RESUMO
BACKGROUND: Autophagy is a highly conserved process for maintaining cellular homeostasis. Upregulation of autophagy promotes metastasis by promoting the cancer stem cell state while also stimulating tumor cell migration and invasion. We hypothesized that autophagy upregulation would be critical for cancer stem cell maintenance as well as cellular migration and invasion in thyroid cancer. METHODS: Validated papillary (MDA-T32, MDA-T68), follicular (FTC-133), and anaplastic (ATC-8505c) human thyroid cancer cell lines in culture were first assessed for autophagic capacity after bafilomycin clamping. Cancer stem cells were quantified by flow cytometry for aldehyde dehydrogenase and thyrosphere formation assay. Scratch migration and Matrigel invasion assays were performed in the presence of known autophagy inhibitors: Lys05, chloroquine, and FIP200siRNA. RESULTS: Autophagy activity was observed across all cell lines. Thyrosphere formation, aldehyde dehydrogenase activity, and CD44 expression were reduced with inhibition of autophagy in MDA-T32, MDA-T68, FTC-133, and 8505c cells. Similarly, cell migration and invasion were attenuated: 42% (FIP200siRNA), 78% (Lys05), P < .001 in MDA-T32 cells; 54% (FIP200siRNA), 67% (Lys05), P < .001 in MDA-T68 cells; 73% (FIP200siRNA), 71% (Lys05), P < .001) in FTC-133 cells; and 60% (FIP200siRNA), 90% (Lys05), P < .001 in 8505c cells. Invasion assays demonstrated a 73%, 39%, 75%, and 65.1% reduction in the presence of Lys05 in T32, T68, FTC-133, and 8505c cells, respectively. We observed similar reductions in invasion with FIP200siRNA: 61%, 62%, 55%, and 81.4% in T32, T68, FTC-133, and 8505c cells. CONCLUSION: Autophagy is upregulated across multiple thyroid cancer subtypes. In thyroid cancer cell lines, inhibition of autophagy attenuates cancer stem cell viability, cell migration, and invasion suggesting a role for autophagy in the progression of thyroid cancer. Greater understanding of autophagy regulation in thyroid cancer will aid in developing targeted therapeutics.
Assuntos
Autofagia/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Cloroquina/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Linhagem Celular Tumoral , Cloroquina/uso terapêutico , Humanos , Macrolídeos/farmacologia , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Neoplasias da Glândula Tireoide/patologiaRESUMO
Despite recent studies showing that inhibition of autophagy depletes the hematopoietic stem cell pool and increases intracellular reactive oxygen species (ROS), it remains unknown whether autophagy is essential in the maintenance of other stem cells. Moreover, it is unclear whether and how the aberrant ROS increase causes depletion of stem cells. Here we report that ablation of FIP200 (also known as Rb1cc1), a gene essential for autophagy induction in mammalian cells, results in a progressive loss of neural stem cells (NSCs) and impairment in neuronal differentiation specifically in the postnatal brain, but not the embryonic brain, in mice. The defect in maintaining the postnatal NSC pool was caused by p53-dependent apoptotic responses and cell cycle arrest. However, the impaired neuronal differentiation was rescued by treatment with the antioxidant N-acetylcysteine but not by p53 inactivation. These data reveal that FIP200-mediated autophagy contributes to the maintenance and functions of NSCs through regulation of oxidative state.
Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/fisiologia , Acetilcisteína/farmacologia , Fatores Etários , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Proteínas Relacionadas à Autofagia , Bromodesoxiuridina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Ventrículos Cerebrais/citologia , Cloroquina/farmacologia , Giro Denteado/citologia , Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Dicarbetoxi-Di-Hidrocolidina/farmacologia , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Marcação In Situ das Extremidades Cortadas , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Degeneração Neural/tratamento farmacológico , Degeneração Neural/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Ácidos Siálicos/metabolismo , Fator de Transcrição TFIIH , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell adhesion, migration, survival, and proliferation. Here we report the identification of the transcription factor KLF8 as a target of FAK in cell cycle regulation. KLF8 is induced by FAK and decreased by FAK dominant-negative mutant DeltaC14. Overexpression of KLF8 increases cell cycle progression, whereas inhibition of endogenous KLF8 by siRNA reduces it. Cyclin D1 promoter is identified as a target of KLF8, which is activated both directly by KLF8 binding to the GT box A and by an indirect mechanism through its repression of a potential inhibitory regulator of cyclin D1. Transcription activation of cyclin D1 by FAK requires both Ets family and KLF8 factors in a temporally differential manner. Together, our data provide further insights into molecular mechanism for FAK to regulate cell cycle progression.
