Article

Timing is critical for an effective anti-metastatic immunotherapy: the decisive role of IFNγ/STAT1-mediated activation of autophagy.

Molecular Immunology and Pharmacology Laboratory, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
PLoS ONE (Impact Factor: 3.53). 01/2011; 6(9):e24705. DOI: 10.1371/journal.pone.0024705
Source: PubMed

ABSTRACT Immunotherapy is often recommended as an adjuvant treatment to reduce the chance of cancer recurrence or metastasis. Interestingly, timing is very important for a successful immunotherapy against metastasis, although the precise mechanism is still unknown.
Using a mouse model of melanoma metastasis induced by intravenous injection of B16-F10 cells, we investigated the mechanism responsible for the diverse efficacy of the prophylactic or therapeutic TLR4 and TLR9 agonist complex against metastasis. We found that the activation of TLR4 and TLR9 prevented, but did not reverse, metastasis because the potency of this combination was neither sufficient to overcome the tumor cell-educated immune tolerance nor to induce efficacious autophagy in tumor cells. The prophylactic application of the complex promoted antimetastatic immunity, leading to the autophagy-associated death of melanoma cells via IFNγ/STAT1 activation and attenuated tumor metastasis. IFNγ neutralization reversed the prophylactic benefit induced by the complex by suppressing STAT1 activation and attenuating autophagy in mice. However, the therapeutic application of the complex did not suppress metastasis because the complex could not reverse tumor cell-induced STAT3 activation and neither activate IFNγ/STAT1 signaling and autophagy. Suppressing STAT3 activation with the JAK/STAT antagonist AG490 restored the antimetastatic effect of the TLR4/9 agonist complex. Activation of autophagy after tumor inoculation by using rapamycin, with or without the TLR4/9 agonist complex, could suppress metastasis.
Our studies suggest that activation of IFNγ/STAT1 signaling and induction of autophagy are critical for an efficacious anti-metastatic immunotherapy and that autophagy activators may overcome the timing barrier for immunotherapy against metastasis.

0 Bookmarks
 · 
102 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To develop a rational immunotherapy against tumor metastasis by combining a Toll-like-receptor 2 (TLR2)-neutralizing antibody with a TLR9 agonist CpG ODN, and then investigate the mechanism of action for this combinational regimen. After mouse melanoma B16-F10 cell inoculation, female C57BL/6 mice were treated with either CpG ODN (0.5 mg/kg) or the anti-TLR2 antibody (200 μg/kg), or with a combination of the two agents. Pulmonary metastases were evaluated by counting metastatic nodes on the lung surface using anatomical microscopy. Flow cytometry was used to evaluate the cytotoxicity of the immune cells in tumor-draining lymph nodes, the cell population in the spleen, and the infiltration of immune cells within the lungs. Cytokine and enzyme expression in the lung tissue was evaluated using ELISA or immunostaining. Anti-metastatic effects were detected in mice treated with either CpG ODN or the anti-TLR2 antibody alone. However, treatment with CpG ODN plus the anti-TLR2 antibody synergistically suppressed the metastasis as compared with treatment with either single agent. The combinational treatment resulted in enhanced infiltration of natural killer cells and cytotoxic T cells, reduced recruitment of type 2 macrophages and Tregs, and decreased expression of immunosuppressive factors including TGF-β1, cyclooxygenase-2 and indoleamine 2,3-dioxygenase, thus stimulated tumor cytotoxicity and suppressed metastasis. The anti-metastatic effect of the combinational regimen was further confirmed in spontaneous metastatic mouse model of Lewis lung carcinoma. Our studies suggest that combining a TLR9 agonist with an anti-TLR2 antibody, which eliminates immunosuppressive factors from the tumor environment, is critical for an effective anti-metastatic immunotherapy.
    Acta Pharmacologica Sinica 03/2012; 33(4):503-12. · 2.35 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND: Expression of toll-like receptor-4 (TLR4) on tumor cells is known to mediate innate immune responses that influence tumor cell growth and migration. This study aimed to characterize TLR4 expression and elucidate its functional significance in human hepatoblastoma (HB) cells. PROCEDURE: Immunohistochemistry (IHC) was used to determine TLR4 expression level and its distribution pattern in HB liver tissues. Transcripts of tumor necrosis factor (TNF)-α, interleukin (IL)-8, matrix metalloproteinase (MMP)-2, MMP-13, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 in HB HepG2 cells with lipopolysacharide (LPS) treatment were measured by quantitative PCR. Soluble cytokines and peptides in conditioned media were measured by ELISA. MMP-2 activity was determined by using gelatin zymography. Cell motility and invasiveness was determined using wound healing migration and Matrigel invasion assays, respectively. RESULTS: TLR4 IHC staining demonstrated that TLR4 overexpression in HB liver tissues dramatically vanished after chemotherapy. In vitro study using an HB cell line, HepG2, showed that TLR4 agonist, LPS, significantly decreased transcripts of IL-8 and TNF-α, but did not affect MMP-13 mRNA level. By contrast, LPS only down-regulated IL-8 production and MMP-2 gelatinolytic activity. The latter might be in part due to the increased levels of MMP-2/TIMP-2 complex in conditioned media, thus leading to the decreased motility and invasiveness of HepG2 cells. CONCLUSIONS: HB cells overexpress TLR4, whereas TLR4 agonistic treatment inhibits migration and invasion of HB HepG2 cells. These findings suggest that TLR4 signaling pathway is a potential therapeutic target for control of HB tumor progression. Pediatr Blood Cancer © 2012 Wiley Periodicals, Inc.
    Pediatric Blood & Cancer 05/2012; · 2.35 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Interferon γ (IFN-γ)-induced cell death is mediated by the BH3-only domain protein, Bik, in a p53-independent manner. However, the effect of IFN-γ on p53 and how this affects autophagy have not been reported. The present study demonstrates that IFN-γ down-regulated expression of the BH3 domain-only protein, Bmf, in human and mouse airway epithelial cells in a p53-dependent manner. p53 also suppressed Bmf expression in response to other cell death-stimulating agents, including ultraviolet radiation and histone deacetylase inhibitors. IFN-γ did not affect Bmf messenger RNA half-life but increased nuclear p53 levels and the interaction of p53 with the Bmf promoter. IFN-γ-induced interaction of HDAC1 and p53 resulted in the deacetylation of p53 and suppression of Bmf expression independent of p53's proline-rich domain. Suppression of Bmf facilitated IFN-γ-induced autophagy by reducing the interaction of Beclin-1 and Bcl-2. Furthermore, autophagy was prominent in cultured bmf(-/-) but not in bmf(+/+) cells. Collectively, these observations show that deacetylation of p53 suppresses Bmf expression and facilitates autophagy.
    The Journal of Cell Biology 04/2013; 201(3):427-37. · 10.82 Impact Factor

Full-text (2 Sources)

View
45 Downloads
Available from
May 17, 2014