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.

  • Original Article
  • Published:

Knockdown of Slingshot 2 (SSH2) serine phosphatase induces Caspase3 activation in human carcinoma cell lines with the loss of the Birt–Hogg–Dubé tumour suppressor gene (FLCN)

Oncogene volume 33, pages 956–965 (2014)Cite this article

Subjects

Abstract

Birt–Hogg–Dubé (BHD) syndrome, is a dominantly inherited familial cancer syndrome associated with susceptibility to renal cell carcinoma (RCC) caused by inactivating mutations in the folliculin (FLCN) gene. The precise functions of the FLCN gene product are still under investigation but RCC from BHD patients show loss of the wild-type allele consistent with a tumor suppressor gene function. In a search for potential synthetic-lethal targets for FLCN using a phosphatase siRNA library screening approach, we found that knockdown of SSH2 serine phosphatase (one of the three members of Slingshot family and previously implicated in actin reorganization) specifically induced Caspase3/7 activity in a dose-dependent manner (up to six-fold increase, 10 nM, 72 h) in two human FLCN-deficient cell lines (BHD-origin renal cell carcinoma UOK257 and thyroid carcinoma FTC133) but not in their folliculin expressing isogenic cell lines. SSH2 siRNA-induced knockdown was accompanied by increased expression of SSH1 and SSH3 (suggesting a compensatory regulatory mechanism among members of SSH family). FLCN-null cells exhibited evidence of dysregulated cofilin de/phosphorylation pathways. Knockdown of SSH2 in FLCN-null cells was associated with an alteration in cell cycle kinetics (20% increase in G1, 30% and 40% decrease in S and G2M, respectively). Combination treatment of multiple SSH family (SSH2 plus SSH1 and/or SSH3) siRNAs potentiated induction of Caspase3/7 activity and changes in the cell cycle kinetics. These data indicate that: (a) apoptotic cell death in FLCN-null cells can be triggered by SSH2 knockdown through cell cycle arrest; (b) SSH2 represents a potential therapeutic target for the development of agents for the treatment of BHD syndrome and, possibly, related tumors.

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
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Latif F, Tory K, Gnarra J, Yao M, Duh FM, Orcutt ML et al. Identification of the von Hippel-Lindau disease tumor suppressor gene. Science 1993; 260: 1317–1320.

    Article  CAS  PubMed  Google Scholar 

  2. Foster K, Prowse A, van den Berg A, Fleming S, Hulsbeek MM, Crossey PA et al. Somatic mutations of the von Hippel-Lindau disease tumour suppressor gene in non-familial clear cell renal carcinoma. Hum Mol Genetic 1994; 3: 2169–2173.

    Article  CAS  Google Scholar 

  3. Gnarra JR, Tory K, Weng Y, Schmidt L, Wei MH, Li H et al. Mutations of the VHL tumour suppressor gene in renal carcinoma. Nat Genet 1994; 7: 85–90.

    Article  CAS  PubMed  Google Scholar 

  4. Banks RE, Tirukonda P, Taylor C, Hornigold N, Astuti D, Cohen D et al. Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer. Cancer Res 2006; 66: 2000–2011.

    Article  CAS  PubMed  Google Scholar 

  5. Patel PH, Chadalavada RS, Chaganti RS, Motzer RJ . Targeting von Hippel-Lindau pathway in renal cell carcinoma. Clin Cancer Res 2006; 12: 7215–7220.

    Article  CAS  PubMed  Google Scholar 

  6. Motzer RJ, Hudes G, Wilding G, Schwartz LH, Hariharan S, Kempin S et al. Phase I trial of sunitinib malate plus interferon-alpha for patients with metastatic renal cell carcinoma. Clin Genitourin Cancer 2009; 7: 28–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Toro JR, Wei MH, Glenn GM, Weinreich M, Toure O, Vocke C et al. BHD mutations, clinical and molecular genetic investigations of Birt-Hogg-Dube syndrome: a new series of 50 families and a review of published reports. J Med Genetic 2008; 45: 321–331.

    Article  CAS  Google Scholar 

  8. Menko FH, van Steensel MA, Giraud S, Friis-Hansen L, Richard S, Ungari S et al. Birt-Hogg-Dube syndrome: diagnosis and management. Lancet Oncol 2009; 10: 1199–1206.

    Article  CAS  PubMed  Google Scholar 

  9. Nickerson ML, Warren MB, Toro JR, Matrosova V, Glenn G, Turner ML et al. Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dube syndrome. Cancer Cell 2002; 2: 157–164.

    Article  CAS  PubMed  Google Scholar 

  10. Schmidt LS, Nickerson ML, Warren MB, Glenn GM, Toro JR, Merino MJ et al. Germline BHD-mutation spectrum and phenotype analysis of a large cohort of families with Birt-Hogg-Dube syndrome. Am J Hum Genetic 2005; 76: 1023–1033.

    Article  CAS  Google Scholar 

  11. Lim DH, Rehal PK, Nahorski MS, Macdonald F, Claessens T, Van Geel M et al. A new locus-specific database (LSDB) for mutations in the folliculin (FLCN) gene. Hum Mutat 2009; 31: E1043–E1051.

    Article  Google Scholar 

  12. da Silva NF, Gentle D, Hesson LB, Morton DG, Latif F, Maher ER . Analysis of the Birt-Hogg-Dube (BHD) tumour suppressor gene in sporadic renal cell carcinoma and colorectal cancer. J Med Genetic 2003; 40: 820–824.

    Article  CAS  Google Scholar 

  13. Baba M, Furihata M, Hong SB, Tessarollo L, Haines DC, Southon E et al. Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys. J Natl Cancer Inst 2008; 100: 140–154.

    Article  CAS  PubMed  Google Scholar 

  14. Chen J, Futami K, Petillo D, Peng J, Wang P, Knol J et al. Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia. PloS One 2008; 3: e3581.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Dedes KJ, Wilkerson PM, Wetterskog D, Weigelt B, Ashworth A, Reis-Filho JS . Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations. Cell Cycle (Georgetown, Tex 2011; 10: 1192–1199.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Tutt A, Robson M, Garber JE, Domchek SM, Audeh MW, Weitzel JN et al. Oral poly(ADP-ribose) polymerase inhibitor olaparib in patients with BRCA1 or BRCA2 mutations and advanced breast cancer: a proof-of-concept trial. Lancet 2010; 376: 235–244.

    Article  CAS  PubMed  Google Scholar 

  17. Rehman FL, Lord CJ, Ashworth A . Synthetic lethal approaches to breast cancer therapy. Nat RevClin Oncol 2010; 7: 718–724.

    Article  CAS  Google Scholar 

  18. Yang Y, Padilla-Nash HM, Vira MA, Abu-Asab MS, Val D, Worrell R et al. The UOK 257 cell line: a novel model for studies of the human Birt-Hogg-Dube gene pathway. Cancer Genetic Cytogenet 2008; 180: 100–109.

    Article  CAS  Google Scholar 

  19. Plastino J, Sykes C . The actin slingshot. Curr Opin Cell Biol 2005; 17: 62–66.

    Article  CAS  PubMed  Google Scholar 

  20. Huang TY, DerMardirossian C, Bokoch GM . Cofilin phosphatases and regulation of actin dynamics. Curr Opin Cell Biol 2006; 18: 26–31.

    Article  CAS  PubMed  Google Scholar 

  21. Mizuno K . Signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation. Cellular Signal 2012; 25: 457–469.

    Article  Google Scholar 

  22. Lu X, Wei W, Fenton J, Nahorski MS, Rabai E, Reiman A et al. Therapeutic targeting the loss of the birt-hogg-dube suppressor gene. Mol Cancer Ther 2011; 10: 80–89.

    Article  CAS  PubMed  Google Scholar 

  23. Bernstein BW, Bamburg JR . ADF/cofilin: a functional node in cell biology. Trends Cell Biol 2010; 20: 187–195.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Posadas I, Perez-Martinez FC, Guerra J, Sanchez-Verdu P, Cena V . Cofilin activation mediates Bax translocation to mitochondria during excitotoxic neuronal death. J Neurochem 2012; 120: 515–527.

    Article  CAS  PubMed  Google Scholar 

  25. Kaelin WG . The von Hippel-Lindau tumor suppressor protein: roles in cancer and oxygen sensing. Cold Spring Harb Symp Quant Biol 2005; 70: 159–166.

    Article  CAS  PubMed  Google Scholar 

  26. Kaelin WG . Treatment of kidney cancer: insights provided by the VHL tumor-suppressor protein. Cancer 2009; 115 (10 Suppl): 2262–2272.

    Article  CAS  PubMed  Google Scholar 

  27. Reinhardt HC, Jiang H, Hemann MT, Yaffe MB . Exploiting synthetic lethal interactions for targeted cancer therapy. Cell Cycle (Georgetown, Tex 2009; 8: 3112–3119.

    Article  CAS  PubMed  Google Scholar 

  28. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M et al. Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 2009; 361: 123–134.

    Article  CAS  PubMed  Google Scholar 

  29. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB . p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 2007; 11: 175–189.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lu X, Errington J, Curtin NJ, Lunec J, Newell DR . The impact of p53 status on cellular sensitivity to antifolate drugs. Clin Cancer Res 2001; 7: 2114–2123.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Myrovlytis Trust for financial support.

Author information

Authors and Affiliations

  1. Centre for Rare Diseases and Personalised Medicine, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

    X Lu, U Boora, L Seabra, J Fenton, A Reiman & E R Maher

  2. Neurodegeneration and Repair, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

    E M Rabai & Z Nagy

Authors
  1. X Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. U Boora
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L Seabra
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E M Rabai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J Fenton
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A Reiman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Z Nagy
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. E R Maher
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to E R Maher.

Ethics declarations

Competing interests

A patent application regarding the use of downregulation of Slingshot expression including siRNA, antisense RNA and relative compounds in Birt–Hogg–Dube related tumours was filed by the Myrovlytis Trust with XL and ERM named as inventors.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lu, X., Boora, U., Seabra, L. et al. Knockdown of Slingshot 2 (SSH2) serine phosphatase induces Caspase3 activation in human carcinoma cell lines with the loss of the Birt–Hogg–Dubé tumour suppressor gene (FLCN). Oncogene 33, 956–965 (2014). https://doi.org/10.1038/onc.2013.27

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2013.27

Keywords

This article is cited by

Search

Advanced search

Quick links