Yuriy Fedorov

3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets

Off-target gene silencing can present a notable challenge in the interpretation of data from large-scale RNA interference (RNAi) screens. We performed a detailed analysis of off-targeted genes identified by expression profiling of human cells transfected with small interfering RNA (siRNA). Contrary to common assumption, analysis of the subsequent off-target gene database showed that overall identity makes little or no contribution to determining whether the expression of a particular gene will be affected by a given siRNA, except for near-perfect matches. Instead, off-targeting is associated with the presence of one or more perfect 3′ untranslated region (UTR) matches with the hexamer or heptamer seed region (positions 2–7 or 2–8) of the antisense strand of the siRNA. These findings have strong implications for future siRNA design and the application of RNAi in high-throughput screening and therapeutic development.

Functional Profiling Reveals Critical Role for miRNA in Differentiation of Human Mesenchymal Stem Cells

Background Mesenchymal stem (MS) cells are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although human MS cells can be isolated from bone marrow and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. Recent reports identify critical roles for microRNAs (miRNAs), regulators of gene expression either by inhibiting the translation or by stimulating the degradation of target mRNAs. Methodology/Principal Findings In this study, we employed a library of miRNA inhibitors to evaluate the role of miRNAs in early osteogenic differentiation of human MS cells. We discovered that miR-148b, -27a and -489 are essential for the regulation of osteogenesis: miR-27a and miR-489 down-regulate while miR-148b up-regulates differentiation. Modulation of these miRNAs induced osteogenesis in the absence of other external differentiation cues and restored osteogenic potential in high passage number human MS cells. Conclusions/Significance Overall, we have demonstrated the utility of the functional profiling strategy for unraveling complex miRNA pathways. Our findings indicate that miRNAs regulate early osteogenic differentiation in human MS cells: miR-148b, -27a, and -489 were found to play a critical role in osteogenesis.

Different delivery methods-different expression profiles.

Recent publications by several groups of researchers have suggested that small interfering RNAs (siRNA) delivered by lipid-mediated transfection induce both sequence-specific effects1 and broad, class-specific changes in gene expression1, 2, 3, 4. These findings challenge convictions previously held in the RNA interference (RNAi) community that assert virtual sequence specificity of siRNA knockdown, and they bring into question the value of this methodology as a research and therapeutic tool.

Assessment of Cr(VI)-Induced Cytotoxicity and Genotoxicity Using High Content Analysis

Oral exposure to high concentrations of hexavalent chromium [Cr(VI)] induces intestinal redox changes, villus cytotoxicity, crypt hyperplasia, and intestinal tumors in mice. To assess the effects of Cr(VI) in a cell model relevant to the intestine, undifferentiated (proliferating) and differentiated (confluent) Caco-2 cells were treated with Cr(VI), hydrogen peroxide or rotenone for 2–24 hours. DNA damage was then assessed by nuclear staining intensity of 8-hydroxydeoxyguanosine (8-OHdG) and phosphorylated histone variant H2AX (γ-H2AX) measured by high content analysis methods. In undifferentiated Caco-2, all three chemicals increased 8-OHdG and γ-H2AX staining at cytotoxic concentrations, whereas only 8-OHdG was elevated at non-cytotoxic concentrations at 24 hr. Differentiated Caco-2 were more resistant to cytotoxicity and DNA damage than undifferentiated cells, and there were no changes in apoptotic markers p53 or annexin-V. However, Cr(VI) induced a dose-dependent translocation of the unfolded protein response transcription factor ATF6 into the nucleus. Micronucleus (MN) formation was assessed in CHO-K1 and A549 cell lines. Cr(VI) increased MN frequency in CHO-K1 only at highly cytotoxic concentrations. Relative to the positive control Mitomycin-C, Cr(VI) only slightly increased MN frequency in A549 at mildly cytotoxic concentrations. The results demonstrate that Cr(VI) genotoxicity correlates with cytotoxic concentrations, and that H2AX phosphorylation occurs at higher concentrations than oxidative DNA damage in proliferating Caco-2 cells. The findings suggest that in vitro genotoxicity of Cr(VI) is primarily oxidative in nature at low concentrations. Implications for in vivo intestinal toxicity of Cr(VI) will be discussed.

Kinome-wide functional genomics screen reveals a novel mechanism of TNFα-induced nuclear accumulation of the HIF-1α transcription factor in cancer cells.

Hypoxia-inducible factor-1 (HIF-1) and its most important subunit, HIF-1α, plays a central role in tumor progression by regulating genes involved in cancer cell survival, proliferation and metastasis. HIF-1α activity is associated with nuclear accumulation of the transcription factor and regulated by several mechanisms including modulation of protein stability and degradation. Among recent advances are the discoveries that inflammation-induced cytokines and growth factors affect protein accumulation of HIF-1α under normoxia conditions. TNFα, a major pro-inflammatory cytokine that promotes tumorigenesis is known as a stimulator of HIF-1α activity. To improve our understanding of TNFα-mediated regulation of HIF-1α nuclear accumulation we screened a kinase-specific siRNA library using a cell imaging–based HIF-1α-eGFP chimera reporter assay. Interestingly, this systematic analysis determined that depletion of kinases involved in conventional TNFα signaling (IKK/NFκB and JNK pathways) has no detrimental effect on HIF-1α accumulation. On the other hand, depletion of PRKAR2B, ADCK2, TRPM7, and TRIB2 significantly decreases the effect of TNFα on HIF-1α stability in osteosarcoma and prostate cancer cell lines. These newly discovered regulators conveyed their activity through a non-conventional RELB-depended NFκB signaling pathway and regulation of superoxide activity. Taken together our data allow us to conclude that TNFα uses a distinct and complex signaling mechanism to induce accumulation of HIF-1α in cancer cells. In summary, our results illuminate a novel mechanism through which cancer initiation and progression may be promoted by inflammatory cytokines, highlighting new potential avenues for fighting this disease.

Abstract 4882: Determining endocrine receptor activity using quantitative automated image analysis and Redistribution® technology

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Endocrine hormone function has been shown to regulate numerous oncogenic processes from tumor progression, promotion, and dependence to prevention, diagnosis, and treatment. Notably, estrogen and androgen activity in breast and prostate cancers have been used to diagnose steroid hormone dependence and determine therapy options. Endocrine disruptors are compounds that mimic estrogen or androgen steroid activity and affect the endocrine system by altering hormone function, providing possible therapies for endocrine-dependent cancers. Screening complex chemical libraries for endocrine disruptors and investigating the molecular and cellular effects of existing treatment strategies is accelerated by using high throughput procedures to assess endocrine activity and potential cancer therapeutic efficacy. Redistribution® technology can be used to monitor the localization of GFP-tagged proteins in response to extra-cellular stimuli such as treatment with steroid compounds or activation of signaling cascades. Redistribution® GFP-tagged steroid receptors such as estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and androgen receptor (AR) form nuclear foci in response to hormone stimulation that can be easily monitored and quantitated by automated image analysis, thereby establishing a high-throughput screen for both agonists and antagonists of ER and AR activation. Additionally, multiplexed cell and nuclear phenotypes such as receptor kinetics, cellular health, and cell cycle effects can be monitored simultaneously following direct or indirect receptor stimulation. Here we utilize Redistribution® cell lines and known endocrine agonist and antagonists to establish a screen for ER and AR disruptors. For ER screens, cells were treated with agonists including 17β-estradiol and bisphenol A with and without antagonists such as fulvestrant (ICI 182,780) and tamoxifen. AR cells were treated with dihydrotesterone (5α-androstan-17β-ol-3-one) or progesterone agonists in the presence or absence of the AR antagonist mifepristone (RU 486). Following fixation and Hoechst staining, plates were analyzed using a quantitative automated imaging platform to evaluate features such as nuclear translocation (for AR only), nuclear GFP foci count, area, and intensity, and overall nuclear morphology on a cell-by-cell basis. EC50 values were determined from dose-dependent response curves of the treatments to establish hierarchical receptor activation comparisons for both ER and AR. Z-factors for these features indicate that using this technology gives a robust assay with high reproducibility. This data suggests that ER and AR Redistribution® cell lines can be effectively utilized with high throughput automated image analysis to screen for potential steroid hormone agonists and antagonists that would increase cancer therapeutic efficacy and specificity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4882. doi:10.1158/1538-7445.AM2011-4882