Downregulation Of Dual Specificity Phosphatase Research Articles

Transcriptomic investigation reveals toxic damage due to tilmicosin and potential resistance against tilmicosin in primary chicken myocardial cells

Tilmicosin is widely used to treat respiratory infections in animals and has been reported to induce cardiac damage and even sudden death. However, its exact mechanisms, especially in chickens, remain unclear. This study confirmed the dose-dependent damaging effect of tilmicosin on primary chicken myocardial cells. Primary chicken myocardial cells treated with tilmicosin (0.5 μg/mL) for 0 h, 12 h, and 48 h were subjected to RNA sequencing and bioinformatics analysis. Transcriptomic analysis revealed that cytokine-cytokine receptor interactions, calcium signaling pathway, peroxisomes, phagosomes, mitogen-activated protein kinase (MAPK) signaling pathway, and oxidative phosphorylation were significantly and differentially affected after 12 h or 48 h of tilmicosin treatment. Further evidence demonstrated consistently increased proinflammatory factors, peroxidation, and ferroptosis, and intracellular ion imbalance was caused by tilmicosin for 12 h, but this imbalance had recovered at 48 h. Meanwhile, intracellular resistance to tilmicosin-induced toxicity involved the active regulation of cyclooxygenase-1 and ATPase H+/K+–transporting beta subunit at 48 h, sustained activation of MAPK12, and downregulation of dual specificity phosphatase 10 at 12 h. In summary, this study suggests that tilmicosin exerts its cardiotoxicity in primary chicken myocardial cells through multiple mechanisms and finds several intracellular molecular targets to resist the toxicity.

MeCP2 regulation of cardiac fibroblast proliferation and fibrosis by down-regulation of DUSP5

Cardiac fibrosis is a complex pathological process that includes the abnormal proliferation of cardiac fibroblasts and deposition of the extracellular matrix (ECM) proteins and collagens. Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional nuclear protein, and plays a key role in the fibrotic diseases. However, the potential role of MeCP2 in cardiac fibrosis remains unclear. We report that MeCP2 modulates cardiac fibrosis via down-regulation of dual-specificity phosphatase 5 (DUSP5), a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. MeCP2 is a critical participant in the epigenetic silencing of regulatory genes. Here, we found that down-regulation of DUSP5 in cardiac fibrosis is associated with MeCP2 over-expression. Treatment of cardiac fibroblasts with MeCP2-siRNA blocked proliferation. Knockdown of MeCP2 elevated DUSP5 expression in activated cardiac fibroblasts. Moreover, we investigated the effect of DUSP5 on the ERK1/2 activation. Our results demonstrated that MeCP2 modulates DUSP5 mediated activation of ERK1/2 in cardiac fibrosis. Taken together, these results indicated that MeCP2 acts as a key regulator of pathological cardiac fibrosis, promotes cardiac fibroblasts proliferation and fibrosis by down-regulation of DUSP5.

Abstract B75: CCL2 promotes epithelial-mesenchymal transition (EMT) in prostate cancer cells via downregulation of dual-specificity phosphatase 22

Abstract CCL2 is a member of the CC chemokine superfamily that plays a crucial role in mediating macrophage recruitment and prostate cancer (PCa) metastasis. We have discovered that inhibiting the function of androgen receptor (AR) in prostate cancer cells results in the upregulation CCL2 and promotes disease progression by recruiting macrophages, activating signal transducer and activator of transcription 3 (STAT3), and by favoring the epithelial-to-mesenchymal transition. However, the molecular switches controlling CCL2 downstream signaling in PCa cells are not well understood. Here, we established an in vitro cell migration screening system using transwell migration chambers, and identified a higher amount of CCL2 in migrating PCa cells, establishing a link between CCL2 and a subpopulation of PCa cells. Importantly, we found that CCL2 expression correlated with the EMT marker, N-cadherin in migrating PCa cells. Importantly, CCL2 stimulation in PCa cells enhanced the expression levels of several EMT markers and induced STAT3 activation. In addition, CCL2 stimulation in PCa cells induced the downregulation of dual-specificity phosphatase 22 (DUSP22), which has been shown to act as a negative regulator of the IL-6-induced STAT3 activation through a direct interaction with STAT3. This supports that CCL2-induced DUSP22 downregulation in PCa cells as a potential mechanism to promote EMT. Therefore, we believe CCL2 may promote PCa metastasis in an autocrine manner through promotion of EMT in PCa cells with more aggressive motility behaviors. Targeting of the CCL2-STAT3 axis may offer new approaches to suppress the multifaceted support (immunosuppression, macrophage recruitment, and angiogenesis) of CCL2 for PCa cells in complex tumor microenvironment. Citation Format: Wen-Jye Lin, Kouji Izumi, Showping Lin, Hui-Min Ho. CCL2 promotes epithelial-mesenchymal transition (EMT) in prostate cancer cells via downregulation of dual-specificity phosphatase 22. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B75.

Abstract 5628: Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6.

Abstract Introduction: As the epidermal growth factor receptor (EGFR) is expressed in up to 90% of all HNSCC tumors and initiates important signaling pathways in carcinogenesis, cetuximab, an EGFR targeting agent, is a promising therapeutic agent. However, many tumors remain non-responsive. Therefore, unraveling the underlying mechanism of resistance is of major importance. Previously, we performed gene expression studies of cetuximab sensitive (LICR-HN2 and SC263) and cetuximab resistant (LICR-HN1 and Cal27) cell lines. These results indicated an upregulation of Aurora kinase B (AURKB) in LICR-HN1 cells and a downregulation of dual specificity phosphatase 6 (DUSP6) in Cal27 cells. Methods: Growth inhibitory experiments for barasertib, an AURKB targeting agent, and apigenin, a MAPK inhibitor, were performed as single-agent (barasertib: 0-25nM and apigenin: 0-40μM), and in combination with 15nM cetuximab for 168h using simultaneous and sequential treatment schedules. Induction of apoptotic cell death was investigated in LICR-HN1 cells using the Annexin V-FITC apoptosis detection kit for all treatment schedules. Immunohistochemical staining for AURKB was performed on formalin fixed, paraffin-embedded tissues from 52 newly diagnosed HNSCC patient samples, using an automated IHC staining platform and will be correlated with clinicopathological parameters. The phosphorylation status of ERK was investigated using the NanoPro technology and the methylation status of DUSP6 will be analyzed using the Pyrosequencing method. Results: The mitosis-targeting drug barasertib induced cytotoxicity in the cetuximab resistant HNSCC cell lines, Cal27 and LICR-HN1, with IC50 values of 13.15 ± 0.60nM and 17.6 ± 1.38nM, respectively. In combination therapy with cetuximab, no additional benefit over barasertib as single agent was observed. However, preliminary results showed that pretreatment of LICR-HN1 cells with 15nM cetuximab followed by barasertib (10 or 20nM) resulted in less viable cells compared to cetuximab or barasertib treatment alone. AURKB expression was present in HNSCC patients, with intensities ranging from 0 - 100%. Apigenin induced cytotoxicity in the two cetuximab resistant cell lines, Cal27 and LICR-HN1, with IC50 values of 22.29 ± 0.60μM and 30.66 ± 0.97μM respectively. Preliminary results showed that the combination of cetuximab with apigenin had a synergistic effect in the Cal27 cell line. Furthermore, increased phosphorylation of ERK was present in the cetuximab-resistant cell lines compared to the cetuximab-sensitive cell lines. Conclusion: These results seem to confirm that overexpression of AURKB and/or downregulation of DUSP6 are involved in mechanisms of cetuximab resistance in HNSCC cell lines and inhibition of these pathways might, therefore, be a new strategy for anticancer therapy. Citation Format: Carolien Boeckx, Vanessa Deschoolmeester, An Wouters, Ken Op de Beeckx, Patrick Pauwels, Olivier Vanderveken, Guy Van Camp, Wim Vanden Berghe, Pol Specenier, Jan B. Vermorken, Marc Peeters, Marc Baay, Filip Lardon. Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP6. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5628. doi:10.1158/1538-7445.AM2013-5628

The synergy in cytokine production through MyD88‐TRIF pathways is co‐ordinated with ERK phosphorylation in macrophages

Although specific single Toll-like receptor (TLR) ligands are known to drive the development of Th1 or Th2 immunity, the outcome of different combinations of TLR ligands on innate immunity is not well defined. Spatiotemporal dynamics are critical in determining the specificity of the immune response, but the mechanisms underlying combinatorial TLR stimulation remain unclear. Here, we tested pairwise combinations of TLR ligands separated by different time intervals for their effect on cytokine production in macrophages. We observed that stimulation via a combination of MyD88- and TRIF-utilizing adaptors leads to a highly synergistic cytokine response. On a timescale of 4-24 h, macrophages pretreated with poly(I:C) (TLR3 ligand) are cross-primed to a second stimulation with R848 (TLR7 ligand) and vice versa, and each condition exhibits different optimal time windows of synergistic response for each cytokine. We show that the synergy resulting from combinatorial stimuli (poly(I:C) and R848 is also regulated by the order and dosage of the TLR agonists. Secondary response genes, which depend on new protein synthesis for transcription, show greater synergy than primary response genes, and such enhancement is abolished when new protein synthesis is inhibited. Synergistic cytokine production appears concordant with sustained ERK phosphorylation, suggesting that the de novo factors act via inhibition of ERK dephosphorylation, for example, by the downregulation of dual specificity phosphatase 6. Taken together, our findings illustrate a checkpoint in the innate immune system, where the synchronization of timing of both MyD88 and TRIF pathways is required for a maximal cytokine response and potential memory effect in macrophages.

Down-Regulation of Dual-Specificity Phosphatase 5 in Gastric Cancer by Promoter CpG Island Hypermethylation and Its Potential Role in Carcinogenesis

Dual-specificity phosphatase 5 (DUSP5), which regulates the duration and magnitude of ERK1/2 phosphoactivation within the mitogen-activated protein kinase (MAPK) cascade, has recently been proposed to be a tumor suppressor. However, the epigenetic regulation of DUSP5 and its critical roles in gastric cancer (GC) remain unknown. We compared differential RNA expression profiles of GC cell lines with or without treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine. DUSP5 expression was dramatically decreased by DNA methylation. Hypermethylation of the DUSP5 promoter was detected in GC tissue samples, but not in normal healthy gastric mucosa samples. Restoring DUSP5 expression in DUSP5-silenced GC cell lines decreased their growth and colony-forming ability by causing arrest in the transition from G1 to S phase in the cell cycle as a result of dephosphorylation of ERK1/2 in the nucleus. Moreover, in a set of surgically resected GC cases (n = 179), GCs with DUSP5 promoter region hypermethylation (30.2%) exhibited significantly shortened survival, compared with GCs without DUSP5 methylation (P = 0.009). These results suggest that silencing of DUSP5 by promoter hypermethylation causes increased maintenance of phosphorylated ERK1/2, driving cell proliferation and contributing to gastric carcinogenesis. Furthermore, DUSP5 methylation may serve as a prognostic marker for GC, but this requires validation in a larger set of GC samples.

Transcriptional silencing of dual specificity phosphatase type 2 (DUSP2) abrogates drug-induced apoptosis and predicts cisplatin resistance in head and neck squamous carcinomas

11030 Background: Deregulated mitogen activated protein kinase (MAPK) signalling occurs in many cancers. Mechanisms underlying deregulation of MAPK remain poorly understood. The Dual Specificity Phosphatases (DUSP) are negative regulators of MAPK, but there is little if any evidence of DUSP gene inactivation in human neoplasia. Methods: We used methylation reversal with 5’deazacytidine combined with genome-wide micro-array to identify epigenetically regulated genes in 146 cases of locally advanced, stage III-IV, head and neck squamous cell carcinomas (HNSCC) treated with cisplatin-based chemo-radiotherapy. Analysis of methylation was performed using bisulphite sequencing and methylation specific PCR (MSP). Results: We identified down-regulation of Dual Specificity Phosphatase 2 (DUSP2), a negative regulator of MAPK activity, in HNSCC. Down-regulation of DUSP2 is associated with aberrant methylation in the CpG island located at the 5’end of the gene and is reversible by treatment with demethylating agents....