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CD95 (APO-1/Fas) is a death receptor used by immune cells to kill cancer cells through induction of apoptosis. However, the elimination of CD95 or its ligand, CD95L, from cancer cells results in death induced by CD95R/L elimination (DICE), a type of cell death that resembles a necrotic form of mitotic catastrophe suggesting that CD95 protects cancer cells from cell death. We now report that stimulation of CD95 on cancer cells or reducing miR-200c levels increases the number of cancer stem cells (CSCs), which are more sensitive to induction of DICE than non-CSC, while becoming less sensitive to CD95-mediated apoptosis. In contrast, induction of DICE or overexpression of miR-200c reduces the number of CSCs. We demonstrate that CSCs and non-CSCs have differential sensitivities to CD95-mediated apoptosis and DICE, and that killing of cancer cells can be maximized by concomitant induction of both cell death mechanisms. The death receptor CD95/Fas induces apoptosis of many normal cells but prevents necrotic death of cancer cells. Here the authors demonstrate that CD95 activation promotes a cancer stem cell (CSC) phenotype, and that CSCs but not differentiated cancer cells are resistant to CD95-mediated apoptosis and depend on CD95 signalling to prevent necrosis.

microRNAs (miRNAs) are (18-22nt long) noncoding short (s)RNAs that suppress gene expression by targeting the 3’ untranslated region of target mRNAs. This occurs through the seed sequence located in position 2-7/8 of the miRNA guide strand, once it is loaded into the RNA induced silencing complex (RISC). G-rich 6mer seed sequences can kill cells by targeting C-rich 6mer seed matches located in genes that are critical for cell survival. This results in induction of Death Induced by Survival gene Elimination (DISE), through a mechanism we have called 6mer seed toxicity. miRNAs are often quantified in cells by aligning the reads from small (sm)RNA sequencing to the genome. However, the analysis of any smRNA Seq data set for predicted 6mer seed toxicity requires an alternative workflow, solely based on the exact position 2–7 of any short (s)RNA that can enter the RISC. Therefore, we developed SPOROS, a semi-automated pipeline that produces multiple useful outputs to predict and compare 6mer seed toxicity of cellular sRNAs, regardless of their nature, between different samples. We provide two examples to illustrate the capabilities of SPOROS: Example one involves the analysis of RISC-bound sRNAs in a cancer cell line (either wild-type or two mutant lines unable to produce most miRNAs). Example two is based on a publicly available smRNA Seq data set from postmortem brains (either from normal or Alzheimer’s patients). Our methods (found at https://github.com/ebartom/SPOROS and at Code Ocean: https://doi.org/10.24433/CO.1732496.v1 ) are designed to be used to analyze a variety of smRNA Seq data in various normal and disease settings.

Alzheimer’s disease (AD) is characterized by progressive neurodegeneration, but the specific events that cause cell death remain poorly understood. Death Induced by Survival gene Elimination (DISE) is a cell death mechanism mediated by short (s) RNAs acting through the RNA-induced silencing complex (RISC). DISE is thus a form of RNA interference, in which G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich 6mer seed matches in genes essential for cell survival, resulting in the activation of cell death pathways. Here, using Argonaute precipitation and RNAseq (Ago-RP-Seq), we analyze RISC-bound sRNAs to quantify 6mer seed toxicity in several model systems. In mouse AD models and aging brain, in induced pluripotent stem cell-derived neurons from AD patients, and in cells exposed to Aβ42 oligomers, RISC-bound sRNAs show a shift to more toxic 6mer seeds compared to controls. In contrast, in brains of “SuperAgers”, humans over age 80 who have superior memory performance, RISC-bound sRNAs are shifted to more nontoxic 6mer seeds. Cells depleted of nontoxic sRNAs are sensitized to Aβ42-induced cell death, and reintroducing nontoxic RNAs is protective. Altogether, the correlation between DISE and Aβ42 toxicity suggests that increasing the levels of nontoxic miRNAs in the brain or blocking the activity of toxic RISC-bound sRNAs could ameliorate neurodegeneration. Events that cause neurons to die in Alzheimer’s disease (AD) are poorly understood. Here, the authors provide evidence for a role of RNA interference in AD. Short RNAs causing neurotoxicity and DNA damage are seen in AD and aged brains, and are counteracted by nontoxic RNAs.

Many small-interfering (si)RNAs are toxic to cancer cells through a 6mer seed sequence (positions 2–7 of the guide strand). Here we performed an siRNA screen with all 4096 6mer seeds revealing a preference for guanine in positions 1 and 2 and a high overall G or C content in the seed of the most toxic siRNAs for four tested human and mouse cell lines. Toxicity of these siRNAs stems from targeting survival genes with C-rich 3′UTRs. The master tumor suppressor miRNA miR-34a-5p is toxic through such a G-rich 6mer seed and is upregulated in cells subjected to genotoxic stress. An analysis of all mature miRNAs suggests that during evolution most miRNAs evolved to avoid guanine at the 5′ end of the 6mer seed sequence of the guide strand. In contrast, for certain tumor-suppressive miRNAs the guide strand contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells. Small interfering (siRNAs) can be toxic to cancer cells. Here the authors investigate the toxicity of microRNA in cancer cells by performing a siRNA screen that tests the miRNA activities of an extensive list of miRNAs with different 6mer seed sequences.

micro(mi)RNAs are short noncoding RNAs that through their seed sequence (pos. 2–7/8 of the guide strand) regulate cell function by targeting complementary sequences (seed matches) located mostly in the 3′ untranslated region (3′ UTR) of mRNAs. Any short RNA that enters the RNA induced silencing complex (RISC) can kill cells through miRNA-like RNA interference when its 6mer seed sequence (pos. 2–7 of the guide strand) has a G-rich nucleotide composition. G-rich seeds mediate 6mer Seed Toxicity by targeting C-rich seed matches in the 3′ UTR of genes critical for cell survival. The resulting Death Induced by Survival gene Elimination (DISE) predominantly affects cancer cells but may contribute to cell death in other disease contexts. This review summarizes recent findings on the role of DISE/6mer Seed Tox in cancer; its therapeutic potential; its contribution to therapy resistance; its selectivity, and why normal cells are protected. In addition, we explore the connection between 6mer Seed Toxicity and aging in relation to cancer and certain neurodegenerative diseases.

6mer seed toxicity is a novel cell death mechanism that kills cancer cells by triggering death induced by survival gene elimination (DISE). It is based on si- or shRNAs with a specific G-rich nucleotide composition in position 2–7 of their guide strand. An arrayed screen of 4096 6mer seeds on two human and two mouse cell lines identified G-rich 6mers as the most toxic seeds. We have now tested two additional cell lines, one human and one mouse, identifying the GGGGGC consensus as the most toxic average 6mer seed for human cancer cells while slightly less significant for mouse cancer cells. RNA Seq and bioinformatics analyses suggested that an siRNA containing the GGGGGC seed (siGGGGGC) is toxic to cancer cells by targeting GCCCCC seed matches located predominantly in the 3′ UTR of a set of genes critical for cell survival. We have identified several genes targeted by this seed and demonstrate direct and specific targeting of GCCCCC seed matches, which is attenuated upon mutation of the GCCCCC seed matches in these 3′ UTRs. Our data show that siGGGGGC kills cancer cells through its miRNA-like activity and points at artificial miRNAs, si- or shRNAs containing this seed as a potential new cancer therapeutics.

Extended CAG trinucleotide repeats (TNR) in the genes huntingtin ( HTT ) and androgen receptor ( AR ) are the cause of two progressive neurodegenerative disorders: Huntington’s disease (HD) and Spinal and Bulbar Muscular Atrophy (SBMA), respectively. Anyone who inherits the mutant gene in the complete penetrance range (>39 repeats for HD and 44 for SBMA) will develop the disease. An inverse correlation exists between the length of the CAG repeat and the severity and age of onset of the diseases. Growing evidence suggests that it is the length of uninterrupted CAG repeats in the mRNA rather than the length of poly glutamine (polyQ) in mutant (m) HTT protein that determines disease progression. One variant of m HTT (loss of inhibition; LOI) causes a 25 year earlier onset of HD when compared to a reference sequence, despite both coding for a protein that contains an identical number of glutamines. Short 21–22 nt CAG repeat (sCAGs)-containing RNAs can cause disease through RNA interference (RNAi). RNA hairpins (HPs) forming at the CAG TNRs are stabilized by adjacent CCG (in HD) or CUG repeats (in SBMA) making them better substrates for Dicer, the enzyme that processes CAG HPs into sCAGs. We now show that cells deficient in Dicer or unable to mediate RNAi are resistant to the toxicity of the HTT and AR derived HPs. Expression of a small HP that mimics the HD LOI variant is more stable and more toxic than a reference HP. We report that the LOI HP is processed by Dicer, loaded into the RISC more efficiently, and gives rise to a higher quantity of RISC-bound 22 nt sCAGs. Our data support the notion that RNAi contributes to the cell death seen in HD and SBMA and provide an explanation for the dramatically reduced onset of disease in HD patients that carry the LOI variant.

micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.

Over 80% of multiple-tested siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death characterized by simultaneous activation of multiple cell death pathways preferentially killing transformed and cancer stem cells. We now show these si/shRNAs kill cancer cells through canonical RNAi by targeting the 3’UTR of critical survival genes in a unique form of off-target effect we call DISE (death induced by survival gene elimination). Drosha and Dicer-deficient cells, devoid of most miRNAs, are hypersensitive to DISE, suggesting cellular miRNAs protect cells from this form of cell death. By testing 4666 shRNAs derived from the CD95 and CD95L mRNA sequences and an unrelated control gene, Venus, we have identified many toxic sequences - most of them located in the open reading frame of CD95L. We propose that specific toxic RNAi-active sequences present in the genome can kill cancer cells. Cells store their genetic code within molecules of DNA. Some of this information will be copied into chemically similar molecules called RNAs, from which the sequence of letters in the genetic code can be translated to build proteins. However, these messenger RNAs are not the only RNA molecules that cells can make. MicroRNAs are other short pieces of RNA that closely match sequences in parts of certain messenger RNAs. The messenger RNAs targeted by microRNAs are broken down inside the cell, which reduces how much protein can be produced from them. Since its discovery, scientists have exploited this process – called RNA interference (or RNAi for short) – and designed microRNA-like small interfering RNAs (siRNAs) to target particular messenger RNAs and decrease the levels of the corresponding proteins in countless experiments. Two proteins that have been studied in RNAi experiments are CD95 and its interaction partner CD95L. Both of these proteins are important in human cancer cells, and targeting them via RNAi killed cancer cells in an unknown mechanism that the cancer cells were unable to resist. RNAi experiments are designed to be specific, but sometimes they can accidently target other non-target messenger RNAs. Putzbach, Gao, Patel et al. have now analyzed all of the siRNAs that can be made from the messenger RNAs for CD95 and CD95L to mediate RNAi in cancer cells. This revealed that several messenger RNAs, other than those for CD95 and CD95L, were unintentionally being targeted, including many that code for proteins that cells need to survive. Further examination of the messenger RNA for CD95 and CD95L showed that they contain short sequences that are similar to those in the messenger RNAs of the genes that encode these survival proteins. Putzbach et al. were able to study and then predict which siRNA sequences would be toxic to cancer cells. These findings indicate that an RNAi off-target effect may actually be used to kill cancer cells. Future studies will determine whether this effect could be exploited to shrink tumors in animal models of cancer. If successful, this in turn could lead to new treatments for cancer patients.

CD95/Fas is an apoptosis inducing death receptor. However, it also has multiple nonapoptotic activities that are tumorigenic. Chronic stimulation of CD95 on breast cancer cells can increase their cancer initiating capacity through activation of a type I interferon (IFN-I)/STAT1 pathway when caspases are inhibited. We now show that this activity relies on the canonical components of the CD95 death-inducing signaling complex, FADD and caspase-8, and on the activation of NF-κB. We identified caspase-2 as the antagonistic caspase that downregulates IFN-I production. Once produced, IFN-Is bind to their receptors activating both STAT1 and STAT2 resulting in upregulation of the double stranded (ds)RNA sensor proteins RIG-I and MDA5, and a release of a subset of endogenous retroviruses. Thus, CD95 is part of a complex cell autonomous regulatory network that involves activation of innate immune components that drive cancer stemness and contribute to therapy resistance.