RESUMO
MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK-/- mice. Transplantation of MerTK-/- bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b+ cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK-/- leukocytes exhibited lower tumor cell-induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8+ T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK-/- mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8+ T lymphocyte depletion restored tumor growth in MerTK-/- mice. These data demonstrate that MerTK signaling in tumor-associated CD11b+ leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.
Assuntos
Neoplasias do Colo/enzimologia , Leucócitos/enzimologia , Neoplasias Mamárias Experimentais/enzimologia , Melanoma Experimental/enzimologia , Animais , Linfócitos T CD8-Positivos/enzimologia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Citocinas/genética , Citocinas/metabolismo , Resistência à Doença/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Masculino , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transplante de Neoplasias , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Transcriptoma , Carga Tumoral , Microambiente Tumoral , c-Mer Tirosina QuinaseRESUMO
BACKGROUND: Mammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs) to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis. RESULTS: ACs induced MerTK expression in MECs, resulting in elevated MerTK levels at the earliest stages of involution. Loss of MerTK resulted in AC accumulation in post-lactational MerTK-deficient mammary glands, but not in Axl and Tyro3-deficient mammary glands. Increased vascularization, fibrosis, and epithelial hyperproliferation were observed in MerTK-deficient mammary glands through at least 60 days post-weaning, due to failed efferocytosis after lactation, but did not manifest in nulliparous mice. WT host-derived macrophages failed to rescue efferocytosis in transplanted MerTK-deficient mammary epithelium. CONCLUSION: Efferocytosis by MECs through MerTK is crucial for mammary gland homeostasis and function during the post-lactational period. Efferocytosis by MECs thus limits pathologic consequences associated with the apoptotic load following lactation.
Assuntos
Apoptose , Células Epiteliais/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/metabolismo , Fagocitose , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Feminino , Homeostase , Lactação , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
Data concerning the prognostic value of ErbB4 in breast cancer and effects on cell growth have varied in published reports, perhaps due to the unknown signaling consequences of expression of the intracellular proteolytic ErbB4 s80(HER4) fragment or due to differing signaling capabilities of alternatively spliced ErbB4 isoforms. One isoform (Cyt1) contains a 16-residue intracellular sequence that is absent from the other (Cyt2). We expressed s80(Cyt1) and s80(Cyt2) in HC11 mammary epithelial cells, finding diametrically opposed effects on the growth and organization of colonies in three-dimensional matrices. Whereas expression of s80(Cyt1) decreased growth and increased the rate of three-dimensional lumen formation, that of s80(Cyt2) increased proliferation without promoting lumen formation. These results were recapitulated in vivo, using doxycycline-inducible, mouse breast-transgenic expression of s80(Cyt1) amd s80(Cyt2). Expression of s80(Cyt1) decreased growth of the mammary ductal epithelium, caused precocious STAT5a activation and lactogenic differentiation, and increased cell surface E-cadherin levels. Remarkably, ductal growth inhibition by s80(Cyt1) occurred simultaneously with lobuloalveolar growth that was unimpeded by s80(Cyt1), suggesting that the response to ErbB4 may be influenced by the epithelial subtype. In contrast, expression of s80(Cyt2) caused epithelial hyperplasia, increased Wnt and nuclear beta-catenin expression, and elevated expression of c-myc and cyclin D1 in the mammary epithelium. These results demonstrate that the Cyt1 and Cyt2 ErbB4 isoforms, differing by only 16 amino acids, exhibit markedly opposing effects on mammary epithelium growth and differentiation.
Assuntos
Processamento Alternativo/genética , Aminoácidos/metabolismo , Epitélio/metabolismo , Receptores ErbB/metabolismo , Glândulas Mamárias Animais/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células , Receptores ErbB/química , Feminino , Regulação da Expressão Gênica , Humanos , Glândulas Mamárias Animais/citologia , Camundongos , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Fosforilação , Gravidez , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Puberdade/metabolismo , Receptor ErbB-4 , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , beta Catenina/metabolismoRESUMO
The ErbB receptor tyrosine kinase family has often been associated with increased growth of breast epithelial cells, as well as malignant transformation and progression. In contrast, ErbB4/HER4 exhibits unique attributes from a two step proteolytic cleavage which releases an 80 kilodalton, nuclear localizing, tyrosine kinase to a signal transduction mechanism that slows growth and stimulates differentiation of breast cells. This review provides an overview of ErbB4/HER4 in growth and differentiation of the mammary epithelium, including its physiologic role in development, the contrasting growth inhibition/tumor suppression and growth acceleration of distinct ErbB4/HER4 isoforms and a description of the unique cell cycle regulated pattern of nuclear HER4 ubiquitination and destruction.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Receptores ErbB/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Animais , Neoplasias da Mama/fisiopatologia , Diferenciação Celular , Feminino , Inibidores do Crescimento/metabolismo , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/metabolismo , Camundongos , Isoformas de Proteínas , Receptor ErbB-4 , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Heregulin-mediated activation of HER4 initiates receptor cleavage (releasing an 80-kDa HER4 intracellular domain, s80(HER4), containing nuclear localization sequences) and results in G(2)-M delay by unknown signaling mechanisms. We report herein that s80(HER4) contains a functional cyclin B-like sequence known as a D-box, which targets proteins for degradation by anaphase-promoting complex (APC)/cyclosome, a multisubunit ubiquitin ligase. s80(HER4) ubiquitination and proteasomal degradation occurred during mitosis but not during S phase. Inhibition of an APC subunit (APC2) using short interfering RNA knockdown impaired s80(HER4) degradation. Mutation of the s80(HER4) D-box sequence stabilized s80(HER4) during mitosis, and s80(HER4)-dependent growth inhibition via G(2)-M delay was significantly greater with the D-box mutant. Polyomavirus middle T antigen-transformed HC11 cells expressing s80(HER4) resulted in smaller, less proliferative, more differentiated tumors in vivo than those expressing kinase-dead s80(HER4) or the empty vector. Cells expressing s80(HER4) with a disrupted D-box did not form tumors, instead forming differentiated ductal structures. These results suggest that cell cycle-dependent degradation of s80(HER4) limits its growth-inhibitory action, and stabilization of s80(HER4) enhances tumor suppression, thus providing a link between HER4-mediated growth inhibition and cell cycle control.
Assuntos
Núcleo Celular/metabolismo , Receptores ErbB/fisiologia , Mitose , Neuregulina-1/metabolismo , Motivos de Aminoácidos , Antígenos Transformantes de Poliomavirus/metabolismo , Divisão Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Fase G2 , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Estrutura Terciária de Proteína , Receptor ErbB-4 , Ubiquitina/metabolismoRESUMO
HER4 expression in human breast cancers correlates with a positive prognosis. While heregulin inhibits the growth of HER4-positive breast cancer cells, it does so by undefined mechanisms. We demonstrate that heregulin-induced HER4 activity inhibits cell proliferation and delays G(2)/M progression of breast cancer cells. While investigating pathways of G(2)/M delay, we noted that heregulin increased the expression of BRCA1 in a HER4-dependent, HER2-independent manner. Induction of BRCA1 by HER4 occurred independently of the cell cycle. Moreover, BRCA1 expression was elevated in HER4-postive human breast cancer specimens. Heregulin stimulated c-Jun N-terminal kinase (JNK), and pharmacologic inhibition of JNK impaired heregulin-enhanced expression of BRCA1 and mitotic delay; inhibition of Erk1/2 did not. Knockdown of BRCA1 with small interfering RNA in a human breast cancer cell line interfered with HER4-mediated mitotic delay. Heregulin/HER4-dependent mitotic delay was examined further with an isogenic pair of mouse mammary epithelial cells (MECs) derived from mice harboring homozygous LoxP sites flanking exon 11 of BRCA1, such that one cell line expressed BRCA1 while the other cell line, after Cre-mediated excision, did not. BRCA1-positive MECs displayed heregulin-dependent mitotic delay; however, the isogenic BRCA1-negative MECs did not. These results suggest that heregulin-mediated growth inhibition in HER4-postive breast cancer cells requires BRCA1.
Assuntos
Proteína BRCA1/metabolismo , Receptores ErbB/metabolismo , Mitose/efeitos dos fármacos , Neuregulina-1/farmacologia , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Neoplasias da Mama/patologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Receptores ErbB/genética , Éxons/genética , Fase G2/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Glândulas Mamárias Animais/citologia , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-4 , Células Tumorais CultivadasRESUMO
Mice heterozygous for the N-ethyl-N-nitrosourea-induced Waved-5 (Wa5) mutation, isolated in a screen for dominant, visible mutations, exhibit a wavy coat similar to mice homozygous for the recessive Tgfa wa1 or Egfr wa2 alleles. In this study, we show that Wa5 is a new allele of Egfr (Egfr Wa5) containing a missense mutation within the coding region for the highly conserved DFG motif of the tyrosine kinase domain. In vivo analysis of placental development, modification of Apc Min tumorigenesis, and levels of EGF-dependent EGFR phosphorylation demonstrates that Egfr Wa5 functions as an antimorphic allele, recapitulating many abnormalities associated with reduced EGFR activity. Furthermore, Egfr wa5 enhances Egfr Wa2 compound or Tgfa tm1Dcl double mutants exposing additional EGFR-dependent phenotypes. In vitro characterization shows that the antimorphic property of Egfr Wa5 is caused by a kinase-dead receptor acting as a dominant negative.
Assuntos
Alelos , Receptores ErbB/genética , Alquilantes/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Cricetinae , Análise Mutacional de DNA , Etilnitrosoureia , Genes Dominantes , Teste de Complementação Genética , Genótipo , Heterozigoto , Homozigoto , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Mutagênicos , Mutação , Mutação de Sentido Incorreto , Fenótipo , Fosforilação , Placenta/metabolismo , Placenta/patologia , Mutação Puntual , Estrutura Secundária de Proteína , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The timing of lethality caused by homozygosity for a null allele of the epidermal growth factor receptor (Egfrtm1Mag) in mice is strongly dependent on genetic background. Initial attempts to genetically map background modifiers using Swiss-derived, outbred CD-1 mice were unsuccessful. To investigate the genetic architecture contributing to survival of Egfrtm1Mag homozygous embryos, the genetic variability segregating within the outbred population was partitioned by surveying viability of Egfrtm1Mag mutants using intercrosses between 129S6/SvEvTAC-Egfrtm1Mag and nine Swiss-derived, inbred strains: ALR/LtJ, ALS/LtJ, APN, APS, ICR/HaRos, NOD/LtJ, NON/LtJ, SJL/J, and SWR/J. The observations showed that these strains support varying levels of survival of Egfrtm1Mag homozygous embryos, suggesting that genetic heterogeneity within the CD-1 stock contributed to the original lack of Egfrtm1Mag modifier detection. Similar to the Swiss-derived intercrosses, nine congenic strains, derived from 129S6/SvEvTAC, AKR/J, APN, BALB/cJ, BTBR-T+ tf/tf, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ inbred backgrounds, also supported varying levels of survival of Egfrtm1Mag mutants. By intercrossing the congenic lines to create hybrid F1 embryos, different genetic backgrounds were found to have complementary modifiers. Analysis of the congenic lines argues against heterosis of outbred backgrounds contributing to Egfrtm1Mag phenotypic variability. A detailed analysis of the crosses suggests that modifiers function at three distinct stages of development. One class of modifiers supports survival of Egfrtm1Mag homozygous embryos to mid-gestation, another class supports development through the mid-gestation transition from yolk-sac to placental-derived nutrient sources, and a third class supports survival through later stages of gestation. Data from microarray analysis using RNA from wild-type and Egfrtm1Mag mutant placentas support the existence of extensive genetic heterogeneity and suggest that it can be molecularly partitioned. This method should be generally useful to partition heterogeneity contributing to other complex traits.