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1.
Nature ; 608(7923): 609-617, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35948633

RESUMO

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer1. However, clinical responses to FGFR inhibitors have remained variable1-9, emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening10,11 and tumour modelling in mice12,13, and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2ΔE18). Functional in vitro and in vivo examination of a compendium of FGFR2ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies.


Assuntos
Éxons , Deleção de Genes , Terapia de Alvo Molecular , Neoplasias , Oncogenes , Inibidores de Proteínas Quinases , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Animais , Éxons/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Oncogenes/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo
2.
Nature ; 572(7770): 538-542, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367040

RESUMO

Cancer-associated systemic inflammation is strongly linked to poor disease outcome in patients with cancer1,2. For most human epithelial tumour types, high systemic neutrophil-to-lymphocyte ratios are associated with poor overall survival3, and experimental studies have demonstrated a causal relationship between neutrophils and metastasis4,5. However, the cancer-cell-intrinsic mechanisms that dictate the substantial heterogeneity in systemic neutrophilic inflammation between tumour-bearing hosts are largely unresolved. Here, using a panel of 16 distinct genetically engineered mouse models for breast cancer, we uncover a role for cancer-cell-intrinsic p53 as a key regulator of pro-metastatic neutrophils. Mechanistically, loss of p53 in cancer cells induced the secretion of WNT ligands that stimulate tumour-associated macrophages to produce IL-1ß, thus driving systemic inflammation. Pharmacological and genetic blockade of WNT secretion in p53-null cancer cells reverses macrophage production of IL-1ß and subsequent neutrophilic inflammation, resulting in reduced metastasis formation. Collectively, we demonstrate a mechanistic link between the loss of p53 in cancer cells, secretion of WNT ligands and systemic neutrophilia that potentiates metastatic progression. These insights illustrate the importance of the genetic makeup of breast tumours in dictating pro-metastatic systemic inflammation, and set the stage for personalized immune intervention strategies for patients with cancer.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Inflamação/genética , Inflamação/patologia , Metástase Neoplásica/patologia , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Proteínas Wnt/metabolismo , Animais , Neoplasias da Mama/complicações , Modelos Animais de Doenças , Feminino , Inflamação/complicações , Inflamação/imunologia , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Camundongos , Neutrófilos/imunologia
3.
Genes Dev ; 30(12): 1470-80, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27340177

RESUMO

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Carcinoma Lobular/genética , Carcinoma Lobular/fisiopatologia , Edição de Genes , Glândulas Mamárias Humanas/fisiopatologia , Animais , Sistemas CRISPR-Cas , Caderinas/genética , Modelos Animais de Doenças , Feminino , Inativação Gênica , Genes Supressores de Tumor , Humanos , Camundongos
5.
J Mammary Gland Biol Neoplasia ; 24(4): 305-321, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31729597

RESUMO

Approximately 75% of all breast cancers express the nuclear hormone receptor estrogen receptor α (ERα). However, the majority of mammary tumors from genetically engineered mouse models (GEMMs) are ERα-negative. To model ERα-positive breast cancer in mice, we exogenously introduced expression of mouse and human ERα in an existing GEMM of p53-deficient breast cancer. After initial ERα expression during mammary gland development, expression was reduced or lost in adult glands and p53-deficient mammary tumors. Chromatin immunoprecipitation (ChIP)-sequencing analysis of primary mouse mammary epithelial cells (MMECs) derived from these models, in which expression of the ERα constructs was induced in vitro, confirmed interaction of ERα with the DNA. In human breast and endometrial cancer, and also in healthy breast tissue, DNA binding of ERα is facilitated by the pioneer factor FOXA1. Surprisingly, the ERα binding sites identified in primary MMECs, but also in mouse mammary gland and uterus, showed an high enrichment of ERE motifs, but were devoid of Forkhead motifs. Furthermore, exogenous introduction of FOXA1 and GATA3 in ERα-expressing MMECs was not sufficient to promote ERα-responsiveness of these cells. Together, this suggests that species-specific differences in pioneer factor usage between mouse and human are dictated by the DNA sequence, resulting in ERα-dependencies in mice that are not FOXA1 driven. These species-specific differences in ERα-biology may limit the utility of mice for in vivo modeling of ERα-positive breast cancer.


Assuntos
Células Epiteliais/patologia , Receptor alfa de Estrogênio/metabolismo , Fator de Transcrição GATA3/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Neoplasias Mamárias Animais/patologia , Proteína Supressora de Tumor p53/deficiência , Animais , Células Cultivadas , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Fator de Transcrição GATA3/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Camundongos , Proteína Supressora de Tumor p53/genética
6.
Nucleic Acids Res ; 45(12): 7064-7077, 2017 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-28575524

RESUMO

Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes.


Assuntos
Neoplasias da Mama/genética , Elementos de DNA Transponíveis , Leucemia de Células B/genética , Mutagênese Insercional , Proteínas de Neoplasias/genética , Software , Doença Aguda , Animais , Neoplasias da Mama/metabolismo , Mapeamento Cromossômico/métodos , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Ensaios de Triagem em Larga Escala , Humanos , Leucemia de Células B/metabolismo , Camundongos , Proteínas de Neoplasias/metabolismo
7.
Int J Cancer ; 136(6): 1434-44, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25080865

RESUMO

The multikinase inhibitor sorafenib is under clinical investigation for the treatment of many solid tumors, but in most cases, the molecular target responsible for the clinical effect is unknown. Furthermore, enhancing the effectiveness of sorafenib using combination strategies is a major clinical challenge. Here, we identify sorafenib as an activator of AMP-activated protein kinase (AMPK), in a manner that involves either upstream LKB1 or CAMKK2. We further show in a phase II clinical trial in KRAS mutant advanced non-small cell lung cancer (NSCLC) with single agent sorafenib an improved disease control rate in patients using the antidiabetic drug metformin. Consistent with this, sorafenib and metformin act synergistically in inhibiting cellular proliferation in NSCLC in vitro and in vivo. A synergistic effect of both drugs is also seen on phosphorylation of the AMPKα activation site. Our results provide a rationale for the synergistic antiproliferative effects, given that AMPK inhibits downstream mTOR signaling. These data suggest that the combination of sorafenib with AMPK activators could have beneficial effects on tumor regression by AMPK pathway activation. The combination of metformin or other AMPK activators and sorafenib could be tested in prospective clinical trials.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/congênito , Neoplasias Pulmonares/tratamento farmacológico , Metformina/farmacologia , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Transdução de Sinais , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/fisiologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Niacinamida/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Sorafenibe , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética
8.
Genome Res ; 21(12): 2181-9, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21852388

RESUMO

Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.


Assuntos
DNA de Neoplasias/genética , Bases de Dados Genéticas , Vírus do Tumor Mamário do Camundongo , Mutagênese Insercional , Infecções por Retroviridae/genética , Infecções Tumorais por Vírus/genética , Animais , Análise Mutacional de DNA/métodos , Camundongos
9.
J Pathol ; 220(1): 34-44, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19927317

RESUMO

The contribution of cancer cell-intrinsic and -extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP-based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time-consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell-intrinsic and -extrinsic factors. Although the latter can be achieved by cleared fat-pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild-type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat-pad transplantations with MMECs isolated from K14cre;Cdh1(F/F); Trp53(F/F) mice expressing Cre recombinase under control of the cytokeratin-14 promoter and carrying conditional null alleles for p53 and E-cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell-intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding.


Assuntos
Transformação Celular Neoplásica/patologia , Glândulas Mamárias Animais/transplante , Neoplasias Mamárias Experimentais/patologia , Tecido Adiposo/transplante , Animais , Caderinas/metabolismo , Transformação Celular Neoplásica/metabolismo , Células Epiteliais/transplante , Feminino , Queratina-8/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Processos Estocásticos , Vimentina/metabolismo
10.
BMC Cancer ; 10: 455, 2010 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-20735817

RESUMO

BACKGROUND: Genomic gains and losses are a result of genomic instability in many types of cancers. BRCA1- and BRCA2-mutated breast cancers are associated with increased amounts of chromosomal aberrations, presumably due their functions in genome repair. Some of these genomic aberrations may harbor genes whose absence or overexpression may give rise to cellular growth advantage. So far, it has not been easy to identify the driver genes underlying gains and losses. A powerful approach to identify these driver genes could be a cross-species comparison of array comparative genomic hybridization (aCGH) data from cognate mouse and human tumors. Orthologous regions of mouse and human tumors that are commonly gained or lost might represent essential genomic regions selected for gain or loss during tumor development. METHODS: To identify genomic regions that are associated with BRCA1- and BRCA2-mutated breast cancers we compared aCGH data from 130 mouse Brca1Δ/Δ;p53Δ/Δ, Brca2Δ/Δ;p53Δ/Δ and p53Δ/Δ mammary tumor groups with 103 human BRCA1-mutated, BRCA2-mutated and non-hereditary breast cancers. RESULTS: Our genome-wide cross-species analysis yielded a complete collection of loci and genes that are commonly gained or lost in mouse and human breast cancer. Principal common CNAs were the well known MYC-associated gain and RB1/INTS6-associated loss that occurred in all mouse and human tumor groups, and the AURKA-associated gain occurred in BRCA2-related tumors from both species. However, there were also important differences between tumor profiles of both species, such as the prominent gain on chromosome 10 in mouse Brca2Δ/Δ;p53Δ/Δ tumors and the PIK3CA associated 3q gain in human BRCA1-mutated tumors, which occurred in tumors from one species but not in tumors from the other species. This disparity in recurrent aberrations in mouse and human tumors might be due to differences in tumor cell type or genomic organization between both species. CONCLUSIONS: The selection of the oncogenome during mouse and human breast tumor development is markedly different, apart from the MYC gain and RB1-associated loss. These differences should be kept in mind when using mouse models for preclinical studies.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Perfilação da Expressão Gênica , Mutação/genética , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/classificação , Aberrações Cromossômicas , Hibridização Genômica Comparativa , Feminino , Instabilidade Genômica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie
11.
Clin Cancer Res ; 14(12): 3916-25, 2008 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-18559613

RESUMO

PURPOSE: To assess efficacy of the novel, selective poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor AZD2281 against newly established BRCA2-deficient mouse mammary tumor cell lines and to determine potential synergy between AZD2281 and cisplatin. EXPERIMENTAL DESIGN: We established and thoroughly characterized a panel of clonal cell lines from independent BRCA2-deficient mouse mammary tumors and BRCA2-proficient control tumors. Subsequently, we assessed sensitivity of these lines to conventional cytotoxic drugs and the novel PARP inhibitor AZD2281. Finally, in vitro combination studies were done to investigate interaction between AZD2281 and cisplatin. RESULTS: Genetic, transcriptional, and functional analyses confirmed the successful isolation of BRCA2-deficient and BRCA2-proficient mouse mammary tumor cell lines. Treatment of these cell lines with 11 different anticancer drugs or with gamma-irradiation showed that AZD2281, a novel and specific PARP inhibitor, caused the strongest differential growth inhibition of BRCA2-deficient versus BRCA2-proficient mammary tumor cells. Finally, drug combination studies showed synergistic cytotoxicity of AZD2281 and cisplatin against BRCA2-deficient cells but not against BRCA2-proficient control cells. CONCLUSION: We have successfully established the first set of BRCA2-deficient mammary tumor cell lines, which form an important addition to the existing preclinical models for BRCA-mutated breast cancer. The exquisite sensitivity of these cells to the PARP inhibitor AZD2281, alone or in combination with cisplatin, provides strong support for AZD2281 as a novel targeted therapeutic against BRCA-deficient cancers.


Assuntos
Proteína BRCA2/genética , Proliferação de Células/efeitos dos fármacos , Cisplatino/administração & dosagem , Neoplasias Mamárias Animais/tratamento farmacológico , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores Enzimáticos/farmacologia , Feminino , Neoplasias Mamárias Animais/genética , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos da radiação , Inibidores de Poli(ADP-Ribose) Polimerases , Rad51 Recombinase/genética
12.
Cancer Res ; 78(19): 5668-5679, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30115694

RESUMO

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified Fgfr2 as a key driver of invasive lobular carcinoma (ILC) in an in vivo insertional mutagenesis screen using the Sleeping Beauty transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our in vivo approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK-ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from de novo transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies.Significance: These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis in vivo is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. Cancer Res; 78(19); 5668-79. ©2018 AACR.


Assuntos
Benzamidas/química , Elementos de DNA Transponíveis , Resistencia a Medicamentos Antineoplásicos , Mutagênese , Piperazinas/química , Pirazóis/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Carcinoma Lobular/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Amplificação de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Mutação , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Transplante de Neoplasias , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Análise de Sequência de RNA , Transcriptoma , Proteína p120 Ativadora de GTPase/metabolismo
13.
Nat Genet ; 49(8): 1219-1230, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28650484

RESUMO

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.


Assuntos
Neoplasias da Mama/genética , Carcinoma Lobular/genética , Mutagênese Insercional , Animais , Caderinas/genética , Linhagem Celular , Sobrevivência Celular/genética , Transformação Celular Neoplásica/genética , Feminino , Haplótipos , Humanos , Masculino , Camundongos , Cadeias Pesadas de Miosina , Fosfatase de Miosina-de-Cadeia-Leve/genética , Miosina não Muscular Tipo IIA/genética , Transposases/genética , Proteínas Supressoras de Tumor/genética
14.
Cell Rep ; 16(8): 2087-2101, 2016 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-27524621

RESUMO

Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC.


Assuntos
Caderinas/genética , Carcinoma Lobular/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Neoplasias Mamárias Experimentais/genética , PTEN Fosfo-Hidrolase/genética , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Antineoplásicos/farmacologia , Caderinas/deficiência , Carcinoma Lobular/tratamento farmacológico , Carcinoma Lobular/mortalidade , Carcinoma Lobular/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Perfilação da Expressão Gênica , Imidazóis/farmacologia , Integrases/genética , Integrases/metabolismo , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/mortalidade , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Knockout , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/deficiência , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Quinolinas/farmacologia , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais , Análise de Sobrevida , Microambiente Tumoral
15.
J Clin Invest ; 126(8): 2903-18, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27454287

RESUMO

Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos , Deleção de Genes , Neoplasias Mamárias Animais/genética , Alelos , Animais , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Cruzamentos Genéticos , Dano ao DNA , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Efeito Fundador , Mutação da Fase de Leitura , Engenharia Genética , Humanos , Masculino , Neoplasias Mamárias Animais/tratamento farmacológico , Camundongos , Mutação , Transplante de Neoplasias , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Recombinação Genética
16.
Cancer Cell ; 20(6): 797-809, 2011 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-22172724

RESUMO

Hereditary breast cancers are frequently caused by germline BRCA1 mutations. The BRCA1(C61G) mutation in the BRCA1 RING domain is a common pathogenic missense variant, which reduces BRCA1/BARD1 heterodimerization and abrogates its ubiquitin ligase activity. To investigate the role of BRCA1 RING function in tumor suppression and therapy response, we introduced the Brca1(C61G) mutation in a conditional mouse model for BRCA1-associated breast cancer. In contrast to BRCA1-deficient mammary carcinomas, tumors carrying the Brca1(C61G) mutation responded poorly to platinum drugs and PARP inhibition and rapidly developed resistance while retaining the Brca1(C61G) mutation. These findings point to hypomorphic activity of the BRCA1-C61G protein that, although unable to prevent tumor development, affects response to therapy.


Assuntos
Proteína BRCA1/genética , Resistencia a Medicamentos Antineoplásicos , Animais , Antineoplásicos/uso terapêutico , Apoptose , Proteína BRCA1/metabolismo , Carcinoma/tratamento farmacológico , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Proliferação de Células , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Inativação de Genes , Instabilidade Genômica , Queratina-8/metabolismo , Masculino , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Transplante de Neoplasias , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Estrutura Terciária de Proteína , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Transplante Heterólogo , Carga Tumoral/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
17.
Clin Cancer Res ; 16(1): 99-108, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20008842

RESUMO

PURPOSE: Hereditary breast cancer is partly explained by germline mutations in BRCA1 and BRCA2. Although patients carry heterozygous mutations, their tumors have typically lost the remaining wild-type allele. Selectively targeting BRCA deficiency may therefore constitute an important therapeutic approach. Clinical trials applying this principle are underway, but it is unknown whether the compounds tested are optimal. It is therefore important to identify alternative compounds that specifically target BRCA deficiency and to test new combination therapies to establish optimal treatment strategies. EXPERIMENTAL DESIGN: We did a high-throughput pharmaceutical screen on BRCA2-deficient mouse mammary tumor cells and isogenic controls with restored BRCA2 function. Subsequently, we validated positive hits in vitro and in vivo using mice carrying BRCA2-deficient mammary tumors. RESULTS: Three alkylators-chlorambucil, melphalan, and nimustine-displayed strong and specific toxicity against BRCA2-deficient cells. In vivo, these showed heterogeneous but generally strong BRCA2-deficient antitumor activity, with melphalan and nimustine doing better than cisplatin and the poly-(ADP-ribose)-polymerase inhibitor olaparib (AZD2281) in this small study. In vitro drug combination experiments showed synergistic interactions between the alkylators and olaparib. Tumor intervention studies combining nimustine and olaparib resulted in recurrence-free survival exceeding 330 days in 3 of 5 animals tested. CONCLUSIONS: We generated and validated a platform for identification of compounds with specific activity against BRCA2-deficient cells that translates well to the preclinical setting. Our data call for the re-evaluation of alkylators, especially melphalan and nimustine, alone or in combination with the poly-(ADP-ribose)-polymerase inhibitors, for the treatment of breast cancers with a defective BRCA pathway.


Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína BRCA2/deficiência , Cisplatino/uso terapêutico , Neoplasias Mamárias Animais/dietoterapia , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Sinergismo Farmacológico , Feminino , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/genética , Camundongos , Mutação , Ftalazinas/farmacologia , Piperazinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Genome Biol ; 11(10): R100, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20942901

RESUMO

BACKGROUND: Here we present the first paired-end sequencing of tumors from genetically engineered mouse models of cancer to determine how faithfully these models recapitulate the landscape of somatic rearrangements found in human tumors. These were models of Trp53-mutated breast cancer, Brca1- and Brca2-associated hereditary breast cancer, and E-cadherin (Cdh1) mutated lobular breast cancer. RESULTS: We show that although Brca1- and Brca2-deficient mouse mammary tumors have a defect in the homologous recombination pathway, there is no apparent difference in the type or frequency of somatic rearrangements found in these cancers when compared to other mouse mammary cancers, and tumors from all genetic backgrounds showed evidence of microhomology-mediated repair and non-homologous end-joining processes. Importantly, mouse mammary tumors were found to carry fewer structural rearrangements than human mammary cancers and expressed in-frame fusion genes. Like the fusion genes found in human mammary tumors, these were not recurrent. One mouse tumor was found to contain an internal deletion of exons of the Lrp1b gene, which led to a smaller in-frame transcript. We found internal in-frame deletions in the human ortholog of this gene in a significant number (4.2%) of human cancer cell lines. CONCLUSIONS: Paired-end sequencing of mouse mammary tumors revealed that they display significant heterogeneity in their profiles of somatic rearrangement but, importantly, fewer rearrangements than cognate human mammary tumors, probably because these cancers have been induced by strong driver mutations engineered into the mouse genome. Both human and mouse mammary cancers carry expressed fusion genes and conserved homozygous deletions.


Assuntos
Animais Geneticamente Modificados , Neoplasias da Mama/genética , Rearranjo Gênico , Mutação , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Sequência de Bases , Caderinas/metabolismo , Linhagem Celular Tumoral , Feminino , Fusão Gênica , Biblioteca Genômica , Humanos , Camundongos , Receptores de LDL/genética , Deleção de Sequência , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
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