Cancer stem cells (CSCs) are a small subset of cancer cells with indefinite potential for self-renewal and the capacity to drive tumorigenesis. Brefeldin A (BFA) is an antibiotic that is known to block protein transport and induce endoplasmic reticulum (ER) stress in eukaryotic cells, but its effects on colorectal CSCs are unknown. We investigated the inhibitory effect of BFA on human colorectal cancer Colo 205 cells. We found that BFA effectively reduced the survival of suspension Colo 205 cells (IC₅₀ = ~15 ng/mL) by inducing apoptosis, and inhibited the clonogenic activity of Colo 205 CSCs in tumorsphere formation assay and soft agar colony formation assay in the same nanogram per milliliter range. We also discovered that at such low concentrations, BFA effectively induced endoplasmic reticulum (ER) stress response as indicated by the increased mRNA expression of ER stress-related genes, such as glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP). Finally, we found that BFA reduced the activity of matrix metallopeptidase 9 (MMP-9). These findings suggest that BFA can effectively suppress the progression of colorectal cancer during the tumorigenesis and metastasis stages. These results may lead to the development of novel therapies for the treatment of colorectal cancer.

Background: Nrf1 [p45 nuclear factor-erythroid 2 (p45 NF-E2)-related factor 1], a member of the CNC-bZIP (CNC basic region leucine zipper) family, is known to be a transcriptional activator by dimerization with distinct partners, such as Maf, FosB, c-Jun, JunD, etc. The transcriptional roles of CNC-bZIP family are demonstrated to be involved in globin gene expression as well as the antioxidant response. For example, CNC-bZIP factors can regulate the expression of detoxification proteins through AREs, such as expression of human gamma-glutamylcysteine synthetases (GCS), glutathione S-transferases (GST), UDP-glucuronosyl transferase (UDP-GT), NADP (H) quinone oxidoreductase (NQOs), etc. To further explore other factor(s) in cells related to the function of Nrf1, we performed a yeast two-hybrid screening assay to identify any Nrf1-interacting proteins. In this study, we isolated a cDNA encoding residues 126–475 of MCRS2 from the HeLa cell cDNA library. Some functions of MCRS1 and its splice variant-MSP58 and MCRS2 have been previously identified, such as transforming, nucleolar sequestration, ribosomal gene regulation, telomerase inhibition activities, etc. Here, we demonstrated MCRS2 can function as a repressor on the Nrf1-mediated transactivation using both in vitro and in vivo systems. Results: To find other proteins interacting with the CNC bZIP domain of Nrf1, the CNC-bZIP region of Nrf1 was used as a bait in a yeast two-hybrid screening assay. MCRS2, a splicing variant of p78/MCRS1, was isolated as the Nrf1-interacting partner from the screenings. The interaction between Nrf1 and MCRS2 was confirmed in vitro by GST pull-down assays and in vivo by co-immunoprecipitation. Further, the Nrf1-MCRS2 interaction domains were mapped to the residues 354–447 of Nrf1 as well as the residues 314–475 of MCRS2 respectively, by yeast two-hybrid and GST pull-down assays. By immunofluorescence, MCRS2-FLAG was shown to colocalize with HA-Nrf1 in the nucleus and didn't result in the redistribution of Nrf1. This suggested the existence of Nrf1-MCRS2 complex in vivo. To further confirm the biological function, a reporter driven by CNC-bZIP protein binding sites was also shown to be repressed by MCRS2 in a transient transfection assay. An artificial reporter gene activated by LexA-Nrf1 was also specifically repressed by MCRS2. Conclusion: From the results, we showed MCRS2, a new Nrf1-interacting protein, has a repression effect on Nrf1-mediated transcriptional activation. This was the first ever identified repressor protein related to Nrf1 transactivation.

Background: Eosinophil cationic protein is a clinical asthma biomarker that would be released into blood, especially gathered in bronchia. The signal peptide of eosinophil cationic protein (ECPsp) plays an important role in translocating ECP to the extracellular space. We previously reported that ECPsp inhibits microbial growth and regulates the expression of mammalian genes encoding tumor growth factor-α (TGF-α) and epidermal growth factor receptor (EGFR).Results: In the present study, we first generated a DNA microarray dataset, which showed that ECPsp upregulated proinflammatory molecules, including chemokines, interferon-induced molecules, and Toll-like receptors. The levels of mRNAs encoding CCL5, CXCL10, CXCL11, CXCL16, STAT1, and STAT2 were increased in the presence of ECPsp by 2.07-, 4.21-, 7.52-, 2.6-, 3.58-, and 1.67-fold, respectively. We then constructed a functional linkage network by integrating the microarray dataset with the pathway database of Kyoto Encyclopedia of Genes and Genomes (KEGG). Follow-up analysis revealed that STAT1 and STAT2, important transcriptional factors that regulate cytokine expression and release, served as hubs to connect the pathways of cytokine stimulation (TGF-α and EGFR pathways) and inflammatory responses. Furthermore, integrating TGF-α and EGFR with the functional linkage network indicated that STAT1 and STAT2 served as hubs that connect two functional clusters, including (1) cell proliferation and survival, and (2) inflammation. Finally, we found that conditioned medium in which cells that express ECPsp had been cultured could chemoattract macrophages. Experimentally, we also demonstrated that the migration of macrophage could be inhibited by the individual treatment of siRNAs of STAT1 or STAT2. Therefore, we hypothesize that ECPsp may function as a regulator for enhancing the migration of macrophages through the upregualtion of the transcriptional factors STAT1 and STAT2.Conclusion: The increased expression and release of various cytokines triggered by ECPsp may attract macrophages to bronchia to purge damaged cells. Our approach, involving experimental and computational systems biology, predicts pathways and potential biological functions for further characterization of this novel function of ECPsp under inflammatory conditions.