Wentao Qi

Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity

BackgroundSoy consumption is associated with a lower incidence of colon cancer which is believed to be mediated by one of its of components, genistein. Genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, but mechanisms are not well understood. Epidermal growth factor (EGF)-induced proliferation of colon cancer cells plays an important role in colon cancer progression and is mediated by loss of tumor suppressor FOXO3 activity. The aim of this study was to assess if genistein exerts anti-proliferative properties by attenuating the negative effect of EGF on FOXO3 activity.MethodsThe effect of genistein on proliferation stimulated by EGF-mediated loss of FOXO3 was examined in human colonic cancer HT-29 cells. EGF-induced FOXO3 phosphorylation and translocation were assessed in the presence of genistein. EGF-mediated loss of FOXO3 interactions with p53 (co-immunoprecipitation) and promoter of p27kip1 (ChIP assay) were examined in presence of genistein in cells with mutated p53 (HT-29) and wild type p53 (HCT116). Silencing of p53 determined activity of FOXO3 when it is bound to p53.ResultsGenistein inhibited EGF-induced proliferation, while favoring dephosphorylation and nuclear retention of FOXO3 (active state) in colon cancer cells. Upstream of FOXO3, genistein acts via the PI3K/Akt pathway to inhibit EGF-stimulated FOXO3 phosphorylation (i.e. favors active state). Downstream, EGF-induced disassociation of FOXO3 from mutated tumor suppressor p53, but not wild type p53, is inhibited by genistein favoring FOXO3-p53(mut) interactions with the promoter of the cell cycle inhibitor p27kip1 in colon cancer cells. Thus, the FOXO3-p53(mut) complex leads to elevated p27kip1 expression and promotes cell cycle arrest.ConclusionThese novel anti-proliferative mechanisms of genistein suggest a possible role of combining genistein with other chemoreceptive agents for the treatment of colon cancer.

Tumor suppressor FOXO3 participates in the regulation of intestinal inflammation

Inflammatory bowel disease (IBD), including Crohns disease and ulcerative colitis, is characterized by chronic mucosal injury and the infiltration of inflammatory cells. Tumor suppressor FOXO3 regulates gene expression and its translocation to the cytosol leads to the abrogation of its transcriptional function. We have previously shown that bacterial infection regulates FOXO3 in intestinal epithelial cells and increases cytokine levels. As TNFα is a major contributor in intestinal inflammation, the aim of this study was to assess its effect on FOXO3 and FOXO3s contribution to intestinal inflammation in vitro and in vivo. TNFα induces the translocation of nuclear FOXO3 into the cytosol where it undergoes proteasomal degradation in human intestinal HT-29 cells. Proximally, the PI3K and IKK pathways mediate TNFα-induced FOXO3 phosphorylation. In FOXO3-silenced HT-29 cells, TNFα-induced IL-8 expression is increased ∼83%. In vivo, Foxo3 is present in the nuclei and cytosol of colonic crypt epithelia. In DSS-induced colonic inflammation, Foxo3s nuclear localization is lost and it is only found in the cytosol. Consistent with a role for Foxo3 in colitis, Foxo3-deficient mice treated with DSS developed more severe colonic inflammation with an increased number of intraepithelial lymphocytes and PMNs infiltrated in the epithelia, than wild-type mice. In summary, TNFα inactivates FOXO3 in intestinal epithelia through the PI3K and IKK pathways and FOXO3 inactivation leads to the upregulation of IL-8 in vitro; in vivo Foxo3 is in the cytosol of inflamed colonic epithelia and Foxo3 deficiency leads to severe intestinal inflammation.

FOXO3 Growth Inhibition of Colonic Cells Is Dependent on Intraepithelial Lipid Droplet Density

Background: The loss of FOXO3 is critical for tumor growth. Results: FOXO3 and lipid droplets (LDs) have feedback regulation, and the loss of FOXO3 leading to increased LDs is key in the growth of colonic cells. Conclusion: FOXO3-dependent LDs provide lipid energy critical for cellular growth. Significance: Identifying regulators of cellular lipid energy could provide new targets for colon cancer treatment. Forkhead transcription factor FOXO3 plays a critical role in suppressing tumor growth, in part, by increasing the cell cycle inhibitor p27kip1, and Foxo3 deficiency in mice results in marked colonic epithelial proliferation. Here, we show in Foxo3-deficient colonic epithelial cells a striking increase in intracytoplasmic lipid droplets (LDs), a dynamic organelle recently observed in human tumor tissue. Although the regulation and function of LDs in non-adipocytes is unclear, we hypothesize that the anti-proliferative effect of FOXO3 was dependent on lowering LD density, thus decreasing fuel energy in both normal and colon cancer cells. In mouse colonic tumors, we found an increased expression of LD coat protein PLIN2 compared with normal colonic epithelial cells. Stimulation of LD density in human colon cancer cells led to a PI3K-dependent loss of FOXO3 and a decrease in the negative regulator of lipid metabolism in Sirtuin6 (SIRT6). Foxo3 deficiency also led to a decrease in SIRT6, revealing the existence of LD and FOXO3 feedback regulation in colonic cells. In parallel, LD-dependent loss of FOXO3 led to its dissociation from the promoter and decreased expression of the cell cycle inhibitor p27kip1. Stimulation of LD density promoted proliferation in colon cancer cells, whereas silencing PLIN2 or overexpression of FOXO3 inhibited proliferation. Taken together, FOXO3 and LDs might serve as new targets for therapeutic intervention of colon cancer.

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.

Polyethylene glycol diminishes pathological effects of Citrobacter rodentium infection by blocking bacterial attachment to the colonic epithelia

Infections from enteric bacteria such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are a public health threat worldwide. EPEC and EHEC are extracellular pathogens, and their interaction with host surface receptors is critical to the infection process. We previously demonstrated that polyethylene glycol (PEG) downregulates surface receptors in intestinal cells. Here we show that PEG decreases β1-integrin, the surface receptor in intestinal cells that is critical for EPEC and EHEC attachment. We hypothesized that PEG would inhibit the attachment of these enteric pathogens to host cells and improve clinical signs of infection. We found that attachment of the mouse enteric pathogen Citrobacter rodentium, which belongs to the same group of pathogens as EPEC and EHEC, was attenuated by the concurrent presence of PEG. Pretreatment with PEG, without concurrent presence during infection, also reduced bacterial attachment. This finding was further supported in vivo such as that PEG administered by gavage daily during infection as well as prior to infection significantly decreased C. rodentium in the colon and improved the appearance of the infected colon in mice. In addition, PEG decreased the β1-integrin in colonic mucosa and reduced the C. rodentium-induced activation of epidermal growth factor receptors. PEG also significantly reduced infection-induced colonic inflammation. Finally, PEG efficiently reduced C. rodentium shedding from the colon during infection. In conclusion, PEG can be an efficient and safe preventive agent against EPEC and EHEC infections.

Lipid Droplet Accumulation Promotes Proliferation of Colon Cancer Cells via Loss of Tumor Suppressor FoxO3

Introduction: Epidemiological studies demonstrate that obesity is associated with colon cancer progression. Intracellular lipids are stored in lipid droplets (LDs) in both adipocytes and non-adipocytes cells. LDs consist of a core of neutral lipids surrounded by numerous proteins including perilipin 2 (ADRP). Since tumor cells require excessive energy to serve their high metabolic needs, limited data suggest that LDs can act as a local source of energy. We have previously demonstrated that loss of FOXO3 leads to lipid accumulation in mice, whereas overexpression of FOXO3 inhibits proliferation of colon cancer cells. Aim: We hypothesized that LD accumulation stimulates progression of colon cancer cells via loss of FOXO3 activity. Methods: Cells: colon cancer HT-29 and DLD1 cells and untransformed YAMC. Treatments: EGF (100ng/ml) and Oleic Acid (OA) (1mg/ml). LDs inhibitors: C75 (fatty acid synthase inhibitor; 50 μg/ml) and TOFA (inhibitor of acetyl-CoA carboxylase; 50 μg/ml). LDs quantification: Oil Red-O staining and expression of ADRP. FOXO3 activity and downstream target p27kip1: immunoblots, immunofluorescence, and Chip assay. Proliferation: MTS assay and flow cytometry. Results: OA, a known inducer of LD accumulation in adipocytes, promoted LD accumulation (enlarged LDs: 3-fold in YAMC, 2-fold in HT29 cells) and ADRP expression in colonic cells (non-transformed and cancer). Similarly, EGF, a critical inducer of proliferation in colon cancer cells, increased LD size and expression of ADRP in HT-29 cells. Moreover, proliferation stimulated by OA (47.5±16%) or EGF was effectively suppressed by LD inhibitors (C75 and TOFA), suggesting that LDs are key regulators of the proliferative process in colon cancer cells. OA-stimulated LD accumulation resulted in FOXO3 degradation (79±13%) in the first 6 hours and de novo synthesis of inactive FOXO3 (at 48 hours) within the cytosol. Overexpression of FOXO3 suppressed the proliferative effect of LDs, suggesting that LD mediated loss of FOXO3 activity is a critical modulator of cell cycle. Additionally, LD accumulation led to disassociation of FOXO3 from the promoter and decreased expression of p27kip1, an inhibitor of the cell cycle G0G1 checkpoint. Conclusion: These data support a novel signaling mechanism in colon cancer cells. They point to the loss of FOXO3 activity as a critical step in LD-induced proliferation and suggest that LDs may act as an intracellular source of energy to fuel colon cancer progression.

134 Brahma-Related Gene 1 (BRG1) as a Novel Epigenetic Modulator of Gene Dysregulation in Early Colorectal Carcinogenesis: Implications for Chemoprevention

Understanding the early molecular events in colorectal carcinogenesis is critical for designing novel diagnostic and chemopreventive strategies. One of the key early events is the diffuse dysregulation of gene expression prior to morphological lesions (field carcinogenesis). The mechanisms are believed to be largely epigenetic with methylation and microRNA being well explored. Recently, interest has focused on the SWI/SNF complex, chromatin remodeling proteins that have been implicated in carcinogenesis. Indeed, the complex member Brahmarelated gene 1 (BRG-1) has been implicated in lung and pancreatic cancer. However, colorectal carcinogenesis is largely unexplored. We therefore wanted to explore the role of BRG-1 in colon carcinogenesis and reversal during chemoprevention. Methods: To study the expression of BRG-1, immunohistochemistry studies were performed using different rat colorectal cancer models: the well-established 40-week azoxymethane treated (AOM) model and polyposis in rat colon (Pirc) model. We used the Pirc rat that harbor germline mutations in the APC mutation, the initiating genetic events in most sporadic colorectal cancer. These animals spontaneously develop colonic adenomas at 10 weeks. We utilized sulindac as a chemopreventive agent that was started at 5-6 weeks of age. Furthermore, BRG-1 expression at a message level was studied using human colon cancer cell line HCT116 with and without celecoxib treatment. Results: Immunohistochemistry revealed significantly reduced nuclear expression of BRG-1 in AOM treated colonic mucosa (50% compared to control). Immunohistochemistry of our Pirc rat model revealed reduced nuclear expression of BRG-1 in colonic mucosa (80% compared to wildtype). (Figure 1). Furthermore, Pirc rats treated with sulindac revealed an increase in BRG-1 expression (139% compared to untreated Pirc). (Figure 1) Finally, PCR data revealed that celecoxib treated HCT 116 cells expressed higher message levels of BRG-1 (137% compared to untreated). (Figure 2) Conclusions: We demonstrate, herein, for the first time that BRG-1 is suppressed early during colorectal carcinogenesis. This occurred both in a novel animal model and humans implicating its role as an important epigenetic regulator of early gene expression alterations in the premalignant mucosa. This suggests a role as a biomarker for risk stratification. Furthermore, treatment with an established chemopreventive agent reversed this process supporting the role that BRG-1 may represent a novel therapeutic target.

Su1871 Lactate Dehydrogenase-a Induction as an Early Metabolomic Marker of Colon Carcinogenesis: Potential Target for Chemoprevention

Our current study investigated the effect of PHLPP expression on the efficacy of chemotherapeutic drugs in colon cancer cells. Both PHLPP isoforms, PHLPP1 and PHLPP2, were either knocked down or overexpressed in SW480 and HT29 human colon cancer cells, and the rate of cell proliferation and apoptosis were measured upon drug treatment. Specifically, the cells were treated with different concentrations of oxaliplatin, PI3K inhibitor (LY294002), or rapamycin for 48 hours and cell proliferation was determined using MTS assays. Our results showed that knockdown of both PHLPP isoforms resulted in a decrease in the sensitivity to all drugs tested, whereas overexpression of PHLPP enhanced the anti-proliferation effect of these drugs. Moreover, we assessed the effect of the chemotherapy drugs in inducing cell death by monitoring the appearance of apoptotic markers including cleaved PARP and Caspase-3. Similarly, knockdown of both PHLPP isoforms rendered the cells resistant to apoptosis upon drug treatment. In summary, we have identified PHLPP as a critical factor in determining the drug sensitivity in the chemotherapeutic treatment of colon cancer. Adding yet another novel function of PHLPP and reasoning for the possible use of PHLPP as a therapeutic target in colon cancer.

885 Polyethylene Glycol (PEG) Suppresses Adenomas and Aberrant Crypt Foci (ACF) in the Polyposis in Rat Colon Model (Pirc): Implications for Colorectal Cancer Prevention

G A A b st ra ct s with regular aspirin use was 0.60 (95% CI, 0.47-0.76) among those with GT genotypes and 0.55 (95% CI, 0.38-0.81) with the TT genotypes. In contrast, regular aspirin use was not associated with lower risk among individuals with GG genotypes (multivariate OR, 0.99; 95% CI, 0.72-1.38). Among those with GT/TT genotypes, regular aspirin use appeared more strongly associated with risk of colorectal cancer with positive nuclear CTNNB1 expression (multivariate OR, 0.44; 95% CI, 0.26-0.75), but not negative nuclear CTNNB1 expression (multivariate OR, 0.86; 95% CI, 0.57-1.31). Conclusions: This molecular pathological epidemiology (MPE) study suggests that aspirin reduces risk of colorectal cancer, particularly tumors with activated CTNNB1, among individuals with rs6983267 GT/TT genotypes, but not among individuals with GG genotypes. Our results support an influence of aspirin on WNT signaling and suggest that aspirin chemoprevention may be tailored according to rs6983267 genotype.

886 Polyposis in Rat Colon (Pirc): A Robust Preclinical Model for Chemoprevention of Colorectal Cancer

G A A b st ra ct s with regular aspirin use was 0.60 (95% CI, 0.47-0.76) among those with GT genotypes and 0.55 (95% CI, 0.38-0.81) with the TT genotypes. In contrast, regular aspirin use was not associated with lower risk among individuals with GG genotypes (multivariate OR, 0.99; 95% CI, 0.72-1.38). Among those with GT/TT genotypes, regular aspirin use appeared more strongly associated with risk of colorectal cancer with positive nuclear CTNNB1 expression (multivariate OR, 0.44; 95% CI, 0.26-0.75), but not negative nuclear CTNNB1 expression (multivariate OR, 0.86; 95% CI, 0.57-1.31). Conclusions: This molecular pathological epidemiology (MPE) study suggests that aspirin reduces risk of colorectal cancer, particularly tumors with activated CTNNB1, among individuals with rs6983267 GT/TT genotypes, but not among individuals with GG genotypes. Our results support an influence of aspirin on WNT signaling and suggest that aspirin chemoprevention may be tailored according to rs6983267 genotype.