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In harsh radiation environments, nanoscale CMOS latches have become more and more vulnerable to triple-node upsets (TNUs). This paper first proposes a latch design that can self-recover from any possible TNU for aerospace applications in the 16-nm CMOS technology. The proposed latch is mainly constructed from seven mutually feeding-back soft-error-interceptive modules (SIMs), any of which consists of two three-input C-elements and one two-input C-element. Due to the mutual feedback mechanism of SIMs and the dual-level soft-error interception of each SIM, the latch can self-recover from any possible TNU. Simulation results demonstrate the TNU self-recoverability from any key TNU for the proposed latch using redundant silicon area. Furthermore, using a high-speed path, the proposed latch saves about 95.45% transmission delay and 86.97% delay-power-area product, compared with the state-of-the-art TNU-tolerant latch that cannot provide complete TNU self-recoverability at all.

International audience; In this paper, a highly reliable SRAM cell, namely SESRS cell, is proposed. Since the cell has a special feedback mechanism among its internal nodes and has more access transistors compared to a standard SRAM cell, the SESRS cell provides the following advantages: (1) it can self-recover from single node upsets (SNUs) and double-node upsets (DNUs); (2) it can reduce power consumption by 49.78% and silicon area by 7.92%, compared with the only existing SRAM cell which can self-recover from all possible DNUs. Simulation results validate the robustness of the proposed SESRS cell. Moreover, compared with the state-of-the-art hardened SRAM cells, the proposed SESRS cell can reduce read access time by 61.93% on average.

International audience; The continuous advancement of CMOS technologies makes SRAMs more and more sensitive to soft errors. This paper presents two novel radiation-hardened SRAM cell designs, namely S4P8N and S8P4N, with enhanced self-recoverability from single-node upsets (SNUs) and Double-node upsets (DNUs). First, the S4P8N cell that has more redundant nodes and more access transistors is proposed. The cell has the following advantages: (1) it can self-recover from all possible SNUs; (2) it can self-recover from a part of DNUs; (3) it has small overhead in terms of power dissipation. Then, to reduce read and write access time, the S8P4N cell that uses a special feedback mechanism among its internal nodes is proposed. The cell has similar soft error tolerability as the S4P8N cell. Simulation results validate the high robustness of the proposed SRAM cells. These results also show that the write access time, read access time, and power dissipation of the S8P4N cell are reduced approximately by 29%, 20%, and 21% on average, at the cost of moderate silicon area, when compared with the state-of-the-art radiation-hardened SRAM cells.

International audience; In nano-scale CMOS technologies, storage cells such as latches are becoming increasingly sensitive to triple-node-upset (TNU) errors caused by harsh radiation effects. In the context of information assurance through redundant design, this article proposes a novel low-cost and TNU on-line self-recoverable latch design which is robust against harsh radiation effects. The latch mainly consists of a series of mutually interlocked 3-input Muller C-elements (CEs) that forms a circular structure. The output of any CE in the latch respectively feeds back to one input of some specified downstream CEs, making the latch completely self-recoverable from any possible TNU, i.e., the latch is completely TNU-resilient. Simulation results demonstrate the complete TNU-resiliency of the proposed latch. In addition, due to the use of fewer transistors and a high-speed path, the proposed latch reduces the delay-power-area product by approximately 91 percent compared with the state-of-the-art TNU hardened latch (TNUHL), which cannot provide a complete TNU-resiliency.

In this paper, a novel self-recoverable SRAM cell, namely SRS14T cell, is proposed in 22nm CMOS technology. Since the cell has a special feedback mechanism among its internal nodes and has more access transistors, the cell provides the following advantages: (1) It can self-recover from single node upsets (SNUs) and partial double-node upsets (DNUs); (2) it can reduce access time and power consumption. Simulation results validate the robustness of the proposed SRS14T cell. Moreover, compared with the state-of-the-art hardened SRAM cells, the proposed SRS14T cell can reduce read access time, write access time and power dissipation by 56.64%, 21.03% and 19.26% on average, respectively, at the cost of moderate silicon area.

De-regulated EFEMP1 gene expression in solid tumors has been widely reported with conflicting roles. We dissected EFEMP1 to identify domains responsible for its cell context-dependent dual functions, with the goal being to construct an EFEMP1-derived tumor-suppressor protein (ETSP) that lacked tumor-promoting function. Exon/intron boundaries of EFEMP1 were used as boundaries of functional modules in constructing EFEMP1 variants, with removal of various module(s), and/or mutating an amino acid residue to convert a weak integrin binding-site into a strong one. A series of in vitro assays on cancerous features, and subcutaneous and intracranial xenograft-formation assays, were carried out for effects from overexpression of wild-type and variant forms of EFEMP1 in two glioma subpopulations characterized as tumor mass-forming cells (TMCs) or stem-like tumor initiating cells (STICs), where EFEMP1 showed cellcontext- dependent dual functions. One of the EFEMP1 variants was identified as the sought-after ETSP, which had a stronger tumor-suppression function in TMCs by targeting EGFR and angiogenesis, and a new tumor-suppression function in STICs by targeting NOTCH signaling and MMP2-mediated cell invasion. Therefore, ETSP may form the basis for further important research to develop a novel cancer therapy to treat many types of cancer by its tumor suppressor effect in the extracellular matrix compartment.

International audience; This paper presents two novel quadruple cross-coupled memory cell designs, namely QCCM10T and QCCM12T, with protection against single event upsets (SEUs) and double-node upsets (DNUs). First, the QCCM10T cell consisting of four cross-coupled input-split inverters is proposed. The cell achieves full SEU tolerance and partial DNU tolerance through a novel feedback mechanism among its internal nodes. It also has a low cost in terms of area and power dissipation mainly due to the use of only a few transistors. Next, based on the QCCM10T cell, the QCCM12T cell is proposed that uses two extra access transistors. The QCCM12T cell has a reduced read-and-write access time with the same soft error tolerance when compared to the QCCM10T cell. Simulation results demonstrate the robustness of the proposed memory cells. Moreover, compared with the state-of-the-art hardened memory cells, the proposed QCCM12T cell saves 28.59% write access time, 55.83% read access time, and 4.46% power dissipation at the cost of 4.04% silicon area on average. INDEX TERMS Double-node upset, memory cell, radiation hardening, single event upset, soft error.

Conflicting functions of EFEMP1 in cancer have been reported. Using two syngeneic glioma cell lines (U251 and U251-NS) carrying two different principal cell subpopulations that express high or low EGFR, and that are able to interconvert via mis-segregation of chromosome 7 (Chr7), we studied EFEMP1's cell-context-dependent functions in regulating subpopulation equilibrium, here defined by the percentage of cells carrying different copies of Chr7. We found that EFEMP1 attenuated levels of EGFR and cellular respiration in high-EGFR-expressing cells, but increased levels of NOTCH1, MMP2, cell invasiveness, and both oxidative phosphorylation and glycolytic respiration in low-EGFR-expressing cells. Consistently, EFEMP1 suppressed intracranial xenograft formation in U251 and promoted its formation in U251-NS. Interestingly, subpopulation equilibria in xenografts of U251-NS without EFEMP1 overexpression were responsive to inoculum size (1, 10 and 100 thousand cells), which may change the tumor-onset environment. It was not observed in xenografts of U251-NS with EFEMP1 overexpression. The anti-EGFR function of EFEMP1 suppressed acceleration of growth of U251-NS, but not the subpopulation equilibrium, when serially passed under a different (serum-containing adherent) culture condition. Overall, the data suggest that the orthotopic environment of the brain tumor supports EFEMP1 in carrying out both its anti-EGFR and pro-invasive/cancer stem cell-transforming functions in the two glioma cell subpopulations during formation of a single tumor, where EFEMP1 stabilizes the subpopulation equilibrium in response to alterations of the growth environment. This finding implies that EFEMP1 may restrain cancer plasticity in coping with ever-changing tumor microenvironments and/or therapeutic-intervention stresses.

Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer and there exists a need to non-invasively identify functional tumor subpopulations responsible for tumor recurrence. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. Using FLIM, we identified a significant shift towards longer NADH fluorescence lifetimes, suggesting an increase in the fraction of protein-bound NADH, in the invasive stem-like tumor-initiating cell (STIC) subpopulation relative to the tumor mass-forming cell (TMC) subpopulation of malignant gliomas. By applying our previously studied model to transition glioma from a majority of STIC to a majority of TMC in serum-adherent culture conditions following serial passages, we compared changes in NADH states, cellular respirations (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth speed over passages. We identified a significant positive correlation between free-NADH fraction and cell growth, which was related to an increase of TMC fraction. In comparison, the increase of EGFR and cellular respirations preceded all these changes. In conclusion, FLIM of NADH provides a non-invasive method to monitor the dynamics of tumor heterogeneity before and after treatment.

EGFR amplification in cells having double minute chromosomes (DM) is commonly found in glioblastoma multiforme (GBM); however, how much it contributes to the current failure to treat GBM successfully is unknown. We studied two syngeneic primary cultures derived from a GBM with and without cells carrying DM, for their differential molecular and metabolic profiles, in vivo growth patterns, and responses to irradiation (IR). Each cell line has a distinct molecular profile consistent with an invasive "go" (with DM) or angiogenic "grow" phenotype (without DM) demonstrated in vitro and in intracranial xenograft models. Cells with DM were relatively radio-resistant and used higher glycolytic respiration and lower oxidative phosphorylation in comparison to cells without them. The DM-containing cell was able to restore tumor heterogeneity by mis-segregation of the DM-chromosomes, giving rise to cell subpopulations without them. As a response to IR, DM-containing cells switched their respiration from glycolic metabolism to oxidative phosphorylation and shifted molecular profiles towards that of cells without DM. Irradiated cells with DM showed the capacity to alter their extracellular microenvironment to not only promote invasiveness of the surrounding cells, regardless of DM status, but also to create a pro-angiogenic tumor microenvironment. IR of cells without DM was found primarily to increase extracellular MMP2 activity. Overall, our data suggest that the DM-containing cells of GBM are responsible for tumor recurrence due to their high invasiveness and radio-resistance and the mis-segregation of their DM chromosomes, to give rise to fast-growing cells lacking DM chromosomes.

The phenotypic and genetic diversity that define tumor subpopulations within high-grade glioma can lead to therapeutic resistance and tumor recurrence. Given that cranial irradiation is a frontline treatment for malignant glioma, understanding how irradiation selectively effects different cellular subpopulations within these heterogeneous cancers should help identify interventions targeted to better combat this deadly disease. To analyze the radiation response of distinct glioma subpopulations, 2 glioma cells lines (U251, A172) were cultured under conditions that promoted either adherence or non-adherent spheroids. Past work has demonstrated that subpopulations derived from defined culture conditions exhibit differences in karyotype, proliferation, gene expression and tumorigenicity. Spheroid cultures from each of the glioma cell lines were found to be more radiosensitive, which was consistent with higher levels of oxidative stress and lower levels of both oxidative phosphorylation and glycolytic metabolism 1 week following irradiation. In contrast, radioresistant non-spheroid parental cultures showed increased glycolytic activity in response to irradiation, while oxidative phosphorylation was affected to a lesser extent. Overall these data suggest that prolonged radiation-induced oxidative stress can compromise the metabolic state of certain glioma subpopulations thereby altering their sensitivity to an important therapeutic intervention used routinely for the control of glioma.

For the population genetics analysis of the naturally grown brown seaweed Laminaria japonica (Laminariales, Phaeophyta) sampled from Dalian, Yantai, Weihai, Rongcheng and Qingdao in China, ten primers were employed to produce 88 bands as revealed by randomly amplified polymorphic DNA (RAPD) markers, and all these bands were polymorphic. According to these band patterns, there were 94 distinct phenotypes occurred in 100 samples indicating the high heterozygosity of this kelp. Dalian population samples showed the highest percentage of polymorphism (71.67%), and also the higher diversity estimated on the basis of the Shannon’s index (8.498), suggesting that this population could be chosen as the best resource for genetic breeding. The highest diversity of Yantai population possibly resulted from the introduction of L. longissima used for interspecific cross breeding with L. japonica cultivated in China. From Dalian southwards to Qingdao, the genetic variation of the five populations became less with a decrease in latitude, possibly due to the natural selection especially of high temperature. The genetic distance (ΦST values) of the five populations was a little significantly correlated with the geographical distance (r=0.496) at P=0.05 by Mantel’s test. Weihai, Rongcheng and Yantai populations were closely grouped genetically together by Neighbor-joining cluster analysis probably in that the dispersal of the kelp by propagules more easily occurring in the range of relatively short distance. The analysis of molecular variance (AMOVA) also demonstrated that the relatively higher variation occurred among populations (71.49%) at an extremely significant level (P

Edge to interior gradients in forest ecosystems can influence the species composition and community structure as a result of variations in micro-environment. In this study, the edge effects on stand productivity and plant species diversity were investigated in two adjacent types of forests in central-southern China: Chinese fir adjacent with pine forests and Chinese fir adjacent with broad-leaved forests. A total of 48 sample plots for overstory and 240 sample quadrates for understory in different stand ages were set up. The tree growth parameters were measured in the overstory while the plant species diversity was measured in both overstory and understory of the examined forests. The results showed that DBH (tree diameter at the breast height), tree height and stand volume in overstory increased from the central areas (CA) of forests to the edge areas (EA), and the plant species richness and diversity in understory were greater in EA than in CA. The edge effect was greater in Chinese fir paired with broad-leaved forests than in Chinese fir paired with pine forests. The edge effect decreased in an order as young aged stands > middle aged stands > mature aged stands in the studied forests. The changes in tree growth and species diversity in the edge area compared to the interior were mainly attributed to the alteration of micro-environmental factors such as light density, temperature and moisture. Our results suggest increasing forest productivity and plant species diversity could be achieved by making more edges in the forests.

De-regulated EFEMP1 gene expression in solid tumors has been widely reported with conflicting roles. We dissected EFEMP1 to identify domains responsible for its cell context-dependent dual functions, with the goal being to construct an EFEMP1-derived tumor-suppressor protein (ETSP) that lacked tumor-promoting function. Exon/intron boundaries of EFEMP1 were used as boundaries of functional modules in constructing EFEMP1 variants, with removal of various module(s), and/or mutating an amino acid residue to convert a weak integrin binding-site into a strong one. A series of in vitro assays on cancerous features, and subcutaneous and intracranial xenograft-formation assays, were carried out for effects from overexpression of wild-type and variant forms of EFEMP1 in two glioma subpopulations characterized as tumor mass-forming cells (TMCs) or stem-like tumor initiating cells (STICs), where EFEMP1 showed cellcontext- dependent dual functions. One of the EFEMP1 variants was identified as the sought-after ETSP, which had a stronger tumor-suppression function in TMCs by targeting EGFR and angiogenesis, and a new tumor-suppression function in STICs by targeting NOTCH signaling and MMP2-mediated cell invasion. Therefore, ETSP may form the basis for further important research to develop a novel cancer therapy to treat many types of cancer by its tumor suppressor effect in the extracellular matrix compartment.

Glioblastoma multiforme (GBM) is an incurable malignant glioma which is very resistant to radiation and alkylating agent-based chemotherapy. Necrosis is a hallmark for GBM and the layer surrounding necrotic area is packed with cells which are now believed to be those migrating out of the necrotic area. Oxidative stress is a condition that GBM cells encounter in the necrotic zone, which is one of the stressful conditions that GBM cells need to resist in order to survive. Our previous studies revealed that low PAX6 expression is a favorable molecular trait for survival acquired by GBM cells because PAX6 could suppress cell invasion and tumorigenicity. Since detachment of cells from GBM is an early event for cell migration and subsequent invasion, we examined whether PAX6 is involved in cell survival after detachment. PAX6 over-expression was achieved in glioma cells transiently (by adenoviral-mediated transient over-expression) or stably (by the establishment of stable cell lines after transfection). The effect of PAX6 over-expression on the survival and growth of glioma cells after detachment from the culture was determined. Our data revealed that GBM cells (with their low PAX6 levels) survived the detachment procedure well. However, PAX6 over-expression attenuated GBM cell recovery of growth after detachment-induced stress. Importantly, intracellular reactive oxygen species (ROS) levels increased following cell detachment and that PAX6 over-expressing cells retained higher level of ROS than control cells. This may be partially responsible for the impaird growth rate after cell detachment. Addition of anti-oxidant improved the cell viability of PAX6 over-expressing cells, but did not restore their ability to proliferate. To survive, GBM cells must resist oxidative stress in the necrotic zone as well as the intracellular ROS generated during detachment. Since PAX6 over-expression in low PAX6-expressing glioma cells attenuated cell survival and growth after detachment, these results suggest that a reduced PAX6 expression may be a molecular trait that gives glioma cells a real selection advantage over other cell types to survive in stressful conditions, thus resulting in expansion of their population.

Glioblastoma multiforme (GBM) is the most invasive brain tumor. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of GBM cells. By an in vitro Matrigel invasion assay on two GBM cell lines stably transfected with wild-type and/or two mutant forms of PAX6, this study displays the first evidence that PAX6 inhibits the invasiveness of GBM cells and that the DNA-binding domain of PAX6 is required for this function. Using real-time quantitative reverse transcription-PCR (RT-PCR), gelatin zymography, and immunohistochemistry assays, the expression of the gene encoding matrix metalloproteinase-2 (MMP2) in GBM cell lines grown in vitro or in intracranial xenografts in nude mice was shown to be repressed by either stable or adenoviral-mediated overexpression of PAX6. Luciferase promoter assays revealed PAX6-mediated suppression of MMP2 promoter activity. Electrophoretic mobility shift assays showed direct binding of PAX6 to the MMP2 promoter. A significant reverse correlation (P < 0.05) occurred between PAX6 and MMP2 expression quantified by real-time quantitative RT-PCR in 41 GBMs, 43 anaplastic astrocytomas, and 7 adjacent normal tissues. Interestingly, the degree and significance of the reverse correlation increased after excluding astrocytomas, whereas it became insignificant after excluding GBMs. In GBM cells stably transfected with a dominant negative mutant PAX6 showing increased MMP2 expression and invasiveness, knock-down of MMP2 revealed that MMP2 is one of the PAX6 target genes mediating its suppression of invasion. Overall data delineated a mechanism for the suppressive function of PAX6 in GBM: suppression of cell invasion by repressing the expression of proinvasive genes such as MMP2. (Cancer Res 2006; 66(20): 9809-17)

Human hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, in which the genetic mechanisms of oncogenesis are still unclear. To investigate whether the genomic DNA copy number alterations may contribute to primary HCC, the cDNA microarray-based comparative genomic hybridization (CGH) analysis was here performed in 41 primary HCC infected by hepatitis B virus and 12 HCC cell lines. The resulting data showed that, on average, 7.25% of genome-wide DNA copy numbers was significantly altered in those samples (4.61±2.49% gained and 2.64±1.78% lost). Gains involving 1q, 6p, 8q and 9p were frequently observed in these cases; and whilst, losses involving Ip, 16q and 19p occurred in most patients. To address the correlation between the alteration of genomic DNA copy numbers and transcriptional expression, the same cDNA microarray was further applied in 20 HCC specimens and all available cell lines to figure out the gene expression profiles of those samples. Interestingly, the genomic DNA copy number alterations of most genes appeared not to be in generally parallel with the corresponding transcriptional expression. However, the transcriptional deregulation of a few genes, such as osteopontin (SPP1), transgelin 2 (TAGLN2) and PEG10, could be ascribed partially to their genomic aberrations, although the many alternative mechanisms could be involved in the deregulation of these genes. In general, this work would provide new insights into the genetic mechanisms in hepatocarcinogenesis associated with hepatitis B virus through the comprehensive survey on correlation between genomic DNA copy number alterations and transcriptional expression.

EFEMP1 exerts both tumor suppressive and oncogenic effects in glioma. EFEMP1's binding to EGFR accounts for its suppression of the intracranial (i.c.) tumorigenicity of glioma cells expressing high levels of EGFR. This finding is consistent with an overall favorable prognosis value of EFEMP1 expression in gliomas, specifically high EGFR-expression ones. We also found that in gliomas without EGFR overexpression, heightened levels of EFEMP1 expression were associated with unfavorable patient outcomes in prognosis models. To delineate the cell context-dependent function of EFEMP1 in glioma, we compared the effect of EFEMP1 to EGFR and NOCTH signaling activities in different glioma cell lines/primary cultures and their derived subpopulation lines. The results showed a common effect of EFEMP1 on blocking EGFR signaling, but variations on NOTCH signaling. In U87 glioma cell line, EFEMP1 blocked EGFR while activated NOTCH, with overall suppression of i.c. tumorigenicity. In U251 glioma cell line, EFEMP1 blocked EGFR but no effect on NOTCH signaling, and overall suppression of i.c. tumorigenicity. In U251-derived subculture (U251-NS) which was enriched with stem-like tumor initiating cells where EGFR expression level was low, EFEMP1 up-regulated NOTCH1, HES1, and HEY1 gene expressions. The effect of EFEMP1 to U251-NS i.c. tumorigenicity was contradictory. It changes the effect of tumor promoting to tumor suppressing by increasing inoculum size. With increase of inoculum size in i.c. xenograft model or exposing cells to serum in in vitro culture, there was an increase of chromosome 7 copy score (total chromosome 7 number per cell in xenografts/cell culture) and increase of EGFR expression. EFEMP1 changed pro-invasion function to anti-growth, hence reduced the alteration on equilibrium of tumor heterogeneity in responding to changes of tumor microenvironment. In conclusion, the effect of EFEMP1 up or down-regulation in malignant glioma needs to be studied in respect to the status of EGFR and NOTCH signaling activities.

A new extraction protocol has been developed to obtain high quality DNAfrom Laminaria japonica, which involves enzymatic dissociation ofsporophyte tissues and subsequent elimination of the remainingpolysaccharides with cetyltrimethyl ammonium bromide. Unicells isolatedfrom frozen kelp tissues with alginate lyase prepared from the abalone Haliotis diversicolor were used to extract total DNA; the yield wasapproximately 13 to 22.5 μg DNA g-1 (wet sporophyteweight). The average size of genomic DNA was around 23 kb estimated byagarose gel electrophoresis, and the purity of total DNA determinedspectrophotometrically as the ratio of OD260/OD280 wasabout 1.7. The extracted kelp DNA (20–40 ng) could be usedsuccessfully as a template for polymerase chain reaction (PCR) under theoptimized conditions (100 μM dNTP, 0.2 μM primer, 1.0unit Taq DNA polymerase). The random amplified polymorphicDNA (RAPD) patterns were highly reproducible. These results suggest thatthe contamination by soluble polysaccharides which interferes with RAPDreproducibility was largely controlled. This RAPD-suited method for DNAextraction from kelp sporophytes using enzyme treatment providedsufficient material, and was inexpensive and convenient to carry out.

In order to establish a novel artificial muscle model, an ionized fibrous hydrogel assembly (FHM) was prepared by means of surface-grafting acrylonitrile onto cellulose fiber and consequent hydrolysis. The structure and character of FHM was investigated. The FHM shows excellent chemomechanical character. This feature is closely interrelated to its molecular, morphological structure and texture.

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation (CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers. Published version

In case-control profiling studies, increasing the sample size does not always improve statistical power because the variance may also be increased if samples are highly heterogeneous. For instance, tumor samples used for gene expression assay are often heterogeneous in terms of tissue composition or mechanism of progression, or both; however, such variation is rarely taken into account in expression profiles analysis. We use a prostate cancer prognosis study as an example to demonstrate that solely recruiting more patient samples may not increase power for biomarker detection at all. In response to the heterogeneity due to mixed tissue, we developed a sample selection strategy termed Stepwise Enrichment by which samples are systematically culled based on tumor content and analyzed with t-test to determine an optimal threshold for tissue percentage. The selected tissue-percentage threshold identified the most significant data by balancing the sample size and the sample homogeneity; therefore, the power is substantially increased for identifying the prognostic biomarkers in prostate tumor epithelium cells as well as in prostate stroma cells. This strategy can be generally applied to profiling studies where the level of sample heterogeneity can be measured or estimated.

Background. HER2 positive (HER2+) breast cancer (BCa) occurs in 25-30% of cases and is associated with an aggressive phenotype. Multiple HER2-stimulated pathways are known but the genes that are specifically controlled by HER2 are poorly understood. We previously used RNA polymerase II (POL II) immunoprecipitation (ChIP) to identify 737 genes that bind POL II in HER2+ but not in HER2- BCa cell lines. 51 of these genes were differentially expressed in a HER2+ dependent manner and 113 genes were differentially expressed in a HER2-dependent manner in breast tumors from 812 patients. The 113 genes are not differentially expressed in cell lines but expressed in BCa cases can be considered “poised” for expression, and activated when in the context of a breast tumor. Methods. The 737 genes were examined using pathway finding tools and the MIT database for Super-enhancers and the dbSUPER database (2). The expression of certain genes in attached cell cultures and in mammosphere cultures were examined by qPCR. Results. Some of the 113 genes that are “poised” for expression in cell lines in a HER2-dependent manner, and then expressed in breast cancers in a HER2-dependent manner, encode proteins that commonly occur in Super-enhancers, which are a variety of DNA regulatory structures formed by looping that associate multiple bound transcription and DNA-modifying factors in proximity to target promoters. Examples include Mediator12 (MED12) and the Bromdomain protein 2 (BRD2), and DNA binding factors such as HDAC and CREB1-cofactors. Moreover, many pluripotency genes are also found in the list of 113 genes; NANOG, OCT3/4, and SOX2 (NOS genes). These genes commonly interact with SEs as associated factors and as targets. We confirmed by qPCR that the three NOS genes exhibit increased expression in mammosphere of HER2+ MCF7 cells but not in control cells. Using the MIT and dbSUPER (2) database we determined that 70 genes of the 113 genes (62%) that are located in or near SEs, including some present in the HER2+ BCa cell line HCC1954. Similarly, 33 (65%) of the 51 differentially expressed genes in HER2+ cell lines were associated with SEs in many diverse cell types. For comparison, the class of 573 genes that are bound to POL II in HER2+ cell lines, but are not differentially expressed in cell lines or breast cancer tissues, have a much lower fraction of genes, 213 or 37%, that encode potential members or targets of SE. The difference is highly significant, p = 0.008. Pathway analyses of the 113 genes indicates that major pathways enriched in these genes (p < 0.001) include EGFR and Map Kinase signaling, notch pathway signaling, hedgehog, Wnt, Integrin-mediated cell and cytokine signaling Interleukin 1 and Interleukin 14. Conclusion. These results suggest that the pathways associated with HER2 over-expression in BCa include genes associated with Super-enhancer assembly, as well as many genes encoded close to the location of Super-enhancers. 1.Rahmatpanah et al,. (2015) Oncotarget 2. Aziz Khan, Nucleic Acids Res., 2015 Citation Format: Farah Rahmatpanah, Zhenyu Jia, Yuanjie Hu, Frank Jones, Michael McClelland, Dan Mercola. HER2 promotes super enhancer formation in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1973.

Gliomas are invasive cancers that resist all forms of attempted therapy. Immunotherapy using Ag-pulsed dendritic cells has improved survival in some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes toward gliomas. Atomic force microscopy of four different glioma types—human U251 and rat T9 and F98 glioma cells, including freshly isolated human glioblastoma multiforme neurosphere cultures (containing “stem cell-like cells”)—revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional CTLs, and chimeric Ag-receptor–redirected T cells) better than their nonmicrovilli-expressing counterparts. Killer lymphocytes released perforin, which was detected within the glioma’s microvilli/filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Air-dried gliomas revealed nodes within the microvilli/filopodia. The microvilli that penetrated 0.4-μm transwell chamber’s pores resisted the actions of CTLs and physical damage. Those nodelike structures may represent a compartmentalization that resists physical damage. These microvilli may play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.

We reported that PAX6 suppresses glioblastoma cell growth in vivo and anchorage-independent growth without significant alteration of cell proliferation in vitro, suggesting that PAX6 may alter the tumor microenvironment. Because we found that PAX6 downregulates expression of the gene encoding vascular endothelial growth factor A (VEGFA) in glioma cells, we used a subcutaneous xenograft model to verify PAX6 suppression of VEGFA-induced angiogenesis based on CD31-immunostaining of endothelial cells. The results showed a significant reduction of VEGFA at the transcription level in PAX6-transfected cells in xenografts and PAX6 has a suppressive effect on the microvascular amplification typically seen in glioblastoma. We showed that PAX6 suppression of VEGFA expression requires its DNA binding-domain. The C-terminal truncation mutant of PAX6, however, did not show the dominant negative function in regulating VEGFA expression that it showed previously in regulating MMP2 expression. In the glioma cell line U251HF, we further determined that blocking the PI3K/Akt signaling pathway with either adenoviral-mediated PTEN expression or LY294002 enhanced PAX6-mediated suppression of VEGFA in an additive manner; thus, PAX6-mediated suppression of VEGFA is not via the canonical pathway through HIF1A. These two VEGFA-regulatory pathways can also be similarly modulated in another malignant glioma cell line, U87, but not in LN229 where the basal VEGFA level is low and PTEN is wild-type. PAX6 suppression of VEGFA appears to be blocked in LN229. In conclusion, our data showed that PAX6 can initiate in glioma cells a new signaling pathway independent of PI3K/Akt-HIF1A signaling to suppress VEGFA expression.

The identification of cancer genes differentially expressed in hepatocellular carcinoma (HCC) plays an important role in understanding the molecular mechanisms of hepatocarcinogenesis. Here, ARHI gene expression was analyzed by real-time RT-PCR and it was significantly downregulated in 33 of the 42 (78.6%, more than two folds) HCC specimens compared with adjacent noncancerous livers (P < 0.01). In addition, ARHI expression was reduced in some HCC samples at protein level confirmed by immunohistochemistry. Furthermore, our data suggested that the overexpression of ARHI can significantly inhibit cell growth and colony formation of Hep3B cells (P < 0.01), whilst silencing endogenous ARHI gene by RNAi could promote cell growth of Huh-7 and Focus. LOH of microsatellite markers D1S2806 and D1S2803 was only found in 2.4% (1 of 42 HCCs) of HCC cases. The expression of ARHI was obviously re-expressed in some HCC cells, Bel-7402, Bel-7405, QGY-7703 and Hep3B, by a demethylation agent, 5-aza-2'-deoxycytidine (DAC). DNA hypermethylation within ARHI promoter was identified in 47.1% of HCC specimens without ARHI expression. Our current observations provide evidences that ARHI downregulated in HCCs could play a role in liver cancer via acting as a tumor suppressor gene, which mainly was triggered by the epigenetic events in HCC specimens.

5-Hydroxypyrrolidin-2-one, along with (2R)-pterosin B, shikimic acid, kaempferol-3-O-beta-D-glucopyranoside, transtiliroside, beta-sitosterol, daucosterol, glycerol 1-stearate and benzoic acid, were isolated from the young fronds of the bracken fern Pteridium aquilinum. 5-Hydroxypyrrolidin-2-one, shikimic acid and glycerol 1-stearate were isolated from the plant for the first time.

Background: The incidence of prostate cancer (PCa) is approximately 60% higher and the mortality rate 2 to 3 times greater among African American men (AA) in the U.S. compared to men with a European background, European Americans (EA). Men of West African ancestry from the Caribbean and South America share a similar increase in incidence and mortality to AA suggesting a genetic contribution to these outcomes. We have observed that expression of hundreds of genes in the tumor microenvironment is strikingly different for AA and EA patients1. The majority of significant differences in the stroma of AA patients were down-regulated compared to EA patients. These expression alterations could be related to somatically acquired DNA changes in the stroma but non-tumor tissue in cancer usually exhibits far fewer genetic changes than tumors. Thus, it may be that inherited or distinct environmental changes are responsible for the differences seen between AA and EA stroma. Whatever the basis of the large number of differences observed, the distinct patterns for AA and EA argue strongly, and for the first time, that there are many molecular distinctions between PCa in these races that are resident in the tumor-adjacent microenvironment. We hypothesize that the stroma of AA PCa exhibits a large-scale lower expression of protein mediators of cellular antitumor immunity and reduction in factors involved in cell-cell and cell-extracellular matrix (ECM) contacts. Such differences may, thereby, favor aggressive cancer including early metastases in AA. Methods: In order to test the basis of the relative decreased expression in AA stroma, ChIP methods for isolating methylated and unmethylated DNA combined with NGS have been used to define methylated sites in two PCa EA cells lines as well as FFPE samples of stroma from EA and AA PCa. To validate the microarray data, we are using Tumor Microarrays from both CA (n=443) and AA (n=105) patients to confirm in immunohistochemistry (IHC) the differential protein expression of many immune and ECM mediators identified in our initial studies. Results: We validated the ChIP-NGS methods by quantitatively confirming significant methylation within the promoter regions of LnCAP and DU135 cells compared to immortalized normal p69 cells for 35 reported methylated genes including GSTPi. Of the 965 largest down-regulated transcripts observed in AA stroma (1), 237 were observed to exhibit increased promoter methylation in AA stroma tissue compared to EA stroma. These 237 genes identified four main pathways (WNT, TGFβ, integrin mediated cell matrix adhesion, and EMT, all with p Conclusions: These preliminary studies support the hypothesis that the stroma of AA PCa exhibits a large-scale lower expression of protein mediators of cellular antitumor immunity and reduction in factors involved in cell-cell and cell-ECM contacts. The results suggest a role for the microenvironment in the aggressive features of PCa of AA. Acknowledgements: This study was funded by the NCI Comprehensive Partnerships to Reduce Cancer Health Disparities (CPRCHD) grants (U54CA132384 and U54CA132379) and by 1U01CA162147 (KLM), NCI 3UL1TR000004-07S2 (DAM) and NCI UO1CA152738A, P30CA62203 and the Congressionally Directed Medical Research Programs W81XWH-12-PCRP-IDA (DAM, MMCC). 1. Kinseth M, A., Z. Jia, et al., 2014. Int. J. Cancer 134: 81-91. DOI: 10.1002/ijc.28326. Citation Format: Farah Rahmatpanah, Tracy Luu, Pardis Zaeri, Xin Chen, Yuanjie Hu, Joe Adams, Harmony Saunders, Sam Takahashi, Michael McClelland, Kathleen L. McGuire, Dan Mercola. The role of tumor microenvironment in prostate cancer of African Americans. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A78.

We recently developed a combination method using RNA polymerase II immunoprecipitation together with expression analysis in order to identify genes regulated by the HER2 oncogene: the HER2 Regulon. We identified 737 genes which are poised for transcription. 113 of these genes are differentially expressed (p < 0.05) in HER2+/- as determined by analysis of available expression arrays of 812 breast cancer tissues. Thus for these genes, expression may depend upon features of the in situ organization and/or features of the microenvironment in addition to the action of high HER2. We further noted that 58/113 genes are members of pathways related by four “nodes”: NANOG, SOX2, OCT3/4, and CREB1.We hypothesize that high HER2 expression and factors of breast cancer cells in situ, possibly including microenvironment factors, determine the concerted expression of the four node proteins and the 58 associated genes. These genes may participate in the formation of CSC cells or cells with enhanced “stemness” in high HER2-expressing breast cancer. Methods The HER2+/- breast cancer cell lines were grown in medium supplied with FBS, the sub-cultures were depleted from FBS and cultured on ultra-low adherent plate supplemented with EGF/FGF/B27. The formed spheres were collected and used for expression analysis of ALDH1, SOX2, NANOG and OCT3/4 and 58 microenvironment dependent genes employing quantitative real time PCR. Immunofluorescence staining was used to measure protein expression levels of ALDH1, NANOG, SOX2 and OCT3/4. Results We used PCR to compare the amount of transcripts formed in high- and low-HER2 expression cells both as attached cultures and as “mammospheres”. We observed all four node transcript levels were strikingly higher in mammospheres of HER2-expressing cells compared to attached cultures or compared to HER2-null control cells. We used immunofluorescence to confirm that the corresponding protein levels were also increased. Thus, in the context of mammospheres, node genes exhibit HER2-dependent and greatly increased expression analogous to the conditions required for the 113 genes. This observation suggests that the anchorage-independent growth mode of mammospheres growing in factor-supplemented medium may replicate key features of cancer cells in situ not observed in attached cultures. If so, mammospheres may provide an in vitro model for the dissection of the key in situ and microenvironment features influencing HER2-dependent expression in breast cancer. Conclusion A “poised” class of genes in HER2+ cell lines has POLII binding and low RNA expression and is differentially expressed in primary tumors in a HER2-dependent manner. These genes include key genes with a role in stem cell proliferation. These genes become expressed when cell lines are grown as three dimensional mammospheres. These observations strongly suggest a role of 3D structure. Note: This abstract was not presented at the meeting. Citation Format: Farah Rahmatpanah, Yuanjie Hu, Xin Chen, Michael McClelland, Frank Jones, Dan Mercola. The HER2 Regulon:Identification of 113 genes that are directly controlled by HER2 and define four nodes of cancer stem cell networks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1982. doi:10.1158/1538-7445.AM2015-1982

Granzyme B expression is essential for eliciting NK cell cytotoxicity and T cell function. However, its transcriptional regulatory mechanism is not well understood. In this report, we demonstrate in human NK cells and T cells that the NF-κB-signaling pathway is involved in such control. Furthermore, a novel downstream human granzyme B gene sequence (GGAGATTCCC) was identified for NF-κB binding. EMSA, luciferase, and chromatin immunoprecipitation assays in vitro and in vivo indicated that this NF-κB binding site is functional in an NK cell line and its primary counterpart. Our data also demonstrate that this binding site is functional in Jurkat T cells. Taken together, we identified a novel NF-κB binding site, which plays a pivotal role in controlling human granzyme B gene transcription.

Purpose There are conflicting reports regarding the function of EFEMP1 in different cancer types. In this study, we sought to evaluate the role of EFEMP1 in malignant glioma biology. Experimental Design Real-time qRT-PCR was used to quantify EFEMP1 expression in 95 glioblastoma multiforme (GBM). Human high-grade glioma cell lines and primary cultures were engineered to express ectopic EFEMP1, a small hairpin RNA of EFEMP1, or treated with exogenous recombinant EFEMP1 protein. Following treatment, growth was assayed both in vitro and in vivo (subcutaneous (s.c.) and intracranial (i.c.) xenograft model systems). Results Cox regression revealed that EFEMP1 is a favorable prognostic marker for patients with GBM. Over-expression of EFEMP1 eliminated tumor development and suppressed angiogenesis, cell proliferation, and VEGFA expression, while the converse was true with knock-down of endogenous EFEMP1 expression. The EFEMP1 suppression of tumor onset time was nearly restored by ectopic VEGFA expression; however, overall tumor growth rate remained suppressed. This suggested that inhibition of angiogenesis was only partly responsible for EFEMP1's impact on glioma development. In glioma cells that were treated by exogenous EFEMP1 protein or over-expressed endogenous EFEMP1, the EGFR level was reduced and AKT signaling activity attenuated. Mixing of EFEMP1 protein with cells prior to s.c. and i.c. implantations or injection of the protein around the established s.c. xenografts, both significantly suppressed tumorigenicity. Conclusions Overall, our data reveals that EEFEMP1 suppresses glioma growth in vivo, both by modulating the tumor extracellular microenvironment and by altering critical intracellular oncogenic signaling pathways.