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NFκB-Activated COX2/PGE₂/EP4 Axis Controls the Magnitude and Selectivity of BCG-Induced Inflammation in Human Bladder Cancer Tissues.

ABSTRACT: Bacillus Calmette-Guérin (BCG) is commonly used in the immunotherapy of bladder cancer (BlCa) but its effectiveness is limited to only a fraction of patients. To identify the factors that regulate the response of human BlCa tumor microenvironment (TME) to BCG, we used the ex vivo whole-tissue explant model. The levels of COX2 in the BCG-activated explants closely correlated with the local production of Treg- and MDSCS attractants and suppressive factors, while the baseline COX2 levels did not have predictive value. Accordingly, we observed that BCG induced high levels of MDSC- and Treg-attracting chemokines (CCL22, CXCL8, CXCL12) and suppressive factors (IDO1, IL-10, NOS2). These undesirable effects were associated with the nuclear translocation of phosphorylated NFκB, induction of COX2, the key enzyme controlling PGE₂ synthesis, and elevation of a PGE₂ receptor, EP4. While NFκB blockade suppressed both the desirable and undesirable components of BCG-driven inflammation, the inhibitors of PGE₂ synthesis (Celecoxib or Indomethacin) or signaling (EP4-selective blocker, ARY-007), selectively eliminated the induction of MDSC/Treg attractants and immunosuppressive factors but enhanced the production of CTL attractants, CCL5, CXCL9 and CXCL10. PGE₂ blockade allowed for the selectively enhanced migration of CTLs to the BCG-treated BlCa samples and eliminated the enhanced migration of Tregs. Since the balance between the CTLs and suppressive cells in the TME predicts the outcomes in patients with BlCa and other diseases, our data help to elucidate the mechanisms which limit the effectiveness of BCG therapies and identify new targets to enhance their therapeutic effects.

SUBMITTER: Ibrahim OM

PROVIDER: S-EPMC7998891 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundProstaglandin E2 (PGE2) is the major product of Cyclooxygenase-2 (COX-2) in colorectal cancer (CRC). We aimed to assess PGE2 cell surface receptors (EP 1-4) to examine the mechanisms by which PGE2 regulates tumour progression.MethodsGene expression studies were performed by quantitative RT-PCR. Cell cycle was analysed by flow cytometry with cell proliferation quantified by BrdU incorporation measured by enzyme immunoassay. Immunohistochemistry was employed for expression studies on formalin fixed paraffin embedded tumour tissue.ResultsEP4 was the most abundant subtype of PGE2 receptor in HT-29 and HCA7 cells (which show COX-2 dependent PGE2 generation) and was consistently the most abundant transcript in human colorectal tumours (n = 8) by qRT-PCR (ANOVA, p = 0.01). G0/G1 cell cycle arrest was observed in HT-29 cells treated with SC-236 5 microM (selective COX-2 inhibitor) for 24 hours (p = 0.02), an effect abrogated by co-incubation with PGE2 (1 microM). G0/G1 arrest was also seen with a specific EP4 receptor antagonist (EP4A, L-161982) (p = 0.01). Treatment of HT-29 cells with either SC-236 or EP4A caused reduction in intracellular cAMP (ANOVA, p = 0.01). Early induction in p21WAF1/CIP1 expression (by qRT-PCR) was seen with EP4A treatment (mean fold increase 4.4, p = 0.04) while other genes remained unchanged. Similar induction in p21WAF1/CIP1 was also seen with PD153025 (1 microM), an EGFR tyrosine kinase inhibitor, suggesting EGFR transactivation by EP4 as a potential mechanism. Additive inhibition of HCA7 proliferation was observed with the combination of SC-236 and neutralising antibody to amphiregulin (AR), a soluble EGFR ligand. Concordance in COX-2 and AR localisation in human colorectal tumours was noted.ConclusionCOX-2 regulates cell cycle transition via EP4 receptor and altered p21WAF1/CIP1 expression. EGFR pathways appear important. Specific targeting of the EP4 receptor or downstream targets may offer a safer alternative to COX-2 inhibition in the chemoprevention of CRC.

Project description:BACKGROUND:Interleukin-33 (IL-33) participates in various types of diseases including cancers. Previous studies of this cytokine in cancers mainly focused on its regulation on immune responses by which IL-33 modulated cancer progression. The IL-33 triggered signals in cancer cells remain unclear. METHODS:We analyzed IL-33 gene expression in human colorectal cancer (CRC) tissues and carried out gene enrichment analysis with TCGA Data Portal. We studied CRC proliferation in vivo by inoculating MC38 tumors in IL-33 transgenic mice. We investigated the cell proliferation in vitro with primary CRC cells isolated from fresh human CRC tissues, human CRC cell line HT-29 and mouse CRC cell line MC38. To evaluate the proliferation modulating effects of recombinant IL-33 incubation and other administrated factors, we measured tumor growth, colony formation, cell viability, and the expression of Ki67 and proliferating cell nuclear antigen (PCNA). We used several inhibitors, prostaglandin E2 (PGE2) neutralizing antibody, ST2 blocking antibody and specific shRNA expressing plasmid to study the pathway mediating IL-33-induced CRC proliferation. The IL-33 receptor ST2 in human CRC tissues was detected by immunohistochemistry staining and western blotting. The ST2-positive or negative subsets of primary CRC cells were acquired by flow cytometry sorting. RESULTS:We found that IL-33 expression was correlated with the gene signature of cell proliferation in 394 human CRC samples. The MC38 tumors grew more rapidly and the tumor Ki67 and PCNA were expressed at higher levels in IL-33 transgenic mice than in wild-type mice. IL-33 promoted cell growth, colony formation and expression of Ki67 and PCNA in primary CRC cells as well as CRC cell lines. IL-33 activated cycloxygenase-2 (COX2) expression and increased PGE2 production, whereas the COX2 selective inhibitor and PGE2 neutralizing antibody abolished the proliferation promoting effect of IL-33. ST2 blockade, ST2-negative sorting, NF-?B specific inhibitor and NF-?B specific shRNA (shP65) abrogated the COX2 induction caused by IL-33. CONCLUSION:IL-33 facilitates proliferation of colorectal cancer dependent on COX2/PGE2. IL-33 functions via its receptor ST2 and upregulates COX2 expression through NF-?B signaling. Understanding the IL-33 signal transduction in CRC cells provides potential therapeutic targets for clinical treatment.

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NFκB-Activated COX2/PGE₂/EP4 Axis Controls the Magnitude and Selectivity of BCG-Induced Inflammation in Human Bladder Cancer Tissues.

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NFκB-Activated COX2/PGE2/EP4 Axis Controls the Magnitude and Selectivity of BCG-Induced Inflammation in Human Bladder Cancer Tissues.

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OmicsDI is part of the ELIXIR infrastructure

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NFκB-Activated COX2/PGE₂/EP4 Axis Controls the Magnitude and Selectivity of BCG-Induced Inflammation in Human Bladder Cancer Tissues.