D. Lee Gorden

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.

Matrix Metalloproteinase-9 from Bone Marrow–Derived Cells Contributes to Survival but not Growth of Tumor Cells in the Lung Microenvironment

The role of specific stromal-derived matrix metalloproteinases (MMPs) was analyzed in experimental metastasis assays in wild-type and either MMP-9, MMP-7, or MMP-2 null mice. MMP-9 null mice showed an 81% reduction in Lewis lung carcinoma tumor number, whereas MMP-7 null mice showed a 42% increase in tumor number, and there was no difference in tumor number in MMP-2 null mice compared with wild-type controls. Similarly, in an orthotopic model of lung cancer, 50% fewer MMP-9 null mice were able to establish tumors in the lung compared with control mice, although the size of the tumors was not different. The effect of MMP-9 on lung tumor colonization was dependent on the expression of MMP-9 from bone marrow-derived cells and is most likely contributed by neutrophils. To examine temporal effects of stromal MMP-9, bioluminescence imaging from luciferase-expressing human lung cancer-derived A549 cells revealed that there were fewer tumor cells in the lungs of MMP-9 null mice as early as 19 hours after injection compared with control mice, with no difference in subsequent growth rates. Six hours after injection of tumor cells, MMP-9 null mice showed a 4-fold increase in the percent of tumor cells undergoing apoptosis compared with control mice. We conclude that MMP-9 from the bone marrow contributes to the early survival and establishment of tumors in the lung and has no effect on subsequent growth. These results provide insights into the failure of MMP inhibitors in clinical trials in patients with late-stage lung cancer.

Antimetastatic Role of Smad4 Signaling in Colorectal Cancer

BACKGROUND & AIMS Transforming growth factor (TGF)-beta signaling occurs through Smads 2/3/4, which translocate to the nucleus to regulate transcription; TGF-beta has tumor-suppressive effects in some tumor models and pro-metastatic effects in others. In patients with colorectal cancer (CRC), mutations or reduced levels of Smad4 have been correlated with reduced survival. However, the function of Smad signaling and the effects of TGF-beta-receptor kinase inhibitors have not been analyzed during CRC metastasis. We investigated the role of TGF-beta/Smad signaling in CRC progression. METHODS We evaluated the role of TGF-beta/Smad signaling on cell proliferation, migration, invasion, tumorigenicity, and metastasis in Smad4-null colon carcinoma cell lines (MC38 and SW620) and in those that transgenically express Smad4. We also determined the effects of a TGF-beta-receptor kinase inhibitor (LY2109761) in CRC tumor progression and metastasis in mice. RESULTS TGF-beta induced migration/invasion, tumorigenicity, and metastasis of Smad4-null MC38 and SW620 cells; incubation with LY2109761 reversed these effects. In mice, LY2109761 blocked metastasis of CRC cells to liver, inducing cancer cell expression of E-cadherin and reducing the expression of the tumorigenic proteins matrix metalloproteinase-9, nm23, urokinase plasminogen activator, and cyclooxygenase-2. Transgenic expression of Smad4 significantly reduced the oncogenic potential of MC38 and SW620 cells; in these transgenic cells, TGF-beta had tumor suppressor, rather than tumorigenic, effects. CONCLUSIONS TGF-beta/Smad signaling suppresses progression and metastasis of CRC cells and tumors in mice. Loss of Smad4 might underlie the functional shift of TGF-beta from a tumor suppressor to a tumor promoter; inhibitors of TGF-beta signaling might be developed as CRC therapeutics.

Selective Visualization of Cyclooxygenase-2 in Inflammation and Cancer by Targeted Fluorescent Imaging Agents

Effective diagnosis of inflammation and cancer by molecular imaging is challenging because of interference from nonselective accumulation of the contrast agents in normal tissues. Here, we report a series of novel fluorescence imaging agents that efficiently target cyclooxygenase-2 (COX-2), which is normally absent from cells, but is found at high levels in inflammatory lesions and in many premalignant and malignant tumors. After either i.p. or i.v. injection, these reagents become highly enriched in inflamed or tumor tissue compared with normal tissue and this accumulation provides sufficient signal for in vivo fluorescence imaging. Further, we show that only the intact parent compound is found in the region of interest. COX-2-specific delivery was unambiguously confirmed using animals bearing targeted deletions of COX-2 and by blocking the COX-2 active site with high-affinity inhibitors in both in vitro and in vivo models. Because of their high specificity, contrast, and detectability, these fluorocoxibs are ideal candidates for detection of inflammatory lesions or early-stage COX-2-expressing human cancers, such as those in the esophagus, oropharynx, and colon.

Effective treatment of biliary cystadenoma.

Objective:Evaluate experience over 15 years with treatment of this lesion. Summary Background Data:Biliary cystadenoma, a benign hepatic tumor arising from Von Meyenberg complexes, usually present as septated intrahepatic cystic lesions. Methods:Data were collected concurrently and retrospectively on patients identified from hospital medical records reviewed for pertinent International Classification of Diseases, Ninth Revision, Clinical Modification and CPT codes, pathology logs, and from operative case logs. Pathology specimens were rereviewed to confirm the diagnosis of biliary cystadenoma or biliary cystadenocarcinoma by 2 GI pathologists. Results:From October 1989 to April 2004 at our institution, 19 (18F:1M) patients had pathologically confirmed biliary cystadenomas, including one with a biliary cystadenocarcinoma. The mean age was 48 ± 15 years at initial evaluation. Complaints included abdominal pain in 74%, abdominal distension in 26%, and nausea/vomiting in 11%. Only 1 patient presented with an incidental finding. Symptoms had been present for 3 ± 5 years, with 1 to 4 different surgeons and many other physicians involved in the diagnosis or treatment prior to definitive ablation. Eight patients had undergone 20 previous treatments, including multiple percutaneous aspirations in 4 and 11 operative procedures. CT or US was diagnostic in 95%, with internal septations present in the hepatic cysts. Definitive operative intervention consisted of hepatic resection in 12 patients, enucleation in 6 patients, and fenestration and complete fulguration in 1 patient. There were no perioperative deaths. No recurrences were observed after definitive therapy, with follow-up of 4 ± 4 years. Conclusions:Biliary cystadenoma must be recognized and treated differently than most hepatic cysts. There remains a need for education about the imaging findings for biliary cystadenoma to reduce the demonstrated delay in appropriate treatment. Traditional treatment of simple cysts such as aspiration, drainage, and marsupialization results in near universal recurrence and occasional malignant degeneration. This experience demonstrates effective options include total ablation by standard hepatic resection and cyst enucleation.

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model.

Background and Aims The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD. Methods Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts. Results Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts. Conclusions Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.

Biomarkers of NAFLD progression: a lipidomics approach to an epidemic

The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis. Recognition and timely diagnosis of these different stages, particularly NASH, is important for both potential reversibility and limitation of complications. Liver biopsy remains the clinical standard for definitive diagnosis. Diagnostic tools minimizing the need for invasive procedures or that add information to histologic data are important in novel management strategies for the growing epidemic of NAFLD. We describe an “omics” approach to detecting a reproducible signature of lipid metabolites, aqueous intracellular metabolites, SNPs, and mRNA transcripts in a double-blinded study of patients with different stages of NAFLD that involves profiling liver biopsies, plasma, and urine samples. Using linear discriminant analysis, a panel of 20 plasma metabolites that includes glycerophospholipids, sphingolipids, sterols, and various aqueous small molecular weight components involved in cellular metabolic pathways, can be used to differentiate between NASH and steatosis. This identification of differential biomolecular signatures has the potential to improve clinical diagnosis and facilitate therapeutic intervention of NAFLD.

Warm hepatic ischemia-reperfusion promotes growth of colorectal carcinoma micrometastases in mouse liver via matrix metalloproteinase-9 induction

Surgical resection remains the best treatment for colorectal metastases isolated to the liver; however, 5-year survival rates following liver resection are only 40% to 50%, with liver recurrence being a significant reason for treatment failure. The ischemia-reperfusion (I/R) injury incurred during liver surgery can lead to cellular dysfunction and elevations in proinflammatory cytokines and matrix metalloproteinases (MMP). In rodents, I/R injury to the liver has been shown to accelerate the outgrowth of implanted tumors. The mechanism for increased tumor growth in the setting of liver I/R injury is unknown. To investigate the effect of I/R on tumor growth, an experimental model was used whereby small hepatic metastases form after 28 days. Mice subjected to 30 min of 70% liver ischemia at the time of tumor inoculation had significantly larger tumor number and volume, and had elevated MMP9 serum and liver tissue MMP9 as evidenced by zymography and quantitative real-time PCR. Mice treated with doxycycline, a broad-spectrum MMP inhibitor, had reduced MMP9 levels and significantly smaller tumor number and volume in the liver. MMP9-null mice were used to determine if the effects of doxycycline were due to the absence of stromal-derived MMP9. The MMP9-null mice, with or without doxycycline treatment, had reduced tumor number and volume that was equivalent to wild-type mice treated with doxycycline. These findings indicate that hepatic I/R-induced elevations in MMP9 contribute to the growth of metastatic colorectal carcinoma in the liver and that postresection MMP9 inhibition may be clinically beneficial in preventing recurrence following hepatic surgery.

Resident stromal cell-derived MMP-9 promotes the growth of colorectal metastases in the liver microenvironment

Colorectal cancer, the second leading cause of cancer deaths in the United States, has a high probability of metastasizing to the liver. Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases that are implicated in cancer metastasis and many aspects of tumor progression. Using a splenic injection experimental metastasis model, mice that are genetically deficient in MMP‐9 demonstrated a nearly 2‐fold decrease in liver weight compared with wild type (WT) mice following injection with MC38 syngeneic colorectal cancer cells. Bioluminesence imaging data demonstrates an early negative effect on tumor growth in MMP‐9 null mice when compared with WT controls. Reconstitution of the bone marrow in MMP‐9−/− mice with cells competent to produce MMP‐9 did not recapitulate the WT phenotype of overwhelming burden of metastatic disease in the liver. In situ hybridization revealed the presence of MMP‐9 mRNA in both MC38 tumor cells and in surrounding stromal cells, and immunostaining with anti MMP‐9 was consistent with MMP‐9 expression by resident liver Kupffer cells. Stromal‐derived MMP‐9 contributes to the establishment and growth of colorectal metastases in the liver. Stromal MMP‐9 was derived from resident cells, most likely Kupffer cells, as opposed to infiltrating bone marrow‐derived cells and the effect of stromal MMP‐9 was independent of expression of MMP‐9 by tumor cells. Stromal‐derived MMP‐9 may represent an important target for selective inhibition in the treatment of metastases.

Use of short tandem repeats for DNA fingerprinting to rapidly diagnose graft-versus-host disease in solid organ transplant patients

Background. Graft-versus-host disease (GVHD) is a rare complication following liver transplantation and carries a poor prognosis with mortality approaching 90–95%. Diagnosis of GVHD is often delayed due to early symptoms mimicking more common, entities such as drug reactions and viral syndromes. To date, definitive diagnosis has been difficult and has relied on a constellation of clinical and histopathologic variables. We present the use of short tandem repeat DNA “fingerprinting” technology as a method of early, definitive diagnosis of GVHD in patients after liver transplantation. Methods. A patient status-postorthotopic cadaveric-liver transplant, with an uncomplicated immediate posttransplant course, presented 4 weeks after transplant with fever, diarrhea, and maculopapular rash on her palms, soles, and back. The patient’s condition worsened despite empiric treatment for an infectious etiology. Skin and rectal biopsies were suspicious for GVHD. Results. DNA was isolated from the skin and rectal biopsies as well as from a donor lymph node. PCR amplification was done for nine highly polymorphic short tandem repeats for each specimen and a unique DNA “fingerprint” was obtained from each. DNA from skin and rectum demonstrated mixed chimerism with both donor and recipient alleles detected. Thorough analysis confirmed GVHD. Conclusion. Short tandem repeats for DNA fingerprinting represents an efficient and reproducible method for the definitive diagnosis of GVHD after liver transplantation. Rapid detection of GVHD using this technology, coupled with early initiation of therapy, may lead to improved survival for patients with GVHD after solid organ transplant.