F. Gregory Buchanan

Role of β-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 E2 (PGE2)-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 β-arrestin 1 may act as an important mediator in G protein-coupled receptor-induced activation of c-Src. Whether β-arrestin 1 serves a functional role in these events is, however, unknown. We investigated the effects of PGE2 on colorectal cancer cells expressing WT and mutant β-arrestin 1. Here we report that PGE2 induces the association of a prostaglandin E receptor 4/β-arrestin 1/c-Src signaling complex resulting in the transactivation of the EGFR and downstream Akt (PKB) signaling. The interaction of β-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/β-arrestin 1/c-Src signaling complex is a crucial step in PGE2-mediated transactivation of the EGFR and may play a pivotal role in tumor metastasis. Furthermore, our data implicate a functional role for β-arrestin 1 as a mediator of cellular migration and metastasis.

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.

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.

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.

Emerging Roles of β-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 β-arrestin including its nuclear function and roles in development, cellular migration, and metastasis.

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

BackgroundConstitutive 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.ResultsMicroarray 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).ConclusionFollowing 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.

Altered activation of phospholipase D by lysophosphatidic acid and endothelin-1 in mouse embryo fibroblasts lacking phospholipase C-γ1

Lysophosphatidic acid (LPA) and endothelin-1 (ET-1) activate phospholipase D (PLD) in many cell types. To see if phospholipase C-gamma1 plays a role, we used embryonic fibroblasts from mice in which the PLCgamma1 gene was disrupted. Surprisingly, the effect of LPA on inositol phosphate accumulation was increased in these PLCgamma1-/- cells, whereas that of ET-1 was completely abrogated. When PLD activity was measured, the response to LPA was also enhanced and the response to ET-1 lost in the PLCgamma1-/- cells. Treatment of these cells with ionomycin and oleoyl acetyl glycerol to mimic PLC stimulation restored PLD activity. Treatment of either PLCgamma1+/+ and PLCgamma1-/- cells with tyrosine kinase inhibitors did not inhibit LPA- or ET-1-induced PLD activity. Moreover, LPA and ET-1 treatment of PLCgamma1+/+ and PLCgamma1-/- cells did not cause tyrosine phosphorylation of PLC-gamma1 or PLC-gamma2. In summary, these results show that the altered PLD responses to LPA and ET-1 in PLCgamma1-/- are due to changes in PLC activity and do not involve tyrosine kinase activity.