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PDF file - 1778K, Data on fibroblast characterization, array and transfection validation, function role of individual microRNAs and mouse xenograft images.

PDF file - 198K, Analysis of microRNA array and gene array data.

All Suppl. Figures

Two suppl. tables

Lists the most abundant miRNAs that are differentially expressed between Pt-R and Pt-S patients.

Ovarian cancer remains one of the deadliest gynecologic malignancies affecting women, and development of resistance to platinum remains a major barrier to achieving a cure. Multiple mechanisms have been identified to confer platinum resistance. Numerous miRNAs have been linked to platinum sensitivity and resistance in ovarian cancer. miRNA activity occurs mainly when the guide strand of the miRNA, with its seed sequence at position 2–7/8, is loaded into the RNA-induced silencing complex (RISC) and targets complementary short seed matches in the 3′ untranslated region of mRNAs. Toxic 6mer seeds, which target genes critical for cancer cell survival, have been found in tumor-suppressive miRNAs. Many siRNAs and short hairpin RNAs (shRNA) can also kill cancer cells via toxic seeds, the most toxic of which carry G-rich 6mer seed sequences. We showed here that treatment of ovarian cancer cells with platinum led to increased RISC-bound miRNAs carrying toxic 6mer seeds and decreased miRNAs with nontoxic seeds. Platinum-tolerant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in tumors from patients with ovarian cancer revealed that the ratio between miRNAs with toxic versus nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses.Significance:These findings demonstrate that the balance of miRNAs that carry toxic and nontoxic 6mer seeds contributes to platinum resistance in ovarian cancer.

Three suppl. tables

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 recently discovered powerful cell death mechanism mediated by short (s) RNAs including micro (mi) RNAs acting through the RNA induced silencing complex (RISC). G-rich 6mer seed sequences in the sRNAs (position 2-7) target hundreds of C-rich seed matches in genes essential for cell survival resulting in the simultaneous activation of multiple cell death pathways. Using Argonaute precipitation and RNAseq (Ago-RP-Seq) we analyzed RISC bound sRNAs (R-sRNAs) inin vitrocell line models and in the brains of twoin vivoAD mouse models and aged mice. In cell line studies we find evidence for a contribution of RNAi to the cell death and DNA damage induced by toxic Aβ42 oligomers. In addition, in AD mouse models and in the aging brain R-sRNAs RISC bound sRNAs show a shift to more toxic seeds. In contrast, in cells of "SuperAgers", individuals over age 80 who have superior memory performance, R-sRNAs are shifted to more nontoxic seeds, supporting a protective function of miRNAs. Cell death induced by Aβ42 oligomers can be rescued by adding back protective miRNAs. Our data provide first evidence of a contribution of RNAi to the neurotoxicity and DNA damage seen in AD suggesting that increasing the levels of protective miRNAs in the brain or blocking the activity of toxic R-sRNAs could lead to a novel way of treating the disease.

Ovarian cancer remains one of the deadliest gynecologic malignancies affecting women, and development of resistance to platinum remains a major barrier to achieving a cure. Multiple mechanisms have been identified to confer platinum resistance. Numerous miRNAs have been linked to platinum sensitivity and resistance in ovarian cancer. miRNA activity occurs mainly when the guide strand of the miRNA, with its seed sequence at position 2–7/8, is loaded into the RNA-induced silencing complex (RISC) and targets complementary short seed matches in the 3′ untranslated region of mRNAs. Toxic 6mer seeds, which target genes critical for cancer cell survival, have been found in tumor-suppressive miRNAs. Many siRNAs and short hairpin RNAs (shRNA) can also kill cancer cells via toxic seeds, the most toxic of which carry G-rich 6mer seed sequences. We showed here that treatment of ovarian cancer cells with platinum led to increased RISC-bound miRNAs carrying toxic 6mer seeds and decreased miRNAs with nontoxic seeds. Platinum-tolerant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in tumors from patients with ovarian cancer revealed that the ratio between miRNAs with toxic versus nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses. Significance: These findings demonstrate that the balance of miRNAs that carry toxic and nontoxic 6mer seeds contributes to platinum resistance in ovarian cancer.

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.

The apoptosis inducing receptor CD95/Fas has multiple tumorigenic activities. Stimulation by its cognate ligand CD95L on many cancer cells increases their growth, motility, ability to invade and/or their cancer stemness. Using genetically engineered mouse models of ovarian and liver cancer, we previously reported that deletion of CD95 in the tumor cells strongly reduced their ability to grow in vivo [1, 2]. Using a combination of immune-deficient and immune-competent mouse models, we now establish that loss of CD95 in metastatic triple negative breast cancer cells prevents tumor growth by modulating the immune landscape. CD95 deficient but not wild-type tumors barely grow in an immune-competent environment and show an increase in immune infiltrates into the tumor. This growth reduction is caused by NK cells and does not involve CD8+ T cells. On the other hand, in immune compromised mice CD95 k.o. cells are not growth inhibited, but they fail to form metastases. In summary, we demonstrate that in addition to its tumor and metastasis promoting activities, CD95 expression by tumor cells can exert immune suppressive activities providing a new target for immune therapy.

Numerous micro(mi)RNAs (short noncoding RNAs that negatively regulate gene expression) have been linked to platinum (Pt) sensitivity and resistance in ovarian cancer (OC). miRNA activity occurs when the guide strand of the miRNA, with its seed sequence (pos. 2-7/8), is loaded into the RNA induced silencing complex (RISC) and targets complementary short seed matches in the 3’ untranslated region of mRNAs. Toxic seeds, targeting genes critical for cancer cell survival, have been found in tumor suppressive miRNAs. Many si- and shRNAs can also kill cancer cells via toxic seeds, the most toxic carrying G-rich 6mer seed sequences. We now show that treatment of OC cells with Pt leads to an increase in RISC-bound miRNAs carrying toxic 6mer seeds and a decrease in miRNAs with nontoxic seeds. Pt-resistant cells did not exhibit this toxicity shift but retained sensitivity to cell death mediated by siRNAs carrying toxic 6mer seeds. Analysis of RISC-bound miRNAs in OC patients revealed that the ratio between miRNAs with toxic versus miRNAs with nontoxic seeds was predictive of treatment outcome. Application of the 6mer seed toxicity concept to cancer relevant miRNAs provides a new framework for understanding and predicting cancer therapy responses.

6mer seed toxicity is a novel anti-cancer 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 a consensus GGGGGC as the most toxic seed. After testing two more cell lines, one human and one mouse, we found that the GGGGGC seed while also toxic to murine cells, is more toxic to human cells, suggesting that the evolution to use of Gs as part of the toxic seeds is still slowly evolving, with Gs more common in the human toxic seeds. While new RNA Seq and bioinformatics analyses suggest that the GGGGGC seed is toxic to cancer cells by targeting GCCCCC seed matches in the 3’ UTR of a set of genes critical for cell survival, we now directly confirm this by identifying a number of genes targeted by this seed. Furthermore, by using a luciferase reporter fused to the 3’ UTR of these genes we confirm direct and specific on-targeting of GCCCCC seed matches. Targeting is strongly attenuated after mutating the GCCCCC seed matches in these 3’ UTRs. Our data confirm that an siRNA containing the GGGGGC seed 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.

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.

Many neurodegenerative diseases are caused by unstable trinucleotide repeat (TNR) expansions located in disease-associated genes. siRNAs based on CAG repeat expansions effectively kill cancer cell lines in vitro through RNA interference (RNAi). They also cause significant reduction in tumor growth in a human ovarian cancer mouse model with no toxicity to the treated mice. This suggests that cancer cells are particularly sensitive to CAG TNR-derived siRNAs and explains a reported inverse correlation between the length of CAG TNRs and reduced global cancer incidences in some CAG TNR diseases. This review discusses both mutant proteins and mutant RNAs as a cause of TNR diseases with a focus on RNAi and its role in contributing to disease pathology and in suppressing cancer.

Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs). We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1) and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95high-expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs) that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95.

Summary MicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells., Graphical Abstract, Highlights • Tumor suppressive miR-15/16-5p with a toxic 6mer seed targets survival genes • kshv-miR-K12-6-5p, a paralog of hsa-miR-15/16-5p carries an offset toxic 6mer seed • A screen of 215 viral miRNAs identifies miRNAs that contain a toxic 6mer seed • Many human viral miRNAs have the capacity to kill through 6mer seed toxicity, Molecular Genetics; Virology; Bioinformatics

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.

SUMMARYMany siRNAs and shRNAs are toxic to cancer cells through a 6mer seed sequence (position 2-7 of the guide strand). A siRNA screen with all 4096 possible 6mer seed sequences in a neutral RNA backbone revealed a preference for guanine in positions 1-3 and a GC content of >80% of the 6mer seed in the most toxic siRNAs. These 6mer seed containing siRNAs exert their toxicity by targeting survival genes which contain GC-rich 3’UTRs. The master tumor suppressor miRNA miR-34a was found to be toxic through such a G-rich 6mer seed suggesting that certain tumor suppressive miRNAs use a toxic 6mer seed to kill cancer cells. An analysis of all mature miRNAs suggests that most miRNAs evolved to avoid guanine at the 5′ end of the 6mer seed sequence of the predominantly expressed arm. In contrast, for many tumor suppressive miRNAs the predominant arm contains a G-rich toxic 6mer seed, presumably to eliminate cancer cells.

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington9s disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA‐sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complementary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the six members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR‐based siRNAs induce cell death in vitro in all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR‐based siRNAs as a novel form of anticancer reagents.

Trinucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington’s disease (HD). Pathology is caused by protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR are toxic to cancer cells by targeting genes that contain long reverse complimentary TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the 6 members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG TNR-based siRNAs induce cell deathin vitroin all tested cancer cell lines and slow down tumor growth in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anti-cancer reagents.

CD95 (Fas/APO-1) and its ligand, CD95L, have long been viewed as a death receptor/death ligand system that mediates apoptosis induction to maintain immune homeostasis. In addition, these molecules are important in the immune elimination of virus-infected cells and cancer cells. CD95L was, therefore, considered to be useful for cancer therapy. However, major side effects have precluded its systemic use. During the last 10 years, it has been recognized that CD95 and CD95L have multiple cancer-relevant nonapoptotic and tumor-promoting activities. CD95 and CD95L were discovered to be critical survival factors for cancer cells, and were found to protect and promote cancer stem cells. We now discuss five different ways in which inhibiting or eliminating CD95L, rather than augmenting, may be beneficial for cancer therapy alone or in combination with standard chemotherapy or immune therapy.

Off-target effects (OTEs) represent a significant caveat for RNAi caused by substantial complementarity between siRNAs and unintended mRNAs. We now discuss the existence of three types of seed-dependent OTEs (sOTEs). Type I involves unintended targeting through the guide strand seed of an siRNA. Type II is caused by the activity of the seed on the designated siRNA passenger strand when loaded into the RNA-induced silencing complex (RISC). Both type I and II sOTEs will elicit unpredictable cellular responses. By contrast, in sOTE type III the guide strand seed preferentially targets essential survival genes resulting in death induced by survival gene elimination (DISE). In this Opinion article, we discuss DISE as a consequence of RNAi that may preferentially affect cancer cells.

// Andrea E. Murmann 1 , Kaylin M. McMahon 3,6 , Ashley Haluck-Kangas 1 , Nandini Ravindran 1 , Monal Patel 1 , Calvin Y. Law 1 , Sonia Brockway 1 , Jian-Jun Wei 4 , C. Shad Thaxton 3,5,6,7 and Marcus E. Peter 1,2,5 1 Department of Medicine/Division of Hematology/Oncology, Feinberg School of Medicine, Chicago, IL, USA 2 Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Chicago, IL, USA 3 Department of Urology, Feinberg School of Medicine, Chicago, IL, USA 4 Department of Pathology and Obstetrics and Gynecology, Feinberg School of Medicine, Chicago, IL, USA 5 Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Northwestern University, Chicago, IL, USA 6 Simpson Querrey Institute (SQI) for BioNanotechnology, Chicago, IL, USA 7 International Institute for Nanotechnology, Evanston, IL, USA Correspondence to: Marcus E. Peter, email: // Keywords : RNAi, Fas, ovarian cancer, TLP nanoparticles, cell death Received : January 23, 2017 Accepted : September 13, 2017 Published : October 04, 2017 Abstract The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3’UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nanoparticles (TLP). In vitro , TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo , two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

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., eLife digest 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.

The death receptor CD95/Fas can be activated by immune cells to kill cancer cells. shRNAs and siRNAs derived from CD95 or CD95 ligand (CD95L) are highly toxic to most cancer cells. We recently found that these sh/siRNAs kill cancer cells in the absence of the target by targeting the 3’UTRs of critical survival genes through canonical RNAi. We have named this unique form of off-target effect DISE (for death induced by survival gene elimination). DISE preferentially kills transformed cells and cancer stem cells. We demonstrate that DISE induction occurs in cancer cells in vivo after introducing a lentiviral CD95L derived shRNA (shL3) into HeyA8 ovarian cancer cells grown as i.p. xenografts in mice, when compared to a scrambled shRNA. To demonstrate the possibility of therapeutically inducing DISE, we coupled siRNAs to templated lipoprotein nanoparticles (TLP). In vitro, TLPs loaded with a CD95L derived siRNA (siL3) selectively silenced a biosensor comprised of Venus and CD95L ORF and killed ovarian cancer cells. In vivo, two siRNA-TLPs (siL2-TLP and siL3-TLP) reduced tumor growth similarly as observed for cells expressing the shL3 vector. These data suggest that it is possible to kill ovarian cancer cells in vivo via DISE induction using siRNA-TLPs.

SummaryCD95 (Fas/APO-1), when bound by its cognate ligand CD95L, induces cells to die by apoptosis. We now show that elimination of CD95 or CD95L results in a form of cell death that is independent of caspase-8, RIPK1/MLKL, and p53, is not inhibited by Bcl-xL expression, and preferentially affects cancer cells. All tumors that formed in mouse models of low-grade serous ovarian cancer or chemically induced liver cancer with tissue-specific deletion of CD95 still expressed CD95, suggesting that cancer cannot form in the absence of CD95. Death induced by CD95R/L elimination (DICE) is characterized by an increase in cell size, production of mitochondrial ROS, and DNA damage. It resembles a necrotic form of mitotic catastrophe. No single drug was found to completely block this form of cell death, and it could also not be blocked by the knockdown of a single gene, making it a promising way to kill cancer cells.

Ovarian cancer patients frequently develop resistance to chemotherapy regiments utilizing Taxol and carboplatin. One of the resistance factors that protects cancer cells from Taxol-based therapy is multi-drug resistance 1 (MDR1). micro(mi)RNAs are small noncoding RNAs that negatively regulate protein expression. Members of the let-7 family of miRNAs are downregulated in many human cancers, and low let-7 expression has been correlated with resistance to microtubule targeting drugs (Taxanes), although little is known that would explain this activity. We now provide evidence that, while let-7 is not a universal sensitizer of cancer cells to Taxanes, it affects acquired resistance of cells to this class of drugs by targeting IMP-1, resulting in de-stabilization of the mRNA of MDR1. Introducing let-7g into ADR-RES cells expressing both IMP-1 and MDR1 reduced expression of both proteins rendering the cells more sensitive to treatment with either Taxol or vinblastine without affecting the sensitivity of the cells to carboplatin, a non-MDR1 substrate. This effect could be reversed by reintroducing IMP-1 into let-7g high/MDR1 low cells causing MDR1 to again become stabilized. Consistently, many relapsed ovarian cancer patients tested before and after chemotherapy were found to downregulate let-7 and to co-upregulate IMP-1 and MDR1, and the increase in the expression levels of both proteins after chemotherapy negatively correlated with disease-free time before recurrence. Our data point at IMP-1 and MDR1 as indicators for response to therapy, and at IMP-1 as a novel therapeutic target for overcoming multidrug resistance of ovarian cancer.

Many siRNAs and shRNAs induce a form of cell death in all cancer cells that involves silencing a set of critical survival genes, a process called death induced by survival gene elimination (DISE). Mechanistically a 6mer seed sequence (position 2-7 of the guide strand) is sufficient to confer DISE-inducing activity. We have now performed a strand specific screen testing the toxicity of all 4096 possible 6mer seed sequences in a neutral double stranded siRNA backbone. We found an asymmetric preference for guanine in positions 1-3 of the 6mer seed in the most toxic siRNAs which target survival genes that are GC rich in their 3'UTR. During over 800 million years of evolution miRNAs have evolved to avoid guanine in their seed sequences. However, two tumor suppressive miRNAs were found to be killing cancer cells through DISE using G rich toxic seeds in cells exposed to genotoxic stress: 1) the p53 inducible miR-34 family and most of its cell death inducing activity comes from its 6mer seed; and 2) miR-320a, a noncanonical miRNA that is still expressed in cancer cells when miRNA processing genes are mutated. Ago bound upregulated miR-320a is converted from a poorly to a highly toxic miRNA by removal of two adenine nucleotides from its 5' end. Our data suggest that most miRNAs have evolved to avoid induction of DISE but certain tumor suppressive miRNAs utilize this mechanism to kill cancer cells. Citation Format: Quan Q. Gao, William T. Putzbach, Andrea E. Murmann, Siquan Chen, Aishe A. Sarshad, Giovanna Ambrosini, Elizabeth T. Bartom, Markus Hafner, Marcus E. Peter. Induction of DISE by tumor suppressive microRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-401.

Triplet nucleotide repeat (TNR) expansions in the genome cause a number of degenerative diseases. A prominent TNR expansion involves the triplet CAG in the huntingtin (HTT) gene responsible for Huntington's disease (HD). Pathology is caused by both protein and RNA generated from the TNR regions including small siRNA-sized repeat fragments. An inverse correlation between the length of the repeats in HTT and cancer incidence has been reported for HD patients. We now show that siRNAs based on the CAG TNR can be used to kill cancer cells by targeting genes containing reverse complimentary long TNRs in their open reading frames. Of the 60 siRNAs based on the different TNRs, the 6 members in the CAG/CUG family of related TNRs are the most toxic to both human and mouse cancer cells. siCAG/CUG-induced cell death occurs in vitro in all tested cancer cell lines and in a preclinical mouse model of ovarian cancer with no signs of toxicity to the mice. We propose to explore TNR-based siRNAs as a novel form of anticancer reagents. Citation Format: Andrea E. Murmann, Will Putzbach, Quan Gao, Monal Patel, Elizabeth T. Bartom, Calvin Law, Bryan Bridgeman, Siquan Chen, Kaylin M. McMahon, Shad C. Thaxton, Marcus E. Peter. Triplet nucleotide repeat-based siRNAs are highly toxic to cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4387.

Tumor progression shares many characteristics with the process of epithelial-to-mesenchymal transition (EMT). Cells that have undergone an EMT are known to have an increased resistance to apoptosis. CD95/Fas is an apoptosis-inducing receptor expressed on many tissues and tumor cells. During tumor progression CD95 is frequently downregulated, and tumor cells lose apoptosis sensitivity. miR-200 microRNAs repress both the EMT-inducing ZEB1 and ZEB2 transcription factors. We now demonstrate that miR-200c sensitizes cells to apoptosis mediated by CD95. We have identified the apoptosis inhibitor FAP-1 as a target for miR-200c. FAP-1 was demonstrated to be responsible for the reduced sensitivity to CD95-mediated apoptosis in cells with inhibited miR-200. The identification of FAP-1 as an miR-200c target provides a molecular mechanism to explain both the downregulation of CD95 expression and the reduction in sensitivity of cells to CD95-mediated apoptosis that is observed in the context of reduced miR-200 expression during tumor progression.

MicroRNAs (miRNAs or miRs) are small noncoding RNAs capable of regulating gene expression at the translational level. Current evidence suggests that a significant portion of the human genome is regulated by microRNAs, and many reports have demonstrated that microRNA expression is deregulated in human cancer. The let-7 family of microRNAs, first discovered in Caenorhabditis elegans, is functionally conserved from worms to humans. The human let-7 family contains 13 members located on nine different chromosomes, and many human cancers have deregulated let-7 expression. A growing body of evidence suggests that restoration of let-7 expression may be a useful therapeutic option in cancers, where its expression has been lost. In this review, we discuss the role of let-7 in normal development and differentiation, and provide an overview of the relationship between deregulated let-7 expression and tumorigenesis. The regulation of let-7 expression, cancer-relevant let-7 targets, and the relationship between let-7 and drug sensitivity are highlighted.

Acentric inverted duplication (inv dup) markers, the largest group of chromosomal abnormalities with neocentromere formation, are found in patients both with idiopathic mental retardation and with cancer. The mechanism of their formation has been investigated by analyzing the breakpoints and the genotypes of 12 inv dup marker cases (three trisomic, six tetrasomic, two polysomic and one X chromosome derived marker) using a combination of fluorescence in situ hybridization, quantitative SNP array and microsatellite analysis. Inv dup markers were found to form either symmetrically with one breakpoint or asymmetrically with two distinct breakpoints. Genotype analyses revealed that all inv dup markers formed from one single chromatid end. This observation is incompatible with the previously suggested model by which the acentric inv dup markers form through inter-chromosomal U-type exchange. On the basis of the identification of DNA sequence motifs with inverted homologies within all observed breakpoint regions, a new general mechanism is proposed for the acentric inv dup marker formation: following a double-strand break an acentric fragment forms, during either meiosis or mitosis. The open DNA end of the acentric fragment is stabilized by the formation of an intra-chromosomal loop promoted by the presence of sequences with inverted homologies. Likely coinciding with the neocentromere formation, this stabilized fragment is duplicated during an early mitotic event, insuring the marker’s survival during cell division and its presence in all cells.

The Indian muntjac (Muntiacus muntjak vaginalis) has a karyotype of 2n = 6 in the female and 2n = 7 in the male. The karyotypic evolution of Indian muntjac via extensive tandem fusions and several centric fusions are well documented by molecular cytogenetic studies mainly utilizing chromosome paints. To achieve higher resolution mapping, a set of 42 different genomic clones coding for 37 genes and the nucleolar organizer region were used to examine homologies between the cattle (2n = 60), human (2n = 46), Indian muntjac (2n = 6/7) and Chinese muntjac (2n = 46) karyotypes. These genomic clones were mapped by fluorescence in situ hybridization (FISH). Localization of genes on all three pairs of M. m. vaginalis chromosomes and on the acrocentric chromosomes of M. reevesi allowed not only the analysis of the evolution of syntenic regions within the muntjac genus but also allowed a broader comparison of synteny with more distantly related species, such as cattle and human, to shed more light onto the evolving genome organization.

Inflammation is a localized, protective response to trauma or microbial invasion that destroys the injurious agent and the injured tissue. Neutrophil elastase (NE), a serine protease stored in the azurophil granules of polymorphonuclear neutrophils, digests microbes after phagocytosis. NE can also digest microbes extracellularly but is associated with tissue damage and inflammatory disease. In this study, we show that polymorphonuclear neutrophils from mice deficient in serine protease inhibitor 6, a weak intracellular NE inhibitor, had increased susceptibility to self-inflicted lysis because of increased NE activity. The resulting transient increase in local extracellular NE activity was within a narrow range that resulted in the clearance of Pseudomonas aeruginosa but did not damage the lung. Therefore, deficiency in a weak intracellular inhibitor of NE results in an acute inflammatory response that protects from P. aeruginosa but does not cause lung disease.

Specific chromosomal translocations are hallmarks of many human leukemias. The basis for these translocation events is poorly understood, but it has been assumed that spatial positioning of genes in the nucleus of hematopoietic cells is a contributing factor. Analysis of the nuclear 3D position of the gene MLL, frequently involved in chromosomal translocations and five of its translocation partners (AF4, AF6, AF9, ENL and ELL), and two control loci revealed a characteristic radial distribution pattern in all hematopoietic cells studied. Genes in areas of high local gene density were found positioned towards the nuclear center, whereas genes in regions of low gene density were detected closer to the nuclear periphery. The gene density within a 2 Mbp window was found to be a better predictor for the relative positioning of a genomic locus within the cell nucleus than the gene density of entire chromosomes. Analysis of the position of MLL, AF4, AF6 and AF9 in cell lines carrying chromosomal translocations involving these genes revealed that the position of the normal genes was different from that of the fusion genes, and this was again consistent with the changes in local gene density within a 2 Mbp window. Thus, alterations in gene density directly at translocation junctions could explain the change in the position of affected genes in leukemia cells.

FADD is essential for death receptor (DR)-induced apoptosis. However, it is also critical for cell cycle progression and proliferation, activities that are regulated by phosphorylation of its C-terminal Ser194, which has also been implicated in sensitizing cancer cells to chemotherapeutic drugs and in regulating FADD's intracellular localization. We now demonstrate that casein kinase Ialpha (CKIalpha) phosphorylates FADD at Ser194 both in vitro and in vivo. FADD-CKIalpha association regulates the subcellular localization of FADD, and phosphorylated FADD was found to colocalize with CKIalpha on the spindle poles in metaphase. Inhibition of CKIalpha diminished FADD phosphorylation, prevented the ability of Taxol to arrest cells in mitosis, and blocked mitogen-induced proliferation of mouse splenocytes. In contrast, a low level of cycling splenocytes from mice expressing FADD with a mutated phosphorylation site was insensitive to CKI inhibition. These data suggest that phosphorylation of FADD by CKI is a crucial event during mitosis.

The functional organization of chromatin in cell nuclei is a fundamental question in modern cell biology. Individual chromosomes occupy distinct chromosome territories in interphase nuclei. Nuclear bodies localize outside the territories and colocalize with ectopically expressed proteins in a nuclear subcompartment, the interchromosomal domain compartment. In order to investigate the structure of this compartment in mammalian cells with distinctly different karyotypes, we analyzed human HeLa cells (3n+ = 71 chromosomes) and cells of two closely related muntjac species, the Chinese muntjac (2n = 46 chromosomes) and the Indian muntjac (2n = 6/7 chromosomes). The distribution of ectopically expressed intermediate filament proteins (vimentin and cytokeratins) engineered to contain a nuclear localization sequence (NLS) and a nuclear particle forming protein (murine Mx1) fused to a yellow fluorescent protein (YFP) was compared. The proteins were predominantly localized in regions with poor DAPI staining independent of the cells' karyotype. In contrast to NLS-vimentin, the NLS-modified cytokeratins were also found close to the nuclear periphery. In Indian muntjac cells, NLS-vimentin colocalized with Mx1-YFP as well as the NLS-cytokeratins. Since the distribution of the ectopically expressed protein markers is similar in cells with distinctly different chromosome numbers, the property of the delineated, limited compartment might indeed depend on chromatin organization.