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Supplementary Data from Cyclooxygenase-2 Up-Regulates Ataxia Telangiectasia and Rad3 Related through Extracellular Signal-Regulated Kinase Activation

Cyclooxygenase-2 (COX-2) overexpression caused prolonged G2 arrest after exposure to ionizing radiation (IR) in our previous study. We were therefore interested in investigating the function of COX-2 in the G2 checkpoint pathway. Interestingly, we found that cells in which COX-2 is overexpressed showed up-regulated ataxia telangiectasia and Rad3 related (ATR) expression compared with control cells. In this study, we investigated the mechanism of ATR up-regulation by COX-2 and tested our hypothesis that COX-2–induced extracellular signal-regulated kinase (ERK) activation mediates up-regulation of ATR by COX-2. To investigate the relationship between COX-2 and ATR, we used two stable COX-2–overexpressing cancer cell lines (HCT116–COX-2 and H460–COX-2), a COX-2 knockdown A549 lung cancer cell line (AS), and an ATR knockdown HCT116 cell line. Cells were treated with various drugs [celecoxib, prostaglandin E2 (PGE2), PD98059, U0126, and hydroxyurea] and were then analyzed using reverse transcription-PCR, confocal microscopy, Western blotting, and clonogenic assay. COX-2–overexpressing cells were shown to have increased ERK phosphorylation and ATR expression compared with control cells, whereas AS cells were shown to have decreased levels of phospho-ERK and ATR. In addition, exogenously administered PGE2 increased ERK phosphorylation. Inhibition of ERK phosphorylation decreased ATR expression in both HCT116–COX-2 and A549 cells. HCT116–COX-2 cells were resistant to IR or hydroxyurea compared with HCT116-Mock cells, whereas administration of ATR shRNA showed the opposite effect. COX-2 stimulates ERK phosphorylation via PGE2. This COX-2–induced ERK activation seems to increase ATR expression and activity in endogenous COX-2–overexpressing cancer cells as well as in COX-2–overexpressing stable cell lines. (Mol Cancer Res 2009;7(7):1158–68)

Supplementary Data from Cyclooxygenase-2 (COX-2) Negatively Regulates Expression of Epidermal Growth Factor Receptor and Causes Resistance to Gefitinib in COX-2–Overexpressing Cancer Cells

Overexpression of cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) has been detected in many types of cancer. Although COX-2 and EGFR are closely related to each other, the exact mechanism of COX-2 in tumors has not been well understood. In this study, we investigated the relationship between COX-2 and EGFR in cancer cells. Using two cell lines stably overexpressing COX-2 (HCT-116-COX-2 and H460-COX-2) and a stable line of COX-2 knockdown MOR-P cells, we analyzed patterns of COX-2 and EGFR expression. To observe the effects of COX-2 on EGFR expression and activity, we did comparative analyses after treatment with various drugs (EGF, celecoxib, prostaglandin E2, gefitinib, Ro-31-8425, PD98059, and SP600125) in HCT-116-Mock versus HCT-116-COX-2 cells and H460-Mock versus H460-COX-2 cells. Overexpression of COX-2 specifically down-regulated EGFR expression at the level of transcription. COX-2–overexpressing cells have a decreased sensitivity to gefitinib. COX-2 induced activation of extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) but suppressed Akt activation. JNK inhibition by SP600125, a specific JNK inhibitor, resulted in restoration of EGFR levels in COX-2–overexpressing cells, whereas ERK inhibition by PD98059 did not. Overexpressed COX-2 negatively regulates EGFR expression via JNK activation, leading to gefitinib resistance. COX-2 may also regulate ERK activity independently of EGFR. Therefore, resistance of COX-2–overexpressing cells to gefitinib may be due to decreased expression of EGFR by JNK activation and EGFR-independent elevation of ERK activity by COX-2. The ability of COX-2 to inhibit EGFR expression and gefitinib effects may have significance in clinical cancer therapy. (Mol Cancer Res 2009;7(8):1367–77)

Purpose:Because of disease heterogeneity, limited studies on effective chemotherapies and therapeutic agents for advanced gastric cancer are available. Erythrocyte membrane protein band 4.1-like 5 (EPB41L5) has critical roles in renal and breast cancer metastasis. However, its role in metastatic gastric cancer remains unknown.Experimental Design:The specimens of 78 gastric cancer patients were analyzed by oligonucleotide microarray and survival analysis. In vitro experiments and metastatic mice models were used to assess the effects of EPB41L5 on gastric cancer metastasis.Results:Gastric cancer patients with high EPB41L5 levels had poor prognosis and low survival rate. Further, TGFβ1-induced EPB41L5 expression promoted gastric cancer cell migration and invasion by Smad-dependent TGFβ signaling. Phospho-Smad3 recruitment to the EPB41L5 promoter was significantly inhibited by a TGFβ inhibitor. EPB41L5 overexpression increased lung metastasis of gastric cancer cells in nude mice, which was completely reversed by anti-EPB41L5 monoclonal antibody treatment. Importantly, p120-catenin knockdown abolished EPB41L5-enhanced gastric cancer cell metastasis. Anti-EPB41L5 monoclonal antibody treatment blocked the association of EPB41L5 with p120-catenin.Conclusions:TGFβ/EPB41L5/p120-catenin axis regulates gastric cancer cell metastasis, and EPB41L5 is a promising therapeutic target for advanced gastric cancer.

Supplementary Data_Tracked Changes

Purpose: Because of disease heterogeneity, limited studies on effective chemotherapies and therapeutic agents for advanced gastric cancer are available. Erythrocyte membrane protein band 4.1-like 5 (EPB41L5) has critical roles in renal and breast cancer metastasis. However, its role in metastatic gastric cancer remains unknown. Experimental Design: The specimens of 78 gastric cancer patients were analyzed by oligonucleotide microarray and survival analysis. In vitro experiments and metastatic mice models were used to assess the effects of EPB41L5 on gastric cancer metastasis. Results: Gastric cancer patients with high EPB41L5 levels had poor prognosis and low survival rate. Further, TGFβ1-induced EPB41L5 expression promoted gastric cancer cell migration and invasion by Smad-dependent TGFβ signaling. Phospho-Smad3 recruitment to the EPB41L5 promoter was significantly inhibited by a TGFβ inhibitor. EPB41L5 overexpression increased lung metastasis of gastric cancer cells in nude mice, which was completely reversed by anti-EPB41L5 monoclonal antibody treatment. Importantly, p120-catenin knockdown abolished EPB41L5-enhanced gastric cancer cell metastasis. Anti-EPB41L5 monoclonal antibody treatment blocked the association of EPB41L5 with p120-catenin. Conclusions: TGFβ/EPB41L5/p120-catenin axis regulates gastric cancer cell metastasis, and EPB41L5 is a promising therapeutic target for advanced gastric cancer.

Although hypoxia is accepted as an important microenvironmental factor influencing tumor progression and treatment response, it is usually regarded as a static global phenomenon. Consequently, less attention is given to the impact of dynamic changes in tumor oxygenation in regulating the behavior of cancer cells. Androgen receptor (AR) signaling plays a critical role in prostate cancer. We previously reported that hypoxia/reoxygenation, an in vitro condition used to mimic an unstable oxygenation climate in a tumor, stimulates AR activation. In the present study, we showed that peroxiredoxin 1 (Prx1), a member of the peroxiredoxin protein family, acts as a key mediator in this process. We found that the aggressive LN3, C4-2, and C4-2B prostate cancer cell lines derived from LNCaP possess constitutively elevated Prx1 compared with parental cells, and display greater AR activation in response to hypoxia/reoxygenation. Although the cell survival–enhancing property of Prx1 has traditionally been attributed to its antioxidant activity, the reactive oxygen species–scavenging activity of Prx1 was not essential for AR stimulation because Prx1 itself was oxidized and inactivated by hypoxia/reoxygenation. Increased AR transactivation was observed when wild-type Prx1 or mutant Prx1 (C52S) lacking antioxidant activity was introduced into LNCaP cells. Reciprocal immunoprecipitation, chromatin immunoprecipitation, and in vitro pull-down assays corroborated that Prx1 interacts with AR and enhances its transactivation. We also show that Prx1 is capable of sensitizing a ligand-stimulated AR. Based on the above information, we suggest that disrupting the interaction between Prx1 and AR may serve as a fruitful new target in the management of prostate cancer. [Cancer Res 2007;67(19):9294–303]