RE S EAR CH | R E P O R T S
RE FE RENCES AND N OT ES
1. D. W. Stephens, J. R. Krebs, Foraging Theory (Princeton Univ. Press, Princeton, NJ, 1986). 2. R. P. Wilson et al., J. Anim. Ecol. 75, 1081–1090 (2006). 3. W. J. Gonyea, Acta Anat. 96, 81–96 (1976). 4. M. Sunquist, F. Sunquist, in Carnivore Behavior, Ecology and Evolution, J. Gittleman, Ed. (Cornell Univ. Press, Ithaca, NY, 1989), pp. 283–301. 5. B. VanValkenburgh, Paleobiology 11, 406–428 (1985). 6. D. W. Macdonald, A. J. Loveridge, K. Nowell, in Biology and Conservation of Wild Felids, D. Macdonald, A. Loveridge, Eds. (Oxford Univ. Press, Oxford, 2010), pp. 3–58. 7. A. M. Wilson et al., Nature 498, 185–189 (2013). 8. J. W. Wilson et al., Biol. Lett. 9, 20130620 (2013). 9. G. B. Schaller, The Serengeti Lion (Univ. of Chicago Press, Chicago, 1972). 10. M. L. Gorman, M. G. Mills, J. P. Raath, J. R. Speakman, Nature 391, 479–481 (1998). 11. I. Girard, Physiol. Biochem. Zool. 74, 191–202 (2001). 12. J. B. Williams, D. Lenain, S. Ostrowski, B. I. Tieleman, P. J. Seddon, Physiol. Biochem. Zool. 75, 479–488 (2002). 13. See supplementary materials on Science Online. 14. T. M. Williams et al., Comp. Biochem. Physiol. A 133, 203–212 (2002). 15. L. Qasem et al., PLOS ONE 7, e31187 (2012). 16. C. R. Taylor, N. C. Heglund, G. M. O. Maloiy, J. Exp. Biol. 97, 1–21 (1982). 17. N. C. Heglund, C. R. Taylor, T. A. McMahon, Science 186, 1112–1113 (1974). 18. P. S. Chassin, C. R. Taylor, N. C. Heglund, H. Seeherman, Physiol. Zool. 49, 1–10 (1976). 19. L. G. Halsey et al., Comp. Biochem. Physiol. A 152, 197–202 (2009). 20. C. R. Taylor et al., Respir. Physiol. 44, 25–37 (1981). 21. C. S. Miller et al., Biol. Conserv. 170, 120–129 (2014). 22. J. Laundré, L. Hernandez, J. Arid Environ. 55, 675–689 (2003). 23. J. Laundre, J. Wildl. Manage. 69, 723–732 (2005). 24. C. R. Taylor, K. Schmidt-Nielsen, J. L. Raab, Am. J. Physiol. 219, 1104–1107 (1970). 25. B. K. McNab, Can. J. Zool. 78, 2227–2239 (2000). 26. O. R. Bidder, L. A. Qasem, R. P. Wilson, PLOS ONE 7, e50556 (2012). 27. D. L. Kramer, R. McLaughlin, Am. Zool. 41, 137–153 (2001). 28. R. P. Wilson et al., Ecol. Lett. 16, 1145–1150 (2013). 29. C. C. Wilmers et al., PLOS ONE 8, e60590 (2013). 30. K. Corts, F. Lindzey, J. Wildl. Manage. 48, 1456–1458 (1984).
Department of Fish and Wildlife. We thank P. Houghtaling, Y. Shakeri, C. Wylie, and D. Tichenor for assistance in catching wild pumas and finding kill sites, and J. A. Estes and the anonymous referees for critical review of this manuscript. Animal procedures were approved by the UCSC IACUC. Statistical data are tabulated in the supplementary materials, with electronic versions available upon request from the senior author through the UCSC Mammal Physiology Project database.
www.sciencemag.org/content/346/6205/81/suppl/DC1 Materials and Methods Tables S1 to S3 References (31–38) 16 April 2014; accepted 30 July 2014 10.1126/science.1254885
PROSTATE CANCER
Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer Jean-Philippe P. Theurillat,1,2,3* Namrata D. Udeshi,1* Wesley J. Errington,4,5* Tanya Svinkina,1 Sylvan C. Baca,2,3 Marius Pop,1,2,3 Peter J. Wild,6 Mirjam Blattner,7 Anna C. Groner,3 Mark A. Rubin,7,8 Holger Moch,6 Gilbert G. Privé,4,5 Steven A. Carr,1 Levi A. Garraway1,2,3,9† Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer–associated mutations of SPOP, an E3 ubiquitin ligase substrate-binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, which implicated DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.
G
enome sequencing studies have revealed unanticipated roles for the ubiquitylation machinery in cancer. For example, the cullin-RING ubiquitin ligase adaptor protein speckle-type POZ protein (SPOP) is mutated in 8 to 14% of prostate and endometrial cancers (1–4). In prostate cancer, SPOP mutations are confined to specific amino acid residues within the substrate-binding cleft, which mediates substrate interaction and ubiquitin transfer (5). This exquisite localization suggests that SPOP mutations have undergone positive selection during tumorigenesis by altering binding and ubiquitylation of distinct effector substrates in a protumorigenic manner. However, the mecha-
1
ACKN OW LEDG MEN TS
The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. 2Harvard Medical School, Boston, MA 02115, USA. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA. 4Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. 5 Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5G 2M9, Canada. 6Institute of Surgical Pathology, University Hospital Zurich, ZH 8091 Zurich, Switzerland. 7Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY 10065, USA. 8Institute for Precision Medicine of Weill Cornell and New York Presbyterian Hospital, New York, NY 10065, USA. 9Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Supported by NSF grants DBI-0963022 and 1255913 (T.M.W., C.C.W., and G.H.E.) with in-kind support from the California
*These authors contributed equally to this work. †Corresponding author. E-mail: [email protected]
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nisms and substrates underlying this phenomenon remain incompletely understood. In principle, mutant SPOP could enhance ubiquitylation of SPOP substrates (gain-of-function effect) or ubiquitylate new substrates (neomorphic effect). Alternatively, SPOP mutants could dimerize with their wild-type counterparts (e.g., through the BTB and BACK domains), and so repress wild-type SPOP function (dominantnegative effect). In support of the latter model, SPOP mutations typically occur in a heterozygous state with a retained wild-type allele and are able to dysregulate known substrates [e.g., nuclear receptor coactivator 3 (NCOA3)] in a dominant-negative manner (1, 6). Characterizing the ubiquitin landscape (or “ubiquitylome”) that results from cancer genomic alterations affecting ubiquitin ligase components may provide new insights into tumorigenesis. To interrogate changes in ubiquitylation conferred by prostate cancer SPOP mutations, we stably overexpressed vector control (C); wild-type SPOP (SPOP-WT); or a mutated variant (SPOPF133L or SPOP-Y87N, in which Phe133 is replaced by Leu or Tyr87 is replaced by Asn (SPOP-MT)], in immortalized prostate epithelial cells expressing endogenous SPOP (fig. S1, A and B) (7). In each case, we characterized the resulting ubiquitylome by measuring glycine-glycine remnants of ubiquitylated lysines (K-e-GG) after 3 OCTOBER 2014 • VOL 346 ISSUE 6205
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Although our study highlights one energetically expensive activity (hunting) for a single carnivore, there are more than 245 mammalian carnivore species (www.iucnredlist.org) and numerous omnivores that pursue and kill mobile, elusive prey. Most have specialized hunting styles and would be expected to display highly variable instantaneous rates of energy expenditure that differ widely among predator and prey, habitats, and even individuals. The laboratory-to-field approach used here, which pairs basic physiological attributes of the animal with standard wildlife monitoring, provides a powerful way of quantifying such species-specific energetic demands. Some activities, including hunting and locomotion across complex habitats, are energetically costly; most are woefully underestimated by many predictive algorithms in common use. Correcting this will become increasingly important for the preservation of large carnivores as foraging becomes progressively affected by continued habitat degradation and loss of vegetative cover essential for cryptic movements (4, 29).
R ES E A RC H | R E PO R TS
D1). Differentially regulated (more than twofold) K-e-GG peptides generally followed a dominantnegative pattern, including those corresponding to WIZ, SCAF1, GLYR1, DEK, and TRIM24 proteins (Fig. 1B and figs. S1, C and D, and S2, A to E). K-e-GG peptides from two proteins (corresponding to G3BP1 and CAPRIN1) showed a loss-offunction pattern (figs. S1, C and D, and S2, F and G), and two K-e-GG peptides from SPOP itself showed up-regulation in the setting of both SPOPWT and SPOP-MT overexpression, suggestive of autoubiquitylation (fig. S1, C and D). Although we observed no evidence for gain-of-function or neomorphic effects, we cannot exclude the existence of such mechanisms. We next determined whether SPOP-binding motifs were enriched in the differentially regulated proteins. Indeed, of the seven proteins described above (excluding SPOP itself), three (43%) contained a previously characterized SPOP-binding sequence (DEK, SCAF1, and CAPRIN1) (5). In contrast, the frequency of SPOP-binding sites was significantly lower (~3%) across the ubiquitylome as a whole (P < 0.001) (table S2). We identified one
Fig. 1. K-e-GG (ubiquitin)–profiling and validation for DEK in immortalized prostate epithelial cells (LHMAR) overexpressing vector control, SPOP-WT and SPOP-F133L/Y87N mutants (MT). (A) Schematic showing the design of the proteomics experiments. Isogenic cell line expressing either vector control, SPOP-WT or SPOP-MT were isotopically labeled (see fig. S1, A and B). K-e-GG ratios were normalized to protein expression changes, assessed in parallel. (B) Scatter plot of protein normalized K-e-GG level changes between SPOP-MT and SPOP-WT (depicted as log2 ratio of MT/WT) shows coordinate down-regulation of distinct K-e-GG peptides across replicates (see also figs. S1, C and D, and S2, A to G). (C) To visualize K-e-GG sites
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K-e-GG peptide corresponding to DAXX, a known SPOP substrate (8). As expected, this peptide was enriched—and the corresponding total protein was down-regulated—after overexpression of wildtype SPOP (fig. S3A). Expression of the transcriptional coactivator NCOA3, another SPOP substrate, followed a dominant-negative pattern, as reported previously (6, 9) (fig. S3B). Because protein ubiquitylation is often a prerequisite for proteasomal degradation, we next sought to ascertain which differentially expressed K-e-GG peptides showed an inverse correlation with total protein expression (database D1). Peptides corresponding to TRIM24, SCAF1, and GLYR1 fit this pattern (one peptide each) (Fig. 1C and fig. S3, C and D). TRIM24 has been shown to mediate ligand-dependent activation of the androgen receptor—a key prostate cancer driver—and to degrade TP53 through its E3 ligase activity (10, 11). This observation is of interest given that primary prostate cancers with SPOP mutations typically lack TP53 alterations (1). However, the K-e-GG peptides that exhibited the most profound down-regulation coupled with
that undergo inverse changes at the protein level (e.g., more than twofold), normalized K-e-GG data shown in (B) was inverted and multiplied by corresponding total protein expression changes (see also fig. S3, C and D, for expression changes). (D) DEK protein expression across isogenic LHMAR cells assessed by immunoblotting with and without proteasome inhibition by MG132. DMSO, diemthyl sulfoxide. Error bars represent means T SD, n = 4 (n.s., not significant; ∗∗P < 0.01, Student's t test). (E) Degradation of Flag-DEK over time. Flag-DEK was induced by doxycycline (Dox) and protein decay measured by immunoblotting after doxycycline withdrawal in inducible LHMAR cells (P value, Friedman test). See also fig. S3E for DEK mRNA expression.
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trypsin digestion and stable isotope labeling of amino acids in cell culture (SILAC)–based mass spectrometry (Fig. 1A). To account for ubiquitylationrelated changes in protein expression, all K-e-GG values were normalized to protein abundance (Fig. 1A) (database D1 in supplementary materials). We reasoned that putative K-e-GG peptide substrates might show distinct patterns of protein abundance depending on whether mutant SPOP caused a dominant-negative, loss-of-function, gainof-function, or neomorphic effect. In a dominantnegative context, substrate peptides should exhibit a [SPOP-WT > control > SPOP-MT] pattern of abundance, whereas a pure loss-of-function effect might yield a [SPOP-WT > control = SPOP-MT] pattern. In contrast, a gain-of-function effect may result in a [SPOP-MT > SPOP-WT > control] pattern, and a neomorphic effect should cause a [SPOP-MT > SPOP-WT = control] pattern. Only 12 of 7181 K-e-GG peptides detected exhibited more than twofold changes in peptide abundance in the SPOP-mutant contexts (z score >3 or 80% of mutations identified in this tumor type (1)] conferred increased DEK protein expression, whereas two recurrent endometrial cancer–associated SPOP mutants (S80R and E50K) (2, 3) had no effect (Fig. 4A and figs. S8A, S9A, and S10A) in prostate cells. As expected, DEK expression correlated with sphere-forming capacity and cell invasion without affecting baseline cell proliferation or viability (figs. S8, B and C, and S9B). Finally, we considered whether other putative SPOP effector proteins—both known substrates and novel ones nominated by our ubiquitylome screen—might also undergo dysregulated expression in the setting of prostate cancer SPOP mutations (8, 9, 21–23). Regarding the latter, we noted that TRIM24 represented another putative SPOP substrate from our screen with potential biological relevance to prostate cancer (Fig. 1, B and C, and fig. S3C) (10, 11). As expected several known or putative SPOP substrates were downregulated in response to SPOP-WT overexpression (including DEK, TRIM24, NCOA3, DAXX, BRMS1, and GLI1 and 2) (Fig. 4A and fig. S10A). However, only DEK, TRIM24, and NCOA3 also became up-regulated upon overexpression of prostate cancer SPOP mutants in this setting (6). Moreover, nuclear expression of these proteins correlated significantly with SPOP mutations in 181/178 primary prostate cancers analyzed by immunohistochemistry (Fig. 4, B and C, and fig. S10B, table S3). These data are consistent with the ubiquitylome and experimental studies, which supports a model in which SPOP mutations may engage overlapping routes of transformation in prostate cancer by dysregulation of DEK, TRIM24, and NCOA3. These data support a model wherein prostate cancer SPOP mutants impair ubiquitylation and degradation of a subset of SPOP substrates in a dominant-negative manner to promote tumorigenesis. Future studies of DEK dysregulation and its biological functions in normal and tumor tissue may therefore promote new biological insights relevant to many human cancers. More generally, large-scale profiling of the ubiquitin landscape linked to tumor genomic alterations in protein homeostasis genes may catalyze the identification of downstream effector mechanisms. Given the growing number of such genes
Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer Jean-Philippe P. Theurillat, Namrata D. Udeshi, Wesley J. Errington, Tanya Svinkina, Sylvan C. Baca, Marius Pop, Peter J. Wild, Mirjam Blattner, Anna C. Groner, Mark A. Rubin, Holger Moch, Gilbert G. Privé, Steven A. Carr and Levi A. Garraway
Science 346 (6205), 85-89. DOI: 10.1126/science.1250255
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REFERENCES
This article cites 30 articles, 11 of which you can access for free http://science.sciencemag.org/content/346/6205/85#BIBL
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Mutant protein in tumors hits the DEK Cancer genome sequencing projects have uncovered a multitude of mutations in human tumors. Understanding whether and how these mutations contribute to tumor development and progression could ultimately lead to new therapies. Theurillat et al. studied the protein product of a gene that is recurrently mutated in prostate cancer. Normally this protein helps attach a biochemical tag to cellular proteins that marks them for degradation. The new work shows that the tumor-associated mutant protein loses this tagging ability, which results in the stabilization of a handful of cellular proteins that would otherwise be degraded. One of the most intriguing of these proteins was DEK, which helps prostate cancer cells invade into surrounding tissue. Science, this issue p. 85
RE FE RENCES AND N OT ES
1. D. W. Stephens, J. R. Krebs, Foraging Theory (Princeton Univ. Press, Princeton, NJ, 1986). 2. R. P. Wilson et al., J. Anim. Ecol. 75, 1081–1090 (2006). 3. W. J. Gonyea, Acta Anat. 96, 81–96 (1976). 4. M. Sunquist, F. Sunquist, in Carnivore Behavior, Ecology and Evolution, J. Gittleman, Ed. (Cornell Univ. Press, Ithaca, NY, 1989), pp. 283–301. 5. B. VanValkenburgh, Paleobiology 11, 406–428 (1985). 6. D. W. Macdonald, A. J. Loveridge, K. Nowell, in Biology and Conservation of Wild Felids, D. Macdonald, A. Loveridge, Eds. (Oxford Univ. Press, Oxford, 2010), pp. 3–58. 7. A. M. Wilson et al., Nature 498, 185–189 (2013). 8. J. W. Wilson et al., Biol. Lett. 9, 20130620 (2013). 9. G. B. Schaller, The Serengeti Lion (Univ. of Chicago Press, Chicago, 1972). 10. M. L. Gorman, M. G. Mills, J. P. Raath, J. R. Speakman, Nature 391, 479–481 (1998). 11. I. Girard, Physiol. Biochem. Zool. 74, 191–202 (2001). 12. J. B. Williams, D. Lenain, S. Ostrowski, B. I. Tieleman, P. J. Seddon, Physiol. Biochem. Zool. 75, 479–488 (2002). 13. See supplementary materials on Science Online. 14. T. M. Williams et al., Comp. Biochem. Physiol. A 133, 203–212 (2002). 15. L. Qasem et al., PLOS ONE 7, e31187 (2012). 16. C. R. Taylor, N. C. Heglund, G. M. O. Maloiy, J. Exp. Biol. 97, 1–21 (1982). 17. N. C. Heglund, C. R. Taylor, T. A. McMahon, Science 186, 1112–1113 (1974). 18. P. S. Chassin, C. R. Taylor, N. C. Heglund, H. Seeherman, Physiol. Zool. 49, 1–10 (1976). 19. L. G. Halsey et al., Comp. Biochem. Physiol. A 152, 197–202 (2009). 20. C. R. Taylor et al., Respir. Physiol. 44, 25–37 (1981). 21. C. S. Miller et al., Biol. Conserv. 170, 120–129 (2014). 22. J. Laundré, L. Hernandez, J. Arid Environ. 55, 675–689 (2003). 23. J. Laundre, J. Wildl. Manage. 69, 723–732 (2005). 24. C. R. Taylor, K. Schmidt-Nielsen, J. L. Raab, Am. J. Physiol. 219, 1104–1107 (1970). 25. B. K. McNab, Can. J. Zool. 78, 2227–2239 (2000). 26. O. R. Bidder, L. A. Qasem, R. P. Wilson, PLOS ONE 7, e50556 (2012). 27. D. L. Kramer, R. McLaughlin, Am. Zool. 41, 137–153 (2001). 28. R. P. Wilson et al., Ecol. Lett. 16, 1145–1150 (2013). 29. C. C. Wilmers et al., PLOS ONE 8, e60590 (2013). 30. K. Corts, F. Lindzey, J. Wildl. Manage. 48, 1456–1458 (1984).
Department of Fish and Wildlife. We thank P. Houghtaling, Y. Shakeri, C. Wylie, and D. Tichenor for assistance in catching wild pumas and finding kill sites, and J. A. Estes and the anonymous referees for critical review of this manuscript. Animal procedures were approved by the UCSC IACUC. Statistical data are tabulated in the supplementary materials, with electronic versions available upon request from the senior author through the UCSC Mammal Physiology Project database.
www.sciencemag.org/content/346/6205/81/suppl/DC1 Materials and Methods Tables S1 to S3 References (31–38) 16 April 2014; accepted 30 July 2014 10.1126/science.1254885
PROSTATE CANCER
Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer Jean-Philippe P. Theurillat,1,2,3* Namrata D. Udeshi,1* Wesley J. Errington,4,5* Tanya Svinkina,1 Sylvan C. Baca,2,3 Marius Pop,1,2,3 Peter J. Wild,6 Mirjam Blattner,7 Anna C. Groner,3 Mark A. Rubin,7,8 Holger Moch,6 Gilbert G. Privé,4,5 Steven A. Carr,1 Levi A. Garraway1,2,3,9† Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer–associated mutations of SPOP, an E3 ubiquitin ligase substrate-binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, which implicated DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation.
G
enome sequencing studies have revealed unanticipated roles for the ubiquitylation machinery in cancer. For example, the cullin-RING ubiquitin ligase adaptor protein speckle-type POZ protein (SPOP) is mutated in 8 to 14% of prostate and endometrial cancers (1–4). In prostate cancer, SPOP mutations are confined to specific amino acid residues within the substrate-binding cleft, which mediates substrate interaction and ubiquitin transfer (5). This exquisite localization suggests that SPOP mutations have undergone positive selection during tumorigenesis by altering binding and ubiquitylation of distinct effector substrates in a protumorigenic manner. However, the mecha-
1
ACKN OW LEDG MEN TS
The Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. 2Harvard Medical School, Boston, MA 02115, USA. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA. 4Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada. 5 Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5G 2M9, Canada. 6Institute of Surgical Pathology, University Hospital Zurich, ZH 8091 Zurich, Switzerland. 7Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY 10065, USA. 8Institute for Precision Medicine of Weill Cornell and New York Presbyterian Hospital, New York, NY 10065, USA. 9Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Supported by NSF grants DBI-0963022 and 1255913 (T.M.W., C.C.W., and G.H.E.) with in-kind support from the California
*These authors contributed equally to this work. †Corresponding author. E-mail: [email protected]
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3
nisms and substrates underlying this phenomenon remain incompletely understood. In principle, mutant SPOP could enhance ubiquitylation of SPOP substrates (gain-of-function effect) or ubiquitylate new substrates (neomorphic effect). Alternatively, SPOP mutants could dimerize with their wild-type counterparts (e.g., through the BTB and BACK domains), and so repress wild-type SPOP function (dominantnegative effect). In support of the latter model, SPOP mutations typically occur in a heterozygous state with a retained wild-type allele and are able to dysregulate known substrates [e.g., nuclear receptor coactivator 3 (NCOA3)] in a dominant-negative manner (1, 6). Characterizing the ubiquitin landscape (or “ubiquitylome”) that results from cancer genomic alterations affecting ubiquitin ligase components may provide new insights into tumorigenesis. To interrogate changes in ubiquitylation conferred by prostate cancer SPOP mutations, we stably overexpressed vector control (C); wild-type SPOP (SPOP-WT); or a mutated variant (SPOPF133L or SPOP-Y87N, in which Phe133 is replaced by Leu or Tyr87 is replaced by Asn (SPOP-MT)], in immortalized prostate epithelial cells expressing endogenous SPOP (fig. S1, A and B) (7). In each case, we characterized the resulting ubiquitylome by measuring glycine-glycine remnants of ubiquitylated lysines (K-e-GG) after 3 OCTOBER 2014 • VOL 346 ISSUE 6205
85
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Although our study highlights one energetically expensive activity (hunting) for a single carnivore, there are more than 245 mammalian carnivore species (www.iucnredlist.org) and numerous omnivores that pursue and kill mobile, elusive prey. Most have specialized hunting styles and would be expected to display highly variable instantaneous rates of energy expenditure that differ widely among predator and prey, habitats, and even individuals. The laboratory-to-field approach used here, which pairs basic physiological attributes of the animal with standard wildlife monitoring, provides a powerful way of quantifying such species-specific energetic demands. Some activities, including hunting and locomotion across complex habitats, are energetically costly; most are woefully underestimated by many predictive algorithms in common use. Correcting this will become increasingly important for the preservation of large carnivores as foraging becomes progressively affected by continued habitat degradation and loss of vegetative cover essential for cryptic movements (4, 29).
R ES E A RC H | R E PO R TS
D1). Differentially regulated (more than twofold) K-e-GG peptides generally followed a dominantnegative pattern, including those corresponding to WIZ, SCAF1, GLYR1, DEK, and TRIM24 proteins (Fig. 1B and figs. S1, C and D, and S2, A to E). K-e-GG peptides from two proteins (corresponding to G3BP1 and CAPRIN1) showed a loss-offunction pattern (figs. S1, C and D, and S2, F and G), and two K-e-GG peptides from SPOP itself showed up-regulation in the setting of both SPOPWT and SPOP-MT overexpression, suggestive of autoubiquitylation (fig. S1, C and D). Although we observed no evidence for gain-of-function or neomorphic effects, we cannot exclude the existence of such mechanisms. We next determined whether SPOP-binding motifs were enriched in the differentially regulated proteins. Indeed, of the seven proteins described above (excluding SPOP itself), three (43%) contained a previously characterized SPOP-binding sequence (DEK, SCAF1, and CAPRIN1) (5). In contrast, the frequency of SPOP-binding sites was significantly lower (~3%) across the ubiquitylome as a whole (P < 0.001) (table S2). We identified one
Fig. 1. K-e-GG (ubiquitin)–profiling and validation for DEK in immortalized prostate epithelial cells (LHMAR) overexpressing vector control, SPOP-WT and SPOP-F133L/Y87N mutants (MT). (A) Schematic showing the design of the proteomics experiments. Isogenic cell line expressing either vector control, SPOP-WT or SPOP-MT were isotopically labeled (see fig. S1, A and B). K-e-GG ratios were normalized to protein expression changes, assessed in parallel. (B) Scatter plot of protein normalized K-e-GG level changes between SPOP-MT and SPOP-WT (depicted as log2 ratio of MT/WT) shows coordinate down-regulation of distinct K-e-GG peptides across replicates (see also figs. S1, C and D, and S2, A to G). (C) To visualize K-e-GG sites
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K-e-GG peptide corresponding to DAXX, a known SPOP substrate (8). As expected, this peptide was enriched—and the corresponding total protein was down-regulated—after overexpression of wildtype SPOP (fig. S3A). Expression of the transcriptional coactivator NCOA3, another SPOP substrate, followed a dominant-negative pattern, as reported previously (6, 9) (fig. S3B). Because protein ubiquitylation is often a prerequisite for proteasomal degradation, we next sought to ascertain which differentially expressed K-e-GG peptides showed an inverse correlation with total protein expression (database D1). Peptides corresponding to TRIM24, SCAF1, and GLYR1 fit this pattern (one peptide each) (Fig. 1C and fig. S3, C and D). TRIM24 has been shown to mediate ligand-dependent activation of the androgen receptor—a key prostate cancer driver—and to degrade TP53 through its E3 ligase activity (10, 11). This observation is of interest given that primary prostate cancers with SPOP mutations typically lack TP53 alterations (1). However, the K-e-GG peptides that exhibited the most profound down-regulation coupled with
that undergo inverse changes at the protein level (e.g., more than twofold), normalized K-e-GG data shown in (B) was inverted and multiplied by corresponding total protein expression changes (see also fig. S3, C and D, for expression changes). (D) DEK protein expression across isogenic LHMAR cells assessed by immunoblotting with and without proteasome inhibition by MG132. DMSO, diemthyl sulfoxide. Error bars represent means T SD, n = 4 (n.s., not significant; ∗∗P < 0.01, Student's t test). (E) Degradation of Flag-DEK over time. Flag-DEK was induced by doxycycline (Dox) and protein decay measured by immunoblotting after doxycycline withdrawal in inducible LHMAR cells (P value, Friedman test). See also fig. S3E for DEK mRNA expression.
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trypsin digestion and stable isotope labeling of amino acids in cell culture (SILAC)–based mass spectrometry (Fig. 1A). To account for ubiquitylationrelated changes in protein expression, all K-e-GG values were normalized to protein abundance (Fig. 1A) (database D1 in supplementary materials). We reasoned that putative K-e-GG peptide substrates might show distinct patterns of protein abundance depending on whether mutant SPOP caused a dominant-negative, loss-of-function, gainof-function, or neomorphic effect. In a dominantnegative context, substrate peptides should exhibit a [SPOP-WT > control > SPOP-MT] pattern of abundance, whereas a pure loss-of-function effect might yield a [SPOP-WT > control = SPOP-MT] pattern. In contrast, a gain-of-function effect may result in a [SPOP-MT > SPOP-WT > control] pattern, and a neomorphic effect should cause a [SPOP-MT > SPOP-WT = control] pattern. Only 12 of 7181 K-e-GG peptides detected exhibited more than twofold changes in peptide abundance in the SPOP-mutant contexts (z score >3 or 80% of mutations identified in this tumor type (1)] conferred increased DEK protein expression, whereas two recurrent endometrial cancer–associated SPOP mutants (S80R and E50K) (2, 3) had no effect (Fig. 4A and figs. S8A, S9A, and S10A) in prostate cells. As expected, DEK expression correlated with sphere-forming capacity and cell invasion without affecting baseline cell proliferation or viability (figs. S8, B and C, and S9B). Finally, we considered whether other putative SPOP effector proteins—both known substrates and novel ones nominated by our ubiquitylome screen—might also undergo dysregulated expression in the setting of prostate cancer SPOP mutations (8, 9, 21–23). Regarding the latter, we noted that TRIM24 represented another putative SPOP substrate from our screen with potential biological relevance to prostate cancer (Fig. 1, B and C, and fig. S3C) (10, 11). As expected several known or putative SPOP substrates were downregulated in response to SPOP-WT overexpression (including DEK, TRIM24, NCOA3, DAXX, BRMS1, and GLI1 and 2) (Fig. 4A and fig. S10A). However, only DEK, TRIM24, and NCOA3 also became up-regulated upon overexpression of prostate cancer SPOP mutants in this setting (6). Moreover, nuclear expression of these proteins correlated significantly with SPOP mutations in 181/178 primary prostate cancers analyzed by immunohistochemistry (Fig. 4, B and C, and fig. S10B, table S3). These data are consistent with the ubiquitylome and experimental studies, which supports a model in which SPOP mutations may engage overlapping routes of transformation in prostate cancer by dysregulation of DEK, TRIM24, and NCOA3. These data support a model wherein prostate cancer SPOP mutants impair ubiquitylation and degradation of a subset of SPOP substrates in a dominant-negative manner to promote tumorigenesis. Future studies of DEK dysregulation and its biological functions in normal and tumor tissue may therefore promote new biological insights relevant to many human cancers. More generally, large-scale profiling of the ubiquitin landscape linked to tumor genomic alterations in protein homeostasis genes may catalyze the identification of downstream effector mechanisms. Given the growing number of such genes
Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer Jean-Philippe P. Theurillat, Namrata D. Udeshi, Wesley J. Errington, Tanya Svinkina, Sylvan C. Baca, Marius Pop, Peter J. Wild, Mirjam Blattner, Anna C. Groner, Mark A. Rubin, Holger Moch, Gilbert G. Privé, Steven A. Carr and Levi A. Garraway
Science 346 (6205), 85-89. DOI: 10.1126/science.1250255
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Mutant protein in tumors hits the DEK Cancer genome sequencing projects have uncovered a multitude of mutations in human tumors. Understanding whether and how these mutations contribute to tumor development and progression could ultimately lead to new therapies. Theurillat et al. studied the protein product of a gene that is recurrently mutated in prostate cancer. Normally this protein helps attach a biochemical tag to cellular proteins that marks them for degradation. The new work shows that the tumor-associated mutant protein loses this tagging ability, which results in the stabilization of a handful of cellular proteins that would otherwise be degraded. One of the most intriguing of these proteins was DEK, which helps prostate cancer cells invade into surrounding tissue. Science, this issue p. 85
