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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 29, 2005 as doi:10.1096/fj.04-3482fje. |
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INSERM U563 CPTP, Département Innovation thérapeutique et Oncologie Moléculaire, Toulouse; and Institut Claudius Regaud, Toulouse F-31052; Université Paul Sabatier, Toulouse, France
1Correspondence: Institut Claudius Regaud, 20-24 Rue du pont Saint-Pierre, 31052 Toulouse Cedex, France. E-mail: tilkin{at}icr.fnclcc.fr
SPECIFIC AIMS
Deficiencies in membrane expression of tumor-derived peptide/MHC Class I complexes are often correlated with tumor progression. The primary aim of this work was to examine the effects of statins on IFN-
-induced expression of MHC Class I in melanoma cell membranes. The latter aim was to test statin capacity to favor immunogenicity due to overexpression of molecules implicated in the setting of anti-tumor response (namely MHC Class I and costimulatory molecules expression) and finally to identify the check-point activity of statins for these effects on the mevalonate pathway.
PRINCIPAL FINDINGS
1. Statins and geranylgeranyl transferase I inhibitor (GGTI-298), but not farnesyl transferase inhibitor (FTI-277), enhance MHC Class I expression induced by mIFN-
Murine B16F10 melanoma cell line, which has deficient MHC Class I (H-2 Class I) antigen surface expression that can be corrected by mIFN- administration, was chosen for this study. The expression of H-2Kb molecules was analyzed by cytofluorometry on B16F10 cells cultivated for 4 days in presence of statins (Atorvastatine 10 µM or Lovastatin data not shown), mevalonolactone (800 µM) or inhibitors of protein prenylation (GGTI-298 5 or 10 µM and FTI-277 5 or 10 µM). As shown in Fig. 1
A treatment of B16F10 with Atorvastatinee and GGTI-298 enhanced the mIFN--induced expression of H-2Kb. Statins, being inhibitors of HMG CoA reductase, their effect on H-2Kb expression was reversed by addition of mevalonate (mevalonolactone), the first product of the reduction of HMG CoA. Statins inhibit isoprenoid pathways by preventing the reduction of HMG CoA to mevalonate, which is the precursor of isopentenyl pyrophosphate, then successively converted to geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. The use of GGTI-298 and FTI-277 showed that the statin effect occurs because of geranylgeranyl pyrophosphate depletion but not of farnesyl pyrophosphate depletion (Fig. 1A
).
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Analysis of H-2Kb expression after B16F10 culture in the presence of increasing doses of mIFN- or GGTI-298 or both is illustrated in Fig. 1B
. It shows that GGTI-298 has no effect on H-2Kb expression whereas this expression increases in a dose-dependent manner in mIFN--treated cells, reaching a steady-state level at 50 IU/mL, and a dramatic dose-dependent increase is noted in B16F10 cells treated with the association of mIFN- plus GGTI-298. The mIFN- reversible defect of MHC Class I expression in B16F10 cells had been attributed to a coordinated down-regulation of multiple components of the MHC Class I antigen-processing pathway, including the peptide transporter TAP1 and subunits of the multicatalytic proteasome, such as LMP2 and LMP7. We determined TAP-1, LMP2, and LMP7 expression after 4 days of treatment with mIFN- (25 IU/mL) combined or not with GGTI-298 (10 µM). As expected, mIFN- up-regulated the expression of TAP-1, LMP2, and LMP7 while GGTI-298 had no significant effect. The combination of GGTI-298 and mIFN- enhanced mIFN--induced expression of TAP1 but not of LMP2 and LMP7 proteins (Fig. 1C
). These results show that the GGTase I inhibition enhances IFN--induced MHC Class I expression through a mechanism involving TAP-1 overexpression.
2. GGTI-298 increases the anti-tumor effect of mIFN- against B16F10 growth in immunocompetent mice but not in nu/nu mice
To test whether these in vitro combined treatments of B16F10 cells with GGTI-298 and mIFN- before injection could improve the anti-tumor response against B16F10 tumor growth in mice, B16F10 cells were treated for 4 days, washed extensively, and 1 x 105 viable cells were subcutaneously injected in parallel into syngeneic immunocompetent or athymic C57BL/6 mice. In immunocompetent mice B16F10 cells treated with 50 IU/mL mIFN- or 10 µM GGTI-298 grew slower than untreated (control) cells, but this was not statistically significant as shown by a Student’s t test (P<0.1) (Fig. 2
A). In contrast, a statistically significant reduction of tumor growth (P<0.05) was noted between untreated cells and those treated with GGTI-298 (10 µM) plus mIFN- (50 IU/mL) (Fig. 2A
). Moreover, we observed a 2 day delay in the appearance of tumors after injecting cells treated with GGTI-298 plus mIFN- compared with untreated cells (Fig. 2A
).
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To evaluate the involvement of T lymphocytes and/or NK cells in these in vivo anti-tumor responses, the same populations of untreated B16F10 cells or those treated with mIFN- or GGTI-298 alone or combined were injected in parallel into athymic (nu/nu) C57BL/6 mice. Untreated or treated B16F10 cells gave rise to similar tumor growth kinetics in nu/nu mice (Fig. 2B
). These results showed that in vitro combined treatment with GGTI-298 and mIFN- can improve the in vivo anti-tumor response against B16F10 tumor growth and that T lymphocyte responses are implicated in this response.
3. GGTI-298 induces membrane expression of CD80 and CD86 costimulatory molecules
The anti-tumor response inducing the slowing down of GGTI-298-treated B16F10 tumor growth in immunocompetent, but not in nu/nu mice, implies the involvement of an additional immunological mechanism independent of MHC Class I expression (Fig. 2A
). Therefore, the role of other relevant molecules known to favor anti-tumor immune responses (namely, CD80 and CD86 costimulatory molecules) was investigated. It was shown by cytofluorometry that expression of these costimulatory molecules was induced in a dose-dependent manner on B16F10 membrane after in vitro culture in the absence of mIFN- and in the presence of GGTI-298.
4. Specific CD8 T lymphocytes are involved in the growth control of B16F10 tumor cells treated with mIFN- and GGTI-298
Analysis of spleen cells harvested from the immunocompetent mice injected with B16F10-treated cells and restimulated in vitro with the same treated tumor cells, showed an increase of the activated CD8 T lymphocyte subpopulation and of specific anti-tumor CD8 T lymphocytes labeled by the B16F10 specific TRP-2/H-2Kb tetramers.
5. Inhibition of Rho protein function mimics GGTI-298 effects on MHC Class-I, CD80, CD86 molecules expression, and on in vivo antitumor response
GGTI-298 efficiently prevents geranylgeranylation of several proteins, among which the Rho GTPases involved in cell signaling and transcription regulation. So we tested the hypothesis that Rho proteins are involved in these events in the B16F10 melanoma model. We therefore incubated B16F10 cells with the Clostridium Botulinum C3 exoenzyme, which specifically and totally inhibits the function of Rho (RhoA, RhoB, RhoC) by ADP ribosylation. Incubation for 4 days with 7,5 µg/mL of the permeant TAT-C3 fusion protein instead of GGTI-298 enhanced mIFN- (50 IU/mL) -induced expression of H-2Kb and induced the expression of the CD80 and CD86 costimulatory molecules. These results show that Rho proteins are involved in the control of IFN--induced expression of MHC Class I and B7 costimulatory molecules expression.
Furthermore, TAT-C3 phenocopy GGTI-298 for the induction of an in vivo anti-B16F10 tumor response in syngeneic immunocompetent mice.
CONCLUSIONS AND SIGNIFICANCE
Our study provides evidence that statins potentiate the IFN--induced expression of MHC Class I in murine and human melanoma cell lines and that geranylgeranyl transferase I inhibition, but not farnesyl transferase inhibition, accounts for this immunomodulatory effect. In vitro treatment of these tumor cells with a geranylgeranyl transferase I inhibitor (GGTI-298) also induces membrane expression of costimulatory molecules of the B7 family (CD80 and CD86). MHC Class I and costimulatory molecules expression being important for the setting of anti-tumor immune response, such ex vivo treatments of melanoma cells by IFN- associated with GGTI-298 favor the development of a specific anti-tumor immune T cell response detected by CD8 antibodies and TRP-2/H-2Kb tetramers.
The similar effects obtained with GGTI-298 or C3 exoenzyme strongly suggest that the GGTI-298 effects act through an inhibition of the Rho proteins functions and implies the involvement of Rho proteins in the negative control of MHC Class I and costimulatory molecules expression. Our data identify Rho proteins as new targets for therapeutic strategies to stimulate specific immune response in melanomas.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3482fje;
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