Reg4 Interacts with CD44 to Regulate Proliferation and Stemness of Colorectal and Pancreatic Cancer Cells.

Regenerating Gene 4 (Reg4) is highly upregulated in gastrointestinal (GI) malignancies including colorectal and pancreatic cancers. Numerous studies demonstrated an association between higher Reg4 expression and tumor aggressiveness, intrinsic resistance to apoptotic death, and poor outcomes from GI malignancies. However, the precise receptor and underlying signaling mechanism have remained unknown. Although we previously reported a Reg4-mediated induction of EGFR activity in colorectal cancer cells, a direct interaction between Reg4 and EGFR was not observed. This study is focused on identifying the cell surface binding partner of Reg4 and dissecting its role in colorectal cancer and pancreatic cancer growth and stem cell survival. In vitro models of human colorectal cancer and pancreatic cancer were used to evaluate the results. Results of this study find: (i) Reg4 interacts with CD44, a transmembrane protein expressed by a population of colorectal cancer and pancreatic cancer cells; (ii) Reg4 activates regulated intramembrane proteolysis of CD44 resulting in γ-secretase-mediated cleavage and release of the CD44 intracytoplasmic domain (CD44ICD) that functions as a transcriptional activator of D-type cyclins involved in the regulation of cancer cell proliferation and Klf4 and Sox2 expression involved in regulating pluripotency of cancer stem cells; and (iii) Reg4 significantly increases colorectal cancer and pancreatic cancer cell proliferation and their clonogenic potential in stem cell assays. IMPLICATIONS: These results suggest that pro-proliferative and pro-stemness effects of Reg4 are mediated through γ-secretase-mediated CD44/CD44ICD signaling, hence strategies to disrupt Reg4-CD44-γ-secretase-CD44ICD signaling axis may increase cancer cell susceptibility to chemo- and radiotherapeutics.

Reg4-induced mitogenesis involves Akt-GSK3ß-ß-Catenin-TCF-4 signaling in human colorectal cancer.

Upregulation of regenerating gene 4 (Reg4) is observed in many human gastrointestinal malignancies including colorectal cancer (CRC). We previously reported a Reg4-mediated induction of epidermal growth factor receptor-Akt-AP1 signaling regulating CRC cell apoptosis. However, the role of Reg4 in the regulation of CRC cell division is poorly understood. This study tests the hypothesis that Reg4 induces Akt-GSK3ß-ß-Catenin-TCF-4 signaling to regulate CRC cell division. In vitro models of human CRC were used to determine the role of Reg4 in regulation of CRC cell division. Cell cycle studies demonstrated that Reg4 treatment significantly decreased CRC cell number in G1 phase and increased in G2 phase. Subsequently Reg4 significantly increased the mitotic index of CRC cells. As assessed by real-time RT-PCR and Western blot analyses, Reg4 significantly increased the expression of cell cycle regulatory genes Cyclin D1 and D3, and associated Cyclin-dependent kinases (CDK4 and CDK6). Reg4-mediated increase in these genes involved a pathway that included an induced Akt activity by increasing phosphorylation of Thr308 and Ser473, a reduced glycogen synthase kinase 3ß (GSK-3ß) activity by increasing phosphorylation of Ser9, an induced nuclear translocation of ß-Catenin by decreasing phosphorylation of Ser33/37/Thr41, and an increased TCF-4 transcriptional activity. Furthermore, antagonism of Reg4-signaling using Reg4-specific mAbs (2H6 and 3E5) and Akt inhibitor significantly decreased, whereas agonism using GSK-3ß antagonist (SB216763) significantly increased mitotic index and proliferation of CRC cells. These results identify Reg4 as a key regulator of the CRC cell division and proliferation, hence a potential target of human CRC treatment.

Toll-like receptor-7 ligand Imiquimod induces type I interferon and antimicrobial peptides to ameliorate dextran sodium sulfate-induced acute colitis.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) is associated with a dysregulated mucosal immune response. Certain stimulators of innate immunity (CpG DNA or GM-CSF) are reported to be anti-inflammatory in IBD. Toll-like receptor-7 (TLR7) is an important regulator of innate immunity and its activation plays a key role in induction of type I interferon (IFN). The present study tests the hypothesis that the TLR7 agonists Imiquimod has therapeutic efficacy in IBD. METHODS: Acute colitis was induced in Balb/c mice by giving 5% dextran sodium sulfate (DSS) in drinking water for 7 days. Mice were treated with Imiquimod either orally or topically and its therapeutic effects on disease activity were examined. Isolated mouse CD11c+ dendritic cells and human intestinal epithelial cells (HT29, HCT116) were treated with Imiquimod (10 µg/mL) and their susceptibility to intracellular Salmonella typhimurium infection was assessed by gentamicin protection assay. RESULTS: Oral administration of Imiquimod induced type I IFN expression in the gastrointestinal mucosa and ameliorated DSS-induced acute colitis as assessed by clinical parameters, histology, and mRNA expression of proinflammatory cytokines. Topical administration of Imiquimod also ameliorated DSS colitis by inducing the expression of type I IFN in the colonic mucosa. However, no evidence for a systemic IFN response was observed. Imiquimod treatments to both CD11c+ and intestinal epithelial cells significantly increased expression of antimicrobial peptides (AMPs) and reduced survival of intracellular S. typhimurium. CONCLUSIONS: Imiquimod induces type I IFN and AMP to ameliorate DSS-induced acute colitis and prevents Salmonella survival. Therefore, Imiquimod treatments provide a new therapeutic approach for IBD patients.

Reg IV regulates normal intestinal and colorectal cancer cell susceptibility to radiation-induced apoptosis.

BACKGROUND & AIMS: Regenerating (Reg) gene IV is predominantly expressed in gastrointestinal cells and highly up-regulated in many gastrointestinal malignancies, including colorectal cancer (CRC). Human CRC cells expressing higher levels of Reg IV gene and its protein product (Reg IV) are resistant to conventional therapies, including irradiation (IR). However, the underlying mechanism is not well defined. METHODS: A murine model of IR-induced intestinal injury and in vitro and in vivo models of human CRC were used to determine the role of Reg IV in regulation of normal intestinal and colorectal cancer cell susceptibility to IR-induced apoptosis. RESULTS: Treatments of recombinant human Reg IV (rhR4) protein protected normal intestinal crypt cells from IR-induced apoptosis by increasing the expression of antiapoptotic genes Bcl-2, Bcl-XL, and survivin. However, overexpression of Reg IV in human CRC cells was associated with increased resistance to IR-induced apoptosis. Therefore, we used antagonism of Reg IV as a tool to increase CRC cell susceptibility to IR-induced cell death. Two complementary approaches using specific monoclonal antibodies and small interfering RNAs were tested in both in vitro and in vivo models of human CRC. Both approaches resulted in increased apoptosis and decreased cell proliferation, leading to decreased tumor growth and increased animal survival. Furthermore, these approaches increased CRC cell susceptibility to IR-induced apoptosis. CONCLUSIONS: These results implicate Reg IV as an important modulator of gastrointestinal cell susceptibility to IR; hence, it is a potential target for adjunctive treatments for human CRC and other gastrointestinal malignancies.

Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis.

BACKGROUND: Sargramostim, granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, stimulates cells of the intestinal innate immune system. Clinical trials show that sargramostim induces clinical response and remission in patients with active Crohn's disease. To study the mechanism, we examined the effects of GM-CSF in the dextran sulfate sodium (DSS)-induced acute colitis model. We hypothesized that GM-CSF may work through effects on dendritic cells (DCs). METHODS: Acute colitis was induced in Balb/c mice by administration of DSS in drinking water. Mice were treated with daily GM-CSF or phosphate-buffered saline (PBS). To probe the role of plasmacytoid DCs (pDCs) in the response to GM-CSF, we further examined the effects of monoclonal antibody 440c, which is specific for a sialic acid-binding immunoglobulin (Ig)-like lectin expressed on pDCs. RESULTS: GM-CSF ameliorates acute DSS-induced colitis, resulting in significantly improved clinical parameters and histology. Microarray analysis showed reduced expression of proinflammatory genes including TNF-alpha and IL1-beta; the results were further confirmed by real-time reverse-transcriptase polymerase chain reaction and serum Bio-plex analysis. GM-CSF treatment significantly expands pDCs and type 1 IFN production. Administration of mAb 440c completely blocked the therapeutic effect of GM-CSF. GM-CSF is also effective in RAG1(-/-) mice, demonstrating activity-independent effects on T and B cells. IFN-beta administration mimics the therapeutic effect of GM-CSF in DSS-treated mice. GM-CSF increases systemic and mucosal type 1 IFN expression and exhibits synergy with pDC activators, such as microbial cytosine-phosphate-guanosine (CpG) DNA. CONCLUSIONS: GM-CSF is effective in the treatment of DSS colitis in a mechanism involving the 440c(+) pDC population.

PEGylated murine Granulocyte-macrophage colony-stimulating factor: production, purification, and characterization.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates proliferation, differentiation, and function of hematopoietic progenitor cells. Aside from expansion of hematopoietic cells, GM-CSF has shown efficacy in other diseases, including Crohn's disease. While GM-CSF being clinically used in humans, the ability to perform mechanistic studies in murine models is difficult due to the limited availability and rapid clearance of murine GM-CSF in the peripheral blood. To address these issues, we efficiently expressed murine GM-CSF under the control of the AOX1 gene promoter in Pichia pastoris using the Mut(S) strain KM71H. We describe the unique conditions that are required for efficient production by high-density fermentation and purification of mGM-CSF protein. Recombinant mGM-CSF protein was purified by tangential flow ultrafiltration and preparative reverse phase chromatography. To address limited half life or rapid clearance in mice, recombinant murine GM-CSF was modified by lysine-directed polyethylene glycol conjugation (PEGylation). PEG-modified and unmodified proteins were characterized by amino terminus sequence analysis and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Under the mild reaction conditions, the recombinant protein is efficiently modified by PEGylation on an average of 2-3 sites per molecule. In vivo treatment of mice with PEGylated mGM-CSF, but not the unmodified recombinant mGM-CSF, reproduces the potent colony stimulating effects of human GM-CSF in patients on myeloid progenitor populations, as assessed by FACs analysis. This simplified approach for the expression, purification, and modification of a biologically potent form of murine GM-CSF should facilitate the study of central mechanisms of action in murine disease models.