Retraction: Apigenin inhibits epithelial-mesenchymal transition of human colon cancer cells through NF-κB/Snail signaling pathway.

The HH signaling pathway is critical for normal embryonic development, tissue patterning and cell differentiation. Aberrant HH signaling is involved in multiple human cancers. HH signaling involves a multi-protein cascade activating the GLI proteins that transcriptionally regulate HH target genes. We have recently reported that HH signaling is essential for human colon cancer cell survival, and that inhibition of this signal induces DNA damage and extensive cell death. The current study demonstrates that HH signaling regulates human telomerase reverse transcriptase (hTERT), which determines the limitless replication potential of cancer cells. Suppression of both GLI1 and GLI2 functions by exogenous expression of a C-terminus truncated GLI3 repressor mutant (GLI3R), or by GANT61, a pharmacologic inhibitor of GLI1 and GLI2 transcriptional activity, reduced hTERT protein expression in human colon, prostate and brain cancer (glioblastoma, GBM) cell lines. Exogenous expression of a constitutively active mutant of GLI2, GLI2m, significantly increased hTERT transcription, protein expression, and hTERT promoter-luciferase activity, in human colon cancer cells. Exposure to GANT61 inhibited hTERT mRNA expression in human colon cancer cells. Insilico analysis of the hTERT promoter revealed 7 putative GLI binding sites suggesting a direct transcriptional mode of regulation of hTERT by GLI. Chromatin immunoprecipitation (ChIP) analysis with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, indicating a direct interaction between GLI proteins and the hTERT promoter. The binding between GLI2 and the promoter of hTERT was significantly reduced upon exposure to GANT61. Of interest, exogenous expression of GLI1 or GLI2m in non-cancerous 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter-luciferase activity. Further, ChIP analysis of GFP-tagged GLI2 did not precipitate the hTERT promoter in 293T cells, in contrast to events in malignant cells. GLI2m also increased telomerase activity in human colon cancer cells, while GANT61 reduced the telomerase activity in human colon, prostate and GBM cells. These results demonstrate that the HH signaling pathway directly regulates hTERT by direct interaction with GLI in cancer cells in contrast to non-transformed cells, and identify a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding important regulatory mechanisms that determine the role of HH/GLI signaling in cancer cell survival. Citation Format: Tapati Mazumdar, Ranjodh Sandhu, Maha Qadan, Victoria Magloire, Akwasi Agyeman, Bibo Li, Janet A. Houghton. Hedgehog signaling at the level of GLI transcriptionally regulates hTERT in human cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1790A. doi:10.1158/1538-7445.AM2013-1790A

DNA Hypermethylation Contributes to Incomplete Synthesis of Carbohydrate Determinants in Gastrointestinal Cancer

Tightly coordinated Hedgehog (HH) signaling is critical during normal embryonic development. Aberrant HH signaling is involved in driving cell proliferation in multiple human cancers. Classical HH signaling involves the interaction of the soluble HH ligands with their receptor, Patched (Ptch), thereby releasing a transmembrane protein Smoothened (Smo) from Ptch-mediated inhibition. Smo then activates the Gli family of transcription factors that regulate HH target genes. We have previously reported that HH signaling provides essential survival support to human colon cancer cells and inhibition of this signal induces DNA damage and extensive cell death. However the downstream targets that govern the regulation of cell survival by HH/Gli in cancer cells are not completely defined. This study demonstrates that the Gli proteins transcriptionally regulate the expression of human telomerase reverse transcriptase (hTERT) gene in human cancer cells. hTERT is a known regulator of telomere homeostasis, which determines the replicative potential and hence the life span of cells. Suppression of both Gli1 and Gli2 functions by exogenous expression of a C-terminus truncated Gli3 repressor mutant (Gli3R), or by GANT61, a pharmacological inhibitor of Gli1 and Gli2 activity, reduced hTERT protein expression over a period of 72 hr in human colon, prostate and brain cancer cell lines. Further, exogenously expressed Gli2 significantly increased hTERT protein expression in human colon cancer cell lines. Exposure to GANT61 also inhibited hTERT mRNA expression within 24 hr in human colon cancer cell lines. Insilico analysis of the hTERT promoter revealed 7 putative Gli binding sites suggesting a transcriptional mode of regulation of hTERT expression by Gli2. Chromatin immunoprecipitation with Gli2 antibody precipitated fragments of the hTERT promoter in human colon cancer cell lines, indicating a direct interaction between Gli2 and the hTERT promoter. The binding between Gli2 and hTERT promoter was significantly reduced upon exposure to GANT61. Further, overexpression of the wild type hTERT cDNA in cancer cells prevented the cytotoxic effects of blocking the HH signaling pathway with GANT61. These findings demonstrate hTERT to be a direct transcriptional target and a critical mediator of the HH/Gli signaling pathway, and identify a previously unknown role of the HH/Gli axis in regulating the replication potential of cancer cells. These findings are of significance in understanding important regulatory mechanisms that determine the role of HH/Gli signaling in cancer cell survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4699A. doi:1538-7445.AM2012-4699A

Isovitexin, a biologically active flavone C-glycosylated derivative, has a variety of biological activities. We aimed to identify the effect of isovitexin (Isov) on colon cancer. Human colonic epithelial cells (HCECs) and cancer cells were treated with Isov and Cell Counting Kit-8 (CCK8) was used to detect cell proliferation and calculate the half-inhibitory concentration (IC50). The biological activity of cancer cells was assessed. The tumor size and volume were recorded. Protein expression levels were analyzed by western blotting. Isov inhibited cancer cell proliferation but had little cytotoxicity on HCECs. Isov significantly attenuated cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and induced cell apoptosis., This trend was blocked by insulin-like growth factor-1 (IGF-1) treatment. The expression levels of phosphorylated phosphatidylinositol 3-kinasep (p-PI3K), phosphorylated protein kinase B (p-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), and B cell lymphoma-2 (Bcl-2) decreased when treated with Isov, while the levels of Bcl2-associated X (Bax) and caspase-3 significantly increased. After Isov treatment, the tumor volume and weight were decreased, and the levels of p-PI3K, p-Akt, p-mTOR, and Bcl-2 significantly decreased in tumor tissues. Our findings demonstrated that Isov inhibited cancer cell migration, invasion, and EMT. Isov may be a new potential treatment for colon cancer.

We have previously reported inhibition of cell migration and proliferation induced by difluoromethylornithine (DMFO) and metformin (Met) combination in human melanoma and colon cancer cells. We further investigated the mechanism of cell death with the combination treatment in melanoma and colon cancer cells. The in vivo effect of the combination in melanoma was also determined. Human colon cancer (HCT 116, HT 29) and melanoma (MEL1861, SK-23) were treated with Met, DFMO, or combination. Western blotting was performed to determine the expression of AMP kinase, mTOR, p70S6K and 4E-BP1. Apoptosis was measured by Annexin V assay. Autophagy was determined by fluorescence microscopy staining for LC3A/B and Western blot analysis of LC3A/B and Beclin-1 expression. For in vivo evaluation, SK-23 cells were injected SQ into BALB/c nu mice. After tumor nodule was established, 4 groups of 6 mice : group 1 - IP injections of vehicle and normal drinking water, group 2 - 2% (w/v) DFMO in drinking water; group 3 - IP Met (250 mg/kg/day), group 4 - DFMO plus Met. Mice body weight and tumor volume were measured every 3 days. Tumor weight was measured on Day 20 at necropsy. For comparison between groups, the student's t test was used and p< 0.05 was considered to be significant. DFMO and Met induced apoptosis in colon cancer and melanoma cells in a dose- and time-dependent manner. Significant increase in apoptosis was noted in the combined treatment group compared with either one alone. Increased number of autolysosomes was observed in the combination group under fluorescence microscopy. Up-regulation of Beclin-1 and LC3A/B expression were also increased with combination treatment compared with either treatment alone. Increased expression of phosphor-AMPK, decreases expression of p70S6 and 4EBP1 were observed with the combination treatment. Both DFMO and Met alone have significant in vivo anti-proliferative effect on human melanoma cells compared with control. However, the anti-proliferative effect of the combination treatment was significantly better than either regimen alone (p< 0.001). Average SK-23 tumor weight was 100 mg for control group compared with 45 mg for group 2 (p<0.05), 35mg for group 3 (p< 0.05), and 25mg for the combination group (p< 0.001). Our findings suggest that combination of DFMO and Met induced cancer cell death via apoptosis and autophagy though activation of AMPK pathway and suppression of the Akt/mTOR signaling pathway. DFMO and Met combination can have significant anti-tumor effect in vivo. Citation Format: Yanping Zhang, Guangyong Peng, Eddy C. Hsueh. Induction of autophagy and apoptosis with polyamine synthesis inhibition and metformin in human melanoma and colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1418. doi:10.1158/1538-7445.AM2014-1418

Hyaluronan is a major component of the pericellular matrix surrounding tumor cells, including colon carcinomas. Elevated cycooxygenase-2 levels have been implicated in several malignant properties of colon cancer. We now show for the first time a strong link between hyaluronan-CD44 interaction and cyclooxygenase-2 in colon cancer cells. First, we have shown that increased expression of hyaluronan synthase-2 induces malignant cell properties, including increased proliferation, anchorage-independent growth, and epithelial-mesenchymal transition in HIEC6 cells. Second, constitutive hyaluronan-CD44 interaction stimulates a signaling pathway involving ErbB2, phosphoinositide 3-kinase/AKT, beta-catenin, and cyclooxygenase-2/prostaglandin E(2) in HCA7 colon carcinoma cells. Third, the HA/CD44-activated ErbB2 --> phosphoinositide 3-kinase/AKT --> beta-catenin pathway stimulates cell survival/cell proliferation through COX-2 induction in hyaluronan-overexpressing HIEC6 cells and in HCA7 cells. Fourth, perturbation of hyaluronan-CD44 interaction by hyaluronan oligomers or CD44-silencing RNA decreases cyclooxygenase-2 expression and enzyme activity, and inhibition of cyclooxygenase-2 decreases hyaluronan production suggesting the possibility of an amplifying positive feedback loop between hyaluronan and cyclooxygenase-2. We conclude that hyaluronan is an important endogenous regulator of colon cancer cell survival properties and that cyclooxygenase-2 is a major mediator of these hyaluronan-induced effects. Defining hyaluronan-dependent cyclooxygenase-2/prostaglandin E(2)-associated signaling pathways will provide a platform for developing novel therapeutic approaches for colon cancer.

BackgroundWe have previously reported significant change of epithelial to mesenchymal transition (EMT) phenotype of Eca-109 cells upon PD-L1 operation, and the cytoplasmic domain of PD-L1 played an essential role in promoting EMT of esophageal cancer cells. However, the underlying mechanism of how PD-L1 regulated EMT in esophageal cancer remained unclear.MethodsThe overexpression and knockdown expression models of PD-L1 and IFIT2 were established by using lenti-virus transfection and RNAi method. Western blotting, qRT-PCR, CCK8 assay, transwell assay and wound healing assay were chosen to investigate their impact on the cells. The expression levels of IFIT2 and EMT markers in esophageal cancer tissues were examined by immunohistochemical staining. The rescue experiments were further applied to investigate the role of STAT1/IFIT2 signal pathway in the PD-L1-mediated EMT. Luciferase reporter assays were performed to examine the IFIT2 promoter activities upon knockdown expression of PD-L1 to identify the putative targeted region of IFIT2 promoter.ResultsThe STAT1/IFIT2 signal pathway was activated when PD-L1 was knockdown in human esophageal cancer cells. Decreased IFIT2 expression significantly increased the cellular abilities of viability, invasion and migration by using RNAi method in human esophageal cancer cells. Decreased IFIT2 expression in esophageal cancer tissues significantly correlated with EMT status, and could be used as an independent prognostic predictor for the patients. Rescue experiments in PD-L1 knockdown cells further confirmed that STAT1/IFIT2 pathway was involved in the PD-L1 mediated EMT of esophageal cancer cells. Moreover, the luciferase reporter assay also confirmed that in esophageal cancer cells, the promoter region of IFIT2 (-3K~-1K) remains more active in PD-L1 knockdown expression cells compared with controls.ConclusionOur present work reveals a novel mechanism of how PD-L1 regulates EMT of cancer cells, namely STAT1/IFIT2 signal pathway is required in PD-L1 mediated EMT in human esophageal cancer.

The aim of the present study was to determine the mechanism by which advanced glycation end products (AGEs) induce proliferation, invasion and epithelial-mesenchymal transition (EMT) of human colon cancer SW480 cells. SW480 cells were divided into groups as follows: i) Control; ii) cells treated with AGEs alone; and iii) cells treated with AGEs combined with LY294002. Proliferation, cell cycle progression, apoptosis, invasion and migration of SW480 cells were assessed using an MTT assay, flow cytometry, Transwell assays and a wound healing assay, respectively. The protein expression levels of PI3K, AKT and epithelial cadherin (E-cadherin) were examined by western blot analysis in SW480 cells treated with various concentrations of AGEs. Proliferation, invasion and migration were enhanced, cell cycle progression was increased and apoptosis was decreased in SW480 cells treated with AGEs compared with the control. The PI3K inhibitor, LY294002, reversed the effects of AGEs. Western blot analysis data demonstrated that AGEs increased the protein expression levels of PI3K and AKT, and decreased the expression of E-cadherin. The results suggested that AGEs exert a positive effect on the proliferation, invasion and EMT in SW480 cells through the PI3K/AKT signaling pathway.

Galectin-3 Modulates MUC2 Mucin Expression in Human Colon Cancer Cells at the Level of Transcription via AP-1 Activation

Ginsenoside Rb2 inhibits epithelial-mesenchymal transition of colorectal cancer cells by suppressing TGF-β/Smad signaling

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability.

Up-regulation of inflammatory cytokines such as IL-1β in tumor microenvironment has been associated with progression of human colorectal cancer, although the exact molecular mechanisms are still unclear. To understand how IL-1β influences colon cancer cell behaviors, we used a human colon cancer cell line HCT-116 as well as primary human colon cancer cells derived from a patient, which more closely reflect the properties of the primary tumor than established cell lines. Here, we demonstrate that IL-1β promotes epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) development in colon cancer. IL-1β treatment resulted in loss of E-cadherin and gain of a mesenchymal phenotype in both HCT-116 cells and primary colon cancer cells. Among E-cadherin suppressors, Zeb1 was up-regulated in IL-1β-induced EMT cells. Consistent with EMT properties, IL-1β-treated HCT-116 cells exhibited higher cell migratory ability. Moreover, IL-1β induced expression of stem-cell marker Bmi-1 in both HCT-116 cells and primary colon cancer cells. IL-1β increased these cells’ self-renewal ability to form spheres in serum-free conditions and chemo-resistance; properties associated with colon CSCs. Taken together, these results indicate that IL-1β can induce EMT and promote generation of CSCs in colon cancer. More importantly, we found that ZEB1, but not other E-cadherin transcriptional repressors (such as Zeb2, Snail and Twist), was consistently up-regulated in IL-1β-induced EMT cells and sphere cells. This suggests that Zeb1 links IL-1β-induced CSC self-renewal and EMT in colon cancer cells. Moreover, knockdown of Zeb1 in HCT-116 cells using shRNAs reversed the IL-1β effects on these two processes, further indicating that Zeb1 is essential for IL-1β-induced stem cell and EMT phenotypes in colon cancer cells. Our finding indicates that IL-1β promotes colon tumor growth and invasion through activation of CSC self-renewal and EMT, while Zeb1 plays a critical role in activation of these two processes. Thus, targeting tumor microenvironment cytokines or Zeb1 might form the basis of a promising treatment for colon cancer. Citation Format: Lei Wang, Yijing Li, Amy Beckley, Lorretta Pappan, Jishu Shi. Zeb1 regulates inflammation-induced invasion and self-renewal of colon cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-326. doi:10.1158/1538-7445.AM2013-LB-326 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Aurora kinase is a family of serine/threonine kinases intimately associated with mitotic progression and the development of human cancers. Studies have shown that aurora kinases are important for the protein kinase C (PKC)-induced invasion of colon cancer cells. Recent studies have shown that aurora kinase A promotes distant metastasis by inducing epithelial-to-mesenchymal transition (EMT) in colon cancer cells. However, the role of aurora kinase A in colon cancer metastasis remains unclear. In this study, we investigated the effects of aurora kinase A on PKC-induced cell invasion, migration, and EMT in human SW480 colon cancer cells. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) changed the expression levels of EMT markers, increasing α-SMA, vimentin, and MMP-9 expression and decreasing E-cadherin expression, with changes in cell morphology. TPA treatment induced EMT in a PKC-dependent manner. Moreover, the inhibition of aurora kinase A by siRNAs and inhibitors (reversine and VX-680) suppressed TPA-induced cell invasion, migration, and EMT in SW480 human colon cells. Inhibition of aurora kinase A blocked TPA-induced vimentin and MMP-9 expression, and decreased E-cadherin expression. Furthermore, the knockdown of aurora kinase A decreased the transcriptional activity of NF-κB and AP-1 in PKC-stimulated SW480 cells. These findings indicate that aurora kinase A induces migration and invasion by inducing EMT in SW480 colon cancer cells. To the best of our knowledge, this is the first study that showed aurora kinase A is a key molecule in PKC-induced metastasis in colon cancer cells.

Background: Epithelial-mesenchymal transition (EMT) is a tumor progression-related process by which epithelial cells lose their epithelial characteristics and acquire mesenchymal properties. EMT promotes the malignant phenotypes including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Therefore, to prevent tumor progression, EMT targeted therapy is required. Oncolytic virotherapy is a promising strategy for anticancer therapy. Although a variety of oncolytic adenoviruses have been developed to induce tumor-selective cell death, the effect of oncolytic adenovirus on the EMT-induced tumor progression remains unclear. In this study, we investigated the biological effect of oncolytic adenovirus on EMT in human cancer cells. Methods: We previously generated a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. Transforming growth factor-β (TGF-β) was used to induce EMT in human lung (A549) and pancreatic (Panc-1) cancer cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT in these cells, western blot and real-time PCR analysis for EMT-related markers were performed. We also examined cell mobility using transwell migration and invasion assays. In vitro antitumor effect of OBP-301 and chemotherapeutic agents in TGF-β-treated cancer cells were assessed by trypan blue exclusion assay. Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-like cell morphology, down-regulation of E-cadherin, up-regulation of vimentin and high motility, in A549 and Panc-1 cells. When TGF-β-treated cancer cells were infected with OBP-301, suppression of E-cadherin expression was attenuated. Conversely, the expression levels of mesenchymal markers, vimentin and N-cadherin, and EMT-inducing transcription factors, Snail, Slug and ZEB1, were reduced. OBP-301 infection also inhibited TGF-β-induced enhancement of migration and invasion in cancer cells. Moreover, OBP-301 efficiently killed TGF-β-treated cancer cells, whereas TGF-β-treated cancer cells were resistant to cisplatin and docetaxel. Conclusion: Our results suggest that telomerase-specific oncolytic adenovirus OBP-301 inhibits TGF-β-induced EMT in human cancer cells. OBP-301 has therapeutic potential against cancer cells that have undergone EMT. Therefore, OBP-301 might be a promising anticancer agent to suppress EMT-mediated cancer progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 343. doi:1538-7445.AM2012-343

BackgroundEpidemiological studies demonstrate that the incidence and mortality rates of colorectal cancer in women are lower than in men. However, it is unknown if 17β-estradiol treatment is sufficient to inhibit prostaglandin E2 (PGE2)-induced cellular motility in human colon cancer cells.MethodsWe analyzed the protein expression of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), matrix metallopeptidases (MMPs), plasminogen activator inhibitor-1 (PAI-1) and tissue inhibitor of metalloproteinases (TIMPs), and the cellular motility in PGE2-stimulated human LoVo cells. 17β-Estradiol and the inhibitors including LY294002 (Akt activation inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), QNZ (NFκB inhibitor) and ICI 182 780 were further used to explore the inhibitory effects of 17β-estradiol on PGE2-induced LoVo cell motility. Student's t-test was used to analyze the difference between the two groups.ResultsUpregulation of urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA) and matrix metallopeptidases (MMPs) is reported to associate with the development of cancer cell mobility, metastasis, and subsequent malignant tumor. After administration of inhibitors including LY294002, U0126, SB203580, SP600125 or QNZ, we found that PGE2 treatment up-regulated uPA and MMP-9 expression via JNK1/2 signaling pathway, thus promoting cellular motility in human LoVo cancer cells. However, PGE2 treatment showed no effects on regulating expression of tPA, MMP-2, plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinase-1, -2, -3 and -4 (TIMP-1, -2, -3 and -4). We further observed that 17β-estradiol treatment inhibited PGE2-induced uPA, MMP-9 and cellular motility by suppressing activation of JNK1/2 in human LoVo cancer cells.ConclusionsCollectively, these results suggest that 17β-estradiol treatment significantly inhibits PGE2-induced motility of human LoVo colon cancer cells.