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Supplementary data includes IC50 of SAHA and B390 (S1),bioinformatic analysis (S2 and S3), and tumor size (S4)

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.

Table S1. CSC frequency measured by ELDA in vitro. Table S2. Multiple corrections for correlation p value. Table S3. Tumor incidence in limiting dilution assay (I). Table S4. Tumor incidence in limiting dilution assay (II). Table S5. Top 10 list of candidate drugs exert similar actions like DUSP2. Table S6: IC50 of B369, B390, and SAHA determined by different methods.

HDACi abolished hypoxia-induced cancer malignancy

Knockdown of DUSP2 facilitates xenograft tumor growth

Legends for supplementary figures S1-S7

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.

Figure S3: PGE2 mediates loss-of-DUSP2-induced cancer stemness. Figure S4: Hypoxia-induced COX-2 expression is mediated by downregulation of DUSP2.

HDACi inhibits tumor growth and drug resistance

Knockdown of DUSP2 enhanced EMT process and migration/invasion ability

Loss-of-DUSP2 results in the expansion of CSC-like population.

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.

Takayasu’s arteritis (TAK), a chronic inflammatory disorder mainly affecting the aorta and its major branches, can cause vascular injury with thickened walls, luminal stenosis, aneurysmal changes, ...

Background Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by early dissemination and poor drug response. Loss-of-function of dual specificity phosphatase 2 (DUSP2), a critical regulator of MAPKs signaling, is highly associated with cancer malignancies. Therefore, it may provide new therapeutic strategy if the actions of DUSP2 can be restored. Methods The tumor suppressor role of DUSP2 was demonstrated via DUSP2 re-expression in the orthotopic mouse model of pancreatic cancer and knockout of Dusp2 in the pancreas by transgenic mouse model. Immunohistochemical staining and histology analysis was performed to evaluate tumor development and progression. Bioinformatic analysis was utilized to identify potential drug which mimics DUSP2 re-expression. Pancreatic cancer cell survival, migration ability, and the expression and function of extracellular vesicle (EV) associated vascular endothelial growth factor C (VEGF-C) was measured. The effect of the novel HDAC inhibitor on pancreatic cancer progression was evalulated by orthotopic mouse model. Results Forced expression of DUSP2 abrogated tumor formation and loss of Dusp2 facilitated Kras-driven PDAC formation. Increased HDAC1 expression was found in PDAC and inhibition of HDAC showed similar gene profile as Kras knockdown and DUSP2 re-expression. Treatment with B390 inhibited growth and migration abilities of PDAC cells, decreased EV-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. Conclusions Our data provide the proof-of-concept evidence to demonstrate the potential of using novel HDAC inhibitor in PDAC treatment which alleviates loss-of-DUSP2-mediated pathological processes.

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.

How does hypoxia-mediated down-regulation of dual specificity phosphatase-2 (DUSP2) promote endometriotic lesion development?Inhibition of DUSP2 by hypoxia enhances endometriotic lesion growth via promoting interleukin-8 (IL-8)-dependent angiogenesis.Angiogenesis is a prerequisite for the development of endometriosis. DUSP2 is down-regulated in endometriotic stromal cells in a hypoxia inducible factor-1α-dependent manner. Down-regulation of DUSP2 contributes to the pathological process of endometriosis.A laboratory study recruiting 20 patients of reproductive age with endometriosis and normal menstrual cycles, and an autoimplant-induced mouse model of endometriosis using 13 mice in a 28-day treatment.IL-8 mRNA levels were assayed in endometrial stromal cells maintained in normoxic or hypoxic (1% O2) conditions, with or without DUSP2 knockdown. Promoter activity and chromatin immunoprecipitation (ChIP) assays were conducted to characterize the regulation of IL-8 by DUSP2. Conditioned media from cells maintained in normoxic or hypoxic conditions, and cells with/without DUSP2 knockdown were collected to investigate the angiogenic capacity using an in vitro tube formation assay. Reparixin, an IL-8 receptor blocker, was administered to investigate the role of IL-8 in hypoxia-mediated angiogenesis and the development of endometriotic-like lesions in an autotransplanted mouse model.IL-8 mRNA was increased by both hypoxia and DUSP2 knockdown in endometrial stromal cells in an extracellular signal-regulated protein kinase-dependent manner (P0.05 versus control). Promoter activity and ChIP assays demonstrated that expression of IL-8 was regulated by CCAAT/enhancer binding protein α (P0.05 versus control). Furthermore, conditioned media collected from hypoxia-exposed or DUSP2 knockdown endometrial stromal cells promoted tube formation, which was abolished by co-treatment with reparixin (P0.05 versus control). Results from the autotransplanted mouse model demonstrated that number of blood vessels and size of endometriotic-like lesions were markedly reduced in recipient mice treated with reparixin (P0.05 versus control).This study was conducted in primary human cell cultures and a mouse model, therefore may not fully reflect the situation in vivo.This is the first study to highlight the potential application of an IL-8 receptor blocker as a therapeutic target to treat endometriosis. This study demonstrates IL-8 as a key angiogenic factor regulated by hypoxia/DUSP2, which suggests an alternative mechanism through which hypoxia may promote angiogenesis.This study was funded by the National Science Council of Taiwan (NSC101-2314-B-006-043-MY2). The author declares that there is no conflict of interest.

Context: Aberrant activation of MAPK has been implicated to play important roles in pathological processes of endometriosis. However, how MAPK are constitutively activated in endometriotic tissues remains largely unknown. microRNA are small noncoding RNA that regulate the stability or translational efficiency of target mRNA by interacting with the 3′ untranslated region. Thus, miRNA are thought to be modulators of the transcriptional response, fine-tuning gene expression. Objective: The aim of this study was to evaluate the functional roles of microRNA-20a (miR20a) in MAPK activation and the pathogenesis of endometriosis. Design: miR20a expression was analyzed in nonpaired (endometrium = 17; endometriosis = 37) and paired (n = 12) endometriotic tissues by quantitative RT-PCR. Overexpression of miR20a in eutopic endometrial stromal cells or inhibition of miR20a in ectopic endometriotic stromal cells was used to evaluate its impact on ERK phosphorylation and subsequently angiogenesis- and proliferation-related gene expression. Results: Levels of miR20a were up-regulated in endometriotic stromal cells. Elevation of miR20a was up-regulated by hypoxia inducible factor-1α. The up-regulation of miR20a causes the down-regulation of dual-specificity phosphatase-2, which leads to prolonged ERK phosphorylation and an increase in the expression of several angiogenic genes. Furthermore, the up-regulation of miR20a enhances the prostaglandin E2-induced expression of fibroblast growth factor-9, a potent mitogen that stimulates both endothelial and endometrial cell proliferation. Conclusion: Our findings provide the novel mechanism that not only functionally links together hypoxic stress, miR20a expression, aberrant ERK phosphorylation, and angiogenesis but also demonstrates that miR20a is an important modulator in the development of endometriosis.

Hypoxia is a common feature of most cancers and contributes to aggressive phenotype and chemoresistance. Hypoxia inducible factor-1 (HIF-1) not only plays important roles in developmental and physiological hypoxic adaptation, but also a crucial mediator in hypoxiainduced tumor progression and chemoresistance. It has been demonstrated that HIF-1α is overexpressed in tumor cells by different kinds of mechanisms, including the presence of hypoxic stress, mutations in von Hippel Lindau (VHL), and hyperactivation of mitogen activated protein kinase and mammalian target of rapamycin signaling pathways. HIF-1 regulates anaerobic metabolism, drug efflux, and expression of anti-apoptotic genes, thus promoting tumor growth, survival, and drug resistance. Due to the common failure of classic chemotherapeutic agents in treating hypoxic cancers, some strategies have been developed to target tumors under hypoxic conditions including bioreductive drugs and inhibitors against pathways that were depended by tumor cells for their growth and survival. These new strategies may provide more effective and specific methods in targeting hypoxic tumors.

Aberrant production of cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandin E2 (PGE2) have been demonstrated to contribute to tumor development and malignancy in many human cancers. However, the initiating factor responsible for maintaining high levels of COX-2 expression during cancer development remains enigmatic. We hypothesize that aberrant expression of COX-2 in cancer cells is initiated by hypoxia, a common phenomenon occurs in very early stage during cancer development. Herein, we present data demonstrating that levels of COX-2 in cancer cells are induced by hypoxia in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Intriguingly, HIF-1α does not directly induce COX-2 promoter activity. In contrast, induction of COX-2 expression is mediated by HIF-1α-induced downregulation of dual specificity phosphatase-2 (DUSP-2), a MAPK-specific phosphatase. Forced expression of DUSP2 reduces the levels of COX-2, while knockdown of DUSP2 induces COX-2 expression in cancer cells. Moreover, depletion of DUSP2 also induces the expression of microsomal prostaglandin E synthase (mPGES) and consequently increases the production of PGE2. Further study reveals that re-induction of DUSP2 abolishes hypoxia-induced COX-2 expression and PGE2 production. To test the hypothesis that COX-2 mediates loss-of-DUSP2-induced tumorigenesis, a selective COX-2 inhibitor, NS-398, was used to block the function of COX-2 in the DUSP2-knockdown xenograft mouse model. Knockdown of DUSP2 markedly enhances tumor growth while treatment with NS-398 reverses loss-of-DUSP2-induced tumor development in vivo. Taken together, our data indicate that DUSP2, a negative regulator of COX-2, plays a critical tumor suppressive role and hypoxia, a common stress during cancer development, suppresses DUSP2 to initiate COX-2 overexpression and promote cancer progression. Our findings suggest that preventing DUSP2 from hypoxia-mediated downregulation or re-induction of DUSP2 during cancer development may represent a potential approach for cancer therapy. Citation Format: Yo-Hua Li, Shih-Chieh Lin, Shaw-Jenq Tsai. Suppression of cyclooxygenase-2 by dual-specificity phosphatase-2 ameilorates cancer malignancy. [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 1666. doi:10.1158/1538-7445.AM2014-1666