Natural Products and Cancer Biology Research Program: Cancer Research Center of Hawai‘i

André S. Bachmann, PhD, MS
abachmann@crch.hawaii.edu

International Journal of Cancer, 2010, 126, 2012-2024
Molecular Cancer Therapeutics, 2009, 8, 2067-2075	Proc Natl Acad Sci, 2009, 106, 6507-6512	Cancer Research, 2008, 68, 9825-9831
Nature, 2008, 452, 755-758	Clinical Cancer Research, 2007, 13, 6312-6319	Oncogene, 2005, 24, 5606-5618

Lab Members (Present and Past)

Collaborators

Neuroblastoma and Medulloblastoma Translational Research Consortium (NMTRC)

Cell and Molecular Biology Program - Cancer Biology (CMB)

Graduate Program in Molecular Biosciences & Bioengineering (MBBE)

American Cancer Society

1. Regulation of Polyamines in MYCN-Amplified Neuroblastoma
Project #1 examines the role of ornithine decarboxylase (ODC) and polyamines in neuroblastoma (NB) development. NB is an aggressive pediatric cancer of the autonomous nervous system originating from the adrenal medulla and autonomous ganglia in the chest and abdomen. After leukemia and cerebral tumors, NB is the third most frequent malignant tumor of childhood. The incidence in the United States is approximately one in 7,000. The therapy of NB is very difficult, especially in advanced stages of the disease with widespread metastases to liver, bone, lymph nodes, and bone marrow. Current therapies include high-dose chemotherapy, autologous peripheral stem cell transplantation, and radiation therapy. More recently, immunotherapy has been added using monoclonal antibodies to the GD2 glycolipid antigen that is heavily expressed by NB cells. However, despite intensive treatments, the long-term survival of patients with high-risk NB remains poor, since these more aggressive forms of NB respond poorly to hormonal and chemotherapeutic approaches. The response of patients that relapse or do not achieve complete remission is even worse, thus demanding the development of novel drugs that exhibit alternative mechanisms of action. NB has a particularly poor prognosis in patients older than 2 years at diagnosis, advanced stage disease and/or disease characterized by MYCN gene amplification. Therefore, there is an urgent need for a better understanding of the cellular regulation of MYCN-amplified NB tumors.

Although a role for the MYCN oncoprotein has been established in NB pathogenesis, the mechanism by which MYCN contributes to both the development of this disease and its poor prognosis is still unclear. The MYCN oncoprotein functions as a transcriptional regulator and thus may influence tumorigenesis and patient survival by regulating the expression of key genes involved in the NB malignant phenotype. Several studies have identified that the ornithine decarboxylase (ODC) gene is directly regulated by the MYCC and MYCN oncoproteins. Based on this MYCN-ODC connection, we proposed several years ago that ODC, and therefore polyamines, may play a significant role in NB tumorigenesis (Fig. 1).

The goal of this project is to explore the polyamine pathway as a novel target for NB therapy. In particular, we have targeted polyamine biosynthesis in NB using clinically-established pharmacological inhibitors of enzymes ODC and S-adenosylmethionine decarboxylase (AdoMetDC), including inhibitors DFMO and SAM486A, respectively (Oncogene, 2005, 24: 5606-5618; Cancer Res, 2008, 68, 9825-9831). As a direct result of our NCI/R01-funded NB research program, we are currently in the process of organizing human NB clinical trials with the NCI-designated Vermont Cancer Center and other participating hospitals. The goal of this trial is to validate the efficacy of polyamine inhibitor DFMO (in combination with other therapeutics) in relapsed/refractory, high-risk NB patients.

Figure 1: Proposed interplay between polyamines and cell cycle regulatory proteins in MYCN-amplified neuroblastoma (NB) cells. A, Micrographs showing the cell morphology (cytoskeleton, green; nucleus, red) of human NB cells using a laser scanning confocal microscope. B, Polyamines play a key role in cell cycle regulation, but the molecular mechanisms underlying polyamine-promoted cell signaling are still poorly defined. High polyamine titers promote cell cycle progression and cell proliferation, presumably via the blocking of cyclin-dependent kinase inhibitor p27Kip1, which leads to hyperphosphorylation of retinoblastoma protein Rb. The blockade of polyamine biosynthesis with specific inhibitors DFMO and SAM486A leads to G1 cell cycle arrest in response to p27Kip1 upregulation and subsequent hypophosphorylation of Rb. MYCN is strongly downregulated in response to polyamine depletion and is known to regulate p27Kip1 and ODC geneexpression in NB cells.

2. Syrbactins: A New Structural Class of Proteasome Inhibitors
Project #2 examines a new structural class of proteasome inhibitors called syrbactins which we identified together with our collaborators in Switzerland and Germany (Nature, 2008, 452: 755-758). This class includes the plant pathogen-produced natural product syringolin A (SylA) and the structurally-related glidobactin A (GlbA). GlbA was isolated from an unknown species of the order Burkholderiales. Most intriguingly, syrbactins bind the catalytic center of the eukaryotic 20S proteasome by a novel mechanism (Fig. 2). We also showed that SylA induces apoptosis in NB and ovarian cancer cells (Cell Prolif, 2006, 39: 599-609). Inhibition of the proteasome has been recognized as a favorable target in cancer therapy and the identification of a new class of proteasome inhibitors is therefore of significant interest.

The goal of this project is to evaluate SylA and GlbA as potential drug candidates. Together with collaborators at the Chemical Genomics Centre of the Max Planck Society in Dortmund, Germany, we are currently in the process of synthesizing SylA- and GlbA-analogs to identify improved biological activities in both in vitro and in vivo studies. More refined ligand (analog) - target (proteasome) structural studies will be performed with new analogs in collaboration with Prof. M. Groll at the Technical University of Munich. Most importantly, larger quantities will be synthesized and studied in animal tumor models to evaluate the anti-tumor efficacy and pharmacokinetics in vivo.

Figure 2: Syringolin A (SylA) belongs to a newly-identified structural class of proteasome inhibitors and binds to the proteasome by a novel mechanism. Therefore, SylA bears strong potential as an anti-cancer agent. A,Pseudomonas bacteria infect bean plants and can cause serious damage to the leaves (brown spots under the magnifying glass). During infection, the bacteria release the virulence factor SylA (atomic structure in image) which weakens the plant's resistance by means of proteasome inhibition. B, Surface model of SylA bound to subunit β5 of the yeast 20S proteasome. Thr1 is covalently linked to the inhibitor and is shown in white. Colors indicate positive and negative electrostatic potentials contoured from 15kT/e (intense blue) to -15kT/e (intense red). C-D, The natural product Syl A induces apoptotic cell death in human neuroblastoma (NB) SK-N-SH cells (C) while control (H2O)-treated cells are triangular in shape and continue to proliferate (D).

3. PRAF Proteins: Novel Regulators of Small GTPases and Vesicle Traffic
Project #3 examines a newly-emerging family of small (19-21 kD) 4TM proteins referred to as prenylated Rab acceptor 1 domain family (PRAF). In humans, we know of three PRAF proteins, PRAF1-3. Several years ago, we found that PRAF2 (then referred to as JM4) interacts with the C-terminal end of human chemokine receptor CCR5 (FEBS Lett, 2005, 579: 1751-1758). Since then we analyzed and localized the native protein by producing a PRAF2-specific antibody. At the molecular level, we found that PRAF2 is enriched in endosomes/lysosomes (Clin Cancer Res, 2007, 13: 6312-6319) (Fig. 3A) and in synaptic vesicles of the brain (Neurosci Lett, 2008, 436: 171-176), suggesting a role for PRAF2 in vesicle traffic. At the clinical level, we found that PRAF2 is a prognostic marker by analyzing over 110 human NB tumor samples (Clin Cancer Res, 2007, 13: 6312-6319). High levels of PRAF2 indicated poor prognosis and low survival rate of NB patients (Fig. 3B). Interestingly, the Pfeffer lab at Stanford University showed that the PRAF2-related protein PRAF1 acts as a GDI displacement factor (GDF) for endosomal Rab proteins and dissociates Rab-GDI complexes (Nature, 2003, 425: 856-859), and the Rothstein lab at Harvard University demonstrated that the PRAF2-related protein PRAF3 (GTRAP3-18) negatively regulates glutamate transport by interaction with EAAC1 (Nature, 2001, 410: 84-88) and also modulates Rab1 during protein transport and neuronal differentiation (J Cell Mol Med, 2008, in press). These studies are in support of our findings and suggest a role for PRAF proteins as novel regulators of Rab-mediated vesicle traffic and protein transport within the cellular environment.

The goal of this project is to fully unravel the function(s) of the recently identified PRAF protein family and to understand their relevance in context to cancer/disease. While evidence suggests that this protein family regulates Rab proteins, other small GTPases may be modulated by PRAF proteins (for example, Rho, Rac, Ras proteins). Therefore, we aim to expand our assay platform to assess the functional role of PRAF1-3 in the regulation of several small GTPases. We will also continue to study the functional relevance of PRAF2-GDE1 by producing knock-down cell lines and assess the need for GDE1/PRAF2 in promoting Rab-GDI dissociation. The identification of the crystal structure(s) of PRAF1-3 (collaboration, UCSF, Stroud lab) will greatly contribute to a better understanding of the functional importance of the PRAF protein family in a biological and pathophysiological context.

Figure 3: PRAF2 expression in endosomes of human neuroblastoma (NB) cells and clinical relevance as prognostic marker in NB patients. A. Colocalization of endogenous PRAF2 protein (green, Alexa 488) and an endosome-resident protein (red, Alexa 594) in NB cells using a laser-scanning confocal microscope. Yellow punctae indicate the sites of colocalization (selected punctae are highlighted with white arrows). B. Kaplan-Meier graph showing a significant correlation between survival probability of NB patients and the RNA expression levels of PRAF2.