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James M. Phang, M.D.

Laboratory of Comparative Carcinogenesis Head, Metabolism and Cancer Susceptibility Section Senior Investigator NCI-Frederick Metabolism and Cancer Susceptibility Section Building 538, Room 115 Frederick, MD 21702-1201 Phone:   301-846-5367 Fax:   301-846-6093 E-Mail:   phang@mail.ncifcrf.gov

Biography

Dr. James Phang received his M.D. from Loma Linda University School of Medicine and his clinical training in internal medicine from Stanford Medical Center. He was a clinical associate with the NCI's Metabolism Branch. After additional training in biochemistry and molecular biology with the Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Dr. Phang was appointed a senior investigator in the Metabolism Branch, NCI, and later became chief of the Endocrinology Section. From 1989 to 1998, he served as chief of the Laboratory of Nutritional and Molecular Regulation, and in 1998, he formed the Metabolism and Cancer Susceptibility Section in the Basic Research Laboratory. The Section joined the Laboratory of Comparative Carcinogenesis in 2003.

Research

Metabolic Mechanisms for Modulating the Cancer Susceptible Phenotype
An underlying theme is the metabolic modulation of proliferative, apoptotic, and stress-related signaling. Recent advances in three areas have focused our efforts: (1) the identification of cancer susceptibility genes and their products-for example, Apc, which is responsible for familial adenomatous polyposis (FAP) and which is mutated in 85 percent of sporadic human colorectal cancers; (2) the participation of metabolic enzymes in carcinogenesis. A number of metabolic enzymes are induced accompanying p53-mediated apoptosis; and (3) the implication of reactive oxygen species (ROS) in mitogenic and cytokine-mediated signaling and/or programmed cell death.

The Apc Genotype in Colorectal Carcinogenesis and Modulation of the Phenotype by Nitric Oxide
The regulation of PGHS-2 (aka COX-2) by nitric oxide (NO), a proinflammatory signaling factor, occurs at several levels. Using conditionally immortalized murine colonic epithelial cells contrasting in Apc genotype, we showed that NOS II and NO donors increased COX-2 expression and NO donors increased the formation of beta-catenin: Tcf/LEF:DNA complexes. The mechanism of this effect is linked to the degradation of E-cadherin and release of beta-catenin bound to plasma membranes. Although this NO-initiated, beta-catenin-dependent effect was associated with increased expression of COX-2, evidence of direct activation of the COX-2 promoter was lacking. We now have shown that beta-catenin signaling increases the expression of PEA3, a member of the Ets family of transcriptional factors and that PEA3 activates the COX-2 promoter. In a COX-2 promoter luciferase model transfected with a variety of transcriptional factors, we showed that PEA3 acting synergistically with p300 markedly induced COX-2 promoter activity. Nitric oxide, delivered exogenously from NO donors or produced endogenously from transfected iNOS, augmented COX-2 promoter activity of transfected PEA3/p300. The stimulation by NO was not seen with c-jun, beta-catenin, TCF-4, or with various combinations of transcriptional factors, but was limited to PEA3/p300. These studies suggest that NO acts at several levels to stimulate COX-2 expression. It activates metalloproteinases which releases beta-catenin, increases its signaling and thereby increases the expression of PEA3. Furthermore, NO increases the transcriptional activity of PEA3 in combination with p300.


Proline and Pyrroline-5-Carboxylate in Cell Regulation
Pyrroline-5-carboxylate (P5C) is the immediate precursor and product of proline. Catalytic cycling of these two molecules can mediate redox transfers between mitochondria and cytosol and can regulate several pathways: (1) augments the production of ribose and PP-rib-P for the synthesis of ribonucleotides; (2) regulates gene expression, especially of hypoxia-induced genes; and (3) modulates programmed cell death in cells undergoing p53-mediated apoptosis. The mechanisms underlying these effects are being studied. In cells undergoing apoptosis, proline oxidase is induced and added proline stimulates the formation of reactive oxygen species (ROS). Importantly, in p53 negative cells transfected to overexpress proline oxidase, the addition of proline is sufficient to induce apoptosis. Recently, it has been shown that the expression of proline oxidase is silenced in certain tumors in the face of normal expression of p53, p21 and BAX suggesting that proline oxidase may act as a cancer suppressor. We are characterizing the proline oxidase promoter to determine its regulation on a molecular level. To date, we have identified regulation by PPARgamma and its ligands, and by the mTOR-AMPK signaling pathway. In addition, we are monitoring the expression of genes participating in the proline metabolic paradigm using real-time PCR and panels of cancer cells and tumors. Finally, we are translating these in vitro cellular effects of proline metabolism to studies using tumor xenografts in immunodeficient mouse models. Adjunctive enhancement of the effects of cancer drugs by proline-induced apoptosis is an attractive possibility. In summary, these studies show that proline and pyrroline-5-carboxylate are an important mechanism in bioenergetics, metabolic signaling and apoptosis and may offer a novel strategy in cancer treatment.

Prolidase renders proline (hydroxyproline) from extracellular matrix degradation
Proline as substrate for the aforementioned mechanisms derives from nutrition, and, more importantly, from the degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). MMPs increase with tumor invasion and metastasis producing peptides from ECM with high content of proline/hydroxyproline. However, it has not been appreciated that the terminal step of ECM degradation is catalyzed by the enzyme, prolidase, which hydrolyzes imidodipeptides containing carboxyl proline or hydroxyproline. Patients with inherited prolidase deficiency have defective wound healing and angiopathy. In colorectal cancer cells (RKO) stably transfected with prolidase, angiogenic signaling is activated with increased expression of vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT-1). Prolidase-generated products, proline and hydroxyproline, inhibit the in situ prolyl hydroxylation of HIF-1alpha thereby decreasing its degradation via VHL-dependent proteasomal mechanisms. These studies show that products of prolidase from ECM degradation not only serve as bioenergetic substrates but also as mediators of angiogenic signaling.

This page was last updated on 6/12/2008.