hes1-protein--human and Colonic-Neoplasms

The loss of HES1, a canonical Notch signaling target, may cooperate with KRAS mutations to remodel the extracellular matrix and to suppress the anti-tumor immune response. While HES1 expression is normal in benign hyperplastic polyps and normal colon tissue, HES1 expression is often lost in sessile serrated adenomas/polyps (SSAs/SSPs) and colorectal cancers (CRCs) such as those right-sided CRCs that commonly harbor BRAF or KRAS mutations. To develop a deeper understanding of interaction between KRAS and HES1 in colorectal carcinogenesis, we selected microsatellite stable (MSS) and KRAS mutant or KRAS wild type CRCs that show aberrant expression of HES1 by immunohistochemistry. By comparing the transcriptional landscapes of microsatellite stable (MSS) CRCs with or without nuclear HES1 expression, we investigated differentially expressed genes and activated pathways. We identified pathways and markers in the extracellular matrix and immune microenvironment that are associated with mutations in KRAS. We found that loss of HES1 expression positively correlated with matrix remodeling and epithelial-mesenchymal transition but negatively correlated with tumor cell proliferation. Furthermore, loss of HES1 expression in KRAS mutant CRCs correlates with a higher M2 macrophage polarization and activation of IL6 and IL10 immunosuppressive signature. Identifying these HES1-related markers may be useful for prognosis stratification and developing treatment for KRAS-mutant CRCs.

Topics: Adenocarcinoma; Colonic Neoplasms; Extracellular Matrix; Humans; Immunosuppression Therapy; Proto-Oncogene Proteins p21(ras); Transcription Factor HES-1; Tumor Microenvironment

Colon adenocarcinoma (COAD) is the commonest type of colorectal cancer with high morbidity and mortality worldwide. ETS variant 4 (ETV4) is a member of the ETS transcription factors and is frequently involved in the progression of many cancers. This study focused on the relevance of ETV4 to the progression of COAD. ETV4 was highly expressed in the collected COAD tissues and acquired cells and indicated advanced Dukes staging in patients. Knockdown of ETV4 in COAD cells weakened proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) activity of cells. The downstream genes of ETV4 were predicted, and a Gene Ontology (GO) analysis was conducted to identify the key molecule involved. ETV4 bound to the promoter sequence of HES1 and activated its transcription. Further overexpression of HES1 restored the malignant behaviors of COAD cells. HES1 was also found to promote phosphorylation of Stat3. Similar results were reproduced in vivo where downregulation of ETV4 blocked the growth of xenograft tumors in nude mice. This study demonstrated that ETV4 encourages malignant development of COAD through activating HES1 transcription and Stat3 phosphorylation. This study may offer novel insights into COAD therapy.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Cell Proliferation; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Phosphorylation; Prognosis; Proto-Oncogene Proteins c-ets; STAT3 Transcription Factor; Transcription Factor HES-1; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

There is increasing body of evidence to suggest that some colonic serrated polyps do not fit morphologically with any of the proposed categories for serrated polyps recommended by the World Health Organization. Most of these polyps have morphologic features of traditional serrated adenoma (TSA) admixed with areas resembling sessile serrated adenoma (SSA) or hyperplastic polyp (HP). Based on these findings it has been suggested that at least some TSAs may arise in association with precursor HP or SSA lesions, particularly those that develop in right colon. To further evaluate this hypothesis, 39 serrated polyps from right side of the colon (cecum, ascending, and transverse colon) with mixed features of TSA and SSA were evaluated by 2 immunostains previously shown to represent markers of SSA. One is Annexin A10 which shows upregulated expression in SSA and the other is Hes-1 which is shown to be down regulated in SSA. The expression patterns of these markers were evaluated in SSA and TSA components of hybrid polyps and compared with control groups (pure SSAs and TSAs of right colon). SSA component in hybrid polyps did not show any significant difference in staining pattern compared with that seen in TSA component of hybrid polyps or in pure TSA polyps. These findings further support the hypothesis that recognizes SSA as a precursor lesion for TSA in the right colon.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Annexins; Colonic Neoplasms; Colonic Polyps; Female; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Transcription Factor HES-1

Various amino acids regulate cell growth and differentiation. In the present study, we examined the ability of HT-29 cells to differentiate into goblet cells in RPMI and DMEM which are largely different in the amounts of numerous amino acids. Most of the HT-29 cells differentiated into goblet cells downregulating the stem cell marker Lgr5 when cultured in DMEM, but remained undifferentiated in RPMI. The goblet cell differentiation in DMEM was inhibited by 1-methyl-tryptophan (1-MT), an inhibitor of indoleamine 2,3 dioxygenase-1 which is the initial enzyme in tryptophan metabolism along the kynurenine (KN) pathway, whereas tryptophan and KN induced goblet cell differentiation in RPMI. The levels of Notch1 and its activation product Notch intracytoplasmic domain in HT-29 cells were lower in DMEM than those in RPMI and were increased by 1-MT in both media. HT-29 cells grown in both media expressed β-catenin at the same level on day 2 when goblet cell differentiation was not observed. β-catenin expression, which was increased by 1-MT in both media, was decreased by KN. DMEM reduced Hes1 expression while enhancing Hath1 expression. Finally, aryl hydrocarbon receptor (AhR) activation moderately induced goblet cell differentiation. Our results suggest that KN promotes goblet cell differentiation by regulating Wnt, Notch, and AhR signals and expression of Hes1 and Hath1.

Topics: Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Cell Differentiation; Cell Proliferation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Goblet Cells; HT29 Cells; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Kynurenine; Receptor, Notch1; Receptors, Aryl Hydrocarbon; Transcription Factor HES-1; Tryptophan; Wnt Signaling Pathway

Sessile serrated adenoma/polyp (SSA/p) is a precancerous lesion, and its differential diagnosis from hyperplastic polyp (HP) could be challenging in certain circumstances based on morphology alone. Hes1 is a downstream target of Notch-signaling pathway and plays an important role in intestinal development by regulating differentiation of enterocytes. In this study, we evaluated the expression patterns of Hes1 in SSA/p and HP, and determine whether Hes1 immunostaining can help differentiate between these 2 entities. Serrated polyps with cytologic dysplasia (SSA with cytologic dysplasia, tubular adenoma, and traditional serrated adenoma) were also studied. Hes1 is ubiquitously expressed in the nuclei of normal colon epithelial cells. The complete loss or a very weak expression of Hes1 is observed in the majority of the SSA/p in the study (58/63, 92%) compared with the normal expression of Hes1 in HP (35/35,100%). In SSA/p with cytologic dysplasia, dysplastic area demonstrated cytoplasmic and/or nuclear staining for Hes1. Tubular adenoma and traditional serrated adenoma showed variability of Hes1 staining within the polyp with a mixed positive and negative staining pattern. Our study suggests that loss of Hes1 could be used as a sensitive and specific marker to differentiate SSA/p from HP, which helps the diagnosis in morphologically challenging cases.

Topics: Adenomatous Polyps; Adult; Aged; Aged, 80 and over; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Colonic Neoplasms; Colonic Polyps; Diagnosis, Differential; Down-Regulation; Female; Homeodomain Proteins; Humans; Immunohistochemistry; Male; Middle Aged; Predictive Value of Tests; Prognosis; Transcription Factor HES-1

Hes1 is a transcription factor that influences cell proliferation and differentiation. However, the effect of Hes1 on invasiveness and the underlying mechanism remain unknown. In the current study, we found that Hes1 suppressed cell apoptosis, promoted cell growth, induced EMT phenotype and cytoskeleton reconstruction, and enhanced the metastatic potential of colon cancer cells in vitro and in vivo. Furthermore, we indicated that Bmi-1 mediated Hes1-induced cell proliferation and migration, downregulated PTEN and activated the Akt/GSK3β pathway, consequently induced EMT and cytoskeleton reconstruction, ultimately leading to enhanced invasiveness of cancer cells. In addition, we also found that both Hes1 and Bmi-1 could directly regulate PTEN by associating at the PTEN locus, and played important roles in regulating PTEN/Akt/GSK3β pathway. Our results provide functional and mechanistic links between Hes1 and Bmi-1/PTEN/Akt/GSK3β signaling in the development and progression of colon cancer.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Movement; Cell Proliferation; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Homeodomain Proteins; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Polycomb Repressive Complex 1; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Transcription Factor HES-1; Transfection

A small subpopulation of cancer cells with stem cell-like features might be responsible for tumour generation, progression, and chemoresistance. Hes1 influences the maintenance of certain stem cells and progenitor cells and the digestive systems. We found upregulated Hes1 in poorly differentiated cancer samples compared with well-differentiated tumour samples, and most of the adenocarcinomas exhibited significantly higher levels of Hes1 mRNA compared with that observed in matched normal colon samples. Moreover, Hes1 expression was found to be correlated with the expression of stem cell markers in colon cancer samples, and Hes1 upregulates the expression of stemness-related genes in colon cancer cells. In addition, Hes1 enhances the self-renewal properties of the stem-like cells by increasing the sizes of CD133+ cells and SP cells and the ability of tumour sphere formation. Additionally, the Hes1-overexpressing cells formed significantly larger and higher number of colonies, as determined through the colony and the soft agar assays. More importantly, Hes1 enhances the tumourigenicity of colon cancer cell lines in nude mice and exhibits a strong tumour-formation ability at a cell density of 1 × 10(3). Taken together, our data indicate that Hes1 induces stem-like cell self-renewal and increases the number of tumour-initiating cells in colon cancer.

Topics: AC133 Antigen; Adenocarcinoma; Animals; Antigens, CD; Basic Helix-Loop-Helix Transcription Factors; Cell Transformation, Neoplastic; Colonic Neoplasms; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glycoproteins; HCT116 Cells; Homeodomain Proteins; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Peptides; RNA, Messenger; Spheroids, Cellular; Transcription Factor HES-1; Transplantation, Heterologous; Tumor Cells, Cultured; Up-Regulation

The Wnt and Notch1 signaling pathways play major roles in intestinal development and tumorigenesis. Sub-cellular localization of β-catenin has been implicated in colorectal carcinogenesis. However, the β-catenin and Notch intracellular domain (NICD) interaction has to be addressed. Immunohistochemistries of β-catenin, NICD, and dual immunofluorescence of β-catenin and NICD were analyzed in colorectal tissues and HT29 cell line. Moreover, real-time PCR analysis of CyclinD1, Hes1 and MUC2 was done in HT29 cells upon N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment. Dual staining emphasized the strong interaction of β-catenin and NICD in adenoma and adenocarcinoma than in normal tissues. Hes1 transcript levels were decreased 1.5- and 7.1-fold in 12.5 and 25 µM DAPT-treated HT29 cells. CyclinD1 transcript levels decreased 1.2- and 1.6-fold, and MUC2 transcript level increased 4.3- and 7.5-fold in 12.5 and 25 µM DAPT-treated HT29 cells. The results of this study showed that the sub-cellular localization of β-catenin converges with NICD inducing proliferation through the activation of CyclinD1 and Hes1. Moreover, the inhibition of Notch1 signaling by DAPT leads to the arrest of cell proliferation and induces apoptosis leading to the upregulation of MUC2, a secretory cell lineage marker.

Topics: Adenocarcinoma; Adenoma; Basic Helix-Loop-Helix Transcription Factors; beta Catenin; Cell Proliferation; Colonic Neoplasms; Colorectal Neoplasms; Cyclin D1; Dipeptides; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; HT29 Cells; Humans; Mucin-2; Protein Structure, Tertiary; Receptor, Notch1; Reference Values; Signal Transduction; Transcription Factor HES-1

Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

Topics: Adult; Aged; Aged, 80 and over; Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genetic Vectors; Homeodomain Proteins; Humans; Immunoglobulin J Recombination Signal Sequence-Binding Protein; Lentivirus; Lymphatic Metastasis; Lymphoid Enhancer-Binding Factor 1; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Nude; Middle Aged; Neoplasm Transplantation; Signal Transduction; Transcription Factor HES-1

Hes1 is a key transcriptional regulator primarily controlled by the Notch signaling pathway, and recent studies have demonstrated both an oncogenic and tumor suppressor role for Hes1, depending on the cell type. Small molecules that activate and inhibit Hes1 activity hold promise as future anticancer chemotherapeutics. We have utilized a cell-based dual luciferase assay to identify modulators of Hes1 expression in a medium-throughput format. A modest screen was performed in HCT-116 colon cancer cell lines, and two small molecules were identified and characterized as Hes1 regulators. Compound 3 induced Hes1 expression and exhibited anticancer effects in pulmonary carcinoid tumor cells, a cell type in which the upregulated Notch/Hes1 signaling plays a tumor suppressive role. Treatment of HCT-116 cells with compound 12 resulted in Hes1 downregulation and antitumor effects.

Topics: Aminoacridines; Aniline Compounds; Antineoplastic Agents; Basic Helix-Loop-Helix Transcription Factors; Carcinoid Tumor; Cell Line, Tumor; Colonic Neoplasms; Coumarins; Down-Regulation; HCT116 Cells; Homeodomain Proteins; Humans; Receptors, Notch; Signal Transduction; Small Molecule Libraries; Transcription Factor HES-1; Up-Regulation

The transcription factor Atoh1/Hath1 plays crucial roles in the differentiation program of human intestinal epithelium cells (IECs). Although previous studies have indicated that the Notch signal suppresses the differentiation program of IEC, the mechanism by which it does so remains unknown. This study shows that the undifferentiated state is maintained by the suppression of the Hath1 gene in human intestine.. To assess the effect of Notch signaling, doxycycline-induced expression of Notch intracellular domain (NICD) and Hes1 cells were generated in LS174T. Hath1 gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR). Hath1 promoter region targeted by HES1 was determined by both reporter analysis and ChIP assay. Expression of Hath1 protein in ulcerative colitis (UC) was examined by immunohistochemistry.. Hath1 mRNA expression was increased by Notch signal inhibition. However, Hath1 expression was suppressed by ectopic HES1 expression alone even under Notch signal inhibition. Suppression of the Hath1 gene by Hes1, which binds to the 5' promoter region of Hath1, resulted in suppression of the phenotypic gene expression for goblet cells. In UC, the cooperation of aberrant expression of HES1 and the disappearance of caudal type homeobox 2 (CDX2) caused Hath1 suppression, resulting in goblet cell depletion.. The present study suggests that Hes1 is essential for Hath1 gene suppression via Notch signaling. Moreover, the suppression of Hath1 is associated with goblet cell depletion in UC. Understanding the regulation of goblet cell depletion may lead to the development of new therapy for UC.

Topics: Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; CDX2 Transcription Factor; Cell Differentiation; Chromatin Immunoprecipitation; Colitis, Ulcerative; Colonic Neoplasms; Gene Expression Regulation; Goblet Cells; Homeodomain Proteins; Humans; Immunoenzyme Techniques; Intestinal Mucosa; Intestines; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Luciferases; Mucin-2; Promoter Regions, Genetic; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transcription Factor HES-1; Transcription, Genetic; Tumor Cells, Cultured

Notch and Wnt signaling function together to regulate colonic progenitor cell division and differentiation. Studies in mice have also shown that Notch signaling is required for adenoma formation in response to elevated Wnt-pathway signaling that occurs in the APCMin mouse model of human adenomatous polyposis coli. We therefore used in situ hybridization to analyze expression of Notch ligands, receptors and fringe genes, as well as the Notch target gene, HES1, in human colorectal cancer (CRC). In a small cohort of tumors, JAGGED ligands, NOTCH1, LFNG and HES1 were expressed at levels similar to, or higher than, levels observed in the crypt. To explore the possibility that Notch signaling may play a quantitative role in human CRC we next analyzed HES1 mRNA expression in 130 tumors, each associated with outcome data. The vast majority of these tumors expressed HES1, although at varying levels. Absolute expression levels did not correlate with patient survival. These results establish that JAG ligands and NOTCH1, as well as Notch receptor activation are consistent features of human CRC and support the notion that many of these tumors, like the APCMin mouse, may respond to anti-Notch therapeutic regimes.

Topics: Adenocarcinoma; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cell Differentiation; Colonic Neoplasms; Gene Expression Regulation, Neoplastic; Germany; Glycosyltransferases; Homeodomain Proteins; Humans; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Kaplan-Meier Estimate; Membrane Proteins; Ontario; Prognosis; Receptor, Notch1; Receptors, Notch; Registries; RNA, Messenger; Serrate-Jagged Proteins; Signal Transduction; Transcription Factor HES-1

Cyclooxygenase-2 (COX-2) is a key factor in the development of colorectal cancer, and non-steroidal anti-inflammatory drugs (NSAIDs) have anti-colorectal cancer activity. However, the potential molecular mechanism of the COX-2 selective inhibitor effect on proliferation and apoptosis of colon cancer cells is unclear. In this study, we have demonstrated for the first time that the Delta1/Notch1 signal transduction pathway mediates the COX-2 selective inhibitor effect on colorectal cancer cells, and we reveal the mechanism of the Notch1 pathway in terms of regulating the proliferation and apoptosis of colorectal cancer cells.. Colon cancer cell lines HT-29 and SW480 were treated with NS-398 (a COX-2 selective inhibitor) and DAPT (a gamma-secretase inhibitor). The colormetric MTT cell proliferation assay and flow cytometry were used to measure cell proliferation and apoptosis. Reverse transcriptase (RT)-PCR and ELISA analyses were used to detect the levels of COX-2 mRNA expression and prostaglandin E2 (PGE2) concentration from the two cell lines, respectively. The expression of the Notch1, Delta1, ICN, Hes1 and NF-kappaB2 proteins was measured by Western blot. Immunohistochemistry results showed that Notch1 was expressed mainly in the cytoplasm and ICN mainly in the nucleus. COX-2 mRNA was highly expressed in HT-29 cells but not in SW480 cells. Both COX-2 mRNA expression and PGE2 concentration decreased in HT-29 cells treated with NS-398; however, PGE2 levels did not change in SW480 cells treated with NS-398. NS-398 and DAPT inhibited cell proliferation and induced apoptosis in a dose time-dependent manner accompanied by significantly decreased Notch1 activity (P < 0.01), and resulted in a significant down-regulation of Hes1 and NF-kappaB2 (P < 0.01).. Our results show that the selective COX-2 inhibitor may inhibit the proliferation and induce apoptosis in colon cancer cells through the COX-2-dependent pathway (HT-29) by decreasing the COX-2 mRNA/PGE2 levels and the activity of the COX-2-independent pathway (SW480). The Notch1 signal pathway mediates the effects of the COX-2 inhibitor on the proliferation and apoptosis of colon cancer cells. This may be a new target of the selective COX-2 inhibitor effect on colon cancer.

Topics: Amyloid Precursor Protein Secretases; Antineoplastic Agents; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Blotting, Western; Cell Proliferation; Colonic Neoplasms; Colorimetry; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Dinoprostone; Dipeptides; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Homeodomain Proteins; HT29 Cells; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Membrane Proteins; NF-kappa B p52 Subunit; Nitrobenzenes; Receptor, Notch1; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfonamides; Time Factors; Transcription Factor HES-1