Because metastatic melanoma are resistant to all currently used treatments, the discovery of new therapeutic drugs is a very important challenge. We and others have previously shown that ciglitazone, a molecule of the TZD family, is able to induce apoptosis independently of PPARγ
activation and to inhibit proliferation of melanoma cells.7, 19, 20, 21
In this report, we have investigated the molecular mechanism by which ciglitazone exerts its anti-melanoma activity.
Our results showed that conditioned media from ciglitazone-treated melanoma cells inhibit normal human melanocyte growth indicating that the effects of ciglitazone on melanoma cells are mediated by secreted factors. RT-QPCR screening demonstrating an inhibition of CXCL1 chemokine upon ciglitazone treatment prompted us to focus our attention on this cytokine. CXCL1 also named melanoma growth stimulating activity or growth-regulated oncogene α
) belongs to the CXC family. CXCL1 is essential for the establishment and the maintenance of the tumoral potential of melanoma.22, 23
CXCL1 bind with high affinity to CXCR2. Overexpression of CXCL1 in INK4a/ARF−/−
mice favors melanoma development24
and promotes malignant growth of murine squamous cell carcinoma by a CXCR2-dependent pathway.25
Blocking antibodies to either CXCL1 or CXCR2 inhibit melanoma cell growth showing the key role of the CXCL1/CXCR2-signaling pathway in melanoma development.26, 27
In addition to its autocrine role, CXCL1 has been shown to have an important paracrine role by regulating angiogenesis during tumor development including melanoma.28, 29
First, we show that ciglitazone negatively regulates mRNA and protein CXCL1 levels. This inhibition is accompanied by a reduction of CXCL1 in the medium. The inhibition of CXCL1 in response to ciglitazone precedes the decrease in cell viability, indicating that the decrease in CXCL1 expression induced by ciglitazone is not a consequence of cell death. A similar decrease in CXCL1 is also observed in response to troglitazone whereas rosiglitazone and pioglitazone have no significant effect on CXCL1 secretion. This result is in agreement with our previous report showing that both rosiglitazone and pioglitazone have only marginal PPARγ
-dependent effect on viability of melanoma cells.7
Consistent with this finding, PPARγ
silencing by siRNA does not abrogate the effects of ciglitazone on CXCL1 expression, indicating that the regulation of CXCL1 is PPARγ
In normal human melanocytes, there is not basal CXCL1 production. Treatment of normal human melanocytes with TNFα
stimulation increases CXCL1 secretion that is not affected by ciglitazone. Conversely, melanoma cells generally have a constitutive basal CXCL1 expression mainly due to the NF-κ
B pathway constitutive activation.22
Independently of the mutational status, melanoma development stage or CXCL1 basal level, we found that ciglitazone inhibits CXCL1 production in all tested melanoma cell lines. Moreover, there is a positive correlation between the level of basal CXCL1 and the decrease in cell viability mediated by ciglitazone, suggesting that melanoma cells producing CXCL1 are addict to these cytokines.
In the same way, in other cancer cell lines such as prostate carcinoma or neuroblastoma, ciglitazone also decreases CXCL1 level and cell viability. These results support the idea that inhibition of CXCL1 cytokine could have a general role in the anti-tumoral effects of ciglitazone.
It has been proposed that some PPARγ
agonists lead to inhibition of NF-κ
B activation,30, 31, 32
but in our system, the inhibition of CXCL1 by ciglitazone is not mediated by the downregulation of NF-κ
B activity (data not shown). Interestingly, a recent paper showed that ciglitazone led to a decrease in MITF expression17
and in silico
analysis of the CXCL1 promoter showed four potential E boxes that match with the consensus binding site for MITF transcription factor (data not shown), thereby suggesting the possible involvement of MITF in the control of CXCL1
expression. These two observations prompted us to investigate the role of MITF in the regulation of CXCL1 by ciglitazone. First, ChIP experiments and luciferase assays show that MITF binds to and regulates the CXCL1
promoter. MITF silencing decreases CXCL1
messengers and inhibits CXCL1 protein secretion. Additional studies suggest that MITF interacts with the CXCL1
promoter through a responsive element (CAGGTG) at –375.
Furthermore, we have previously found that MITF is cleaved in response to the apoptosis inductor, TRAIL.33
MITF diminution that we observed in response to ciglitazone is not due to a protein cleavage by caspases because MITF decrease is seen before apoptosis induction and is also seen on mRNA level and in the presence of pan-caspase inhibitor Z-VAD-FMK. These interesting results highlight a new specific pathway mediated by ciglitazone in melanoma cells. We have demonstrated that the negative regulation of MITF by ciglitazone is a PPARγ
-independent event. However, the precise mechanism responsible for this negative regulation of MITF is not known. One possible mechanism might involve downregulation of the Wnt/β
-catenin pathway because this pathway is inhibited by some PPARγ
agonists and might mediate the inhibition of MITF expression.17, 18
Thus, considering all our results we show for the first time that the control of MITF expression by ciglitazone is involved in the inhibition of CXCL1 expression and secretion. This new signaling pathway involves MITF in the regulation of CXCL1 and strengthens the importance of this transcription factor in melanoma tumorigenicity.
We have previously shown that ciglitazone has cytostatic and cytotoxic effects.7
We found that both cell cycle arrest and apoptosis evoked by ciglitazone are reversed by addition of rCXCL1 or TNFα
. These data suggest that ciglitazone effects on cell viability are mediated mainly by a decrease in CXCL1 expression and secretion. In addition, rCXCL1 does not protect cells from apoptosis induced by TRAIL and staurosporine, suggesting a potential specificity of rCXCL1 on ciglitazone effects. The depletion of CXCL1 with siRNA is sufficient to mimic the biological effects of ciglitazone on A375 cells. Consequently, these results reinforce the hypothesis that inhibition of CXCL1 synthesis by ciglitazone is implied in the biological effects of this TZD. A second approach by CXCL1-neutralizing antibodies confirmed these results. Therefore, the reduction of secreted form of CXCL1 is involved in ciglitazone biological effects.
Finally, we have evaluated the correlation between potential anti-melanoma activity of ciglitazone and CXCL1 secretion in a mouse model of melanoma xenografts. We have previously demonstrated that ciglitazone prevents tumor growth development.7
Importantly, our present results show that the short-term administration of ciglitazone not only prevents tumor formation but also reduces the volume of already established melanoma tumors. In addition, ciglitazone decreases MITF
mRNA level in tumor xenograft, reinforcing the hypothesis that in vivo
, MITF is also involved in the control of CXCL1 mediated by ciglitazone. In parallel, we observe a drastic reduction of serum CXCL1 level in mice. This decrease is not due to the reduction of tumor growth, because when we expressed the quantity of CXCL1 as a function of tumor size, we still found a reduction of circulating CXCL1 in ciglitazone-treated mice. Moreover, injection of CXCL1 in ciglitazone-treated mice compensates for the loss of endogenous CXCL1, impairing the effects of ciglitazone and preventing the decrease in tumor volume. From these observations, we can propose that the decrease in CXCL1 secretion mediated by ciglitazone might be responsible for the antineoplastic effects of this TZD in vivo
In summary, we demonstrate for the first time that ciglitazone inhibits tumor growth through a negative regulation of the MITF/CXCL1/CXCR2 axis-signaling pathway. This demonstration brings new and additional clues to the mechanism of ciglitazone-induced melanoma cell death. Finally, taking into account the drastic effect of ciglitazone on melanoma cell growth, survival and anti-melanoma xenograft development, it might be worth evaluating ciglitazone treatment in patients with metastatic melanoma. Our reports also highlight the pivotal role of tumor cell-produced CXCL1 in melanoma cell proliferation and support the idea that CXCL1 might be used as a new progression marker in the follow-up of the metastatic melanomas. Because the overexpression of CXCL1 and the CXCL1 regulation by ciglitazone are not specific to the melanomas, our study, besides its interest in melanoma pathology, contributes to better understand the general anti-cancer effects of ciglitazone.