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2.
Sci Transl Med ; 13(620): eabf4969, 2021 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-34788078

RESUMO

Quantifying response to drug treatment in mouse models of human cancer is important for treatment development and assignment, yet remains a challenging task. To be able to translate the results of the experiments more readily, a preferred measure to quantify this response should take into account more of the available experimental data, including both tumor size over time and the variation among replicates. We propose a theoretically grounded measure, KuLGaP, to compute the difference between the treatment and control arms. We test and compare KuLGaP to four widely used response measures using 329 patient-derived xenograft (PDX) models. Our results show that KuLGaP is more selective than currently existing measures, reduces the risk of false-positive calls, and improves translation of the laboratory results to clinical practice. We also show that outcomes of human treatment better align with the results of the KuLGaP measure than other response measures. KuLGaP has the potential to become a measure of choice for quantifying drug treatment in mouse models as it can be easily used via the kulgap.ca website.


Assuntos
Xenoenxertos , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Cell ; 184(1): 226-242.e21, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33417860

RESUMO

Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Diapausa , Resistencia a Medicamentos Antineoplásicos , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Células Clonais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética/efeitos dos fármacos , Humanos , Irinotecano/farmacologia , Irinotecano/uso terapêutico , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nature ; 588(7836): 169-173, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33087935

RESUMO

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor1-6. This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses7,8. However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of 'orphan' CpG islands9. In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA10, which prevents activation of the MDA5 receptor11. We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment.


Assuntos
Adenosina Desaminase/metabolismo , Elementos Alu/efeitos dos fármacos , Elementos Alu/genética , Decitabina/farmacologia , Decitabina/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/efeitos dos fármacos , Imunidade Adaptativa/efeitos dos fármacos , Adenosina Desaminase/deficiência , Elementos Alu/imunologia , Animais , Linhagem Celular Tumoral , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , DNA Intergênico/efeitos dos fármacos , DNA Intergênico/genética , DNA Intergênico/imunologia , DNA-Citosina Metilases/antagonistas & inibidores , Retroalimentação Fisiológica , Humanos , Imunidade Inata/efeitos dos fármacos , Helicase IFIH1 Induzida por Interferon/metabolismo , Íntrons/efeitos dos fármacos , Íntrons/genética , Íntrons/imunologia , Sequências Repetidas Invertidas/efeitos dos fármacos , Sequências Repetidas Invertidas/genética , Sequências Repetidas Invertidas/imunologia , Masculino , Camundongos , Mimetismo Molecular/efeitos dos fármacos , Mimetismo Molecular/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , RNA de Cadeia Dupla/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Vírus/efeitos dos fármacos , Vírus/imunologia
6.
JAMA Oncol ; 5(7): 961-966, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30973610

RESUMO

IMPORTANCE: Chemoradiotherapy (CRT), followed by surgery, is the recommended approach for stage II and III rectal cancer. While CRT decreases the risk of local recurrence, it does not improve survival and leads to poorer functional outcomes than surgery alone. Therefore, new approaches to better select patients for CRT are important. OBJECTIVE: To conduct a phase 2 study to evaluate the safety and feasibility of using magnetic resonance imaging (MRI) criteria to select patients with "good prognosis" rectal tumors for primary surgery. DESIGN, SETTING, AND PARTICIPANTS: Prospective nonrandomized phase 2 study at 12 high-volume colorectal surgery centers across Canada. From September 30, 2014, to October 21, 2016, a total of 82 patients were recruited for the study. Participants were patients newly diagnosed as having rectal cancer with MRI-predicted good prognosis rectal cancer. The MRI criteria for good prognosis tumors included distance to the mesorectal fascia greater than 1 mm; definite T2, T2/early T3, or definite T3 with less than 5 mm of extramural depth of invasion; and absent or equivocal extramural venous invasion. INTERVENTIONS: Patients with rectal cancer with MRI-predicted good prognosis tumors underwent primary surgery. MAIN OUTCOMES AND MEASURES: The primary outcome was the proportion of patients with a positive circumferential resection margin (CRM) rate. Assuming a 10% baseline probability of a positive CRM, a sample size of 75 was estimated to yield a 95% CI of ±6.7%. RESULTS: Eighty-two patients (74% male) participated in the study. The median age at the time of surgery was 66 years (range, 37-89 years). Based on MRI, most tumors were midrectal (65% [n = 53]), T2/early T3 (60% [n = 49]), with no suspicious lymph nodes (63% [n = 52]). On final pathology, 91% (n = 75) of tumors were T2 or greater, 29% (n = 24) were node positive, and 59% (n = 48) were stage II or III. The positive CRM rate was 4 of 82 (4.9%; 95% CI, 0.2%-9.6%). CONCLUSIONS AND RELEVANCE: The use of MRI criteria to select patients with good prognosis rectal cancer for primary surgery results in a low rate of positive CRM and suggests that CRT may not be necessary for all patients with stage II and III rectal cancer. TRIAL REGISTRATION: ISRCTN.com identifier: ISRCTN05107772.


Assuntos
Imageamento por Ressonância Magnética , Neoplasias Retais/diagnóstico por imagem , Neoplasias Retais/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Procedimentos Cirúrgicos do Sistema Digestório , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Retais/patologia
7.
Nat Commun ; 10(1): 1436, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926792

RESUMO

In embryonic stem cells, promoters of key lineage-specific differentiation genes are found in a bivalent state, having both activating H3K4me3 and repressive H3K27me3 histone marks, making them poised for transcription upon loss of H3K27me3. Whether cancer-initiating cells (C-ICs) have similar epigenetic mechanisms that prevent lineage commitment is unknown. Here we show that colorectal C-ICs (CC-ICs) are maintained in a stem-like state through a bivalent epigenetic mechanism. Disruption of the bivalent state through inhibition of the H3K27 methyltransferase EZH2, resulted in decreased self-renewal of patient-derived C-ICs. Epigenomic analyses revealed that the promoter of Indian Hedgehog (IHH), a canonical driver of normal colonocyte differentiation, exists in a bivalent chromatin state. Inhibition of EZH2 resulted in de-repression of IHH, decreased self-renewal, and increased sensitivity to chemotherapy in vivo. Our results reveal an epigenetic block to differentiation in CC-ICs and demonstrate the potential for epigenetic differentiation therapy of a solid tumour through EZH2 inhibition.


Assuntos
Autorrenovação Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas Hedgehog/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Feminino , Fluoruracila/farmacologia , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Piridonas/farmacologia
8.
Sci Rep ; 9(1): 842, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696911

RESUMO

Cell surface antigen discovery is of great interest for biomedical research both for isolation of rare cell populations and therapeutic targeting. We developed a rapid, cost-effective, fully in vitro technology which facilities the simultaneous target discovery and human antibody generation on the surface of virtually any cell population of interest. We apply our technique to human colorectal cancer-initiating cells (CICs) and identify hundreds of unique human antibodies. We characterized the top three antibody candidates targeting these CICs and identify their protein targets as integrin α7 (ITGA7), HLA-A1 and integrin ß6 (ITGB6). We demonstrate that these antibodies can be used to isolate self-renewing colorectal CICs, and that the integrin α7 antibody can prospectively identify glioblastoma brain tumor initiating cells as well as human muscle stem cells. We also demonstrate that genetic ablation of integrin ß6 impedes colorectal CIC function. The methodology can be readily applied to other cell populations including stem cells, cancer, or immune cells to facilitate the rapid identification of novel targets and simultaneous generation of potent and specific antibodies with therapeutic potential.


Assuntos
Anticorpos/imunologia , Antígenos de Superfície/imunologia , Biomarcadores Tumorais/imunologia , Neoplasias Colorretais/imunologia , Glioblastoma/imunologia , Antígenos CD/imunologia , Células CACO-2 , Células HCT116 , Células HEK293 , Antígeno HLA-A1/imunologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Cadeias alfa de Integrinas/imunologia , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/imunologia , Células MCF-7 , Células PC-3 , Interferência de RNA , RNA Interferente Pequeno/genética , Células Tumorais Cultivadas
9.
Cell Metab ; 27(3): 572-587.e6, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29514066

RESUMO

Long-chain acyl-CoA synthetase (ACSL)-dependent upper small intestinal lipid metabolism activates pre-absorptive pathways to regulate metabolic homeostasis, but whether changes in the upper small intestinal microbiota alter specific fatty acid-dependent pathways to impact glucose homeostasis remains unknown. We here first find that upper small intestinal infusion of Intralipid, oleic acid, or linoleic acid pre-absorptively increases glucose tolerance and lowers glucose production in rodents. High-fat feeding impairs pre-absorptive fatty acid sensing and reduces upper small intestinal Lactobacillus gasseri levels and ACSL3 expression. Transplantation of healthy upper small intestinal microbiota to high-fat-fed rodents restores L. gasseri levels and fatty acid sensing via increased ACSL3 expression, while L. gasseri probiotic administration to non-transplanted high-fat-fed rodents is sufficient to restore upper small intestinal ACSL3 expression and fatty acid sensing. In summary, we unveil a glucoregulatory role of upper small intestinal L. gasseri that impacts an ACSL3-dependent glucoregulatory fatty acid-sensing pathway.


Assuntos
Coenzima A Ligases/metabolismo , Ácidos Graxos/metabolismo , Microbioma Gastrointestinal , Glucose/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Lactobacillus gasseri/metabolismo , Animais , Dieta Hiperlipídica/métodos , Emulsões/metabolismo , Transplante de Microbiota Fecal/métodos , Homeostase , Ácido Linoleico/metabolismo , Camundongos Endogâmicos C57BL , Ácido Oleico/metabolismo , Fosfolipídeos/metabolismo , Ratos Sprague-Dawley , Óleo de Soja/metabolismo
10.
Clin Cancer Res ; 24(9): 2116-2127, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29476017

RESUMO

Purpose: Cancer-initiating cells (C-IC) have been described in multiple cancer types, including colorectal cancer. C-ICs are defined by their capacity to self-renew, thereby driving tumor growth. C-ICs were initially thought to be static entities; however, recent studies have determined these cells to be dynamic and influenced by microenvironmental cues such as hypoxia. If hypoxia drives the formation of C-ICs, then therapeutic targeting of hypoxia could represent a novel means to target C-ICs.Experimental Design: Patient-derived colorectal cancer xenografts were treated with evofosfamide, a hypoxia-activated prodrug (HAP), in combination with 5-fluorouracil (5-FU) or chemoradiotherapy (5-FU and radiation; CRT). Treatment groups included both concurrent and sequential dosing regimens. Effects on the colorectal cancer-initiating cell (CC-IC) fraction were assessed by serial passage in vivo limiting dilution assays. FAZA-PET imaging was utilized as a noninvasive method to assess intratumoral hypoxia.Results: Hypoxia was sufficient to drive the formation of CC-ICs and colorectal cancer cells surviving conventional therapy were more hypoxic and C-IC-like. Using a novel approach to combination therapy, we show that sequential treatment with 5-FU or CRT followed by evofosfamide not only inhibits tumor growth of xenografts compared with 5-FU or CRT alone, but also significantly decreases the CC-IC fraction. Furthermore, noninvasive FAZA-PET hypoxia imaging was predictive of a tumor's response to evofosfamide.Conclusions: Our data demonstrate a novel means to target the CC-IC fraction by adding a HAP sequentially after conventional adjuvant therapy, as well as the use of FAZA-PET as a biomarker for hypoxia to identify tumors that will benefit most from this approach. Clin Cancer Res; 24(9); 2116-27. ©2018 AACR.


Assuntos
Neoplasias Colorretais/metabolismo , Hipóxia/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Nitroimidazóis/administração & dosagem , Mostardas de Fosforamida/administração & dosagem , Pró-Fármacos/administração & dosagem , Animais , Biomarcadores , Caspases/metabolismo , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiorradioterapia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/radioterapia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Feminino , Humanos , Masculino , Camundongos , Fenótipo , Tomografia por Emissão de Pósitrons , Padrão de Cuidado , Via de Sinalização Wnt , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Cell Metab ; 27(1): 101-117.e5, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29056513

RESUMO

The gut microbiota alters energy homeostasis. In parallel, metformin regulates upper small intestinal sodium glucose cotransporter-1 (SGLT1), but whether changes of the microbiota or SGLT1-dependent pathways in the upper small intestine mediate metformin action is unknown. Here we report that upper small intestinal glucose sensing triggers an SGLT1-dependent pathway to lower glucose production in rodents. High-fat diet (HFD) feeding reduces glucose sensing and SGLT1 expression in the upper small intestine. Upper small intestinal metformin treatment restores SGLT1 expression and glucose sensing while shifting the upper small intestinal microbiota partly by increasing the abundance of Lactobacillus. Transplantation of upper small intestinal microbiota from metformin-treated HFD rats to the upper small intestine of untreated HFD rats also increases the upper small intestinal abundance of Lactobacillus and glucose sensing via an upregulation of SGLT1 expression. Thus, we demonstrate that metformin alters upper small intestinal microbiota and impacts a glucose-SGLT1-sensing glucoregulatory pathway.


Assuntos
Microbioma Gastrointestinal/efeitos dos fármacos , Glucose/metabolismo , Metformina/farmacologia , Transportador 1 de Glucose-Sódio/metabolismo , Animais , Dieta Hiperlipídica , Comportamento Alimentar , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Análise de Componente Principal , Ratos
12.
Gastroenterology ; 149(3): 705-17.e2, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26026391

RESUMO

BACKGROUND & AIMS: Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer. METHODS: An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 DM colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription polymerase chain reaction, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib after injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis. RESULTS: KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5 associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cells. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44(+) cells indicated that KIT can promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT(+) colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice. CONCLUSIONS: KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors can benefit from KIT RTK inhibitors.


Assuntos
Proliferação de Células , Neoplasias do Colo/enzimologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Animais , Comunicação Autócrina , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Comunicação Parácrina , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Fator de Células-Tronco/genética , Fatores de Tempo , Transcrição Gênica , Transfecção , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Sci Signal ; 7(351): ra107, 2014 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-25389372

RESUMO

Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC). We investigated potential bypass mechanisms to PI3K/mTOR inhibition in KRAS-mutant CRC. Using genetically engineered mouse model cells that had acquired resistance to the dual PI3K/mTOR small-molecule inhibitor PF-04691502, we determined with chemical library screens that inhibitors of the ERBB [epidermal growth factor receptor (EGFR)] family restored the sensitivity to PF-04691502. Although EGFR inhibitors alone have limited efficacy in reducing KRAS-mutant tumors, we found that PF-04691502 induced the abundance, phosphorylation, and activity of EGFR, ERBB2, and ERBB3 through activation of FOXO3a (forkhead box O 3a), a transcription factor inhibited by the PI3K to AKT pathway. PF-04691502 also induced a stem cell-like gene expression signature. KRAS-mutant patient-derived xenografts from mice treated with PF-04691502 had a similar gene expression signature and exhibited increased EGFR activation, suggesting that this drug-induced resistance mechanism may occur in patients. Combination therapy with dacomitinib (a pan-ERBB inhibitor) restored sensitivity to PF-04691502 in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Our findings suggest that combining PI3K/mTOR and EGFR inhibitors may improve therapeutic outcome in patients with KRAS-mutant CRC.


Assuntos
Neoplasias Colorretais/metabolismo , Inibidores Enzimáticos/química , Receptores ErbB/antagonistas & inibidores , Genes ras , Inibidores de Fosfoinositídeo-3 Quinase , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Sobrevivência Celular , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Engenharia Genética , Humanos , Camundongos , Camundongos SCID , Mutação , Transplante de Neoplasias , Fosforilação , Transdução de Sinais , Transcrição Gênica , Proteína Supressora de Tumor p53/metabolismo , beta Catenina/metabolismo , Proteínas ras/genética
14.
Nat Med ; 20(1): 29-36, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24292392

RESUMO

Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichment of cancer-initiating cells (CICs) in tumors that survive therapy. Together with recent reports showing that CIC gene signatures influence patient survival, these studies predict that targeting self-renewal, the key 'stemness' property unique to CICs, may represent a new paradigm in cancer therapy. Here we demonstrate that tumor formation and, more specifically, human colorectal CIC function are dependent on the canonical self-renewal regulator BMI-1. Downregulation of BMI-1 inhibits the ability of colorectal CICs to self-renew, resulting in the abrogation of their tumorigenic potential. Treatment of primary colorectal cancer xenografts with a small-molecule BMI-1 inhibitor resulted in colorectal CIC loss with long-term and irreversible impairment of tumor growth. Targeting the BMI-1-related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Compostos Heterocíclicos com 2 Anéis/farmacologia , Recidiva Local de Neoplasia/metabolismo , Células-Tronco Neoplásicas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Tiazóis/farmacologia , Animais , Western Blotting , Bromodesoxiuridina , Linhagem Celular Tumoral , Citometria de Fluxo , Vetores Genéticos/genética , Compostos Heterocíclicos com 2 Anéis/uso terapêutico , Humanos , Luciferases , Camundongos Endogâmicos NOD , Camundongos SCID , Complexo Repressor Polycomb 1/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Tiazóis/uso terapêutico
15.
Mol Syst Biol ; 9: 696, 2013 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-24104479

RESUMO

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN(-/-) DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.


Assuntos
Neoplasias da Mama/genética , Epistasia Genética , Genes Essenciais , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética , PTEN Fosfo-Hidrolase/genética , Neoplasias Pancreáticas/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Redes Reguladoras de Genes , Genoma Humano , Humanos , Mutação , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , PTEN Fosfo-Hidrolase/deficiência , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
16.
Science ; 339(6119): 543-8, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23239622

RESUMO

Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.


Assuntos
Evolução Clonal/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Animais , Linhagem da Célula , Rastreamento de Células , Células Clonais , Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA , Humanos , Lentivirus , Camundongos , Transplante de Neoplasias , Transcriptoma , Transdução Genética , Células Tumorais Cultivadas
17.
Cancer Cell ; 21(6): 777-92, 2012 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-22698403

RESUMO

There is increasing evidence that some cancers are hierarchically organized, sustained by a relatively rare population of cancer-initiating cells (C-ICs). Although the capacity to initiate tumors upon serial transplantation is a hallmark of all C-ICs, little is known about the genes that control this process. Here, we establish that ID1 and ID3 function together to govern colon cancer-initiating cell (CC-IC) self-renewal through cell-cycle restriction driven by the cell-cycle inhibitor p21. Regulation of p21 by ID1 and ID3 is a central mechanism preventing the accumulation of excess DNA damage and subsequent functional exhaustion of CC-ICs. Additionally, silencing of ID1 and ID3 increases sensitivity of CC-ICs to the chemotherapeutic agent oxaliplatin, linking tumor initiation function with chemotherapy resistance.


Assuntos
Neoplasias do Colo/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Proteína 1 Inibidora de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/genética , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Proteínas Inibidoras de Diferenciação/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia Confocal , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Clin Cancer Res ; 15(9): 3135-42, 2009 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-19366833

RESUMO

PURPOSE: To accurately identify gene expression alterations that differentiate neoplastic from normal prostate epithelium using an approach that avoids contamination by unwanted cellular components and is not compromised by acute gene expression changes associated with tumor devascularization and resulting ischemia. EXPERIMENTAL DESIGN: Approximately 3,000 neoplastic and benign prostate epithelial cells were isolated using laser capture microdissection from snap-frozen prostate biopsy specimens provided by 31 patients who subsequently participated in a clinical trial of preoperative chemotherapy. cDNA synthesized from amplified total RNA was hybridized to custom-made microarrays composed of 6,200 clones derived from the Prostate Expression Database. Expression differences for selected genes were verified using quantitative reverse transcription-PCR. RESULTS: Comparative analyses identified 954 transcript alterations associated with cancer (q < 0.01%), including 149 differentially expressed genes with no known functional roles. Gene expression changes associated with ischemia and surgical removal of the prostate gland were absent. Genes up-regulated in prostate cancer were statistically enriched in categories related to cellular metabolism, energy use, signal transduction, and molecular transport. Genes down-regulated in prostate cancers were enriched in categories related to immune response, cellular responses to pathogens, and apoptosis. A heterogeneous pattern of androgen receptor expression changes was noted. In exploratory analyses, androgen receptor down-regulation was associated with a lower probability of cancer relapse after neoadjuvant chemotherapy followed by radical prostatectomy. CONCLUSIONS: Assessments of tumor phenotypes based on gene expression for treatment stratification and drug targeting of oncogenic alterations may best be ascertained using biopsy-based analyses where the effects of ischemia do not complicate interpretation.


Assuntos
Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Neoplasias da Próstata/genética , Biomarcadores Tumorais/metabolismo , Biópsia por Agulha , Humanos , Lasers , Masculino , Microdissecção , Análise de Sequência com Séries de Oligonucleotídeos , Hiperplasia Prostática/genética , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Nature ; 445(7123): 106-10, 2007 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-17122772

RESUMO

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells--cancer stem cells--possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 x 10(4) unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.


Assuntos
Antígenos CD/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Glicoproteínas/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Peptídeos/metabolismo , Antígeno AC133 , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Transplante de Neoplasias , Transplante Heterólogo
20.
Transplantation ; 76(2): 400-9, 2003 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-12883200

RESUMO

Tolerance induction by CD45RB monoclonal antibody (mAb) in murine allograft models is associated with an alteration in the CD45RBlo/CD45RBhi T-cell ratio in favor of CD45RBlo T cells, which can function as regulatory cells and promote tolerance. It has been proposed that inversion of the CD45RBhi/CD45RBlo normal T-cell ratio by mAb can occur by down-regulation of CD45RB surface molecules expressed by T cells. Because CD45RB mAb infusion can lead to a reduction in peripheral T cells, we tested whether other mechanisms might participate in the inversion of the CD45RBhi/CD45RBlo ratio, including apoptosis of CD45RBhi cells. We report that CD45RB mAb led to rapid elimination of both CD4+ and CD8+ T cells in vitro. Importantly, CD45RB mAb selectively eliminated CD45RBhi T cells without affecting the viability of CD45RBlo T cells. Furthermore, the death of T cells occurred with a reduction in mitochondrial transmembrane potential and DNA fragmentation but with little evidence of nuclear condensation and cell shrinkage typically found with cells undergoing apoptosis. We propose that CD45RB mAb therapy may promote a dominant regulatory T-cell population that has the capacity to inhibit rejection by the selective elimination of CD45RBhi effector T cells. This occurs by a process that does not involve the classic morphologic features of apoptosis. Strategies that facilitate an inversion of the CD45RBhi/CD45RBlo T-cell subset ratio may improve the efficacy of CD45RB mAb, and therapeutic measures that prevent deletion of CD45RBhi T cells may need to be avoided to achieve tolerance clinically.


Assuntos
Anticorpos Monoclonais/farmacologia , Tolerância Imunológica/imunologia , Antígenos Comuns de Leucócito/análise , Linfócitos T/química , Linfócitos T/citologia , Animais , Transporte Biológico/imunologia , Morte Celular/imunologia , Feminino , Sobrevivência de Enxerto/imunologia , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Linfócitos T/imunologia , Imunologia de Transplantes
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