Clifton Stephens

Lysophosphatidic Acid Receptors Determine Tumorigenicity and Aggressiveness of Ovarian Cancer Cells

BACKGROUND Lysophosphatidic acid (LPA) acts through the cell surface G protein-coupled receptors, LPA1, LPA2, or LPA3, to elicit a wide range of cellular responses. It is present at high levels in intraperitoneal effusions of human ovarian cancer increasing cell survival, proliferation, and motility as well as stimulating production of neovascularizing factors. LPA2 and LPA3 and enzymes regulating the production and degradation of LPA are aberrantly expressed by ovarian cancer cells, but the consequences of these expression changes in ovarian cancer cells were unknown. METHODS Expression of LPA1, LPA2, or LPA3 was inhibited or increased in ovarian cancer cells using small interfering RNAs (siRNAs) and lentivirus constructs, respectively. We measured the effects of changes in LPA receptor expression on cell proliferation (by crystal violet staining), cell motility and invasion (using Boyden chambers), and cytokines (interleukin 6 [IL-6], interleukin 8 [IL-8], and vascular endothelial growth factor [VEGF]) production by enzyme-linked immunosorbent assay. The role of LPA receptors in tumor growth, ascites formation, and cytokine production was assessed in a mouse xenograft model. All statistical tests were two-sided. RESULTS SKOV-3 cells with increased expression of LPA receptors showed increased invasiveness, whereas siRNA knockdown inhibited both migration (P < .001, Student t test) and invasion. Knockdown of the LPA2 or LPA3 receptors inhibited the production of IL-6, IL-8, and VEGF in SKOV-3 and OVCAR-3 cells. SKOV-3 xenografts expressing LPA receptors formed primary tumors of increased size and increased ascites volume. Invasive tumors in the peritoneal cavity occurred in 75% (n = 4) of mice injected with LPA1 expressing SKOV-3 and 80% (n = 5) of mice injected with LPA2 or LPA3 expressing SKOV-3 cells. Metastatic tumors expressing LPA1, LPA2, and LPA3 were identified in the liver, kidney, and pancreas; tumors expressing LPA2 and LPA3 were detected in skeletal muscle; and tumors expressing LPA2 were also found in the cervical lymph node and heart. The percent survival of mice with tumors expressing LPA2 or LPA3 was reduced in comparison with animals with tumors expressing beta-galactosidase. CONCLUSIONS Expression of LPA2 or LPA3 during ovarian carcinogenesis contributes to ovarian cancer aggressiveness, suggesting that the targeting of LPA production and action may have potential for the treatment of ovarian cancer.

Programmed cell death and radioresistance.

Whereas apoptosis is a critical mode of cell deletion in normal organism development, apoptotic cells are also observed in tumors, especially following cytotoxic treatments, leading to questions about their role in tumor response to therapy. We have conducted a series of studies using murine tumor models and found that the ability of the tumor cells to undergo apoptosis correlates with tumor response to radiation. The best correlation was with the pretreatment apoptotic index, suggesting that apoptosis in some tumors may govern radiocurability by regulating the number of tumor clonogens. However, other roles for apoptosis in tumor response to radiation have not been ruled out. One of the important observations that has come from this work has been the heterogeneity in apoptosis propensity both within the cell population of a given tumor and among different types of tumors. Such findings underscore the fact that apoptosis is under complex genetic control and that some of the same oncogenes and tumor suppressor genes that are responsible for tumor initiation and progression to malignancy also dictate the apoptotic response to treatment. Understanding the biochemical and molecular pathways that govern this process may ultimately allow the development of strategies for modulating apoptosis for therapeutic benefit.

Selenium and Vitamin E: Cell Type– and Intervention-Specific Tissue Effects in Prostate Cancer

BACKGROUND Secondary analyses of two randomized, controlled phase III trials demonstrated that selenium and vitamin E could reduce prostate cancer incidence. To characterize pharmacodynamic and gene expression effects associated with use of selenium and vitamin E, we undertook a randomized, placebo-controlled phase IIA study of prostate cancer patients before prostatectomy and created a preoperative model for prostatectomy tissue interrogation. METHODS Thirty-nine men with prostate cancer were randomly assigned to treatment with 200 microg of selenium, 400 IU of vitamin E, both, or placebo. Laser capture microdissection of prostatectomy biopsy specimens was used to isolate normal, stromal, and tumor cells. Gene expression in each cell type was studied with microarray analysis and validated with a real-time polymerase chain reaction (PCR) and immunohistochemistry. An analysis of variance model was fit to identify genes differentially expressed between treatments and cell types. A beta-uniform mixture model was used to analyze differential expression of genes and to assess the false discovery rate. All statistical tests were two-sided. RESULTS The highest numbers of differentially expressed genes by treatment were 1329 (63%) of 2109 genes in normal epithelial cells after selenium treatment, 1354 (66%) of 2051 genes in stromal cells after vitamin E treatment, and 329 (56%) of 587 genes in tumor cells after combination treatment (false discovery rate = 2%). Validation of 21 representative genes across all treatments and all cell types yielded Spearman correlation coefficients between the microarray analysis and the PCR validation ranging from 0.64 (95% confidence interval [CI] = 0.31 to 0.79) for the vitamin E group to 0.87 (95% CI = 0.53 to 0.99) for the selenium group. The increase in the mean percentage of p53-positive tumor cells in the selenium-treated group (26.3%), compared with that in the placebo-treated group (5%), showed borderline statistical significance (difference = 21.3%; 95% CI = 0.7 to 41.8; P = .051). CONCLUSIONS We have demonstrated the feasibility and efficiency of the preoperative model and its power as a hypothesis-generating engine. We have also identified cell type- and zone-specific tissue effects of interventions with selenium and vitamin E that may have clinical implications.

Nanoparticle Based Systemic Gene Therapy for Lung Cancer: Molecular Mechanisms and Strategies to Suppress Nanoparticle-Mediated Inflammatory Response

Cancer gene therapy for the treatment of lung cancer has shown promise in the laboratory and in Phase I/II clinical trials. However, it is currently limited to treating localized tumors due to host-immunity against the gene delivery vector and the transgene. Therefore, there is a tremendous effort to develop and test alternate gene delivery vectors that are efficient, non-immunogenic, and applicable for systemic therapy. One such gene delivery vehicle is the non-viral vector, DOTAP: cholesterol (DOTAP:Chol) nanoparticle. Preclinical studies from our laboratory has shown that DOTAP:Chol. nanoparticles are effective systemic gene delivery vectors that efficiently deliver tumor-suppressor genes to disseminated lung tumors. Based on our findings we have recently initiated a Phase-I trial for systemic treatment of lung cancer using a novel tumor suppressor gene, FUS1. Although DOTAP:Chol. nanoparticles complexed to DNA (DNA-nanoparticles) are efficient vectors for systemic therapy, induction of an inflammatory response in a dose-dependent fashion has also been observed thereby limiting its use. A better understanding of the underlying mechanism for DNA-nanoparticles-mediated inflammatory response will allow us to develop strategies to suppress inflammation and expand the therapeutic window in treating human cancer. In the present study we conducted experiments examining the mechanism of nanoparticle-mediated inflammatory response in vitro and in vivo. We demonstrate that systemic administration of DNA-nanoparticles induced multiple signaling molecules both in vitro and in vivo that are associated with inflammation. Use of small molecule inhibitors against the signaling molecules resulted in their suppression and thereby reduced inflammation without affecting transgene expression. Our results provide a rationale to use small molecule inhibitors to suppress nanoparticle-mediated inflammation when administered systemically. Further development and testing will allow us to incorporate this strategy into future clinical trials that is based on systemic non-viral vector gene therapy.

Bcr-Abl-mediated suppression of normal hematopoiesis in leukemia

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.Oncogene advance online publication, 28 February 2005; doi:10.1038/sj.onc.1208500

Preclinical Assessment of a 980-nm Diode Laser Ablation System in a Large Animal Tumor Model

PURPOSE To characterize the performance of a 980-nm diode laser ablation system in an in vivo tumor model. MATERIALS AND METHODS This study was approved by the institutional animal care and use committee. The ablation system consisted of a 15-W, 980-nm diode laser, flexible diffusing-tipped fiber optic, and 17-gauge internally cooled catheter. Ten immunosuppressed dogs were inoculated subcutaneously with canine-transmissible venereal tumor fragments in eight dorsal locations. Laser ablations were performed at 79 sites where inoculations were successful (99%) at powers of 10 W, 12.5 W, and 15 W, with exposure times between 60 and 180 seconds. In 20 cases, multiple overlapping ablations were performed. After the dogs were euthanized, the tumors were harvested, sectioned along the applicator tract, measured, and photographed. Measurements of ablation zone were performed on gross specimen. Histopathology and viability staining was performed with hematoxylin and eosin and nicotinamide adenine dinucleotide hydrogen staining. RESULTS Gross pathologic examination confirmed a well circumscribed ablation zone with sharp boundaries between thermally ablated tumor in the center surrounded by viable tumor tissue. When a single applicator was used, the greatest ablation diameters ranged from 12 mm at the lowest dose (10 W, 60 seconds) to 26 mm at the highest dose (15 W, 180 seconds). Multiple applicators created ablation zones as large as 42 mm in greatest diameter (with the lasers operating at 15 W for 120 seconds). CONCLUSIONS The new 980-nm diode laser and internally cooled applicator effectively create large ellipsoid thermal ablations in less than 3 minutes.

BCR gene expression blocks Bcr-Abl induced pathogenicity in a mouse model

It is well accepted that the Bcr-Abl oncoprotein encoded by the Philadelphia chromosome is responsible for causing chronic myelogenous leukemia (CML). We have previously demonstrated that expression of Bcr interferes with the oncogenic effects of Bcr-Abl. To examine the effects of increased Bcr expression on Bcr-Abl oncogenic effects in a more physiological system, we tested the leukemogenic potential of a clone of K562 cells (K6 K562) containing an inducible BCR gene in NOD/scid mice. In this clone, the BCR gene was placed under the control of a tetracycline (Tet) repression system with a cytomegalovirus (CMV) promoter. Induction of exogenous Bcr protein by removal of Tet from the culture medium caused a dramatic increase in Bcr serine kinase activity, yielding predominantly phosphoserine Bcr, despite the presence of Bcr-Abl in the kinase reaction mixture. Prior to induction, the endogenous Bcr was predominantly in the phosphotyrosine form because of phosphorylation by Bcr-Abl, which we previously have shown suppresses Bcr serine/threonine kinase activity. Injection of K6 K562 cells into NOD/scid mice under conditions where BCR expression was suppressed resulted in death or terminal illness in 100% of the mice within 35 days after injection. These mice had a severe wasting syndrome characterized by atrophy of bone marrow hematopoiesis, and/or neoplasia of liver, bone marrow and spleen. Neoplastic spleens from these mice usually contained b3a2 Bcr-Abl transcripts. In contrast, induction of BCR expression at the time of injection allowed 80% survival; these healthy mice had no detectable microscopic lesions in blood forming organs. This difference in survival was significant with P<0.0001. Of interest, mice that were fed Tet for 19 days to initiate the disease syndrome and then released from the BCR transcriptional block had a significantly better survival pattern than mice exposed to Tet throughout the entire period. Moreover, 30% of these mice (three mice) survived through day 50. We conclude from these findings that BCR gene expression strongly inhibits the oncogenic effects of Bcr-Abl in NOD/scid mice, yielding healthy mice in most cases.

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.

Pharmacokinetics, Tissue Distribution, and Safety of P-Ethoxy Oligonucleotides Incorporated in Liposomes

AbstractP-ethoxy oligonucleotides (oligos) are lipophilic analogs of phospho-diesters. We have used liposomes to increase the intracellular uptake of P-ethoxy oligos, and demonstrated that liposomal P-ethoxy antisense oligos specific for Bcr-Abl, Grb2, Crkl or Bcl-2 mRNA could selectively inhibit the production of the corresponding proteins, thereby inducing growth inhibition in leukemia and lymphoma cell lines. In support of studying the effectiveness of liposomal P-ethoxy antisense oligos in animal models, we had conducted a series of studies to evaluate the pharmacokinetics, tissue distribution and safety of intravenous injection of liposomal P-ethoxy oligos in normal mice. The pharmacokinetics and tissue distribution of liposomal P-ethoxy oligos are very similar to those of other liposomal compounds. The plasma clearance rate of liposomal P-ethoxy oligos was biphasic; the t1/2 α and t1/2 β were approximately 6.7 min and 7 h, respectively. The highest concentrations of liposomal P-ethoxy oligos were fo...