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Tumour Necrosis Factor (TNF) potently induces a transient inflammatory response that must be downregulated once any invasive stimulus has resolved. Yet, how TNF-induced inflammation is shut down in normal cells is incompletely understood. The present study shows that STAT3 was activated in mouse embryo fibroblasts (MEFs) by treatment with TNF or an agonist antibody to TNFR1. STAT3 activation was inhibited by pharmacological inhibition of the Jak2 tyrosine kinase that associates with TNFR1. To identify STAT3 target genes, global transcriptome analysis by RNA sequencing was performed in wild-type MEFs and MEFs from STAT3 knockout (STAT3 KO ) mice that were stimulated with TNF, and the results were validated at the protein level by using multiplex cytokine assays and immunoblotting. After TNF stimulation, STAT3 KO MEFs showed greater gene and protein induction of the inflammatory chemokines Ccl2, Cxcl1 and Cxcl10 than WT MEFs. These observations show that, by activating STAT3, TNF selectively modulates expression of a cohort of chemokines that promote inflammation. The greater induction by TNF of chemokines in STAT3 KO than WT MEFs suggested that TNF induced an inhibitory protein in WT MEFs. Consistent with this possibility, STAT3 activation by TNFR1 increased the expression of Tnfaip3/A20, a ubiquitin modifying enzyme that inhibits inflammation, in WT MEFs but not in STAT3 KO MEFs. Moreover, enforced expression of Tnfaip3/A20 in STAT3 KO MEFs suppressed proinflammatory chemokine expression induced by TNF. Our observations identify Tnfaip3/A20 as a new downstream target for STAT3 which limits the induction of Ccl2, Cxcl1 and Cxcl10 and inflammation induced by TNF., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.

The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR-SOS1-Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras-ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR-RasGRP1 and EGFR-SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma.

Purpose: We sought to determine whether hypoxia-induced radioresistance is mediated by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha)., Methods and Materials: We used 2 mouse embryonic fibroblast cell lines transformed with H-ras and TAg, 1 HIF-1alpha+/+ and the other HIF-1alpha-/-. Cell were exposed to either 95% air and 5% CO2 (normoxic conditions) or 0.2% O2, 94.8% N2, and 5% CO2 (hypoxic conditions) for 4 hours. Cells were then irradiated and subjected to clonogenic survival assays., Results: Whereas neither +/+ ras/TAg nor -/- ras/TAg cells expressed HIF-1alpha under normoxic conditions, hypoxia induced expression of HIF-1alpha only in +/+ ras/TAg cells, confirming the absence of HIF-1alpha in -/- ras/TAg cells. Clonogenic survival curves for +/+ ras/TAg and -/- ras/TAg cells under normoxia and hypoxia demonstrated that hypoxia increased radioresistance in both cell lines to the same degree. At 1-log cell kill, the +/+ ras/TAg and -/- ras/TAg cells had an identical oxygen enhancement ratio of 1.28 +/- 0.09 and nearly identical oxygen enhancement ratios at 2-log cell kill., Conclusion: In our system of transformed mouse embryonic fibroblasts, hypoxia-mediated radiation resistance is independent of HIF-1alpha.

IL-4 has been shown to participate in interregulatory networks with other cytokines including IL-2, IFN-gamma, IL-1 beta, and TNF-alpha. The ability of recombinant IL-4 (rIL-4) to modulate the synthesis and release of IL-6 was investigated in vitro. LPS-stimulated adherent cells (AC) from human peripheral blood secreted IL-6 as determined by ELISA and bioassay with the B9 hybridoma cell line. The secretion of IL-6 peaked between 12-16 h after LPS stimulation. The addition of rIL-4 inhibited LPS-induced IL-6 production measured at 8 h. The inhibitory effect of rIL-4 on IL-6 secretion was dose-dependent and occurred at doses as low as 0.001 U/ml (0.01 pg/ml). Recombinant IL-4 was added to the cultures for various periods and removed by aspiration of the medium and washing the cells. The subsequent LPS-induction of IL-6 secretion was reduced in cultures exposed to rIL-4 for as little as 5 minutes indicating that the effect of rIL-4 on the monocytes was rapid and irreversible. Northern blot analysis revealed that the effect of rIL4 on LPS-induced production of IL-6 by AC occurred at least in part at the level of transcription. In contrast, PMA-induced IL-6 gene transcription was not affected by rIL-4. These findings indicate that rIL-4 can regulate the synthesis of IL-6 by adherent peripheral blood mononuclear cells.

Recombinant human interferon alpha subtype D (RIFN alpha D) was effective in reducing the shedding of herpes simplex virus type-1 (HSV-1) induced by 6-hydroxydopamine iontophoresis followed by topical epinephrine application in previously infected rabbit corneas. A treatment schedule of RIFN alpha D, two drops QID was superior to one drop BID. RIFN alpha A also appeared to be effective in reducing viral shedding. Rabbits treated with RIFN alpha D during two episodes of adrenergically induced HSV-1 shedding, but not during anticipated episodes of spontaneous shedding, did not show a significant reduction in shedding of virus. Interferon was present in significantly higher concentration in tear samples following treatment with RIFN alpha D as compared with RIFN alpha A.

The effects of four subtypes of recombinant human alpha interferon (RIFN alpha), (A,B,D, and the hybrid A/D) were tested on six strains of herpes simplex virus (HSV). RIFN alpha -D was the most effective subtype in rabbit kidney cells, which is consistent with our previous in vivo results in the rabbit herpetic keratitis model. In human corneal cells, however, RIFN alpha -D was one of the least effective IFN subtypes tested. Conversely, RIFN alpha-A appeared to be relatively more effective in the human corneal cells than in the rabbit kidney cells, but RIFN alpha -B and RIFN alpha -A/D were the most effective interferon subtypes in human corneal cells. Different strains of HSV had different susceptibilities to the various IFN subtypes tested.

The use of recombinant human interferon alpha subtype D (RIFN alpha D) was effective in reducing shedding of herpes simplex virus type-1 induced by iontophoresis of 6-hydroxydopamine and epinephrine. A post stimulation treatment schedule of RIFN alpha D, one drop four times a day was as effective as pretreatment, using the same dose regimen. The levels of interferon (IFN) present in the assay system are not sufficient to produce an antiviral effect. The levels of IFN required to suppress cell growth are substantially higher than the concentrations detected in the assay system.