Prognostic implication of MET overexpression in myxofibrosarcomas: an integrative array comparative genomic hybridization, real-time quantitative PCR, immunoblotting, and immunohistochemical analysis

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Purpose: The principal goals were to identify and validate targetable metabolic drivers relevant to myxofibrosarcoma pathogenesis using a published transcriptome.Experimental Design: As the most significantly downregulated gene regulating amino acid metabolism, argininosuccinate synthetase (ASS1) was selected for further analysis by methylation-specific PCR, pyrosequencing, and immunohistochemistry of myxofibrosarcoma samples. The roles of ASS1 in tumorigenesis and the therapeutic relevance of the arginine-depriving agent pegylated arginine deiminase (ADI-PEG20) were elucidated in ASS1-deficient myxofibrosarcoma cell lines and xenografts with and without stable ASS1 reexpression.Results: ASS1 promoter hypermethylation was detected in myxofibrosarcoma samples and cell lines and was strongly linked to ASS1 protein deficiency. The latter correlated with increased tumor grade and stage and independently predicted a worse survival. ASS1-deficient cell lines were auxotrophic for arginine and susceptible to ADI-PEG20 treatment, with dose-dependent reductions in cell viability and tumor growth attributable to cell-cycle arrest in the S-phase. ASS1 expression was restored in 2 of 3 ASS1-deficient myxofibrosarcoma cell lines by 5-aza-2 0 -deoxycytidine, abrogating the inhibitory effect of ADI-PEG20.Conditioned media following ASS1 reexpression attenuated HUVEC tube-forming capability, which was associated with suppression of MMP-9 and an antiangiogenic effect in corresponding myxofibrosarcoma xenografts. In addition to delayed wound closure and fewer invading cells in a Matrigel assay, ASS1 reexpression reduced tumor cell proliferation, induced G 1 -phase arrest, and downregulated cyclin E with corresponding growth inhibition in soft agar and xenograft assays. Conclusions: Our findings highlight ASS1 as a novel tumor suppressor in myxofibrosarcomas, with loss of expression linked to promoter methylation, clinical aggressiveness, and sensitivity to ADI-PEG20.

Section: Cell Culture Transfection and Stable Clonesmentioning

confidence: 99%

“…To standardize transfection efficiency, pEGFP (Promega) were transfected in parallel. Afterwards, cells were cultured for further 24 …”

Section: Cell Culture Transfection and Stable Clonesmentioning

confidence: 99%

ASS1as a Novel Tumor Suppressor Gene in Myxofibrosarcomas: Aberrant Loss via Epigenetic DNA Methylation Confers Aggressive Phenotypes, Negative Prognostic Impact, and Therapeutic Relevance

Huang

Wang

et al. 2013

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126

“…The criteria for diagnosis and parameter assessment have been previously elaborated (5,7,16). We assembled 114 formalinfixed primary myxofibrosarcoma samples from patients who received resection with curative intent, but not neoadjuvant radiation or chemotherapy, into recipient blocks of tissue microarrays (TMA) containing triplicate 1.0-mm tissue cores for each case.…”

Section: Tumor Characteristicsmentioning

confidence: 99%

AMACRAmplification in Myxofibrosarcomas: A Mechanism of Overexpression That Promotes Cell Proliferation with Therapeutic Relevance

Fang

Lan

et al. 2014

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Purpose: Myxofibrosarcomas frequently display arm-level gains on 5p. We characterized the pathogenetic and therapeutic relevance of the a-methylacyl coenzyme A racemase (AMACR) at 5p13.3.Experimental Design: AMACR mRNA expression in myxofibrosarcomas was analyzed using the public transcriptome and laser-microdissected sarcoma cells. We performed florescence in situ hybridization (FISH) and immunohistochemistry in independent samples for clinical correlates. In AMACR-overexpressing myxofibrosarcoma cells and xenografts, we elucidated the biologic function of AMACR using RNA interference and explored the therapeutic effect and mechanism of an AMACR inhibitor, ebselen oxide.Results: AMACR protein overexpression and gene amplification were significantly associated with each other (P < 0.001), with higher tumor grades (both P 0.002), and univariately with worse metastasis-free survival (MFS; both P < 0.0001) and disease-specific survival (DSS; P ¼ 0.0002 for overexpression; P ¼ 0.0062 for amplification). AMACR protein overexpression also independently portended adverse outcome (DSS, P ¼ 0.007; MFS, P ¼ 0.001). However, 39% of AMACR-overexpression cases did not show gene amplification, implying alternative regulatory mechanisms. In myxofibrosarcoma cell lines, stable AMACR knockdown suppressed cell proliferation, anchorage-independent growth, and expression of cyclin D1 and cyclin T2. These growth-promoting attributes of AMACR were corroborated in the AMACR-silenced xenograft model and AMACR-underexpressed myxofibrosarcomas, showing decreased labeling for cyclin D1, cyclin T2, and Ki-67. Compared with fibroblasts, AMACR-expressing myxofibrosarcoma cells were more susceptible to ebselen oxide, which not only decreased viable cells, promoted proteasome-mediated degradation of AMACR protein, and induced cellular apoptosis in vitro, but also dose-dependently suppressed xenografted tumor growth in vivo.

“…The KEL-FIB fibroblast cell line was purchased from Taiwan Bioresource and Collection Research Center. The cell lines were maintained with the medium, nutrients, and antibiotics as previously reported (12,13).…”

Section: Cell Culturementioning

confidence: 99%

“…Twelve fresh tumor specimens were previously analyzed by genome-wide aCGH to evaluate copy number alterations (CNA; ref. 13). These materials, along with 4 additional fresh specimens, were used in real-time reverse transcriptase PCR (RT-PCR) assay to detect mRNA expression of candidate oncogenes on 5p and in quantitative DNA-PCR assay to determine SKP2 gene dosage, respectively.…”

Section: Tumor Specimens and Patient Characteristics Of Myxofibrosarcmentioning

confidence: 99%

Characterization of Gene Amplification–Driven SKP2 Overexpression in Myxofibrosarcoma: Potential Implications in Tumor Progression and Therapeutics

Wang

Kang

et al. 2012

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Purpose: Myxofibrosarcoma remains obscure in molecular determinants of clinical aggressiveness, for which we elucidated implications of SKP2 amplification.Experimental Design: Array comparative genomic hybridization was applied on samples and cell lines (NMFH-1 to OH931) to search causal genes of tumor progression. SKP2 gene dosage was determined in 82 independent tumors for clinical correlates. Stable SKP2 knockdown was achieved in myxofibrosarcoma cells to assess its oncogenic attributes and candidate mediators in prometastatic function. Pharmacologic assays were evaluated in vitro and in vivo for the therapeutic relevance of bortezomib.Results: DNA gains frequently involved 5p in which three amplicons were differentially overrepresented in samples behaving unfavorably, encompassing mRNA-upregulated TRIO, SKP2, and AMACR genes. Detected in NMFH-1 cells and 38% of tumors, SKP2 amplification was associated with SKP2 immunoexpression and adverse prognosticators and independently predictive of worse outcomes. Nevertheless, SKP2-expressing OH931 cells and 14% of such tumors lacked gene amplification. Knockdown of SKP2 suppressed proliferation, anchorage-independent growth, migration, and invasion of sarcoma cells and downregulated motility-promoting genes, including ITGB2, ACTN1, IGF1, and ENAH. In vitro, bortezomib downregulated SKP2 expression at the mRNA level with p27 kip1 accumulation, induced caspase activation, and decreased cell viability in myxofibrosarcoma cells but not in fibroblasts. In vivo, bortezomib inhibited growth of NMFH-1 xenografts, the cells of which displayed decreased SKP2 expression but increased p27 kip1 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Conclusions: As a predominant mechanism driving protein overexpression, SKP2 amplification confers tumor aggressiveness in myxofibrosarcoma. The sensitivity of myxofibrosarcoma cells to bortezomib with SKP2-repressing effect indicates the potentiality of ubiquitin-proteasome pathway as a therapeutic target.