Wen Ren Wu

ASS1 as a Novel Tumor Suppressor Gene in Myxofibrosarcomas: Aberrant Loss via Epigenetic DNA Methylation Confers Aggressive Phenotypes, Negative Prognostic Impact, and Therapeutic Relevance

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′-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 G1-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. Clin Cancer Res; 19(11); 2861–72. ©2013 AACR.

IGFBP-5 overexpression as a poor prognostic factor in patients with urothelial carcinomas of upper urinary tracts and urinary bladder

Background Urothelial carcinoma (UC) is prevalent worldwide. Dysregulation of cell growth is a critical event of tumorigenesis and has not been assessed systemically in UC. We thus assessed the published transcriptome of urinary bladder urothelial carcinoma (UBUC) and identified insulin-like growth factor-binding protein-5 (IGFBP-5) as the most significantly upregulated gene associated with the regulation of cell growth. Moreover, validated by using public domain data set, IGFBP-5 expression also significantly predicted worse outcome. IGFBP-5 is one of the binding proteins that regulate insulin-like growth factors (IGFs) and its significance has not been comprehensively evaluated in UCs. Methods Using immunohistochemistry, we evaluated the IGFBP-5 expression status and its associations with clinicopathological features and survival in 340 cases of upper urinary tract urothelial carcinoma (UTUC) and 295 cases of UBUC. Western blot analysis was used to evaluate IGFBP-5 protein expression in human urothelial cell (HUC) lines. Results IGFBP-5 overexpression was significantly associated with advanced pT stage (p<0.001), high histological grade (UTUC, p<0.001; UBUC, p=0.035), lymph node metastasis (UTUC, p=0.006; UBUC, p=0.004), vascular invasion (UTUC, p<0.001; UBUC, p=0.003), perineural invasion (UTUC, p=0.034; UBUC, p=0.021) and frequent mitosis (UTUC, p<0.001; UBUC, p=0.023). IGFBP-5 overexpression also independently predicted poor disease-specific survival and metastasis-free survival in both groups of patients. Western blot analysis showed IGFBP-5 protein as overexpressed in human urothelial cancer cell lines and not in normal urothelial cancer cells. Conclusions IGFBP-5 plays an important role in tumour progression in UC. Its overexpression is associated with advanced tumour stage and conferred poorer clinical outcome.

Associations of Rsf-1 overexpression with poor therapeutic response and worse survival in patients with nasopharyngeal carcinoma.

Aims Deregulated chromatin remodelling often leads to aberrant gene expression in cells, thereby implicating tumour development and progression. As a subunit of remodelling and spacing factor complex, Rsf-1 (HBXAP), a novel nuclear protein with histone chaperon function, mediates ATPase-dependent chromatin remodelling and confers tumour aggressiveness in common carcinomas. However, the expression of Rsf-1 has never been reported in nasopharyngeal carcinoma (NPC). This study aimed at evaluating the expression status, associations with clincopathological variables and prognostic implications of Rsf-1 in a well-defined cohort of NPC. Methods Rsf-1 immunoexpression was retrospectively assessed in biopsies of 108 consecutive NPC patients without initial distant metastasis and treated with consistent guidelines. The results were correlated with the clinicopathological features, therapeutic response, local recurrence-free survival (LRFS), distant metastasis-free survival (DMFS) and disease-specific survival (DSS). Results Present in 49 cases (45%), Rsf-1 overexpression was associated with N2,3 status (p=0.016), American Joint Committee on Cancer stage 3, 4 (p=0.004), and incomplete therapeutic response (p=0.041). In multivariate analyses, Rsf-1 overexpression not only emerged as the sole independent adverse prognosticator for LRFS (p=0.0002, RR 5.287) but also independently predicted worse DMFS (p=0.0011, RR 3.185) and DSS (p<0.0001, RR 4.442), along with T3,4 (p=0.0454) and N2,3 (p=0.0319), respectively. Conclusion Rsf-1 overexpression is common and is associated with adverse prognosticators and therapeutic response, which confers tumour aggressiveness through chromatin remodelling, and represents a potential prognostic biomarker in NPC.

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

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 p27kip1 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 p27kip1 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. Clin Cancer Res; 18(6); 1598–610. ©2012 AACR.

HuR cytoplasmic expression is associated with increased cyclin A expression and poor outcome with upper urinary tract urothelial carcinoma

BackgroundHuR is an RNA-binding protein that post-transcriptionally modulates the expressions of various target genes implicated in carcinogenesis, such as CCNA2 encoding cyclin A. No prior study attempted to evaluate the significance of HuR expression in a large cohort with upper urinary tract urothelial carcinomas (UTUCs).MethodsIn total, 340 cases of primary localized UTUC without previous or concordant bladder carcinoma were selected. All of these patients received ureterectomy or radical nephroureterectomy with curative intents. Pathological slides were reviewed, and clinical findings were collected. Immunostaining for HuR and cyclin A was performed and evaluated by using H-score. The results of cytoplasmic HuR and nuclear cyclin A expressions were correlated with disease-specific survival (DSS), metastasis-free survival (MeFS), urinary bladder recurrence-free survival (UBRFS), and various clinicopathological factors.ResultsHuR cytoplasmic expression was significantly related to the pT status, lymph node metastasis, a higher histological grade, the pattern of invasion, vascular and perineurial invasion, and cyclin A expression (p = 0.005). Importantly, HuR cytoplasmic expression was strongly associated with a worse DSS (p < 0.0001), MeFS (p < 0.0001), and UBRFS (p = 0.0370) in the univariate analysis, and the first two results remained independently predictive of adverse outcomes (p = 0.038, relative risk [RR] = 1.996 for DSS; p = 0.027, RR = 1.880 for MeFS). Cyclin A nuclear expression was associated with a poor DSS (p = 0.0035) and MeFS (p = 0.0015) in the univariate analysis but was not prognosticatory in the multivariate analyses. High-risk patients (pT3 or pT4 with/without nodal metastasis) with high HuR cytoplasmic expression had better DSS if adjuvant chemotherapy was performed (p = 0.015).ConclusionsHuR cytoplasmic expression was correlated with adverse phenotypes and cyclin A overexpression and also independently predictive of worse DSS and MeFS, suggesting its roles in tumorigenesis or carcinogenesis and potentiality as a prognostic marker of UTUC. High HuR cytoplasmic expression might identify patients more likely to be beneficial for adjuvant chemotherapy.

Rsf-1 expression in rectal cancer: with special emphasis on the independent prognostic value after neoadjuvant chemoradiation

Aims Neoadjuvant chemoradiation therapy (CRT) is an increasingly used therapeutic strategy for rectal cancer. Clinically, it remains a major challenge to predict therapeutic response and patient outcome after CRT. Rsf-1 (HBXAP), a novel nuclear protein with histone chaperon function, mediates ATPase-dependent chromatin remodelling and confers tumour aggressiveness and predicts therapeutic response in certain carcinomas. However, the expression of Rsf-1 has never been reported in rectal cancer. This study examined the predictive and prognostic impacts of Rsf-1 expression in patients with rectal cancer following neoadjuvant CRT. Methods Rsf-1 immunoexpression was retrospectively assessed for pre-treatment biopsies of 172 rectal cancer patients without initial distant metastasis. All of them were treated with neoadjuvant CRT followed by surgery. The results were correlated with the clinicopathological features, therapeutic response, tumour regression grade and metastasis-free survival (MeFS), local recurrent-free survival and disease-specific survival. Results Present in 82 cases (47.7%), high-expression of Rsf-1 was associated with advanced pre-treatment tumour status (T3, T4, p=0.020), advanced post-treatment tumour status (T3, T4, p<0.001) and inferior tumour regression grade (p=0.028). Of note, high-expression of Rsf-1 emerged as an adverse prognosticator for diseases-specific survival (p=0.0092) and significantly predicted worse MeFS (p=0.0006). Moreover, high-expression of Rsf-1 also remained prognostic independent for worse MeFS (HR 2.834; p=0.0214). Conclusions High-expression of Rsf-1 is associated with poor therapeutic response and adverse outcome in rectal cancer patients treated with neoadjuvant CRT, which confers tumour aggressiveness and therapeutic resistance through chromatin remodelling and represents a potential prognostic biomarker in rectal cancer.

Alpha-methylacyl coenzyme A racemase overexpression in gallbladder carcinoma confers an independent prognostic indicator

Aims Increased β-oxidation of branched-chain fatty acids provides an additional metabolic advantage for cancer cells thereby enhancing tumour development and progression. Alpha-methylacyl coenzyme A racemase (AMACR) is an enzyme essential for the catabolism of branched-chain fatty acids that allows their subsequent β-oxidation and thus plays an important role in generating biological energy. However, the expression of AMACR has never been systemically investigated in gallbladder carcinoma. This study evaluated the expression status, associations with clinicopathological variables and prognostic implications of AMACR in a well-defined cohort of gallbladder carcinoma and confirmed their expression status in gallbladder carcinoma cells. Methods AMACR immunostaining was assessable in 89 cases on tissue microarrays of gallbladder carcinoma, and it was correlated with clinicopathological factors and patient survival. In three gallbladder carcinoma cell lines and one non-tumorigenic cholangiocyte, AMACR mRNA expression was measured by real-time reverse transcription PCR and the endogenous expression of AMACR protein was analysed by western blotting. Results AMACR overexpression was significantly associated with an advanced primary tumour status (p=0.027) and American Joint Committee on Cancer stage (p=0.027), an increased histological grade (p=0.002) and vascular invasion (p=0.017). Importantly, AMACR overexpression independently predicted worse disease-specific survival (p=0.0452, RR 1.887). Expression levels of AMACR mRNA and total protein in various cells were comparable. The abundance of AMACR expression increased in tumour cells and was even higher in the metastatic cell line. Conclusions In primary gallbladder carcinoma, AMACR overexpression was correlated with important prognosticators and independently portended worse outcomes, highlighting the potential prognostic and therapeutic utility of AMACR in gallbladder carcinoma.

Loss of epithelial membrane protein-2 expression confers an independent prognosticator in nasopharyngeal carcinoma: a cohort study

Objective To evaluate the expression of epithelial membrane protein-2 (EMP2) protein and its clinicopathological associations in patients with nasopharyngeal carcinoma. Design Retrospective population-based cohort study. Setting This study was based on a biobank in Chi-Mei Medical Center (Tainan, Taiwan) from 1993 to 2002. Participants Biopsies of 124 consecutive nasopharyngeal carcinoma patients without initial distant metastasis and treated with consistent guidelines were assessed. Immunoexpressions of EMP2 were analysed and the outcomes were correlated with clinicopathological features and patient survivals. Primary and secondary outcome measures Immunoexpressions of EMP2 were analyzed and the outcomes were correlated with clinicopathological features and patient survivals. Results Loss of EMP2 expression (49.2%) was correlated with advanced primary tumour (p=0.044), nodal status (p=0.045) and the 7th American Joint Committee on Cancer stage (p=0.027). In multivariate analyses, loss of EMP2 expression emerged as an independent prognosticator for worse disease-specific survival (DSS; p=0.015) and local recurrence-free survival (LRFS; p=0.030), along with the American Joint Committee on Cancer stages III–IV (p=0.034, DSS; p=0.023, LRFS). Conclusions Loss of EMP2 expression is common and associated with adverse prognosticators and might confer tumour aggressiveness through hampering its interaction with specific membrane protein(s) and hence the downstream signal transduction pathway(s).

Upregulation of cyclin-dependent kinase inhibitors CDKN1B and CDKN1C in hepatocellular carcinoma-derived cells via goniothalamin-mediated protein stabilization and epigenetic modifications

Cell cycle deregulation is common in human hepatocellular carcinoma (HCC). To ensure proper cell cycle controlling, cyclin/cyclin-dependent kinases (CDK) complexes are tightly regulated by CDK inhibitors (CKIs) in normal cells. However, insufficient cyclin-dependent kinase inhibitor 1B (CDKN1B, also known as p27Kip1) and CDKN1C (p57Kip2) proteins are characteristics of high-risk HCC. In two HCC-derived cell lines with distinct genetic backgrounds, we identified a small natural compound, goniothalamin (GTN), serving as an inducer of CKIs. In TP53-mutated (Y220C) and retinoblastoma 1 (RB1)-positive Huh-7 cells, GTN stabilized CDKN1B protein levels by targeting the degradation of its specific E3 ubiquitin ligase (S-phase kinase-associated protein 2). Alternatively, in TP53- and RB1-negative Hep-3B cells, GTN increased CDKN1C transcription and its subsequent translation by acting as a histone deacetylase inhibitor. In both cell lines, GTN induced G0/G1 cell cycle arrest, delayed S phase entry of cells and inhibited anchorage-independent cell growth which might be attributed to the upregulation of CKIs and downregulation of several positive cell cycle regulators, including CDC28 protein kinase regulator subunit 1B, cyclin E1 and D1, cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, E2F transcription factor 1 and/or transcription factor Dp-1. Therefore, GTN might represent a novel class of anticancer drug that induces CKIs through post-translational and epigenetic modifications.