RESUMO
To date, no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. In order to elucidate the potential function of Dclk1+ cells, we FACS-sorted Dclk1+ cells from mouse small intestinal epithelium using transgenic mice expressing YFP under the control of the Dclk1 promoter (Dclk1-CreER;Rosa26-YFP). Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFP- cells. Dclk1+ population display ~6 fold enrichment for the putative quiescent stem cell marker Bmi1. We observed significantly greater expression of pluripotency genes, pro-survival genes, and quiescence markers in the Dclk1+ population. A significant increase in self-renewal capability (14-fold) was observed in in vitro isolated Dclk1+ cells. The unique genetic report presented in this manuscript suggests that Dclk1+ cells may maintain quiescence, pluripotency, and metabolic activity for survival/longevity. Functionally, these reserve characteristics manifest in vitro, with Dclk1+ cells exhibiting greater ability to self-renew. These findings indicate that quiescent stem-like functionality is a feature of Dclk1-expressing tuft cells.
Assuntos
Diferenciação Celular , Proliferação de Células , Células Epiteliais/citologia , Intestino Delgado/citologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Apoptose , Células Cultivadas , Quinases Semelhantes a Duplacortina , Células Epiteliais/fisiologia , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Intestino Delgado/fisiologia , Longevidade , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Renal clear cell carcinoma (RCC) is the most common type of kidney cancer and the 8th most common cancer overall in the US. RCC survival rates drop precipitously with regional and distant spread and recent studies have demonstrated that RCC presents an epithelial-mesenchymal transition (EMT) phenotype linked to increased recurrence and decreased survival. EMT is a key characteristic of tumor stem cells (TSCs) along with chemo-resistance and radio-resistance, which are also phenotypic of RCC. Targeting these factors is key to increasing the survival of RCC patients. Doublecortin-like kinase 1 (DCLK1) marks TSCs in pancreatic and colorectal cancer and regulates EMT and stemness. Analysis of the Cancer Genome Atlas' RCC dataset revealed that DCLK1 is overexpressed and dysregulated on the mRNA and epigenetic level in more than 93% of RCC tumors relative to adjacent normal tissue. Immunohistochemistry using α-DCLK1 antibody confirmed overexpression and demonstrated a major increase in immunoreactivity in stage II-III tumors compared to normal kidney and stage I tumors. Small-interfering RNA (siRNA) mediated knockdown of DCLK1 resulted in decreased expression of EMT and pluripotency factors and significantly reduced invasion, migration, focal adhesion, drug-resistance, and clonogenic capacity. These findings suggest that DCLK1 is a novel, overexpressed factor in RCC progression that may be targeted to suppress EMT, metastasis, and stemness in early-stage and advanced RCC to increase patient survival. Moreover, the possibility that DCLK1 may mark a population of tumor stem-like cells in RCC should be further investigated in light of these findings.