- A New, More Accurate Blood Test to Detect Prostate Cancer
- Attacking Cancer: ATCAAs Interfere with Lipid Growth Factor Signaling in Prostate Cancer
- Redeploying PSMA within Cells to Optimize Targeting by Immunotherapies
- Finding NEMO: Guanine-Rich Oligonucleotides (GROs) as a New Type of Cancer Therapy
- A New Genetic Link to Increased Prostate Cancer Risk
- New Biomarkers of Selenium Chemoprevention of Prostate Cancer
- Magnetic Resonance Imaging of Prostate Cancer
- Getting to the Bones of Prostate Cancer
The number of prostate cancer deaths in the United States declined about 25 percent between 1995 and 2005, due largely to improved screening and earlier detection of cancer. Although the routine use of digital rectal examinations and prostate-specific antigen (PSA) tests during physical exams is responsible for much of this improvement, the PSA test is plagued by a high rate of false positives, leading to increased patient anxiety and unnecessary medical procedures, including prostate biopsies. An ideal diagnostic assay would be both highly sensitive (resulting in few false negative results) and highly specific (generating few false positive results). Dr. Xiaoju Wang, Dr. Arun Sreekumar, and Dr. Rohit Mehra of the University of Michigan, whose research was funded in part by Department of Defense Fiscal Years 2002 and 2004 Prostate Cancer Research Program Postdoctoral Traineeship Awards and a Fiscal Year 2004 Health Disparity Training-Prostate Scholar Award, have developed a new blood test that is more accurate than the PSA test. Dr. Wang, Dr. Sreekumar, Dr. Mehra, and colleagues searched for a panel of biomarkers that, when used in combination, would be more accurate than any single marker such as PSA. These researchers identified an optimal set of 22 peptides which can detect the autoantibodies derived from circulating blood. In contrast to PSA, this panel of biomarkers had high specificity, generating false positive rates only 12 percent of the time (compared to 80 percent for PSA). The false negative rates of approximately 20 percent are similar to those of the PSA test. Importantly, the 22-biomarker test was able to identify prostate cancer in samples even when PSA scores fell in an intermediate range (4 to 10 ng/ml or 2.5 to 10 ng/ml). The researchers have performed initial discovery and validation studies; the next step is to perform larger validation studies in community-based screening cohorts, followed by prospective and multi-institutional studies. This new prostate cancer assay offers the hope of earlier and more accurate diagnosis. Notably, by substantially reducing the number of false positive results, patients may benefit from a reduction in unnecessary, costly, and obtrusive procedures.
Wang X, Yu J, Sreekumar A, Varambally S, Shen R, Giacherio D, Mehra R, Montie JE, Pienta KJ, Sanda MG, Kantoff PW, Rubin MA, Wei JT, Ghosh D, and Chinnaiyan AM. 2005. Antibody signatures in prostate cancer. New England Journal of Medicine 353(12):1224-1235.
Sreekumar A, Laxman B, Rhodes DR, Bhagavathula S, Harwood J, Giacherio D, Ghosh D, Sanda MG, Rubin MA, and Chinnaiyan AM. 2004. Humoral immune response to alpha-methylacyl-CoA racemase and prostate cancer. Journal of the National Cancer Institute 96(11):834-843.
Yan F, Sreekumar A, Laxman B, Chinnaiyan AM, Lubman DM, and Barder TJ. 2003. Protein micorarrays using liquid phase fractionation of cell lysates. Proteomics 3(7):1228-1235.
Attacking Cancer: ATCAAs Interfere with Lipid Growth Factor Signaling in Prostate Cancer
Posted November 28, 2005
Duane Miller, Ph.D., University of Tennessee, Knoxville, Tennessee and James T. Dalton, Ph.D., The Ohio State University, Columbus, Ohio
Growth and metastasis of cancer is stimulated by certain lipid molecules, including lysophospholipids (LPL), a group of small lipid growth factors. These molecules bind to a family of G protein-coupled membrane receptors known collectively as LPL receptors (LPL-R). Therefore, novel agents that inhibit proteins in the LPL-stimulated signal transduction pathways hold promise as therapeutic agents for advanced prostate cancer. With funding from a fiscal year 2001 Prostate Cancer Research Program Idea Development Award, Dr. Duane D. Miller, University of Tennessee, and Dr. James T. Dalton, The Ohio State University, collaborated to discover and develop new drugs that target the LPL factors in prostate cancer, particularly the androgen-independent type. Their results showed that LPL analogs potently and specifically kill prostate cancer cells. Three generations of lipid analogs, each with a unique structural moiety, were synthesized and tested in prostate cancer cells in vitro. All three generations of lipid analogs were effective in killing prostate cancer cells. First generation analogs failed to differentiate LPL-R-expressing cells from receptor-deficient cells despite potent cytotoxicity. Thiazolidine was introduced to replace the phosphate group of the first generation analogs to generate compounds with improved selectivity and specificity. These second-generation derivatives afforded selective toxicity against prostate cancer cells while maintaining the potency of first generation analogs. Dr. Miller and Dr. Dalton discovered a new class of cytotoxic agents called 2-arylthiazolidine-4-carboxylic acid amides (ATCAAs) while working on an improved third generation of analogs. These third generation analogs killed prostate cancer cells most effectively without severe toxicity to non-cancerous cells. ATCAAs killed LPL-R-expressing cells selectively while sparing receptor-deficient cells, suggesting receptor-mediated cytotoxicity. Furthermore, ATCAAs were more potent against androgen receptor (AR)-resistant cell lines than AR-sensitive cell lines. Dr. Dalton and Dr. Miller further investigated the mechanism of cytotoxicity. ATCAAs almost completely inhibited a specific AKT phosphorylation signal transduction pathway involved in prostate cancer cells, resulting in programmed cell death, called apoptosis. They believe that ATCAAs kill lipid receptor-positive cells selectively by interfering with lipid growth factor binding to their receptors. These analogs had favorable pharmacokinetic properties and did not produce any signs of toxicity in mice, thereby identifying them as valid lead drug candidates for evaluation in a tumor-bearing mouse model and potential development as novel anticancer agents. Potentially, ATCAAs could provide a nonsurgical alternative to managing localized disease and a more selective and curative pharmacological therapy for advanced disease.
Gududuru V, Hurh E, Sullivan J, Dalton JT, and Miller DD. 2005. SAR studies of 2-arylthiazolidine-4-carboxylic acid amides: a novel class of cytotoxic agents for prostate cancer. Bioorganic and Medicinal Chemistry Letters 15: 4010-4013.
Gududuru V, Hurh E, Dalton JT, and Miller DD. 2005. Discovery of 2-Arylthiazolidine-4-carboxylic acid amides as a New Class of Cytotoxic Agents for Prostate Cancer. Journal of Medicinal Chemistry 48: 2584-2588.
Hurh E, Gududuru V, Miller DD, and Dalton JT. 2005. Effect of lysophosphatidic acid on lysophospholipid analog-induced cytotoxicity in prostate cancer cells. Proceedings of the American Association for Cancer Research Abstract 642.
Gududuru V, Hurh E, Dalton JT, and Miller DD. 2004. Synthesis and antiproliferative activity of 2-Aryl-4-oxo-thiazolidin-3-yl amides for prostate cancer. Bioorganic and Medicinal Chemistry Letters 14: 5289-5293.
Gududuru V, Hurh E, Durgam GG, Hong SS, Sardar VM, Xu H, Dalton JT, and Miller DD. 2004. Synthesis and biological evaluation of novel cytotoxic phospholipids for prostate cancer. Bioorganic and Medicinal Chemistry Letters 14: 4919-4923.
Prostate-specific membrane antigen (PSMA) is an attractive therapeutic target for prostate cancer because its expression is restricted to prostate cancer cells and tumor-associated vasculature of nearly all solid tumors. Also, levels of PSMA are greatest in high-grade, androgen-independent tumors and distant metastases, which are often refractory to standard therapies. Several antibodies that target PSMA have been developed as potential immunotherapies, but their efficacy is limited due to the significant physical barriers that must be overcome before reaching the tumor. One such barrier is that the antibodies must reach the cellular region where PSMA is located. Unfortunately, in well-differentiated early-stage tumors PSMA is predominantly located at the apical plasma membrane in prostate cells, which is the region of the cell least accessible to the bloodstream. With funding from a Department of Defense Prostate Cancer Research Program Fiscal Year 2001 Idea Development Award, Dr. Ayyappan Rajasekaran of UCLA discovered a way to redeploy PSMA to the basolateral region of the cell (the cellular region most accessible to the bloodstream). Dr. Rajasekaran and his colleagues showed that N-glycosylation of PSMA and microtubule integrity were required for localization of PSMA to the apical surface of the cell. The investigators destabilized microtubules with commonly used chemotherapy drugs called Vinca alkaloids and, as a result, PSMA moved to the basolateral surface of the cells. Surprisingly, the investigators also observed that prostate cancer cells derived from some advanced tumors maintained a well-differentiated morphology, with PSMA still hidden in the apical membrane of the cell. Thus, relocalizing PSMA to the basolateral surface prior to immunotherapy could be a useful therapeutic strategy in late-stage tumors. These results suggest that a combination of chemotherapy (with Vinca alkaloids) and immunotherapy (with anti-PSMA antibody) could be effective in both early-stage and advanced prostate cancers.
Christiansen JJ, Rajasekaran S, Chung L, Anilkumar G, Bander N, and Rajasekaran AK. 2005. N-glycosylation and microtubule integrity are involved in apical targeting of prostate-specific membrane antigen: Implications for immunotherapy. Molecular Cancer Therapeutics 4(5):704-714.
Rajasekaran AK, Anilkumar G, and Christiansen J. 2005. Is prostate-specific membrane antigen a multifunctional protein? American Journal of Physiology-Cell Physiology 288(5):C975-981.
Christiansen JJ and Rajasekaran AK. 2004. Biological impediments to antibody based cancer immunotherapy. Molecular Cancer Therapeutics 11:1493-1501.
Rajasekaran SA, Anilkumar G, Oshima E, Bowie JU, Liu H, Heston W, Bander N, and Rajasekaran AK. 2003. A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate cancer specific membrane antigen. Molecular Biology of the Cell 14:4835-4845.
Anilkumar G, Rajasekaran SA, Wang S, Hankinson O, Bander N, and Rajasekaran AK. 2003. Prostate-specific membrane antigen association with filamin A modulates its internalization and NAALADase activity. Cancer Research 63:2645-2648.
The ideal chemotherapeutic agent delivers a killing blow to cancer cells while leaving normal cells unharmed. Past attempts to achieve this level of specificity have relied upon the attachment of a drug to a tumor-homing carrier molecule. With funding received from a Department of Defense Prostate Cancer Research Program New Investigator Award in fiscal year 2000 and an Idea Development Award in fiscal year 2003, Dr. Paula Bates and colleagues at the University of Louisville Brown Cancer Center have found an alternative approach. They discovered a class of oligonucleotides with a natural affinity for cancer cells, which, after attaching to the tumor cell, are drawn inside the cell, triggering its death. These guanine-rich oligonucleotides (GROs) are synthetic molecules that fold to form unusual structures containing clusters of four guanine bases called G-quadruplexes and attach to specific cancer cell molecules. This mechanism is different from any cancer therapy discovered so far. The investigators extended these studies by identifying cellular targets for GROs, one of which is nucleolin. GROs differentiate tumor cells from normal cells by binding nucleolin, which is expressed at high levels on the surface of cancer cells. Inside the cell, GROs are found in a complex with nucleolin and a protein called nuclear factor kappa B (NF-kappaB) essential modulator (NEMO). NEMO, sometimes known as IKKgamma, plays a critical role in the NF-kappaB survival pathway that helps cancer cells avoid death triggered by chemotherapy. Dr. Bates' team found that sequestration of NEMO in complex with one of the GROs (AGRO100) and nucleolin inhibited NEMO-dependent cell survival by inhibiting NF-kappaB signaling. Importantly, these targets are susceptible to GROs even in prostate cancer cells that are resistant to conventional androgen ablation therapy, and thus they hold great promise for the treatment of advanced prostate cancer. Since discovering this GRO capacity 7 years ago, the University of Louisville research team has developed and tested several active versions (https://cdmrp.army.mil/pcrp/research_highlights/2002#16). AGRO100 showed promising antitumor activity with few adverse side effects in a recently conducted Phase I clinical trial. This was the first clinical trial using GROs. Dr. Bates and colleagues are now exploring the interaction of GROs with nucleolin and NEMO at the molecular level to learn more about the process. Their goal is to determine the exact mechanisms required for GROs' cancer-specific activity. This work has great potential to ultimately lead to the development of highly active, very specific therapies for prostate cancer.
Girvan AC, Barve SS, Thongboonkerd V, Klein JB, Pierce WM, and Bates PJ. 2004. Inhibition of NFkB signaling by an oligonucleotide aptamer. Proceedings of the American Association of Cancer Research, Abstract 3047.
Laber DA, Sharma VR, Bhupalam L, Taft B, Hendler FJ, and Barnhart KM. 2005. Update on the First Phase I Study of AGRO100 in Advanced Cancer. American Society of Clinical Oncology Annual Meeting. Abstract 3064.
Dapic V, Abdomerovic V, Marrington R, Peberdy J, Rodger A, Trent JO, and Bates PJ. 2003. Biophysical and biological properties of quadruplex oligodeoxyribonucleotides. Nucleic Acids Research 31:2097-2107.
Mi Y, Thomas SD, Xu X, Casson LK, Miller DM, and Bates PJ. 2003. Apoptosis in leukemia cells is accompanied by alterations in the levels and localization of nucleolin. Journal of Biological Chemistry 278:8572-8579.
Xu X, Hamhouyia F, Thomas SD, Burke TJ, Girvan AC, McGregor WG, Trent JO, Miller DM, and Bates PJ. 2001. Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides. Journal of Biological Chemistry 276:43221-43230.
Identifying the genes that contribute to an increased risk of prostate cancer is critical in developing diagnostic tools and novel therapeutic strategies. Familial prostate cancer is associated with a few "high-penetrance" genetic mutations, which often lead to prostate cancer. Non-familial, or sporadic, prostate cancer may be associated with "low-penetrance" mutations that rarely cause prostate cancer, but are associated with an increased risk of developing the disease. These low-penetrance mutations are predicted to have high prevalence in the population. Drs. John Martignetti and Scott Friedman of the Mount Sinai School of Medicine have proposed that one such candidate is the Krüppel-like factor 6 gene (KLF6), a tumor suppressor that inhibits cell growth through p53-independent activation of p21 (WAF1/CIP1). Previous studies showed that DNA containing the KLF6 gene is deleted and/or mutated in a majority of spontaneous prostate cancers. These studies were expanded to determine whether subtle genetic variations of KLF6, called single nucleotide polymorphisms (SNP), which are present at birth and detectable in blood might represent low-penetrance mutations that contribute to the development of all forms of prostate cancer.
Supported in part by funding from the fiscal year 2002 Department of Defense Prostate Cancer Research Program, the Mount Sinai team collaborated with other researchers to catalog the genetic variants of KLF6. These investigators screened the KLF6 gene for SNPs in blood samples from a cohort of 3,411 men that included patients with sporadic or familial prostate cancer, and a control group of healthy men. These studies confirmed that the presence of a particular variant of this gene increases prostate cancer risk about 50% regardless of family history of the disease. These findings are significant because this KLF6 SNP is the first reported high-prevalence, low-penetrance prostate cancer susceptibility allele. The intronic KLF6 SNP, in contrast to many other SNPs, has functional significance. It results in the generation of a novel, truncated form of KLF6, which no longer suppressed cell growth, but instead, promoted growth. The team then performed targeted inhibition studies of the two KLF6 forms to better understand their biologic effects. As anticipated, inhibition of the truncated cytoplasmic KLF6 isoform caused marked decreases in key hallmarks of cancer growth and spread: cell proliferation, anchorage-independent growth, invasion, tumorigenicity, and angiogenesis. This research strongly supports the hypothesis that a specific variant of the KLF6 tumor suppressor gene is responsible in part for increased prostate cancer risk. These studies also demonstrated that the novel dual growth-promoting/growth-suppressing effects of the KLF6 gene and its variants could provide a target for preventive/diagnostic strategies, ultimately lowering prostate cancer risk for men.
Li D, Yea S, Dolios G, Martignetti JA, Narla G, Wang R, Walsh MJ, and Friedman SL. 2005. Regulation of kruppel-like factor 6 tumor suppressor activity by acetylation. Cancer Research 65(20): 9216-9225.
Narla G, DiFeo A, Yao S, Banno A, Hod E, Reeves HL, Qiao RF, Camacho-Vanegas O, Levine A, Kirschenbaum A, Chan AM, Friedman SL, and Martignetti JA. 2005. Targeted inhibition of the KLF6 splice variant, KLF6 SV!, suppresses prostate cancer cell growth and spread. Cancer Research 65(13):5761-5768.
Narla, G, DiFeo A, Reeves HL, Schaid DJ, Hirshfeld J, Hod E, Katz A, Isaacs WB, Hebbring S, Komiya A, McDonnell SK, Wiley KE, Jacobsen SJ, Isaacs SD, Walsh PC, Zheng SL, Chang B-L, Friedrichsen DM, Stanford JL, Ostrander EA, Chinnaiyan AM, Rubin MA, Xu J, Thibodeau SN, Friedman SL, and Martignetti JA. 2005. A germ line DNA polymorphism enhances alternative splicing of the KLF6 tumor suppressor gene and is associated with increased prostate cancer risk. Cancer Research 65(4):1213-1222.
Kaiser J. 2004. New prostate cancer genetic link. Science 306:1285.
Selenium is a trace mineral that may help to prevent cancer by protecting against the damaging effects of free radicals, boosting the immune system, and inhibiting tumor angiogenesis. Clinical trials testing selenium chemoprevention of prostate cancer are underway. Identifying selenium-responsive biomarkers could be of great use for these trials, because these biomarkers could provide a tool to measure the efficacy of selenium. With funding received from a fiscal year 2001 Prostate Cancer Research Program Postdoctoral Traineeship Award, Dr. Dong, of the Roswell Park Cancer Institute, used microarray analysis to identify a set of selenium-responsive genes in prostate epithelial cells. The results of this work are described at https://cdmrp.army.mil/pcrp/research_highlights/2003#4. Dr. Dong has expanded these studies to validate these candidate selenium-responsive biomarkers and has correlated their expression with cell cycle and apoptotic status. Selenium-responsive genes were found in both the androgen-resistant PC-3 cell line and the androgen-responsive LNCaP cell line. Six of the targets (GADD153, CHK2, p21Waf-1, cyclin A, CDK1, DHFR) were confirmed to be regulated by selenium in the PC-3 cells. These targets play critical roles in regulation of the cell cycle. In the LNCaP cells selenium downregulated the expression and activity of the androgen receptor (AR), as well as the AR-regulated gene prostate specific antigen (PSA). Furthermore, selenium inhibited growth of both PC-3 and LNCaP cells by blocking the cell cycle and inducing apoptosis. Dr. Dong and colleagues developed a novel promoter-based microarray data mining approach to identify key transcription factors that might mediate selenium-induced gene expression changes. The gut-enriched kruppel-like factor (GKLF)-consensus element was identified in the promoters of 39% of the selenium-responsive genes analyzed. Selenium was confirmed to induce GKLF DNA-binding activity and upregulate levels of GKLF transcript. With funding received from a fiscal year 2003 Prostate Cancer Research Program New Investigator Award, Dr. Dong is building on these findings by studying the mechanistic basis for GKLF upregulation by selenium and the effects of GKLF overexpression on the growth of prostate cancer cells. Dr. Dong and colleagues also developed a microarray data mining approach to systematically analyze datasets collected from human tumor specimens, LNCaP cells treated with synthetic androgen, and their own LNCaP and PC-3 studies. They found that selenium reversed the expression of 42 genes implicated in prostate carcinogenesis and that selenium reversed the effect of androgen on the expression of 38 androgen-regulated genes. In conclusion, several candidate selenium-responsive biomarkers that provide exciting clues about selenium action were identified. These targets could potentially be used as diagnostic markers and/or therapeutic targets.
Dong Y, Zhang H, Hawthorn L, Ganther HE, Ip C. 2003. Delineation of the molecular basis for selenium-induced growth arrest in human prostate cancer cells by oligonucleotide array. Cancer Research 63:52-59.
Dong Y, Lee SO, Zhang H, Marshall J, Gao A, Ip C. 2004. Prostate specific antigen (PSA) expression is down-regulated by selenium through disruption of androgen receptor signaling. Cancer Research 64:19-22.
Zhang H, Dong Y, Marshall J, Mowak N, Ip C. 2005. Microarray data mining of potential selenium targets in chemoprevention of prostate cancer. Cancer Genomics and Proteomics, in press.
Advances in the development of effective local therapies for prostate cancer have been hindered by the lack of imaging techniques that can reliably identify the location of the cancer within the prostate. Magnetic resonance imaging (MRI) is a promising candidate for imaging the prostate because it can provide sharp images in soft tissue, give multi-dimensional images, and provide unique biological information not available with other imaging modalities. However, MRI is rarely used as a first-tier imaging modality for prostate cancer because current systems are limited in their sensitivity and specificity for prostate cancer. Prostate cancer growth is accompanied by the development of a large network of immature, leaky blood vessels that may cause an increase in blood flow, blood volume, and permeability in the area of their development. Dr. David Buckley has developed techniques to measure this vascular signature of prostate cancer. He used MRI coupled with computer technology to measure blood flow, blood volume, and vasculature permeability by tracking a contrast agent, a type of MRI "dye," over time. In a study of 22 men with adenocarcinoma of the prostate, the addition of the MRI contrast agent showed that blood flow to the prostate cancer tissue exceeded that to the non-cancerous prostate tissue. Dr. Buckley was able to produce three-dimensional images of the prostates that could distinguish tumor tissue from the surrounding normal tissue with this MRI technique. The areas of the images visualized as tumors were confirmed by pathology reports. He also found that there was little difference in blood volume or vasculature permeability between normal and tumor tissue. This is consistent with pathologists' observations of the differences between tumor vasculature and normal vasculature. This MRI technique represents considerable promise in improving prostate cancer diagnosis and prognosis.
Buckley DL, Roberts C, Parker GJM, et al. 2004. Prostate Cancer: Evaluation of vascular characteristics with dynamic contrast-enhanced T1-weighted MR imaging initial experience. Radiology 233:709-715.
Buckley DL. 2002. Uncertainty in the analysis of tracer kinetics using dynamic contrast-enhanced T1-weighted MRI. Magnetic Resonance in Medicine 47:601-606.
Bones are a common site of spread for many cancers including prostate cancer. Prostate cancer bone metastases cause bone pain and fractures, severely reducing the quality of life for the patient. Treatment for prostate cancer bone metastases has been aimed primarily at reducing the pain and delaying the time to bone fractures through the use of hormone therapy and/or chemotherapy, radiation therapy, or bisphosphonates. Professor Zelig Eshhar and his colleagues at the Weizmann Institute of Science (Rehovot, Israel) and the Sheba Medical Center (Tel Hashomer, Israel) have shown that a common treatment for prostate cancer may help redirect immune cells to prostate cancer in the bones. The treatment uses immune cells from the patient's body that are engineered to detect specific cancer cells and have the capacity to fight and kill the invading cancer cells. Dr. Eshhar called these custom-modified cells T bodies. After developing a strategy to create these cells, he and his colleagues needed to get the T bodies to the bone metastases in the bone. Initial experiments using mice with prostate cancer growing in their leg bones and bone marrow showed that the T bodies did not appear to be able to get to the bone metastases. However, a significant drop in the tumor marker prostate-specific antigen, a reduction in the tumor load, and increased survival time were observed when prostate cancer-bearing mice were "preconditioned" using some common forms of cancer therapy such as low doses of radiation or chemotherapy drugs before T body injection. Dr. Eshhar believes that the preconditioning cancer therapy produces bone marrow disruption, causing the bone marrow to release chemical distress signals to the immune system. These signals not only warn immune cells of potential danger in the bone marrow but also help attacking immune cells pinpoint the problem area and traverse barriers that otherwise might prevent them from getting to the treatment site. This method, developed in an experimental mouse model, holds promise for bone metastases in prostate cancer and other types of disseminated cancers resistant to conventional therapy.
Pinthus JH, Waks T, Malina V, Kaufman-Francis K, Harmelin A, Aizenberg I, Kanety H, Ramon J, Eshhar Z. 2004. Adoptive immunotherapy of prostate cancer bone lesions using redirected effector lymphocytes. J Clin Invest 114:1774-1781.