Dual-specificity Phosphatase-2 Expression Research Articles

The SKA3-DUSP2 Axis Promotes Gastric Cancer Tumorigenesis and Epithelial-Mesenchymal Transition by Activating the MAPK/ERK Pathway

Background: Spindle and kinetochore-related complex subunit 3 (SKA3), a member of the SKA family of proteins, is associated with the progression of multiple cancers. However, the role of SKA3 in gastric cancer has not been studied. Methods: The expression levels of SKA3 and dual-specificity phosphatase 2 (DUSP2) proteins were detected by immunohistochemistry. The effects of SKA3 and DUSP2 on the proliferation, migration, invasion, adhesion, and epithelial-mesenchymal transition of gastric cancer were studied in vitro and in vivo. Results: Immunohistochemical analysis of 164 cases of gastric cancer revealed that high expression of SKA3 was negatively correlated with DUSP2 expression and related to N stage, peritoneal metastasis, and poor prognosis. In vitro studies showed that silencing SKA3 expression inhibited the proliferation, migration, invasion, adhesion and epithelial-mesenchymal transition of gastric cancer. In vivo experiments showed that silencing SKA3 inhibited tumor growth and peritoneal metastasis. Mechanistically, SKA3 negative regulates the tumor suppressor DUSP2 and activates the MAPK/ERK pathway to promote gastric cancer. Conclusion: Our results indicate that the SKA3-DUSP2-ERK1/2 axis is involved in the regulation of gastric cancer progression, and SKA3 is a potential therapeutic target for gastric cancer.

DUSP2-mediated inhibition of tubular epithelial cell pyroptosis confers nephroprotection in acute kidney injury.

Rationale: Acute kidney injury (AKI) is pathologically characterized by renal tubular epithelial cell (RTEC) death and interstitial inflammation, while their pathogenesis remains incompletely understood. Dual-specificity phosphatase 2 (DUSP2) recently emerges as a crucial regulator of cell death and inflammation in a wide range of diseases, but its roles in renal pathophysiology are largely unknown.Methods: The expression of DUSP2 in the kidney was characterized by histological analysis in renal tissues from patients and mice with AKI. The role and mechanism of DUSP2-mediated inhibition of tubular epithelial cell pyroptosis in AKI were evaluated both in vivo and in vitro, and confirmed in RTEC-specific deletion of DUSP2 mice.Results: Here, we show that DUSP2 is enriched in RTECs in the renal tissue of both human and mouse and mainly positions in the nucleus. Further, we reveal that loss-of-DUSP2 in RTECs not only is a common feature of human and murine AKI but also positively contributes to AKI pathogenesis. Especially, RTEC-specific deletion of DUSP2 sensitizes mice to AKI by promoting RTEC pyroptosis and the resultant interstitial inflammation. Mechanistic studies show that gasdermin D (GSDMD), which mediates RTEC pyroptosis, is identified as a transcriptional target of activated STAT1 during AKI, whereas DUSP2 as a nuclear phosphatase deactivates STAT1 to restrict GSDMD-mediated RTEC pyroptosis. Importantly, DUSP2 overexpression in RTECs via adeno-associated virus-mediated gene transfer significantly ameliorates AKI.Conclusion: Our findings demonstrate a hitherto unrecognized role of DUSP2-STAT1 axis in regulating RTEC pyroptosis in AKI, highlighting that DUSP2-STAT1 axis is an attractive therapeutic target for AKI.

Apatinib combined with Keytruda treatment induces apoptosis of gastric carcinoma cells through CES4/miR-616-5p/DUSP2 axis.

Gastric carcinoma (GC) is a highly malignant and heterogeneous tumour. Long non-coding RNA CES4 is down-regulated in GC. However, whether CES4 can participate in GC remains unclear; we have carried out research on this topic. GC cells (HGC-27 and MKN-7) were treated with anti-tumour drugs: apatinib combined with Keytruda. Cell viability and apoptosis were detected by CCK-8 assay and flow cytometry. Gene and protein expression were examined by quantitative real-time PCR and western blot. Luciferase reporter assay was performed to verify the relationship among CES4, miR-616-5p and dual-specificity phosphatase-2 (DUSP2). CES4 was highly expressed in the apatinib combined with Keytruda-treated HGC-27 and MKN-7 cells. Apatinib combined with Keytruda treatment repressed cell viability and promoted apoptosis of HGC-27 and MKN-7 cells, which was abrogated by CES4 knockdown. Furthermore, CES4 promoted DUSP2 expression by sponging miR-616-5p in HGC-27 and MKN-7 cells. CES4 knockdown promoted cell viability and inhibited apoptosis of drug-treated HGC-27 and MKN-7 cells by regulating miR-616-5p/DUSP2 axis. In conclusion, these data demonstrate that apatinib combined with Keytruda treatment induces apoptosis of GC cells through CES4/miR-616-5p/DUSP2 axis. Thus, this work provides the experimental basis for the combination of apatinib and Keytruda as a treatment for GC.

Suppression of Extracellular Vesicle VEGF-C-mediated Lymphangiogenesis and Pancreatic Cancer Early Dissemination By a Selective HDAC1/2 Inhibitor.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by early dissemination and poor drug response. Therefore, it is an unmet medical need to develop new strategies for treatment. As aberrant activation of ERK due to KRAS activating mutation is a driving force for PDAC, a brake system that can terminate ERK signaling represents an ideal druggable target. Herein, we demonstrate that forced expression of dual specificity phosphatase-2 (DUSP2), a specific ERK phosphatase, abrogated tumor formation and loss of Dusp2 facilitated Kras-driven PDAC progression. We report that a selective HDAC1/2 inhibitor (B390) has multifaceted therapeutic potential in PDAC by restoring the expression and function of DUSP2. In vitro study showed that treatment with B390 inhibited growth and migration abilities of PDAC cells, decreased extracellular vesicle-associated VEGF-C expression, and suppressed lymphatic endothelial cell proliferation. In vivo, B390 not only suppressed tumor growth by increasing tumor cell death, it also inhibited lymphangiogenesis and lymphovascular invasion. Taken together, our data demonstrate that B390 was able to alleviate loss of DUSP2-mediated pathologic processes, which provides the proof-of-concept evidence to demonstrate the potential of using selective HDAC1/2 inhibitors in PDAC treatment and suggests reinstating DUSP2 expression may be a strategy to subside PDAC progression.

DUSP2 regulates extracellular vesicle-VEGF-C secretion and pancreatic cancer early dissemination

ABSTRACT Early dissemination is a unique characteristic and a detrimental process of pancreatic ductal adenocarcinoma (PDAC); however, the underlying mechanism remains largely unknown. Here, we investigate the role of dual-specificity phosphatase-2 (DUSP2)-vascular endothelial growth factor-C (VEGF-C) axis in mediating PDAC lymphangiogenesis and lymphovascular invasion. Expression of DUSP2 is greatly suppressed in PDAC, which results in increased aberrant expression of extracellular vesicle (EV)-associated VEGF-C secretion. EV-VEGF-C exerts paracrine effects on lymphatic endothelial cells and autocrine effects on cancer cells, resulting in the lymphovascular invasion of cancer cells. Tissue-specific knockout of Dusp2 in mouse pancreas recapitulates PDAC phenotype and lymphovascular invasion. Mechanistically, loss-of-DUSP2 enhances proprotein convertase activity and vesicle trafficking to promote the release of the mature form of EV-VEGF-C. Collectively, these findings represent a conceptual advance in understanding pancreatic cancer lymphovascular invasion and suggest that loss-of-DUSP2-mediated VEGF-C processing may play important roles in early dissemination of pancreatic cancer. Abbreviations: DUSP2: dual-specificity phosphatase-2; VEGF-C: vascular endothelial growth factor-C; EV: extracellular vesicles; PDAC: pancreatic ductal adenocarcinoma; KD: knockdown

Expression of Dual-Specificity Phosphatase 2 (DUSP2) in Patients with Serous Ovarian Carcinoma and in SKOV3 and OVCAR3 Cells In Vitro.

BackgroundOvarian cancer commonly presents at a late stage and is associated with poor prognosis. The most common histological subtype is serous ovarian carcinoma. Dual-specificity phosphatase 2 (DUSP2) is a protein phosphatase and substrate for mitogen-activated protein kinases (MAPKs) with increased expression levels in malignancy. This study aimed to evaluate the expression of DUSP2 in tumor tissues from patients with serous ovarian carcinoma and the association with tumor grade, stage, and patient survival and to investigate the effects of DUSP2 expression in SKOV3 and OVCAR3 cells in vitro.Material/MethodsTumor tissue and adjacent normal ovarian tissue from 127 patients with histologically confirmed serous ovarian carcinoma underwent quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry to measure DUSP2 mRNA and protein expression, respectively. Tumor grade, stage, and clinicopathological data underwent correlation analysis with DUSP2 expression, and survival data were assessed with Kaplan-Meier and Cox regression analysis. The effects of DUSP2 expression on the proliferation and migration of SKOV3 and OVCAR3 cells were evaluated.ResultsImmunohistochemistry showed that DUSP2 was down-regulated in serous ovarian carcinoma tissues compared with adjacent ovarian tissues, and was significantly correlated with tumor stage. Survival analysis showed that DUSP2 expression was an independent risk factor for patient survival. DUSP2 expression in SKOV3 and OVCAR3 cells in vitro suppressed cell proliferation and migration.ConclusionsDown-regulation of DUSP2 expression in serous ovarian carcinoma was an independent risk factor for patient survival, and its expression in SKOV3 and OVCAR3 cells inhibited cell proliferation and migration in vitro.

Loss of DUSP2 predicts a poor prognosis in patients with bladder cancer

Dual-specificity phosphatase 2 (DUSP2), a member of nuclear type I DUSP family, abolishes the activation of mitogen-activated protein kinases (MAPKs) and plays critical roles in the immune processes, inflammatory responses, and cancer progression. Currently, whether DUSP2 is involved in pathogenesis of bladder cancer remains unclear. In this study, we demonstrate that the expression level of DUSP2 was predominantly downregulated in bladder cancer tissues and cell lines as compared with that of paired normal tissues and benign urothelial cells. Besides, the expression of DUSP2 was significantly associated with pathological grade (P = .009), AJCC stage (P = .017), and subtype (P = .001) in The Cancer Genome Atlas cohort and mainly related to TNM stage (P = .016) in the tissue microarray cohort. Kaplan-Meier analysis suggested that patients with low DUSP2 expression had a shorter 5-year overall survival (P = .018 in The Cancer Genome Atlas; P = .012 in tissue microarray) and lower recurrence-free survival (P = .008). Cox regression analysis indicated that reduced DUSP2 was an independent high risk factor for survival prognosis in both cohorts. Taken together, our findings for the first time suggested DUSP2 as a progression and prognosis biomarker for bladder cancer. Whether DUSP2 functions as a tumor suppressor in bladder cancer deserves further studies.

PO-177 The novel function of DUSP2/VEGF-C axis in pancreatic cancer progression

IntroductionThe rising incidence and extremely poor prognosis makes pancreatic adenocarcinoma (PDAC) becoming one of the most malignant cancers. Dissemination through the lymphatic/vascular system is a critical process causing poor prognosis; however, how this process is regulated remains largely unknown. We aimed to elucidate the novel function of the tumour suppressor dual specificity phosphatase-2 (DUSP2), the master negative regulator of MAPK signalling, in PDAC progression and the mechanism by which DUSP2 mediates lymphovascular invasion (LVI).Material and methodsDUSP2 expression was examined in human pancreatic tumours. Orthotopic pancreatic tumour mouse models were used to determine the gain and loss of DUSP2 in PDAC progression. RT-qPCR and Western blotting were used to measure the expression of VEGF-C regulated by DUSP2. The autocrine and paracrine effects of DUSP2/VEGF-C axis were measured by migration/invasion assays. Proprotein convertase activity assay was performed to investigate the regulation of VEGF-C. Extracellular vesicle (EV) secretion was measured by nanoparticle tracking analysis, confocal imaging and Western blotting.Results and discussionsDUSP2 knockdown (KD) tumours developed increased lymphangiogenesis and increased LVI in mouse model of pancreatic cancer. Forced expression of DUSP2 abolished pancreatic cancer development. A significant increase in the functional form of VEGF-C of DUSP2-KD pancreatic cancer cells promoted survival and migration of lymphatic endothelial cells in a VEGFR dependent manner. In addition, VEGF-C signalling mediated migration/invasion ability of DUSP2-KD pancreatic cancer cells. Knockdown of DUSP2 not only increased VEGFC mRNA level but also enhanced the conversion of proform VEGF-C to become the mature form, which is associated with increased activity of proprotein convertase. Loss-of-DUSP2 enhanced EV secretion thus promoted the release of mature form VEGF-C. Novel histone deacetylase inhibitor, exerting similar effect as DUSP2 re-expression, can not only diminish VEGF-C secretion but also confer synergistic effect with routinely used chemotherapeutic drug for PDAC.ConclusionWe provide new evidence demonstrating that loss of DUSP2 in pancreatic cancer cells increases expression of VEGF-C, which exerts autocrine and paracrine functions to promote early dissemination of pancreatic cancer.

PO-180 Transforming growth factor beta 1activates cell migration through the down-regulation of PRH/HHEX expression

IntroductionThe rising incidence and extremely poor prognosis makes pancreatic adenocarcinoma (PDAC) becoming one of the most malignant cancers. Dissemination through the lymphatic/vascular system is a critical process causing poor prognosis; however, how this process is regulated remains largely unknown. We aimed to elucidate the novel function of the tumour suppressor dual specificity phosphatase-2 (DUSP2), the master negative regulator of MAPK signalling, in PDAC progression and the mechanism by which DUSP2 mediates lymphovascular invasion (LVI).Material and methodsDUSP2 expression was examined in human pancreatic tumours. Orthotopic pancreatic tumour mouse models were used to determine the gain and loss of DUSP2 in PDAC progression. RT-qPCR and Western blotting were used to measure the expression of VEGF-C regulated by DUSP2. The autocrine and paracrine effects of DUSP2/VEGF-C axis were measured by migration/invasion assays. Proprotein convertase activity assay was performed to investigate the regulation of VEGF-C. Extracellular vesicle (EV) secretion was measured by nanoparticle tracking analysis, confocal imaging and Western blotting.Results and discussionsDUSP2 knockdown (KD) tumours developed increased lymphangiogenesis and increased LVI in mouse model of pancreatic cancer. Forced expression of DUSP2 abolished pancreatic cancer development. A significant increase in the functional form of VEGF-C of DUSP2-KD pancreatic cancer cells promoted survival and migration of lymphatic endothelial cells in a VEGFR dependent manner. In addition, VEGF-C signalling mediated migration/invasion ability of DUSP2-KD pancreatic cancer cells. Knockdown of DUSP2 not only increased VEGFC mRNA level but also enhanced the conversion of proform VEGF-C to become the mature form, which is associated with increased activity of proprotein convertase. Loss-of-DUSP2 enhanced EV secretion thus promoted the release of mature form VEGF-C. Novel histone deacetylase inhibitor, exerting similar effect as DUSP2 re-expression, can not only diminish VEGF-C secretion but also confer synergistic effect with routinely used chemotherapeutic drug for PDAC.ConclusionWe provide new evidence demonstrating that loss of DUSP2 in pancreatic cancer cells increases expression of VEGF-C, which exerts autocrine and paracrine functions to promote early dissemination of pancreatic cancer.

Differential expression of DUSP2 in left- and right-sided colon cancer is associated with poor prognosis in colorectal cancer.

Dual-specificity phosphatase-2 (DUSP2), a negative regulator of extracellular-regulated kinase activity, has been identified as an important kinase with emerging roles in cancer. However, the clinical significance of DUSP2 in colorectal cancer (CRC) remains to be fully elucidated. In the present study, the expression of DUSP2 was investigated using immunohistochemistry in 96 patients with CRC. Cell viability was estimated using a cell counting kit-8 assay, and cell apoptosis by flow cytometry. The relationship between DUSP2 expression and patient characteristics, including overall survival, were studied retrospectively in these patients. It was found that DUSP2 was differentially expressed between left-sided colon carcinoma (LSCC) and right-sided colon carcinoma (RSCC). It was also found that decreased expression of DUSP2 was correlated with significantly shorter overall survival (P=0.001) and short distant-metastasis-free survival (P=0.002). In univariate comparisons, the decreased expression of DUSP2 was found to be an independent risk factor for poor survival rate (HR 3.55, CI 1.092–9.896; P=0.002). It was also found that the enforced overexpression of DUSP2 sensitized CRC cells to cetuximab. In conclusion, the findings demonstrated that DUSP2 was differentially expressed between RSCC and LSCC, and that the overexpression of DUSP2 increased the inhibitory effect of cetuximab in CRC, suggesting that DUSP2 may be a novel biomarker and therapeutic target in CRC therapy.

Hypoxia-Induced Downregulation of DUSP-2 Phosphatase Drives Colon Cancer Stemness.

Cancer stem-like cells (CSC) evolve to overcome the pressures of reduced oxygen, nutrients or chemically induced cell death, but the mechanisms driving this evolution are incompletely understood. Here, we report that hypoxia-mediated downregulation of the dual specificity phosphatase 2 (DUSP2) is critical for the accumulation of CSC in colorectal cancer. Reduced expression of DUSP2 led to overproduction of COX-2-derived prostaglandin E2, which promoted cancer stemness via the EP2/EP4 signaling pathways. Genetic and pharmacological inhibition of PGE2 biosynthesis or signal transduction ameliorated loss-of-DUSP2-induced tumor growth and cancer stemness. Genome-wide profile analysis revealed that genes regulated by DUSP2 were similar to those controlled by histone deacetylase. Indeed, treatment with novel histone deacetylase inhibitors abolished hypoxia-induced DUSP2 downregulation, COX-2 overexpression, cancer stemness, tumor growth, and drug resistance. Our findings illuminate mechanisms of cancer stemness and suggest new cancer therapy regimens. Cancer Res; 77(16); 4305-16. ©2017 AACR.

Hypoxia-inhibited DUSP2 expression promotes IL-6/STAT3 signaling in endometriosis.

How does hypoxia-mediated downregulation of dual-specificity phosphatase-2 (DUSP2) promote the development of endometriotic lesions? The levels of IL-6 and DUSP2 were assessed in eutopic stromal cells with DUSP2 knockdown or hypoxia treatment. Bromodeoxyuridine (BrdU) incorporation was applied for evaluating cell proliferation. The protein levels of DUSP2, cleaved caspase-3, phosphorylated STAT3, and STAT3 were analyzed using immunoblot. The genomewide analysis of cells with DUSP2 overexpression indicated IL-6 regulates multiple pathways related to inflammation, proliferation, and apoptosis. DUSP2 overexpression significantly suppressed IL-6 expression, while DUSP2 knockdown promoted IL-6 expression. The hypoxia-treated eutopic stromal cells expressed higher levels of IL-6, recapitulating the elevated levels of IL-6 in ectopic stromal cells. The treatment with IL-6 elicited the phosphorylation of STAT3, mimicking the elevated levels of phosphorylated STAT3 in the ectopic stromal cells. The IL-6-treated eutopic stromal cells showed more BrdU incorporation and less cleaved caspase-3, which can be reversed by STAT3 inhibitor. Hypoxia-induced IL-6 production in endometriotic lesions is mediated via downregulation of DUSP2, which causes aberrant activation of STAT3 signaling pathway and helps the endometriotic cells survive under the ectopic environment.

Loss of dual-specificity phosphatase-2 promotes angiogenesis and metastasis via up-regulation of interleukin-8 in colon cancer.

Dual-specificity phosphatase 2 (DUSP2) is a negative regulator of mitogen-activated protein kinases. Our previous study showed that DUSP2 expression is down-regulated in many human cancers and loss of DUSP2 promotes cancer progression; however, the underlying mechanism remains largely uncharacterized. Herein, we found that loss of DUSP2 induces angiogenesis, while forced expression of DUSP2 inhibits microvessel formation in xenografted mouse tumours. Genome-wide screening of expression profiles, and meta-analysis of clinical data, identified that the level of interleukin-8 (IL-8) correlated negatively with that of DUSP2, suggesting that it may be a downstream target of DUSP2. Molecular characterization revealed that DUSP2 inversely regulates IL-8 expression, mediated by ERK1/2 and C/EBPα-dependent transcriptional regulation. Further study showed that hypoxia-induced IL-8 expression in cancer cells is also mediated via down-regulation of DUSP2. Treatment with the IL-8 receptor inhibitor reparixin or knockdown of IL-8 in cancer cells abolished angiogenesis induced by loss of DUSP2. Functionally, knockdown of DUSP2 enhanced tumour growth and metastasis, which were abolished by treatment with reparixin or knockdown of IL-8 in an orthotopic mouse model. Taken together, our results demonstrate that hypoxia inhibits DUSP2 expression in colon cancer, leading to up-regulation of IL-8, which facilitates angiogenesis and tumour metastasis. Our findings suggest that blocking hypoxia-DUSP2-IL-8 signalling may be a plausible approach for therapeutic intervention in cancer. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The dual specificity phosphatase 2 gene is hypermethylated in human cancer and regulated by epigenetic mechanisms.

BackgroundDual specificity phosphatases are a class of tumor-associated proteins involved in the negative regulation of the MAP kinase pathway. Downregulation of the dual specificity phosphatase 2 (DUSP2) has been reported in cancer. Epigenetic silencing of tumor suppressor genes by abnormal promoter methylation is a frequent mechanism in oncogenesis. It has been shown that the epigenetic factor CTCF is involved in the regulation of tumor suppressor genes.MethodsWe analyzed the promoter hypermethylation of DUSP2 in human cancer, including primary Merkel cell carcinoma by bisulfite restriction analysis and pyrosequencing. Moreover we analyzed the impact of a DNA methyltransferase inhibitor (5-Aza-dC) and CTCF on the epigenetic regulation of DUSP2 by qRT-PCR, promoter assay, chromatin immuno-precipitation and methylation analysis.ResultsHere we report a significant tumor-specific hypermethylation of DUSP2 in primary Merkel cell carcinoma (p = 0.05). An increase in methylation of DUSP2 was also found in 17 out of 24 (71 %) cancer cell lines, including skin and lung cancer. Treatment of cancer cells with 5-Aza-dC induced DUSP2 expression by its promoter demethylation, Additionally we observed that CTCF induces DUSP2 expression in cell lines that exhibit silencing of DUSP2. This reactivation was accompanied by increased CTCF binding and demethylation of the DUSP2 promoter.ConclusionsOur data show that aberrant epigenetic inactivation of DUSP2 occurs in carcinogenesis and that CTCF is involved in the epigenetic regulation of DUSP2 expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2087-6) contains supplementary material, which is available to authorized users.

Abstract 716: Targeting Mek-Erk pathway abrogates hypoxia-mediated lapatinib resistance in ErbB2-positive breast cancer cells

Abstract Overexpression/amplification of the tyrosine kinase ErbB2 is often observed in breast cancers. ErbB2 expression activates EGFR signaling pathway promoting tumorigenesis. Breast cancer ErbB2-positive patients are treated with lapatinib, a dual EGFR/ERBB2 inhibitor. Despite lapatinib clinical efficacy, acquired resistance has been reported and its mechanism is poorly understood. Another factor that is associated with mammary tumors is hypoxia. Hypoxia stabilizes hypoxia inducible factor 1 (HIF-1), a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors are often chemoresistant. Here, we show that hypoxia decreases lapatinib-mediated growth inhibition and apoptosis in three-dimensional cultures. In agreement with this data, hypoxia also promotes activation of signaling pathways downstream from ErbB2/EGFR including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 and can be abrogated by HIF-1 RNAi. Moreover stable overexpression HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to rescue cell proliferation and survival and maintain ERK and AKT signaling in the presence of lapatinib. We also show that activation of ERK signaling is required for hypoxia-mediated lapatinib resistance and inhibition of MEK (a kinase upstream of Erk) reverses hypoxia protective effect on ErbB2-expressing cells. One potential mechanism of how hypoxia/HIF-1 may lead to activation of ERK pathway is via regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of ERK pathway. Here we demonstrate that cells exposed to hypoxia have decreased levels of DUSP2 and it correlate with increased activation of Erk signaling. Moreover, reducing DUSP2 expression alone in ErbB2-positive breast cancer cells is sufficient to reduce lapatinib-mediated effects. And DUSP2 overexpression abrogates hypoxia-mediated protective effect on ErbB2-expressing cells treated with lapatinib. All these data suggests that targeting MEK-Erk signaling pathway may reverse hypoxia mediated lapatinib resistance and might be a viable option for lapatinib resistant ErbB2-posive tumors. Indeed, a combination of lapatinib and an FDA approved MEK inhibitor trametinib abrogates hypoxia protective effect on cell growth inhibition and apoptosis in three dimensional (3D) cultures. Thus targeting MEK/ERK pathway in hypoxic breast cancer may improve an anti-ErbB2 therapy in breast cancer. Citation Format: Sergey Karakashev, Mauricio Reginato. Targeting Mek-Erk pathway abrogates hypoxia-mediated lapatinib resistance in ErbB2-positive breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 716. doi:10.1158/1538-7445.AM2015-716

Curcumin Enhances the Radiosensitivity of U87 Cells by Inducing DUSP-2 Up-Regulation

Glioblastoma multiforme (GBM), an aggressive primary brain tumor, is radioresistant and recurs despite aggressive surgery, chemotherapy, and radiotherapy. Curcumin as a potential radiosensitizer has received extensive attention in cancer treatment. To explore an effectiveness of this radiosensitizer for GBM treatment, we evaluated the radiosensitizing effect of curcumin and investigated its potential molecular mechanisms in the human glioma cell line U87. The cytotoxic effects of curcumin on U87 cells were evaluated using the Cell Counting Kit-8 assay, and the radiosensitivity of U87 cells treated with curcumin was accessed by colony information assay. The effects of curcumin on cell proliferation and cell cycle regulation were determined using the 5-ethynyl-2-deoxyuridine incorporation assay and flow cytometry, respectively. Western blotting was applied to determine the effects of curcumin on protein expression of dual-specificity phosphatase-2 (DUSP-2), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) as well as phosphorylated ERK and JNK. Curcumin significantly inhibited the proliferation of U87 cells in a dose-and time-dependent manner. Curcumin treatment at the concentrations of 5 µM and 10 M could significantly reduce the clonogenic activity and enhance the radiosensitivity of U87 cells with sensitive enhancement ratios (SERs) of 1.71 and 4.65, respectively. Curcumin resulted in G2/M cell cycle arrest in U87 cells, which were radiosensitive. Pre-treatment of U87-MG cells with 5 µM curcumin enhanced radiation-induced cell proliferation inhibition and apoptosis. Furthermore, we observed that curcumin increased DUSP-2 protein expression and decreased the phosphorylation of ERK and JNK. Our results suggest that low-dose curcumin may enhance the radiosensitivity of human glioma U87 cells in vitro by inducing G2/M cell cycle arrest through up-regulation of DUSP-2 expression and inhibition of ERK and JNK phosphorylation.

Abstract 1824: Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2

Abstract Breast cancer is one of the most common cancers among women. Many factors are associated with this disease including overexpression/amplification of the tyrosine kinase HER2/ErbB2. ErbB2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can activate multiple signaling pathways that can promote tumor progression via regulation of cell growth, proliferation, and survival. Breast cancer patients with ErbB2 amplifications are currently treated with lapatinib, a small-molecule kinase inhibitor that blocks the active site of ErbB2 to inhibit EGFR/ErbB2 downstream signaling. While lapatinib has displayed clinical efficacy in breast cancer patients, acquired resistance has been reported and the mechanism of this resistance is poorly understood. Hypoxia or low oxygen tension occurs in many breast cancers and is associated with poor patient survival. One of the major players in the cellular response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors often exhibit weak response to chemotherapeutic agents and are associated with poor prognosis. Here, we show that hypoxia, via HIF-1α, blocks lapatinib-mediated effects on ErbB2-expressing mammary epithelial and ErbB2-positive breast cancer cells. Lapatinib-mediated growth inhibition and apoptosis in three dimensional (3D) cultures are decreased under hypoxic conditions (1%O2) Consistent with these results, we find that hypoxia can maintain activation of signaling pathways downstream from ErbB2 including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 expression as cells expressing HIF-1 RNAi no longer are resistant to lapatinib treatment. Moreover, overexpression of stable HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to inhibit lapatinib-mediated effects on proliferation and survival and maintain ERK and AKT signaling. Moreover, hypoxia-mediated activation of ERK signaling, but not AKT, is required for lapatinib resistance as MEK inhibitors (including clinically approved tramatinib) reverses hypoxia-mediated lapatinib resistance in standard and 3D cultures. HIF-1 may bypass lapatinib-treated inhibition of the ERK pathway via its regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of the ERK pathway and we show that cells exposed to hypoxia contain decreased DUSP2 levels and correlate with increased Erk activation. Indeed, reducing DUSP2 expression via RNAi in ErbB2-positive breast cancer cells is sufficient to reduce lapatanib-mediated effects under normal oxygen. Thus, hypoxia, via HIF-1, causes lapatinib resistance in ErbB2-expressing breast cancer cells and suggests that targeting MEK/ERK pathway in hypoxic breast cancer may increase sensitivity to ErbB2-targeted therapy. Citation Format: Sergey Karakashev. Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1824. doi:10.1158/1538-7445.AM2014-1824

Abstract PR15: Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2

Abstract Breast cancer is one of the most common cancers among women. Many factors are associated with this disease including overexpression/amplification of the tyrosine kinase HER2/ErbB2. ErbB2 belongs to the EGFR-family of receptor tyrosine kinases and its overexpression can activate multiple signaling pathways that can promote tumor progression via regulation of cell growth, proliferation, metabolism, and survival. Breast cancer patients with ErbB2 amplifications are currently treated with lapatinib (Tykerb), a small-molecule kinase inhibitor that specifically blocks the active site of ErbB2 to inhibit EGFR/ErbB2 downstream signaling. While lapatinib has displayed clinical efficacy in breast cancer patients, acquired resistance has been reported and the exact mechanism of this resistance is poorly understood. Hypoxia or low oxygen tension occurs in many breast cancers and is associated with poor patient survival. One of the major players in the cellular response to hypoxia is stabilization of the transcription factor hypoxia inducible factor 1 (HIF-1). HIF-1 is a transcription factor that regulates cell growth, proliferation and survival. As a result, hypoxic tumors often exhibit weak response to different chemotherapeutic agents and poor prognosis. Here, we show that hypoxia, via HIF-1α, blocks lapatinib-mediated effects on ErbB2-expressing mammary epithelial and ErbB2-positive breast cancer cells. Lapatinib-mediated growth inhibition is reduced under hypoxic conditions (1%O2). Consistent with these results, we find that hypoxia can maintain activation of signaling pathways downstream from ErbB2 including AKT and ERK in the presence of lapatinib. This protective effect depends on HIF-1 expression as cells expressing HIF-1 RNAi no longer are resistant to lapatinib treatment. Moreover, overexpression of stable HIF-1 in ErbB2-expressing cells under normoxic conditions is sufficient to inhibit lapatinib-mediated effects on proliferation and survival and maintain ERK and AKT signaling. One possible mechanism of how HIF-1 may bypass lapatinib-treated inhibition of the ERK pathway is via regulation of dual-specificity phosphatase 2 (DUSP2). DUSP2 is a negative regulator of the ERK pathway and we show that cells exposed to hypoxia contain decreased DUSP2 levels and correlate with increased Erk activation. Indeed, reducing DUSP2 expression alone in ErbB2-positive breast cancer cells is sufficient to reduce lapatinib-mediated effects. Thus, hypoxia, via HIF-1, causes lapatinib resistance in ErbB2-expressing breast cancer cells and suggests that targeting ERK/MAPK pathway in hypoxic breast cancer may increase sensitivity to ErbB2-targeted therapy. This abstract is also presented as poster C63. Citation Format: Sergey V. Karakashev, Reginato J. Mauricio. Hypoxia induces lapatinib resistance in ErbB2-positive breast cancer cells via regulation of DUSP2. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR15.

Regulation of epithelial‐mesenchymal transition and apoptosis by dual specificity phosphatase‐2

Our previous findings provide evidence to demonstrate that dual specificity phosphatase‐2 (DUSP2) is markedly reduced or even lost in most solid cancers. To further unravel the effect of DUSP2 downregulation in cancer progression, we overexpressed DUSP2 in cancer cells and performed microarray assay to document gene expression profile. Analysis of microarray data derived from DUSP2 overexpressed cancer cells revealed that many genes involved in angiogenesis, epithelial‐mesenchymal transition (EMT), cell migration, and apoptosis are regulated by DUSP2. Since EMT is a critical process for an increase in metastatic capacity and drug resistance, we tested whether loss‐of‐function of DUSP2 would induce EMT. Knock down of DUSP2 induced the expression of mesenchymal markers (N‐cadherin) and reduced the level of epithelial marker (E‐cadherin). Consistent with this notion, treatment with conditioned media collected from DUSP2 knockdown cells enhanced cancer cells migration while treatment with conditioned medium collected from DUSP2 overexpressed cells inhibited cell migration. Furthermore, we established a stable clone of HCT116 cells carrying doxycycline‐inducible DUSP2 minigene. Treatment of cells with doxycycline to induce DUSP2 expression resulted in upregulation of pro‐apoptotic genes such as Bcl2‐modifying factor and endonuclease/exonuclease/phosphatase family domain‐containing protein 1. As expect, induction of DUSP2 expression increased cell death. These data suggest that restoration of DUSP2 expression in cancer cells represents a potential therapeutic approach. In‐depth investigation of mechanisms responsible for DUSP2‐induced cell death will provide valuable information for designing novel treatment regimen for cancer therapy.

Abstract 3083: Dual-specificity phosphatase-2 (DUSP2) negatively control cyclooxygenase-2 (COX-2) expression in cancer cells

Abstract Cyclooxygenase-2 (COX-2), an inducible form of cyclooxygenase, plays a crucial role in the development of cancer via its enzymatic product, prostaglandin E2 (PGE2). Clinical studies reveal that COX-2 is overexpressed in numerous types of cancers, and is associated with increase of angiogenesis, metastasis, and drug resistance. Although nonsteroidal anti-inflammatory drugs, COX-2 inhibitors, have been used for cancer therapy; they have serious side effects in many aspects. Therefore, to explore the underlying mechanism of COX-2 overexpression during cancer progression is critical for developing more efficient and safer way to block COX-2 function. Previously, we have found that the expression of dual-specificity phosphatase-2 (DUSP2), a MAPK-specific phosphatase, is markedly reduced in many kinds of cancers. Here, we identified that COX-2 was a potential downstream target gene of DUSP2. In vitro studies demonstrate that knockdown of DUSP2 significantly increases COX-2 expression and PGE2 production while overexpression of DUSP2 reduces those in cancer cells. Xenografted mouse study further reveals that levels of DUSP2 are reduced during cancer development, which is inversely correlated with COX-2 expression. Re-expression of DUSP2 during tumor progression inhibits COX-2 expression and suppresses tumor growth while knockdown of DUSP2 increases COX-2 expression and enhances tumor growth. These results implied that COX-2 overexpression was due to loss of DUSP2 expression in cancer. Furthermore, we found that hypoxia represses DUSP2 expression resulting in COX-2 overexpression. Taken together, we have demonstrated for the first time that aberrant expression of COX-2 during tumor development is regulated by hypoxia-mediated DUSP2 downregulation. Our data also suggest that DUSP2 is a good molecular target for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3083. doi:1538-7445.AM2012-3083