Michael F. Verderame

The biology of the opioid growth factor receptor (OGFr).

Opioid peptides act as growth factors in neural and non-neural cells and tissues, in addition to serving for neurotransmission/neuromodulation in the nervous system. The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that plays a role in cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. Assays with radiolabeled OGF have detected specific and saturable binding, with a one-site model of kinetics. Subcellular fractionation studies show that the receptor for OGF (OGFr) is an integral membrane protein associated with the nucleus. Using antibodies generated to a binding fragment of OGFr, this receptor has been cloned and sequenced in human, rat, and mouse. OGFr is distinguished by containing a series of imperfect repeats. The molecular and protein structure of OGFr have no resemblance to that of classical opioid receptors, and have no significant homologies to known domains or functional motifs with the exception of a bipartite nuclear localization signal. Immunoelectron microscopy and immunocytochemistry investigations, including co-localization studies, have detected OGFr on the outer nuclear envelope where it interfaces with OGF. The peptide-receptor complex associates with karyopherin, translocates through the nuclear pore, and can be observed in the inner nuclear matrix and at the periphery of heterochromatin of the nucleus. Signal transduction for modulation of DNA activity is dependent on the presence of an appropriate confirmation of peptide and receptor. This report reviews the history of OGF-OGFr, examines emerging insights into the mechanisms of action of opioid peptide-receptor interfacing, and discusses the clinical significance of these observations.

Shared and distinct functions of RAGS and ELF‐1 in guiding retinal axons

Two ligands for Eph‐related receptor tyrosine kinases, RAGS and ELF‐1, have been implicated in the control of development of the retinotectal projection. Both molecules are expressed in overlapping gradients in the tectum, the target area of retinal ganglion cell axons. In two in vitro assays ELF‐1 is shown to have a repellent axon guidance function for temporal, but apparently not for nasal axons. RAGS on the other hand is repellent for both types of axons, though to different degrees. Thus, RAGS and ELF‐1 share some and differ in other properties. The biological activities of these molecules correlate with the strength of interaction with their receptors expressed on RGC axons. The meaning of these findings for guidance of retinal axons in the tectum is discussed.

Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis.

Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.

Analysis of mechanisms underlying BRMS1 suppression of metastasis

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70–90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1–q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50–90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise, BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30–60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80–89%, but the decrease for MDA-MB-231 was less (13–15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

The OGF–OGFr Axis Utilizes the p16INK4a and p21WAF1/CIP1 Pathways to Restrict Normal Cell Proliferation

Opioid growth factor (OGF) is an endogenous opioid peptide ([Met(5)]enkephalin) that interacts with the OGF receptor (OGFr) and serves as a tonically active negative growth factor in cell proliferation of normal cells. To clarify the mechanism by which OGF inhibits cell replication in normal cells, we investigated the effect of the OGF-OGFr axis on cell cycle activity in human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (NHEKs). OGF markedly depressed cell proliferation of both cell lines by up to 40% of sterile water controls. Peptide treatment induced cyclin-dependent kinase inhibitor (CKI) p16(INK4a) protein expression and p21(WAF1/CIP1) protein expression in HUVECs and NHEKs, but had no effect on p15, p18, p19, or p27 protein expression in either cell type. Inhibition of either p16(INK4a) or p21(WAF1/CIP1) activation by specific siRNAs blocked OGF inhibitory action. Human dermal fibroblasts and mesenchymal stem cells also showed a similar dependence of OGF action on p16(INK4a) and p21(WAF1/CIP1). Collectively, these results indicate that both p16(INK4a) and p21(WAF1/CIP1) are required for the OGF-OGFr axis to inhibit cell proliferation in normal cells.

The Opioid Growth Factor (OGF)–OGF Receptor Axis Uses the p16 Pathway to Inhibit Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) represents 5.5% of malignancies worldwide, with approximately 30,000 new cases and approximately 11,000 deaths reported in the United States annually. The opioid growth factor (OGF; [Met(5)]-enkephalin) and the OGF receptor (OGFr) form an endogenous growth regulating system; the OGF-OGFr axis influences the G(0)-G(1) phase of the cell cycle in HNSCC. Cells treated with small interfering RNA (siRNA) for OGFr no longer responded to the growth inhibitory effects of OGF or the growth stimulatory effects of naltrexone, indicating that these activities are entirely mediated by OGFr. In this investigation, we examined the precise target of OGF in the cell cycle. Using SCC-1 cells, OGF decreased the phosphorylation of retinoblastoma protein. This change was correlated with reduced Cdk4, but not Cdk2, kinase activity. OGF treatment increased cyclin-dependent kinase inhibitor p16 protein expression. Importantly, p16 complexed with Cdk4 was increased by OGF treatment at all time points, consistent with the hypothesis that OGF mediated growth inhibition through p16. Blockade of OGF-OGFr interactions with naloxone abolished the increased expression of p16 protein by OGF. Inhibition of p16 (INK4a) activation by p16-specific siRNA blocked OGFs repressive action on proliferation of SCC-1, CAL-27, and SCC-4 HNSCC cells. These data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human HNSCC is a cyclin-dependent kinase inhibitory pathway, and this may be useful in the diagnosis and treatment of HNSCC.

Identification and characterization of the murine ortholog (brms1) of breast‐cancer metastasis suppressor 1 (BRMS1)

We have cloned a novel metastasis‐suppressor gene (BRMS1) by differential display, comparing metastatic human breast carcinoma cell line MDA‐MB‐435 to its metastasis‐suppressed human chromosome 11 microcell hybrid. Screening of a murine cDNA library led to the identification of a 1.4 kb cDNA with a sequence revealing 85% homology to human BRMS1 within the open reading frame. The predicted protein sequence for the murine ortholog is 95% identical, suggesting that it is strongly conserved across these 2 species. The cloned cDNA was used to screen a murine strain SV129 BAC library to obtain brms1 genomic DNA. Three BAC clones [226(I4), 226(H4) and 239(N7)] were confirmed to encode the entire brms1 gene. Detailed analysis of BAC clone 226(I4) shows that the gene spans 8.5 kb and, like the human gene, is organized into 10 exons and 9 introns. While the exons share a high degree of homology, there are greater differences when comparing intron structures between the human and murine genes. The 5′ upstream region shares about 64% homology with its human counterpart, retaining several of the many putative regulatory elements. Like the human genomic BRMS1, the murine ortholog of the iGnT gene is found upstream of brms1 and the murine ortholog of the RIN1 gene is found downstream of brms1. brms1 was then tested for suppression of metastasis of mouse mammary carcinoma cell line 66cl4 in syngeneic BALB/c mice. Transfection with brms1 did not inhibit 66cl4 primary tumor formation but significantly suppressed its metastatic capability. This suggests that the murine ortholog functions similarly to BRMS1.

Antisense oligonucleotides to gastrin inhibit growth of human pancreatic cancer.

Human pancreatic cancer is stimulated by the autocrine production of gastrin. In this study, the effects of administration of antisense oligonucleotides to gastrin on growth of pancreatic cancer were evaluated in vitro and in vivo. Log phase BxPC-3 human pancreatic cancer cells in culture were exposed to increasing concentrations (0.5-10 microM) of a synthetic 20-mer antisense phosphorothioate oligonucleotide to gastrin for 48 h and growth was assessed by the cellular proliferation assay. Growth was inhibited up to 88% by anti-gastrin oligonucleotides in a dose-related fashion compared to cells treated with diluent or a randomized sequence with the same composition as the anti-gastrin oligonucleotide. In vivo nude mice bearing BxPC-3 xenografts were treated daily for 14 days with a 0.1-ml intratumoral injection of either anti-gastrin (5 microM), the scrambled sequence control phosphorothioate oligonucleotide (5 microM), or buffer. Tumors from the anti-gastrin-treated mice were significantly smaller in volume and weight and had less gastrin detected by radioimmunoassay than either controls. These results support the role of gastrin as a stimulatory peptide for growth of human pancreatic cancer. Antisense oligonucleotide to gastrin may have a role in the future treatment of patients with pancreatic cancer.

The OGF-OGFr axis utilizes the p21 pathway to restrict progression of human pancreatic cancer

BackgroundPancreatic cancer is the 4th leading cause of death from cancer in the U.S. The opioid growth factor (OGF; [Met5]-enkephalin) and the OGF receptor form an inhibitory growth regulatory system involved in the pathogenesis and treatment of pancreatic cancer. The OGF-OGFr axis influences the G0/G1 phase of the cell cycle. In this investigation, we elucidate the pathway of OGF in the cell cycle.ResultsUsing BxPC-3 cells, OGF decreased phosphorylation of retinoblastoma (Rb) protein without changing total Rb. This change was correlated with reduced cyclin-dependent kinase protein (Cdk) 2 kinase activity, but not total Cdk2. OGF treatment increased cyclin-dependent kinase inhibitor (CKI) p21 protein expression in comparison to controls, as well levels of p21 complexed with Cdk2. Naloxone abolished the increased expression of p21 protein by OGF, suggesting a receptor-mediated activity. p21 specific siRNAs blocked OGFs repressive action on proliferation in BxPC-3, PANC-1, and Capan-2 cells; cells transfected with negative control siRNA had no alteration in p21 expression, and therefore were inhibited by OGF.ConclusionThese data are the first to reveal that the target of cell proliferative inhibitory action of OGF in human pancreatic cancer is a p21 CKI pathway, expanding strategies for diagnosis and treatment of these neoplasias.

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.