BERKELEY, CA (UroToday.com) - Gene expression and DNA copy number microarray technology have been applied to profile various tumors over the past decade in the hopes of teasing out the pertinent genes and pathways that can then serve as therapeutic targets. In the past, these approaches have been limited by the relative scarcity of patient samples compared to the very large number of genes arrayed and by various preanalytical variables related to tumor specimen handling that influence mRNA expression leading to a significant false discovery rate. DNA copy number change is less prone to preanalytical factors; however, the low resolution of older generation array comparative genomic hybridization platforms has meant that copy number change could only be approximated to regions spanning hundreds (or thousands) of genes.
|"If successful, this work can be rapidly translated into the clinic for the benefit of patients."|
In our report, we have examined the terminal castration resistant phase of prostate cancer (CRPC) using an unbiased approach to look for candidate genes that encode potentially “druggable” target proteins. We first used a high-resolution array comparative genomic hybridization platform that can resolve copy number change to the single gene level and integrated this data with transcriptomic profiling in order to generate a list of candidate genes. We externally validated our expression data using independent CRPC datasets to arrive at our candidate of interest: asparagine synthetase (ASNS). ASNS is a well-characterized enzyme that synthesizes the amino acid asparagine from aspartic acid and glutamine. It therefore lies at the heart of cellular metabolism. ASNS has also been implicated in the control of the cell cycle. (1) We then validated ASNS at the protein level using two complementary methods: reverse phase protein lysate array (RPPA) and immunohistochemistry. RPPA is better for quantitation of protein abundance whereas immunohistochemistry is better for localization of the protein of interest within a specific cellular compartment.
Subsequently, we assessed functional significance using models of prostate cancer progression to a castration-resistant and chemotherapy-resistant state and found ASNS knockdown to inhibit cell growth in asparagine-deprived media. Thus, we have found a target, ASNS, and elucidated asparagine amino acid depletion as a mechanism mediating its effect. This opens up a new avenue to exploit a vulnerability of castration resistant prostate cancer cells, namely their augmented metabolic requirements at this advanced phase of disease. Interestingly, the 7q21 region harboring the ASNS gene was seen to fall within an area of high frequency copy number gain(2) in a prior molecular profiling study of CRPC. As well, ASNS has recently been shown to be among a handful of genes upregulated by osteoblast-derived factors in prostate cancer.(3) We hypothesize that ASNS may function as a metabolic oncogene and help cancer cells to survive chemotherapy-induced apoptosis. ASNS inhibition could therefore act as a potential chemosensitizing agent as well as arresting the growth of metabolically hyperactive CRPC tumor cells.
Figure 1: Compound 1
Our follow up plans will be aimed at studying the regulation of the ASNS gene in the context of advanced therapy-resistant prostate cancer and especially to examine silencing of this gene in the more physiologic setting of murine xenografts. The CRPC xenografts would presumably already harbor a relative asparagine deficiency given their high metabolic demands that led to the upregulation of ASNS in the first place. We postulate that further knockdown of ASNS would result in tumor regression. For this work to be feasible, however, we will need to employ a potent ASNS inhibitor to treat CRPC tumors growing in mice and deliver it effectively to the prostate. Fortunately, such inhibitors to ASNS have already been synthesized and are ready for application to the foregoing tumor model systems. Thus, recent work using asparaginase-resistant leukemia cell lines has demonstrated that when asparagine is depleted in the local environment, ASNS inhibitors, such as compound 1 (Figure 1), can suppress cell proliferation.(4) This compound mimics the transition state for a key chemical step in the reaction catalyzed by ASNS, and has nanomolar affinity for the enzyme. If successful, this work can be rapidly translated into the clinic for the benefit of patients.
- Gong SS, Basilico C. A mammalian temperature-sensitive mutation affecting G1 progression results from a single amino acid substitution in asparagine synthetase. Nucleic Acids Res. 1990;18:3509-13.
- Holcomb IN, Young JM, Coleman IM, Salari K, Grove DI, Hsu L, et al. Comparative analyses of chromosome alterations in soft-tissue metastases within and across patients with castration-resistant prostate cancer. Cancer Res. 2009;69:7793-802.
- Wang G, Haile S, Comuzzi B, Tien AH, Wang J, Yong TM, et al. Osteoblast-derived factors induce an expression signature that identifies prostate cancer metastasis and hormonal progression. Cancer Res. 2009;69:3433-42.
- Gutierrez JA, Pan YX, Koroniak L, Hiratake J, Kilberg MS, Richards NG. An inhibitor of human asparagine synthetase suppresses proliferation of an L-asparaginase-resistant leukemia cell line. Chem Biol. 2006;13:1339-47.
Kanishka Sircar, MD,1 Nora Navone, MD, PhD,2 Wei Zhang, PhD,1 and Nigel Richards, PhD3 as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.
Departments of 1Pathology and 2Genitourinary Medical Oncology and David H. Koch Center for Applied Research of Genitourinary Cancers, The University of Texas MD Anderson Cancer Center, Houston, TX, United States and the 3Department of Chemistry, University of Florida, Gainesville, FL