Insulin-like Growth Factor II mRNA-Binding Protein 1 Regulates Pancreatic Cancer Cell Growth through the Surveillance of CDC25A mRNA.

A number of data indicate that the sources of different kinds of PDAC may be discovered at the transcription/transduction stage. RNA metabolism is manipulated at various steps by different RNA-binding proteins (RBPs), and the deregulation or irregular activity of RBPs is known to contribute to tumor promotion and progression. The insulin-like growth factor 2 mRNA-binding protein family (IMPs), and IMP1 in particular, has been linked with a poor prognosis in PDAC patients; however, little is known about its contribution in PDAC carcinogenesis. In this study, we investigated the function of IMP1 in PDAC. To evaluate IMP1 expression and correlation with PDAC prognosis, we utilized several public databases. Using a specific siRNA IMP1, we analyzed cell death and cell cycle progression in PDAC cell lines and 3D spheroids. The role of IMP1 was also evaluated in vivo in a Panc-1-derived tumor xenograft murine model. Public data suggest that PDAC patients with higher expression of IMP1 showed poor overall and progression-free survival. IMP1 silencing leads to reduced cell growth in PDAC cells and three-dimensional spheroids. Abrogation of IMP1 in PDAC cells showed lower levels of CDC25A, increased phosphorylation of the cyclin-dependent kinase (CDK)2, and accumulation of PDAC cells in the G1 phase. Immunoprecipitation experiments revealed that IMP1 binds CDC25A mRNA, thus controlling cell-cycle progression. Ultimately, we proved that suppression of IMP1 blocked in vivo growth of Panc-1 transferred into immunodeficient mice. Our results indicate that IMP1 drives the PDCA cell cycle and represents a novel strategy for overcoming PDCA cell proliferation.

Corrigendum: An essential role of adenosine deaminase acting on RNA 1 in coeliac disease mucosa.

[This corrects the article DOI: 10.3389/fimmu.2023.1175348.].

An essential role of adenosine deaminase acting on RNA 1 in coeliac disease mucosa.

Background and aim: Type I interferons (IFNs) are highly expressed in the gut mucosa of celiac disease (CD) gut mucosa and stimulates immune response prompted by gluten ingestion, but the processes that maintain the production of these inflammatory molecules are not well understood. Adenosine deaminase acting on RNA 1 (ADAR1), an RNA-editing enzyme, plays a crucial role in inhibiting self or viral RNAs from activating auto-immune mediated responses, most notably within the type-I IFN production pathway. The aim of this study was to assess whether ADAR1 could contribute to the induction and/or progression of gut inflammation in patients with celiac disease. Material and methods: ADAR1 expression was assessed by Real time PCR and Western blotting in duodenal biopsy taken from inactive and active celiac disease (CD) patients and normal controls (CTR). To analyze the role of ADAR1 in inflamed CD mucosa, lamina propria mononuclear cells (LPMC) were isolated from inactive CD and ADAR1 was silenced in with a specific antisense oligonucleotide (AS) and then incubated with a synthetic analogue of viral dsRNA (poly I:C). IFN-inducing pathways (IRF3, IRF7) in these cells were evaluated with Western blotting and inflammatory cytokines were evaluated with flow cytometry. Lastly, the role of ADAR1 was investigated in a mouse model of poly I:C-driven small intestine atrophy. Results: Reduced ADAR1 expression was seen in duodenal biopsies compared to inactive CD and normal controls. Ex vivo organ cultures of duodenal mucosal biopsies, taken from inactive CD patients, stimulated with a peptic-tryptic digest of gliadin displayed a decreased expression of ADAR1. ADAR1 silencing in LPMC stimulated with a synthetic analogue of viral dsRNA strongly boosted the activation of IRF3 and IRF7 and the production of type-I IFN, TNF-α and IFN-γ. Administration of ADAR1 antisense but not sense oligonucleotide to mice with poly I:C-induced intestinal atrophy, significantly increased gut damage and inflammatory cytokines production. Conclusions: These data show that ADAR1 is an important regulator of intestinal immune homeostasis and demonstrate that defective ADAR1 expression could provide to amplifying pathogenic responses in CD intestinal mucosa.

A novel tumour enhancer function of Insulin-like growth factor II mRNA-binding protein 3 in colorectal cancer.

CRC cells evolve a variety of strategies to limit or circumvent apoptosis cell death. RNA binding proteins (RBPs) regulate many of the molecular mechanisms that underlie the development of cancer. The insulin-like growth factor II mRNA-binding proteins (IMP) family are oncofoetal RBPs, consisting of IMP1, IMP2 and IMP3, which have an important role in RNA metabolism. IMP3 is highly expressed in colorectal cancer (CRC) tissue, where its expression often correlates with poor prognosis. However, the role of IMP3 in CRC is not fully understood. IMP3 expression was analysed using a public database and by Western blotting and immunohistochemistry in human colon samples derived from patients with sporadic CRC and healthy subjects. To address whether IMP3 controls cancer cell survival, we analysed cell death pathways in in vitro and in vivo experiments after IMP3 downregulation by siRNA or an antisense oligonucleotide. IMP3 was highly expressed in CRC samples compared to normal control tissues. The knockdown of IMP3 enhanced a caspase-independent cell death in CRC cell lines. Furthermore, the treatment of CRC cells with IMP3 siRNA did not alter the expression of GSDMD, GPX-4 and the activated form of RIP3, three key molecules that govern pyroptosis, ferroptosis and necroptosis, respectively. Abrogation of IMP3 in CRC significantly reduced Bcl-2 and Bcl-xL mRNA and was associated with an altered mitochondrial membrane potential that allowed the nuclear migration of the apoptosis-inducing factor (AIF). Moreover, specific immunoprecipitation experiments on CRC human cell lines indicated that IMP3 binds Bcl-2 and Bcl-xL mRNA, suggesting that IMP3 acts as a regulator of the intrinsic apoptotic pathway through the surveillance of anti-apoptotic Bcl mRNA metabolism. Finally, we showed that IMP3 block inhibited the growth of CRC cell lines in vivo after transplantation into immunodeficient mice. Altogether, these data support a novel role for IMP3 in controlling the intrinsic caspase-independent apoptotic pathway in CRC.

Hepcidin Upregulation in Colorectal Cancer Associates with Accumulation of Regulatory Macrophages and Epithelial-Mesenchymal Transition and Correlates with Progression of the Disease.

Advanced, metastatic colorectal cancer (CRC) is associated with high rate of mortality because of its poor responsiveness to chemotherapy/immunotherapy. Recent studies have shown that hepcidin, a peptide hormone produced mainly by hepatocytes, is expressed by and enhances the growth of tumor cells. We here assessed whether hepcidin expression helps identify subsets of CRC with advanced and aggressive course. By integrating results of in vitro/ex vivo studies with data of bioinformatics databases, we initially showed that hepcidin RNA and protein expression was more pronounced in tissue samples taken from the tumor area, as compared to the macroscopically unaffected, adjacent, colonic mucosa of CRC patients. The induction of hepcidin in the colonic epithelial cell line HCEC-1ct by interleukin (IL)-6, IL-21 and IL-23 occurred via a Stat3-dependent mechanism and, in primary CRC cells, hepcidin co-localized with active Stat3. In CRC tissue, hepcidin content correlated mainly with macrophage accumulation and IL-10 and CD206 expression, two markers of regulatory macrophages. Consistently, both IL-10 and CD206 were up-regulated by hepcidin in blood mononuclear cells. The highest levels of hepcidin were found in metastatic CRC and survival analysis showed that high expression of hepcidin associated with poor prognosis. Moreover, hepcidin expression correlated with markers of epithelial-to-mesenchymal transition and the silencing of hepcidin in CRC cells reduced epithelial-to-mesenchymal transition markers. These findings indicate that hepcidin is markedly induced in the advanced stages of CRC and suggest that it could serve as a prognostic biomarker in CRC.

Smad7 Sustains Stat3 Expression and Signaling in Colon Cancer Cells.

Colorectal cancer (CRC) cells contain elevated levels of active signal transducer and the activator of transcription (Stat)-3, which exerts proliferative and anti-apoptotic effects. Various molecules produced in the CRC tissue can activate Stat3, but the mechanisms that amplify such an activation are yet to be determined. In this paper, we assessed whether Smad7, an inhibitor of Transforiming Growth Factor (TGF)-ß1 activity, sustains Stat3 expression/activation in CRC cells. Both Smad7 and phosphorylated (p)/activated-Stat3 were more expressed in the tumoral areas of CRC patients, compared to the normal adjacent colonic mucosa of the same patients, and were co-localized in primary CRC cells and CRC cell lines. The knockdown of Smad7 with a Smad7 antisense oligonucleotide (AS) reduced p-Stat3 in both unstimulated and interleukin (IL)-6- and IL-22-stimulated DLD-1 and HCT116 cells. Consistently, reduced levels of BCL-xL and survivin, two downstream signaling targets of Stat3 activation, were seen in Smad7 AS-treated cells. An analysis of the mechanisms underlying Smad7 AS-induced Stat3 inactivation revealed that Smad7 AS reduced Stat3 RNA and protein expression. A chromatin immunoprecipitation assay showed the direct regulatory effect of Smad7 on the Stat3 promoter. RNA-sequencing data from the Tumor, Normal and Metastatic (TNM) plot database showed a positive correlation between Smad7 and Stat3 in 1450 CRC samples. To our knowledge, this is the first evidence supporting the theory that Smad7 positively regulates Stat3 function in CRC.

Rafoxanide sensitizes colorectal cancer cells to TRAIL-mediated apoptosis.

Colorectal cancer (CRC) remains a leading causes of cancer-related death in the world, mainly due to the lack of effective treatment of advanced disease. TNF-related apoptosis-inducing ligand (TRAIL)-driven cell death, a crucial event in the control of tumor growth, selectively targets malignant rather than non-transformed cells. However, the fact that cancer cells, including CRC cells, are either intrinsically resistant or acquire resistance to TRAIL, represents a major hurdle to the use of TRAIL-based strategies in the clinic. Agents able to overcome CRC cell resistance to TRAIL have thus great therapeutic potential and many researchers are making efforts to identify TRAIL sensitizers. The anthelmintic drug rafoxanide has recently emerged as a potent anti-tumor molecule for different cancer types and we recently reported that rafoxanide restrained the proliferation of CRC cells, but not of normal colonic epithelial cells, both in vitro and in a preclinical model mimicking sporadic CRC. As these findings were linked with the induction of endoplasmic reticulum stress, a phenomenon involved in the regulation of various components of the TRAIL-driven apoptotic pathway, we sought to determine whether rafoxanide could restore the sensitivity of CRC cells to TRAIL. Our data show that rafoxanide acts as a selective TRAIL sensitizer in vitro and in a syngeneic experimental model of CRC, by decreasing the levels of c-FLIP and survivin, two key molecules conferring TRAIL resistance. Collectively, our data suggest that rafoxanide could potentially be deployed as an anti-cancer drug in the combinatorial approaches aimed at overcoming CRC cell resistance to TRAIL-based therapies.

The Deubiquitinating Enzyme OTUD5 Sustains Inflammatory Cytokine Response in Inflammatory Bowel Disease.

BACKGROUND AND AIMS: The inflammatory bowel disease [IBD]-associated immune response is marked by excessive production of a variety of inflammatory cytokines, which are supposed to sustain and amplify the pathological process. OTUD5 is a deubiquitinating enzyme, which regulates cytokine production by both innate and adaptive immune cells. Here, we investigated the expression and role of OTUD5 in IBD. METHODS: OTUD5 expression was evaluated in mucosal samples of patients with Crohn's disease [CD], patients with ulcerative colitis [UC], and controls, as well as in mice with trinitrobenzene-sulphonic acid [TNBS]-induced colitis by real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. Moreover, OTUD5 was assessed in lamina propria mononuclear cells [LPMC] stimulated with inflammatory cytokines. TNF-α, IL-6, and IL-10 were evaluated in LPMCs of IBD patients and in colitic mice transfected with a specific OTUD5 antisense oligonucleotide [AS]. RESULTS: OTUD5 protein, but not RNA, expression was increased in inflamed ileal and colonic mucosal samples of patients with CD and patients with UC as compared with controls. In IBD, OTUD5-expressing cells were abundant in both epithelial and lamina propria compartments, and non-CD3+, HLA-DR+ LPMC were one of the major sources of the protein. OTUD5 expression was enhanced by IFN-γ through a p38/MAPK-dependent mechanism, and the AS-induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-α. CONCLUSIONS: Our data show that OTUD5 is overexpressed in both CD and UC and suggest the involvement of such a protein in the amplification of the aberrant cytokine response in IBD.

GATA6 Deficiency Leads to Epithelial Barrier Dysfunction and Enhances Susceptibility to Gut Inflammation.

BACKGROUND AND AIMS: Intestinal barrier dysfunction is a hallmark of inflammatory bowel diseases [IBD], but the mechanisms that lead to such a defect are not fully understood. This study was aimed at characterising the factors involved in the defective barrier function in IBD. METHODS: Transcriptome analysis was performed on colon samples taken from healthy controls [CTR] and IBD patients. Expression of GATA-binding factor 6 [GATA6], a transcription factor involved in intestinal epithelial cell differentiation, was evaluated in colon samples taken from CTR and IBD patients by real-time polymerase chain reaction [PCR] and immunohistochemistry. Intestinal sections of wild-type and Gata6del mice, which exhibit a conditional Gata6 deletion in intestinal epithelial cells and which are either left untreated or receive subcutaneous indomethacin or rectal trinitrobenzene sulphonic acid, were stained with haematoxylin and eosin. In parallel, some Gata6del mice received antibiotics to deplete intestinal flora. Mucosal inflammatory cell infiltration and cytokine production were evaluated by flow cytometry and real-time PCR, respectively, and tight junction proteins were examined by immunofluorescence. Intestinal barrier integrity was assessed by fluorescein isothiocyanate [FITC]-dextran assay. RESULTS: Multiple genes involved in cell commitment/proliferation and wound healing were differentially expressed in IBD compared with CTR. Among these, GATA6 was significantly decreased in the IBD epithelium compared with CTR. In mice, conditional deletion of GATA6 in the intestinal epithelium induced primarily epithelial damage, diminished zonula occludens-1 expression, and enhanced intestinal permeability, ultimately resulting in bacteria-driven local immune response and enhanced susceptibility to gut inflammation. CONCLUSIONS: Reduced expression of GATA6 promotes intestinal barrier dysfunction, thus amplifying intestinal inflammatory pathology.

The Fragile X Mental Retardation Protein Regulates RIPK1 and Colorectal Cancer Resistance to Necroptosis.

BACKGROUND & AIMS: The fragile X mental retardation protein (FMRP) affects multiple steps of the mRNA metabolism during brain development and in different neoplastic processes. However, the contribution of FMRP in colon carcinogenesis has not been investigated. METHODS: FMR1 mRNA transcript and FMRP protein expression were analyzed in human colon samples derived from patients with sporadic colorectal cancer (CRC) and healthy subjects. We used a well-established mouse model of sporadic CRC induced by azoxymethane to determine the possible role of FMRP in CRC. To address whether FMRP controls cancer cell survival, we analyzed cell death pathway in CRC human epithelial cell lines and in patient-derived colon cancer organoids in presence or absence of a specific FMR1 antisense oligonucleotide or siRNA. RESULTS: We document a significant increase of FMRP in human CRC relative to non-tumor tissues. Next, using an inducible mouse model of CRC, we observed a reduction of colonic tumor incidence and size in the Fmr1 knockout mice. The abrogation of FMRP induced spontaneous cell death in human CRC cell lines activating the necroptotic pathway. Indeed, specific immunoprecipitation experiments on human cell lines and CRC samples indicated that FMRP binds receptor-interacting protein kinase 1 (RIPK1) mRNA, suggesting that FMRP acts as a regulator of necroptosis pathway through the surveillance of RIPK1 mRNA metabolism. Treatment of human CRC cell lines and patient-derived colon cancer organoids with the FMR1 antisense resulted in up-regulation of RIPK1. CONCLUSIONS: Altogether, these data support a role for FMRP  in controlling RIPK1 expression and necroptotic activation in CRC.

High Smad7 in the early post-operative recurrence of Crohn's disease.

BACKGROUND: In Crohn's disease (CD), one of the major inflammatory bowel disease (IBD) in human beings, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine TGF-ß1. The aim of this study was to assess whether Smad7 over-expression occurs in the early and/or late phases of CD. METHODS: Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with or without (early CD) post-operative endoscopic recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (late CD). Smad7 was examined by immunohistochemistry and cytokine expression was analysed by flow-cytometry. RESULTS: Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of Smad7-expressing cells in both the epithelial and lamina propria compartments. Transition from this stage to endoscopic recurrence was marked by persistence of high number of Smad7-positive cells, which reduced significantly in the late stages of the disease, where Smad7 expression remained, however, greater than that seen in normal controls. In samples with early lesions, Smad7 expression positively correlated with the number of interferon-γ-secreting cells. CONCLUSIONS: Smad7 induction is an early event in the inflammatory sequence occurring in CD, thus suggesting that knockdown of Smad7 can help prevent post-operative recurrence.

A Novel Smad7 Genetic Variant Mapping on the Genomic Region Targeted by Mongersen Is Associated with Crohn's Disease.

BACKGROUND: Down-regulation of Smad7 with a specific Smad7 antisense (AS) oligonucleotide-containing oral drug (Mongersen) was effective in pre-clinical studies and initial clinical trials in Crohn's disease (CD) patients. A recent phase 3 trial was discontinued due to an apparent inefficacy of the drug, but factors contributing to the failure of this study remain unknown. Here, we analysed the frequency in CD of rs144204026 C/T single nucleotide polymorphism (SNP), which maps on the corresponding region targeted by the Smad7 AS contained in the Mongersen formulation and examined whether such a variant allele affects the ability of Smad7 AS to knockdown Smad7. METHODS: rs144204026 SNP frequency was evaluated in two independent Italian cohorts of Crohn's disease patients and normal controls. Genotyping was performed by allelic discrimination assay. Smad7 expression was evaluated in wild-type or heterozygous PBMCs treated with Smad7 AS. RESULTS: No TT genotype was seen in CD patients and controls. Heterozygous genotype was more frequent in CD patients of both cohort 1 (11/235, 4.68%) and cohort 2 (8/122, 6.56%) as compared to controls (6/363, 1.65%; p = 0.029 and p = 0.01 respectively). Overall, a statistically significant association was observed between the T variant allele and CD patients' susceptibility (p = 0.008; OR = 3.28, 95%CI: 1.3-8.3). Smad7 AS down-regulated Smad7 RNA independently of the presence of the variant allele. CONCLUSIONS: This is the first study to show an association between Smad7 rs144204026 SNP and CD patients. Data indicate that such a variant does not negatively influence the in vitro inhibitory effect of Smad7 AS on Smad7.

Rafoxanide Induces Immunogenic Death of Colorectal Cancer Cells.

Colorectal cancer (CRC) is a major cause of cancer-related death in the world. Emerging evidence suggests that the clinical success of conventional chemotherapy does not merely rely on cell toxicity, but also results from the restoration of tumor immune surveillance. Anti-tumor immune response can be primed by immunogenic cell death (ICD), a form of apoptosis associated with endoplasmic reticulum stress (ERS) induction and the expression/release of specific damage-associated molecular patterns (DAMPs). Unfortunately, a limited number of ICD inducers have been identified so far. The anti-helmintic drug rafoxanide has recently showed anti-tumor activity in different cancer types, including CRC. As such latter effects relied on ERS activation, we here investigated whether rafoxanide could promote ICD of CRC cells. The potential of rafoxanide to induce ICD-related DAMPs in both human and mouse CRC cells was assessed by flow-cytometry, chemiluminescent assay and ELISA. In addition, the immunogenic potential of rafoxanide was assessed in vivo using a vaccination assay. Rafoxanide induced all the main DAMPs (ecto-calreticulin exposure, adenosine triphosphate (ATP)/high mobility group box 1 (HMGB1) release) required for ICD. We observed a marked increase of tumor-free survival among immunocompetent mice immunized with rafoxanide-treated dying tumor cells as compared with sham. Altogether, our data indicate rafoxanide as a bona fide ICD inducer.

Interleukin-34 Stimulates Gut Fibroblasts to Produce Collagen Synthesis.

BACKGROUND AND AIM: The mechanisms underlying the formation of intestinal fibrostrictures [FS] in Crohn's disease [CD] are not fully understood, but activation of fibroblasts and excessive collagen deposition are supposed to contribute to the development of FS. Here we investigated whether interleukin-34 [IL-34], a cytokine that is over-produced in CD, regulates collagen production by gut fibroblasts. METHODS: IL-34 and its receptor macrophage colony-stimulating factor receptor 1 [M-CSFR-1] were evaluated in inflammatory [I], FS CD, and control [CTR] ileal mucosal samples by real-time polymerase chain reaction [RT-PCR], western blotting, and immunohistochemistry. IL-34 and M-CSFR-1 expression was evaluated in normal and FS CD fibroblasts. Control fibroblasts were stimulated with IL-34 in the presence or absence of a MAP kinase p38 inhibitor, and FS CD fibroblasts were cultured with a specific IL-34 antisense oligonucleotide, and collagen production was evaluated by RT-PCR, western blotting, and Sircol assay. The effect of IL-34 on the wound healing capacity of fibroblasts was evaluated by scratch test. RESULTS: We showed enhanced M-CSFR-1 and IL-34 RNA and protein expression in FS CD mucosal samples as compared with ICD and CTR samples. Immunohistochemical analysis showed that stromal cells were positive for M-CSFR-1 and IL-34. Enhanced M-CSFR-1 and IL-34 RNA and protein expression was seen in FS CD fibroblasts as compared with CTR. Stimulation of control fibroblasts with IL-34 enhanced COL1A1 and COL3A1 expression and secretion of collagen through a p38 MAP kinase-dependent mechanism, and wound healing. IL-34 knockdown in FS CD fibroblasts was associated with reduced collagen production and wound repair. CONCLUSIONS: Data indicate a prominent role of IL-34 in the control of intestinal fibrogenesis.

Albendazole negatively regulates keratinocyte proliferation.

BACKGROUND: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. AIM: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. METHODS: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. RESULTS: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1ß, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. CONCLUSIONS: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.

Effect of chemical modulation of toll-like receptor 4 in an animal model of ulcerative colitis.

PURPOSE: The partial ineffectiveness and side effects of inflammatory bowel disease (IBD) current therapies drive basic research to look for new therapeutic target in order to develop new drug lead. Considering the pivotal role played by toll-like receptors (TLRs) in gut inflammation, we evaluate here the therapeutic effect of the synthetic glycolipid TLR4 antagonist FP7. METHODS: The anti-inflammatory effect of FP7, active as TLR4 antagonist, was evaluated on peripheral blood mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) isolated from IBD patients, and in a mouse model of ulcerative colitis. RESULTS: FP7 strongly reduced the inflammatory responses induced by lipopolysaccharide (LPS) in vitro, due to its capacity to compete with LPS for the binding of TLR4/MD-2 receptor complex thus inhibiting both the MyD88- and TRIF-dependent inflammatory pathways. Colitic mice treated with FP7 exhibit reduced colonic inflammation and decreased levels of pro-inflammatory cytokines. CONCLUSIONS: This study suggests that TLR4 chemical modulation can be an effective therapeutic approach to IBD. The selectivity of FP7 on TLR4 makes this molecule a promising drug lead for new small molecules-based treatments.

Cadherin-11 Is a Regulator of Intestinal Fibrosis.

BACKGROUND AND AIMS: Although the mechanisms underlying the formation of intestinal fibrostrictures in Crohn's disease [CD] are not fully understood, activation of fibroblasts and excessive collagen deposition are supposed to contribute to the development of such complications. Here, we investigated the role of cadherin-11 [CDH-11], a fibroblast-derived protein that induces collagen production in various organs, in intestinal fibrosis. METHODS: CDH-11 expression was evaluated in inflammatory [I] and fibrostricturing [FS] CD mucosal samples, ulcerative colitis [UC] mucosal samples, and ileal and colonic control samples, by real-time polymerase chain reaction, western blotting, and immunohistochemistry. CDH-11 expression was evaluated in normal and in CD intestinal fibroblasts stimulated with inflammatory/fibrogenic cytokines. FS CD fibroblasts were cultured either with a specific CDH-11 antisense oligonucleotide [AS], or activating CDH-11 fusion protein and activation of RhoA/ROCK, and TGF-ß pathways and collagen production were evaluated by western blotting. Finally, we assessed the susceptibility of CDH-11-knockout [KO] mice to colitis-induced intestinal fibrosis. RESULTS: CDH-11 RNA and protein expression were increased in both CD and UC as compared with controls. In CD, the greater expression of CDH-11 was seen in FS samples. Stimulation of fibroblasts with TNF-α, interleukin [IL]-6, IFN-γ, IL-13, and IL-1ß enhanced CDH-11 expression. Knockdown of CDH-11 in FS CD fibroblasts impaired RhoA/ROCK/TGF-ß signalling and reduced collagen synthesis, whereas activation of CDH-11 increased collagen secretion. CDH-11 KO mice were largely protected from intestinal fibrosis. CONCLUSIONS: Data show that CDH-11 expression is up-regulated in inflammatory bowel disease [IBD] and suggest a role for this protein in the control of intestinal fibrosis.

Induction of endoplasmic reticulum stress and inhibition of colon carcinogenesis by the anti-helmintic drug rafoxanide.

Colorectal cancer (CRC) remains one of the leading causes of mortality worldwide. Drug repositioning is a promising approach for new cancer therapies, as it provides the opportunity to rapidly advance potentially promising agents into clinical trials. The FDA-approved anti-helminthic drug rafoxanide was recently reported to antagonize the oncogenic function of the BRAF V600E mutant protein, commonly found in CRCs, as well as to inhibit the proliferation of skin cancer cells. These observations prompted us to investigate the potential anti-cancer effects of rafoxanide in CRC models. We found rafoxanide inhibited proliferation in CRC cells, but not in normal colonic epithelial cells. Rafoxanide's anti-proliferative action was associated with marked reduction in cyclin D1 protein levels and accumulation of cells in the G0/G1 phase. These effects relied on selective induction of the endoplasmic reticulum stress (ERS) response in CRC cells and were followed by caspase-dependent cell death. Systemic administration of rafoxanide to Apcmin/+ mice induced to develop CRCs caused ERS activation, proliferation inhibition and apoptosis induction in the neoplastic cells. Collectively, our data suggest rafoxanide might be repurposed as an anti-cancer drug for the treatment of CRC.

Progranulin sustains STAT3 hyper-activation and oncogenic function in colorectal cancer cells.

Persistent activation of Signal Transducer and Activator of Transcription (STAT)3 occurs in a high percentage of tumors, including colorectal cancer (CRC), thereby contributing to malignant cell proliferation and survival. Although STAT3 is recognized as an attractive therapeutic target in CRC, conventional approaches aimed at inhibiting its functions have met with several limitations. Moreover, the factors that sustain hyper-activation of STAT3 in CRC are not yet fully understood. The identification of tumor-specific STAT3 cofactors may facilitate the development of compounds that interfere exclusively with STAT3 activity in cancer cells. Here, we show that progranulin, a STAT3 cofactor, is upregulated in human CRC as compared to nontumor tissue/cells and its expression correlates with STAT3 activation. Progranulin physically interacts with STAT3 in CRC cells, and its knockdown with a specific antisense oligonucleotide (ASO) inhibits STAT3 activation and restrains the expression of STAT3-related oncogenic proteins, thus causing cell cycle arrest and apoptosis. Moreover, progranulin knockdown reduces STAT3 phosphorylation and cell proliferation induced by tumor-infiltrating leukocyte (TIL)-derived supernatants in CRC cell lines and human CRC explants. These findings indicate that CRC exhibits overexpression of progranulin, and suggest a role for this protein in amplifying the STAT3 pathway in CRC.