William Putzbach

The role of CD95 and CD95 ligand in cancer.

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.

Erratum: The role of CD95 and CD95 ligand in cancer (Cell Death and Differentiation (2015) 22 (549-559) (DOI: 10.1038/cdd.2015.3))

Correction to: Cell Death and Differentiation (2015) 22, 549–559; doi:10.1038/cdd.2015.3; published online 6 February 2015 Since the publication of this article the authors have noted that they have inadvertently failed to list an important paper in the field in Table 2. This work (Kleber et al.) has now been added to the revised Table 2 shown below.

Radial intercalation is regulated by the Par complex and the microtubule-stabilizing protein CLAMP/Spef1.

During radial intercalation of epithelial cells, Par3 and aPKC promote the apical positioning of centrioles, whereas CLAMP stabilizes microtubules along the axis of migration.

Many si/shRNAs can kill cancer cells by targeting multiple survival genes through an off-target mechanism

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.

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to 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 Huntingtons 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.

6mer Seed Toxicity Determines Strand Selection in miRNAs

Many 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.

DISE: A Seed-Dependent RNAi Off-Target Effect That Kills Cancer Cells

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.

6mer seed toxicity in tumor suppressive microRNAs

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.

CD95L mRNA is toxic to cells

CD95/Fas ligand binds to the death receptor CD95/Fas to induce apoptosis in sensitive cells. We previously reported the CD95L mRNA is enriched in sequences that, when converted to si/shRNAs, are toxic to cells (Putzbach et al., 2017). These si/shRNAs kill all cancer cells through a RNAi off-target effect by targeting critical survival genes. We now report expression of full-length CD95L mRNA, itself, is highly toxic to cells and induces a similar form of cell death. We demonstrate that small RNAs derived from CD95L are loaded into the RNA induced silencing complex (RISC) and that the RISC is required for the toxicity. Drosha and Dicer knock-out cells are highly sensitive to this toxicity, suggesting that processing of CD95L mRNA into small toxic RNAs is independent of both Dicer and Drosha. The data provide evidence that a higher vertebrate transgene can be processed to RNAi-active small RNAs that elicit cellular responses.

CD95L mRNA And CD95L Derived si- and shRNAs Kill Cancer Cells Through An RNAi Mechanism By Targeting Survival Genes

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 using specific toxic RNAi-active sequences present in the genome can kill cancer cells.>80% of a large number of siRNAs and shRNAs targeting CD95 or CD95 ligand (CD95L) induce a form of cell death that is characterized by the simultaneous activation of multiple death pathways and preferentially affects transformed and cancer stem cells. We now show that 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. By testing 4666 shRNAs derived from the CD95 and CD95L mRNAs and an unrelated control gene, Venus, we have located the most toxic sequences in the open reading frame of CD95L. Consistently, CD95L mRNA is highly toxic to cancer cells after complete deletion of CD95. Our data provide the first evidence for mRNAs to affect cell fate through RNAi in mammalian cells. In addition, they suggest that cancer cells can be targeted with specific toxic RNAi active sequences present in the genome.