RESUMO
BACKGROUND: Hair follicle (HF) regeneration begins when signals from the mesenchyme-derived dermal papilla cells (DPC) reach multipotent epidermal stem cells in the bulge region. Wnt/ß-catenin signalling is known to affect mammalian hair growth positively. In androgenetic alopecia (AGA), androgens cause HF miniaturization through a mechanism that remains unclear. Circulating androgens act on DPC and alter paracrine factors that influence hair epithelial cells. OBJECTIVES: To elucidate the role of androgens in dermal papilla-induced differentiation of HF stem cells. METHODS: HF stem cell differentiation was evaluated in a coculture model with DPC or culturing with media conditioned by DPC after activation of androgen and Wnt/ß-catenin signalling pathways. To study the molecular cross-talk between the androgen and Wnt signalling pathway in DPC, we analysed the expression and activation of downstream Wnt signalling molecules in the presence of androgens. RESULTS: In a coculture model with human DPC from patients with AGA and HF stem cells, we observed that androgens abrogate hair differentiation evaluated by hair-specific keratin 6 expression. Wnt signalling activation restored the ability of androgen-treated DPC to induce differentiation. Androgen treatment revealed a significant decrease in the cytoplasmic/total ß-catenin protein ratio and upregulation of the activity of glycogen synthase kinase-3ß in DPC, indicative of canonical Wnt pathway inhibition. CONCLUSIONS: These results suggest that androgens deregulate DPC-secreted factors involved in normal HF stem cell differentiation via the inhibition of the canonical Wnt signalling pathway.
Assuntos
Alopecia/patologia , Androgênios/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco/patologia , Via de Sinalização Wnt/fisiologia , Androgênios/farmacologia , Células Cultivadas , DNA Complementar/biossíntese , Derme/patologia , Di-Hidrotestosterona/farmacologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Folículo Piloso/patologia , Humanos , Queratinas Específicas do Cabelo/metabolismo , Queratinas Tipo II/metabolismo , Cloreto de Lítio/farmacologia , Masculino , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/fisiologia , Couro Cabeludo/metabolismo , TransfecçãoRESUMO
The cancer stem cell (CSC) hypothesis, predicts that a small subpopulation of cancer cells that possess "stem-like" characteristics, are responsible for initiating and maintaining cancer growth. According to the CSC model the many cell populations found in a tumour might represent diverse stages of differentiation. From the cellular point of view metastasis is considered a highly inefficient process and only a subset of tumour cells is capable of successfully traversing the entire metastatic cascade and eventually re-initiates tumour growth at distant sites. Some similar features of both normal and malignant stem cells suggest that CSCs are not only responsible for tumorigenesis, but also for metastases. The CSC theory proposes that the ability of a tumour to metastasize is an inherent property of a subset of CSCs. The similar biological characteristics shared by normal stem cells (NSCs) and CSCs mainly implicate self-renewal and differentiation potential, survival ability, niche-specific microenvironment requirements and specific homing to metastatic sites and may have important implications in terms of new approaches to cancer therapy in the metastatic setting. There are several agents targeting many of these CSC features that have shown to be effective both in vitro and in vivo. Although clinical trials results are still preliminary and continue under investigation, these new therapies are very promising. The identification of new therapeutic targets and drugs based on CSC model constitutes a great challenge.
Assuntos
Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Células-Tronco Neoplásicas , Animais , Apoptose , Diferenciação Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Humanos , Metástase Neoplásica , Neoplasias/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Especificidade de Órgãos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Most drugs used for treatment of androgen-related dermatological disorders are not completely satisfactory in terms of clinical efficacy and potential secondary effects. There is, therefore, a need for a new generation of specific antiandrogens. This paper focuses on an oligonucleotide antisense pharmacological strategy. Acceptor sites were first disclosed by mapping the human Androgen Receptor (AR) mRNA conformation using an mRNA walking approach, oligonucleotide binding, and S1 protection assays. Antisense-sensitive regions were localized by RNAse H degradation and AR in vitro translation inhibition. Oligonucleotides were then designed and assessed, in primary cultures of human hair dermal papillae and skin derived fibroblasts, for their capability to down-regulate AR expression. Some of them were able to inhibit more than 60 to 80% of the AR expression. These could be a new class of antiandrogen oligonucleotides pharmacologically active in hair and skin derived cells, suitable for the treatment of dermatological disorders.
Assuntos
Antagonistas de Androgênios/farmacologia , Cabelo/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Pele/metabolismo , Antagonistas de Receptores de Andrógenos , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica/efeitos dos fármacos , Cabelo/citologia , Humanos , Immunoblotting , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Pele/citologia , Transcrição Gênica/genéticaRESUMO
The present study focused on interactions between signaling pathways activated by progestins and by type I and II receptor tyrosine kinases (RTKs) in mammary tumors. An experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in Balb/c mice was used. MPA-stimulated proliferation, both in vivo and in vitro, of progestin-dependent tumors induced up-regulation of ErbB-2 protein levels and tyrosine phosphorylation of this receptor. Combinations of antisense oligodeoxynucleotides (ASODNs) directed to ErbB-2 mRNA with ASODNs directed to the insulin-like growth factor-I receptor (IGF-IR) were used to study the effect of the simultaneous block of these receptors on the MPA-induced proliferation of epithelial cells from the progestin-dependent C4HD line. Neither synergistic nor additive effects on the inhibition of MPA-induced proliferation of C4HD cells were observed as a result of the combination of these ASODNs. Suppression of IGF-IR expression by ASODNs resulted in complete abrogation of MPA-induced phosphorylation of ErbB-2 in C4HD cells, whereas blockage of ErbB-2 did not affect IGF-IR phosphorylation. These results show the existence of a hierarchical interaction between IGF-IR and ErbB-2, by means of which IGF-IR directs ErbB-2 phosphorylation. We demonstrated, for the first time, that this hierarchical interaction involves physical association of both receptors, resulting in the formation of a heteromeric complex. Furthermore, confocal laser microscopy experiments demonstrated that MPA was able to induce co-localization of ErbB-2 and IGF-IR. This hetero-oligomer was also found in MCF-7 human breast cancer cells in which association of IGF-IR and ErbB-2 was induced by heregulin and IGF-I. Oncogene (2001) 20, 34 - 47.
Assuntos
Neoplasias Mamárias Experimentais/metabolismo , Receptor ErbB-2/metabolismo , Receptor IGF Tipo 1/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Substâncias Macromoleculares , Neoplasias Mamárias Experimentais/enzimologia , Acetato de Medroxiprogesterona/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Fosforilação/efeitos dos fármacos , Congêneres da Progesterona/farmacologia , Receptor Cross-Talk/efeitos dos fármacos , Receptor ErbB-2/antagonistas & inibidores , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/biossíntese , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Tirosina/antagonistas & inibidores , Tirosina/metabolismoRESUMO
The present study addressed links between progestin and heregulin (HRG) signaling pathways in mammary tumors. An experimental model of hormonal carcinogenesis, in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in female Balb/c mice, was used. MPA induced an in vivo up-regulation of HRG mRNA expression in progestin-dependent (HD) tumor lines. Mammary tumor progression to a progestin-independent (HI) phenotype was accompanied by a high constitutive expression of HRG. The HRG message arose from the tumor epithelial cells. Primary cultures of malignant epithelial cells from a HD tumor line were used to investigate HRG involvement on cell proliferation. HRG induced a potent proliferative effect on these cells and potentiated MPA mitogenic effects. Blocking endogenous HRG synthesis by antisense oligodeoxynucleotides (ASODNs) to HRG mRNA inhibited MPA-induced cell growth, indicating that HRG acts as a mediator of MPA-induced growth. High levels of ErbB-2 and ErbB-3 expression and low ErbB-4 levels were found in HD cells. Treatment of these cells with either MPA or HRG resulted in tyrosine phosphorylation of both ErbB-2 and ErbB-3. Furthermore, both HRG and MPA proliferative effects were abolished when cells were treated with ASODNs to ErbB-2 mRNA, providing evidence for a critical role of ErbB-2 in HRG-induced growth. Finally, blocking type I insulin-like growth factor receptor (IGF-IR) expression with ASODN resulted in the complete inhibition of HRG proliferative effect, demonstrating that a functional IGF-IR is required for HRG mitogenic activity. These results provide the first evidence of interactions between progestins and HRB/ErbB signal transduction pathways in mammary cancer and the first demonstration that IGF-IR is required for HRG proliferative effects.
Assuntos
Adenocarcinoma/genética , Carcinógenos/toxicidade , Neoplasias Mamárias Experimentais/genética , Acetato de Medroxiprogesterona/toxicidade , Proteínas de Neoplasias/fisiologia , Neoplasias Hormônio-Dependentes/genética , Neuregulina-1/fisiologia , Progestinas , Receptor IGF Tipo 1/fisiologia , Transdução de Sinais/efeitos dos fármacos , Adenocarcinoma/induzido quimicamente , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , DNA Antissenso/farmacologia , Feminino , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias Mamárias Experimentais/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Neoplasias Hormônio-Dependentes/induzido quimicamente , Neuregulina-1/genética , RNA Mensageiro/antagonistas & inibidores , RNA Neoplásico/antagonistas & inibidores , Receptor ErbB-2/biossíntese , Receptor ErbB-2/genética , Receptor ErbB-2/fisiologia , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
The role of the insulin-like growth factors (IGFs) system was investigated in hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. IGF-II protein and message showed a three- to four-fold increase in HD lines growing in MPA-treated mice, as compared with HD tumors growing in untreated mice. Progression to a hormone-independent phenotype in all these lines was accompanied by a high constitutive expression of IGF-II. Similar IGF-I mRNA levels were detected in HD and HI lines. Both IGF-I and -II messages arose from the malignant epithelial cells, as shown by in situ hybridization studies. A significant decrease in Man-6P/type II IGF-R content was detected in HD tumors growing in MPA-treated mice as compared with HD lines growing in untreated mice. On the other hand, in HI tumors, notwithstanding high IGF-II synthesis, the levels of Man-6P/type II IGF-R remain high. Competitive inhibition and affinity labeling studies showed an almost exclusive binding of IGF-II to Man-6P/type II IGF-R on tumor membranes. The involvement of IGFs in the growth of epithelial primary cultures of the C4-HD line was evaluated. Exogenous IGF-I potentiated MPA stimulatory effect at concentrations of 50-100 ng/ml. Treatment of C4-HD cells with antisense oligodeoxynucleotides (ASODNs) to type I IGF-R and to IGF-II RNA resulted in a dose-dependent inhibition of MPA-mediated cell proliferation. The inhibition caused by IGF-II ASODNs could not be overcome by the addition of IGF-II up to 150 ng/ml. ASODNs to type I IGF-R at 40 microg/ml reduced by 75% the number of type I IGF-R; ASODNs to IGF-II at 1 microM decreased by 83% the levels of IGF-II protein. Our results provide support for the involvement of IGF-I and -II in MPA-induced mammary tumor growth by autocrine pathways.
Assuntos
Fator de Crescimento Insulin-Like II/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Neoplasias Mamárias Experimentais/patologia , Acetato de Medroxiprogesterona/farmacologia , Receptor IGF Tipo 1/fisiologia , Receptor IGF Tipo 2/fisiologia , Adenocarcinoma/patologia , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Feminino , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Oligonucleotídeos Antissenso/farmacologia , Células Tumorais CultivadasRESUMO
We have studied the involvement of growth factors (GF), their receptors (GF-R) and oncogenes in modulating tumor growth in the medroxyprogesterone acetate (MPA)-induced mammary tumor model in BALB/c mice. We demonstrated the presence of both ligands of the insulin-like growth factor family (IGF-I, IGF-II) and the two types of receptors (IGF-RI, IGF-RII). MPA upregulated IGF-II mRNA and protein levels in hormone-dependent lines (MPA-D). The progression to a hormone-independent phenotype was accompanied by a high constitutive expression of IGF-II and by a significant decrease in IGF-IIR number. An antisense strategy used to evaluate the role of IGF in the MPA-induced growth of epithelial MPA-D cells showed that IGF mediate progestin-induced mammary tumor growth by autocrine/intracrine pathways. We also studied the role of heregulins (HRG), the recently identified ligands for the c-erbB3 and c-erbB4 oncogenes. HRG mRNA expression was restricted to tumors of ductal origin. MPA induced an in vivo up-regulation of HRG expression. Finally, we also found that MPA may be exerting its proliferative effect on MPA-D lines by inhibiting the expression of transforming growth factor beta 1, (TGF-beta 1) and the lack of expression of TGF-beta 1 in hormone-independent tumors may be related to the acquisition of autonomous growth.