Jianhe Shen

MMP13, Birc2 (cIAP1), and Birc3 (cIAP2), Amplified on Chromosome 9, Collaborate with p53 Deficiency in Mouse Osteosarcoma Progression

Osteosarcoma is the primary malignant cancer of bone and particularly affects adolescents and young adults, causing debilitation and sometimes death. As a model for human osteosarcoma, we have been studying p53(+/-) mice, which develop osteosarcoma at high frequency. To discover genes that cooperate with p53 deficiency in osteosarcoma formation, we have integrated array comparative genomic hybridization, microarray expression analyses in mouse and human osteosarcomas, and functional assays. In this study, we found seven frequent regions of copy number gain and loss in the mouse p53(+/-) osteosarcomas but have focused on a recurrent amplification event on mouse chromosome 9A1. This amplicon is syntenic with a similar chromosome 11q22 amplicon identified in several human tumor types. Three genes on this amplicon, the matrix metalloproteinase gene MMP13 and the antiapoptotic genes Birc2 (cIAP1) and Birc3 (cIAP2), show elevated expression in mouse and human osteosarcomas. We developed a functional assay using clonal osteosarcoma cell lines transduced with lentiviral short hairpin RNA vectors to show that down-regulation of MMP13, Birc2, or Birc3 resulted in reduced tumor growth when transplanted into immunodeficient recipient mice. These experiments revealed that high MMP13 expression enhances osteosarcoma cell survival and that Birc2 and Birc3 also enhance cell survival but only in osteosarcoma cells with the chromosome 9A1 amplicon. We conclude that the antiapoptotic genes Birc2 and Birc3 are potential oncogenic drivers in the chromosome 9A1 amplicon.

A systems biology approach reveals common metastatic pathways in osteosarcoma

BackgroundOsteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. The survival rate of patients with metastatic disease remains very dismal. Nevertheless, metastasis is a complex process and a single-level analysis is not likely to identify its key biological determinants. In this study, we used a systems biology approach to identify common metastatic pathways that are jointly supported by both mRNA and protein expression data in two distinct human metastatic OS models.ResultsmRNA expression microarray and N-linked glycoproteomic analyses were performed on two commonly used isogenic pairs of human metastatic OS cell lines, namely HOS/143B and SaOS-2/LM7. Pathway analysis of the differentially regulated genes and glycoproteins separately revealed pathways associated to metastasis including cell cycle regulation, immune response, and epithelial-to-mesenchymal-transition. However, no common significant pathway was found at both genomic and proteomic levels between the two metastatic models, suggesting a very different biological nature of the cell lines. To address this issue, we used a topological significance analysis based on a “shortest-path” algorithm to identify topological nodes, which uncovered additional biological information with respect to the genomic and glycoproteomic profiles but remained hidden from the direct analyses. Pathway analysis of the significant topological nodes revealed a striking concordance between the models and identified significant common pathways, including “Cytoskeleton remodeling/TGF/WNT”, “Cytoskeleton remodeling/Cytoskeleton remodeling”, and “Cell adhesion/Chemokines and adhesion”. Of these, the “Cytoskeleton remodeling/TGF/WNT” was the top ranked common pathway from the topological analysis of the genomic and proteomic profiles in the two metastatic models. The up-regulation of proteins in the “Cytoskeleton remodeling/TGF/WNT” pathway in the SaOS-2/LM7 and HOS/143B models was further validated using an orthogonal Reverse Phase Protein Array platform.ConclusionsIn this study, we used a systems biology approach by integrating genomic and proteomic data to identify key and common metastatic mechanisms in OS. The use of the topological analysis revealed hidden biological pathways that are known to play critical roles in metastasis. Wnt signaling has been previously implicated in OS and other tumors, and inhibitors of Wnt signaling pathways are available for clinical testing. Further characterization of this common pathway and other topological pathways identified from this study may lead to a novel therapeutic strategy for the treatment of metastatic OS.

Implications of Tumor Location on Subtypes of Medulloblastoma

Medulloblastoma (MB) comprises of four molecular subtypes, Sonic hedgehog (SHH), Wingless (WNT), Groups 3 and 4. WNT‐subtype MBs were found to arise from midline of the brainstem occupying the fourth ventricle while SHH‐subtype occupied the cerebellar hemisphere in a small subset of patients.

Optimising the Use of TRIzol-extracted Proteins in Surface Enhanced Laser Desorption/ Ionization (SELDI) Analysis

BackgroundResearch with clinical specimens is always hampered by the limited availability of relevant samples, necessitating the use of a single sample for multiple assays. TRIzol is a common reagent for RNA extraction, but DNA and protein fractions can also be used for other studies. However, little is known about using TRIzol-extracted proteins in proteomic research, partly because proteins extracted from TRIzol are very resistant to solubilization.ResultsTo facilitate the use of TRIzol-extracted proteins, we first compared the ability of four different common solubilizing reagents to solubilize the TRIzol-extracted proteins from an osteosarcoma cell line, U2-OS. Then we analyzed the solubilized proteins by Surface Enhanced Laser Desorption/ Ionization technique (SELDI). The results showed that solubilization of TRIzol-extracted proteins with 9.5 M Urea and 2% CHAPS ([3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate]) (UREA-CHAPS) was significantly better than the standard 1% SDS in terms of solubilization efficiency and the number of detectable ion peaks. Using three different types of SELDI arrays (CM10, H50, and IMAC-Cu), we demonstrated that peak detection with proteins solubilized by UREA-CHAPS was reproducible (r > 0.9). Further SELDI analysis indicated that the number of ion peaks detected in TRIzol-extracted proteins was comparable to a direct extraction method, suggesting many proteins still remain in the TRIzol protein fraction.ConclusionOur results suggest that UREA-CHAPS performed very well in solubilizing TRIzol-extracted proteins for SELDI applications. Protein fractions left over after TRIzol RNA extraction could be a valuable but neglected source for proteomic or biochemical analysis when additional samples are not available.

Plasma proteome predicts chemotherapy response in osteosarcoma patients

Osteosarcoma is the most common malignant bone tumor that affects hundreds of children and young adults every year. The major prognostic factor in patients with localized osteosarcoma is the development of resistance towards pre-operative chemotherapy. However, modifications of post-operative chemotherapy based on the histological response have not significantly improved the outcome of patients. Thus, it would be of tremendous clinical value if the poor responders could be identified at the time of diagnosis, so that ineffective therapy can be prevented and intensified or alternative therapy could be provided to improve their outcome. We hypothesized that plasma proteomic profiles could be used to distinguish good from poor responders prior to the start of treatment. In order to test this hypothesis, we analyzed the proteomic profiles in two sets of plasma samples (n=54) from osteosarcoma patients collected before (n=27) and after (n=27) pre-operative chemotherapy. Using a linear support vector machine algorithm and external leave-one-out cross validation, we developed two classifiers that classified good and poor responders with an equal accuracy of 85% (p<0.01 after 5000 permutations) in both sets of plasma samples. In order to understand the biological basis of the classifiers, we further identified and validated two plasma proteins, serum amyloid protein A and transthyretin, in the classifiers. Our results suggest that plasma proteomic profiles can predict chemotherapy response before treatment as accurately as after treatment. Our study could lead to the development of a simple blood test that can predict chemotherapy response in osteosarcoma patients. Since the two identified proteins are involved in innate immunity, our findings are corroborated by the notion that boosting the innate immunity in conjunction with chemotherapy, achieves a better anti-tumor activity, thus improving the overall survival of osteosarcoma patients.

Biomarker significance of plasma and tumor miR-21, miR-221, and miR-106a in osteosarcoma

Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who have a poor response to chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients. In this study, we examined whether circulating miRNAs can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of osteosarcoma and control plasma samples. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to control plasma samples. miR-21, miR-221 and miR-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in osteosarcoma samples than normal samples in an independent cohort obtained from the Children’s Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a were elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of these miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma.Osteosarcoma is the most common malignant bone tumor in children and young adults. Despite the use of surgery and multi-agent chemotherapy, osteosarcoma patients who have a poor response to chemotherapy or develop relapses have a dismal outcome. Identification of biomarkers for active disease may help to monitor tumor burden, detect early relapses, and predict prognosis in these patients. In this study, we examined whether circulating miRNAs can be used as biomarkers in osteosarcoma patients. We performed genome-wide miRNA profiling on a discovery cohort of osteosarcoma and control plasma samples. A total of 56 miRNAs were upregulated and 164 miRNAs were downregulated in osteosarcoma samples when compared to control plasma samples. miR-21, miR-221 and miR-106a were selected for further validation based on their known biological importance. We showed that all three circulating miRNAs were expressed significantly higher in osteosarcoma samples than normal samples in an independent cohort obtained from the Childrens Oncology Group. Furthermore, we demonstrated that miR-21 was expressed significantly higher in osteosarcoma tumors compared with normal bone controls. More importantly, lower expressions of miR-21 and miR-221, but not miR-106a, significantly correlated with a poor outcome. In conclusion, our results indicate that miR-21, miR-221 and miR-106a were elevated in the circulation of osteosarcoma patients, whereas tumor expressions of miR-21 and miR-221 are prognostically significant. Further investigation of these miRNAs may lead to a better prognostic method and potential miRNA therapeutics for osteosarcoma.

Abstract 3402: Knockdown of TWIST1 increases chemosensitivity of osteosarcoma cells

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Knock-down of TWIST1 increases chemosensitivity of osteosarcoma cells Texas Childrens Cancer Center; Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 TWIST1, a basic helix-loop-helix (bHLH) transcription factor, has previously been reported to be involved in cancer metastasis, invasion, drug response, and osteoblast differentiation induced by bone morphogenic protein. Based on expression profiling, we have previously identified a chemoresistance signature in osteosarcoma that could predict at the time of diagnosis the response to neoadjuvant chemotherapy. One of the 45 genes in the chemoresistance signature is TWIST1 whose expression level was significantly higher in osteosarcomas that were subsequently found to be more chemoresistant. To investigate the involvement of TWIST1 in chemoresistance of osteosarcoma at the cellular level, we first demonstrated that the IC50 of doxorubicin but not cisplatin treatment in eight osteosarcoma cell lines correlated with the endogenous TWIST1 expression level. We then tested the hypothesis that down-regulation of TWIST1 expression through RNAi in osteosarcoma cell lines with high TWIST1 expression could restore the chemosensitivity in these cells. Using two independent shRNAs to knock-down TWIST1 in HOS, SJSA-1 or 143B cells, we were able to show that the transduced cells became more sensitive to doxorubicin treatment as compared with the parental cells or control cells that were transduced with shRNAs with scrambled sequences. The results of the cytotoxicity assay were confirmed by the clonogenic assay using single colonies of stable transfectants with HOS cells. Based on these results, we concluded that TWIST1 is involved in chemoresistance of osteosarcoma cell lines and that down-regulation of its expression through RNAi would sensitize osteosarcoma cells to doxorubicin. However, we did not observe increased chemoresistance with overexpression of TWIST1 in osteosarcoma cell lines that have low levels of TWIST1 expression, suggesting that TWIST1 alone is not sufficient to cause chemoresistance in osteosarcoma cells. Identification of downstream targets of TWIST1 that could mediate chemoresistance by comparing expression profiles of TWIST1-knockdown clones with parental and scrambled controls is ongoing. In summary, these results validate our previous finding that TWIST1 can be used as a potential marker for predicting chemoresistance in osteosarcoma and suggest that modulating TWIST1 or its downstream targets could be considered as a novel strategy to overcome chemoresistance in osteosarcoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3402.

Abstract 3824: Integrated analysis of array-CGH and gene expression profiling identifies HSPA5 as a prognostic marker in SHH pathway-actived Medulloblastoma.

Medulloblastoma (MB) is the most malignant brain tumor in children and is characterized by marked biological and clinical heterogeneity. It includes four distinct molecular subtypes [wingless (WNT), sonic hedgehog (SHH), Group 3 and Group 4]. Previous attempts to identify prognostic markers applicable to all MBs were unsuccessful and we therefore hypothesize that each molecular subtype of MB may be associated with its unique prognostic markers. The aim of this study is to identify prognostic markers specific for SHH-active MB tumors. We analyzed SNP-array CGH data and gene expression profiling for 60 pediatric MBs and compared the molecular results with clinical outcome data. Using unsupervised hierarchical clustering, we identified 18 MBs that have activated SHH signaling. This subtype of SHH-active tumors also have frequent chromosome 9q loss which are associated with more favorable outcome. In an attempt to identify candidate genes on chromosome 9q with survival impact, we found that low expression of Heat Shock 70kDa Protein 5 (HSPA5) was significantly associated with better event free survival (EFS) in SHH MB patients independent of other clinical variables. This finding had been validated in two other independent MB cohorts. By immuohistochemistry and western blotting, we confirmed the negative correlation between patient survival and HSPA5 protein expression in SHH tumors. Furthermore, in vitro study indicated that knockdown of HSPA5 by siRNA caused dramatic growth suppression in SHH active MB cells. These findings suggest that HSPA5 is a potential prognostic marker in predicting survival of SHH active MB patients and this protein may also be exploited as therapeutic target for SHH MBs that do not harbor chromosome 9q loss. Citation Format: Angela Kwok-Fung Lo, Jian Wang, Jianhe Shen, Alexander Yu, Wing-Yuk Chow, Jack Su, Adekunle Adesina, Robert Dauser, William Whitehead, Murali Chintagumpala, Rudy Guerra, Tsz-Kwong Man, Ching C. Lau. Integrated analysis of array-CGH and gene expression profiling identifies HSPA5 as a prognostic marker in SHH pathway-actived Medulloblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3824. doi:10.1158/1538-7445.AM2013-3824

Abstract 3155: High-throughput profiling of miRNA expression in plasma and cerebrospinal fluid using OpenArray® platform for biomarker discovery

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL OpenArray® platform can be used for expression profiling using TaqMan® assays that enables higher throughput, less assay consumption, and lower sample input than other low-density assay platforms. One OpenArray® plate has 3,072 through-holes, each of which requires only 33nl of reaction volume. We combined the workflow of Megaplex™ RT primer pools and pre-amplification with TaqMan® MicroRNA assays on OpenArray® plates. Each OpenArray® plate provides the capacity of 3 samples, and each run can take up to 3 plates with a run time about 2.5 hours. Therefore, each typical work day can have 4 instrument runs generating expression data of 754 miRNAs from 36 samples. This platform is ideal to identify potential miRNA biomarkers within a short period of time by screening a large cohort of clinical specimens that might have limitations in sample size, contamination of PCR inhibitors, or low abundance of target miRNAs. We optimized the original workflow of Megaplex™ RT primer pools and pre-amplification before applying to the OpenArray® plates to ensure increasing detection sensitivity while maintaining the linearity in plasma samples. In a proof-of-principle investigation, we applied the new protocol to a small panel of plasma and cerebrospinal fluid (CSF) specimens derived from pediatric patients diagnosed with either germinoma or non-germinoma germ cell tumors of the brain, and benchmarked with another miRNA expression profiling platform, TaqMan® Array Card (TAC), and OpenArray® plates demonstrated good concordance with the TAC. Expression patterns of miRNAs detected in our CSF samples are largely consistent with previously published miRNAs differentially expressed in primary cancerous tissues between these two types of intracranial germ cell tumors. (For Research Use Only. Not for use in diagnostic procedures.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3155. doi:1538-7445.AM2012-3155