Yu-Wen E. Chang

Cyclooxygenase 2 Promotes Cell Survival by Stimulation of Dynein Light Chain Expression and Inhibition of Neuronal Nitric Oxide Synthase Activity

ABSTRACT Cyclooxygenase 2 (COX-2) inhibits nerve growth factor (NGF) withdrawal apoptosis in differentiated PC12 cells. The inhibition of apoptosis by COX-2 was concomitant with prevention of caspase 3 activation. To understand how COX-2 prevents apoptosis, we used cDNA expression arrays to determine whether COX-2 regulates differential expression of apoptosis-related genes. The expression of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase [PIN]) was significantly stimulated in PC12 cells overexpressing COX-2. The COX-2-dependent stimulation of DLC expression was, at least in part, mediated by prostaglandin E2. Overexpression of DLC also inhibited NGF withdrawal apoptosis in differentiated PC12 cells. Stimulation of DLC expression resulted in an increased association of DLC/PIN with neuronal nitric oxide synthase (nNOS), thereby reducing nNOS activity. Furthermore, nNOS expression and activity were significantly increased in differentiated PC12 cells after NGF withdrawal. This increased nNOS activity as well as increased nNOS dimer after NGF withdrawal were inhibited by COX-2 or DLC/PIN overexpression. An nNOS inhibitor or a membrane-permeable superoxide dismutase (SOD) mimetic protected differentiated PC12 cells from NGF withdrawal apoptosis. In contrast, NO donors induced apoptosis in differentiated PC12 cells and potentiated apoptosis induced by NGF withdrawal. The protective effects of COX-2 on apoptosis induced by NGF withdrawal were also overcome by NO donors. These findings suggest that COX-2 promotes cell survival by a mechanism linking increased expression of prosurvival genes coupled to inhibition of NO- and superoxide-mediated apoptosis.

RhoA Mediates Cyclooxygenase-2 Signaling to Disrupt the Formation of Adherens Junctions and Increase Cell Motility

Cyclooxygenase-2 (COX-2) represents an important target for treatment and prevention of colorectal cancer. Although COX-2 signaling is implicated in promoting tumor cell growth and invasion, the molecular mechanisms that mediate these processes are largely unknown. In this study, we show that the RhoA pathway mediates COX-2 signaling to disrupt the formation of adherens junctions and increase cell motility. Disruption of adherens junctions promotes tumor cell invasion and metastasis and is often associated with tumor progression. We detected high levels of RhoA activity in HCA-7 colon carcinoma cells that constitutively express COX-2. Inhibition of COX-2 significantly reduced the levels of RhoA activity in HCA-7 cells, suggesting that constitutive expression of COX-2 stimulates RhoA activity. Interestingly, inhibition of COX-2 or silencing of COX-2 expression with small interfering RNA (siRNA) stimulated the formation of adherens junctions, concomitant with increased protein levels of E-cadherin and alpha-catenin. Furthermore, inhibition of RhoA or silencing of RhoA expression with siRNA increased the levels of E-cadherin and alpha-catenin. Inhibition of Rho kinases (ROCK), the RhoA effector proteins, also increased levels of E-cadherin and alpha-catenin and stimulated formation of adherens junctions. The motility of HCA-7 cells was significantly decreased when COX-2 or RhoA was inhibited. Therefore, our data reveal a novel molecular mechanism that links COX-2 signaling to disrupt the formation of adherens junctions; COX-2 stimulates the RhoA/ROCK pathway, which reduces levels of E-cadherin and alpha-catenin leading to disruption of adherens junction formation and increased motility. Understanding of COX-2 downstream signaling pathways that promote tumor progression is crucial for the development of novel therapeutic strategies.

Targeting RhoA/Rho kinase and p21-activated kinase signaling to prevent cancer development and progression.

Elevated RhoA/Rho kinase and p21-activated kinase signaling have been shown to promote cancer development and metastasis and have drawn much attention as potential targets of anti-cancer therapy. Elevated RhoA and Rho kinase activity promote cancer cell invasion and eventually lead to metastasis by disrupting E-cadherin-mediated adherens junctions and degradation of the extracellular matrix. Elevated p21-activated kinase activity promotes invasion by stimulating cell motility but also promotes cancer cell survival and growth. In this review we describe normal functions of RhoA/Rho kinase and p21-activated kinase signaling, mechanisms that lead to constitutive activation of RhoA/Rho kinase and p21-activated kinase pathways, and processes by which constitutive RhoA/Rho kinase and p21-activated kinase activity promote cancer development and progression to more aggressive and metastatic phenotypes. In addition, we summarize relevant patents on RhoA/Rho kinase and p21-activated kinase as targets of anti-cancer therapy and discuss the clinical potential of different approaches to modulate RhoA/Rho kinase and p21-activated kinase signaling.

Cyclooxygenase 2 inhibits SAPK activation in neuronal apoptosis.

Cyclooxygenase 2 (COX-2) expressed in cultured neuronal PC12 cells under inducible promoter protects cells from trophic withdrawal apoptosis. Stimulation of SAPK is thought to play a significant role in initiation of PC12 cell death. We have therefore examined whether COX-2 expression inhibits trophic withdrawal-mediated activation of SAPK. SAPK activity increased during the first 6h after NGF removal in mock-transfected PC12 cells. COX-2 expression attenuated the increase of SAPK, as detected by Western blot analysis with phosphorylation state specific anti-SAPK antibodies and by SAPK activity assays. We propose that COX-2 attenuated SAPK activation by preventing activation of nNOS, which occurs, as we have shown before, via COX-2-mediated expression of dynein light chain (DLC). Activation of SAPK in neuronal cell death was attenuated by DLC expression. These observations support a role for NO production and SAPK activation in the neuronal death mechanisms.

Caspase Activation of p21-Activated Kinase 2 Occurs During Cisplatin-Induced Apoptosis of SH-SY5Y Neuroblastoma Cells and in SH-SY5Y Cell Culture Models of Alzheimer’s and Parkinson’s Disease

p21-activated kinase 2 (PAK-2) appears to have a dual function in the regulation of cell survival and cell death. Activation of full-length PAK-2 by the p21 G-proteins Rac or Cdc42 stimulates cell survival. However, PAK-2 is unique among the PAK family because it is also activated through proteolytic cleavage by caspase 3 or similar caspases to generate the constitutively active PAK-2p34 fragment. Caspase activation of PAK-2 correlates with the induction of apoptosis in response to many stimuli and recombinant expression of PAK-2p34 has been shown to stimulate apoptosis in several human cell lines. Here, we show that caspase activation of PAK-2 also occurs during cisplatin-induced apoptosis of SH-SY5Y neuroblastoma cells as well as in SH-SY5Y cell culture models for Alzheimer’s and Parkinson’s disease. Inhibition of mitochondrial complex I or of ubiquitin/proteasome-mediated protein degradation, which both appear to be involved in Parkinson’s disease, induce apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Overexpression of the amyloid precursor protein, which results in accumulation and aggregation of β-amyloid peptide, the main component of β-amyloid plaques in Alzheimer’s disease, also induces apoptosis and caspase activation of PAK-2 in SH-SY5Y cells. Expression of the PAK-2 regulatory domain inhibits caspase-activated PAK-2p34 and prevents apoptosis in 293T human embryonic kidney cells, indicating that caspase activation of PAK-2 is directly involved in the apoptotic response. This is the first evidence that caspase activation of PAK-2 correlates with apoptosis in cell culture models of Alzheimer’s and Parkinson’s disease and that selective inhibition of caspase-activated PAK-2p34 could prevent apoptosis.

Abstract 3042: Investigating microRNA expression profiles in colon cancer cells in response to cyclooxygenase 2 signaling

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC MicroRNAs (miRNAs) are emerging as central players in cancer progression. Aberrant expression of miRNAs leads to malignant transformation and correlates with staging, progression and prognosis of cancers. Unfortunately, signaling pathways leading to altered expression of miRNAs are largely unknown. During early development of colon cancer, the expression of cyclooxygenase 2 (COX-2) is constitutively induced. We and others have shown that constitutive expression of COX-2 promotes colon carcinogenesis. However, the underlying mechanisms are not completely understood. To determine if COX-2 signaling alters expression of miRNAs, we used the Human Cancer RT2-miRNA PCR arrays to profile miRNA expression in HCA-7 human colon cancer cells treated with or without the COX-2 selective inhibitor NS-398. HCA-7 cells constitutively express endogenous COX-2. We found that expression levels of 39 miRNAs are different in HCA-7 cells as compared to HCA-7 cells treated with NS-398; 8 miRNAs were up-regulated and 31 miRNAs were down-regulated. To confirm the data from the PCR array, we used quantitative RT-PCR to quantify expression of 8 miRNAs in HCA-7 cells with or without NS-398 treatment. These 8 miRNAs were selected due to their relevance to cancer invasion and metastasis. We also compared the expression of these miRNAs in HCT-15 human colon cancer cells that were engineered to stably express COX-2 (HCT-15-COX-2) to the parental HCT-15 cells. Expression levels of 6 of the 8 examined miRNAs (miR-23b, 124, 146b-5p, 155, 183, 184) were significantly lower in HCT-15-COX-2 or HCA-7 cells than in parental HCT-15 or HCA-7 cells that were treated with NS-398. Therefore, we identified 6 miRNAs that are modulated by COX-2 and may play a role in mediating COX-2 signaling to promote colon carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3042.

Abstract 5214: Role of mitogen-activated protein kinase (ERK) signaling in cyclooxygenase 2-mediated colon cancer cell migration

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Colon cancer is the second most common cause of cancer-related deaths. Many patients present with colon cancer in advanced stages of metastasis, where the outcome is poor and mortality is high. Invasion is an essential capability acquired by cancer cells to metastasize to distant organs. Currently there are no effective strategies to prevent invasion because the underlying mechanisms are poorly understood. During early development of colon cancer, cyclooxygenase 2 (COX-2) is constitutively induced. We have shown that COX-2 promotes invasion by disrupting cell adhesion and increasing motility in HCA-7 colon cancer cells that constitutively express endogenous COX-2. To further determine the mechanisms by which COX-2 stimulates motility and to confirm this cellular behavior in other colon epithelial cells, we established HCT-15 colon cancer cells that stably express recombinant COX-2 (HCT-15-COX-2). Scratch wound gap assays were used to monitor cell motility and proliferation in HCA-7 and HCT-15-COX-2 cells. HT-29 colon cancer cells that do not express COX-2 and parental HCT-15 cells were used as controls. HCA-7 and HCT-15-COX-2 cells closed the gap significantly faster than HT-29 and HCT-15 cells. Inhibition of COX-2 in HCA-7 and HCT-15-COX-2 cells by the COX-2 selective inhibitor NS-398 decreased the gap closing. Interestingly, the enhanced gap closing ability of HCA-7 and HCT-15-COX-2 cells correlated with activation of ERKs, mitogen-activated protein kinases that are known to mediate COX-2 signaling to stimulate colon cancer cell growth. Activation of ERK depended on COX-2 activity. Inhibition of ERK signaling in HCA-7 and HCT-15-COX-2 cells reduced gap closing as effectively as NS-398. Furthermore, inhibition of ERK signaling prevented migration of both HCA-7 and HCT-15-COX-2 cells in modified Boyden chamber assays. Interestingly, reduced migration by inhibition of ERK signaling correlated with increased protein levels of adherens junction protein alpha-catenin. Our data reveal for the first time a role for ERK to promote cell migration in response to COX-2 signaling in addition to its stimulation of cancer cell growth. Since cardiovascular toxicity caused by long-term use of COX-2 inhibitors raises several concerns, targeting ERK can be an alternative approach for preventing colon cancer growth and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5214.