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Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma.

ABSTRACT: Cancer-promoting inflammation is an important event in cancer development. Canine urothelial carcinoma (cUC) overexpresses prostaglandin E2 (PGE2) and has a unique sensitivity to cyclooxygenase 2 (COX2)-inhibiting therapy. In addition, majority of cUC harbour BRAFV595E mutation. However, mechanisms underlying aberrant PGE2 production in BRAFV595E cUC patients remain unclear. Drug screening revealed that inhibition of RAF/MEK/ERK pathway, p38 and JNK pathway reduced PGE2 production in cUC cells. By pharmacological inhibition of the multiple components in the pathway, activation of the ERK MAPK pathway was shown to mediate overexpression of COX2 and production of PGE2 in BRAFV595E cUC cells. In silico gain-of-function analysis of the BRAF mutation also implicated involvement of mutation in the process. The positive association between ERK activation and COX2 expression was further validated in the clinical patients. Moreover, it was also suggested that p38 and JNK regulates PGE2 production independently of ERK pathway, possibly through COX2-dependent and COX1-/COX2- independent manner, respectively. In conclusion, this study demonstrated that activation of ERK induces production of PGE2 in BRAFV595E cUC cells, which is also independently regulated by p38 and JNK. With its unique vulnerability to COX-targeted therapy, BRAFV595E cUC may serve as a valuable model to study the tumour-promoting inflammation.

SUBMITTER: Yoshitake R

PROVIDER: S-EPMC7210937 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma.

Yoshitake Ryohei R Saeki Kohei K Eto Shotaro S Shinada Masahiro M Nakano Rei R Sugiya Hiroshi H Endo Yoshifumi Y Fujita Naoki N Nishimura Ryohei R Nakagawa Takayuki T

Scientific reports 20200508 1

Cancer-promoting inflammation is an important event in cancer development. Canine urothelial carcinoma (cUC) overexpresses prostaglandin E2 (PGE2) and has a unique sensitivity to cyclooxygenase 2 (COX2)-inhibiting therapy. In addition, majority of cUC harbour BRAFV595E mutation. However, mechanisms underlying aberrant PGE2 production in BRAFV595E cUC patients remain unclear. Drug screening revealed that inhibition of RAF/MEK/ERK pathway, p38 ...[more]

PMID: 32385388

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Project description:BackgroundLymphangioleiomyomatosis (LAM) is a female-predominant interstitial lung disease, characterized by progressive cyst formation and respiratory failure. Clinical treatment with the mTORC1 inhibitor rapamycin could relieve partially the respiratory symptoms, but not curative. It is urgent to illustrate the fundamental mechanisms of TSC2 deficiency to the development of LAM, especially mTORC1-independent mechanisms. Glutaredoxin-1 (Glrx), an essential glutathione (GSH)-dependent thiol-oxidoreductase, maintains redox homeostasis and participates in various processes via controlling protein GSH adducts. Redox signalling through protein GSH adducts in LAM remains largely elusive. Here, we demonstrate the underlying mechanism of Glrx in the pathogenesis of LAM.Methods1. Abnormal Glrx expression in various kinds of human malignancies was identified by the GEPIA tumour database, and the expression of Glrx in LAM-derived cells was detected by real-time quantitative reverse transcription (RT-qPCR) and immunoblot. 2. Stable Glrx knockdown cell line was established to evaluate cellular impact. 3. Cell viability was determined by CCK8 assay. 4. Apoptotic cell number and intracellular reactive oxygen species (ROS) level were quantified by flow cytometry. 5. Cox2 expression and PGE2 production were detected to clarify the mechanism of Bim expression modulated by Glrx. 6. S-glutathionylated p65 was enriched and detected by immunoprecipitation and the direct regulation of Glrx on p65 was determined. 7. The xenograft animal model was established and photon flux was analyzed using IVIS Spectrum.ResultsIn LAM, TSC2 negatively regulated abnormal Glrx expression and activation in a mTORC1-independent manner. Knockdown of Glrx increased the expression of Bim and the accumulation of ROS, together with elevated S-glutathionylated proteins, contributing to the induction of apoptotic cell death and inhibited cell proliferation. Knockdown of Glrx in TSC2-deficient LAM cells increased GSH adducts on nuclear factor-kappa B p65, which contributed to a decrease in the expression of Cox2 and the biosynthesis of PGE2. Inhibition of PGE2 metabolism attenuated phosphorylation of ERK, which led to the accumulation of Bim, due to the imbalance of its phosphorylation and proteasome degradation. In xenograft tumour models, knockdown of Glrx in TSC2-deficient LAM cells inhibited tumour growth and increased tumour cell apoptosis.ConclusionsCollectively, we provide a novel redox-dependent mechanism in the pathogenesis of LAM and propose that Glrx may be a beneficial strategy for the treatment of LAM or other TSC-related diseases.

Project description:Urothelial carcinoma (UC), also referred to as transitional cell carcinoma (TCC), is the most common bladder malignancy in both human and canine populations. In human UC, numerous studies have demonstrated the prevalence of chromosomal imbalances. Although the histopathology of the disease is similar in both species, studies evaluating the genomic profile of canine UC are lacking, limiting the discovery of key comparative molecular markers associated with driving UC pathogenesis. In the present study, we evaluated 31 primary canine UC biopsies by oligonucleotide array comparative genomic hybridization (oaCGH). Results highlighted the presence of three highly recurrent numerical aberrations: gain of dog chromosome (CFA) 13 and 36 and loss of CFA 19. Regional gains of CFA 13 and 36 were present in 97 % and 84 % of cases, respectively, and losses on CFA 19 were present in 77 % of cases. Fluorescence in situ hybridization (FISH), using targeted bacterial artificial chromosome (BAC) clones and custom Agilent SureFISH probes, was performed to detect and quantify these regions in paraffin-embedded biopsy sections and urine-derived urothelial cells. The data indicate that these three aberrations are potentially diagnostic of UC. Comparison of our canine oaCGH data with that of 285 human cases identified a series of shared copy number aberrations. Using an informatics approach to interrogate the frequency of copy number aberrations across both species, we identified those that had the highest joint probability of association with UC. The most significant joint region contained the gene PABPC1, which should be considered further for its role in UC progression. In addition, cross-species filtering of genome-wide copy number data highlighted several genes as high-profile candidates for further analysis, including CDKN2A, S100A8/9, and LRP1B. We propose that these common aberrations are indicative of an evolutionarily conserved mechanism of pathogenesis and harbor genes key to urothelial neoplasia, warranting investigation for diagnostic, prognostic, and therapeutic applications.

Dataset Information

Aberrant expression of the COX2/PGE2 axis is induced by activation of the RAF/MEK/ERK pathway in BRAFV595E canine urothelial carcinoma.

Publications

Aberrant expression of the COX2/PGE<sub>2</sub> axis is induced by activation of the RAF/MEK/ERK pathway in BRAF<sup>V595E</sup> canine urothelial carcinoma.

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