This was accomplished using a recently published site-specific ZFN-mediated knockin strategy (Fig. cancer (25,C27). GalNAc-Ts control the initiation of or a combination of the two in the LS174T colon cancer cell line demonstrated that GalNAc-T6 expression was essential for the acquisition of oncogenic features such as hyperproliferation, loss of normal colonic epithelial architecture, and the disruption of cellCcell adhesion. Deferasirox Thus, LS174T knockout cells showed terminal differentiation traits and formed crypt-like structures that resembled the tissue architecture of a healthy colon, features that were reverted upon reintroduction of exogenous GalNAc-T6. Differential transcriptomic analysis confirmed that the expression profile of the GalNAc-T6-expressing LS174T cells resembled that of colon cancer cells, whereas LS174T knockout cells had an expression profile that was more similar to that of normal colon tissue. Furthermore, differential and Fig. S1). Of the 20 GalNAc-T isoforms, GalNAc-T6 was the only GalNAc-T that was expressed in colon cancer, was absent from healthy colon tissue. In contrast, the majority of GalNAc-Ts was either unregulated or Rabbit Polyclonal to A20A1 down-regulated in colon cancer (Fig. 1and Fig. S1). To confirm the cancer-specific up-regulation of GalNAc-T6 at the protein level, we evaluated the expression of GalNAc-T6 in 39 cases of colorectal carcinomas and in healthy colorectal mucosa by immunostaining. The expression pattern of GalNAc-T6 was compared with the expression of its close homolog GalNAc-T3 (Fig. 1TCGA IlluminaHiSeq RNAseq data obtained from https://genome-cancer.ucsc.edu/proj/site/hgHeatmap3 show the expression of GalNAc-Ts in 288 colon adenocarcinomas and 44 Deferasirox healthy colon tissue samples. >0, = 0, <0, = no data. The data are normalized by subtracting the mean of the RNAseq values from each sample value for each of the 20 GalNAc-T and shown in or color. GalNAc-T6 is specifically up-regulated in colon adenocarcinoma, whereas GalNAc-T3 expression is unchanged. immunofluorescence staining of GalNAc-T6 (mAb 2F3) and GalNAc-T3 (mAb 2D10) (DAPI). GalNAc-T6 is strongly expressed in tumor tissue and absent in normal tissue, whereas GalNAc-T3 is expressed in both types of tissue. Hematoxylin and eosin (H&E) staining shows the morphology of tumor tissue compared with normal tissue in the present sample. 50 m. Table 1 GalNAc-T6 and GalNAc-T3 expression in colon adenocarcinoma Tissues were evaluated as positive when more than 25% of the cells were labeled. Labeling intensities were scored from 0 (negative) to 3 (high intensity staining). = 22)1231695% (21/22)00022100% (22/22)????Moderately differentiated (= 10)202680% (8/10)0009100% (9/9)????Poorly differentiated (= 1)10000% (0/1)0001100% (1/1)????No information (= 6)113183% (5/6)0006100% (6/6)Total5382387% (34/39)00039100% (39/39)Healthy40000% (0/4)0002100% (2/2) Open in a separate window GalNAc-T6 disrupts the formation of actin-lined lumens and is associated with the expression of cancer-associated genes in vitro We next used the well-differentiated human LS174T colon adenocarcinoma cell line as a cell model to evaluate colon cell growth in the presence and absence of GalNAc-T6. LS174T cells exhibit unrestricted growth and grow as separate clusters of cells, supposedly due to inhibited p21WAF1 expression (69). and were knocked out in LS174T cells, individually or combined, using zinc finger nuclease (ZFN)-based genome editing to produce T6 and T3 cells. Successful out-of-frame mutagenesis was confirmed in individual single-cell clones (Table S1). RNAseq verified that non-senseCmediated RNA decay had removed the targeted transcripts (Fig. 2was accompanied by an increase in transcripts, and similarly, the knockout of was associated with an increase in transcripts, which suggests that these two enzymes can compensate for each other (Fig. 2or and/or knockout cells at the RNA level in the transcriptomic analysis (GalNAc-T; DAPI. 10 m in all images. In accordance with previous reports (69), wildtype (WT) LS174T cells formed multilayered colonies, thereby replicating colon cancer growth. Phalloidin staining, to detect F-actin cytoskeletal protein, showed that WT LS174T colon cancer cells, expressing high levels of GalNAc-T6, grew as clusters of cells with dense tubular structures and multiple small, actin-lined lumens, which could resemble the disordered crypts seen in colon cancer tissue (Fig. 3, resulted in cells that grew as Deferasirox colonies with one large actin-lined lumen surrounded by a wall of cells of varying thickness. Staining of healthy colon tissue revealed similarity of these luminal.