Tipifarnib sensitizes cells to proteasome inhibition by blocking degradation of bortezomib-induced aggresomes

Abstract

In this report, we investigated the mechanism responsible for synergistic induction of myeloma cell apoptosis induced by the combination of tipifarnib and bortezomib. Immunofluorescence studies revealed that bortezomib alone resulted in an accumulation of puncta of ubiquitinated proteins that was further enhanced by the addition of tipifarnib. These data suggest inhibition of the degradation of bortezomib-induced aggresomes; and consistent with this possibility, we also observed an increase in p62SQSTM1 in cells treated with the combination. However, autophagy in these cells appears to be normal as LC3BII is present, and auto- IntroductionWe have previously reported that the farnesyl transferase inhibitor (FTI) lonafarnib (SCH66336) combined with the proteasome inhibitor bortezomib induced synergistic apoptosis in myeloma cell lines and primary patient cells; however, the mechanism responsible for this synergy was unclear. 1-5 Whereas many groups have speculated that FTIs induce their effect via ras signaling, 6-8 others have suggested alternative pathways by which FTIs exert their anticancer effects. 9-11 Marcus et al first reported that FTIs inhibit histone deacetylase 6 expression, resulting in destabilization of the microtubule assembly structure, and impaired cell survival in nonsmall cell lung cancer cell lines. 12 Based on published preclinical data demonstrating that histone deacetylase inhibitors synergize with proteasome inhibition via inhibition of aggresome formation, we sought to determine whether the in vitro synergy we have previously observed using the combination of an FTI with bortezomib was a result of dual blockade of the proteasome and aggresome pathways of protein catabolism. MethodsThe MM.1S (provided by S. Rosen, Chicago, IL) and RPMI8226 (ATCC) cell lines were used in this study. Tipifarnib (R115777) was provided by Johnson & Johnson Pharmaceuticals, and bortezomib was provided by Millennium Pharmaceuticals. Methyl-thiazol-tetrazolium assays, annexin V staining, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were performed according to previously published methods. 1,5,13 The antibodies used included antiubiquitin, anticaspase-8, anticaspase-9, anticaspase-3, and anti-poly-adenosine diphosphate or PARP ribose polymerase (Cell Signaling). p62SQSTM1 antibody (MBL International), bafilomycin A 1 , and LC3B antibody (Sigma-Aldrich) were used to assess autophagy. Confocal microscopy was performed using standard methods with minor modifications to assess aggresome formation. 12,13 Vimentin (SigmaAldrich) and ␥-tubulin (Abcam) antibodies were used in the evaluation of aggresome formation by confocal immunofluorescence microscopy. Results and discussionConsistent with our previous findings using lonafarnib in myeloma cells, the combination of tipifarnib and bortezomib resulted in greater growth inhibition than when either agent was used separately in both MM.1S as well as in RPMI8826 with concentrations up to 10nM of bortezomib and 5M of tipifarnib (Figure 1). Combination therapy...

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