RESUMO
Pancreatic cancer is the fourth leading cause of cancer-related mortality in the world. Pancreatic cancer can be localized, locally advanced, or metastatic. The median 1- and 5-year survival rates are 25% and 6%, respectively. Epigenetic modifications such as DNA methylation play a significant role during both normal human development and cancer progression. To investigate epigenetic regulation of genes in the tumor-initiating population of pancreatic cancer cells, which are also termed cancer stem cells (CSCs), we conducted epigenetic arrays in PANC1 and HPAC pancreatic cancer cell lines and compared the global DNA methylation status of CpG promoters in invasive cells, demonstrated to be CSCs, to their noninvasive counterparts, or non-CSCs. Our results suggested that the NF-κB pathway is one of the most activated pathways in pancreatic CSCs. In agreement with this, we determined that upon treatment with NF-κB pathway inhibitors, the stem cell-like properties of cells are significantly disrupted. Moreover, SOX9, demethylated in CSCs, is shown to play a crucial role in the invasion process. Additionally, we found a potential NF-κB binding site located in the SOX9 promoter and determined that the NF-κB subunit p65 positively regulates SOX9 expression by binding to its promoter directly. This interaction can be efficiently blocked by NF-κB inhibitors. Thus, our work establishes a link between the classic NF-κB signaling transduction pathway and the invasiveness of pancreatic CSCs, which may result in the identification of novel signals and molecules that function at an epigenetic level, and could potentially be targeted for pharmaceutical investigations and clinical trials.
Assuntos
NF-kappa B/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Animais , Linhagem Celular Tumoral , Metilação de DNA , Epigenômica , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/genética , Invasividade Neoplásica , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The existence of "tumor-initiating cells" (TICs) has been a topic of heated debate for the last few years within the field of cancer biology. Their continuous characterization in a variety of solid tumors has led to an abundance of evidence supporting their existence. TICs are believed to be responsible for resistance against conventional treatment regimes of chemotherapy and radiation, ultimately leading to metastasis and patient demise. This review summarizes DNA repair mechanism(s) and their role in the maintenance and regulation of stem cells. There is evidence supporting the hypothesis that TICs, similar to embryonic stem (ES) cells and hematopoietic stem cells (HSCs), display an increase in their ability to survive genotoxic stress and injury. Mechanistically, the ability of ES cells, HSCs and TICs to survive under stressful conditions can be attributed to an increase in the efficiency at which these cells undergo DNA repair. Furthermore, the data presented in this review summarize the results found by our lab and others demonstrating that TICs have an increase in their genomic stability, which can allow for TIC survival under conditions such as anticancer treatments, while the bulk population of tumor cells dies. We believe that these data will greatly impact the development and design of future therapies being engineered to target and eradicate this highly aggressive cancer cell population.
Assuntos
Dano ao DNA , Reparo do DNA/genética , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Predisposição Genética para Doença/genética , Humanos , Modelos Genéticos , Neoplasias/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
BACKGROUND: Polycomb repressive complex 2 (PRC2) mediates gene silencing through histone H3K27 methylation. PRC2 components are over-expressed in metastatic prostate cancer (PC), and are required for cancer stem cell (CSC) self-renewal. 3-Dezaneplanocin-A (DZNeP) is an inhibitor of PRC2 with broad anticancer activity. METHOD: we investigated the effects of DZNeP on cell proliferation, tumorigenicity and invasive potential of PC cell lines (LNCaP and DU145). RESULTS: Exploring GEO and Oncomine databases, we found that specific PRC2 genes (EED, EZH2, SUZ12) predict poor prognosis in PC. Non-toxic DZNeP concentrations completely eradicated LNCaP and DU145 prostatosphere formation, and significantly reduced the expression of CSC markers. At comparable doses, other epigenetic drugs were not able to eradicate CSCs. DZNeP was also able to reduce PC cell invasion. Cells pre-treated with DZNeP were significantly less tumorigenic (LNCaP) and formed smaller tumors (DU145) in immunocompromised mice. CONCLUSION: DZNeP is effective both in vitro and in vivo against PC cells. DZNeP antitumor activity is in part mediated by inhibition of CSC tumorigenic potential.
Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Repressoras/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Metilação/efeitos dos fármacos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Proteínas do Grupo Polycomb , RNA Mensageiro/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Esferoides Celulares/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The niche is the environment in which stem cells reside and is responsible for the maintenance of unique stem cell properties such as self-renewal and an undifferentiated state. The heterogeneous populations which constitute a niche include both stem cells and surrounding differentiated cells. This network of heterogeneity is responsible for the control of the necessary pathways that function in determining stem cell fate. The concept that cancer stem cells, a subpopulation of cells responsible for tumor initiation and formation, reside in their own unique niche is quickly evolving and it is of importance to understand and identify the processes occurring within this environment. The necessary intrinsic pathways that are utilized by this cancer stem cell population to maintain both self-renewal and the ability to differentiate are believed to be a result of the environment where cancer stem cells reside. The ability of a specific cancer stem cell niche to provide the environment in which this population can flourish is a critical aspect of cancer biology that mandates intense investigation. This review focuses on current evidence demonstrating that homeostatic processes such as inflammation, epithelial to mesenchymal transition, hypoxia and angiogenesis contribute to the maintenance and control of cancer stem cell fate by providing the appropriate signals within the microenvironment. It is necessary to understand the key processes occurring within this highly specialized cancer stem cell niche to identify potential therapeutic targets that can serve as the basis for development of more effective anticancer treatments.
Assuntos
Células-Tronco Neoplásicas/fisiologia , Nicho de Células-Tronco/fisiologia , Microambiente Tumoral/fisiologia , Animais , Homeostase , Humanos , Transdução de SinaisRESUMO
BACKGROUND: Recently, much attention has been focused on gaining a better understanding of the different populations of cells within a tumor and their contribution to cancer progression. One of the most commonly used methods to isolate a more aggressive sub-population of cells utilizes cell sorting based on expression of certain cell adhesion molecules. A recently established method we developed is to isolate these more aggressive cells based on their properties of increased invasive ability. These more invasive cells have been previously characterized as tumor initiating cells (TICs) that have a stem-like genomic signature and express a number of stem cell genes including Oct3/4 and Nanog and are more tumorigenic compared to their 'non-invasive' counterpart. They also have a profile reminiscent of cells undergoing a classic pattern of epithelial to mesenchymal transition or EMT. Using this model of invasion, we sought to investigate which genes are under epigenetic control in this rare population of cells. Epigenetic modifications, specifically DNA methylation, are key events regulating the process of normal human development. To determine the specific methylation pattern in these invasive prostate cells, and if any developmental genes were being differentially regulated, we analyzed differences in global CpG promoter methylation. RESULTS: Differentially methylated genes were determined and select genes were chosen for additional analyses. The non-receptor tyrosine kinase BMX and transcription factor SOX1 were found to play a significant role in invasion. Ingenuity pathway analysis revealed the methylated gene list frequently displayed genes from the IL-6/STAT3 pathway. Cells which have decreased levels of the targets BMX and SOX1 also display loss of STAT3 activity. Finally, using Oncomine, it was determined that more aggressive metastatic prostate cancers in humans also have higher levels of both Stat3 and Sox1. CONCLUSIONS: Using this method we can begin to understand which genes are epigenetically regulated in the invasive population compared to the bulk tumor cells. These aggressive sub-populations of cells may be linked to the cancer stem cell hypothesis, making their patterns of epigenetic regulation very attractive for biomarker analysis.
Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/genética , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Transição Epitelial-Mesenquimal/genética , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunoprecipitação , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Proteína Homeobox Nanog , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Reação em Cadeia da Polimerase , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismoRESUMO
Through the classic study of genetics, much has been learned about the regulation and progression of human disease. Specifically, cancer has been defined as a disease driven by genetic alterations, including mutations in tumor-suppressor genes and oncogenes, as well as chromosomal abnormalities. However, the study of normal human development has identified that in addition to classical genetics, regulation of gene expression is also modified by 'epigenetic' alterations including chromatin remodeling and histone variants, DNA methylation, the regulation of polycomb group proteins, and the epigenetic function of non-coding RNA. These changes are modifications inherited during both meiosis and mitosis, yet they do not result in alterations of the actual DNA sequence. A number of biological questions are directly influenced by epigenetics, such as how does a cell know when to divide, differentiate or remain quiescent, and more importantly, what happens when these pathways become altered? Do these alterations lead to the development and/or progression of cancer? This review will focus on summarizing the limited current literature involving epigenetic alterations in the context of human cancer stems cells (CSCs). The extent to which epigenetic changes define cell fate, identity, and phenotype are still under intense investigation, and many questions remain largely unanswered. Before discussing epigenetic gene silencing in CSCs, the different classifications of stem cells and their properties will be introduced. This will be followed by an introduction to the different epigenetic mechanisms. Finally, there will be a discussion of the current knowledge of epigenetic modifications in stem cells, specifically what is known from rodent systems and established cancer cell lines, and how they are leading us to understand human stem cells.
Assuntos
Epigênese Genética , Neoplasias/genética , Células-Tronco/metabolismo , Metilação de DNA , Regulação da Expressão Gênica , Inativação Gênica , Histonas/metabolismo , Humanos , Modelos GenéticosRESUMO
Estrogen and progestins are essential for mammary growth and differentiation but also enhance the activity of the p53 tumor suppressor protein in the mammary epithelium. However, the pathways by which these hormones regulate p53 activity are unknown. Microarrays were used to profile the transcriptional changes within the mammary gland after administration of either vehicle, 17beta-estradiol (E), or progesterone (P) individually and combined (EP). Treatment with EP yielded 1182 unique genes that were differentially expressed compared to the vehicle-treated group. Although 30% of genes were responsive to either E or P individually, combined treatment with both EP had a synergistic effect accounting for 60% of the differentially regulated genes. Analysis of protein-protein interactions identified p53, RelA, Snw1, and Igfals as common targets of genes regulated by EP. RelA and p53 form hubs within a network connected by genes that are regulated by EP and that may coordinate the competing functions of RelA and p53 in proliferation and survival of cells. Induction of early growth response 1 (Egr1) and Stratifin (Sfn) (also known as 14-3-3sigma) by EP was confirmed by reverse transcription-quantitative PCR and shown to be p53 independent. In luciferase reporter assays, Egr1 was shown to enhance transcriptional activation by p53 and inhibit nuclear factor kappaB activity. These results identify a gene expression network that provides redundant activation of RelA to support proliferation as well as sensitize p53 to ensure proper surveillance and integration of their competing functions through factors such as Egr1, which both enhance p53 and inhibit RelA.
Assuntos
Estradiol/farmacologia , Glândulas Mamárias Animais/fisiologia , Progesterona/farmacologia , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas 14-3-3/genética , Animais , Neoplasias da Mama , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proteína 1 de Resposta de Crescimento Precoce/genética , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Feminino , Perfilação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ovariectomia , Transcrição Gênica/fisiologiaRESUMO
DNA damage normally induces p53 activity, but responses to ionizing radiation in the mammary epithelium vary among developmental stages. The following studies examined the hormones and growth factors that regulate radiation-responsiveness of p53 in mouse mammary epithelium. Immunoreactive p21/WAF1 and TUNEL staining were used as indicators of p53 activity following exposure to ionizing radiation. In ovariectomized mice, radiation-induced accumulation of p21/WAF1 was minimal in the mammary epithelial cells (<1%). Systemic injections of estrogen and progesterone (E+P) for 72 h were necessary to recover maximal expression of p21/WAF1 following ionizing radiation (55%). The effects of E+P on radiation-induced p21/WAF1 were p53-dependent as responses were absent in Trp53-/- mice. Though hormonal treatments stimulated increases in the proportion of cycling cells (PCNA-positive), this was not directly correlated with p53 activity. Whole organ cultures were used to determine whether E+P act directly upon the mammary gland. Treatment with E+P was sufficient to render p53 responsive to radiation, but TGF-beta-neutralizing antibodies blocked responsiveness. In the absence of E+P, TGF-beta1 alone did not alter p53 activity. These results demonstrate that estrogen and progesterone together with TGF-beta signaling are necessary for maintenance of p53 activity in the mammary epithelium.
Assuntos
Estrogênios/farmacologia , Glândulas Mamárias Animais/efeitos dos fármacos , Progesterona/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/genética , Animais , Northern Blotting , Western Blotting , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Imuno-Histoquímica , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Técnicas de Cultura de ÓrgãosRESUMO
Recent evidence indicates that tumor-initiating cells (TICs), also called cancer stem cells (CSCs), are responsible for tumor initiation and progression, therefore representing an important cell population that may be used as a target for the development of future anticancer therapies. In the present study, Cryptotanshinone (CT), a traditional Chinese herbal medicine, was demonstrated to regulate the behaviors of LNCaP prostate cells and prostate LNCaP TICs. The results demonstrate that treatment with CT alters cellular proliferation, cell cycle status, migration, viability, colony formation and notably, sphere formation and down-regulation of stemness genes (Nanog, OCT4, SOX2, ß-catenin, CXCR4) in TICs. The present study demonstrates that CT targets the LNCaP CD44+CD24- population that is representative of prostate TICs and also affects total LNCaP cells as well via down-regulation of stemness genes. The strong effect with which CT has on prostate TICs suggests that CT may potentially function as a novel natural anticancer agent that specifically targets TICs.
RESUMO
BACKGROUND: Human bone marrow derived mesenchymal stem cells (hMSCs) are capable of differentiation into multiple cell lineages and demonstrate a wide variety of use in various therapeutic applications. Only recently has research begun to understand the gene expression profiles of hMSCs and their differentiated counterparts in vivo and ex vivo. PURPOSE: The research presented here aimed at gaining a better understanding of gene expression patterns present during hMSC invasion through a basement membrane. METHODS: Changes in gene expression were evaluated between invasive and non-invasive cells using Agilent's gene expression arrays and Matrigel invasion chambers. The cells were specifically attracted to a defined stem cell media called SCM. RESULTS: A total 435 genes were up-regulated by 2- fold or more in the invasive population of cells and classified into developmental programs and immunological/inflammatory signaling pathways determined by Ingenuity Pathway Analysis (IPA). This list included a variety of regulators of growth and differentiation including NANOG, STAT3 and STAT5A and members of the polycomb repressive complex-2 (PCRC2) EZH2 and SUZ12. The known regulator of inflammation and hypoxia HIF-1α was also increased suggesting that regulation of the microenvironment is important during this process. Finally, the invasion process could be reversed using the STAT3 inhibitor Static. CONCLUSIONS: Overall these data will increase the understanding of the genetic pathways functioning during hMSC invasion and aid in the development of their therapeutic applications.
RESUMO
OBJECTIVE: Pancreatic cancer is a leading cancer type and its molecular pathology is poorly understood. The only potentially curative therapeutic option available is complete surgical resection; however, this is inadequate as most of the patients are diagnosed at an advanced or metastatic stage. Tumor-initiating cells (TICs) constitute a subpopulation of cells within a solid tumor that sustain tumor growth, metastasis, and chemo/radioresistance. Within pancreatic cancer, TICs have been identified based on the expression of specific cell surface markers. METHODS: We use a sphere formation assay to enrich putative TICs and use human serum as a driver of differentiation. We demonstrate by using specific blocking reagents that we can inhibit the differentiation process and maintain TIC-associated markers and genes. RESULTS: We can induce differentiation of pancreatospheres with the addition of human serum, and we identified vitronectin as an inducer of differentiation. We inhibit differentiation by human serum using an arginine-glycine-aspartate-specific peptide, which is Cilengitide; hence, demonstrating this differentiation is mediated via specific integrin receptors. CONCLUSIONS: Overall, our studies further the definition of pancreatic TICs and provide further insight into both the maintenance and differentiation of this lethal population.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Venenos de Serpentes/farmacologia , Vitronectina/farmacologia , Biomarcadores Tumorais/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Integrina alfaVbeta3/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Receptores de Vitronectina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro/fisiologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células Tumorais CultivadasRESUMO
High-content flow (HCF) screening systems, such as the iQue Screener and HTFC Screening System from IntelliCyt, have facilitated the implementation of flow cytometry assays for high-throughput screening. HCF screening systems enable the use of smaller sample volumes and multiplexed assays to simultaneously assess different cellular parameters from a single well. This becomes invaluable when working with cells or compounds that are available in limited quantities or when conducting large-scale screens. When assays can be miniaturized to a 384- or 1536-well microplate format, it is possible to implement dose-response-based high-throughput screens, also known as quantitative HTS or qHTS. This article describes how qHTS at the new National Center for Advancing Translational Science (NCATS) has been systematically coupled with the HTFC Screening System and Multimetric Apoptosis Screening Kit from IntelliCyt to biologically validate active compounds from primary cell proliferation screens using a model of diffuse large B cell lymphoma (DLBCL).
Assuntos
Anexina A5/biossíntese , Caspase 3/metabolismo , Caspase 7/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Membranas Mitocondriais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , HumanosRESUMO
The identification of novel combinations of effective cancer drugs is required for the successful treatment of cancer patients for a number of reasons. First, many "cancer specific" therapeutics display detrimental patient side-effects and second, there are almost no examples of single agent therapeutics that lead to cures. One strategy to decrease both the effective dose of individual drugs and the potential for therapeutic resistance is to combine drugs that regulate independent pathways that converge on cell death. BCL2-like family members are key proteins that regulate apoptosis. We conducted a screen to identify drugs that could be combined with an inhibitor of anti-apoptotic BCL2-like proteins, ABT-263, to kill human leukemia cells lines. We found that the combination of D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) hydrochloride, an inhibitor of glucosylceramide synthase, potently synergized with ABT-263 in the killing of multiple human leukemia cell lines. Treatment of cells with PDMP and ABT-263 led to dramatic elevation of two pro-apoptotic sphingolipids, namely ceramide and sphingosine. Furthermore, treatment of cells with the sphingosine kinase inhibitor, SKi-II, also dramatically synergized with ABT-263 to kill leukemia cells and similarly increased ceramides and sphingosine. Data suggest that synergism with ABT-263 requires accumulation of ceramides and sphingosine, as AMP-deoxynojirimycin, (an inhibitor of the glycosphingolipid pathway) did not elevate ceramides or sphingosine and importantly did not sensitize cells to ABT-263 treatment. Taken together, our data suggest that combining inhibitors of anti-apoptotic BCL2-like proteins with drugs that alter the balance of bioactive sphingolipids will be a powerful combination for the treatment of human cancers.
Assuntos
Ceramidas/metabolismo , Compostos de Anilina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Humanos , Morfolinas/farmacologia , Sulfonamidas/farmacologiaRESUMO
Myelodysplastic syndromes (MDSs) arise from a defective hematopoietic stem/progenitor cell. Consequently, there is an urgent need to develop targeted therapies capable of eliminating the MDS-initiating clones. We identified that IRAK1, an immune-modulating kinase, is overexpressed and hyperactivated in MDSs. MDS clones treated with a small molecule IRAK1 inhibitor (IRAK1/4-Inh) exhibited impaired expansion and increased apoptosis, which coincided with TRAF6/NF-κB inhibition. Suppression of IRAK1, either by RNAi or with IRAK1/4-Inh, is detrimental to MDS cells, while sparing normal CD34(+) cells. Based on an integrative gene expression analysis, we combined IRAK1 and BCL2 inhibitors and found that cotreatment more effectively eliminated MDS clones. In summary, these findings implicate IRAK1 as a drugable target in MDSs.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Síndromes Mielodisplásicas/tratamento farmacológico , Animais , Apoptose , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Camundongos , Síndromes Mielodisplásicas/patologia , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Fator 6 Associado a Receptor de TNF/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Camundongos , Proteína Homeobox Nanog , Neoplasias/tratamento farmacológicoRESUMO
Knowledge of oncogenic mutations can inspire therapeutic strategies that are synthetically lethal, affecting cancer cells while sparing normal cells. Lenalidomide is an active agent in the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), but its mechanism of action is unknown. Lenalidomide kills ABC DLBCL cells by augmenting interferon ß (IFNß) production, owing to the oncogenic MYD88 mutations in these lymphomas. In a cereblon-dependent fashion, lenalidomide downregulates IRF4 and SPIB, transcription factors that together prevent IFNß production by repressing IRF7 and amplify prosurvival NF-κB signaling by transactivating CARD11. Blockade of B cell receptor signaling using the BTK inhibitor ibrutinib also downregulates IRF4 and consequently synergizes with lenalidomide in killing ABC DLBCLs, suggesting attractive therapeutic strategies.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Adaptadoras de Transdução de Sinal , Adenina/análogos & derivados , Animais , Western Blotting , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon beta/genética , Interferon beta/metabolismo , Interferon beta/farmacologia , Lenalidomida , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/genética , NF-kappa B/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Piperidinas , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Talidomida/administração & dosagem , Talidomida/análogos & derivados , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carga Tumoral/genética , Ubiquitina-Proteína LigasesRESUMO
OBJECTIVE: Pancreatic cancer was the fourth leading cause of cancer death in the United States in 2010. Recurrence of disease after resection occurs because of neoplastic cell survival. To better understand these highly aggressive cells, gene expression microarrays were performed. METHODS: Using the established lines HPAC and PANC1 and a Matrigel assay, genome expression arrays were performed to analyze patterns between invasive and total cells. RESULTS: Significant increases in the expression of genes related to DNA repair were observed. A number of the same genes also demonstrated an increase in expression when comparing bulk cells to a putative tumor-initiating cell (TIC) population. The TIC population was isolated using the spheroid technique, and compared with bulk cells, spheroid cells functionally repair breaks in DNA faster after challenge with the drug gemcitabine. Finally, using Oncomine, we observed a significant increase in DNA copy number of BRCA1 and RAD51 in tissue isolated from metastatic pancreatic cancer compared with tissue isolated from the primary site. CONCLUSIONS: From these data, we conclude that the most invasive cells within a pancreatic tumor are able to thrive because of their increased genomic stability. These cells have also been linked to the TIC population in a tumor.
Assuntos
Reparo do DNA/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Bases de Dados Factuais , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Dosagem de Genes , Expressão Gênica , Genes BRCA1 , Instabilidade Genômica , Humanos , Invasividade Neoplásica/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Rad51 Recombinase/genética , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , GencitabinaRESUMO
Cancer stem cells are the sub-population of cells present within tumors responsible for tumorigenesis. These cells have unique biological properties including self-renewal and the ability to differentiate. Furthermore, it is thought that these cells are more resistant to conventional chemotherapy and, as a result, are responsible for patient relapse. We will discuss the identification of prostate cancer stem cells, their unique properties and how these cells may be targeted for more efficacious therapies.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/química , Sistemas de Liberação de Medicamentos/tendências , Humanos , Masculino , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Development of metastasis is a leading cause of cancer-induced death. Acquisition of an invasive tumor cell phenotype suggests loss of cell adhesion and basement membrane breakdown during a process termed epithelial-to-mesenchymal transition (EMT). Recently, cancer stem cells (CSC) were discovered to mediate solid tumor initiation and progression. Prostate CSCs are a subpopulation of CD44(+) cells within the tumor that give rise to differentiated tumor cells and also self-renew. Using both primary and established prostate cancer cell lines, we tested the assumption that CSCs are more invasive. The ability of unsorted cells and CD44-positive and -negative subpopulations to undergo Matrigel invasion and EMT was evaluated, and the gene expression profiles of these cells were analyzed by microarray and a subset confirmed using QRT-PCR. Our data reveal that a subpopulation of CD44(+) CSC-like cells invade Matrigel through an EMT, while in contrast, CD44(-) cells are non-invasive. Furthermore, the genomic profile of the invasive cells closely resembles that of CD44(+)CD24(-) prostate CSCs and shows evidence for increased Hedgehog signaling. Finally, invasive cells from DU145 and primary prostate cancer cells are more tumorigenic in NOD/SCID mice compared with non-invasive cells. Our data strongly suggest that basement membrane invasion, an early and necessary step in metastasis development, is mediated by these potential cancer stem cells.
Assuntos
Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Antígeno CD24/biossíntese , Epitélio/patologia , Genômica , Humanos , Receptores de Hialuronatos/biossíntese , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Células-Tronco/metabolismoRESUMO
The pathways and key players that regulate parity-induced protection against breast cancer, conferred by estrogen (E) and progesterone (P), have not fully been explained. Interestingly, in rodents, high levels of circulating insulin-like growth factor-I (IGF-I) appear to block this parity-induced protection. Using an in-vitro mouse mammary gland whole organ culture system, we investigated the mechanisms by which IGF-I affects the protective effects of E+P. Earlier studies have demonstrated that important changes induced by parity include both the enhanced sensitivity to p53 activation, and the cell death that occurs in response to DNA damage. Remarkably, when IGF-I was added to cultures containing E+P and irradiated, both cell death and expression of the tumor suppressor protein p53 were inhibited (P<0.03 and <0.006, respectively). In addition, quantitative real-time PCR analysis of numerous genes identified by microarray as being upregulated in response to prolonged exposure to pregnancy levels of E+P suggested that a subset was affected by the addition of IGF-I. IGF-I suppressed the hormone-induced expression of insulin-like growth factor-binding protein-3, transforming growth factor beta-1, and cellular retinol-binding protein-1 (P<0.01 for all genes); all of which are genes that are important in growth suppression and the regulation of survival of cells. Overall, the observed reductions in p53 sensitivity and hormone-induced gene expression in response to IGF-I in vitro suggest a mechanism for the blockade in parity-induced protection previously seen in response to IGF-I in vivo.