Rolf Jakobi

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.

Differential regulation of cyclooxygenase 2 expression by small GTPases Ras, Rac1, and RhoA

Cyclooxygenase 2 (COX‐2) is an immediate early gene induced by a variety of stimuli and its expression is stimulated by individual activation of Ras or Rho GTPases. Here we investigate the role of coordinate activation of Ras and Rho GTPases in the induction of COX‐2. Individual expression of constitutively active Ras, RhoA, or Rac1 was capable of stimulating COX‐2 expression in NIH3T3 cells, but co‐expression of constitutively active RhoA with either constitutively active Ras or Rac1 was required for full stimulation of COX‐2 expression. Serum growth factors differentially activated Ras, RhoA, and Rac1, which correlated with the activation of Raf‐1, ERK, and c‐Jun as well as with induction of COX‐2. Inhibition of Ras significantly blocked the activation of Raf‐1, ERK, and c‐Jun and the stimulation of COX‐2 expression in response to serum. In contrast, inhibition of Rho family GTPases partially blocked serum induction of ERK activation but had little effects on COX‐2 expression. Both inhibitors of MEK (PD098059) and JNK (SP600125) inhibited serum induction of COX‐2. PD98059 only inhibited constitutively active Ras‐induced COX‐2 expression, while SP600125 significantly inhibited both constitutively active Ras‐ and RhoA‐induced COX‐2 expression. Together, our data suggest that constitutively active oncogenic Ras and Rho coordinately stimulate COX‐2 expression whereas transient activation of Ras but not RhoA or Rac1 mediates the induction of COX‐2 in response to serum. Furthermore, ERK and JNK activation are both required for serum‐ and oncogenic Ras‐mediated COX‐2 expression whereas only JNK activation is required for oncogenic RhoA‐mediated stimulation of COX‐2 expression.

Identification and Characterization of PS-GAP as a Novel Regulator of Caspase-activated PAK-2

p21-activated protein kinase (PAK)-2 is a member of the PAK family of serine/threonine kinases. PAKs are activated by the p21 G-proteins Rac and Cdc42 in response to a variety of extracellular signals and act in pathways controlling cell growth, shape, motility, survival, and death. PAK-2 is unique among the PAK family members because it is also activated through proteolytic cleavage by caspase-3 or similar proteases to generate the constitutively active PAK-2p34 fragment. Activation of full-length PAK-2 by Rac or Cdc42 stimulates cell survival and protects cells from cell death, whereas caspase-activated PAK-2p34 induces a cell death response. Caspase-activated PAK-2p34 is rapidly degraded by the 26 S proteasome, but full-length PAK-2 is not. Stabilization of PAK-2p34 by preventing its polyubiquitination and degradation results in a dramatic stimulation of cell death. Although many proteins have been shown to interact with and regulate full-length PAK-2, little is known about the regulation of caspase-activated PAK-2p34. Here, we identify PS-GAP as a regulator of caspase-activated PAK-2p34. PS-GAP is a GTPase-activating protein for Cdc42 and RhoA that was originally identified by its interaction with the tyrosine kinase PYK-2. PS-GAP interacts specifically with caspase-activated PAK-2p34, but not active or inactive full-length PAK-2, through a region between the GAP and SH3 domains. The interaction with PS-GAP inhibits the protein kinase activity of PAK-2p34 and changes the localization of PAK-2p34 from the nucleus to the perinuclear region. Furthermore, PS-GAP decreases the stimulation of cell death induced by stabilization of PAK-2p34.

Functional PAK-2 knockout and replacement with a caspase cleavage-deficient mutant in mice reveals differential requirements of full-length PAK-2 and caspase-activated PAK-2p34

Abstractp21-Activated protein kinase 2 (PAK-2) has both anti- and pro-apoptotic functions depending on its mechanism of activation. Activation of full-length PAK-2 by the monomeric GTPases Cdc42 or Rac stimulates cell survival, whereas caspase activation of PAK-2 to the PAK-2p34 fragment is involved in the apoptotic response. In this study we use functional knockout of PAK-2 and gene replacement with the caspase cleavage-deficient PAK-2D212N mutant to differentiate the biological functions of full-length PAK-2 and caspase-activated PAK-2p34. Knockout of PAK-2 results in embryonic lethality at early stages before organ development, whereas replacement with the caspase cleavage-deficient PAK-2D212N results in viable and healthy mice, indicating that early embryonic lethality is caused by deficiency of full-length PAK-2 rather than lack of caspase activation to the PAK-2p34 fragment. However, deficiency of caspase activation of PAK-2 decreased spontaneous cell death of primary mouse embryonic fibroblasts and increased cell growth at high cell density. In contrast, stress-induced cell death by treatment with the anti-cancer drug cisplatin was not reduced by deficiency of caspase activation of PAK-2, but switched from an apoptotic to a nonapoptotic, caspase-independent mechanism. Homozygous PAK-2D212N primary mouse embryonic fibroblasts that lack the ability to generate the proapoptotic PAK-2p34 show less activation of the effector caspase 3, 6, and 7, indicating that caspase activation of PAK-2 amplifies the apoptotic response through a positive feedback loop resulting in more activation of effector caspases.

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.