Ovidiu C. Trifan

Overexpression of Cyclooxygenase-2 Is Sufficient to Induce Tumorigenesis in Transgenic Mice

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene inMin mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the β-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-xL and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.

Localization of the fourth locus (GLC1E) for adult-onset primary open-angle glaucoma to the 10p15-p14 region.

One of the major causes of blindness is primary open-angle glaucoma, which affects millions of elderly people worldwide. Genetic studies have so far mapped three loci for the adult-onset form of this condition to the 2cen-q13, 3q21-q24, and 8q23 regions. Herein, we report the localization of a fourth locus, to the 10p15-p14 region, in one large British family with a classical form of normal-tension open-angle glaucoma. Of the 42 meioses genotyped in this pedigree, 39 subjects (16 affected) inherited a haplotype compatible with their prior clinical designation, whereas the remaining 3 were classified as unknown. Although a maximum LOD score of 10.00 at a recombination fraction of straight theta=.00 was obtained with D10S1216, 21 other markers provided significant values, varying between 3.77 and 9.70. When only the affected meioses of this kindred were analyzed, LOD scores remained statistically significant, ranging from 3.16 (D10S527) to 3.57 (D10S506). Two critical recombinational events in the affected subjects positioned this new locus to a region of approximately 21 cM, flanked by D10S1729 and D10S1664. However, an additional recombination in a 59-year-old unaffected female suggests that this locus resides between D10S585 (or D10S1172) and D10S1664, within a genetic distance of 5-11 cM. However, the latter minimum region must be taken cautiously, because the incomplete penetrance has previously been documented for this group of eye conditions. A partial list of genes that positionally are considered as candidates includes NET1, PRKCT, ITIH2, IL2RA, IL15RA, IT1H2, hGATA3, the mRNA for open reading frame KIAA0019, and the gene for D123 protein.

Cyclooxygenase-1 and -2 isoenzymes.

The cyclooxygenase isoenzymes (COX-1 and -2) catalyze the rate-limiting steps in prostanoid biosynthesis. COX-1 and -2 genes encode two isoenzymes with overlapping yet distinct expression patterns and functions. Physiologically, various extracellular stimuli such as growth factors, cytokines and tumor promoters regulate the expression of COX-1 and -2 genes at both transcriptional and post-transcriptional levels. COX-2 is overexpressed in rheumatoid arthritis, colorectal and breast cancer. Prostanoids produced by the COX pathway signal via plasma membrane-localized, G-protein-coupled receptors as well as via nuclear receptors. Currently, several COX-2-selective inhibitors are developed to control the anti-inflammatory and anti-neoplastic activities of the COX-2 isoenzyme. Inhibition of the COX isoenzyme activity and/or expression may be the basis of future generation of anti-inflammatory and anti-neoplastic drugs.

Cyclooxygenase-2 modulates cellular growth and promotes tumorigenesis.

Cyclooxygenase (COX)‐2 and the prostaglandins resulting from its enzymatic activity have been shown to play a role in modulating cell growth and development of human neoplasia. Evidence includes a direct relationship between COX‐2 expression and cancer incidence in humans and animal models, increased tumorigenesis after genetic manipulation of COX‐2, and significant anti‐tumor properties of non‐steroidal anti‐inflammatory drugs in animal models and in some human cancers. Recent data showed that COX‐2 and the derived prostaglandins are involved in control of cellular growth, apoptosis, and signal through a group of nuclear receptors named peroxisome proliferator‐activated receptors (PPARs). In this article we will review some of the findings suggesting that COX‐2 is involved in multiple cellular mechanisms that lead to tumorigenesis.

A third locus (GLC1D) for adult-onset primary open-angle glaucoma maps to the 8q23 region

PURPOSE Two genes for adult-onset primary open-angle glaucoma have been mapped to chromosomes 2cen-q13 and 3q21-q24. We studied a family with adult-onset primary open-angle glaucoma in which the disease did not map to these two chromosomal regions. METHODS We ascertained a four-generation family with adult-onset primary open-angle glaucoma in which the disease status of individuals was objectively assigned using defined criteria. Complete ophthalmologic examinations, visual field testing, optic nerve head photographs, and venous blood samples were obtained. Family members were genotyped using polymerase chain reaction amplification of microsatellite polymorphic markers. Linkage analysis was performed and lod scores were calculated. Haplotype transmission data were analyzed. RESULTS A total of 20 subjects in three successive generations agreed to participate in the study. This included samples from eight affected subjects, one glaucoma suspect, one normal individual, and two spouses in generations II and III, and an additional eight individuals in generation IV. The phenotype in this family appears to be variable, with onset of visual field loss in middle age, followed by modest elevation of intraocular pressure and progression of the disease in older individuals. Linkage was established with a group of DNA markers located in the 8q23 region. A lod score value of 3.61 was obtained using marker D8S1471. Three other markers from the same region gave lod score values of over 3.0. Haplotype transmission data identified two recombination events that placed the gene in a 6.3-cM region flanked by D8S1830 and D8S592. The disease-bearing haplotype was inherited by eight affected subjects and three glaucoma suspects. CONCLUSION We present evidence for a third adult-onset primary open-angle glaucoma locus (GLC1D) on chromosome 8q23. The genetic heterogeneity of adult-onset glaucoma is evident from the multiplicity of chromosomal loci associated with this disease.

Overexpression of Cyclooxygenase-2 Induces Cell Cycle Arrest EVIDENCE FOR A PROSTAGLANDIN-INDEPENDENT MECHANISM

The immediate-early gene cyclooxygenase 2 (Cox-2) is induced in a variety of hyperplastic pathological conditions, including rheumatoid arthritis and colorectal cancer. Although a causal role for Cox-2 has been proposed, mechanisms by which Cox-2 function contributes to the pathogenesis of hyperplastic disease are not well defined. We constructed a green fluorescent protein-tagged Cox-2 (Cox-2-GFP) to examine its effects on a variety of cell types upon overexpression. Subcellular localization and enzymatic and pharmacological properties of Cox-2-GFP polypeptide were indistinguishable from those of the wild-type Cox-2 polypeptide. Overexpression of the Cox-2-GFP or the Cox-2 polypeptide by transient transfection suppressed the population of cells in the S phase of the cell cycle, with a concomitant increase in G0/G1 population. In contrast, transient overexpression of GFP had no effect on cell cycle distribution, whereas endoplasmic reticulum-retained GFP (GFP-KDEL) overexpression was associated with only a minor decrease of cells in S phase. Interestingly, neither NS-398 (a Cox-2-specific inhibitor) nor indomethacin could reverse the effect of Cox-2-GFP overexpression on cell cycle progression. Furthermore, two mutants of Cox-2, S516Q and S516M, which lack the cyclooxygenase activity, exhibited the same effect as Cox-2-GFP. The cell cycle effect of Cox-2-GFP was observed in ECV-304, NIH 3T3, COS-7, bovine microvascular endothelial cells, and human embryonic kidney 293 cells. These findings suggest that Cox-2 inhibits cell cycle progression in a variety of cell types by a novel mechanism that does not require the synthesis of prostaglandins.

Serum withdrawal-induced post-transcriptional stabilization of cyclooxygenase-2 mRNA in MDA-MB-231 mammary carcinoma cells requires the activity of the p38 stress-activated protein kinase.

Overexpression of thecyclooxygenase-2 (COX-2) gene is observed in several neoplastic diseases. However, molecular mechanisms involved in the regulation of expression of COX-2 are not well understood. In this report, we describe a unique post-transcriptional regulatory mechanism of COX-2 mRNA stabilization in MDA-MB-231 cells, a highly metastatic cell line derived from a human mammary tumor. High levels of COX-2 mRNA, protein, and enzyme activity were induced by serum withdrawal, which were potently inhibited by the addition of serum or >100-kDa serum factor. Nuclear run-on analysis and actinomycin D chase experiments indicate that regulation is primarily at the level of post-transcriptional mRNA stability. Interestingly, SB203580, an inhibitor of the p38 stress-activated protein kinase (SAPK), and overexpression of the dominant-negative p38α construct potently inhibited the serum withdrawal-induced COX-2 mRNA levels. Indeed, the half-life of COX-2 mRNA decreased from 9 to 4.5 h after SB203580 treatment, suggesting that signal transduction by the p38 SAPK pathway is required for COX-2 mRNA stability.

COX-2 inhibitors as radiosensitizing agents for cancer therapy

Prostaglandins have long been known to impact the radiosensitivity of cells and tissues, and many studies have centered on exploiting nonspecific prostaglandin inhibitors such as NSAIDs for therapeutic gain. These studies have ultimately been unsuccessful due to the lack of targeted specificity against the tumor. The discovery of the inducible cyclooxygenase enzyme (COX-2) and development of some highly selective inhibitors (which spare the constitutive COX-1 activity) has renewed excitement for modulating tumor prostaglandins as a method of specific radiosensitization of tumors, while sparing normal tissues. This review discusses these new data and generates a rationale for use of COX-2 inhibitors as radiosensitizing agents in cancer therapy.

Abstract 5004: APX005M, a humanized anti-CD40 antibody with strong immune-modulatory activities capable of tumor eradication in vivo

The success of immune checkpoint inhibitors validates the concept that immunotherapy is an effective approach for the treatment of solid tumors. In addition to reversing tumor-induced immune suppression, immune activating antibodies are being explored as the next generation of immuno-oncology therapeutics. CD40, which is expressed on antigen presenting cells such as dendritic and B cells initiates and regulates both innate and adaptive immunity and is essential for the activation of antigen-specific T cells. APX005M is a humanized IgG1 CD40 agonistic antibody developed using Apexigen9s APXiMAB™ discovery platform. APX005M binds with high affinity to human (Kd = 0.12nM) and monkey (Kd = 0.37nM) CD40. It recognizes a unique epitope that overlaps with the CD40 ligand binding sites and thus blocks the binding of CD40 to CD40L. APX005M is a potent CD40 agonistic antibody capable of activating antigen presenting B cells (EC50 = 12pM) and dendritic cells (EC50 = 0.49nM). Its CD40 agonistic activity requires crosslinking by Fc-gamma receptors since F(ab)’2 fragment of APX005M loses the agonistic activity. Upon binding to CD40 expressing tumor cells APX005M induces antibody-dependent cellular phagocytosis (ADCP) and apoptosis. In vivo APX005M completely eradicates CD40+ lymphoma tumors and inhibits the growth of rituximab-resistant tumors. The data suggest that a CD40 agonistic mAb such as APX005M, that activates CD40 through binding to the CD40L binding site while being dependent on crosslinking via Fc-gamma receptors, may represent an ideal immune activating antibody drug candidate for stimulating effective immune responses against tumors. APX005M is currently being evaluated in clinical trials for the treatment of patients with solid tumors. Citation Format: Pia Bjorck, Erin Filbert, Xiaodong Yang, Ovidiu C. Trifan. APX005M, a humanized anti-CD40 antibody with strong immune-modulatory activities capable of tumor eradication in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5004.

Abstract 4867: The CD40 agonistic antibody APX005M ‘licenses’ antigen presenting cells to promote tumor-specific T-cell responses

Modulation of co-stimulatory and co-inhibitory molecules on immune cells has become a promising approach for cancer immunotherapy. CD40 engagement on antigen presenting cells (APCs) by its ligand CD154 leads to maturation and expression of co-stimulatory molecules such as CD80, CD86, OX-40L, and 4-1BBL that are requisite for optimal antigen-specific T-cell activation, an essential component of the anti-tumor immune response. To evaluate the therapeutic potential of targeting the CD40-CD154 pathway, Apexigen has developed APX005M - a humanized IgG1 CD40 agonistic antibody that binds with high affinity to human CD40. APX005M mimics CD154, has potent CD40 agonistic activity and promotes activation of APCs. Monocyte-derived dendritic cells treated with APX005M display a mature phenotype characterized by upregulation of CD80, CD86 and HLA-DR and increased secretion of IL-12. In mixed lymphocyte reaction and viral antigen recall assays, APX005M significantly enhances proliferation of and IFN-a secretion from both naive and memory T cells. Since CD40-CD154 signaling is involved in the priming phase of T cell activation and acts upstream of many critical costimulatory pathways, CD40 agonistic antibodies may represent an essential component of combination immunotherapy. In support of this, we show that APX005M synergizes with checkpoint inhibitors to promote antigen-specific T-cell responses. Importantly, in an ex vivo tumor assay APX005M induces T-cell proliferation and cytokine secretion. These data demonstrate that APX005M binds to APCs and induces their activation and maturation to ultimately drive a potent tumor-specific T-cell response. T cell activation in cancer patients receiving APX005M will be assessed in current and future clinical studies. Citation Format: Erin L. Filbert, Pia Bjorck, Xiaodong Yang, Ovidiu C. Trifan. The CD40 agonistic antibody APX005M ‘licenses’ antigen presenting cells to promote tumor-specific T-cell responses. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4867.