Increased neurotensin receptor-1 expression during progression of colonic adenocarcinoma

The high affinity neurotensin receptor (NTSR1) mediates most of the biologic effects of neurotensin (NT), a 13-amino acid peptide that stimulates growth in certain cell types. NT is expressed in fetal but not differentiated colonic epithelium and is re-expressed in colonic adenocarcinoma. The cognate receptor, NTSR1, is also not expressed or is present at a low level in adult colonic epithelial cells but is expressed in most colon cancer cell lines. These observations suggest that altered NT-NTSR1 signaling may be associated with malignant transformation in the colon. To further understand the possible role of NTSR1 expression in colonic tumorigenesis and progression, we examined NTSR1 mRNA by in situ hybridization in normal colonic mucosa, adenomas, and colonic adenocarcinomas. NTSR1 mRNA expression was undetectable or weak in superficial differentiated epithelial cells of normal colonic epithelium, but adenomas and adenocarcinomas showed moderate to strong expression (p<0.05). Adenocarcinomas showed a higher level of expression compared to adenomas (p<0.05). Furthermore, adenocarcinomas that infiltrated into and beyond the muscularis propria showed a higher intensity of NTSR1 expression compared with tumors that were localized to the mucosa or submucosa. In some cases, infiltrating margins and foci of lymphovascular invasion showed a higher intensity of expression than the main mass of the tumor. These results suggest that increased NTSR1 expression may be an early event during colonic tumorigenesis and also contribute to tumor progression and aggressive behavior in colonic adenocarcinomas. NTSR1 may thus be a potential target for preventive or therapeutic strategies in colon cancer.     

Comparative study of the expression of Rb and p53 genes in human colorectal cancers,colon carcinoma cell lines and synchronized human fibroblasts

We have compared the expression of the retinoblastoma (Rb) and p53 genes in normal human fibroblasts, colon carcinoma cell lines, matched pairs of colorectal tumor tissues and adjacent normal mucosa and in synchronized human diploid fibroblast cell line W138. The increased expression of Rb and p53 RNA was observed in a majority of colorectal cancers in comparison to adjacent normal mucosa and is accompanied by proportional increase in the expression of histone H3 gene. The Rb and p53 RNA levels varied significantly between the various colon carcinoma cell lines. However, we found that the expression of Rb and p53 RNA is regulated differently in cell cycle synchronized normal human fibroblasts. The Rb mRNA level did not change with the position in the cell cycle and did not differ significantly whether the cells were serum deprived or in 10% serum. But p53 mRNA expression follows the same pattern as histone H3 mRNA.     

Pathways of mucin O-glycosylation in normal and malignant rat colonic epithelial cells reveal a mechanism for cancer-associated Sialyl-Tn antigen expression

The Sialyl-Tn antigen (Sialyl alpha-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells. Its presence is associated with a poor prognosis in patients with colorectal and other cancers. We previously reported that Sialyl-Tn expression in LSC human colon cancer cells could be explained by a specific lack of the activity of core 1 beta3-Gal-transferase (Brockhausen et al., Glycoconjugate J. 15, 595-603, 1998) and an inability to synthesize the common O-glycan core structures. To support this mechanism, or find other mechanisms to explain Sialyl-Tn antigen expression, we investigated the O-glycosylation pathways in clonal rat colon cancer cell lines that were selected for positive or negative expression of Sialyl-Tn antigen, and compared these pathways to those in normal rat colonic mucosa. Normal rat colonic mucosa had very active glycosyltransferases synthesizing O-glycan core structures 1 to 4. Several sialyl-, sulfo- and fucosyltransferases were also active. An M type core 2 beta6-GlcNAc-transferase was found to be present in rat colon mucosa and all of the rat colon cancer cells. O-glycosylation pathways in rat colon cancer cells were significantly different from normal rat colonic mucosa; for example, rat colon cancer cells lost the ability to synthesize O-glycan core 3. All rat colon cancer cell lines, regardless of the Sialyl-Tn phenotype, expressed glycosyltransferases assembling complex O-glycans of core 1 and core 2 structures (unlike human LSC colon cancer cells which lack core 1 beta3-Gal-transferase activity). It was the activity of CMP-sialic acid:GalNAc-mucin alpha6-sialyltransferase that coincided with Sialyl-Tn expression. Sialyl-Tn negative cells had a several fold higher activity of core 2 beta6-GlcNAc-transferase which synthesizes complex O-glycans that may mask adjacent Sialyl-Tn epitopes. The results suggest a new mechanism controlling Sialyl-Tn expression in cancer cells.     

Synthesis of type 1 and 2 lacto series glycolipid antigens in human colonic adenocarcinoma and derived cell lines is due to activation of a normally unexpressed beta 1----3N-acetylglucosaminyltransferase

Human colonic adenocarcinoma tissue and derived cell lines have been characterized by an abundance of different type 1 and 2 lacto series glycolipid antigens which are either low or not found in normal colonic mucosa. The enzymatic basis for the expression of contrasting glycolipid compositions between adenocarcinomas and normal colonic mucosa, as well as between derived cell lines, has been studied. The following results were of particular interest. (i) Abundant activities of beta 1----4galactosyltransferase associated with synthesis of both lactosylceramide and lactoneotetraosylceramide, beta 1----3galactosyltransferase for synthesis of lactotetraosylceramide, and an alpha 1----3/4fucosyltransferase responsible for synthesis of Lex and Lea antigens were found in normal colonic mucosa or in a normal mucosal epithelial cell line HCMC, or in both. Variable levels of these activities were found in adenocarcinoma tissues and in various established adenocarcinoma cell lines. In striking contrast, significant activity of a beta 1----3N-acetylglucosaminyltransferase responsible for synthesis of lactotriaosylceramide (Lc3) was found in various cases of colonic adenocarcinoma and cell lines, but was undetectable in normal colonic epithelial cells. (ii) In situ transfer of galactose to Lc3 was performed on histologic sections by preincubation of the tissue with acceptor glycolipid followed by incubation with UDP-galactose. The biosynthesized glycolipid was revealed by indirect immunofluorescence with the monoclonal antibody 1B2 which defines lactoneotetraosylceramide antigen. In these studies, histologic sections prepared from frozen normal proximal colon tissue were shown to lack native type 2 chain structures. However, transfer of galactose from UDP-galactose could be demonstrated in the epithelial cells of normal proximal colon after incorporation of Lc3 into the membranes, indicating the ability of normal colonic epithelial cells to synthesize type 2 chain core structures if the precursor Lc3 is available. In contrast, adenocarcinoma tissues showed significant native immunofluorescence with the antibody. These data suggest that an accumulation of both type 1 and 2 chain lacto series glycolipids with alpha 1----3- or alpha 1----4fucosyl substitution in human adenocarcinoma is due to enhanced beta 1----3N-acetylglucosaminyltransferase rather than enhancement of other enzymes. This enzyme may play a key role in regulating the level of various types of lacto series tumor-associated antigens with the lacto type 1 or 2 chain.     

miRNA expression in colon polyps provides evidence for a multihit model of colon cancer

Changes in miRNA expression are a common feature in colon cancer. Those changes occurring in the transition from normal to adenoma and from adenoma to carcinoma, however, have not been well defined. Additionally, miRNA changes among tumor subgroups of colon cancer have also not been adequately evaluated. In this study, we examined the global miRNA expression in 315 samples that included 52 normal colonic mucosa, 41 tubulovillous adenomas, 158 adenocarcinomas with proficient DNA mismatch repair (pMMR) selected for stage and age of onset, and 64 adenocarcinomas with defective DNA mismatch repair (dMMR) selected for sporadic (n = 53) and inherited colon cancer (n = 11). Sporadic dMMR tumors all had MLH1 inactivation due to promoter hypermethylation. Unsupervised PCA and cluster analysis demonstrated that normal colon tissue, adenomas, pMMR carcinomas and dMMR carcinomas were all clearly discernable. The majority of miRNAs that were differentially expressed between normal and polyp were also differentially expressed with a similar magnitude in the comparison of normal to both the pMMR and dMMR tumor groups, suggesting a stepwise progression for transformation from normal colon to carcinoma. Among the miRNAs demonstrating the largest fold up- or down-regulated changes (≥4), four novel (miR-31, miR-1, miR-9 and miR-99a) and two previously reported (miR-137 and miR-135b) miRNAs were identified in the normal/adenoma comparison. All but one of these (miR-99a) demonstrated similar expression differences in the two normal/carcinoma comparisons, suggesting that these early tumor changes are important in both the pMMR- and dMMR-derived cancers. The comparison between pMMR and dMMR tumors identified four miRNAs (miR-31, miR-552, miR-592 and miR-224) with statistically significant expression differences (≥2-fold change).     

Bone morphogenetic protein-4 is overexpressed in colonic adenocarcinomas and promotes migration and invasion of HCT116 cells

Bone morphogenetic protein (BMP), a member of the TGF-beta superfamily, is involved in development, morphogenesis, cell proliferation and apoptosis. Dysregulation of BMP signaling has been suggested in tumorigenesis. In an analysis of human colon normal mucosa and tumors at different stages by immunohistochemistry, we observed that the intensity of BMP-4 staining in late-adenocarcinomas was stronger than that in normal mucosa and adenomas, while there was no difference in the staining of its receptors (BMPR-IA and BMPR-II) at all stages. The up-regulation of BMP-4 was further validated in another panel of tumor tissues by real-time RT-PCR, showing that BMP-4 mRNA levels in primary colonic carcinomas with liver metastasis were significantly higher than that in the matched normal mucosa. In order to understand the functional relevance of BMP-4 expression in colon cancer progression, BMP-4-overexpressing cell clones were generated from HCT116 cells. Overexpression of BMP-4 did not affect the HCT116 cell growth. The cells overexpressing BMP-4 became resistant to serum-starvation-induced apoptosis and exhibited enhanced migration and invasion characteristics. Overexpression of BMP-4 changed cell morphology to invasive spindle phenotype and induced the expression and activity of urokinase plasminogen activator (uPA). These results indicate that BMP-4 confers invasive phenotype during progression of colon cancer.     

Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus.

Thirteen adherent human non-lymphocyte cell lines were tested for their susceptibility to infection by human immunodeficiency virus. Productive infection could be demonstrated in three of five colorectal carcinoma cell lines examined; the other eight human non-lymphocyte cell lines were uninfectible. A susceptible colon carcinoma cell line (HT29), as well as normal colonic mucosa, was shown to contain a 3.0-kilobase species of poly(A)+ CD4 RNA, whereas uninfectible colon carcinoma and rhabdomyosarcoma cell lines synthesized no detectable T4 RNA. A persistently infected colon carcinoma cell line was established that continued to produce progeny human immunodeficiency virus for more than 10 weeks postinfection.     

Post-translational processing of gastrin in neoplastic human colonic tissues.

Gastrin has been postulated to stimulate proliferation in colorectal neoplasms. Although gastrin mRNA has been demonstrated to be present in colon cancer cell lines, the intact peptide had not been recovered from human colorectal neoplasms. We demonstrate that gastrin and its precursors are present in both colorectal neoplasia and adjacent normal-appearing colonic mucosa. In colonic tissue, the glycine-extended precursor form of the peptide is over 10-fold more abundant than the amidated gastrin, and progastrin is more than 700-fold more abundant. In contrast, amidated gastrin in the human antrum is the predominant form of gastrin by a factor of 10. Furthermore, the ratio of gastrin precursors to gastrin is significantly increased in neoplastic colonic mucosa when compared with normal colonic tissue. These data suggest that the processing of gastrin is unique in the human colon and that further differences in processing occur in neoplastic colonic tissue.     

Distinct E2F-mediated transcriptional program regulates p14ARF gene expression

The tumor suppressor p14(ARF) gene is induced by ectopically expressed E2F, a positive regulator of the cell cycle. The gene is expressed at low levels in normally growing cells in contrast to high levels in varieties of tumors. How p14(ARF) gene is regulated by E2F in normally growing cells and tumor cells remains obscure. Here we show that regulation of p14(ARF) gene by E2F is distinct from that of classical E2F targets. It is directly mediated by E2F through a novel E2F-responsive element that varies from the typical E2F site. The element responds to E2F activity resulting from ectopic E2F1 expression, inactivation of pRb by adenovirus E1a or shRNA, but not to phosphorylation of pRb by serum stimulation or ectopic cyclin D1/cyclin-dependent kinase-4 expression in normal human fibroblasts. The element has activity in various tumor cells with defective pRb, but not in normally growing cells. These results indicate that the distinct regulation constitutes the basis of p14(ARF) function as a tumor suppressor, discriminating abnormal growth signals caused by defects in pRb function from normal growth signals.     

Detection of dysplastic intestinal adenomas using a fluorescent folate imaging probe

Macrophages have long been recognized as a prominent component of tumors. Activated macrophages overexpress folate receptors and we used this phenomenon to image inflammatory reactions in colon dysplasia using a fluorescent folate probe (FFP). APC(Delta468) mice injected with FFP showed fluorescent adenomas (target-to-background ratio, adenoma vs. adjacent normal mucosa, of 2.46 +/- 0.41), significantly higher (p < .001) than adenomas in animals injected with a non-folate-containing control probe. Fluorescence-activated cell-sorting analysis revealed a 3-fold higher content of Mac1-positive cells in colonic adenomas compared with normal adjacent mucosa (6.8% vs. 2.2%), and confirmed the source of FFP-positive cells to be primarily an F4/80-positive macrophage subpopulation. Taken together, these results indicate that probe potentially can be used to image dysplastic intestinal adenomas in vivo.     

Tumor suppressor FOXO3 mediates signals from the EGF receptor to regulate proliferation of colonic cells

Epithelial proliferation, critical for homeostasis, healing, and colon cancer progression, is in part controlled by epidermal growth factor receptor (EGFR). Proliferation of colonic epithelia can be induced by Citrobacter rodentium infection, and we have demonstrated that activity of tumor suppressor FOXO3 was attenuated after this infection. Thus the aim of this study was to determine the contribution of FOXO3 in EGFR-dependent proliferation of intestinal epithelia and colon cancer cell lines. In this study we show that, during infection with C. rodentium, EGFR was significantly phosphorylated in colonic mucosa and Foxo3 deficiency in this model lead to an increased number of bromodeoxyuridine-positive cells. In vitro, in human colon cancer cells, increased expression and activation of EGFR was associated with proliferation that leads to FOXO3 phosphorylation (inactivation). Following EGFR activation, FOXO3 was phosphorylated (via phosphatidylinositol 3-kinase/Akt) and translocated to the cytosol where it was degraded. Moreover, inhibition of proliferation by overexpressing FOXO3 was not reversed by the EGFR signaling, implicating FOXO3 as one of the regulators downstream of EGFR. FOXO3 binding to the promoter of the cell cycle inhibitor p27kip1 was decreased by EGFR signaling, suggesting its role in EGFR-dependent proliferation. In conclusion, we show that proliferation in colonic epithelia and colon cancer cells, stimulated by EGFR, is mediated via loss of FOXO3 activity and speculate that FOXO3 may serve as a target in the development of new pharmacological treatments of proliferative diseases.     

Myb expression is higher in malignant human colonic carcinoma and premalignant adenomatous polyps than in normal mucosa.

Expression of the protooncogene c-Myb protein was assessed in normal mucosa and in tumor samples resected from six patients. We found that the tumor samples always expressed higher levels of full length Myb protein than the normal tissue. This contrasts with the situation in c-myb-associated hemopoietic malignancies of the mouse and chicken, in which Myb proteins are generally amino or carboxyl truncated. Tissues from five patients with colonic adenomatous polyps were also examined and found to express levels of Myb that were, in general, intermediate between those found in normal tissues and tumors. Of particular interest is that the more dysplastic polyps displayed higher Myb levels. In one patient with carcinoma and multiple colonic polyps, some polyps had intermediate levels of Myb, whereas one polyp with carcinoma in situ expressed tumor-like levels of Myb. To directly test the hypothesis that Myb expression may be important in determining the rate of colonic cell proliferation, we examined three colonic carcinoma cell lines and one polyp cell line. We found that the cell lines with the most rapid doubling times exhibited the highest Myb levels. In addition, we show that antisense myb oligonucleotides retard the proliferation of one of these colonic cell lines which expresses the highest level of Myb.