Molecular Vision 2024; 30:160-166
<http://www.molvis.org/molvis/v30/160>
Received 22 October 2023 | Accepted 20 March 2024 | Published 22 March 2024
Molecular Vision 2024; 30:160-166
<http://www.molvis.org/molvis/v30/160>
Received 22 October 2023 |
Accepted 20 March 2024 |
Published 22 March 2024
Coralie Doudnikoff,1 Delphine Leclerc,2 Gaëlle Angenard,3 David Gilot,2 Cédric Coulouarn,2 Frederic Mouriaux1,2
The first two authors contributed equally to this work and are considered as first authors.
The last two authors contributed equally and considered as senior authors.
1Service d’Ophtalmologie, CHU Rennes, Rennes, France; 2INSERM U1242, OSS, Université Rennes, Rennes, France; 3INSERM U1317, NuMeCan, INRAE, Université de Rennes, Rennes, France
Correspondence to: Frederic Mouriaux, INSERM U1242 OSS, Service d’Ophtalmologie, CHU Rennes, 2 Rue Henri le Guilloux, 35000 Rennes, France; email: frederic.mouriaux@chu-rennes.fr
Purpose: Uveal melanoma (UM) is a deadly cancer with limited therapeutic options. At advanced stages, UM cells metastasize almost exclusively into the liver, where targeting metastatic UM cells remain a clinical challenge. Transforming growth factor beta (TGF-β) exhibits a functional duality in cancer, with one arm limiting tumor growth at an early stage and the second arm promoting metastasis at an advanced stage, notably by inducing an epithelial-to-mesenchymal transition. Thus, we hypothesized that targeting the TGF-β pathway could be relevant in the treatment of metastatic UM.
Methods: In this study, we first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines Mel270 and 92.1. We then treated the cell lines with TGF-β to evaluate their responsiveness to the cytokine and to characterize the functional impact of TGF-β on their cell viability.
Results: The results demonstrated, first, that the UM cell lines exhibited a mesenchymal phenotype and responded to TGF-β treatment in vitro and, second, that TGF-β promoted a cytostatic effect on the UM cell lines.
Conclusions: Our findings indicate that UM cells are sensitive to the two arms of TGF-β signaling, which suggests that targeting the TGF-β pathway could be challenging in UM and would require a precise selection of patients in which only the prometastatic arm of TGF-β is activated.
Uveal melanoma (UM) is the most common primary intraocular tumor in adults and represents approximately 5% of all melanomas [1]. This aggressive cancer affects approximately 1 to 9 persons per million population per year [2]. This tumor localizes preferentially in the choroid [2,3]. However, despite effective treatment of the primary tumor, patients may still develop metastases within a few years, almost exclusively in the liver [2]. Currently, the approved treatments for metastatic UM are limited; thus, UM often lead to fatality within 1 to 2 years. While tebentafusp is effective, its application is restricted to HLA-A*02:01-positive patients [4], and selective hepatic perfusion treatment is constrained to cases with isolated liver metastases. The challenges in developing new therapeutic susceptibilities include better understanding of the reason why UM metastasizes preferentially to the liver and identification of the key drivers of this process.
Cytokines are soluble proteins that act through cell-cell communication. By remodeling the tumor microenvironment, cytokines support cancer cell growth, differentiation, migration, and metastasis [5]. Transforming growth factor beta (TGF-β) is a pleiotropic cytokine that exerts a wide range of functions from embryogenesis to tissue homeostasis in adults. In addition, TGF-β contributes to several hallmarks of cancer [6,7]. TGF-β activity is highly dependent on the cellular context and tissue microenvironment. Thus, at the early stages of carcinogenesis, TGF-β exerts cytostatic functions, while in later stages, its activity shifts to tumor-promoting functions, including metastasis, as a potent inducer of epithelial-to-mesenchymal transition (EMT) [8]. Previous works of our team demonstrated that specific TGF-β gene expression signatures can predict patient prognosis in hepatocellular carcinoma (HCC) [9] and induce the expression of EMT-associated transcription factor FOXS1 [10]. Several inhibitors are currently under clinical evaluation in several cancers, including HCC (NCT01246986) [11,12]. Moreover, previous works have demonstrated that TGF-β controls metastasis development, notably in breast cancer and cutaneous melanoma [13,14]. Thus, inhibition of the TGF-β pathway in choroidal melanoma may represent a promising therapeutic approach.
In this study, we hypothesized that as UM metastasizes, especially in the liver, where the microenvironment facilitates TGF-β signaling, the TGF-β pathway might be activated in the UM cells. Thus, the aim of this study was to characterize the TGF-β activity in UM cells.
Mel270 [15] and 92.1 [16] cell lines originate from primary uveal melanoma (UM) tumors diagnosed in patients who have developed metastases [17]. The Mel270 cell line was kindly provided by Dr. Martine Jager (University of Leiden, The Netherlands), and the 92.1 cell
line was obtained from the European Collection of Authenticated Cell Cultures. Cell line authentication was confirmed using
short tandem repeat analysis. UM cells were cultivated in a humidified atmosphere (37 °C, 5% CO2) with RPMI 1640 medium supplemented with 10% (Mel270) or 20% (92.1) fetal bovine serum (FBS; HyClone, Logan, UT), 1%
The cells were seeded in 96-well plates. When the cells reached confluency, they were fixed with 4% paraformaldehyde for 15 min, washed 3 times in phosphate-buffered saline (PBS), and blocked for 1 h in PBS/5% FBS/0.3% Triton X-100. The primary antibodies used for immunofluorescent staining were rabbit anti-vimentin (1:200; No. 5741S, Cell Signaling Technology, Danvers, MA) and rabbit anti-E-cadherin (1:100, No. 3195, Cell Signaling Technology). After overnight incubation with primary antibodies, the cells were washed 3 times with PBS and then incubated with the secondary antibody Dylight 488 goat anti-rabbit (1:400; Eurobio Scientific, Les Ulis, France) for 1 h in the presence of Hoechst 333,442 (1:5000; ThermoScientific, Waltham, MA). The cells were observed under a fluorescence microscope at 470 nm for the secondary antibody and 365 nm for Hoechst. Acquired images were analyzed with the ImageJ software.
UM cells were seeded in 6-well plates. When the cells reached approximately 70% confluency, FBS starvation was performed for 8 h. Four different treatments were then performed in a FBS-free medium as follows: 1) control (solvent alone), 2) TGF-β (1 ng/ml TGF-β1; R&D Systems, Minneapolis, MN), 3) galunisertib (10 mM; Interchim, Montluçon, France), and 4) TGF-β + galunisertib (1 ng/ml TGF-β1 + 10 mM galunisertib). TGF-β1 was diluted in 0.1% bovine serum albumin (BSA), 4 mM HCl, and galunisertib with dimethyl sulfoxide (DMSO). Unless otherwise indicated, the treatments were performed for 16 h.
Mel270 and 92.1 were seeded in triplicate in 96-well plates at a density of 5.0 × 103 and 1.0 × 104 cells/well, respectively. Cell lines were treated with TGF-β (1 ng/ml TGF-β), galunisertib (10 mM LY2157299), or combined treatments for 16 h, 8 h after cell seeding. When cells were incubated for more than 2 days in culture, the same treatment was re-applied on day 2. Cell viability was assessed using a colorimetric assay on the basis of the enzymatic reduction of a yellow tetrazolium salt substrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT; Sigma M2128–250MG] into purple insoluble formazan by metabolically active cells, in accordance with the manufacturer's instructions. Briefly, after removing the cell medium, the cells were incubated with 100-μL MTT substrate for 4 h at 37 °C. Subsequently, the solution was aspirated, and the resulting formazan product was dissolved with 100 μl of DMSO. After 5 min of shaking, absorbance was measured at 535 nm (Polar Star, BMG Labtec).
The cells were lysed chemically using a radioimmunoprecipitation assay lysis buffer (ThermoScientific) supplemented with phosphatase and protease inhibitors (Roche, Bâle, Switzerland). A western blot analysis was performed as previously described [10]. Membranes were initially incubated for 2 h in a blocking solution (TBS-Tween-0.1% + 5% BSA) followed by overnight incubation at 4 °C with a primary antibody: β-Actin (1:1000; No. 8457; Cell Signaling Technology), E-cadherin (1:1000; No. 3195, Cell Signaling Technology), vimentin (1:1000; No. 5741S, Cell signaling Technology), P-SMAD3 (1:1000; No. 9520T, Cell Signaling Technology), P-SMAD2 (1:1000; No. 3108P, Cell Signaling Technology), SMAD3 (1:100; No. 9523P, Cell Signaling Technology), or SMAD2 (1:100; No. 5339P, Cell Signaling Technology). After three washes with TBS-Tween-0.1%, the membrane was incubated for 1 h with an HRP-linked secondary antibody (anti-rabbit IgG, 1:1000; No. 7074, Cell Signaling Technology), and the membrane was incubated for 1 h. Detection was performed using a chemiluminescent substrate reagent (Western ECL Select, GE Healthcare, Chicago, IL).
RNA was directly extracted from the cell culture plates using a miRNAeasy kit (Qiagen, Hilden, Germany) and quantified using a nanodrop. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed as previously described [10], starting from 1-µg total RNA. Briefly, reverse transcription was performed using a Superscript III RT enzyme (Invitrogen), and quantitative PCR was performed using the SYBR Green Master Mix (Applied Biosystems). The primers used (Eurogentec) are listed in Appendix 1, and the TATA-binding protein was used as the internal control for normalization. The results were analyzed using the StepOne v2.3 software. Relative expressions were compared with the control group using the 2−ΔΔCt method.
We first characterized the pseudoepithelial/mesenchymal phenotype of UM cell lines to evaluate their degree of tumor progression. The presence of Mel270 and 92.1 expressions were established in primary UMs from patients who developed metastases [17]. Immunofluorescent staining demonstrated that Mel270 and 92.1 cells do not express E-cadherin (CDH1−) but express vimentin (VIM+; Figure 1A). These results were confirmed by the results of western blot experiments that showed the presence of VIM expression but the absence of CDH1 expression (Figure 1B) in both cell lines. Thus, Mel270 and 92.1 already lost their pseudoepithelial identity and switched to a mesenchymal state, consistent with the results of previous studies that associated UM cell line aggressivity [18] and UM metastatic phenotype with high vimentin and low E-cadherin expression levels [19,20].
We next performed TGF-β treatments to evaluate if the TGF-β pathway is active in these cell lines in vitro. First, we conducted a time course experiment to analyze the TGF-β activation. Our results demonstrated that Mel270 responded to TGF-β and that the highest activation rate was obtained after 2 h of treatment, as exemplified by SMAD2/3 phosphorylation (Figure 2A). We next tested the specificity of the response by performing a galunisertib treatment (LY2157299, an inhibitor of the type 1 TGF-β receptor, TGFBR1). Our results indicated that SMAD2/3 phosphorylation was dependent on TGF-β activation through TGFBR1, as expected (Figure 2B–D). The results obtained from the 92.1 cells showed that P-SMAD2 and P-SMAD3 appeared to be poorly regulated by TGF-β compared with the Mel270 cells (Figure 2B). However, the pathway was still activated, as the canonical TGF-β target genes (SMAD-dependent) were induced in both cell lines. Our experiment results showed that the expression of SERPINE1, a serine protease inhibitor transcriptionally activated by SMAD2/SMAD3, was induced more than tenfold after TGF-β treatment. The expression of SMAD7, a TGF-β antagonist acting through negative feedback, was also clearly induced in the 92.1 cells (Figure 2E), supporting the activation of the SMAD-dependent TGF-β pathway. Even if the SMAD7 expression in Mel270 cells was unclear (Figure 2E), differences in the origins and genetic backgrounds of the cell lines likely explain these phenotypic variations. We then performed a functional analysis (viability assay) to evaluate whether the cell lines might be sensitive to the cytostatic effect of TGF-β. Surprisingly, we observed a slight decrease in the cell density after 3 or 4 days of treatment (Figure 2F).
Taken together, our results demonstrate that although the 92.1 and Mel270 cell lines exhibited a mesenchymal phenotype, they not only responded to the TGF-β treatment but also were sensitive to its cytostatic effects in vitro.
In this study, we evaluated the impact of TGF-β, a pleiotropic cytokine involved in numerous cancers, including liver tumors [9,10], in primary UM cell lines in vitro (92.1 and Mel270). In summary, our results show that Mel270 and 92.1 expressed a mesenchymal phenotype, consistent with their ability to invade and develop metastases. Functional analyses demonstrated that the TGF-β pathway can be activated in these cell lines through the SMAD-dependent canonical pathway, and this activation leads to a slight reduction in cell viability in vitro. While further experiments are needed to confirm these findings in other UM cell lines, replicating the complexity of liver metastasis regulation in vitro is challenging because of multiple influencing factors. While we observed a weak antitumorigenic effect on cell density, whether TGF-β reduces proliferation or induces cell death in these cells and whether this observation is true at different UM progression steps remain to be clarified. Questions regarding adhesion, invasion, and migration properties might be interesting to investigate. One other interesting point is whether TGF-β can induce EMT in UM, as already demonstrated in HCC by our team [10].
Other research groups have studied the functional effect of TGF-β on UM cells. For instance, a previous study demonstrated that TGF-β inhibits the invasion of UM cell lines in vitro [21], thus confirming its antitumoral role, as we also observed in the present study. It was also reported that paradoxically, TGF-β1 secretion by UM cells promotes liver metastatic colonization through crosstalk with hepatic stellate cells and extracellular matrix remodeling, highlighting a protumoral effect of TGF-β1 [22]. These observations were also validated in vivo. In a mouse model of intra-splenically inoculated Mel270-luc cells, mice treated with SB525334 (an inhibitor of TGFBR1) presented less bioluminescence signal and metastatic nodules in the liver than mice treated with the vehicle [22]. These conflicting results raise the question of whether the TGF-β ligands from the liver microenvironment act directly on cancer cells and/or indirectly through stromal cells and illustrate the complexity of studying metastasis. Thus, TGF-β may exert tumor-suppressive or tumor-promoting functions depending on the tumor stage of UM, highlighting its functional duality, as observed in other types of cancer [8]. In conclusion, targeting the TGF-β pathway in metastatic UM should take into account the sensitivity of tumor cells to the cytostatic effects of TGF-β. In this context, specific biomarkers should also be developed in the future to identify patients with UM who may benefit from inhibition of the TGF-β pathway.
Authorship contribution: Coralie Doudnikoff: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Validation, Writing – review & editing Delphine Leclerc: Visualization, Validation, Writing – original draft, Writing – review & editing Gaëlle Angenard: Data curation, Investigation – review & editing David Gilot: Resources, Investigation, Writing – review & editing Cédric Coulouarn: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Validation, Writing – original draft, Writing – review & editing, Project administration. Frederic Mouriaux: Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Validation, Writing – original draft, Writing – review & editing, Project administration. Funding information: Authors would like to thanks their sponsors: CD is funded by funded by Fonds de recherche en opthtalmologie (CHU Rennes). DL is funded by Ministère de l’enseignement Supérieur, de la Recherche et de l’Innovation (PhD fellowship). CC is funded by Inserm, University of Rennes 1, Ligue Contre le Cancer (CD22, CD35, CD44, CD85), Fondation ARC, French Ministry of Health and the French National Cancer Institute (PRT-K20–136, CHU Rennes, CLCC Eugène Marquis, Rennes). DG is funded by Aviesan Plan Cancer and Canceropôle Grand-Ouest.