COX-2-dependent PGE2 acts as a growth factor in mycosis fungoides (MF)

Abstract

Cancer often originates from a site of persistent inflammation, and the mechanisms turning chronic inflammation into a driving force of carcinogenesis are intensely investigated. Cyclooxygenase-2 (COX-2) is an inducible key modulator of inflammation that carries out the rate-limiting step in prostaglandin synthesis. Aberrant COX-2 expression and prostaglandin E 2 (PGE 2 ) production have been implicated in tumorigenesis. In this study we show that COX-2 is ectopically expressed in malignant T-cell lines from patients with cutaneous T-cell lymphoma (CTCL) as well as in situ in lymphocytic cells in 21 out of 22 patients suffering from mycosis fungoides (MF) in plaque or tumor stage. COX-2 is not expressed in lymphocytes of 11 patients with patch-stage MF, whereas sporadic COX-2 staining of stromal cells is observed in the majority of patients. COX-2 expression correlates with a constitutive production of PGE 2 in malignant T cells in vitro. These cells express prostaglandin receptors EP3 and EP4 and the receptor antagonist as well as small interfering RNA (siRNA) directed against COX-2, and specific COX-2 inhibitors strongly reduce their spontaneous proliferation. In conclusion, our data indicate that COX-2 mediated PGE 2 exerts an effect as a tumor growth factor in MF.

STAT5-mediated expression of oncogenic miR-155 in cutaneous T-cell lymphoma

“…mRNA was purified with mRNeasy Kit (Qiagen) and transcribed to cDNA as described previously. 49 Quantitative analysis of BIC expression was performed using DyNAmo™ Flash SYBR ® Green qPCR Kit (Finnzymes) with BIC (pri-miR-155)-specific primers (forward: 5'-CCC AAT GGA GAT GGC TCT AA-3'; reverse: 5'-AGG AGT CAG TTG GAG GCA AA-3') according to the manufacturer's instructions. Amplification was performed in an Mx3000P real-time thermal cycler (Stratagene).…”

“…41 All cell lines were cultured as described previously. 26,29,30,[49][50][51] Peripheral blood mononuclear cells (PBMCs) from a healthy donor were isolated by density gradient centrifugation using Lymphoprep (Axis-Shield, PoC AS) as CTCL patients as well as PBMCs from healthy individuals also display a significant upregulation of BIC/miR-155 upon STAT5 activation by IL-2Rβγ cytokines (IL-2 and IL-15), indicating that transcriptional regulation of BIC by STAT5 is not a unique feature of malignant T cells, but a common regulatory pathway.…”

“…Malignant T cells lines (MyLa2059, PB2B, SeAx and SeZ4.1) and non-malignant T cell lines (MySi, MyLa1850) were obtained from CTCL patients and have been described perviously. 26,29,30,49 MAC2A is an anaplastic large T-cell lymphoma (ALCL) cell line established from a skin tumor in the progressive phase of the disease. 50 The Jurkat cell line (J-Tag) has been described elsewhere.…”

“…Amplification with BIC (pri-miR-155) (forward: 5'-CTC TAA TGG TGG CAC AAA-3', reverse: 5'-TGA TAA AAA CAA ACA TGG GCT TGA C-3') and GAPDH (forward: 5'-CCA TGG AGA AGG CTG GGG-3', reverse: 5'-CAA AGT TGT CAT GGA TGA CC-3')-specific primers was performed as described previously. 49 …”

“…Cells were transfected with small interfering RNA (siRNA) against STAT3, STAT5A, STAT5B and nontargeting control #1 (ONtarget PLUS, smart pool, Dharmacon) or siRNA directed against BIC and universal negative control #1 (Sigma) using 0.5 nmol of the respective siRNA on 2 × 10 6 cells with an Amaxa Nucleofector as described previously. 49 For proliferation assays, the same protocol was applied to transfect cells with miR-155 antagomiRs and negative control (mirVana™ miRNA Inhibitors, Ambion ® ) using a concentration of 300 nM. described before.…”