Connecting COX-2 and Wnt in cancer.

Both the cyclooxygenase-2 (COX-2) and Wnt signaling cascades are active in the majority of colorectal cancers. Nevertheless, a direct link between these two key pathways has remained elusive. Recent reports show that one of the bioactive products of COX-2, prostaglandin E2, activates components of the canonical Wnt signaling system. The findings reviewed below reveal important crosstalk between these pathways, which may provide opportunities for the development of new drugs for treatment and/or prevention of colorectal cancer.

Targeting cyclooxygenase-2 and the epidermal growth factor receptor for the prevention and treatment of intestinal cancer.

Clinical and animal studies indicate a role for cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) in the development and progression of intestinal polyps and cancers. Although this combination of enzyme inhibition has shown synergy in intestinal polyp and tumor models, the exact mechanism for these effects remains undefined. Therefore, we sought to define the molecular mechanisms through which this process occurs. We observed a significant reduction in the number and size of small intestinal polyps in APC(min+/-) mice treated with either celecoxib (a selective COX-2 inhibitor) or erlotinib (Tarceva, an EGFR inhibitor). However, in combination, there was an overall prevention in the formation of polyps by over 96%. Furthermore, we observed a 70% reduction of colorectal xenograft tumors in mice treated with the combination and microarray analysis revealed genes involved in cell cycle progression were negatively regulated. Although we did not observe significant changes in mRNAs of genes with known apoptotic function, there was a significant increase of apoptosis in tumors from animals treated with the combination. The inhibition of EGFR also induced the down-regulation of COX-2 and further inhibited prostaglandin E2 formation. We observed similar effects on the prevention of intestinal adenomas and reduction of xenograft tumor volume when nonselective COX inhibitors were used in combination with erlotinib. Together, these findings suggest that the inhibition of both COX-2 and EGFR may provide a better therapeutic strategy than either single agent through a combination of decreased cellular proliferation and prostaglandin signaling as well as increased apoptosis.

Repression of prostaglandin dehydrogenase by epidermal growth factor and snail increases prostaglandin E2 and promotes cancer progression.

Prostaglandin E(2) (PGE(2)), a proinflammatory bioactive lipid, promotes cancer progression by modulating proliferation, apoptosis, and angiogenesis. PGE(2) is a downstream product of cyclooxygenase (COX) and is biochemically inactivated by prostaglandin dehydrogenase (PGDH). In the present study, we investigated the mechanisms by which PGDH is down-regulated in cancer. We show that epidermal growth factor (EGF) represses PGDH expression in colorectal cancer cells. EGF receptor (EGFR) signaling induces Snail, which binds conserved E-box elements in the PGDH promoter to repress transcription. Induction of PGE(2) catabolism through inhibition of EGFR signaling blocks cancer growth in vivo. In human colon cancers, elevated Snail expression correlates well with down-regulation of PGDH. These data indicate that PGDH may serve a tumor suppressor function in colorectal cancer and provide a possible COX-2-independent way to target PGE(2) to inhibit cancer progression.

Prostaglandin E2 enhances intestinal adenoma growth via activation of the Ras-mitogen-activated protein kinase cascade.

A large body of clinical, genetic, and biochemical evidence indicates that cyclooxygenase-2 (COX-2), a key enzyme for prostanoid biosynthesis, contributes to the promotion of colorectal cancer. COX-2-derived prostaglandin E2 (PGE2) is the most abundant prostaglandin found in several gastrointestinal malignancies. Although PGE2 enhances intestinal adenoma growth in Apcmin mice, the mechanism(s) by which it accelerates tumor growth is not completely understood. Here we investigated how PGE2 promotes intestinal tumor growth and the signaling pathways responsible for its effects. We observed that PGE2 treatment leads to increased epithelial cell proliferation and induces COX-2 expression in intestinal adenomas. Furthermore, we show that PGE2 regulation of COX-2 expression is mediated by activation of a Ras-mitogen-activated protein kinase signaling cascade. One intriguing finding is that COX-2-derived PGE2 mimics the effects of constitutively active Ras through a self-amplifying loop that allows for a distinct growth advantage.

Discovery and Characterization of Novel Nonsubstrate and Substrate NAMPT Inhibitors.

Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.

Gene expression profiling following constitutive activation of MEK1 and transformation of rat intestinal epithelial cells.

BACKGROUND: Constitutive activation of MEK1 (caMEK) can induce the oncogenic transformation of normal intestinal epithelial cells. To define the genetic changes that occur during this process, we used oligonucleotide microarrays to determine which genes are regulated following the constitutive activation of MEK in normal intestinal epithelial cells. RESULTS: Microarray analysis was performed using Affymetrix GeneChip and total RNA from doxycycline inducible RIEtiCAMEK cells in the presence or absence of doxycycline. MEK-activation induced at least a three-fold difference in 115 gene transcripts (75 transcripts were up-regulated, and 40 transcripts were down-regulated). To verify whether these mRNAs are indeed regulated by the constitutive activation of MEK, RT-PCR analysis was performed using the samples from caMEK expressing RIE cells (RIEcCAMEK cells) as well as RIEtiCAMEK cells. The altered expression level of 69 gene transcripts was confirmed. Sixty-one of the differentially expressed genes have previously been implicated in cellular transformation or tumorogenesis. For the remaining 8 genes (or their human homolog), RT-PCR analysis was performed on RNA from human colon cancer cell lines and matched normal and tumor colon cancer tissues from human patients, revealing three novel targets (rat brain serine protease2, AMP deaminase 3, and cartilage link protein 1). CONCLUSION: Following MEK-activation, many tumor-associated genes were found to have significantly altered expression levels. However, we identified three genes that were differentially expressed in caMEK cells and human colorectal cancers, which have not been previously linked to cellular transformation or tumorogenesis.

15-LOX-1 inhibits p21 (Cip/WAF 1) expression by enhancing MEK-ERK 1/2 signaling in colon carcinoma cells.

Currently, some controversy exists regarding the precise role of 15-lipoxygenase-1 (15-LOX-1) in colorectal carcinogenesis and other aspects of cancer biology. The aim of this study was to evaluate the effect of 15-LOX-1 on p21 (Cip/WAF 1) expression and growth regulation in human colon carcinoma cells. The effect of 13-S-hydroxyoctadecadienoic acid (HODE), a product of 15-LOX-1, on p21 (Cip/WAF 1) expression was evaluated in Caco-2 cells treated with sodium butyrate (NaBT) and/or nordihydroguaiarectic acid (NDGA), a LOX inhibitor. The effect of transfecting HCT-116 cells with 15-LOX-1 was also examined. NaBT-induced p21 (Cip/WAF 1) expression was enhanced by treatment with NDGA and 13-S-HODE reversed NaBT-induced p21 (Cip/WAF 1) expression in Caco-2 cells. Overexpression of 15-LOX-1 induced extracellular signal-related kinase (ERK) 1/2 phosphorylation, decreased p21 (Cip/WAF 1) expression, and increased HCT-116 cell growth. Treatment with NDGA decreased ERK 1/2 phosphorylation, and increased p21 (Cip/WAF 1) expression in 15-LOX-1 overexpressing HCT-116 cells. Our experimental results support the hypothesis that 15-LOX-1 may have "pro-neoplastic" effects during the development of colorectal cancer.

Emerging roles of beta-arrestins.

Arrestins were originally characterized as structural adaptor proteins which modulate the desensitization and trafficking of seven-membrane-spanning receptors. From these seminal observations a multitude of novel functions for this gene family have arisen. Here we review the recently identified roles for beta-arrestin including its nuclear function and roles in development, cellular migration, and metastasis.

Role of beta-arrestin 1 in the metastatic progression of colorectal cancer.

G protein-coupled receptor ligand-dependent transactivation of growth factor receptors has been implicated in human cancer cell proliferation, migration, and cell survival. For example, prostaglandin E(2) (PGE(2))-induced transactivation of the EGF receptor (EGFR) in colorectal carcinoma cells is mediated by means of a c-Src-dependent mechanism and regulates cell proliferation and migration. Recent evidence indicates that beta-arrestin 1 may act as an important mediator in G protein-coupled receptor-induced activation of c-Src. Whether beta-arrestin 1 serves a functional role in these events is, however, unknown. We investigated the effects of PGE(2) on colorectal cancer cells expressing WT and mutant beta-arrestin 1. Here we report that PGE(2) induces the association of a prostaglandin E receptor 4/beta-arrestin 1/c-Src signaling complex resulting in the transactivation of the EGFR and downstream Akt (PKB) signaling. The interaction of beta-arrestin 1 and c-Src is critical for the regulation of colorectal carcinoma cell migration in vitro as well as metastatic spread of disease to the liver in vivo. These results show that the prostaglandin E/beta-arrestin 1/c-Src signaling complex is a crucial step in PGE(2)-mediated transactivation of the EGFR and may play a pivotal role in tumor metastasis. Furthermore, our data implicate a functional role for beta-arrestin 1 as a mediator of cellular migration and metastasis.

Cyclooxygenase-1-derived PGE2 promotes cell motility via the G-protein-coupled EP4 receptor during vertebrate gastrulation.

Gastrulation is a fundamental process during embryogenesis that shapes proper body architecture and establishes three germ layers through coordinated cellular actions of proliferation, fate specification, and movement. Although many molecular pathways involved in the specification of cell fate and polarity during vertebrate gastrulation have been identified, little is known of the signaling that imparts cell motility. Here we show that prostaglandin E(2) (PGE(2)) production by microsomal PGE(2) synthase (Ptges) is essential for gastrulation movements in zebrafish. Furthermore, PGE(2) signaling regulates morphogenetic movements of convergence and extension as well as epiboly through the G-protein-coupled PGE(2) receptor (EP4) via phosphatidylinositol 3-kinase (PI3K)/Akt. EP4 signaling is not required for proper cell shape or persistence of migration, but rather it promotes optimal cell migration speed during gastrulation. This work demonstrates a critical requirement of PGE(2) signaling in promoting cell motility through the COX-1-Ptges-EP4 pathway, a previously unrecognized role for this biologically active lipid in early animal development.

Requirement of phospholipase D1 activity in H-RasV12-induced transformation.

The ability of the Ras oncogene to transform normal cells has been well established. One downstream effector of Ras is the lipid hydrolyzing enzyme phospholipase D. Recent evidence has emerged indicating a role for phospholipase D in cell proliferation, membrane trafficking, and migration. To study the potential importance of phospholipase D in the oncogenic ability of Ras, we used Rat-2 fibroblasts with reduced phospholipase D1 activity (Rat-2V25). Here, we show that H-Ras transformation of Rat-2 fibroblasts requires normal phospholipase D1 activity. WT Rat-2 fibroblasts transfected with the H-RasV12 oncogene grew colonies in soft agar and tumors in nude mice. However, Rat-2V25 cells when transfected with the H-RasV12 oncogene did not form colonies in soft agar or produce tumors when xenografted onto nude mice. Interestingly, in the presence of phosphatidic acid, the product of phospholipase D, growth in soft agar and tumor formation was restored. We also observed a dramatic increase in the expression of phospholipase D1 in colorectal tumors when compared with adjacent normal mucosa. Our studies identify phospholipase D1 as a critical downstream mediator of H-Ras-induced tumor formation.

Oncogenic potential of MEK1 in rat intestinal epithelial cells is mediated via cyclooxygenase-2.

BACKGROUND & AIMS: The mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase (MEK) pathway plays an important role in the regulation of cell growth and differentiation. Constitutively active components of the MEK signaling cascade can induce oncogenic transformation in many cell systems. Downstream MEK signaling also plays an important role in the regulation of cyclooxygenase-2 (COX-2), which is known to be involved in colorectal cancer. Therefore, we determined the role of COX-2 on the oncogenic potential of MEK1 in nontransformed rat intestinal epithelial cells. METHODS: Constitutively active MEK1 (CA-MEK) mutant transfected rat intestinal epithelial cells were established and tested for their ability to grow in soft agar and form tumors in vivo. The effect of CA-MEK on sodium butyrate (NaB)-induced apoptosis was evaluated by the Annexin V assay. The transcriptional activity and posttranscriptional stability of the COX-2 gene was determined by transient transfection with COX-2 reporter variants and by Northern analysis. To address the role of COX-2 in tumor growth in vivo, xenografted mice were treated with celecoxib (100 mg/kg) or vehicle. RESULTS: CA-MEK transfected RIE-1 and IEC-6 cells formed colonies in soft agar and tumors in nude mice. These cells showed resistance to NaB-induced apoptosis and cell cycle arrest. MEK activation led to increased expression of COX-2, Bcl-X(L), Mcl-1, and phosphorylated Bad and decreased expression of Bak. Along with elevated COX-2 levels, PGI(2) and PGE(2) levels were also increased. Pharmacologic inhibition of COX-2 inhibited MEK-induced tumor growth in vivo through enhanced apoptosis. CONCLUSIONS: COX-2 and its bioactive lipid products may play an important role in MEK-induced transformation.

15-Hydroxyprostaglandin dehydrogenase is down-regulated in colorectal cancer.

Prostaglandin E2 (PGE2) can stimulate tumor progression by modulating several proneoplastic pathways, including proliferation, angiogenesis, cell migration, invasion, and apoptosis. Although steady-state tissue levels of PGE2 stem from relative rates of biosynthesis and breakdown, most reports examining PGE2 have focused solely on the cyclooxygenase-dependent formation of this bioactive lipid. Enzymatic degradation of PGE2 involves the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The present study examined a range of normal tissues in the human and mouse and found high levels of 15-PGDH in the large intestine. By contrast, the expression of 15-PGDH is decreased in several colorectal carcinoma cell lines and in other human malignancies such as breast and lung carcinomas. Consistent with these findings, we observe diminished 15-Pgdh expression in ApcMin+/- mouse adenomas. Enzymatic activity of 15-PGDH correlates with expression levels and the genetic disruption of 15-Pgdh completely blocks production of the urinary PGE2 metabolite. Finally, 15-PGDH expression and activity are significantly down-regulated in human colorectal carcinomas relative to matched normal tissue. In summary, these results suggest a novel tumor suppressive role for 15-PGDH due to loss of expression during colorectal tumor progression.

Prostaglandin E2 regulates cell migration via the intracellular activation of the epidermal growth factor receptor.

Over the past decade cyclooxygenase-2-derived prostaglandins have been implicated in the development and progression of many types of cancer. Recently our laboratory has shown that treatment with prostaglandin E2 (PGE2) induces increased proliferation, migration, and invasiveness of colorectal carcinoma cells (Sheng, H., Shao, J., Washington, M. K., and DuBois, R. N. (2001) J. Biol. Chem. 276, 18075-18081). The stimulatory effects of PGE2 were dependent upon the activation of the phosphatidylinositol 3-kinase/Akt pathway. However, the exact signaling cascade responsible for phosphatidylinositol 3-kinase/Akt activation by PGE2 remains poorly defined. In the present study, we demonstrate that the PGE2-induced migration and invasion occurs via rapid transactivation and phosphorylation of the epidermal growth factor receptor (EGFR). Within minutes following treatment, PGE2 induces the activation of Akt. This effect was completely abolished by EGFR-specific tyrosine kinase inhibitors providing evidence for the role of the EGFR in this response. The rapid transactivation of the EGFR occurs via an intracellular Src-mediated event but not through the release of an extracellular epidermal growth factor-like ligand. EGFR transactivation was also observed in vivo by the direct comparison of normal and malignant human colorectal samples. These results suggest that in developing colonic carcinomas, the early effects of cyclooxygenase-2-derived PGE2 are in part mediated by the EGFR, and this transactivation is responsible for subsequent down-stream effects including the stimulation of cell migration and invasion.

Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor.

BACKGROUND AND AIMS: The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). METHODS: Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A(2) [cPLA(2)]). RESULTS: The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2 alone also induced the expression of VEGF. CONCLUSIONS: Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.