound collection identified a class of acyl hydrazones to be selectively lethal to breast cancer stem cell (CSC) enriched populations. Medicinal chemistry efforts were undertaken to optimize potency and selectivity of this class of compounds. The optimized compound was declared as a probe (ML239) with the NIH Molecular Libraries Program and displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control line (HMLE_sh_GFP).


Assuntos
Neoplasias da Mama/tratamento farmacológico , Hidrazonas/farmacologia , Células-Tronco Neoplásicas/citologia , Pirróis/farmacologia , Neoplasias da Mama/patologia , Feminino , Humanos
20.
Nat Commun ; 12(1): 7116, 2021 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-34893587

RESUMO

Mammary morphogenesis is an orchestrated process involving differentiation, proliferation and organization of cells to form a bi-layered epithelial network of ducts and lobules embedded in stromal tissue. We have engineered a 3D biomimetic human breast that makes it possible to study how stem cell fate decisions translate to tissue-level structure and function. Using this advancement, we describe the mechanism by which breast epithelial cells build a complex three-dimensional, multi-lineage tissue by signaling through a collagen receptor. Discoidin domain receptor tyrosine kinase 1 induces stem cells to differentiate into basal cells, which in turn stimulate luminal progenitor cells via Notch signaling to differentiate and form lobules. These findings demonstrate how human breast tissue regeneration is triggered by transmission of signals from the extracellular matrix through an epithelial bilayer to coordinate structural changes that lead to formation of a complex ductal-lobular network.


Assuntos
Mama/citologia , Mama/fisiologia , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Receptor com Domínio Discoidina 1/metabolismo , Materiais Biocompatíveis , Engenharia Biomédica , Linhagem Celular , Receptor com Domínio Discoidina 1/genética , Células Epiteliais/citologia , Matriz Extracelular , Humanos , Regeneração , Transdução de Sinais , Células-Tronco/citologia
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