RESUMO
Purpose: Insulin-like growth factor 1 (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (CDH1) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R/insulin receptor (InsR) therapies in breast cancer.Experimental Design: Breast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R/InsR therapies. In situ proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data were used to compare IGF1R/InsR activation in estrogen receptor-positive (ER+) invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) tumors. ER+ ILC cell lines and xenograft tumor explant cultures were used to evaluate efficacy to IGF1R pathway inhibition in combination with endocrine therapy.Results: Diminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R/InsR therapies. PLA demonstrated a direct endogenous interaction between IGF1R and E-cadherin at points of cell-cell contact. Increased expression of IGF1 ligand and levels of IGF1R/InsR phosphorylation were observed in E-cadherin-deficient ER+ ILC compared with IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R/InsR inhibition reduced proliferation in ILC tumor explant culture.Conclusions: We provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R/InsR targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin-deficient breast cancers. Clin Cancer Res; 24(20); 5165-77. ©2018 AACR.
Assuntos
Antineoplásicos/farmacologia , Caderinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fator de Crescimento Insulin-Like I/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Camundongos , RNA Interferente Pequeno/genética , Receptor IGF Tipo 1 , Receptores de Somatomedina/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer development and progression are influenced by insulin-like growth factor receptor 1 (IGF1R) and insulin receptor (InsR) signaling, which drive cancer phenotypes such as cell growth, proliferation, and migration. IGF1R and InsR form IGF1R/InsR hybrid receptors (HybRs) consisting of one molecule of IGF1R and one molecule of InsR. The specific signaling and functions of HybR are largely unknown, as HybR is activated by both IGF1 and insulin, and no cellular system expresses HybR in the absence of holo-IGF1R or holo-InsR. Here we studied the role of HybR by constructing inducible chimeric receptors and compared HybR signaling with that of holo-IGF1R and holo-InsR. We cloned chemically inducible chimeric IGF1R and InsR constructs consisting of the extracellular domains of the p75 nerve growth factor receptor fused to the intracellular ß subunit of IGF1R or InsR and a dimerization domain. Dimerization with the drugs AP20187 or AP21967 allowed specific and independent activation of holo-IGF1R, holo-InsR, or HybR, resulting in activation of the PI3K pathway. Holo-IGF1R and HybR both promoted cell proliferation and glucose uptake, whereas holo-InsR only promoted glucose uptake, and only holo-IGF1R showed anti-apoptotic effects. We also found that the three receptors differentially regulated gene expression: holo-IGF1R and HybR up-regulated EGR3; holo-InsR specifically down-regulated JUN and BCL2L1; holo-InsR down-regulated but HybR up-regulated HK2; and HybR specifically up-regulated FHL2, ITGA6, and PCK2. Our findings suggest that, when expressed and activated in mammary epithelial cells, HybR acts in a manner similar to IGF1R and support further investigation of the role of HybR in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Glândulas Mamárias Humanas/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Receptor de Insulina/metabolismo , Receptores de Somatomedina/metabolismo , Animais , Neoplasias da Mama/patologia , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Indicadores e Reagentes/farmacologia , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Células MCF-7 , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/patologia , Camundongos , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica/efeitos dos fármacos , Receptor de Insulina/agonistas , Receptor de Insulina/química , Receptor de Insulina/genética , Receptores de Somatomedina/agonistas , Receptores de Somatomedina/química , Receptores de Somatomedina/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Tacrolimo/análogos & derivados , Tacrolimo/farmacologiaRESUMO
Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. The two top candidates, chosen for experimental validation, were shown to regulate IGF1/insulin induced phosphorylation events. First, acetyl-CoA carboxylase (ACC) knock-down was shown to increase the level of mitogen-activated protein kinase (MAPK) phosphorylation. Second, stable knock-down of E-Cadherin increased the phospho-Akt protein levels. Both of the knock-down perturbations incurred phosphorylation responses stronger in IGF1 stimulated cells compared with insulin. Overall, the time-translation modeling coupled to wet-lab experiments has proven to be powerful in inferring differential interactions downstream of IGF1 and insulin signaling, in vitro.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Insulina/farmacologia , Proteômica/métodos , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Células MCF-7 , Análise de Regressão , Transdução de Sinais/efeitos dos fármacosRESUMO
Increased AMP-activated protein kinase (AMPK) activity leads to enhanced fatty acid utilization, while also promoting increased ubiquitin-dependent proteolysis (UDP) in mammalian skeletal muscle. ß-guanidinopropionic acid (ßGPA) is a commercially available dietary supplement that has been shown to promote an AMPK-dependent increase in fatty acid utilization and aerobic capacity in mammals by compromising creatine kinase function. However, it remains unknown if continuous ßGPA supplementation can negatively impact skeletal muscle growth in a rapidly growing juvenile. The current study was conducted to examine the effect of ßGPA supplementation on whole-body and skeletal muscle growth in juvenile and young adult mice. Three-week old, post weanling CD-1 mice were fed a standard rodent chow that was supplemented with either 2% (w/w) α-cellulose (control) or ßGPA. Control and ßGPA-fed mice (n = 6) were sampled after 2, 4, and 8 weeks. Whole-body and hindlimb muscle masses were significantly (P < 0.05) reduced in ßGPA-fed mice by 2 weeks. The level of AMPK (T172) phosphorylation increased significantly (P < 0.05) in the gastrocnemius of ßGPA-fed versus control mice at 2 weeks, but was not significantly different at the 4- and 8-week time points. Further analysis revealed a significant (P < 0.05) increase in the skeletal muscle-specific ubiquitin ligase MAFbx/Atrogin-1 protein and total protein ubiquitination in the gastrocnemius of ßGPA versus control mice at the 8-week time point. Our data indicate that feeding juvenile mice a ßGPA-supplemented diet significantly reduced whole-body and skeletal muscle growth that was due, at least in part, to an AMPK-independent increase in UDP.