Silencing LY6D Expression Inhibits Colon Cancer in Xenograft Mice and Regulates Colon Cancer Stem Cells' Proliferation, Stemness, Invasion, and Apoptosis via the MAPK Pathway.

This study explored the role of lymphocyte antigen 6 family member D (LY6D) in colon cancer stem cells' (CCSCs) proliferation and invasion. LY6D was knocked down using siRNA, and the down-regulation of LY6D was verified using Western blotting. After LY6D knockdown, CCSCs' proliferation, stemness, and invasion were suppressed, whereas apoptosis was increased. Gene Ontology (GO) enrichment analysis revealed that the differentially expressed genes (DEGs) between siLY6D and the negative control groups were significantly enriched in the cell-substrate adherens junction, focal adhesion, and cell-substrate junction terms. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DEGs were significantly enriched in the MAPK pathway. In addition, Western blotting results showed that pBRAF and pERK1/2, cascade kinases of the MAPK pathway, were significantly down-regulated after LY6D knockdown. In addition, nude mice xenograft experiments showed that the siLY6D treatment decreased tumor sizes and weights and improved tumor-bearing mice survival rates compared with the control group. In conclusion, these findings indicate that LY6D, which is highly expressed in CCSCs, is a key factor involved in tumor growth and development and might be a potential cancer marker and therapeutic target for colon cancer.

Generation of a GFI1-flag knock-in human embryonic stem cell line using CRISPR-Cas9 technology.

GFI1 is a DNA binding transcriptional repressor, it is shown to be an important gene associated with blood cells development and many blood diseases (Möröy et al., 2015). But the role of GFI1 in human hematopoieticdevelopment has not been known (Thambyrajah et al., 2016). To illustrate the function of GFI1 in human hematopoieticdevelopment, we constructed a GFI1-2 × flag-tag knock-in human embryonic stem cell line by CRISPR/Cas9 mediated gene targeting, and it would be the effective tool to study GFI1. The cell line could express GFI1-2 × flag-tag and can be identified with western blot and immunofluorescence. This cell line maintains stem cell morphology, and displays normal karyotype, pluripotent stem cell marker expression and differentiation potential.

Therapeutic Antitumor Efficacy of Cancer Stem Cell-Derived DRibble Vaccine on Colorectal Carcinoma.

Dendritic cell (DC)-based immunotherapy has been a promising strategy for colon cancer therapy, but the efficacy of dendritic cell vaccines is in part limited by immunogenicity of loaded antigens. In this study, we aimed to identify a putative tumor antigen that can generate or enhance anti-tumor immune responses against colon cancer. CD44+ colon cancer stem cells (CCSCs) were isolated from mouse colorectal carcinoma CT-26 cell cultures and induced to form defective ribosomal products-containing autophagosome-rich blebs (DRibbles) by treatment with rapamycin, bortezomib, and ammonium chloride. DRibbles were characterized by western blot and transmission electron microscopy. DCs generated from the mice bone marrow monocytes were cocultured with DRibbles, then surface markers of DCs were analyzed by flow cytometry. Meanwhile, the efficacy of DRibble-DCs was examined in vivo. Our results showed that CCSC-derived DRibbles upregulated CD80, CD86, major histocompatibility complex (MHC)-I, and MHC-II on DCs and induced proliferation of mouse splenic lymphocytes and CD8+ T cells. In a model of colorectal carcinoma using BALB/c mice with robust tumor growth and mortality, DC vaccine pulsed with CCSC-derived DRibbles suppressed tumor growth and extended survival. A lactate dehydrogenase test indicated a strong cytolytic activity of cytotoxic T-cells derived from mice vaccinated with CCSC-derived DRibbles against CT-26 cells. Furthermore, flow cytometry analyses showed that the percentages of IFN-γ-producing CD8+ T-cells were increased in SD-DC group compare with the other groups. These findings provide a rationale for novel immunotherapeutic anti-tumor approaches based on DRibbles derived from colon cancer stem cells.

RNAi mediated silencing of Nanog expression suppresses the growth of human colorectal cancer stem cells.

Colorectal cancer (CRC) is the third most common cancer in the world known for its poor recurrence-free prognosis. Previous studies have shown that it is closely linked with cancer stem cells (CSCs), which have self-renewal potential and the capacity to differentiate into diverse populations. Nanog is an important transcription factor that functions to maintain the self-renewal and proliferation of embryonic stem cells; however, many recent studies have shown that Nanog is also highly expressed in many cancer stem cells. To investigate whether Nanog plays a crucial role in maintaining the stemness of colorectal CSCs, RNA interference was used to downregulate Nanog expression in the CRC stem cell line, EpCAM+CD44+HCT-116 cells (CCSCs). We examined the anti-tumor function of Nanog in vitro and in vivo, using small interfering RNA. Our results revealed that the Nanog mRNA expression level in CCSCs was higher than that in HCT-116 cells. We found that the depletion of Nanog inhibited proliferation and promoted apoptosis in CCSCs. In addition, the invasive ability of CCSCs was markedly restricted when Nanog was silenced by small interfering RNA. Furthermore, we found that the silencing of Nanog decreased tumor size and weight and improved the survival rate of tumor-bearing mice. In conclusion, these findings collectively demonstrate that Nanog, which is highly expressed in CRC stem cells, is a key factor in the development of tumor growth, and it may serve as a potential marker of prognosis and a novel and effective therapeutic target for the treatment of CRC.

NRF2 regulates the sensitivity of human NSCLC cells to cystine deprivation-induced ferroptosis via FOCAD-FAK signaling pathway.

Transcription factor nuclear factor-erythroid 2-like 2 (NRF2) mainly regulates cellular antioxidant response, redox homeostasis and metabolic balance. Our previous study illustrated the translational significance of NRF2-mediated transcriptional repression, and the transcription of FOCAD gene might be negatively regulated by NRF2. However, the detailed mechanism and the related significance remain unclear. In this study, we mainly explored the effect of NRF2-FOCAD signaling pathway on ferroptosis regulation in human non-small-cell lung carcinoma (NSCLC) model. Our results confirmed the negative regulation relationship between NRF2 and FOCAD, which was dependent on NRF2-Replication Protein A1 (RPA1)-Antioxidant Response Elements (ARE) complex. In addition, FOCAD promoted the activity of focal adhesion kinase (FAK), which further enhanced the sensitivity of NSCLC cells to cysteine deprivation-induced ferroptosis via promoting the tricarboxylic acid (TCA) cycle and the activity of Complex I in mitochondrial electron transport chain (ETC). However, FOCAD didn't affect GPX4 inhibition-induced ferroptosis. Moreover, the treatment with the combination of NRF2 inhibitor (brusatol) and erastin showed better therapeutic action against NSCLC in vitro and in vivo than single treatment, and the improved therapeutic function partially depended on the activation of FOCAD-FAK signal. Taken together, our study indicates the close association of NRF2-FOCAD-FAK signaling pathway with cysteine deprivation-induced ferroptosis, and elucidates a novel insight into the ferroptosis-based therapeutic approach for the patients with NSCLC.

Dendritic cells loaded with CD44+ CT-26 colon cell lysate evoke potent antitumor immune responses.

Increasing evidence supports the concept that cancer stem cells (CSCs) are responsible for cancer progression and metastasis, therapy resistance and relapse. In addition to conventional therapies for colon cancer, the development of immunotherapies targeting cancer stem cells appears to be a promising strategy to suppress tumor recurrence and metastasis. In the present study, dendritic cells (DCs) were pulsed with whole-tumor cell lysates or total RNA of CD44+ colon cancer stem cells (CCSCs) isolated from mouse colon adenocarcinoma CT-26 cell cultures and investigated for their antitumor immunity against CCSCs in vivo and in vitro. In a model of colon adenocarcinoma using BALB/c mice, a sequential reduction in tumor volume and weight was associated with an extended survival in tumor-bearing mice vaccinated with DCs pulsed with RNA or CCSC lysate. In addition, a lactate dehydrogenase assay indicated that cytotoxic T-cells derived from the treated mice exhibited strong cytotoxic activity. Additionally, an enzyme-linked immunosorbent assay revealed that the cytotoxic T-cells of the treated mice released higher levels of interferon-γ against CCSCs compared with those of the control group. In all experiments, the antitumor efficacy of the lysate-pulsed DC-treated and RNA-pulsed DC-treated groups were significantly higher compared with that of the DC-treated and control groups. The results of the present study indicated the potential use of DCs pulsed with cancer stem cell lysates as a potent therapeutic antigen to target CSCs in colon cancer. Additionally, the results provided a rationale for using lysate-pulsed DCs in vivo to eliminate residual tumor deposits in post-operative patients.

Long Non-coding RNA XIST Promotes Glioma Tumorigenicity and Angiogenesis by Acting as a Molecular Sponge of miR-429.

Glioma is a worldwide malignancy, which displays significantly active metastasis and angiogenesis. Interaction between long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) has been shown to play crucial role in regulating tumor properties. However, the potential of lncRNA X-inactive specific transcript (XIST) to function as a miRNA regulator and its relevance in glioma tumorigenicity and angiogenesis have so far remained unclear. Expression analysis of lncRNA XIST in glioma cells revealed its significant up-regulation. Interestingly, silencing of XIST repressed both metastatic and pro-angiogenic ability in vitro as well as in vivo. Subsequent studies revealed that lncRNA XIST expression inversely correlated with miR-429 expression in glioma cells; miR-429 modulated XIST expression by directly targeting the XIST gene sequence. In addition, miR-429 inhibitor restored metastatic and pro-angiogenic ability of gliomas abolished by silencing XIST. Our data provide insight into the key roles of the lncRNA-miRNA functional network in gliomas, which can aid in developing new therapeutic strategies for gliomas through clinical trials.