Induction of DISE in ovarian cancer cellsin vivo

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 nano particles (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.

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

“…Overall our data allow us to predict that any small RNA with DISE inducing RNAi activity that does not require Dicer processing can kill cancer cells regardless of its Dicer or Drosha status. In fact, in an accompanying manuscript we demonstrate that DISE can be triggered in vivo to treat ovarian cancer in xenografted mice by delivering CD95L-derived siRNAs using nanoparticles 34 . No toxicity was observed in the treated mice.…”

“…We now demonstrate that DISE is a unique form of OTE that results in the simultaneous activation of multiple cell death pathways in cancer cells. The discovery that DISE involves loss of multiple survival genes now provides an explanation for the unique properties we described for this form of cell death, especially the observation that cancer cells have a hard time developing resistance to this cell death mechanism 9,34 .…”

“…We now demonstrate DISE is a unique form of OTE that results in the simultaneous 532 activation of multiple cell death pathways in cancer cells. The discovery that DISE involves loss 533 of multiple survival genes now provides an explanation for the unique properties we described 534 for this form of cell death, especially the observation that cancer cells have a hard time 535 developing resistance to this cell death mechanism (Hadji et al, 2014;Murmann et al, 2017). The following arguments and evidence support our prediction that 543 DISE is a manifestation of a novel, functionally important, conserved mechanism of genome 544 regulation, and not the result of one of the above-mentioned effects: 545…”

“…The HeyA8 cells used in Fig. 2d carried a lentiviral Venus-siL3 sensor vector 34 and were infected with NucLight Red lentivirus (Essen Bioscience #4476) with 8 μg/ml polybrene and selected with 3 μg/ml puromycin and sorted for high Venus expression 48 hours later. HeyA8 ΔshR6 clone #2 sensor cells used in Fig.…”

Introductory Chapter: Insight into the OMICS Technologies and Molecular Medicine

“…Peter and colleagues have identified suicide/killer RNA molecules (siRNA, shRNA, miRNA, siRNA+miRNA complex) on numerous cancer types. In addition, they have shown that specific toxic RNAi-active sequences present in the genome can kill cancer cells [40][41][42][43][44]. Rozowsky and colleagues have generated a comprehensive analytic platform for extracellular RNA profiling called "exceRpt" [45].…”