ACCOMPLISHMENTS
1. Scientific Accomplishments
Highlights of accomplishments by theme
1. Regulation of cell proliferation by vitamin D and soy isoflavones
As indicated in the previous section, a major emphasis of research on regulation of cell growth is the role of dietary factors. Dr. Scott Cramer and his collaborators have used genetic models for investigating the signaling pathways involved in vitamin D growth inhibition, and also have demonstrated strong synergistic growth inhibition between vitamin D and the soy isoflavone genistein on prostatic cells. Their data demonstrate that vitamin D and genistein cooperatively induce p21 protein and that genistein upregulates vitamin D receptor (VDR) content by modulating protein stability. The effect of genistein on VDR content leads to the hypothesis that one mechanism of synergism between vitamin D and genistein is by enhanced VDR signaling. They will test this hypothesis in a recently funded R01 using a novel mouse model of prostate cells in culture. During the course of their studies with the mouse prostate culture system, they have identified cells with properties like putative prostate stem cells (or progenitor cells), and have developed an in vitro 3-dimensional culture system to evaluate the effects on ductal morphogenesis and differentiation. The goal is to make fully functional prostatic structures in vitro that can be used to study mechanisms of differentiation and oncogenesis and the role of epithelial-stromal cell signaling in prostate cancer. Dr. Cramer collaborates with Drs. Koumenis (CGS Program) and Schwartz (Cancer Control Program) on vitamin D in prostate and pancreatic cancer and with Dr. Mark Cline (CGS Program) on the synergy with genistein. This work complements in vivo studies by Dr. Cline, who is continuing his work on the role of hormones and hormonally active dietary components (e.g. isoflavone phytoestrogens) on normal and neoplastic breast, prostate, and uterine tissues. Dr. Cline has shown that dietary soy reduces the cancer-promoting stimulatory effect of estradiol on the breast funded by an R01 grant from NCCAM. In addition, he has shown that novel hormone replacement agents (tibolone, new estrogen-progestin combinations, and SERMs) may provide health benefits of postmenopausal hormone therapy without increasing breast and endometrial cancer risk. He has recently been extending these observations to prostate in a new translational project funded by an R03 grant. He collaborates with Drs. Hall, Clark, and Lee (Clinical Research Program) on this project. Dr. Cline has also been actively engaged in developing nonhuman primate models to study effects of dietary and environmental factors in cancer. He has studied the effects of soy on proliferative signaling in breast, and more recently he has shown that monkeys suffer from papillomavirus-induced cervical cancer, resembling women. This represents a promising animal model for human cervical cancer.
2. Role of dietary lipids in regulating cancer cell proliferation and metastasis
Building on our institutional strengths in lipid signaling, Dr. Yong Chen has assembled an interdisciplinary team of CGS investigators consisting of Drs. Isabel Berquin, Joseph O’Flaherty, Larry Daniel, Iris Edwards and Robert Wykle who are collaborating to study mechanisms through which dietary lipids regulate breast and prostate cancer cell growth. For more than 2 decades, epidemiologic studies have reported associations between dietary fat and risk of prostate cancer. Experimental data indicate that n-3 polyunsaturated fatty acids (PUFAs) inhibit (whereas n-6 PUFAs stimulate) prostate cancer cell growth. The group is pursuing two parallel approaches to investigate the molecular mechanism involved in the growth inhibition of prostate cells by n-3PUFA. First, they are using a candidate pathway approach, focusing on the phosphoinositide signaling pathway which is affected by a fish oil diet, and which plays a central role in regulating prostate cell growth and survival. Secondly, they are performing general unbiased screening to identify candidate genes and phospholipids regulated by a fish oil diet. This work complements strengths in other CCCWFU programs, notably the Cancer Control program, where behavioral, epidemiological and population genetics studies on the effects of dietary lipids on cancer risk are underway, such as Dr. Xu’s studies on phytanic acid deficiency (a lipid derivative) in African-American populations. An exciting and unique characteristic of these studies is the use of LDL particles isolated from nonhuman primates to deliver fatty acids to the tumor cells. The normal physiological mechanism by which most fatty acids are delivered to cells is as conjugates with LDL that are taken up through the LDL receptor. Most investigators do not use this method, which is difficult and requires a source of LDL from animals or people fed a defined diet for extensive periods of time, and instead deliver fatty acids as conjugates with albumin. Dr. Edwards has purified LDL particles with defined ratios of n-3 and n-6PUFAs from cynomolgus monkeys fed diets rich in particular oils. This approach, which is made possible by the fact that Wake Forest has a unique colony of nonhuman primates that have been fed diets with defined lipid compositions for long periods, has opened up new avenues of research. In collaboration with Dr. Chen, Dr. Edwards has made the remarkable discovery that when fatty acids are delivered to breast cancer cells via the physiologically-relevant, lipoprotein-mediated mechanism, the gene expression changes that occur are much more extensive than when fatty acids are delivered as albumin conjugates. They have also discovered that the gene expression pattern induced by LDL rich in n-3 fatty acids is completely different from that induced by n-6 fatty acids. They are testing the hypothesis that the inhibitory effects of n-3 PUFA on cell growth are mediated in part through altered expression of the cell surface heparan sulfate proteoglycan, syndecan 1. They have shown syndecan 1 is the major proteoglycan produced by human breast cancer cell lines and that syndecan 1 production is significantly lower in breast cancer cell lines than in non-tumorigenic cells. A major new finding is that syndecan 1 is able to induce apoptosis in breast cancer cells. This suggests that the lower syndecan 1 expression in tumor cells may favor cell growth by reducing an apoptotic stimulus.
The relationship between lipids and cancer is also a focus of Dr. Kridel’s research. His research of the past year has focused on the mechanism of action of the novel fatty acid synthase (FAS) inhibitor orlistat. Included in these studies is a crystallographic analysis of FAS in collaboration with Dr. Todd Lowther (DDCD program). Recent work suggests that FAS may regulate tumor cell metabolism. This is supported by evidence linking FAS inhibition to activation of AMP-activated kinase, suggesting that ATP levels are depleted when FAS is inhibited. Further, they have found that the mTor pathway is activated while eIF2α is apparently inactivated. These last two pieces of data indicate that FAS activity may regulate translation in tumor cells, an indication that FAS provides a metabolic advantage in tumor cells. In a second project, Dr. Kridel has found that activators of PPAR γ, a member of the nuclear-hormone receptor superfamily that acts as a regulator of lipogenic enzymes, can induce apoptosis in prostate tumor cells. This finding contrasts what has been described in the literature. The mechanism behind this finding is currently being investigated in the lab. Dr. Kridel collaborates with Dr. Mike Robbins (DDCD Program) on fatty acid synthase as a novel target for glioma therapy. Dr. Robbins also collaborates on the PPAR γ project.
3. Role of reactive oxygen species in regulating cell proliferation
Drs. Larry Daniel and Jason Grayson collaborate with Dr. Leslie Poole (DDCD program) in studies focused on the role of ROS in proliferative signaling. This is a very active area of cancer research in which cells have been found to produce ROS as a type of “second messenger” system that appears to amplify proliferative signaling. The mechanism appears to involve reversible oxidation of specific cysteine residues on target proteins, including phosphatases such as PTEN and PPIIa, to the relatively unstable cysteine-sulfenic acid form. The group has found that the response to tumor promoters is inhibited by compounds that block cysteine-sulfenic acids, and are using novel proteomics approaches to determine targets involved in the signaling and its inhibition. Similar approaches are being developed with Dr. Grayson to analyze the role of ROS in proliferative signaling in T cells. This newly emerging collaboration involves interaction with chemists (King) and computational biologists (Fetrow, VBI group), and is supported by pilot funding and a recently awarded R21 grant from NCI [R21 CA112145, Profiling of Redox-Sensitive Signaling Proteins (Poole, PI; Daniel, King, Fetrow, Co-Is)]. The group of investigators in the Virginia Bioinformatics Institute (VBI) at Virginia Tech is also collaborating with investigators from Wake Forest to develop mathematical models of redox pathways in cancer cells, based on initial work done by the Virginia Tech group in yeast. This multi-disciplinary, systems biology approach is an excellent example of how the NIH “roadmap” ideas have been applied.
4. Translational control and endoplasmic reticulum-based stress responses in hypoxia
Tumor cell adaptation to hypoxic stress is an important determinant of malignant progression. Dr. Koumenis and his collaborators have shown that cells cultured under hypoxic conditions, as well as transformed cells in hypoxic areas of tumors, activate a translational control program known as the integrated stress response (ISR), which adapts cells to endoplasmic reticulum stress. Inactivation of ISR signaling by mutations in the endoplasmic reticulum kinase PERK and the translation initiation factor eIF2alpha, or by a dominant-negative PERK, impairs cell survival under extreme hypoxia. Tumors derived from these mutant cell lines are smaller and exhibit higher levels of apoptosis in hypoxic areas compared to tumors with an intact ISR. Moreover, expression of the ISR targets ATF4 and CHOP was noted in hypoxic areas of human tumor biopsy samples. These results show that activation of the ISR is required for tumor cell adaptation to hypoxia, and suggest that this pathway is an attractive target for antitumor modalities. The focus on translational control and stress responses shows strong synergism with the work on translational control and oncolytic viruses conducted by Dr. Lyles (CGS Program) and his colleagues. Dr. Lyles’ group has shown that vesicular stomatitis virus can replicate in glioma cells under conditions of severe hypoxia. This discovery is important for a potential clinical application of the virus, because most other oncolytic viruses (e.g. adenoviruses) do not replicate efficiently under the hypoxic conditions that are often found in tumors. Thus, vesicular stomatitis virus may be an especially useful agent to treat hypoxic tumors. Dr. Koumenis has also been collaborating with Dr. Kridel to show that the FAS inhibitor orlistat induces an endoplasmic reticulum-based stress response similar to that seen in the induction of apoptosis in prostate cancer cells.
5. Genetic control of innate antitumor immune mechanisms
Regulation of the balance between inflammation and tumor genesis upon signaling by Toll-like receptors (TLRs) is being studied in the laboratory of Dr. Liwu Li. He has shown that the Toll-like Receptor 4 (TLR4), which is well known for its role in response to bacterial lipopolysaccharide is activated by the widely-used chemotherapeutic agent Taxol. Dr. Li has further shown that IRAK1, a protein kinase that is recruited to activated TR4 is required for Taxol-induced apoptosis of lymphoma cells. This effect, which is separate from Taxol’s well known activities as a microtubule stabilizer suggest that novel apoptotic pathways activated through TR4 and IRAK1 signaling may contribute to the anti-tumor effect of taxanes.
Dr. Li’s expertise in TLR signaling also provides a framework for understanding the results of Dr. Jianfeng Xu (Cancer Control program), who has mapped prostate cancer susceptibility genes to several genes encoding inflammatory mediators, including TLRs. The genetic link between prostate cancer and signaling pathways involved in inflammation has emerged as a major thrust of Dr. Xu’s group. The origins and expertise of research that supports this area includes the work of the members of the CGS Program. Dr. Li recently moved to Virginia Tech, and is one of the select group of Virginia Tech faculty included as members of the program, based on his continued close collaboration with CCCWFU investigators.
Dr. Zheng Cui recently identified a previously unrecognized, genetically determined cancer resistance trait that is mediated through cytolytic death of cancer cells. Tumor cell death occurs through an innate immune response that is T cell independent. Along with program members Drs. Willingham and Alexander-Miller, they are studying how the innate immune system induces this unique anti-tumor response. In collaboration with members of the Cancer Control Program, they are mapping the gene that is responsible for this trait. They have mapped resistance in congenic mice to 2 independent loci, one on chromosome 10 and one on chromosome 4. Sequencing of candidate genes in these two loci is ongoing. Further they have shown a unique multi-pronged immune response of resistant mice against cancer cells with macrophages, neutrophils, natural killer cells and cytotoxic T lymphocytes that all have independent killing activities. Adoptive transfer of leukocytes offers normal mice a life-long cancer resistance indistinguishable from the donor mice.
6. Antagonism between antiviral signals and cell proliferation and development of novel oncolytic viruses
Several members of the CGS Program are seeking to understand the relationship between proliferative signaling and antiviral signaling with the goal of developing novel oncolytic viruses for tumor therapy. It has recently been discovered that many cancers down-regulate their antiviral pathways during oncogenesis. This makes them correspondingly more susceptible to virus infection, which can be exploited to develop novel oncolytic viruses. The Centers of Excellence have provided a critical forum to integrate the efforts of virologists and cancer biologists. The Prostate Center of Excellence has played a major role in the efforts of Drs. Douglas Lyles and Maryam Ahmed (Research Assistant Professor in Dr. Lyles’ group) collaborating with Drs. Cramer, Chen, and Kulik (CGS Program) to study the mechanism of cell killing by the RNA virus vesicular stomatitis virus in prostate cancer cells and uncover the reasons why normal prostate cells are comparatively resistant to this killing. They are also collaborating with Dr. Willingham (CGS Program) to determine the mechanisms of virus-induced apoptosis.
Dr. Griffith Parks is developing the paramyxovirus SV5 as an oncolytic agent. He has engineered this otherwise non-pathogenic virus to selectively kill tumor cells by activating apoptosis, in collaboration with Drs. Cramer and Lyles. It may also be possible to combine the direct viral oncolytic effect of the simian virus 5 (SV5) with strategies to increase host immune-mediated anti-tumor responses. In this approach, in collaboration with Dr. Alexander-Miller (CGS Program) who has identified SV5 proteins that induce high avidity T cell responses, Dr. Parks is modifying the design of recombinant SV5s as cancer vaccine vectors.
The Breast Center of Excellence has played a similar role in stimulating research on oncolytic viruses for the treatment of breast cancer. In a new project funded from the Susan Komen Breast Cancer Foundation, Dr. Parks is pursuing a new approach to develop SV5 as a therapeutic vector for controlled killing of breast cancer cells. They will test the hypothesis that an SV5 mutant expressing hyper-fusogenic variants of the viral fusion protein F will be more efficient at killing breast cancer cells than rSV5 expressing WT F protein. Secondly, they will test the hypothesis that fusion activity and cell killing by an rSV5 vector can be controlled by engineering the SV5 F protein to have a cleavage site for matrix metalloproteinases that are overexpressed in metastatic breast cancer cells. This project includes Dr. Steve Kridel (CGS Program) as a co-investigator.
Dr. Maryam Ahmed has begun a new research program in collaboration with immunologists in the CGS Program, funded by the DoD breast cancer program, to explore the ability of mutant vesicular stomatitis viruses to activate dendritic cells to overcome the immune suppression induced by factors released by breast cancers.
Research in the Brain Tumor Center of Excellence has focused on oncolytic viruses for the treatment of glioblastoma multiforme. Dr. David Ornelles’ research has focused for many years on E1B-55k-mutantadenoviruses (recently commercialized as ONYX-015 as a therapy for brain tumors and other cancers). Dr. Ornelles’ research seeks to identify cellular products targeted by adenovirus oncogenes and to investigate the molecular basis by which oncolytic adenoviruses selectively replicate in tumor cells. The Ornelles lab has established that the E1B-55K-mutant virus selectively replicates in and kills S phase cells better than G1 cells.
Evidence suggests that this selectivity is due to the cell cycle-dependent control of late viral translation, which is mediated by another viral oncogene, E4orf1. A third viral oncogene, E4orf3, has been shown to be necessary for the selective S phase replication of the 55K-mutant virus. Remarkably, although the E4orf3/55Kdouble-mutant virus replicates more poorly than either parent virus, it kills cells at a greater rate than either parent virus. This unusual finding is being explored as a means of modifying E4orf3 activity to create tumor-restricted oncolytic viruses with enhanced cytolytic properties. Ongoing work has identified the cellular geneAML1 as being important for adenovirus gene expression. The activity of the E1B-55K and E4orf6 protein complex appears to be dependent on this cellular product. Studies performed in collaboration with Linda Gooding (Emory University) have demonstrated that certain adenovirus-infected human T cells continue to replicate while expressing adenovirus genes. Because adenovirus blocks double-stranded DNA-break repair, these results raise the possibility that adenovirus is mutagenic in lymphoid cells. The ability of adenovirus to cause the mutations and the connection between adenovirus replication and AML1, the most frequently mutated gene in childhood leukemia, will be explored through a new research program.
In collaboration with Dr. Koumenis (CGS Program), Dr. Ornelles is also investigating mechanisms of radiosensitization by the adenovirus E4orf6 protein, which inhibits double strand break repair and these investigators have constructed a mutant virus that acts as an effective radiosensitizer in glioma cells.