In this report, we analyzed the specific role played by GLI2 in human melanoma. We demonstrated that GLI2 expression in melanoma cells is highly variable. High GLI2 expression was associated with an invasive and metastatic phenotype in vitro and in vivo and with mesenchymal traits, such as loss of E-cadherin expression and enhanced MMP secretion. Likewise, in human melanoma tumors, increased GLI2 expression was associated with loss of E-cadherin protein expression. Taken together, these data implicate GLI2 as an important modulator of melanoma progression and metastasis.
Over the past several years, major advances have been made in the identification of genetic and environmental factors that contribute to melanoma development. Defining biomarkers that are predictive of the risk of metastasis remains a major challenge. In this report, we demonstrate that GLI2 is a potent modulator of melanoma invasion and metastasis. Its expression is highly variable among human melanoma cell lines and tissue samples and is inversely correlated with the loss of E-cadherin protein expression. In functional studies, we unveil a direct role for GLI2 in driving melanoma cell invasiveness and metastasis. We previously identified GLI2 as a direct target of TGF-β signaling (23
). Consistently, we found that high GLI2 expression in aggressive melanoma cell lines is driven, at least in part, by either autocrine or paracrine TGF-β signaling. Exogenous TGF-β is a potent inducer of GLI2 expression in a variety of cell types including melanoma cells (23
), irrespective of their basal GLI2 expression status (data not shown). Incubation of the GLI2high cell lines 1205Lu and WM852 with the TGF-β receptor-I kinase inhibitor SB431542 resulted in a three- to fourfold reduction in steady-state levels of GLI2 mRNA (data not shown). We previously demonstrated that either systemic inhibition of TGF-β signaling with the small molecule TGF-β receptor-I inhibitor, SD-208, or overexpression of an inhibitory signaling component, SMAD7, can inhibit melanoma cell invasion in vitro and reduce the occurrence of experimental bone metastases in a mouse model (22
). In the assays of bone metastasis in mice, both SD-208 and SMAD7 overexpression dramatically reduced GLI2 expression as retrospectively evaluated by real-time RT-PCR (data not shown). Likewise, in breast cancer cells, inhibition of TGF-β signaling also efficiently abrogated bone metastasis (33
), whereas overexpression of GLI2 increased bone metastasis (44
). Targeting GLI2
expression in melanoma cells by means of a specific shRNA dramatically reduced invasion of extracellular matrix in vitro () and experimental metastasis to bone in mice (), two processes that are highly dependent on TGF-β signaling (21
). Our findings thus provide new support for a critical role played by GLI2 downstream of TGF-β signaling to drive melanoma metastasis.
In epithelial cancers, loss of E-cadherin protein expression is a hallmark of EMT. This phenomenon is a complex phenotypic conversion that involves changes in morphology, differentiation markers, and cell–cell adhesion molecules, and acquisition of a motile behavior (42
) that is functionally associated with poor prognosis in various cancers (41
). Likewise, a mesenchymal transition is characteristic of melanoma switch from an early radial growth phase to vertical growth phase and subsequent metastasis (47
). In this report, we determined that melanoma cell lines that exhibit high GLI2 at either the mRNA or the protein levels do not express E-cadherin. Reduction of GLI2 protein levels using shRNA provided evidence for a direct role for GLI2 in controlling melanoma invasion and metastasis to bone. We establish that high GLI2 expression in melanoma cell lines is associated with their metastatic potential and inversely correlated with loss of E-cadherin. Our expression data clearly indicate that loss of CDH1 expression is not necessarily associated with changes in SNAIL, SLUG, or TWIST expression, whereas it is consistently associated with elevated steady-state levels of GLI2 mRNA (see ). Interestingly, the GLI1 protein, a direct GLI2 target (37
), was shown to induce EMT in rat kidney epithelial cells via induction of the E-cadherin repressor, SNAIL (49
). Future investigations will focus on the molecular events by which GLI2 controls the transition of melanoma cells to a mesenchymal phenotype.
Remarkably, opposite expression of GLI2 and CDH1 was also found in human melanoma samples. Using three independent sets of samples with distinct experimental approaches, we found that melanoma lesions express GLI2 and CDH1 at variable levels and that melanoma lesions are heterogeneous for GLI2 and E-cadherin expression and/or distribution. These findings are consistent with recent reports in the literature that have identified new regulators of melanoma progression, such as Brn2 or Dia-1, whose expression is also heterogeneous within tumors (50
). E-cadherin plays a critical role in maintaining melanocyte interactions with epidermal keratinocytes and thus keeps progression to a metastatic state at bay. It is therefore reasonable to hypothesize that GLI2 may be a marker of melanoma aggressiveness; first, GLI2high melanoma cell lines are more invasive and metastatic than GLI2low cells, and second, high GLI2
expression in primary melanoma lesions was localized in the deeper part of the tumors and was consistent with a possible role for GLI2 in the progression toward an invasive phenotype. Larger cohorts of samples will be necessary to validate this observation.
Increased GLI2 expression was accompanied with statistically significant reduction of CDH1 expression in distant metastases as compared with primary melanoma tumors (, C). In two sets of samples, we also examined whether more aggressive tumors expressed GLI2 at higher levels than less aggressive ones.
In making this assessment, we used clinical observations of time to metastasis and the extent of initial metastatic spread to define tumor aggressiveness. We found that the two most aggressive primary tumors and the two most aggressive distant metastatic lesions in this study expressed higher levels of GLI2 mRNA compared with their less aggressive counterparts. Inversely, in these two groups, the tumors that expressed the lowest levels of GLI2 mRNA were much more indolent.
This study has several limitations. First, we demonstrated a role for GLI2 in the metastatic activity of melanoma cells in vitro and in a mouse model of bone metastasis in which the experiments were conducted with immunocompromised mice. Immune function may affect the growth and dissemination of melanoma in human patients. In future investigations, it will be important to study whether GLI2 targeting interferes with melanoma metastasis in animal models with spontaneous metastasis. Second, we were not able to measure GLI2 protein levels in human tissue because of the lack of a suitable antibody when these studies were performed, and the number of human specimens that we analyzed was rather small. Newly released commercial antibodies are currently being tested for their specificity. Once adequate antibodies become available, immunohistochemical analyses will be performed on tissues from a larger cohort of patients, with sufficient sample size to allow us to determine whether there is a direct correlation between GLI2 protein expression and histopathological staging of human melanoma.
Taken together, our findings provide a strong rationale to further investigate the role of GLI2 in the initiation and progression of melanoma. Our future goals include the generation of genetic mouse models in which GLI2 function is altered specifically in the melanocyte lineage. It will also be very important to investigate whether GLI2 expression, or the GLI2 to CDH1 ratio, may be a marker of a more aggressive melanoma phenotype in a large cohort of human melanoma samples.