Alexander Dubeykovskiy

The Murine Gastrin Promoter Is Synergistically Activated by Transforming Growth Factor-β/Smad and Wnt Signaling Pathways

The transforming growth factor-β (TGF-β) and Wnt/wingless pathways play critical roles in the specification of cell fate during development and also contribute to cancer formation and progression. Whereas Wnt signaling is clearly pro-oncogenic, TGF-β signaling is cell- and context-dependent, manifesting both inhibitory and proliferative effects. The growth factor, gastrin, has previously been shown to be a downstream target of the Wnt pathway and a promoter of gastrointestinal cancer. In this study, we show that the mouse gastrin promoter is regulated synergistically by TGF-β/Smads and β-catenin/T-cell factor (TCF). Co-transfection of Smad3/Smad4 and β-catenin expression constructs synergistically activated mouse gastrin promoter activity 30–60-fold in AGS cells with minimal effect seen with either construct alone. This activation was further potentiated by TGF-β1 treatment. Mutating either the TCF binding site or the Smad-binding element (SBE) diminished the activation of gastrin expression by Smad3/Smad4 and β-catenin and led to a loss of gastrin promoter responsiveness to TGF-β1 treatment. Wnt and TGF-β regulated endogenous gastrin mRNA levels in AGS cells in a similar fashion, as revealed by small interference RNA studies or overexpression of Smads and TCF4/β-catenin. Electrophoretic mobility shift assays and DNA affinity precipitation assays showed that the putative SBE and T-cell factor (TCF) sites were able to bind a complex containing Smads and β-catenin/TCF4. In addition, the synergy between Smads and β-catenin/TCF4 was dependent on CREB-binding protein (CBP)/P300, as demonstrated by overexpression of CBP or E1A. Moreover, by using a heterogeneous promoter reporter system, we showed that this complex containing Smads/TCF4/β-catenin complex was able to up-regulate transcription at isolated SBE or TCF sites. Thus, the Wnt signaling pathway is able to activate some target genes through its actions as a co-activator at non-TCF sites and has the potential to profoundly alter transcriptional responses to TGF-β signaling.

Mice That Express Human Interleukin-8 Have Increased Mobilization of Immature Myeloid Cells, Which Exacerbates Inflammation and Accelerates Colon Carcinogenesis

BACKGROUND & AIMS Interleukin (IL)-8 has an important role in initiating inflammation in humans, attracting immune cells such as neutrophils through their receptors CXCR1 and CXCR2. IL-8 has been proposed to contribute to chronic inflammation and cancer. However, mice do not have the IL-8 gene, so human cancer cell lines and xenograft studies have been used to study the role of IL-8 in colon and gastric carcinogenesis. We generated mice that carry a bacterial artificial chromosome that encompasses the entire human IL-8 gene, including its regulatory elements (IL-8Tg mice). METHODS We studied the effects of IL-8 expression in APCmin(+/-) mice and IL-8Tg mice given azoxymethane and dextran sodium sulfate (DSS). We also examined the effects of IL-8 expression in gastric cancer in INS-GAS mice that overexpress gastrin and IL-8Tg mice infected with Helicobacter felis. RESULTS In IL-8Tg mice, expression of human IL-8 was controlled by its own regulatory elements, with virtually no messenger RNA or protein detectable under basal conditions. IL-8 was strongly up-regulated on systemic or local inflammatory stimulation, increasing mobilization of immature CD11b(+)Gr-1(+) myeloid cells (IMCs) with thioglycolate-induced peritonitis, DSS-induced colitis, and H. felis-induced gastritis. IL-8 was increased in colorectal tumors from patients and IL-8Tg mice compared with nontumor tissues. IL-8Tg mice developed more tumors than wild-type mice following administration of azoxymethane and DSS. Expression of IL-8 increased tumorigenesis in APCmin(+/-) mice compared with APCmin(+/-) mice that lack IL-8; this was associated with increased numbers of IMCs and angiogenesis in the tumors. CONCLUSIONS IL-8 contributes to gastrointestinal carcinogenesis by mobilizing IMCs and might be a therapeutic target for gastrointestinal cancers.

Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines

The secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at ∼0.5 μm) activate Ca2+ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1α) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca2+ rise and chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo.

Neural innervation stimulates splenic TFF2 to arrest myeloid cell expansion and cancer

CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) expand in the spleen during cancer and promote progression through suppression of cytotoxic T cells. An anti-inflammatory reflex arc involving the vagus nerve and memory T cells is necessary for resolution of acute inflammation. Failure of this neural circuit could promote procarcinogenic inflammation and altered tumour immunity. Here we show that splenic TFF2, a secreted anti-inflammatory peptide, is released by vagally modulated memory T cells to suppress the expansion of MDSCs through CXCR4. Splenic denervation interrupts the anti-inflammatory neural arc, resulting in the expansion of MDSCs and colorectal cancer. Deletion of Tff2 recapitulates splenic denervation to promote carcinogenesis. Colorectal carcinogenesis could be suppressed through transgenic overexpression of TFF2, adenoviral transfer of TFF2 or transplantation of TFF2-expressing bone marrow. TFF2 is important to the anti-inflammatory reflex arc and plays an essential role in arresting MDSC proliferation. TFF2 offers a potential approach to prevent and to treat cancer.

Flow cytometric detection of progastrin interaction with gastrointestinal cells

The unprocessed gastrin precursor, progastrin (PG), is often overexpressed in colon cancer and other malignancies where it appears to stimulate colonic growth. Overexpression of progastrin also stimulates proliferation of normal colonic mucosa, but the receptors mediating these effects have not been identified. Here we report the development of a non-radioactive assay for assessment of PG binding to normal and transformed cells. Progastrin was labeled using biotinylation, and binding of biotinylated PG to cells was assessed using flow cytometry. Using this approach, we show strong and specific binding of PG to some cell lines (IEC-6, IEC-18, HT-29, COLO320) and minimal binding to others (HeLa, DC2.4, Jurkat). We also found PG binding to several non-gut epithelial lines, such as CHO-K1, COS-6 and HEK293 cells. The specificity of binding was confirmed by competition with cold, unlabeled PG but not with glycine-extended gastrin or amidated gastrin-17. Binding was not influenced by the presence of the classical CCK-2 receptor, but was partially dependent on the charged glycosaminoglycans (GAG). The analysis of primary colonic tissues isolated from wild type C57BL/6 mouse, revealed a small epithelial subpopulation of non-hematopoietic (CD45-negative) cells that strongly interacted with PG. Surprisingly, this population was greatly expanded in gastrin knockout mice. This non-radioactive, FACS-based assay should prove useful for further characterization of cells expressing the progastrin receptor.

P53 Gene Mutation Increases Progastrin Dependent Colonic Proliferation and Colon Cancer Formation in Mice

ABSTRACT Transgenic mice overexpressing human progastrin (hGAS) show colonic crypt hyper-proliferation and elevated susceptibility to colon carcinogenesis. We aimed to investigate effects of p53 mutation on colon carcinogenesis in hGAS mice. We show that introducing a p53 gene mutation further increases progastrin dependent BrdU labeling and results in markedly elevated number of aberrant crypt foci (ACF) and colonic tumors. We demonstrate that hGAS/Lgr5-GFP mice have higher number of Lgr5+ colonic stem cells per crypt when compared to Lgr5-GFP mice indicating that progastrin changes crypt biology through increased stem cell numbers and additional p53 mutation leads to more aggressive phenotype in this murine colon cancer model.

The G-protein coupled receptor 56, expressed in colonic stem and cancer cells, binds progastrin to promote proliferation and carcinogenesis

Overexpression of human progastrin increases colonic mucosal proliferation and colorectal cancer progression in mice. The G-protein coupled receptor 56 (GPR56) is known to regulate cell adhesion, migration, proliferation and stem cell biology, but its expression in the gut has not been studied. We hypothesized that the promotion of colorectal cancer by progastrin may be mediated in part through GPR56. Here, we found that GPR56 expresses in rare colonic crypt cells that lineage trace colonic glands consistent with GPR56 marking long-lived colonic stem-progenitor cells. GPR56 was upregulated in transgenic mice overexpressing human progastrin. While recombinant human progastrin promoted the growth and survival of wild-type colonic organoids in vitro, colonic organoids cultured from GPR56−/− mice were resistant to progastrin. We found that progastrin directly bound to, and increased the proliferation of, GPR56-expressing colon cancer cells in vitro, and proliferation was increased in cells that expressed both GPR56 and the cholecystokinin-2 receptor (CCK2R). In vivo, deletion of GPR56 in the mouse germline abrogated progastrin-dependent colonic mucosal proliferation and increased apoptosis. Loss of GPR56 also inhibited progastrin-dependent colonic crypt fission and colorectal carcinogenesis in the azoxymethane (AOM) mouse model of colorectal cancer. Overall, we found that progastrin binds to GPR56 expressing colonic stem cells, which in turn promotes their expansion, and that this GPR56-dependent pathway is an important driver and potential new target in colorectal carcinogenesis.

Transgenic Mice Expressing Human IL-8 Show Enhanced Inflammatory Responses and Accelerated Colonic and Gastric Carcinogenesis

G A A b st ra ct s polyp number (WT-placebo=3±0.82, WT-DAPT=6.4±1.39). In addition, increased erosion of mucosal layer, infiltration of neutrophils and dysplasia were seen in the polyps of DAPT treated mice compared to placebo both induced with CAC. Western blot analysis indicated significantly decreased protein expression (in percent) of NICD (89.0±20.0), p53 (35.0±20.0), p21WAF1/Cip1 (57.0±9.0), Bax-1 (65.0±7.0) and caspase-3 (90.0±10.0) with Notch-1 inhibition compared to placebo group mice both induced with CAC. TUNEL staining showed decreased apoptosis in the colons of mice treated with DAPT (0.61±0.26) compared to placebo group (14.36±1.12) mice both induced with CAC. MMP-9 overexpression in HCT116 cells resulted in significantly increased protein expression of NICD, p53 and p21WAF1/Cip1 expression (2±0.05, 2.8±0.3, 4.5±0.05 fold increase compared to vector transfection, respectively). Conclusion: MMP-9 via activation of Notch-1 signaling mediates p53 expression and modulates apoptosis, thereby acts as a tumor suppressor in CAC.