Combinatorial Ubiquitination REal-time PROteolysis (CURE-PROs): A Modular Platform for Generating Reversible, Self-Assembling Bifunctional Targeted Degraders.

Proteolysis-Targeting Chimeras (PROTACs) are bifunctional molecules that bring a target protein and an E3 ubiquitin ligase into proximity to append ubiquitin, thus directing target degradation. Although numerous PROTACs have entered clinical trials, their development remains challenging, and their large size can produce poor drug-like properties. To overcome these limitations, we have modified our Coferon platform to generate Combinatorial Ubiquitination REal-time PROteolysis (CURE-PROs). CURE-PROs are small molecule degraders designed to self-assemble through reversible bio-orthogonal linkers to form covalent heterodimers. By modifying known ligands for Cereblon, MDM2, VHL, and BRD with complementary phenylboronic acid and diol/catechol linkers, we have successfully created CURE-PROs that direct degradation of BRD4 both in vitro and in vivo. The combinatorial nature of our platform significantly reduces synthesis time and effort to identify the optimal linker length and E3 ligase partner to each target and is readily amenable to screening for new targets.

Multi-CpG linear regression models to accurately predict paclitaxel and docetaxel activity in cancer cell lines.

The microtubule-targeting paclitaxel (PTX) and docetaxel (DTX) are widely used chemotherapeutic agents. However, the dysregulation of apoptotic processes, microtubule-binding proteins, and multi-drug resistance efflux and influx proteins can alter the efficacy of taxane drugs. In this review, we have created multi-CpG linear regression models to predict the activities of PTX and DTX drugs through the integration of publicly available pharmacological and genome-wide molecular profiling datasets generated using hundreds of cancer cell lines of diverse tissue of origin. Our findings indicate that linear regression models based on CpG methylation levels can predict PTX and DTX activities (log-fold change in viability relative to DMSO) with high precision. For example, a 287-CpG model predicts PTX activity at R2 of 0.985 among 399 cell lines. Just as precise (R2=0.996) is a 342-CpG model for predicting DTX activity in 390 cell lines. However, our predictive models, which employ a combination of mRNA expression and mutation as input variables, are less accurate compared to the CpG-based models. While a 290 mRNA/mutation model was able to predict PTX activity with R2 of 0.830 (for 546 cell lines), a 236 mRNA/mutation model could calculate DTX activity at R2 of 0.751 (for 531 cell lines). The CpG-based models restricted to lung cancer cell lines were also highly predictive (R2≥0.980) for PTX (74 CpGs, 88 cell lines) and DTX (58 CpGs, 83 cell lines). The underlying molecular biology behind taxane activity/resistance is evident in these models. Indeed, many of the genes represented in PTX or DTX CpG-based models have functionalities related to apoptosis (e.g., ACIN1, TP73, TNFRSF10B, DNASE1, DFFB, CREB1, BNIP3), and mitosis/microtubules (e.g., MAD1L1, ANAPC2, EML4, PARP3, CCT6A, JAKMIP1). Also represented are genes involved in epigenetic regulation (HDAC4, DNMT3B, and histone demethylases KDM4B, KDM4C, KDM2B, and KDM7A), and those that have never been previously linked to taxane activity (DIP2C, PTPRN2, TTC23, SHANK2). In summary, it is possible to accurately predict taxane activity in cell lines based entirely on methylation at multiple CpG sites.

Biomarker Discovery for Meta-Classification of Melanoma Metastatic Progression Using Transfer Learning.

Melanoma is considered to be the most serious and aggressive type of skin cancer, and metastasis appears to be the most important factor in its prognosis. Herein, we developed a transfer learning-based biomarker discovery model that could aid in the diagnosis and prognosis of this disease. After applying it to the ensemble machine learning model, results revealed that the genes found were consistent with those found using other methodologies previously applied to the same TCGA (The Cancer Genome Atlas) data set. Further novel biomarkers were also found. Our ensemble model achieved an AUC of 0.9861, an accuracy of 91.05, and an F1 score of 90.60 using an independent validation data set. This study was able to identify potential genes for diagnostic classification (C7 and GRIK5) and diagnostic and prognostic biomarkers (S100A7, S100A7, KRT14, KRT17, KRT6B, KRTDAP, SERPINB4, TSHR, PVRL4, WFDC5, IL20RB) in melanoma. The results show the utility of a transfer learning approach for biomarker discovery in melanoma.

A Unified Transcriptional, Pharmacogenomic, and Gene Dependency Approach to Decipher the Biology, Diagnostic Markers, and Therapeutic Targets Associated with Prostate Cancer Metastasis.

Our understanding of metastatic prostate cancer (mPrCa) has dramatically advanced during the genomics era. Nonetheless, many aspects of the disease may still be uncovered through reanalysis of public datasets. We integrated the expression datasets for 209 PrCa tissues (metastasis, primary, normal) with expression, gene dependency (GD) (from CRISPR/cas9 screen), and drug viability data for hundreds of cancer lines (including PrCa). Comparative statistical and pathways analyses and functional annotations (available inhibitors, protein localization) revealed relevant pathways and potential (and previously reported) protein markers for minimally invasive mPrCa diagnostics. The transition from localized to mPrCa involved the upregulation of DNA replication, mitosis, and PLK1-mediated events. Genes highly upregulated in mPrCa and with very high average GD (~1) are potential therapeutic targets. We showed that fostamatinib (which can target PLK1 and other over-expressed serine/threonine kinases such as AURKA, MELK, NEK2, and TTK) is more active against cancer lines with more pronounced signatures of invasion (e.g., extracellular matrix organization/degradation). Furthermore, we identified surface-bound (e.g., ADAM15, CD276, ABCC5, CD36, NRP1, SCARB1) and likely secreted proteins (e.g., APLN, ANGPT2, CTHRC1, ADAM12) that are potential mPrCa diagnostic markers. Overall, we demonstrated that comprehensive analyses of public genomics data could reveal potentially clinically relevant information regarding mPrCa.

Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression.

BACKGROUND: The molecular regulation of increased MGMT expression in human brain tumors, the associated regulatory elements, and linkages of these to its epigenetic silencing are not understood. Because the heightened expression or non-expression of MGMT plays a pivotal role in glioma therapeutics, we applied bioinformatics and experimental tools to identify the regulatory elements in the MGMT and neighboring EBF3 gene loci. RESULTS: Extensive genome database analyses showed that the MGMT genomic space was rich in and harbored many undescribed RNA regulatory sequences and recognition motifs. We extended the MGMT's exon-1 promoter to 2019 bp to include five overlapping alternate promoters. Consensus sequences in the revised promoter for (a) the transcriptional factors CTCF, NRF1/NRF2, GAF, (b) the genetic switch MYC/MAX/MAD, and (c) two well-defined p53 response elements in MGMT intron-1, were identified. A putative protein-coding or non-coding RNA sequence was located in the extended 3' UTR of the MGMT transcript. Eleven non-coding RNA loci coding for miRNAs, antisense RNA, and lncRNAs were identified in the MGMT-EBF3 region and six of these showed validated potential for curtailing the expression of both MGMT and EBF3 genes. ChIP analysis verified the binding site in MGMT promoter for CTCF which regulates the genomic methylation and chromatin looping. CTCF depletion by a pool of specific siRNA and shRNAs led to a significant attenuation of MGMT expression in human GBM cell lines. Computational analysis of the ChIP sequence data in ENCODE showed the presence of NRF1 in the MGMT promoter and this occurred only in MGMT-proficient cell lines. Further, an enforced NRF2 expression markedly augmented the MGMT mRNA and protein levels in glioma cells. CONCLUSIONS: We provide the first evidence for several new regulatory components in the MGMT gene locus which predict complex transcriptional and posttranscriptional controls with potential for new therapeutic avenues.

MGMT Epigenetics: The Influence of Gene Body Methylation and Other Insights Derived from Integrated Methylomic, Transcriptomic, and Chromatin Analyses in Various Cancer Types.

BACKGROUND: MGMT (O6-methylguanine-DNA methyltransferase) is primarily responsible for limiting the activity of some widely used chemotherapeutic agents, including temozolomide (TMZ) and carmustine (BCNU). The gene encoding this protein is epigenetically regulated, and assessment of methylation at its promoter region is used to predict glioma patients' response to TMZ. METHODS: In this report, we employed a bioinformatic approach to elucidate MGMT's epigenetic regulation. Integrated for the analysis were genome-wide methylation and transcription datasets for > 8,600 human tissue (representing 31 distinct cancer types) and 500 human cancer cell line samples. Also crucial to the interpretation of results were publicly available data from the ENCODE Project: tracks for histone modifications (via ChIP-seq) and DNase I hypersensitivity (via DNaseseq), as well as methylation and transcription data for representative cell lines (HeLa-S3, HMEC, K562). RESULTS AND DISCUSSION: We were able to validate (perhaps more comprehensively) the contrasting influences of CpG methylation at promoter region and at gene body on MGMT transcription. While the MGMT promoter is populated by CpG sites whose methylation levels displayed high negative correlation (R) with MGMT mRNA counts, the gene body harbors CpG sites exhibiting high positive R values. The promoter CpG sites with very high negative R's across cancer types include cg12981137, cg12434587, and cg00618725. Among the notable gene body CpG sites (high positive R's across cancer types) are cg00198994 (Intron 1), cg04473030 (Intron 2), and cg07367735 (Intron 4). For certain cancer types, such as melanoma, gene body methylation appears to be a better predictor of MGMT transcription (compared to promoter methylation). In general, the CpG methylation v. MGMT expression R values are higher in cell lines relative to tissues. Also, these correlations are noticeably more prominent in certain cancer types such as colorectal, adrenocortical, esophageal, skin, and head and neck cancers, as well as glioblastoma. As expected, hypomethylation at the promoter region is associated with more open chromatin, and enrichment of histone marks H3K4m1, H3K4m2, H3K4m3, and H3K9ac. CONCLUSION: Overall, our analysis illustrated the contrasting influence of promoter and gene body methylation on MGMT expression. These observations may help improve diagnostic assays for MGMT.

Application of Multiplex Bisulfite PCR-Ligase Detection Reaction-Real-Time Quantitative PCR Assay in Interrogating Bioinformatically Identified, Blood-Based Methylation Markers for Colorectal Cancer.

The analysis of CpG methylation in circulating tumor DNA fragments has emerged as a promising approach for the noninvasive early detection of solid tumors, including colorectal cancer (CRC). The most commonly employed assay involves bisulfite conversion of circulating tumor DNA, followed by targeted PCR, then real-time quantitative PCR (alias methylation-specific PCR). This report demonstrates the ability of a multiplex bisulfite PCR-ligase detection reaction-real-time quantitative PCR assay to detect seven methylated CpG markers (CRC or colon specific), in both simulated (approximately 30 copies of fragmented CRC cell line DNA mixed with approximately 3000 copies of fragmented peripheral blood DNA) and CRC patient-derived cell-free DNAs. This scalable assay is designed for multiplexing and incorporates steps for improved sensitivity and specificity, including the enrichment of methylated CpG fragments, ligase detection reaction, the incorporation of ribose bases in primers, and use of uracil DNA glycosylase. Six of the seven CpG markers (located in promoter regions of PPP1R16B, KCNA3, CLIP4, GDF6, SEPT9, and GSG1L) were identified through integrated analyses of genome-wide methylation data sets for 31 different types of cancer. These markers were mapped to CpG sites at the promoter region of VIM; VIM and SEPT9 are established epigenetic markers of CRC. Additional bioinformatics analyses show that the methylation at these CpG sites negatively correlates with the transcription of their corresponding genes.

Prediction of blood-based biomarkers and subsequent design of bisulfite PCR-LDR-qPCR assay for breast cancer detection.

BACKGROUND: Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR. METHODS: In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood. RESULTS: Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27). CONCLUSION: This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.

Single-molecule detection of cancer mutations using a novel PCR-LDR-qPCR assay.

Detection of low-abundance mutations in cell-free DNA is being used to identify early cancer and early cancer recurrence. Here, we report a new PCR-LDR-qPCR assay capable of detecting point mutations at a single-molecule resolution in the presence of an excess of wild-type DNA. Major features of the assay include selective amplification and detection of mutant DNA employing multiple nested primer-binding regions as well as wild-type sequence blocking oligonucleotides, prevention of carryover contamination, spatial sample dilution, and detection of multiple mutations in the same position. Our method was tested to interrogate the following common cancer somatic mutations: BRAF:c.1799T>A (p.Val600Glu), TP53:c.743G>A (p.Arg248Gln), KRAS:c.35G>C (p.Gly12Ala), KRAS:c.35G>T (p.Gly12Val), KRAS:c.35G>A (p.Gly12Asp), KRAS:c.34G>T (p.Gly12Cys), and KRAS:c.34G>A (p.Gly12Ser). The single-well version of the assay detected 2-5 copies of these mutations, when diluted with 10,000 genome equivalents (GE) of wild-type human genomic DNA (hgDNA) from buffy coat. A 12-well (pixel) version of the assay was capable of single-molecule detection of the aforementioned mutations at TP53, BRAF, and KRAS (specifically p.Gly12Val and p.Gly12Cys), mixed with 1,000-2,250 GE of wild-type hgDNA from plasma or buffy coat. The assay described herein is highly sensitive, specific, and robust, and potentially useful in liquid biopsies.

Regulation of neuroblastoma migration, invasion, and in vivo metastasis by genetic and pharmacological manipulation of MDA-9/Syntenin.

Despite multi-modality treatments, prognosis for advanced stage neuroblastoma (NB) remains challenging with residual long-term disabilities in survivors. Advanced stage NB is metastatic, which is a principal cause of cancer-related deaths. We presently document a primary role of MDA-9 in NB progression and define the molecular mechanisms by which MDA-9 promotes transformed phenotypes. NB cell lines and clinical samples display elevated MDA-9 expression and bioinformatic analysis supports an association between elevated MDA-9 and bone metastasis and poor prognosis. Genetic (shmda-9, mda-9 siRNA) or pharmacological (small molecule inhibitor of protein-protein interactions; PDZ1i) blockade of MDA-9 decreases NB migration, invasion, and metastasis. Blocking mda-9 expression or disrupting MDA-9 partner protein interactions downregulates integrin α6 and ß4, diminishing Src activity and suppressing Rho-Rac-Cdc42 activity. These signaling changes inhibit cofilin and matrix metalloproteinases reducing in vitro and in vivo NB cell migration. Overexpression of integrin α6 and ß4 rescues the invasion phenotype and increases Src activity, supporting integrins as essential regulators of MDA-9-mediated NB migration and invasion. We identify MDA-9 as a key contributor to NB pathogenesis and show that genetic or pharmacological inhibition suppresses NB pathogenesis by an integrin-mediated Src-disruption pathway.

Can CpG methylation serve as surrogate markers for immune infiltration in cancer?

Recent reports describe how genome-wide transcriptional analysis of cancer tissues can be exploited to identify molecular signatures of immune infiltration in cancer. We hypothesize that immune infiltration in cancer may also be defined by changes in certain epigenetic signatures. In this context, a primary objective is to identify site-specific CpG markers whose levels of methylation may be highly indicative of known transcriptional markers of immune infiltration such as GZMA, PRF1, T cell receptor genes, PDCD1, and CTLA4. This has been accomplished by integrating genome-wide transcriptional expression and methylation data for different types of cancer (melanoma, kidney cancers, lung cancers, gliomas, head and neck cancer). Our findings establish that cancers of related histology also have a high degree of similarity in immune-infiltration CpG markers. For example, the epigenetic immune infiltration signatures in lung adenocarcinoma (LUAD), mesothelioma (MESO), lung squamous cell carcinoma (LUSC), and head and neck squamous cell carcinoma (HNSC) are distinctly similar. So are glioblastoma multiforme (GBM) and brain lower grade glioma (LGG); and kidney renal papillary cell carcinoma (KIRP) and kidney renal clear cell carcinoma (KIRC). Kidney chromophobe (KICH), on the other hand has markers that are unique to this cohort. The strong relationships between immune infiltration and CpG methylation (for certain sites) in cancer tissues were not observed upon integrated analysis of publicly available cancer cell line datasets. Results from comparative pathways analyses offer further justification to methylation at certain CpG sites as being indicators of cancer immune infiltration, and possibly of predicting patient response to immunotherapeutic drugs. Achieving this target objective would significantly enhance therapeutic outcomes employing immunotherapy through focused patient-centric personalized medicine.

Pathways- and epigenetic-based assessment of relative immune infiltration in various types of solid tumors.

Recent clinical studies document the power of immunotherapy in treating subsets of patients with advanced cancers. In this context and with multiple cancer immunotherapeutics already evaluated in the clinic and a large number in various stages of clinical trials, it is imperative to comprehensively examine genomics data to better comprehend the role of immunity in different cancers in predicting response to therapy and in directing appropriate therapies. The approach we chose is to scrutinize the pathways and epigenetic factors predicted to drive immune infiltration in different cancer types using publicly available TCGA transcriptional and methylation datasets, along with accompanying clinico-pathological data. We observed that the relative activation of T cells and other immune signaling pathways differs across cancer types. For example, pathways related to activation and proliferation of helper and cytotoxic T cells appear to be more highly enriched in kidney, skin, head and neck, and esophageal cancers compared to those of lung, colorectal, and liver or bile duct cancers. The activation of these immune-related pathways positively associated with prognosis in certain cancer types, most notably melanoma, head and neck, and cervical cancers. Integrated methylation and expression data (along with publicly available, ENCODE-generated histone ChIP Seq and DNAse hypersensitivity data) predict that epigenetic regulation is a primary factor driving transcriptional activation of a number of genes crucial to immunity in cancer, including T cell receptor genes (e.g., CD3D, CD3E), CTLA4, and GZMA. However, the extent to which epigenetic factors (primarily methylation at promoter regions) affect transcription of immune-related genes may vary across cancer types. For example, there is a high negative correlation between promoter CpG methylation and CD3D expression in renal and thyroid cancers, but not in brain tumors. The types of analyses we have undertaken provide insights into the relationships between immune modulation and cancer etiology and progression, offering clues into ways of therapeutically manipulating the immune system to promote immune recognition and immunotherapy.

Examination of Epigenetic and other Molecular Factors Associated with mda-9/Syntenin Dysregulation in Cancer Through Integrated Analyses of Public Genomic Datasets.

mda-9/Syntenin (melanoma differentiation-associated gene 9) is a PDZ domain containing, cancer invasion-related protein. In this study, we employed multiple integrated bioinformatic approaches to identify the probable epigenetic factors, molecular pathways, and functionalities associated with mda-9 dysregulation during cancer progression. Analyses of publicly available genomic data (e.g., expression, copy number, methylation) from TCGA, GEO, ENCODE, and Human Protein Atlas projects led to the following observations: (a) mda-9 expression correlates with both copy number and methylation level of an intronic CpG site (cg1719774) located downstream of the CpG island, (b) cg1719774 methylation is a likely prognostic marker in glioma, (c) among 22 cancer types, melanoma exhibits the highest mda-9 level, and lowest level of methylation at cg1719774, (d) cg1719774 hypomethylation is also associated with histone modifications (at the mda-9 locus) indicative of more active transcription, (e) using Gene Set Enrichment Analysis (GSEA), and the Virtual Gene Overexpression or Repression (VIGOR) analytical scheme, we were able to predict mda-9's association with extracellular matrix organization (e.g., MMPs, collagen, integrins), IGFBP2 and NF-κB signaling pathways, phospholipid metabolism, cytokines (e.g., interleukins), CTLA-4, and components of complement cascade pathways. Indeed, previous publications have shown that many of the aforementioned genes and pathways are associated with mda-9's functionality.

Astrocyte elevated gene-1 interacts with Akt isoform 2 to control glioma growth, survival, and pathogenesis.

The oncogene astrocyte elevated gene-1 (AEG-1; MTDH) is highly expressed in glioblastoma multiforme (GBM) and many other types of cancer, where it activates multiple signaling pathways that drive proliferation, invasion, angiogenesis, chemoresistance, radioresistance, and metastasis. AEG-1 activates the Akt signaling pathway and Akt and c-Myc are positive regulators of AEG-1 transcription, generating a positive feedback loop between AEG-1 and Akt in regulating tumorigenesis. Here, we describe in GBM cells a direct interaction between an internal domain of AEG-1 and the PH domain of Akt2, a major driver in GBM. Expression and interaction of AEG-1 and Akt2 are elevated in GBM and contribute to tumor cell survival, proliferation, and invasion. Clinically, in silico gene expression and immunohistochemical analyses of patient specimens showed that AEG-1 and Akt2 expression correlated with GBM progression and reduced patient survival. AEG-1-Akt2 interaction prolonged stabilization of Akt2 phosphorylation at S474, regulating downstream signaling cascades that enable cell proliferation and survival. Disrupting AEG-1-Akt2 interaction by competitive binding of the Akt2-PH domain led to reduced cell viability and invasion. When combined with AEG-1 silencing, conditional expression of Akt2-PH markedly increased survival in an orthotopic mouse model of human GBM. Our study uncovers a novel molecular mechanism by which AEG-1 augments glioma progression and offers a rationale to block AEG-1-Akt2 signaling function as a novel GBM treatment.

Analysis of global changes in gene expression induced by human polynucleotide phosphorylase (hPNPase(old-35)).

As a strategy to identify gene expression changes affected by human polynucleotide phosphorylase (hPNPase(old-35)), we performed gene expression analysis of HeLa cells in which hPNPase(old-35) was overexpressed. The observed changes were then compared to those of HO-1 melanoma cells in which hPNPase(old-35) was stably knocked down. Through this analysis, 90 transcripts, which positively or negatively correlated with hPNPase(old-35) expression, were identified. The majority of these genes were associated with cell communication, cell cycle, and chromosomal organization gene ontology categories. For a number of these genes, the positive or negative correlations with hPNPase(old-35) expression were consistent with transcriptional data extracted from the TCGA (The Cancer Genome Atlas) expression datasets for colon adenocarcinoma (COAD), skin cutaneous melanoma (SKCM), ovarian serous cyst adenocarcinoma (OV), and prostate adenocarcinoma (PRAD). Further analysis comparing the gene expression changes between Ad.hPNPase(old-35) infected HO-1 melanoma cells and HeLa cells overexpressing hPNPase(old-35) under the control of a doxycycline-inducible promoter, revealed global changes in genes involved in cell cycle and mitosis. Overall, this study provides further evidence that hPNPase(old-35) is associated with global changes in cell cycle-associated genes and identifies potential gene targets for future investigation.

Evolutionary dynamics of Polynucelotide phosphorylases.

Polynucleotide phosphorylase (PNPase) is an evolutionarily conserved 3'→5' phosphate-dependent exoribonucease belonging to the PDX family of proteins. It consists of two catalytic RNase PH domains (PNP1 and PNP2), an α-helical domain and two RNA-binding domains. The PNP1 and PNP2 domains share substantial sequence and structural homology with RNase PH (RPH), which is another PDX family member found in all the three major kingdoms of life, suggesting that these three domains originated from a common ancestor. Phylogenetic analysis (based on the PNPase/RNase PH sequence information for 43 vertebrate taxa) shows that PNP2 and RPH are sister taxa which arose through duplication of the ancestral PNP1 domain. Also, all three domains (PNP1, PNP2 and RPH), along with the KH and S1 domains have undergone significant and directional sequence change, as determined by branch and site-specific dN/dS analyses. In general, codons that show dN/dS ratios that are significantly greater than 1.0 are outside the ordered regions (α-helices and ß-sheets) of these protein domains. In addition, sites that have been selected for mutagenesis in these proteins lie embedded in regions where there is a preponderance of codons with dN/dS values that are not significantly different from 0.0. Overall, this report is an attempt to further our understanding of the evolutionary history of these three protein domains, and define the evolutionary events that led to their refinement in the vertebrate lineage leading to mammals.

MDA-9/syntenin is a key regulator of glioma pathogenesis.

BACKGROUND: The extraordinary invasiveness of human glioblastoma multiforme (GBM) contributes to treatment failure and the grim prognosis of patients diagnosed with this tumor. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in vitro and in vivo in human melanoma models. To determine whether MDA-9/syntenin is a relevant target in GBM, we investigated its expression in tumor samples and involvement in GBM invasion and angiogenesis. MATERIALS: We assessed MDA-9/syntenin levels in available databases, patient tumor samples, and human-derived cell lines. Through gain-of-function and loss-of-function studies, we analyzed changes in invasion, angiogenesis, and signaling in vitro. We used orthotopic xenografts with GBM6 cells to demonstrate the role of MDA-9/syntenin in GBM pathogenesis in vivo. RESULTS: MDA-9/syntenin expression in high-grade astrocytomas is significantly higher than normal tissue counterparts. Forced overexpression of MDA-9/syntenin enhanced Matrigel invasion, while knockdown inhibited invasion, migration, and anchorage-independent growth in soft agar. Moreover, overexpression of MDA-9/syntenin increased activation of c-Src, p38 mitogen-activated protein kinase, and nuclear factor kappa-B, leading to elevated expression of matrix metalloproteinase 2 and secretion of interleukin-8 with corresponding changes observed upon knockdown. GBM6 cells that stably express small hairpin RNA for MDA-9/syntenin formed smaller tumors and had a less invasive phenotype in vivo. CONCLUSIONS: Our findings indicate that MDA-9/syntenin is a novel and important mediator of invasion in GBM and a key regulator of pathogenesis, and we identify it as a potential target for anti-invasive treatment in human astrocytoma.

Identification of genes potentially regulated by human polynucleotide phosphorylase (hPNPase old-35) using melanoma as a model.

Human Polynucleotide Phosphorylase (hPNPase(old-35) or PNPT1) is an evolutionarily conserved 3'→ 5' exoribonuclease implicated in the regulation of numerous physiological processes including maintenance of mitochondrial homeostasis, mtRNA import and aging-associated inflammation. From an RNase perspective, little is known about the RNA or miRNA species it targets for degradation or whose expression it regulates; except for c-myc and miR-221. To further elucidate the functional implications of hPNPase(old-35) in cellular physiology, we knocked-down and overexpressed hPNPase(old-35) in human melanoma cells and performed gene expression analyses to identify differentially expressed transcripts. Ingenuity Pathway Analysis indicated that knockdown of hPNPase(old-35) resulted in significant gene expression changes associated with mitochondrial dysfunction and cholesterol biosynthesis; whereas overexpression of hPNPase(old-35) caused global changes in cell-cycle related functions. Additionally, comparative gene expression analyses between our hPNPase(old-35) knockdown and overexpression datasets allowed us to identify 77 potential "direct" and 61 potential "indirect" targets of hPNPase(old-35) which formed correlated networks enriched for cell-cycle and wound healing functional association, respectively. These results provide a comprehensive database of genes responsive to hPNPase(old-35) expression levels; along with the identification new potential candidate genes offering fresh insight into cellular pathways regulated by PNPT1 and which may be used in the future for possible therapeutic intervention in mitochondrial- or inflammation-associated disease phenotypes.