A heterochromatin inducing protein differentially recognizes self versus foreign genomes.

Krüppel-associated box-domain zinc finger protein (KRAB-ZFP) transcriptional repressors recruit TRIM28/KAP1 to heterochromatinize the mammalian genome while also guarding the host by silencing invading foreign genomes. However, how a KRAB-ZFP recognizes target sequences in the natural context of its own or foreign genomes is unclear. Our studies on B-lymphocytes permanently harboring the cancer-causing Epstein-Barr virus (EBV) have shown that SZF1, a KRAB-ZFP, binds to several lytic/replicative phase genes to silence them, thereby promoting the latent/quiescent phase of the virus. As a result, unless SZF1 and its binding partners are displaced from target regions on the viral genome, EBV remains dormant, i.e. refractory to lytic phase-inducing triggers. As SZF1 also heterochromatinizes the cellular genome, we performed in situ footprint mapping on both viral and host genomes in physically separated B-lymphocytes bearing latent or replicative/active EBV genomes. By analyzing footprints, we learned that SZF1 recognizes the host genome through a repeat sequence-bearing motif near centromeres. Remarkably, SZF1 does not use this motif to recognize the EBV genome. Instead, it uses distinct binding sites that lack obvious similarities to each other or the above motif, to silence the viral genome. Virus mutagenesis studies show that these distinct binding sites are not only key to maintaining the established latent phase but also silencing the lytic phase in newly-infected cells, thus enabling the virus to establish latency and transform cells. Notably, these binding sites on the viral genome, when also present on the human genome, are not used by SZF1 to silence host genes during latency. This differential approach towards target site recognition may reflect a strategy by which the host silences and regulates genomes of persistent invaders without jeopardizing its own homeostasis.

Real-world progression, treatment, and survival outcomes during rapid adoption of immunotherapy for advanced non-small cell lung cancer.

BACKGROUND: Despite the rapid adoption of immunotherapies in advanced non-small cell lung cancer (advNSCLC), knowledge gaps remain about their real-world (rw) performance. METHODS: This retrospective, observational, multicenter analysis used the Flatiron Health deidentified electronic health record-derived database of rw patients with advNSCLC who received treatment with PD-1 and/or PD-L1 (PD-[L]1) inhibitors before July 1, 2017 (N = 5257) and had ≥6 months of follow-up. The authors investigated PD-(L)1 line of treatment and PD-L1 testing rates and the relationship between overall survival (OS) and rw intermediate endpoints: progression-free survival (rwPFS), rw time to progression (rwTTP), rw time to next treatment (rwTTNT), and rw time to discontinuation (rwTTD). RESULTS: First-line PD-(L)1 inhibitor use increased from 0% (in the third quarter of 2014 [Q3 2014]) to 42% (Q2 2017) over the study period. PD-L1 testing also increased (from 3% in Q3 2015 to 70% in Q2 2017). The estimated median OS was 9.3 months (95% CI, 8.9-9.8 months), and the estimated rwPFS was 3.2 months (95% CI, 3.1-3.3 months). Longer OS and rwPFS were associated with ≥50% PD-L1 percentage staining results. Correlations (⍴) between OS and intermediate endpoints were ⍴ = 0.75 (95% CI, 0.73-0.76) for rwPFS and ⍴ = 0.60 (95% CI, 0.57-0.63) for rwTTP, and, for treatment-based intermediate endpoints, correlations were ⍴ = 0.60 (95% CI, 0.56-0.64) for rwTTNT (N = 856) and ⍴ = 0.81 (95% CI, 0.80-0.82) for rwTTD. CONCLUSIONS: The use of first-line PD-(L)1 inhibitors and PD-L1 testing has substantially increased, with better outcomes for patients who have ≥50% PD-L1 percentage staining. Intermediate rw tumor-dynamics estimates were moderately correlated with OS in patients with advNSCLC who received immunotherapy, highlighting the need for optimizing and standardizing rw endpoints to enhance the understanding of patient outcomes outside clinical trials.

Real-World Outcomes of Patients with Metastatic Non-Small Cell Lung Cancer Treated with Programmed Cell Death Protein 1 Inhibitors in the Year Following U.S. Regulatory Approval.

BACKGROUND: Evidence from cancer clinical trials has strong internal validity but can be difficult to generalize to real-world patient populations. Here we analyzed real-world outcomes of patients with metastatic non-small cell lung cancer (mNSCLC) treated with programmed cell death protein 1 (PD-1) inhibitors in the first year following U.S. regulatory approval. MATERIALS AND METHODS: This retrospective study leveraged electronic health record (EHR) data collected during routine patient care in community cancer care clinics. The cohort included patients with mNSCLC who had received nivolumab or pembrolizumab for metastatic disease (n = 1,344) with >1 EHR-documented visit from January 1, 2011, to March 31, 2016. Patients with a > 90-day gap between advanced disease diagnosis and first EHR structured data entry were excluded. RESULTS: Estimated median overall survival (OS) was 8.0 months (95% confidence interval 7.4-9.0 months). Estimated median OS was 4.7 months (3.4-6.6) for patients with anaplastic lymphoma kinase rearrangement- and epidermal growth factor receptor mutation-positive tumors, and 8.6 months (7.7-10.6) for patients without such mutations. Age at PD-1 inhibitor initiation or line of therapy did not impact OS. CONCLUSION: This analysis suggests OS in real-world patients may be shorter than in conventional clinical trial patient cohorts, potentially due to narrow trial eligibility criteria. The lack of difference in OS by line of therapy or age at immunotherapy initiation suggests sustained benefit of PD-1 inhibitors in multitreated patients with mNSCLC and that age is not a predictor of outcome. Further studies are underway in patients with comorbidities, organ dysfunction, and multiple prior therapies. IMPLICATIONS FOR PRACTICE: This study evaluated data derived from electronic health records of patients with metastatic non-small cell lung cancer treated with programmed cell death protein 1 (PD-1) inhibitors in the year following regulatory approval. This real-world cohort had shorter overall survival (OS) indexed to PD-1 inhibitor initiation than reported in clinical trials. Late-line treatment did not influence OS, and patients aged >75 at immunotherapy initiation did not have worse outcomes than younger patients. As new therapies enter clinical practice, real-world data can complement clinical trial evidence providing information on generalizability and helping inform clinical treatment decisions.

KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses.

Cancer-causing herpesviruses infect nearly every human and persist indefinitely in B lymphocytes in a quiescent state known as latency. A hallmark of this quiescence or latency is the presence of extrachromosomal viral genomes with highly restricted expression of viral genes. Silencing of viral genes ensures both immune evasion by the virus and limited pathology to the host, yet how multiple genes on multiple copies of viral genomes are simultaneously silenced is a mystery. In a unifying theme, we report that both cancer-causing human herpesviruses, despite having evolved independently, are silenced through the activities of two members of the Krüppel-associated box (KRAB) domain-zinc finger protein (ZFP) (KRAB-ZFP) epigenetic silencing family, revealing a novel STAT3-KRAB-ZFP axis of virus latency. This dual-edged antiviral strategy restricts the destructive ability of the lytic phase while promoting the cancer-causing latent phase. These findings also unveil roles for KRAB-ZFPs in silencing of multicopy foreign genomes with the promise of evicting herpesviruses to kill viral cancers bearing clonal viral episomes.IMPORTANCE Despite robust immune responses, cancer-causing viruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) persist for life. This persistence is accomplished partly through a stealth mechanism that keeps extrachromosomal viral genomes quiescent. Quiescence, or latency, ensures that not every cell harboring viral genomes is killed directly through lytic activation or indirectly via the immune response, thereby evicting virus from host. For the host, quiescence limits pathology. Thus, both virus and host benefit from quiescence, yet how quiescence is maintained through silencing of a large set of viral genes on multiple viral genomes is not well understood. Our studies reveal that members of a gene-silencing family, the KRAB-ZFPs, promote quiescence of both cancer-causing human viruses through simultaneous silencing of multiple genes on multicopy extrachromosomal viral genomes.

Characteristics of Real-World Metastatic Non-Small Cell Lung Cancer Patients Treated with Nivolumab and Pembrolizumab During the Year Following Approval.

BACKGROUND: Evidence from cancer clinical trials can be difficult to generalize to real-world patient populations, but can be complemented by real-world evidence to optimize personalization of care. Further, real-world usage patterns of programmed cell death protein 1 (PD-1) inhibitors following approval can inform future studies of subpopulations underrepresented in clinical trials. MATERIALS AND METHODS: We performed a multicenter analysis using electronic health record data collected during routine care of patients treated in community cancer care clinics in the Flatiron Health network. Real-world metastatic non-small cell lung cancer (NSCLC) patients who received nivolumab or pembrolizumab in the metastatic setting (n = 1,344) were selected from a starting random sample of 55,969 NSCLC patients with two or more documented visits from January 1, 2011, through March 31, 2016. The primary study outcome measurement was demographic and treatment characteristics of the cohort. RESULTS: Median age at PD-1 inhibitor initiation was 69 years (interquartile range 61-75). Patients were 56% male, 88% smokers, 65% nonsquamous histology, and 64% diagnosed at stage IV. Of 1,344 patients, 112 (8%) were tested for programmed death-ligand 1 expression. Overall, 50% received nivolumab or pembrolizumab in the second line, with a substantial proportion of third and later line use that began to decline in Q4 2015. CONCLUSION: During the year following U.S. regulatory approval of PD-1 inhibitors for treatment of NSCLC, real-world patients receiving nivolumab or pembrolizumab were older at treatment initiation and more had smoking history relative to clinical trial cohorts. Studies of outcomes in underrepresented subgroups are needed to inform real-world treatment decisions. IMPLICATIONS FOR PRACTICE: Evidence gathered in conventional clinical trials used to assess safety and efficacy of new therapies is not necessarily generalizable to real-world patients receiving these drugs following regulatory approval. Real-world evidence derived from electronic health record data can yield complementary evidence to enable optimal clinical decisions. Examined here is a cohort of programmed cell death protein 1 inhibitor-treated metastatic non-small cell lung cancer patients in the first year following regulatory approval of these therapies in this indication. The analysis revealed how the real-world cohort differed from the clinical trial cohorts, which will inform which patients are underrepresented and warrant additional studies.

BRCA1 is a negative modulator of the PRC2 complex.

The Polycomb-repressive complex 2 (PRC2) is important for maintenance of stem cell pluripotency and suppression of cell differentiation by promoting histone H3 lysine 27 trimethylation (H3K27me3) and transcriptional repression of differentiation genes. Here we show that the tumour-suppressor protein BRCA1 interacts with the Polycomb protein EZH2 in mouse embryonic stem (ES) and human breast cancer cells. The BRCA1-binding region in EZH2 overlaps with the noncoding RNA (ncRNA)-binding domain, and BRCA1 expression inhibits the binding of EZH2 to the HOTAIR ncRNA. Decreased expression of BRCA1 causes genome-wide EZH2 re-targeting and elevates H3K27me3 levels at PRC2 target loci in both mouse ES and human breast cancer cells. BRCA1 deficiency blocks ES cell differentiation and enhances breast cancer migration and invasion in an EZH2-dependent manner. These results reveal that BRCA1 is a key negative modulator of PRC2 and that loss of BRCA1 inhibits ES cell differentiation and enhances an aggressive breast cancer phenotype by affecting PRC2 function.

The novel agent phospho-glycerol-ibuprofen-amide (MDC-330) inhibits glioblastoma growth in mice: an effect mediated by cyclin D1.

Given that glioblastoma multiforme (GBM) is associated with poor prognosis, new agents are urgently needed. We developed phospho-glycerol-ibuprofen-amide (PGIA), a novel ibuprofen derivative, and evaluated its safety and efficacy in preclinical models of GBM, and its mechanism of action using human GBM cells and animal tumor models. Furthermore, we explored whether formulating PGIA in polymeric nanoparticles could enhance its levels in the brain. PGIA was 3.7- to 5.1-fold more potent than ibuprofen in suppressing the growth of human GBM cell lines. PGIA 0.75× IC50 inhibited cell proliferation by 91 and 87% in human LN-229 and U87-MG GBM cells, respectively, and induced strong G1/S arrest.In vivo, compared with control, PGIA reduced U118-MG and U87-MG xenograft growth by 77 and 56%, respectively (P< 0.05), and was >2-fold more efficacious than ibuprofen. Normal human astrocytes were resistant to PGIA, indicating selectivity. Mechanistically, PGIA reduced cyclin D1 levels in a time- and concentration-dependent manner in GBM cells and in xenografts. PGIA induced cyclin D1 degradation via the proteasome pathway and induced dephosphorylation of GSK3ß, which was required for cyclin D1 turnover. Furthermore, cyclin D1 overexpression rescued GBM cells from the cell growth inhibition by PGIA. Moreover, the formulation of PGIA in poly-(L)-lactic acid poly(ethylene glycol) polymeric nanoparticles improved its pharmacokinetics in mice, delivering PGIA to the brain. PGIA displays strong efficacy against GBM, crosses the blood-brain barrier when properly formulated, reaching the target tissue, and establishes cyclin D1 as an important molecular target. Thus, PGIA merits further evaluation as a potential therapeutic option for GBM.

Cellular STAT3 functions via PCBP2 to restrain Epstein-Barr Virus lytic activation in B lymphocytes.

UNLABELLED: A major hurdle to killing Epstein-Barr virus (EBV)-infected tumor cells using oncolytic therapy is the presence of a substantial fraction of EBV-infected cells that does not support the lytic phase of EBV despite exposure to lytic cycle-promoting agents. To determine the mechanism(s) underlying this refractory state, we developed a strategy to separate lytic from refractory EBV-positive (EBV(+)) cells. By examining the cellular transcriptome in separated cells, we previously discovered that high levels of host STAT3 (signal transducer and activator of transcription 3) curtail the susceptibility of latently infected cells to lytic cycle activation signals. The goals of the present study were 2-fold: (i) to determine the mechanism of STAT3-mediated resistance to lytic activation and (ii) to exploit our findings to enhance susceptibility to lytic activation. We therefore analyzed our microarray data set, cellular proteomes of separated lytic and refractory cells, and a publically available STAT3 chromatin immunoprecipitation sequencing (ChIP-Seq) data set to identify cellular PCBP2 [poly(C)-binding protein 2], an RNA-binding protein, as a transcriptional target of STAT3 in refractory cells. Using Burkitt lymphoma cells and EBV(+) cell lines from patients with hypomorphic STAT3 mutations, we demonstrate that single cells expressing high levels of PCBP2 are refractory to spontaneous and induced EBV lytic activation, STAT3 functions via cellular PCBP2 to regulate lytic susceptibility, and suppression of PCBP2 levels is sufficient to increase the number of EBV lytic cells. We expect that these findings and the genome-wide resources that they provide will accelerate our understanding of a longstanding mystery in EBV biology and guide efforts to improve oncolytic therapy for EBV-associated cancers. IMPORTANCE: Most humans are infected with Epstein-Barr virus (EBV), a cancer-causing virus. While EBV generally persists silently in B lymphocytes, periodic lytic (re)activation of latent virus is central to its life cycle and to most EBV-related diseases. However, a substantial fraction of EBV-infected B cells and tumor cells in a population is refractory to lytic activation. This resistance to lytic activation directly and profoundly impacts viral persistence and the effectiveness of oncolytic therapy for EBV(+) cancers. To identify the mechanisms that underlie susceptibility to EBV lytic activation, we used host gene and protein expression profiling of separated lytic and refractory cells. We find that STAT3, a transcription factor overactive in many cancers, regulates PCBP2, a protein important in RNA biogenesis, to regulate susceptibility to lytic cycle activation signals. These findings advance our understanding of EBV persistence and provide important leads on devising methods to improve viral oncolytic therapies.

Direct reprogramming by oncogenic Ras and Myc.

Genetically or epigenetically defined reprogramming is a hallmark of cancer cells. However, a causal association between genome reprogramming and cancer has not yet been conclusively established. In particular, little is known about the mechanisms that underlie metastasis of cancer, and even less is known about the identity of metastasizing cancer cells. In this study, we used a model of conditional expression of oncogenic KrasG12D allele in primary mouse cells to show that reprogramming and dedifferentiation is a fundamental early step in malignant transformation and cancer initiation. Our data indicate that stable expression of activated KrasG12D confers on cells a large degree of phenotypic plasticity that predisposes them to neoplastic transformation and acquisition of stem cell characteristics. We have developed a genetically tractable model system to investigate the origins and evolution of metastatic pancreatic cancer cells. We show that metastatic conversion of KrasG12D-expressing cells that exhibit different degrees of differentiation and malignancy can be reconstructed in cell culture, and that the proto-oncogene c-Myc controls the generation of self-renewing metastatic cancer cells. Collectively, our results support a model wherein non-stem cancer cells have the potential to dedifferentiate and acquire stem cell properties as a direct consequence of oncogene-induced plasticity. Moreover, the disturbance in the normally existing dynamic equilibrium between cancer stem cells and non-stem cancer cells allows the formation of cancer stem cells with high metastatic capacity at any time during cancer progression.

Signal transducer and activator of transcription 3 limits Epstein-Barr virus lytic activation in B lymphocytes.

Lytic activation of Epstein-Barr virus (EBV) is central to its life cycle and to most EBV-related diseases. However, not every EBV-infected B cell is susceptible to lytic activation. This lack of uniform susceptibility to lytic activation also directly impacts the success of viral oncolytic therapy for EBV cancers, yet determinants of susceptibility to lytic induction signals are not well understood. To determine if host factors influence susceptibility to EBV lytic activation, we developed a technique to separate lytic from refractory cells and reported that EBV lytic activation occurs preferentially in cells with lower levels of signal transducer and activator of transcription 3 (STAT3). Using this tool to detect single cells, we now extend the correlation between STAT3 and lytic versus refractory states to EBV-infected circulating B cells in patients with primary EBV infection, leading us to investigate whether STAT3 controls susceptibility to EBV lytic activation. In loss-of-function and gain-of-function studies in EBV-positive B lymphoma and lymphoblastoid cells, we found that the levels of functional STAT3 regulate susceptibility to EBV lytic activation. This prompted us to identify a pool of candidate cellular genes that might be regulated by STAT3 to limit EBV lytic activation. From this pool, we confirmed increases in transcript levels in refractory cells of a set of genes known to participate in transcription repression. Taken together, our findings place STAT3 at a critical crossroads between EBV latency and lytic activation, processes fundamental to EBV lymphomagenesis.

Tiled microarray identification of novel viral transcript structures and distinct transcriptional profiles during two modes of productive murine gammaherpesvirus 68 infection.

We applied a custom tiled microarray to examine murine gammaherpesvirus 68 (MHV68) polyadenylated transcript expression in a time course of de novo infection of fibroblast cells and following phorbol ester-mediated reactivation from a latently infected B cell line. During de novo infection, all open reading frames (ORFs) were transcribed and clustered into four major temporal groups that were overlapping yet distinct from clusters based on the phorbol ester-stimulated B cell reactivation time course. High-density transcript analysis at 2-h intervals during de novo infection mapped gene boundaries with a 20-nucleotide resolution, including a previously undefined ORF73 transcript and the MHV68 ORF63 homolog of Kaposi's sarcoma-associated herpesvirus vNLRP1. ORF6 transcript initiation was mapped by tiled array and confirmed by 5' rapid amplification of cDNA ends. The ∼1.3-kb region upstream of ORF6 was responsive to lytic infection and MHV68 RTA, identifying a novel RTA-responsive promoter. Transcription in intergenic regions consistent with the previously defined expressed genomic regions was detected during both types of productive infection. We conclude that the MHV68 transcriptome is dynamic and distinct during de novo fibroblast infection and upon phorbol ester-stimulated B cell reactivation, highlighting the need to evaluate further transcript structure and the context-dependent molecular events that govern viral gene expression during chronic infection.

The Association Between Baseline Hepatic or Renal Function and Clinical Outcomes for Patients With Non-Small Cell Lung Cancer Treated With a PD-1/PD-L1 Blocking Antibody Using Real-World and Trial Data.

Clinical trials have demonstrated the benefit of PD-1/PD-L1 blocking antibodies for the treatment of patients with advanced non-small cell lung cancer (NSCLC) in defined patient populations that often exclude patients with moderate or severe hepatic or renal impairment. We assessed the association between overall survival (OS) and baseline organ function in patients with advanced NSCLC treated with PD-1/PD-L1 blocking antibodies in real-world data (RWD; patient-level data from electronic health records) and pooled clinical trial data submitted to the US Food and Drug Administration (FDA). The Kaplan-Meier estimator was used to estimate OS in different subgroups based on organ function. Unadjusted and adjusted Cox proportional hazards models were used to estimate the association between OS and organ function. In this hypothesis-generating study, baseline renal impairment did not appear to be associated with OS, while patients with baseline liver impairment had shorter OS. RWD provided information on a broader range of renal and hepatic function than was evaluated in clinical trials and hold promise to complement trial data in better understanding populations not represented in clinical trials.

Inhibition of matrix metalloproteinase 14 (MMP-14)-mediated cancer cell migration.

Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination.

Human MLPA Probe Design (H-MAPD): a probe design tool for both electrophoresis-based and bead-coupled human multiplex ligation-dependent probe amplification assays.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) is an efficient and reliable technique for gene dosage analysis. Currently MLPA can be conducted on two platforms: traditional electrophoresis-based, and FlexMAP bead-coupled. Since its introduction in 2002, MLPA has been rapidly adopted in both clinical and research situations. However, MLPA probe design is a time consuming process requiring many steps that address multiple criteria. There exist only one or two commercial software packages for traditional electrophoresis-based MLPA probe design. To our knowledge, no software is yet available that performs bead-coupled MLPA probe design. RESULTS: We have developed H-MAPD, a web-based tool that automates the generation and selection of probes for human genomic MLPA. The software performs physical-chemical property tests using UNAFold software, and uniqueness tests using the UCSC genome browser. H-MAPD supports both traditional electrophoresis-based assays, as well as FlexMAP bead-coupled MLPA. CONCLUSION: H-MAPD greatly reduces the efforts for human genomic MLPA probe design. The software is written in Perl-CGI, hosted on a Linux server, and is freely available to non-commercial users.

KRAS-dependent suppression of MYC enhances the sensitivity of cancer cells to cytotoxic agents.

KRAS is the most commonly mutated oncogene, frequently associated with some of the deadliest forms of cancer. However, the need for potent and specific KRAS inhibitors remains unmet. Here, we evaluated the effects of selected cytotoxic agents on oncogenic KRAS signaling and drug response. The data provided new insights into the functional interaction between the KRAS and MYC pathways and revealed key differences between WT and mutant KRAS expressing cells. Systematic investigation of non-small cell lung cancer cell lines revealed that KRAS mutation can paradoxically increase the sensitivity of cells to cytotoxic agents. We identify MYC as a key regulator of the cellular stress responses and tumor cell viability as MYC expression was suppressed in drug-sensitive but not resistant cells. Furthermore, this suppression was driven by hyperactive KRAS/MAPK signaling. Our findings support a direct link between MYC and cancer cell viability, and raise the possibility that inactivation of MYC may be an effective therapeutic strategy for KRAS mutant tumors across various cancer types.

Identification of the microRNA transcriptome during the early phases of mammalian fracture repair.

Fracture repair is a complex process that involves multiple biological processes requiring spatiotemporal expression of thousands of genes. The molecular regulation of this process is not completely understood. MicroRNAs (miRNAs) regulate gene expression by promoting mRNA degradation or blocking translation. To identify miRNAs expressed during fracture repair, we generated murine bone fractures and isolated miRNA-enriched RNA from intact and post-fracture day (PFD) 1, 3, 5, 7, 11, and 14 femurs. RNA samples were individually hybridized to mouse miRNA microarrays. Results indicated that 959 (51%) miRNAs were absent while 922 (49%) displayed expression in at least one sample. Of the 922 miRNAs, 306 (33.2%) and 374 (40.6%) were up- and down-regulated, respectively, in the calluses in comparison to intact bone. Additionally, 20 (2.2%) miRNAs displayed combined up- and down-regulated expression within the time course and the remaining 222 (24%) miRNAs did not exhibit any changes between calluses and intact bone. Quantitative-PCR validated the expression of several miRNAs. Further, we identified 2048 and 4782 target genes that were unique to the up- and down-regulated miRNAs, respectively. Gene ontology and pathway enrichment analyses indicated relevant biological processes. These data provide the first complete analysis of the miRNA transcriptome during the early phases of fracture repair.

Analysis techniques for microarray time-series data.

We address possible limitations of publicly available data sets of yeast gene expression. We study the predictability of known regulators via time-series analysis, and show that less than 20% of known regulatory pairs exhibit strong correlations in the Cho/Spellman data sets. By analyzing known regulatory relationships, we designed an edge detection function which identified candidate regulations with greater fidelity than standard correlation methods. We develop general methods for integrated analysis of coarse time-series data sets. These include 1) methods for automated period detection in a predominately cycling data set and 2) phase detection between phase-shifted cyclic data sets. We show how to properly correct for the problem of comparing correlation coefficients between pairs of sequences of different lengths and small alphabets. Finally, we note that the correlation coefficient of sequences over alphabets of size two can exhibit very counterintuitive behavior when compared with the Hamming distance.

Small-molecule anticancer compounds selectively target the hemopexin domain of matrix metalloproteinase-9.

Lack of target specificity by existing matrix metalloproteinase (MMP) inhibitors has hindered antimetastatic cancer drug discovery. Inhibitors that bind to noncatalytic sites of MMPs and disrupt protease signaling function have the potential to be more specific and selective. In this work, compounds that target the hemopexin (PEX) domain of MMP-9 were identified using an in silico docking approach and evaluated using biochemical and biological approaches. Two of the selected compounds interfere with MMP-9-mediated cancer cell migration and proliferation in cells expressing exogenous or endogenous MMP-9. Furthermore, these inhibitors do not modulate MMP-9 catalytic activity. The lead compound, N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, specifically binds to the PEX domain of MMP-9, but not other MMPs. This interaction between the compound and the PEX domain results in the abrogation of MMP-9 homodimerization and leads to blockage of a downstream signaling pathway required for MMP-9-mediated cell migration. In a tumor xenograft model, this pyrimidinone retarded MDA-MB-435 tumor growth and inhibited lung metastasis. Thus, we have shown for the first time that a novel small-molecule interacts specifically with the PEX domain of MMP-9 and inhibits tumor growth and metastasis by reducing cell migration and proliferation.

MAPD: a probe design suite for multiplex ligation-dependent probe amplification assays.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) was originally described as an efficient and reliable technique for gene dosage or DNA copy number variation (CNV) analysis. Due to its low cost, reliability, sensitivity, and relative simplicity, MLPA has rapidly gained acceptance in research and diagnostic laboratories, and fills the gap between genome-wide analysis and single gene analysis. A number of new applications have been developed shortly after the introduction of MLPA, including methylation-specific MLPA (MS-MLPA), the use of MLPA in SNP genotyping, copy number analysis in segmentally duplicated regions, etc. However, probe design is time consuming and error prone. Recently software has been developed to help human genomic MLPA probe selection and optimization. For other genomes and MS-MLPA, probe design remains a challenge. FINDINGS: This paper describes a number of new features added to the previous H-MAPD software, which include: 1) probe selection for MS-MLPA; 2) support of mouse and rat genomes; 3) a set of new stuffer sequences. In addition, a physical-chemical property verification tool was implemented to verify user defined probes. CONCLUSIONS: MAPD is a web-based tool which is freely available to non-commercial users. The previous H-MAPD software has been used by about 200 users from more than 30 countries. With the new features, the author hopes MAPD will bring more convenience to the MLPA community.

The lipogenic gene spot 14 is activated in bone by disuse yet remains unaffected by a mechanical signal anabolic to the skeleton.

There is increasing evidence of the interaction of fat and bone metabolism and the role mechanical signals may have in regulating the adaptation of these tissues. The rat hindlimb suspension model of disuse osteoporosis was used to identify genes differentially expressed relative to normal weight-bearing bones and whether the relative expression of these genes is sensitive to anabolic mechanical stimuli. Ten days of hindlimb suspension suppressed percent labeled surface and bone volume/trabecular volume of the proximal tibia by 46% and 69%, respectively, compared to controls. Differential display polymerase chain reaction (DD-PCR) and Northern blot analysis identified and verified, respectively, that expression of Spot 14 (S14), an important gene in lipogenesis, was upregulated fourfold in tibiae of tail-suspended animals compared to long-term controls. Anabolic mechanical stimulation (45 Hz, 10 min/day at 0.25 g) did not show a statistically significant effect on S14 expression. These results indicate a potential role for lipogenic genes during bone loss caused by disuse, further supporting a link between bone and fat tissue, and, considering the insensitivity of these genes to mechanical signals which promote bone formation in the skeleton, the independence of resorptive and formative processes in bone.