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Bcl2l12-Mediated Inhibition of Effector Caspase-3 and Caspase-7 Via Distinct Mechanisms in Glioblastoma

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Bcl2l12-Mediated Inhibition of Effector Caspase-3 and Caspase-7 Via Distinct Mechanisms in Glioblastoma Bcl2L12-mediated inhibition of effector caspase-3 and caspase-7 via distinct mechanisms in glioblastoma Alexander H. Stegh*, Santosh Kesari*†, John E. Mahoney‡, Harry T. Jenq§, Kristin L. Forloney*, Alexei Protopopov*‡, David N. Louis¶, Lynda Chin*‡ʈ, and Ronald A. DePinho*‡**†† *Department of Medical Oncology, and ‡Center for Applied Cancer Science of the Belfer Foundation Institute for Innovative Cancer Science, Dana–Farber Cancer Institute, Boston, MA 02115; †Center for Neuro-Oncology and Department of Neurology, Brigham and Women’s Hospital, Boston, MA 02115; §Division of Health Sciences and Technology, Harvard Medical School and Massachusetts Institute of Technology, Boston, MA 02114; ¶Department of Pathology and Cancer Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; and Departments of ʈDermatology and **Medicine and Genetics, Harvard Medical School, Boston, MA 02115 Edited by William A. Weiss, University of California, San Francisco, CA, and accepted by the Editorial Board May 22, 2008 (received for review January 7, 2008) Glioblastoma multiforme (GBM) is a highly aggressive brain cancer matched well with direct physical interaction in vitro and in vivo with that is characterized by the paradoxical features of intense apoptosis the pro-caspase. The means through which Bcl2L12 inhibited resistance yet a marked propensity to undergo necrosis. Bcl2L12 (for caspase-3 was not defined and did not involve physical interaction Bcl2-Like12) is a nuclear and cytoplasmic oncoprotein that is univer- between Bcl2L12 and caspase-3. sally overexpressed in primary GBM and functions to block postmi- In this study, we sought to elucidate the mechanism of Bcl2L12- tochondrial apoptosis signaling by neutralizing effector caspase-3 mediated neutralization of caspase-3. Transcriptome analyses of and caspase-7 maturation. This postmitochondrial block in apoptosis Bcl2L12-expressing astrocytic cultures revealed prominent up- engenders the alternate cell fate of cellular necrosis, thus providing a regulation of ␣B-crystallin expression. The previously described molecular explanation for GBM’s classical features. Whereas Bcl2L12- capacity of recombinant ␣B-crystallin protein to inhibit caspase-3 mediated neutralization of caspase-7 maturation involves physical activation in a cell-free system (14) prompted a detailed analysis of interaction, the mechanism governing Bcl2L12-mediated inhibition of a potential Bcl2L12-␣B-crystallin signaling axis in cultured astro- caspase-3 activity is not known. The nuclear localization of Bcl2L12 cytes and glioma cell lines, orthotopic glioma xenotransplants, and prompted expression profile studies of primary astrocytes engi- primary GBM specimens. Gain- and loss-of-function assays and neered to overexpress Bcl2L12. The Bcl2L12 transcriptome revealed a detailed biochemical studies in both normal and neoplastic glial striking induction of the small heat shock protein ␣-basic-crystallin model systems establish a pathway comprised of Bcl2L12–␣B- (␣B-crystallin/HspB5), a link reinforced by robust ␣B-crystallin expres- crystallin–caspase-3 in the regulation of apoptosis and necrosis in sion in Bcl2L12-expressing orthotopic glioma and strong coexpression vitro and in vivo. Together, these studies reveal the multifunctional of ␣B-crystallin and Bcl2L12 proteins in human primary GBMs. On the nature of the Bcl2L12 oncoprotein, operating in both the cytoplasm functional level, enforced ␣B-crystallin or Bcl2L12 expression en- and nucleus to effect survival signaling in gliomagenesis. SCIENCES hances orthotopic tumor growth. Conversely, RNAi-mediated knock- ␣ Results down of B-crystallin in Bcl2L12-expressing astrocytes and glioma cell APPLIED BIOLOGICAL lines with high endogenous ␣B-crystallin showed enhanced apopto- Bcl2L12 Drives Up-Regulation of ␣B-Crystallin. The cytoplasmic pres- sis, yet decreased necrotic cell death with associated increased ence of Bcl2L12 is consistent with its physical and inhibitory actions caspase-3 but not caspase-7 activation. Mirroring this specific effect on caspase-7 (13). At the same time, abundant Bcl2L12 in the on effector caspase-3 activation, ␣B-crystallin selectively binds pro- nucleoplasm prompted transcriptional profiling studies to assess caspase-3 and its cleavage intermediates in vitro and in vivo. Thus, whether the range of Bcl2L12’s antiapoptotic activities might ␣B-crystallin is a Bcl2L12-induced oncoprotein that enables Bcl2L12 to extend to more direct effects on gene expression. Strikingly, block the activation of both effector caspases via distinct mechanisms, Bcl2L12V5-expressing Ink4a/Arf-deficient cortical astrocytes thereby contributing to GBM pathogenesis and its hallmark biological showed marked up-regulation of ␣B-crystallin by micorarray, quan- properties. titative RT-PCR, and Western blot analyses [Fig. 1A, supporting information (SI) Table S1, and data not shown]. In contrast, heat shock protein ͉ apoptosis/necrosis balance ͉ glial cells ␣B-crystallin was unchanged in the context of Bcl-2 expression (Fig. 1A), suggesting that up-regulation of ␣B-crystallin is not a general function of Bcl-2 family proteins. The specific physiological link lioblastoma multiforme (GBM) is a highly aggressive brain ␣ Gcancer characterized by rapid tumor cell proliferation, intense between Bcl2L12 and B-crystallin is supported further by the apoptosis resistance, and marked necrosis, tumor biological fea- inability of Bcl2L12 to influence expression of other apoptosis tures underlying its neurologically destructive course and its high modulators including postmitochondrial caspases and their inhib- lethality typically within 12–24 months of diagnosis. The mecha- itors and other members of the heat shock protein family such as nisms underlying GBM’s intense apoptosis resistance and associ- Hsp70 and Hsp27 (Fig. 1A and Table S1). Table S2 provides a ated poor therapeutic responsiveness relate in part to the reinforc- complete list of differentially expressed genes by 1.3-fold relative to ing activities of coactivated RTKs (1) and defective PTEN and p53 empty vector controls. proteins (2, 3) and modulation of classical apoptosis regulators such as Bcl-2 family proteins (4–7), soluble decoy receptor 3 (DcR3) (8), Author contributions: A.H.S. and R.A.D. designed research; A.H.S., S.K., J.E.M., H.T.J., K.L.F., APRIL (9), and PEA-15 (10–12). and A.P. performed research; A.H.S., S.K., J.E.M., A.P., D.N.L., and L.C. analyzed data; and In GBM oncogenomic studies, we identified and functionally A.H.S., L.C., and R.A.D. wrote the paper. characterized Bcl2L12 (for Bcl2-Like-12), a proline-rich and BH2 The authors declare no conflict of interest. domain-containing protein that is robustly expressed in virtually all This article is a PNAS Direct Submission. W.A.W. is a guest editor invited by the Editorial human primary GBM specimens (13). Enforced Bcl2L12 expres- Board. sion in primary cortical astrocytes conferred marked apoptosis ††To whom correspondence should be addressed. E-mail: ron࿝[email protected]. resistance and engendered cellular necrosis through inhibition of This article contains supporting information online at www.pnas.org/cgi/content/full/ effector caspase-3 and caspase-7 maturation downstream of mito- 0712034105/DCSupplemental. chondria (13). The capacity of Bcl2L12 to block caspase-7 activation © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0712034105 PNAS ͉ August 5, 2008 ͉ vol. 105 ͉ no. 31 ͉ 10703–10708 Downloaded by guest on September 24, 2021 A C αB-crystallinhigh αB-crystallinhigh αB-crystallinlow GBM-1 GBM-2 GBM-3 pBabe Bcl-2 kDa LacZ Bcl2L12 75- -Hsp70 40x 105- -Hsp90 Bcl2L12 B-crystallin 25- -αB-crystallin α -Hsp27 zoom 25- D 50- -XIAP p<0.01 B 400 Bcl2L12 B-crystallin ␣ α α Fig. 1. Bcl2L12 up-regulates B-crystallin protein levels in vitro and in vivo.(A) Western blot analysis of 350 postmitochondrial apoptosis effectors in Ink4a/ArfϪ/Ϫ astrocytes ectopically expressing a pBabe control, 300 Bcl2L12V5, LacZV5, and Bcl-2. The migration positions of p=0.018 Hsp70, Hsp90, ␣B-crystallin, Hsp27, and XIAP are indi- ␣ 250 cated. (B) Quantification of B-crystallin positivity in U98MG xenograft tumor sections. A total of 10–15 low 0% 57% 0% 44% 0% 5% power fields (LPFs) per genotype were counted. Error 200 bars represent SDs, and P values were calculated by using Student’s t test. (C) Coexpression analysis of 12% 31% 16% 40% 37% 58% ␣ 150 B-crystallin B-crystallin and Bcl2L12 in primary human GBM. GBM α Bcl2L12 cores were stained with a monoclonal anti-␣B- crystallin antibody (red) and a polyclonal Bcl2L12 (anti- B-crystallin-postivie100 cells/LPF L12–2) antiserum (brown). Shown are three represen- α E ρ=0.62 ρ=0.62 50,000 tative cores at ϫ40 magnifications. (D) Heat map (Top; 50 rel. intensity yellow dots ϭ Bcl2L12 positive; red dots ϭ ␣B-crystallin 40,000 positive) and density plot analyses (Middle) of Bcl2L12/ ␣B-crystallin double stainings in three representative 0 30,000 2 GBM cores shown in C. The percentages of Bcl2L12 and e 1 (+) b 20,000 ␣ L a B-crystallin-positive and -negative tumor cells are in- 2 l B c dicated (Bottom) with Bcl2L12 intensities plotted on p 10,000 B the x axis and ␣B-crystallin intensities on the y axis. (E) U87MG xenograft 0 Analysis of coexpression across 61 GBM cores in com- 46 51 56 61 31 36 41 parison with normal brain. Shown are intensity levels 16 21 26 6 11 Bcl2L12pos of Bcl2L12 (yellow), ␣B-crystallin (red), and levels of Tumor # αB-crystallinpos coexpression (green). Pearson correlation coefficient normal Double pos. ␳ ϭ 0.62. To assess whether Bcl2L12V5-dependent up-regulation of ␣B- detailed staining protocol and analysis methodology). Triply crystallin in cell culture is also operative in glioma cells in vivo,we stained for Bcl2L12 [diaminobenzidine (DAB), brown], ␣B- examined ␣B-crystallin protein expression in intracranial xeno- crystallin (FastRed), and hematoxylin, TMA sections were scanned transplants derived from the well established U87MG human with three lasers in distinct wavelength channels, demonstrating the glioma model system. Bcl2L12V5 and EGFRvIII overexpression coexpression of both proteins (Fig.
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