Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

DEPDC1/LET-99 participates in an evolutionarily conserved pathway for anti-tubulin drug-induced apoptosis

Nature Cell Biology volume 16, pages 812–820 (2014)Cite this article

Subjects

Abstract

Microtubule-targeting chemotherapeutics induce apoptosis in cancer cells by promoting the phosphorylation and degradation of the anti-apoptotic BCL-2 family member MCL1. The signalling cascade linking microtubule disruption to MCL1 degradation remains however to be defined. Here, we establish an in vivo screening strategy in Caenorhabditis elegans to uncover genes involved in chemotherapy-induced apoptosis. Using an RNAi-based screen, we identify three genes required for vincristine-induced apoptosis. We show that the DEP domain protein LET-99 acts upstream of the heterotrimeric G protein alpha subunit GPA-11 to control activation of the stress kinase JNK-1. The human homologue of LET-99, DEPDC1, similarly regulates vincristine-induced cell death by promoting JNK-dependent degradation of the BCL-2 family protein MCL1. Collectively, these data uncover an evolutionarily conserved mediator of anti-tubulin drug-induced apoptosis and suggest that DEPDC1 levels could be an additional determinant for therapy response upstream of MCL1.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Four chemotherapeutic compounds induce germline apoptosis.
Figure 2: Identification of let-99 as a gene mediating vincristine-induced apoptosis.
Figure 3: The Gα GPA-11 and the stress-kinase JNK regulate apoptosis downstream of LET-99/DEPDC1.
Figure 4: Human DEPDC1 participates in vincristine-induced cell death.
Figure 5: DEPDC1 regulates MCL1 degradation.

Similar content being viewed by others

References

  1. Jackson, J. R., Patrick, D. R., Dar, M. M. & Huang, P. S. Targeted anti-mitotic therapies: Can we improve on tubulin agents? Nat. Rev. Cancer 7, 107–117 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Jordan, M. A. & Wilson, L. Microtubules as a target for anticancer drugs. Nat. Rev. Cancer 4, 253–265 (2004).

    Article  CAS  PubMed  Google Scholar 

  3. Jordan, M. A., Thrower, D. & Wilson, L. Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis. J. Cell Sci. 102, 401–416 (1992).

    CAS  PubMed  Google Scholar 

  4. Inuzuka, H. et al. SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature 471, 104–109 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wertz, I. E. et al. Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature 471, 110–114 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Johnstone, R. W., Ruefli, A. A. & Lowe, S. W. Apoptosis: A link between cancer genetics and chemotherapy. Cell 108, 153–164 (2002).

    Article  CAS  PubMed  Google Scholar 

  7. Horvitz, H. R. Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res. 59, 1701s–1706s (1999).

    CAS  PubMed  Google Scholar 

  8. Gumienny, T. L., Lambie, E., Hartwieg, E., Horvitz, H. R. & Hengartner, M. O. Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline. Development 126, 1011–1022 (1999).

    CAS  PubMed  Google Scholar 

  9. Gartner, A., Milstein, S., Ahmed, S., Hodgkin, J. & Hengartner, M. O. A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans. Mol. Cell 5, 435–443 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Lant, B. & Derry, W. B. Methods for detection and analysis of apoptosis signaling in the C. elegans germline. Methods 61, 174–182 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Derry, W. B., Putzke, A. P. & Rothman, J. H. Caenorhabditis elegans p53: Role in apoptosis, meiosis, and stress resistance. Science 294, 591–595 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Conradt, B. & Horvitz, H. R. The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519–529 (1998).

    Article  CAS  PubMed  Google Scholar 

  13. Schumacher, B., Hofmann, K., Boulton, S. & Gartner, A. The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis. Curr. Biol. 11, 1722–1727 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Schumacher, B. et al. C. elegans ced-13 can promote apoptosis and is induced in response to DNA damage. Cell Death Differ. 12, 153–161 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Rieder, C. L. & Maiato, H. Stuck in division or passing through: What happens when cells cannot satisfy the spindle assembly checkpoint. Dev. Cell 7, 637–651 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Vilpo, J. A., Koski, T. & Vilpo, L. M. Selective toxicity of vincristine against chronic lymphocytic leukemia cells in vitro. Eur. J. Haematol. 65, 370–378 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Pazdernik, N. & Schedl, T. Introduction to germ cell development in Caenorhabditis elegans. Adv. Exp. Med. Biol. 757, 1–16 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Rocheleau, C. E. et al. The Caenorhabditis elegans ekl (enhancer of ksr-1 lethality) genes include putative components of a germline small RNA pathway. Genetics 178, 1431–1443 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Siegert, R., Leroux, M. R., Scheufler, C., Hartl, F. U. & Moarefi, I. Structure of the molecular chaperone prefoldin: Unique interaction of multiple coiled coil tentacles with unfolded proteins. Cell 103, 621–632 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Kemphues, K. J., Priess, J. R., Morton, D. G. & Cheng, N. S. Identification of genes required for cytoplasmic localization in early C. elegans embryos. Cell 52, 311–320 (1988).

    Article  CAS  PubMed  Google Scholar 

  21. Etemad-Moghadam, B., Guo, S. & Kemphues, K. J. Asymmetrically distributed PAR-3 protein contributes to cell polarity and spindle alignment in early C. elegans embryos. Cell 83, 743–752 (1995).

    Article  CAS  PubMed  Google Scholar 

  22. Wu, J-C. & Rose, L. S. PAR-3 and PAR-1 inhibit LET-99 localization to generate a cortical band important for spindle positioning in Caenorhabditis elegans embryos. Mol. Biol. Cell 18, 4470–4482 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rose, L. S. & Kemphues, K. The let-99 gene is required for proper spindle orientation during cleavage of the C. elegans embryo. Development 125, 1337–1346 (1998).

    CAS  PubMed  Google Scholar 

  24. Bringmann, H., Cowan, C. R., Kong, J. & Hyman, A. A. LET-99, GOA-1/GPA-16, and GPR-1/2 are required for aster-positioned cytokinesis. Curr. Biol. 17, 185–191 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Bellaiche, Y. & Gotta, M. Heterotrimeric G proteins and regulation of size asymmetry during cell division. Curr. Opin. Cell Biol. 17, 658–663 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Krueger, L. E., Wu, J-C., Tsou, M-F. B. & Rose, L. S. LET-99 inhibits lateral posterior pulling forces during asymmetric spindle elongation in C. elegans embryos. J. Cell Biol. 189, 481–495 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Tsou, M-F. B., Hayashi, A., DeBella, L. R., McGrath, G. & Rose, L. S. LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos. Development 129, 4469–4481 (2002).

    CAS  PubMed  Google Scholar 

  28. Tsou, M-F. B., Hayashi, A. & Rose, L. S. LET-99 opposes Galpha/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling. Development 130, 5717–5730 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Chen, S. & Hamm, H. E. DEP domains: More than just membrane anchors. Dev. Cell 11, 436–438 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Consonni, S. V., Maurice, M. M. & Bos, J. L. DEP domains: Structurally similar but functionally different. Nat. Rev. Mol. Cell Biol. 15, 357–362 (2014).

    Article  CAS  PubMed  Google Scholar 

  31. Ponting, C. P. & Bork, P. Pleckstrin’s repeat performance: A novel domain in G-protein signaling? Trends Biochem. Sci. 21, 245–246 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Boutros, M., Paricio, N., Strutt, D. I. & Mlodzik, M. Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling. Cell 94, 109–118 (1998).

    Article  CAS  PubMed  Google Scholar 

  33. Wang, T. H. et al. Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways. J. Biol. Chem. 273, 4928–4936 (1998).

    Article  CAS  PubMed  Google Scholar 

  34. Zhu, B. et al. Activation of Jun N-terminal kinase is a mediator of vincristine-induced apoptosis of melanoma cells. Anticancer Drugs 19, 189–200 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Vucic, D., Dixit, V. M. & Wertz, I. E. Ubiquitylation in apoptosis: A post-translational modification at the edge of life and death. Nat. Rev. Mol. Cell Biol. 12, 439–452 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Praitis, V., Casey, E., Collar, D. & Austin, J. Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, 1217–1226 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kamath, R. S. et al. Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, 231–237 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Follenzi, A. & Naldini, L. Generation of HIV-1 derived lentiviral vectors. Methods Enzymol. 346, 454–465 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Hengartner and Bano laboratory members for help and discussions. We thank L. Rose for the LET-99 antibody. We are grateful to L. Rose and M. Gotta for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation, the Kanton of Zurich and the Josef-Steiner Foundation. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR).

Author information

Author notes

  1. Ataman Sendoel & Lilli Stergiou

    Present address: Present addresses: The Rockefeller University, New York, New York 10065, USA (A.S.); Redbiotec, Wagistrasse 23, CH-8952 Schlieren-Zurich, Switzerland (L.S.).,

Authors and Affiliations

  1. Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190 CH-8057 Zurich, Switzerland,

    Ataman Sendoel, Xue Zheng, Youjin Teo, Lilli Stergiou, Deni Subasic, Sergio M. Pinto, Moyin Shi & Michael O. Hengartner

  2. Division of Hematology, University Hospital Zurich, Raemistrasse 190 CH-8091 Zurich, Switzerland,

    Ataman Sendoel, Steffen Boettcher & Markus G. Manz

  3. German Center for Neurodegenerative Diseases (DZNE) e.V. Ludwig-Erhard-Allee 2, D-53175 Bonn, Germany

    Simona Maida, Carlo-Alberto Rossi & Daniele Bano

  4. Center for Cell Clearance, University of Virginia, Charlottesville, Virginia 22908, USA

    Jason M. Kinchen

  5. Nicholas School of the Environment, Duke University, Durham, North Carolina 27708, USA

    Joel N. Meyer

Authors
  1. Ataman Sendoel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Simona Maida
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xue Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Youjin Teo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lilli Stergiou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carlo-Alberto Rossi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Deni Subasic
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sergio M. Pinto
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jason M. Kinchen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Moyin Shi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Steffen Boettcher
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Joel N. Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Markus G. Manz
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Daniele Bano
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Michael O. Hengartner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.S., X.Z., Y.T., L.S. and S.M.P. performed C. elegans experiments. S.M., C-A.R. and D.B. performed cell culture experiments. D.S., S.M.P., J.M.K., M.S., S.B. and J.N.M. helped with experimental design and procedures. A.S., M.G.M., D.B. and M.O.H. designed experiments. A.S. and M.O.H. wrote the manuscript. All authors provided detailed comments.

Corresponding author

Correspondence to Michael O. Hengartner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 2 Cisplatin, doxorubicin, etoposide and vincristine do not induce cell cycle arrest at the given concentrations.

(a) Synchronized young adult animals were treated with cisplatin, doxorubicin, etoposide, vincristine or ionizing radiation (IR, 60 Gy). Cells per area of the mitotic germline region were quantified 12 h, 24 h and 36 h post-treatment. Ionizing radiation (IR) results in a drop in the number of germ cells in the mitotic region as a result of cell cycle arrest within the mitotic germline compartment. By contrast, chemotherapeutic drugs did not affect the number of mitotic germ cells. Data shown represent the average of three independent experiments ± s.d. (n = 30 quantified germ lines for each time point). (b) PAR-3, LET-99 and PFD-1(C08F8.1) are specifically mediating vincristine-induced germline apoptosis. In contrast, loss of EKL-1 results in hypersensitivity also to IR. Synchronized young adult animals were treated with either IR 60 Gy or UV-C 100 J m−2 and germline apoptosis was quantified 24 h post-treatment. Data shown represent the average of three independent experiments ± s.d. (n = 60 animals for each time point). (c–d) LET-99 mediates apoptosis also in response to other antitubulin chemotherapeutics. Synchronized young adult animals were treated with 0.05 mM nocodazole or 0.05 mM docetaxel and germline apoptosis was quantified 24 h post-treatment. Data represent the average of two independent experiments ± s.d. (n = 40 animals). (e) Vincristine dose-response in the germ line of C. elegans 36 h post-treatment. Data shown represent the average of n = 40 animals from two independent experiments (for 0.1 mM, 0.2 mM and 0.5 mM) or n = 20 (for 0 mM and 0.05 mM) from one experiment ± s.d. (f) Vincristine response in rrf-1(pk1417) mutant animals 36 h post-treatment. Data represent the average of n = 20 animals± s.d. from one experiment. (g) Time course for doxorubicin uptake. Synchronized young adults were treated with doxorubicin 0.1 mM and imaged (doxorubicin fluorescence λex = 470 nm, λem = 590 nm) at the indicated time points. All results are representative images from one experiment. (h–i) The BH3-only domain protein EGL-1 and CED-13 partially mediate vincristine-induced apoptosis. Synchronized young adult animals were exposed to vincristine and germline apoptosis was quantified at the indicated time points. Data shown represent the average of three independent experiments ± s.d. (n = 60 animals for each time point). (j) egl-1 and ced-13 transcripts are induced upon vincristine treatment. Synchronized young adult animals were treated with 0.1 mM vincristine and mRNA levels were evaluated by qRT-PCR. Data shown represent the average ± s.d. from a full 10 cm plate of animals pooled from one experiment.

Supplementary Figure 3 LET-99 localization in embryos and the germ line.

(a–d) LET-99::GFP cortical localization (arrows) in embroys. Scale bars, 10 μm. All results are representative of at least three independent experiments. (e–g) Immunostaining using the LET-99 antibody in wild-type embryos confirms nuclear localization (arrows) of LET-99. Scale bars, 10 μm. (h–l) LET-99 localization does not change in response to vincristine treatment (h-j: LET-99 antibody staining; k-l: LET-99::GFP). Scale bars, 10 μm.

Supplementary Figure 4 An RNAi based screen for G proteins or interactors of LET-99 involved in vincristine-induced germline apoptosis.

(a) Synchronized young adult rrf-1 mutant animals (partially defective in somatic RNAi) were treated with vincristine and germline apoptosis was assessed 36 h post-treatment. Data shown represent the average of n = 20 or n = 40 (gpa-11) animals ± s.d. from one experiment. (b) Synchronized young adult animals were treated with vincristine and germline apoptosis was assessed 36 h following vincristine, 0.1 mM, treatment. Data shown represents the average of n = 20 (control) or n = 30 animals (lin-5, goa-1, gpr-1/2) ± s.d. from one experiment. (c) siRNA directed against DEPDC1A and DEPDC1B attenuates MCL1 degradation also in HeLa cells. Western blot analysis of MCL1 in response to vincristine treatment 12 h and 24 h post-treatment. Results are representative of at least three independent experiments. (d) DEPDC1 does not regulate doxorubicin-induced cell death in HeLa cells. Data shown represent the average ± s.d. from one representative experiment, three independent experiments to assess repeatability (Scramble n = 1741; DEPDC1B n = 1844; DEPDC1A n = 1494; MCL1 n = 1844; Scramble Doxo n = 699; DEPDC1B Doxo n = 782; DEPDC1A Doxo n = 643; Mcl1 Dox n = 507). (e) DEPDC1 mediates vincristine-induced cell death also in SH-SY5Y neuroblastoma cells. Data shown represent the average ± s.d. from one representative experiment, three independent experiments to assess repeatability (Scramble n = 1139; DEPDC1B n = 1168; DEPDC1A n = 1521; Scramble 6 h vin n = 1119; DEPDC1B vin 6 h n = 852; DEPDC1A vin 6 h n = 954; Scramble vin 12 h n = 1115; DEPDC1B vin 12 h n = 989; DEPDC1A vin 12 h n = 1071). (f) Knock-down efficiency by DEPDC1A siRNA and DEPDC1B siRNA versus scramble control siRNA. DEPDC1A and DEPDC1B transcript levels were quantified by qRT-PCR ± s.d. in HeLa and MCF-7 cells treated with siRNA directed either against scramble control siRNA, DEPDC1A siRNA or DEPDC1B siRNA. Data shown are from one experiment, at least three independent experiments to assess repeatability.

Supplementary Figure 5 Multiple alignment of DEPDC1 and LET-99.

(a) Multiple alignment of DEPDC1A (isoforms 1 and 2), DEPDC1B and LET-99. Depicted in red is the DEP domain, in green the RhoGAP domain.

Supplementary Figure 6 The role of JNK-1 in germline apoptosis.

(a) Time course analysis of wild-type and jnk-1(gk7) animals upon IR 60 Gy. Data shown represent the average of three independent experiments ± s.d. (n = 60 animals for each time point). (b) Western blot time-course analysis of phosphorylated JNK-1 upon ionizing radiation (IR) 60 Gy. Results are representative of two independent experiments. (c) The MAP kinase kinases MEK-1 and SEK-1 are dispensable for JNK-1 phosphorylation upon vincristine treatment. Western blot analysis of phosphorylated JNK-1 in synchronized mek-1(ks54) and sek-1(km4) animals 6 h post vincristine treatment (0.1 mM). Results are representative of two independent experiments. (d) Time-course analysis of wild-type and mkk-4(km23) animals upon vincristine treatment. Data shown represent the average of three independent experiments ± s.d. (n = 60 animals for each time point). (e) MKK-4 is required for UV-C-induced apoptosis. Synchronized wild-type and mkk-4(km23) animals were treated with UV-C or IR and germ cell apoptosis was quantified 24 h post-treatment. Data shown represent the average of three independent experiments ± s.d. (n = 60 animals). (f–h) Time-course analysis of wild-type and jnk-1(gk7) animals following chemotherapy treatment. Data shown represent the average of three independent experiments ± s.d. (n = 60 animals for each time point). (i) Western blot analysis of phosphorylated JNK in MCF-7 cells. Results are representative of at least three independent experiments.

Supplementary Figure 7 Uncropped images of blots.

Uncropped versions of key electrophoretic data have been provided. These scans correspond to data in Figs 3d–f, 4h–i, 5a, b, d, h, and Supplementary Fig. 3c.

Supplementary Table 1 A candidate-based RNAi screen for genes involved in vincristine-mediated apoptosis.
Full size table

Supplementary information

Supplementary Information

Supplementary Information (PDF 2060 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sendoel, A., Maida, S., Zheng, X. et al. DEPDC1/LET-99 participates in an evolutionarily conserved pathway for anti-tubulin drug-induced apoptosis. Nat Cell Biol 16, 812–820 (2014). https://doi.org/10.1038/ncb3010

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3010

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer