RESUMO
Acquired resistance to endocrine therapy is responsible for half of the therapeutic failures in the treatment of breast cancer. Recent findings have implicated increased expression of the ETS transcription factor ELF5 as a potential modulator of estrogen action and driver of endocrine resistance, and here we provide the first insight into the mechanisms by which ELF5 modulates estrogen sensitivity. Using chromatin immunoprecipitation sequencing we found that ELF5 binding overlapped with FOXA1 and ER at super enhancers, enhancers and promoters, and when elevated, caused FOXA1 and ER to bind to new regions of the genome, in a pattern that replicated the alterations to the ER/FOXA1 cistrome caused by the acquisition of resistance to endocrine therapy. RNA sequencing demonstrated that these changes altered estrogen-driven patterns of gene expression, the expression of ER transcription-complex members, and 6 genes known to be involved in driving the acquisition of endocrine resistance. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, and proximity ligation assays, we found that ELF5 interacted physically with members of the ER transcription complex, such as DNA-PKcs. We found 2 cases of endocrine-resistant brain metastases where ELF5 levels were greatly increased and ELF5 patterns of gene expression were enriched, compared to the matched primary tumour. Thus ELF5 alters ER-driven gene expression by modulating the ER/FOXA1 cistrome, by interacting with it, and by modulating the expression of members of the ER transcriptional complex, providing multiple mechanisms by which ELF5 can drive endocrine resistance.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Células MCF-7 , Camundongos , Ligação ProteicaRESUMO
BACKGROUND: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice. METHODS: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry. RESULTS: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present. CONCLUSIONS: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy.
Assuntos
2',5'-Oligoadenilato Sintetase , Neoplasias da Mama , 2',5'-Oligoadenilato Sintetase/genética , Nucleotídeos de Adenina , Animais , Neoplasias da Mama/genética , Feminino , Humanos , Interferons , Ligases , Camundongos , Oligorribonucleotídeos , GravidezRESUMO
BACKGROUND: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms. METHODS: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq. RESULTS: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency. CONCLUSIONS: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Proteínas Inibidoras de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/metabolismo , Animais , Apoptose/fisiologia , Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Prognóstico , Proteogenômica , Células Tumorais CultivadasRESUMO
We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , Lactação/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Técnicas de Cultura de Células , Endorribonucleases/metabolismo , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Camundongos , Leite , Mutação/genética , Oligorribonucleotídeos/metabolismo , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais/genéticaRESUMO
During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias Pulmonares/secundário , Pulmão/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Animais , Neoplasias da Mama/imunologia , Neoplasias da Mama/fisiopatologia , Neoplasias da Mama/virologia , Permeabilidade Capilar , Proliferação de Células , Proteínas de Ligação a DNA , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemorragia/etiologia , Hemorragia/prevenção & controle , Humanos , Leucócitos/imunologia , Leucócitos/patologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/prevenção & controle , Depleção Linfocítica , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/patologia , Proteínas de Neoplasias/genética , Neovascularização Patológica/etiologia , Neovascularização Patológica/prevenção & controle , Infiltração de Neutrófilos , Polyomavirus/patogenicidade , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sobrevida , Fatores de Transcrição , Carga TumoralRESUMO
Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.
Assuntos
Guanilato Quinases/metabolismo , Infertilidade Masculina/metabolismo , Espermatogênese , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Corpos Basais/metabolismo , Membrana Celular/metabolismo , Guanilato Quinases/química , Guanilato Quinases/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Espermatozoides/citologia , Testículo/citologia , Testículo/metabolismoRESUMO
piRNAs are critical for transposable element (TE) repression and germ cell survival during the early phases of spermatogenesis, however, their role in adult germ cells and the relative importance of piRNA methylation is poorly defined in mammals. Using a mouse model of HEN methyltransferase 1 (HENMT1) loss-of-function, RNA-Seq and a range of RNA assays we show that HENMT1 is required for the 2' O-methylation of mammalian piRNAs. HENMT1 loss leads to piRNA instability, reduced piRNA bulk and length, and ultimately male sterility characterized by a germ cell arrest at the elongating germ cell phase of spermatogenesis. HENMT1 loss-of-function, and the concomitant loss of piRNAs, resulted in TE de-repression in adult meiotic and haploid germ cells, and the precocious, and selective, expression of many haploid-transcripts in meiotic cells. Precocious expression was associated with a more active chromatin state in meiotic cells, elevated levels of DNA damage and a catastrophic deregulation of the haploid germ cell gene expression. Collectively these results define a critical role for HENMT1 and piRNAs in the maintenance of TE repression in adult germ cells and setting the spermatogenic program.
Assuntos
Infertilidade Masculina/genética , Metiltransferases/genética , Estabilidade de RNA/genética , RNA Interferente Pequeno/genética , Espermatogênese/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cromatina/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/crescimento & desenvolvimento , Humanos , Infertilidade Masculina/patologia , Masculino , CamundongosRESUMO
BACKGROUND: E74-like factor 5 (ELF5) is an epithelial-specific member of the E26 transforming sequence (ETS) transcription factor family and a critical regulator of cell fate in the placenta, pulmonary bronchi, and milk-producing alveoli of the mammary gland. ELF5 also plays key roles in malignancy, particularly in basal-like and endocrine-resistant forms of breast cancer. Almost all genes undergo alternative transcription or splicing, which increases the diversity of protein structure and function. Although ELF5 has multiple isoforms, this has not been considered in previous studies of ELF5 function. METHODS: RNA-sequencing data for 6757 samples from The Cancer Genome Atlas were analyzed to characterize ELF5 isoform expression in multiple normal tissues and cancers. Extensive in vitro analysis of ELF5 isoforms, including a 116-gene quantitative polymerase chain reaction panel, was performed in breast cancer cell lines. RESULTS: ELF5 isoform expression was found to be tissue-specific due to alternative promoter use but altered in multiple cancer types. The normal breast expressed one main isoform, while in breast cancer there were subtype-specific alterations in expression. Expression of other ETS factors was also significantly altered in breast cancer, with the basal-like subtype demonstrating a distinct ETS expression profile. In vitro inducible expression of the full-length isoforms 1 and 2, as well as isoform 3 (lacking the Pointed domain) had similar phenotypic and transcriptional effects. CONCLUSIONS: Alternative promoter use, conferring differential regulatory responses, is the main mechanism governing ELF5 action rather than differential transcriptional activity of the isoforms. This understanding of expression and function at the isoform level is a vital first step in realizing the potential of transcription factors such as ELF5 as prognostic markers or therapeutic targets in cancer.
Assuntos
Processamento Alternativo/genética , Proteínas de Ligação a DNA/genética , Neoplasias/genética , Isoformas de Proteínas/genética , Proteínas Proto-Oncogênicas c-ets/genética , Animais , Proteínas de Ligação a DNA/biossíntese , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Glândulas Mamárias Humanas/patologia , Neoplasias/patologia , Especificidade de Órgãos , Gravidez , Regiões Promotoras Genéticas , Isoformas de Proteínas/biossíntese , Proteínas Proto-Oncogênicas c-ets/biossíntese , Fatores de TranscriçãoRESUMO
BACKGROUND: Metastatic disease is largely resistant to therapy and accounts for almost all cancer deaths. Myeloid cell leukemia-1 (MCL-1) is an important regulator of cell survival and chemo-resistance in a wide range of malignancies, and thus its inhibition may prove to be therapeutically useful. METHODS: To examine whether targeting MCL-1 may provide an effective treatment for breast cancer, we constructed inducible models of BIMs2A expression (a specific MCL-1 inhibitor) in MDA-MB-468 (MDA-MB-468-2A) and MDA-MB-231 (MDA-MB-231-2A) cells. RESULTS: MCL-1 inhibition caused apoptosis of basal-like MDA-MB-468-2A cells grown as monolayers, and sensitized them to the BCL-2/BCL-XL inhibitor ABT-263, demonstrating that MCL-1 regulated cell survival. In MDA-MB-231-2A cells, grown in an organotypic model, induction of BIMs2A produced an almost complete suppression of invasion. Apoptosis was induced in such a small proportion of these cells that it could not account for the large decrease in invasion, suggesting that MCL-1 was operating via a previously undetected mechanism. MCL-1 antagonism also suppressed local invasion and distant metastasis to the lung in mouse mammary intraductal xenografts. Kinomic profiling revealed that MCL-1 antagonism modulated Src family kinases and their targets, which suggested that MCL-1 might act as an upstream modulator of invasion via this pathway. Inhibition of MCL-1 in combination with dasatinib suppressed invasion in 3D models of invasion and inhibited the establishment of tumors in vivo. CONCLUSION: These data provide the first evidence that MCL-1 drives breast cancer cell invasion and suggests that MCL-1 antagonists could be used alone or in combination with drugs targeting Src kinases such as dasatinib to suppress metastasis.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Dasatinibe/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Progesterone-RankL paracrine signaling has been proposed as a driver of stem cell expansion in the mammary gland, and Elf5 is essential for the differentiation of mammary epithelial progenitor cells. We demonstrate that Elf5 expression is induced by progesterone and that Elf5 and progesterone cooperate to promote alveolar development. The progesterone receptor and Elf5 are expressed in a mutually exclusive pattern, and we identify RankL as the paracrine mediator of the effects of progesterone on Elf5 expression in CD61+ progenitor cells and their consequent differentiation. Blockade of RankL action prevented progesterone-induced side branching and the expansion of Elf5(+) mature luminal cells. These findings describe a mechanism by which steroid hormones can produce the expansion of steroid hormone receptor-negative mammary epithelial cells.
Assuntos
Proteínas de Ligação a DNA/genética , Glândulas Mamárias Animais/efeitos dos fármacos , Progesterona/farmacologia , Ligante RANK/farmacologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Ligante RANK/metabolismo , Ligante RANK/fisiologia , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Receptores de Progesterona/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/metabolismo , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women's Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49f(hi) stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer.
Assuntos
Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/patologia , Progestinas/efeitos adversos , Ligante RANK/metabolismo , Animais , Apoptose/efeitos da radiação , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Feminino , Raios gama , Integrina alfa6/metabolismo , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Acetato de Medroxiprogesterona/administração & dosagem , Acetato de Medroxiprogesterona/efeitos adversos , Camundongos , NF-kappa B/metabolismo , Osteoclastos/citologia , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Progestinas/administração & dosagem , Ligante RANK/deficiência , Ligante RANK/genética , Receptor Ativador de Fator Nuclear kappa-B/deficiência , Receptor Ativador de Fator Nuclear kappa-B/genética , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismoRESUMO
Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.
Assuntos
Processamento Alternativo/genética , Diferenciação Celular/genética , Infertilidade Masculina/genética , Motivos de Nucleotídeos/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células Germinativas/patologia , Humanos , Infertilidade Masculina/patologia , Masculino , Camundongos , Modelos Moleculares , Mutação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Espermátides/metabolismo , Espermátides/patologia , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance.
Assuntos
Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estrogênios/farmacologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Animais , Sítios de Ligação , Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Adesão Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano/genética , Humanos , Camundongos , Modelos Biológicos , Fenótipo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Proteínas Proto-Oncogênicas c-ets/genética , Análise de Sequência de DNA , Fatores de Transcrição , Transcrição Gênica/efeitos dos fármacosRESUMO
Advances in the study of hematopoietic cell maturation have paved the way to a deeper understanding the stem and progenitor cellular hierarchy in the mammary gland. The mammary epithelium, unlike the hematopoietic cellular hierarchy, sits in a complex niche where communication between epithelial cells and signals from the systemic hormonal milieu, as well as from extra-cellular matrix, influence cell fate decisions and contribute to tissue homeostasis. We review the discovery, definition and regulation of the mammary cellular hierarchy and we describe the development of the concepts that have guided our investigations. We outline recent advances in in vivo lineage tracing that is now challenging many of our assumptions regarding the behavior of mammary stem cells, and we show how understanding these cellular lineages has altered our view of breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Humanas/citologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem da Célula , Feminino , Heterogeneidade Genética , Humanos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.
Assuntos
Infertilidade Masculina/genética , Infertilidade Masculina/metabolismo , Espermatozoides/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Fertilidade/genética , Expressão Gênica , Ordem dos Genes , Células Germinativas/metabolismo , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Fenótipo , Ligação Proteica , Transporte Proteico , Alinhamento de Sequência , Motilidade dos Espermatozoides/genética , Cauda do Espermatozoide/metabolismoRESUMO
INTRODUCTION: Although aberrant tyrosine kinase signalling characterises particular breast cancer subtypes, a global analysis of tyrosine phosphorylation in mouse models of breast cancer has not been undertaken to date. This may identify conserved oncogenic pathways and potential therapeutic targets. METHODS: We applied an immunoaffinity/mass spectrometry workflow to three mouse models: murine stem cell virus-Neu, expressing truncated Neu, the rat orthologue of human epidermal growth factor receptor 2, Her2 (HER2); mouse mammary tumour virus-polyoma virus middle T antigen (PyMT); and the p53-/- transplant model (p53). Pathways and protein-protein interaction networks were identified by bioinformatics analysis. Molecular mechanisms underpinning differences in tyrosine phosphorylation were characterised by Western blot analysis and array comparative genomic hybridisation. The functional role of mesenchymal-epithelial transition factor (Met) in a subset of p53-null tumours was interrogated using a selective tyrosine kinase inhibitor (TKI), small interfering RNA (siRNA)-mediated knockdown and cell proliferation assays. RESULTS: The three models could be distinguished on the basis of tyrosine phosphorylation signatures and signalling networks. HER2 tumours exhibited a protein-protein interaction network centred on avian erythroblastic leukaemia viral oncogene homologue 2 (Erbb2), epidermal growth factor receptor and platelet-derived growth factor receptor α, and they displayed enhanced tyrosine phosphorylation of ERBB receptor feedback inhibitor 1. In contrast, the PyMT network displayed significant enrichment for components of the phosphatidylinositol 3-kinase signalling pathway, whereas p53 tumours exhibited increased tyrosine phosphorylation of Met and components or regulators of the cytoskeleton and shared signalling network characteristics with basal and claudin-low breast cancer cells. A subset of p53 tumours displayed markedly elevated cellular tyrosine phosphorylation and Met expression, as well as Met gene amplification. Treatment of cultured p53-null cells exhibiting Met amplification with a selective Met TKI abrogated aberrant tyrosine phosphorylation and blocked cell proliferation. The effects on proliferation were recapitulated when Met was knocked down using siRNA. Additional subtypes of p53 tumours exhibited increased tyrosine phosphorylation of other oncogenes, including Peak1/SgK269 and Prex2. CONCLUSION: This study provides network-level insights into signalling in the breast cancer models utilised and demonstrates that comparative phosphoproteomics can identify conserved oncogenic signalling pathways. The Met-amplified, p53-null tumours provide a new preclinical model for a subset of triple-negative breast cancers.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Proteoma/metabolismo , Animais , Feminino , Dosagem de Genes , Humanos , Indóis/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Oncogenes , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Sulfonas/farmacologiaRESUMO
PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminished PRL-induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE-A in PRL signalling in vivo, we generated PIKE knockout (PIKE-/-) mice. PIKE-/- mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE-/- mice. The defective mammary gland development in PIKE-/- mice was rescued by overexpression of a mammary-specific cyclin D1 transgene. These data establish a critical function for PIKE-A in mediating PRL functions.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Prolactina/farmacologia , Fator de Transcrição STAT5/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Linhagem Celular , Proliferação de Células , Ciclina D1/metabolismo , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Feminino , GTP Fosfo-Hidrolases/genética , Expressão Gênica , Genótipo , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Lactação/genética , Lactação/fisiologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Gravidez , Ligação Proteica/efeitos dos fármacos , Receptores da Prolactina/metabolismoRESUMO
Grb10 is an intracellular adaptor protein that acts as a negative regulator of insulin and insulin-like growth factor 1 (IGF1) receptors. Since global deletion of Grb10 in mice causes hypermuscularity, we have characterized the skeletal muscle physiology underlying this phenotype. Compared to wild-type (WT) controls, adult mice deficient in Grb10 have elevated body mass and muscle mass throughout adulthood, up to 12 mo of age. The muscle enlargement is not due to increased myofiber size, but rather an increase in myofiber number (142% of WT, P<0.01). There is no change in myofiber type proportions between WT and Grb10-deficient muscles, nor are the metabolic properties of the muscles altered on Grb10 deletion. Notably, the weight and cross-sectional area of hindlimbs from neonatal mice are increased in Grb10-deficient animals (198 and 137% of WT, respectively, both P<0.001). Functional gene signatures for myogenic signaling and proliferation are up-regulated in Grb10-deficient neonatal muscle. Our findings indicate that Grb10 plays a previously unrecognized role in regulating the development of fiber number during murine embryonic growth. In addition, Grb10-ablated muscle from adult mice shows coordinate gene changes that oppose those of muscle wasting pathologies, highlighting Grb10 as a potential therapeutic target for these conditions.
Assuntos
Proteína Adaptadora GRB10/fisiologia , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/crescimento & desenvolvimento , Animais , Imunofluorescência , Proteína Adaptadora GRB10/genética , Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismoRESUMO
Recent characterization of mammary stem and progenitor cells has improved our understanding of the transcriptional network that coordinates mammary development; however, little is known about the mechanisms that enforce lineage commitment and prevent transdifferentiation in the mammary gland. The E-twenty six transcription factor Elf5 forces the differentiation of mammary luminal progenitor cells to establish the milk producing alveolar lineage. Methylation of the Elf5 promoter has been proposed to act as a lineage gatekeeper during embryonic development. We used bisulphite sequencing to investigate in detail whether Elf5 promoter methylation plays a role in lineage commitment during mammary development. An increase in Elf5 expression was associated with decreasing Elf5 promoter methylation in differentiating HC11 mammary cells. Similarly, purified mammary epithelial cells from mice had increased Elf5 expression and decreased promoter methylation during pregnancy. Finally, analysis of epithelial subpopulations revealed that the Elf5 promoter is methylated and silenced in the basal, stem cell-containing population relative to luminal cells. These results demonstrate that Elf5 promoter methylation is lineage-specific and developmentally regulated in the mammary gland in vivo, and suggest that loss of Elf5 methylation specifies the mammary luminal lineage, while continued Elf5 methylation maintains the stem cell and myoepithelial lineages.