Acquired resistance to reoviral oncolysis in Ras-transformed fibrosarcoma cells

Reovirus shows considerable potential as an oncolyticagentforRas-activatedtumorsandiscurrentlyinclinicaltrials. Here we ask whether such tumor cell lines canacquire resistance to reoviral oncolysis. We challengedhuman HT1080 fibrosarcoma cells that carry a Rasmutation by prolonged exposure to reovirus, therebyyieldinghighlyvirus-resistantHTR1cells.Thesecellsarepersistently infected with reovirus, exhibit high Rasactivity and retain the original Ras gene mutation,showingthatresistancetoreoviruscanbedisplayedincellswithactiveRas.TheHTR1cellsalsoexhibitreducedcellularcathepsinBactivity,whichnormallycontributestoviralentryandactivation.PersistentlyinfectedHTR1cellswerenottumorigenicin vivo,whereasimmunologi-callycuredvirus-freeHTR1cellswerehighlytumorigenic.Thus,acquisitionofresistancetoreovirusmayconstraintherapeutic strategies. To determine whether reoviralresistance is associated with a general reduction inapoptoticpotential,wechallengedtheHTR1cellswithapoptoticinducersandE1B-defectiveadenovirus,result-ing in significant apoptosis and cell death followingboth approaches. Therefore, even if resistance to reo-viraloncolysisshouldariseintumorcellsin vivo,othertherapeuticstrategiesmayneverthelessremaineffective.Oncogene (2007) 26,4124–4134;doi:10.1038/sj.onc.1210189;published online 8 January 2007Keywords: reovirus; Ras; oncolysis; resistanceIntroductionThe human reovirus is a ubiquitous, non-envelopedvirus containing 10 segments of double-stranded RNA(dsRNA) asits genome, withinfections thatare generallymild,restrictedtotheupperrespiratoryandgastrointesti-nal tractsand oftenasymptomatic (Tyler,2001). Evenso,ithas been recognized for many years thatreovirusdisplays striking cytocidal activity when it infects certaintypes of transformed cells (Hashiro et al., 1977; Duncanet al., 1978). The underlying basis for reoviral killing ofsome tumor cell types but not others remained unknownuntil it was shown that transformed cells containingactivated Ras-dependent pathways were preferentiallysusceptible to reovirus (type 3 Dearing strain) viainactivation of PKR (dsRNA-activated protein kinase)phosphorylation (Coffey et al., 1998; Strong et al., 1998).In untransformed cells that are virus infected, viraltranscripts stimulate PKR phosphorylation, leading toinhibition of viral translation and replication. This eventis blocked in Ras-transformed cells, however, promotingactive viral replication and subsequent cytolysis of thehost cell. Thus, activation of the oncogenic Ras-signalingpathway enhances reoviral oncolytic targeting of Ras-transformed cells. As Ras gene mutations are frequentlyobserved in various types of human cancers (Duursmaand Agami, 2003), these results have led to the currentuse of reovirus in clinical trials (Norman and Lee, 2005).Other investigators have shown that reoviral oncolysis isassociated with the induction of apoptosis in variouscancer cell types (Clarke et al., 2001; Connolly andDermody, 2002; Kominsky et al., 2002). Because cellulartransformation may arise through a variety of pathways,and because acquired resistance to other cancer thera-peutic agents is commonly observed in vitro and in vivo,we speculated that resistance to reoviral oncolysis inRas-transformed cells might also arise in some cases.Here, we show that a human fibrosarcoma cell line thatcontains a well-defined activating mutation in Ras canindeed acquire resistance to reovirus following prolongedculture in vitro. The resistance to reovirus is associatedwithpersistentinfectionandviralandcellularchangesthatnevertheless permit apoptotic responses to other selectiveagents. These results will provide useful insights into thedesign of effective therapeutic strategies for use in vivo.ResultsSelection for reoviral resistance in Ras-transformedcancer cellsAlthough resistance to reoviral infection in vitro haspreviously been demonstrated (Taber et al., 1976;