Transformation-Selective Apoptotic Program Triggered by Farnesyltransferase Inhibitors Requires Bin1
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Oncogene (2003) 22, 3578–3588 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc Transformation-selective apoptotic program triggered by farnesyltransferase inhibitors requires Bin1 James B DuHadaway1, Wei Du2, Scott Donover1, Judith Baker2,5, Ai–xue Liu3,6, Diane M Sharp2, Alexander J Muller1 and George C Prendergast*,1,4 1Lankenau Institute for Medical Research, Wynnewood, PA, USA; 2Cancer Research Group, The DuPont Pharmaceuticals Company, Glenolden, PA, USA; 3The Wistar Institute, Philadelphia, PA, USA; 4Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA Neoplastic transformation sensitizes many cells to apop- therapeutic agents that exploit the heightened sensitivity tosis. This phenomenon may underlie the therapeutic of neoplastic cells to apoptosis. benefit of many anticancer drugs, but its molecular basis is Oncogene (2003) 22, 3578–3588. doi:10.1038/sj.onc.1206481 poorly understood. We have used a selective and potent farnesyltransferase inhibitor (FTI) to probe a mechanism Keywords: FTI; farnesyltransferase; Ras; RhoB; SV40 T of apoptosis that is peculiarly linked to neoplastic antigen; amphiphysin transformation. While nontoxic to untransformed mouse cells, FTI triggers a massive RhoB-dependent, p53- independent apoptosis in mouse cells that are neoplasti- cally transformed. Here we offer evidence that the BAR Introduction adapter-encoding tumor suppressor gene Bin1 is required for this transformation-selective death program. Targeted Cell suicide processes play a significant role in cancer deletion of Bin1 in primary mouse embyro fibroblasts suppression and therapeutic response. Of particular (MEFs) transformed by E1A+Ras did not affect FTI- interest are processes that are connected specially to the induced reversion, actin fiber formation, or growth death decisions made by cells harboring preneoplastic inhibition, but it abolished FTI-induced apoptosis. The or neoplastic lesions. Losses in suicide processes that are previously defined requirement for RhoB in these effects germane to cancer pathophysiology would be expected suggests that Bin1 adapter proteins act downstream or in to promote cancer progression and to blunt therapeutic parallel to RhoB in cell death signaling. The death defect responses to cancer drugs. While great progress has been in Bin1 null cells was significant insofar as it abolished made in identifying the basic mechanisms of apoptosis, FTI efficacy in tumor xenograft assays. p53 deletion did it remains unclear in many cases exactly how these not phenocopy the effects of Bin1 deletion. However, mechanisms impact cancer and therapy as they occur in MEFs transformed by SV40 large T antigen+Ras were humans. There has also been little progress is uncover- also resistant to apoptosis by FTI, consistent with other ing apoptotic mechanisms that are specially associated evidence that large T inhibits Bin1-dependent cell death by with cancer pathophysiology (that is, mechanisms that a p53-independent mechanism. Taken together, the results are germane to cancer as a disease, rather than to define a function for Bin1 in apoptosis that is conditional apoptosis generally). Such processes are of great on transformation stress. This study advances under- interest, as they may help distinguish cancers that will standing of the functions of BAR adapter proteins, which become clinically troublesome and help improve treat- are poorly understood, by revealing genetic interactions ment for those that do so. with an Rho small GTPase that functions in stress We have investigated the proapoptotic properties of signaling. The frequent losses of Bin1 expression that farnesyltransferase inhibitors (FTIs) as a way to gain occur in human breast and prostate cancers may promote novel insight into cancer cell suicide processes. FTIs are tumor progression and limit susceptibility to FTI or other a class of experimental chemotherapeutic agents that potently trigger p53-independent apoptosis in trans- formed rodent cells, but that often have little to no effect on the survival of nontransformed cells (reviewed in *Correspondence: GC Prendergast, Lankenau Institute for Medical Prendergast, 2000; Cox, 2001). Preclinical tests have Research, 100 Lancaster Avenue, Wynnewood PA 19096, USA; established a variety of antineoplastic properties of FTIs E-mail: [email protected] in various model systems, including proapoptotic 5Current address: Johnson and Johnson Pharmaceutical Research effects; this class of drugs has moved to clinical testing, Institute, Spring House, PA, USA with some promising results emerging recently in breast 6Current address: GlaxoSmithKline Pharmaceuticals Co., Inc., King of Prussia, PA, USA cancer trials. Received 29 September 2002; revised 29 January 2003; accepted 12 FTIs were developed originally as a strategy to attack February 2003 the farnesylation requirement of oncogenic Ras in Transformation stress in apoptosis by Bin1 JB DuHadaway et al 3579 human cancers. Recent work, however, has made it clear This finding bears on the functions of BAR adapter that FTI responsiveness is based upon factors beyond proteins, which are poorly understood, by supporting Ras targeting. Our laboratory has developed a model the idea that these proteins function in signal transduc- for the FTI mechanism of action that features tion. One clinical implication of our work is the the small GTPase RhoB as a critical gain-of-function potential explanation it offers for why FTI susceptibility target of these drugs (Prendergast, 2001). A genetic is limited in human cancers where Bin1 expression is proof of the crucial and transformation-selective role for frequently lost, such as breast cancers. RhoB in apoptosis by FTI and other agents used in the clinic to attack cancer cells has been demonstrated recently using a knockout mouse model system (Liu Materials and methods et al., 2000, 2001a, b). Identifying genes that act downstream of RhoB may reveal further insights into Tissue culture this novel cell death mechanism. Loss of such genes would be expected to promote cancer progression and to The generation and characterization of Bin1 nullizygous limit the action of therapeutic agents that exploit the animals will be described elsewhere. The heads, limbs, and internal organs were removed from E13.5 embryos from heightened sensitivity of most cancer cells to cell death. pregnant mice. Carcasses were individually minced in DMEM This issue is particularly germane to FTI, where clinical and trypsinized 20 min at 371C. Fetal bovine serum (FBS) was studies have not recapitulated the promising results of added to stop the trypsin and the cell suspension was seeded preclinical testing, perhaps due to secondary events that into 25 cm2 flasks containing DMEM and 10% calf serum. A occur in frank clinical cancers but are absent in model portion of this dispersed tissue was used for genotypic analysis. systems. Early passage fibroblasts were derived similarly from embryos In this study, we have identified a role for the tumor from p53–/– mice (a gift of A Levine). Embyro fibroblasts suppressor gene Bin1 in the transformation-selective were transformed at passage 2–3by transfection with 10 mg apoptotic program triggered by FTI. Bin1 is an adapter- each pT22 (mutant human H-Ras vector) and p1A/neo encoding gene that was initially identified via c-Myc (adenovirus E1A) or pCMVneo Tag (SV40 T antigen) (Pre- ndergast et al., 1991), essentially as described previously (Liu interaction (Sakamuro et al., 1996). Bin1 can induce cell et al., 2000). p1A/neo contains a fragment of the adenovirus death in human cancer cells and in c-Myc-transformed genome that expresses the 13S, 12S, and 9S E1A polypeptides. primary cells (DuHadaway et al., 2001; Elliott et al., In some experiments, we also examined the response of a 2000). However, the breadth of studies on Bin1 to date mutant H-Ras-transformed mouse embryo fibroblast (MEF) suggest a much more complex and integrative signaling cell clone that arose from a focus but that did not express E1A function which extends beyond the Myc network. The (presumably established by spontaneous immortalization). two ubiquitous Bin1 adapter proteins generated by Cells were passaged at a 1 : 5 ratio the day after transfection alternate splicing localize to the nuclear and/or cytosolic and transformed cell foci were cloned and expanded into compartments of the cell. Bin1 splice isoforms, some of E1A+Ras (ER)- or T+Ras (TR)-transformed cell lines 12–14 which are alternately termed amphiphysin II isoforms, days later. The status of Ras farnesylation in ER and TR cells was documented by a Western mobility shift assay that has have been suggested to control actin organization, been described (Liu et al., 2000). A derivative of ER +/+ cells endocytosis, and transcription (Wigge and McMahon, that overexpressed Bcl-2 was generated by transfection of a 1998; Prendergast, 1999). Roles in myoblast differentia- human Bcl-2 vector (Sakamuro et al., 1995). Transgene tion and membrane deformation related to cytosolic expression was confirmed by Western analysis with a Bcl-2 localization have been suggested (Wechsler-Reya et al., antibody (Santa Cruz Biochemicals). 1998; Lee et al., 2002). Notably, isoforms of Bin1 that localize to the nucleus can suppress transformation by Cell assays Myc and other oncogenes, and trigger p53-independent apoptosis in human breast cancers, prostate cancers, Cell morphology was documented by photography