Implications of MTAP Gene Deletion in Frequency, Prognosis, Biological and Radiation Response for Myxoid Sarcoma by Array Comparative Genomic Hybridization: Evaluation of L-Alanosine as a Potential Radiosensitizer

The common types of myxoid soft tissue sarcomas are myxofibrosarcoma and liposarcoma (LPS) with myxoid change. The latter encompass three variants, i.e., well-differentiated/dedifferentiated LPS, myxoid/round cell LPS, and pleomorphic LPS. These myxoid sarcomas have histopathological overlap but difference in behavior, posing challenges in diagnosis and risk stratification. Although well-differentiated and myxoid LPSs are characterized by tumor-specific oncogenic events, little is known about the molecular mechanisms responsible for their progression into high-grade forms, i.e., dedifferentiated and round cell LPS, respectively. Similarly, it remains elusive in the tumorigenesis of myxofibrosarcoma and pleomorphic LPS, for which there’s no known tumor-specific aberration. High-resolution array comparative genomic hybridization (aCGH) is promising to narrow down imbalanced chromosomal regions for fine mapping cancer-associated tumor suppressor genes (TSGs) and oncogenes. Using myxofibrosarcoma as a model, we have applied aCGH to analyze genomewide DNA copy number alterations (CNAs) of 12 primary tumors and 2 sarcoma cell lines.We identified 4 major regions of deletion in 9p, some of which are known to be frequently lost in common carcinomas and harbor several putative TSGs. Among the potential candidates, MTAP gene at 9p21.3 recurrently showed DNA losses and homozygous co-deletion with two established TSGs (i.e., p16INK4a and p15INK4b). Its encoding enzyme, methylthioadenosine phosphorylase, is essential in the methionine and adenine salvage pathways. Validating the reliability of aCGH, we have used quantitative RT-PCR and Western blotting to prove complete or partial loss of MTAP expression in OH931 (homozygous deletion) and NMFH1 (hemizygous deletion) myxofibrosarcoma cell lines, respectively. Moreover, loss of MTAP immuoexpression was observed in a subset of myxofibrosarcomas and various LPS types. Recently, functional role of MTAP as a genuine TSG has been proposed, since DNAmethylation may also silence MTAP gene expression, which affects the tumorigenesis and invasiveness in a tumor type-dependent context.We hypothesize that MTAP protein deficiency can result from genetic and/or epigenetic inactivation to promote tumorigenesis in myxoid sarcomas. Noticeably, MTAP-deficient tumors may be susceptible to inhibitors of de novo AMP synthesis, connoting a potential therapeutic application. In this 3-year project, we raise the following specific aims to be elucidated: (A) Genomic profiling of various LPS types will be characterized by aCGH, with special attention given to CNAs in 9p harboring MTAP gene. (B) To assess its clinicopathological relevance, MTAP immunostain and in situ hybridization will be performed on tissue microarrays of well-characterized cohorts of myxofibrosarcomas and LPSs of various types. (C) Genomic DNA dosage and mRNA expression of MTAP will be quantified by real-time quantitative PCR and RT-PCR for pure sarcoma cells isolated by laser capture microdissection. (D) Methylation-specific PCR will be used to determine the methylation status of MTAP promoter for myxoid sarcoma samples with loss of protein expression but no homozygous deletion of gene. (E) MTAP-homozygously deleted OH931 cells will be stably transfected with expression vectors and MTAP-hemizygously deleted NMFH1 cells treated with demethylating agent to analyze its implications in the regulation of ornithine decarboxylase and various cellular processes essential to cancerous state. (F) Conversely, MTAP-specific shRNA will be applied to non-transformed fibroblasts to knock down endogenous MTAP transcript to assess its consequence in tumorigenicity. (G) MTAP-deficient sarcoma cells with be treated with L-alanosine to evaluate its anticancer effect and underlying pharmacological mechanisms. (H) The myxoid sarcoma cells undergoing various treatments will be evaluated for the role of MTAP in regulating the cellular response to ionizing radiation. (I) The sarcoma cells in (H) will also be pretreated with L-alanosine before radiation to assess whether this MTAP-directed agent can act as a potential radiosensitizer. The findings will provide important insight into cell pathobiology of myxoid soft tissue sarcomas and may aid in future identification of novel prognostic biomarkers and/or molecular therapeutic targets.

Project ID:PC9902-1659
External Project ID:NSC98-2314-B182-035-MY3