CD95 promotes tumour growth

Journal name:
Nature
Volume:
465,
Pages:
492–496
Date published:
DOI:
doi:10.1038/nature09075
Received
Accepted

CD95 (also called Fas and APO-1) is a prototypical death receptor that regulates tissue homeostasis mainly in the immune system through the induction of apoptosis1, 2, 3. During cancer progression CD95 is frequently downregulated or cells are rendered apoptosis resistant4, 5, raising the possibility that loss of CD95 is part of a mechanism for tumour evasion. However, complete loss of CD95 is rarely seen in human cancers4 and many cancer cells express large quantities of CD95 and are highly sensitive to CD95-mediated apoptosis in vitro. Furthermore, cancer patients frequently have elevated levels of the physiological ligand for CD95, CD95L6. These data raise the possibility that CD95 could actually promote the growth of tumours through its non-apoptotic activities7. Here we show that cancer cells in general, regardless of their CD95 apoptosis sensitivity, depend on constitutive activity of CD95, stimulated by cancer-produced CD95L, for optimal growth. Consistently, loss of CD95 in mouse models of ovarian cancer and liver cancer reduces cancer incidence as well as the size of the tumours. The tumorigenic activity of CD95 is mediated by a pathway involving JNK and Jun. These results demonstrate that CD95 has a growth-promoting role during tumorigenesis and indicate that efforts to inhibit its activity rather than to enhance it should be considered during cancer therapy.

At a glance

Figures

  1. Reducing CD95 or CD95L expression inhibits proliferation of cancer cells.
    Figure 1: Reducing CD95 or CD95L expression inhibits proliferation of cancer cells.

    af, Growth of different cell lines infected with R6. Insets show the total CD95-expression levels of cells expressing scrambled control (Vec) or CD95shRNA6 (R6) as determined by western blot analysis. A similar effect was also found with a CD95-expressing variant of the neuroblastoma cell line NB4 (not shown). Apoptosis sensitivity of HeyA8 vec and R6 cells by leucine zipper-tagged (Lz)CD95L treatment was determined by quantifying DNA fragmentation (a). OD, optical density. g, HepG2 cells were stably infected with three different CD95L-specific shRNA lentiviruses (L1, L2 and L4) and CD95L mRNA was quantified using real-time PCR. h, Growth of cells in g over 5days. i, Growth of different cell lines infected with the L4 virus. Proliferation of cells was examined by SRB assay (af, h and i). *P<0.05, **P<0.01, ***P<0.001. Values in graphs represent mean ± s.d. from three independent experiments. d, day.

  2. Loss of CD95 expression inhibits ovarian cancer in vivo.
    Figure 2: Loss of CD95 expression inhibits ovarian cancer in vivo.

    a, Tumour weight, number of tumour colonies and ascites volume from mice injected with SKOV3.ip1 vec or R6 cells. Lysates of cells and tumour tissues were examined for CD95 level by western blot analysis. b, The same parameters as in a were measured from mice injected with SKOV3.ip1 vec or R4 cells. c, Histology and immunohistochemistry staining for Ki67, TUNEL and CD31 of SKOV3.ip1 vec and R6 tumours. Scale bar, 100μm. **P<0.001. d, Tumour load and number of tumour colonies of mice treated with neutralizing mAb for murine CD95L (MFL3), human CD95L (NOK-1) or corresponding isotype control mAbs (hamster IgG and mouse IgG1, respectively) were measured. Inset, western blot analysis of SKOV3.ip1 cell lysate for CD95L. e, Surface CD95 staining (upper left) and apoptosis sensitivity by LzCD95L (100ngml−1) treatment (lower left) of MONTY-1 vec and R6 cells. FL2, fluorescence channel 2. Weight and number of colonies of tumours formed by MONTY-1 vec and R6 cells are shown (right). The horizontal bars in the right part of e represent the mean of six mice. f, The staining intensity for Ki67, TUNEL and CD31 of tumours from MONTY-1 vec and R6 cells were quantified. *P<0.05. Values in graphs represent mean ± s.d. from three independent experiments (af). kDa, kilodaltons.

  3. Deletion of CD95 leads to a reduction in tumour formation in a spontaneous model of endometrioid ovarian cancer.
    Figure 3: Deletion of CD95 leads to a reduction in tumour formation in a spontaneous model of endometrioid ovarian cancer.

    a, Staining for CD95 in three primary human endometrioid ovarian cancers. Scale bar, 50μm. b, Number of mice that formed visible tumours either 8 or 14weeks after injection of Cre into the right ovarian bursa. Asterisk indicates one mouse that died from ovarian cancer 42days after Cre injection. c, Representative image and histology of right ovaries from wild-type (WT) and knockout (KO) mice 8weeks after injection of Cre. Arrowhead indicates ovarian tumour. Scale bar, 100μm. d, CD95 staining of ovary from untreated wild-type, Cre-treated wild-type or Cre-treated knockout mice. Scale bar, 50μm. Wild type, LSL-KrasG12D/+PtenloxP/loxPCD95WT/WT; knockout, LSL-KrasG12D/+PtenloxP/loxPCD95loxP/loxP.

  4. Deletion of CD95 in the liver leads to a decrease in tumour formation caused by the reduced ability of hepatocytes to proliferate and to activate JNK.
    Figure 4: Deletion of CD95 in the liver leads to a decrease in tumour formation caused by the reduced ability of hepatocytes to proliferate and to activate JNK.

    a, Haematoxylin and eosin and BrdU staining of livers from wild-type and liver-specific CD95-knockout mice 48h after partial hepatectomy. Scale bars,100μm. b, Quantification of relative BrdU staining intensity of the mice in a. c, Wild-type and liver-specific CD95 knockout mice were injected with a single dose of DEN i.p. to induce liver tumour formation. Eight months later, all mice were killed and parameters of total liver weight, number of liver surface nodules and maximum nodule diameter were recorded. d, Intact livers (scale bar, 1cm), haematoxylin and eosin staining and immunohistochemistry of TUNEL, Ki67 and CD31 from mice in c. Scale bar, 100μm. e, Quantification of Ki67 and CD31 staining for liver samples in d. There was no detectable TUNEL staining. f, Western blot for phospho-JNK and phospho-Jun in three untreated wild-type and three CD95 knockout mouse livers. g, Wild-type mice with or without partial hepatectomy (PH) were injected i.p. with 10μg of murine CD95-specific agonistic antibody, Jo2- or isotype-matched control mAb. After 6h, phospho-JNK (p-JNK), phospho-Jun (p-Jun) levels and cleavage of caspase-3 (CASP-3) in livers were measured by western blot analysis. Concentration of the liver enzyme ALT in the serum of injected mice is given. h, Immunohistochemistry staining of ovarian tumours from wild-type (LSL-KrasG12D/+PtenloxP/loxPCD95WT/WT) or knockout (LSL-KrasG12D/+PtenloxP/loxPCD95loxP/loxP) mice 8weeks after injection of Cre for CD95 and phospho-Jun. Scale bar, 20 μm. Values in graphs in b and e represent mean ± s.d. from three independent experiments. The horizontal bars in c represent the mean.

References

  1. Nagata, S. Fas ligand-induced apoptosis. Annu. Rev. Genet. 33, 2955 (1999)
  2. Krammer, P. H. CD95’s deadly mission in the immune system. Nature 407, 789795 (2000)
  3. Strasser, A., Jost, P. J. & Nagata, S. The many roles of FAS receptor signaling in the immune system. Immunity 30, 180192 (2009)
  4. Peter, M. E., Legembre, P. & Barnhart, B. C. Does CD95 have tumor promoting activities? Biochim. Biophys. Acta 1755, 2536 (2005)
  5. Debatin, K. M. & Krammer, P. H. Death receptors in chemotherapy and cancer. Oncogene 23, 29502966 (2004)
  6. Barnhart, B. C. et al. CD95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells. EMBO J. 23, 31753185 (2004)
  7. Peter, M. E. et al. The CD95 receptor: apoptosis revisited. Cell 129, 447450 (2007)
  8. Lavrik, I. N. et al. Analysis of CD95 threshold signaling: triggering of CD95 (FAS/APO-1) at low concentrations primarily results in survival signaling. J. Biol. Chem. 282, 1366413671 (2007)
  9. Baldwin, R. L., Tran, H. & Karlan, B. Y. Primary ovarian cancer cultures are resistant to Fas-mediated apoptosis. Gynecol. Oncol. 74, 265271 (1999)
  10. Abrahams, V. M. et al. Epithelial ovarian cancer cells secrete functional Fas ligand. Cancer Res. 63, 55735581 (2003)
  11. Rabinowich, H. et al. Lymphocyte apoptosis induced by Fas ligand-expressing ovarian carcinoma cells. Implications for altered expression of T cell receptor in tumor-associated lymphocytes. J. Clin. Invest. 101, 25792588 (1998)
  12. Taylor, D. D., Lyons, K. S. & Gercel-Taylor, C. Shed membrane fragment-associated markers for endometrial and ovarian cancers. Gynecol. Oncol. 84, 443448 (2002)
  13. Sawada, K. et al. C-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion. Cancer Res. 67, 16701679 (2007)
  14. Kaur, S. et al. β3-integrin expression on tumor cells inhibits tumor progression, reduces metastasis, and is associated with a favorable prognosis in patients with ovarian cancer. Am. J. Pathol. 175, 21842196 (2009)
  15. Dinulescu, D. M. et al. Role of K-ras and Pten in the development of mouse models of endometriosis and endometrioid ovarian cancer. Nature Med. 11, 6370 (2004)
  16. Stranges, P. B. et al. Elimination of antigen-presenting cells and autoreactive T cells by Fas contributes to prevention of autoimmunity. Immunity 26, 629641 (2007)
  17. Desbarats, J. & Newell, M. K. Fas engagement accelerates liver regeneration after partial hepatectomy. Nature Med. 6, 920923 (2000)
  18. Maeda, S., Kamata, H., Luo, J. L., Leffert, H. & Karin, M. IKKβ couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 121, 977990 (2005)
  19. Schmidt-Supprian, M. & Rajewsky, K. Vagaries of conditional gene targeting. Nature Immunol. 8, 665668 (2007)
  20. Hui, L., Zatloukal, K., Scheuch, H., Stepniak, E. & Wagner, E. F. Proliferation of human HCC cells and chemically induced mouse liver cancers requires JNK1-dependent p21 downregulation. J. Clin. Invest. 118, 39433953 (2008)
  21. Sakurai, T., Maeda, S., Chang, L. & Karin, M. Loss of hepatic NF-κB activity enhances chemical hepatocarcinogenesis through sustained c-Jun N-terminal kinase 1 activation. Proc. Natl Acad. Sci. USA 103, 1054410551 (2006)
  22. Chang, Q. et al. Sustained JNK1 activation is associated with altered histone H3 methylations in human liver cancer. J. Hepatol. 50, 323333 (2009)
  23. Lim, C. P., Jain, N. & Cao, X. Stress-induced immediate-early gene, egr-1, involves activation of p38/JNK1. Oncogene 16, 29152926 (1998)
  24. Cavigelli, M., Dolfi, F., Claret, F. X. & Karin, M. Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation. EMBO J. 14, 59575964 (1995)
  25. Corsini, N. S. et al. The death receptor CD95 activates adult neural stem cells for working memory formation and brain repair. Cell Stem Cell 5, 178190 (2009)
  26. Kleber, S. et al. Yes and PI3K bind CD95 to signal invasion of glioblastoma. Cancer Cell 13, 235248 (2008)
  27. O’ Reilly, L. A. et al. Membrane-bound Fas ligand only is essential for Fas-induced apoptosis. Nature 461, 659663 (2009)
  28. Romero, I. L. et al. Effects of oral contraceptives or a gonadotropin-releasing hormone agonist on ovarian carcinogenesis in genetically engineered mice. Cancer Prev. Res. 2, 792799 (2009)
  29. Greene, A. K. & Puder, M. Partial hepatectomy in the mouse: technique and perioperative management. J. Invest. Surg. 16, 99102 (2003)
  30. Mitchell, C. & Willenbring, H. A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice. Nature Protocols 3, 11671170 (2008)

Download references

Author information

  1. These authors contributed equally to this work.

    • Lina Chen &
    • Sun-Mi Park

Affiliations

  1. The Ben May Department for Cancer Research, The University of Chicago, 924 E 57th Street, Chicago, Illinois 60637, USA

    • Lina Chen,
    • Sun-Mi Park,
    • Annika Hau,
    • Christine Feig &
    • Marcus E. Peter
  2. Department of Pathology, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637, USA

    • Alexei V. Tumanov,
    • Jerrold R. Turner &
    • Yang-Xin Fu
  3. Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, The University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637, USA

    • Kenjiro Sawada,
    • Iris L. Romero &
    • Ernst Lengyel
  4. Present addresses: Osaka University Graduate School of Medicine, Department of Obstetrics & Gynecology, 2-2, Yamadaoka, Suita, Osaka 565-0879, Japan (K.S.); Cambridge Research Institute/Cancer Research UK, Li Ka Shing Centre, Robinson Way, Cambridge, CB2 0RE, UK (C.F.); Northwestern University Feinberg School of Medicine, 303 East Superior Street, Chicago, Illinois 60611, USA (L.C., A.H. and M.E.P.).

    • Lina Chen,
    • Annika Hau,
    • Kenjiro Sawada,
    • Christine Feig &
    • Marcus E. Peter

Contributions

L.C. and S.M.P performed the experiments; A.V.T. performed the partial hepatectomy; A.H., K.S., C.F. and I.L.R. performed some experiments; J.R.T. performed pathology analyses; Y.-X.F. and E.L. supervised some experiments; M.E.P designed experiments and supervised the project.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Information (3.7M)

    This file contains Supplementary Figures 1-13 with legends, Supplementary Results, Supplementary Materials and Methods and additional references. Supplementary Fig. 8 and Supplementary Methods were replaced on 9 March 2011.

Additional data