RESUMO
In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of ß1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.
Assuntos
Neoplasias do Colo/genética , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/metabolismo , Sindecana-1/genética , Via de Sinalização Wnt/efeitos dos fármacos , Antígeno AC133/genética , Antígeno AC133/metabolismo , Família Aldeído Desidrogenase 1/genética , Família Aldeído Desidrogenase 1/metabolismo , Animais , Benzotiazóis/farmacologia , Células CACO-2 , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/metabolismo , Células HT29 , Humanos , Indóis/farmacologia , Integrina beta1/genética , Integrina beta1/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Oligopeptídeos/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Sulfonamidas/farmacologia , Sindecana-1/antagonistas & inibidores , Sindecana-1/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Recent studies suggest that human tumors are generated from cancer cells with stem cell (SC) properties. Spontaneously occurring cancers in dogs contain a diversity of cells that like for human tumors suggest that certain canine tumors are also generated from cancer stem cells (CSCs). CSCs, like normal SCs, have the capacity for self-renewal as mammospheres in suspension cultures. To understand how cells with SC properties contribute to canine mammary gland tumor development and progression, comparative analysis between normal SCs and CSCs, obtained from the same individual, is essential. We have utilized the property of sphere formation to develop culture conditions for propagating stem/progenitor cells from canine normal and tumor tissue. We show that cells from dissociated mammospheres retain sphere reformation capacity for several serial passages and have the capacity to generate organoid structures ex situ. Utilizing various culture conditions for passaging SCs and CSCs, fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) were found to positively or negatively regulate mammosphere regeneration, organoid formation, and multi-lineage differentiation potential. The response of FGF2 and EGF on SCs and CSCs was different, with increased FGF2 and EGF self-renewal promoted in SCs and repressed in CSCs. Our protocol for propagating SCs from normal and tumor canine breast tissue will provide new opportunities in comparative mammary gland stem cell analysis between species and anticancer treatment and therapies for dogs. J. Cell. Biochem. 118: 570-584, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Neoplasias Mamárias Animais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides/metabolismo , Animais , Cães , Feminino , Neoplasias Mamárias Animais/patologia , Células-Tronco Neoplásicas/patologia , Organoides/patologia , Células Tumorais CultivadasRESUMO
MicroRNAs (miRNAs) are a class of small RNAs (18-22 nt) that post transcriptionally regulate gene expression by binding to complementary sequences on target mRNAs, resulting in translational repression or target degradation and gene silencing. As aberrant expression of miRNAs is implicated in important diseases including cancer miRNA-based therapies are under intensive investigation. We optimized strategies to stably or conditionally generate miRNA inhibitors for a continuous block of miRNA activity that allows for probing miRNA function in long-term cell culture experiments, cancer xenografts, 3D tissue models and for in vivo studies with transgenic organisms.