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RESEARCH
Trabectedin impairs invasiveness and metastasis in adrenocortical carcinoma preclinical models
Andrea Abate 1, Mariangela Tamburello1, Elisa Rossini1, Ram Manohar Basnet1, Giovanni Ribaudo1, Alessandra Gianoncelli1, Constanze Hantel2,3, Deborah Cosentini4, Marta Laganà4, Salvatore Grisanti4, Guido Alberto Massimo Tiberio5, Maurizio Memo1, Alfredo Berruti4 and Sandra Sigala1
1Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
2Department of Endocrinology, Diabetology and Clinical Nutrition, University Hospital Zurich (USZ) and University of Zurich (UZH), Zurich, Switzerland 3Medizinische Klinik und Poliklinik III, University Hospital Carl Gustav Carus Dresden, Dresden, Germany
4Oncology Unit, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia and ASST Spedali Civili di Brescia, Brescia, Italy
5Surgical Clinic, Department of Clinical and Experimental Sciences, University of Brescia at ASST Spedali Civili di Brescia, Brescia, Italy
Correspondence should be addressed to A Abate: a.abate005@unibs.it
Abstract
The pharmacological approach to adrenocortical carcinoma (ACC) is based on mitotane with/without etoposide, doxorubicin, and cisplatin, according to the disease stage. Considering the limited efficacy and toxicity of this treatment, new strategies are required. Trabectedin is a marine-derivated antitumoral agent that inhibits oncogenic transcription. We have already demonstrated trabectedin cytotoxic activity at sub- nanomolar concentrations in ACC cells. Here, we expanded the investigation of trabectedin effect on ACC preclinical models, evaluating whether trabectedin could affect ACC cells’ invasiveness and metastasis formation. NCI-H295R, MUC-1, and TVBF-7 cell lines were used. Cell tumor xenografts in Danio rerio embryos were performed. The tumor mass areas and the number of embryos with metastasis were evaluated. The in vitro invasiveness of cells was evaluated. Effects of trabectedin of MMP2, TIMP1, and TIMP2 were evaluated at gene level qRT-PCR. MMP2 secreted in the cell medium was evaluated by Western blot and by zymography. Xenograft experiments demonstrated that trabectedin significantly reduced the tumor area in each ACC cell model and metastasis formation in embryos injected with metastasis-derived cell lines. Trabectedin treatment reduced the invasiveness of ACC cells across the matrix, which was greater at baseline for the metastatic models. In metastatic cell models, protein analysis demonstrated a reduction of MMP2 secretion and activity in the culture medium after treatment. Our results indicate that trabectedin interferes with invasiveness and metastasis processes, both dramatic features of ACC. Furthermore, these results support those previously published in providing the rationale for a clinical evaluation of the efficacy of trabectedin in ACC patients.
Key Words
ACC
cell invasiveness
metastasis
cell cultures
· zebrafish embryos
Endocrine-Related Cancer (2022) 30, e220273
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Introduction
Adrenocortical carcinoma (ACC) is a rare tumor with an annual incidence of 0.7-2.0 cases/million people per year. The disease often has an aggressive behavior, and the overall 5-year survival rate of metastatic patients is less than 15% (Fassnacht et al. 2020). To date, the greatest chance of survival for patients with an early-stage disease is provided by radical surgery in experienced centers, while those with locally advanced ACC could respond to neoadjuvant treatment. However, in about half of patients, the diagnosis of ACC occurs when the disease is already metastatic and not eligible for surgery. Moreover, 30-70% of radically operated patients recur within 2 years, often with metastatic disease (Terzolo et al. 2014). The current pharmacotherapy for ACC is based on the adrenolytic drug mitotane (M) as adjuvant therapy in radically operated patients who are at high risk of relapse (Berruti et al. 2017, Fassnacht et al. 2020), and mitotane combined with the chemotherapeutic drugs etoposide (E), doxorubicin (D), and cisplatin (C) in advanced/metastatic ACC patients is not eligible for surgery (Berruti et al. 2005, Fassnacht et al. 2020). The EDP-mitotane (EDP-M) approach, however, has limited efficacy with penalizing toxicity (Fassnacht et al. 2012, Laganà et al. 2020). To date, no efficacious second- line therapies are available in the clinical armamentarium (Grisanti et al. 2019, Cremaschi et al. 2022), and the identification of new therapeutic strategies is an unsolved need. Preclinical (Abate et al. 2020, Cremaschi et al. 2022) and clinical (Cosentini et al. 2019) evidence supports the role of DNA binders against ACC. Trabectedin is an antitumor drug, isolated from Ecteinascidia turbinata, approved for the treatment of soft tissue sarcoma (STS) and relapsed platinum-sensitive ovarian cancer (OC) (https:// www.micromedexsolutions.com/home/dispatch/). Several clinical studies are currently ongoing, with the aim of expanding the therapeutic indications of this molecule (https://www.clinicaltrials.gov). In addition to the DNA- binding activity, the mechanism of action of trabectedin is more complex and can affect both tumor cells processes (the most relevant effect is the transcription inhibition of activated genes, through direct and indirect mechanisms) and tumor microenvironment (for an extensive discussion refer to Larsen et al. 2016). The Wnt/ß-catenin pathway upregulation is one of the major tumor drivers of ACC pathogenesis (Tissier et al. 2005, Gaujoux et al. 2011) and contributes to the resistance of ACC to immunotherapies (Cosentini et al. 2018, Fiorentini et al. 2019). Trabectedin was also found to inhibit the Wnt signaling pathway in human biliary tract carcinoma (Peraldo-Neia et al.
2014) and in ACC (Abate et al. 2020) preclinical cell models, providing a further mechanism of antineoplastic activity. In the present study, we investigated the effect of trabectedin on ACC cells invasiveness and metastasis formation, using both cell models and zebrafish embryos engrafted with ACC cells.
Materials and methods
Cell lines and culture conditions
Both primary and metastatic ACC cell lines were used. The worldwide used primary ACC cell model, namely NCI- H295R cells (Nanba et al. 2021), were purchased from ATCC and cultured as indicated by the company. As metastatic ACC models, MUC-1 and TVBF-7 cells were used and maintained as suggested (Hantel et al. 2016, Sigala et al. 2022a). Both cell lines were derived from ACC patients in progression after EDP-M. Cell lines were periodically tested for mycoplasma and authenticated using short tandem repeats profile by BMR Genomics S.r.l (Padova, Italy). Media and supplements were purchased from Merck.
Chemicals
Trabectedin was kindly given by Pharma Mar S.A. (Madrid, Spain), dissolved in 100% dimethyl sulfoxide, and stored at -20°℃ in 10 mM aliquots. Cimetidine was purchased from Sigma Aldrich, resuspended in 100 % dimethyl sulfoxide, and stored at -20°℃ in 10 mM aliquots. For high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) experiments, drugs were subsequently diluted in methanol, purchased from CARLO ERBA Reagents S.r.l. (Milan, Italy).
Cell treatment, cell viability, and cell proliferation
TVBF-7 cells (20,000 cells/well) were seeded in 24-well plates and treated with increasing concentrations of trabectedin for up to 4 days. The length of treatment was chosen based on doubling time calculation, according to the ATCC method. In particular, the cells were treated for a time corresponding to approximately 2× doubling time, in order to allow the completion of a cell cycle for the entire cell population. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye reduction assay according to the manufacturer protocol (Merck). Cell proliferation rate was evaluated with TC20 automated cell counter (Bio-Rad
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Laboratories). NCI-H295R and MUC-1 cells were treated with trabectedin as described by Abate et al. (2020). Briefly, NCI-H295R and TVBF-7 cells were treated for 4 days, while MUC-1 cells were treated for 5 days. All the subsequent sets of experiments were conducted with the reported trabectedin IC50 and the length of the treatment indicated for each cell line, unless otherwise specified.
Invasion assay
The in vitro invasiveness of cells was evaluated using ECMatrix Cell Invasion Assay™ (Merck). Cells were seeded in a culture dish at an appropriate cell density. After 24 h, cells were exposed to trabectedin or vehicled in fresh medium for the appropriate time of treatment, determined for each cell line as described above. Then, cells were detached using trypsin/EDTA, resuspended, and added to the inserts according to manufacturer protocol. Invasive cells were stained after 3 days of incubation, and after washing, inserts were air-dried. Images were acquired using an Olympus IX51 optical microscope (Olympus) equipped with 10x objective. Subsequently, staining was eluted and absorbance was detected using an EnSight Multimode Plate Reader (PerkinElmer) at 560 nm.
Gene expression
Gene expression was evaluated by q-RT-PCR (ViiA7, Applied Biosystems), and SYBR Green was used as fluorochrome as previously described (Fragni et al. 2016). Sequences of oligonucleotide primers were listed in Supplementary Table 1 (see section on supplementary materials given at the end of this article). ACTB was chosen as housekeeping gene. Differences in the threshold cycle Ct values between the housekeeping gene and the genes of interest (ACt) were calculated as an indicator of the amount of mRNA expressed. The Livak method was applied to analyze the relative changes in gene expression (Livak & Schmittgen 2001).
Western blot
The conditioned medium was obtained as described in Fiorentini et al. (2016), stored at -80℃ for at least 24 h, and subsequently freeze-dried. The residue was resuspended in PBS and total protein concentrations were determined by Bio-Rad Protein Assay (Bio-Rad Laboratories). Proteins were separated by electrophoresis on a 4-12% NuPAGEbis- tris gel system (Life Technologies) and electroblotted to a PVDF membrane, incubated with anti-MMP2 (final
concentration: 0.9 µg/mL. Proteintech) primary antibody. Secondary HRP-labeled anti-rabbit antibody (Promega) was used, and the signal was visualized using Odyssey® Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
Zymography
Different aliquots of the same conditioned medium samples were used for both Western blot and zymography. Proteins were separated by electrophoresis on a 7.5% SDS- PAGE gel. Subsequently, the gel was incubated for 1 h with 2.5% Triton X-100 solution at room temperature. Gel was incubated overnight at 37℃ with collagenase buffer (NaCl 11.68 g/L, CaCl2 1.47 g/L, Tris 4.84 g/L and Brij® 35 0.06% v/v, brought to pH 7.5). Next, the gel was stained with a 0.5% Coomassie R-250 solution (50% water, 40% methanol, and 10% acetic acid) for 1 h and then a decoloring step was performed for 5 min with a solution with the same composition without dye. Reagents for buffer and solution preparation were purchased from Merck.
Fish maintenance and egg collection
Zebrafish were handled according to national and international guidelines (EU Directive 2010/63/EU), following protocols approved by the local committee (Organismo Preposto al Benessere Animale, Università degli Studi di Brescia, protocol no. 211B5.24) and authorized by the Ministry of Health (authorization no. 393/2017-PR). The experiments were performed in compliance with the ARRIVE guidelines (https://arriveguidelines.org). Fish were maintained under standard laboratory conditions as indicated (Westerfield 2000), particularly at 28℃ on a constant 14 h light:10 h darkness cycle. Fish were fed thrice a day with granular dry food and fresh artemia (Special Diet Services, SDS Diets; LBS Biotech, Horley, Surrey, UK). Healthy adult WT zebrafish (AB) and Tg(kdrl:EGFP) zebrafish were used for egg production. The developing embryos were incubated at 28℃ and maintained in 0.003% (w/v) 1-phenyl-2-thiourea (PTU; Merck) to prevent pigmentation.
ACC cell xenograft
ACC cells were exposed overnight with the vital red fluorescent dye CellTrackerTM CM-DiI (Thermo Fisher Scientific), according to manufacturer protocol. Cells were detached with trypsin/EDTA, washed in PBS, resuspended in 50 µL of PBS, and kept at 4℃ until use. Cell xenografts were performed as described in (Gianoncelli et al. 2019)
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with minor modifications. Briefly, zebrafish embryos at 48 h post-fertilization (hpf) were dechorionated, anesthetized with 0.042 mg/mL tricaine (Sigma Aldrich), and microinjected with the labeled ACC cells into the subperidermal space of the yolk sac. Microinjections were performed with a FemtoJet electronic microinjector coupled with an InjectMan N12 manipulator (Eppendorf Italia, Milan, Italy). Approximately 250 cells/4 nL were injected into each embryo; embryos were maintained in PTU/fish water in a 32℃ incubator. Trabectedin (15 nM) or vehicle was directly added to the PTU/fish water in respectively treated and untreated experimental groups 3 h after cell injection. Pictures of injected embryos were acquired using Zeiss Axiozoom V13 (Zeiss) fluorescence microscope, equipped with Zen pro software, 2 h (TO) and 3 days (T3) after cell injection. The tumor areas of treated and untreated groups at TO and T3 were measured with Noldus DanioScopeTM software (Noldus Information Technology, Wageningen, Netherlands). Representative embryos at T3 stage were fixed, embedded in low melting agarose, and acquired using a Zeiss LSM 510 META confocal laser-scanning microscope (Carl Zeiss AG) equipped with a 10× objective.
Trabectedin absorption quantification by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)
Trabectedin absorption from embryos was evaluated by quantifying the concentration of trabectedin by LC-MS/ MS. Ultra-performance liquid chromatography (UPLC) was performed using a DionexTM UltiMateTM 3000 Thermo Fisher Scientific S.p.A equipped with an LPG- 3400SD quaternary analytical pump, a WPS-3000SL analytical autosampler, and a TCC-3000SD thermostatted column compartment. Chromatographic separation was performed using an Eclipse Plus C18 column (100 mm × 2.1 mm ID, particle size: 1.8 um) (Agilent Technologies). The chromatographic system was set up with a flow rate of 0.150 mL/min. The column was kept at 25℃ and equilibrated with 90% mobile phase A (water containing 0.05% formic acid) and 10% mobile phase B (acetonitrile). The gradient was described in Supplementary Fig. 1. Reagent-grade acetonitrile for LC-MS and formic acid (98%) were purchased from CARLO ERBA Reagents S.r.l .. Ultra-pure water was prepared using a Millipore Milli-Q purification system (Millipore Corporation). The UPLC system was coupled with an electrospray ionization mass spectrometer (LCQ Fleet Ion Trap MSn, Thermo Fisher Scientific). The positive ESI conditions were set
as reported in Supplementary Table 2. Data were treated with the Xcalibur software (Version 4.0, Thermo Fisher Scientific). The calibration curves for the quantification of trabectedin and cimetidine (used as internal standard (IS) (Lee et al. 2008)) were obtained both as described later. Fifty embryos for each batch (up to 120 hpf) were put at 4°℃. One hundred microliters of IS (200 nM) was added to each batch with 100 µL of trabectedin at different dilutions in order to obtain the final concentrations 5-500 nM of trabectedin in methanol. Samples were extracted following the protocol previously reported (Gianoncelli 2019). Five microliters of each sample were analyzed by LC-MS/MS (LOD: 1 nM; LOQ: 2.5 nM). The SRM quantified transitions were m/z 744.4-495.4 for trabectedin and m/z 253.3-156.9 for cimetidine.
Statistical analysis
The analysis of the data was carried out by the GraphPad Prism version 5.02 software (GraphPad Software) using the one-way ANOVA with Bonferroni’s multiple comparisons test.
P < 0.05 was considered the threshold for a significant difference. Unless otherwise specified, data are expressed as mean ± S.E.M. of at least three experiments run in triplicate.
Results
Trabectedin-induced cytotoxicity in ACC experimental cell models
Trabectedin reduces cell viability in NCI-H295R and MUC-1 cell lines with an IC50 of 0.15 and 0.8 nM, respectively (Abate et al. 2020). The efficacy of trabectedin on the new- established ACC cell line TVBF-7 cells was then evaluated. Exposure of TVBF-7 cells to increasing concentrations of trabectedin (0.0625-1.5 nM) for 4 days led to a concentration-dependent reduction of cell viability. The sigmoidal concentration-response function was applied to calculate the IC50 value of trabectedin, which was 0.68 nM (95% CI: 0.46-1.01 nM). The drug was highly active in inducing cytotoxicity, indeed its efficacy reached about 90% at the highest concentration tested (Supplementary Fig. 2A). The effect of drug-treatment discontinuation on TVBF-7 cell viability was evaluated as well. Cells were exposed to trabectedin for 4 days, and then, the medium was replaced with drug-free medium for additional 4 days. Results indicated a long-lasting effect of trabectedin, as reported in Supplementary Figure 2B.
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Besides the cytotoxic effect, trabectedin affected the cell proliferation rate as well. The impact of drug treatment on this process was evaluated in each ACC cell model. NCI-H295R, MUC-1, and TVBF-7 cells were treated with three concentrations of trabectedin, corresponding to 0.5x, 1×, and 2× of the respective IC50 trabectedin values obtained with the cytotoxicity experiments. Cell count revealed a concentration-dependent reduction of the cell proliferation rate in each ACC cell model (Fig. 1). Trabectedin displayed a higher potency in the inhibition of the ACC cell proliferation compared to the cytotoxic effect, in particular, the MUC-1 cells seemed to be more sensitive to the antiproliferative effect of trabectedin.
Trabectedin induced a reduction of ACC cell xenograft area and metastasis formation in zebrafish embryos
The effect of trabectedin on cell proliferation and cytotoxicity was then evaluated in an in vivo preclinical model using zebrafish embryos xenografted with ACC cells and randomly assigned to be exposed to vehicle or to 15 nM of trabectedin. Trabectedin concentration was chosen accordingly to preliminary experiments aimed at evaluating the concentration of drug in the embryos. LC-MS/MS analysis revealed in embryos exposed to 15 nM of trabectedin dissolved in fish water for 72 h that the concentration (±S.D.) of trabectedin was 0.22 ± 0.05 nM. Trabectedin induced a statistically significant reduction of cell xenograft area compared to vehicle for each experimental cell model, as reported in Fig. 2A. Figure 2B shows representative figures of Tg (kdrl:EGFP) zebrafish embryos xenografted with the three cell lines and exposed to vehicle or trabectedin. As MUC-1 and TVBF-7 cells derive from an ACC metastatic localization, we then
investigated whether these cell lines retain the ability to migrate from the embryo injection site. The presence of metastasis (defined as cells at sites other than the injection site) at T3 was evaluated. MUC-1 cells were found to be able to metastasize in the tail of the embryos. Trabectedin exposure reduced the percentage of metastasis-positive embryos xenografted with MUC-1 cells treated from (mean ± S.D.) 72.15 ± 9.65% to 24.20 ± 3.52% (P <0.01). TVBF-7 cells were found to be able to form metastasis as well, although at lower rate. Indeed, metastasis was observed in the pericardial area and the percentage of metastasis-positive embryos was 12.07 ± 7.31% in vehicle- exposed embryos and 1.19 ± 2.06% in trabectedin-treated embryos, although the reduction did not reach a statistical significance. Figure 3 showed a representative image of MUC-1 and TVBF-7 metastatic cells. No metastasis was observed in embryos xenografted with NCI-H295R cells, in line with their primary ACC origin.
Trabectedin reduces cell invasiveness
Results obtained in the zebrafish embryo model were then confirmed and deepened with an in vitro approach, studying the invasiveness capability of the ACC cell models. NCI-H295R, MUC-1, and TVBF-7 cells were exposed to the respective trabectedin IC50 values. As shown in Fig. 4A, trabectedin treatment significantly reduced the invasiveness through the matrix of both cell lines established from patients in progression after EDP-M, as MUC-1 cells (untreated: mean OD ± S.D .= 0.390 ± 0.029; trabectedin-treated: 0.114 ± 0.014, P=0.0070) and TVBF-7 cells (untreated: 0.319 ± 0.062; trabectedin-treated: 0.210 ± 0.034, P=0.029). NCI-H295R cells displayed a low invasiveness capability, accordingly to their origin, that was not significantly modified by trabectedin
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treatment (untreated: 0.134 ± 0.039; trabectedin-treated: 0.079 ± 0.008, ns). Figure 4B shows a representative image of the cell invasion assay.
Trabectedin affects metalloprotease 2 (MMP2) activity in metastatic ACC cell lines
It is known that matrix metalloproteinase type 2 (MMP2) is involved in metastasis processes in the context of ACC (Volante et al. 2006). To investigate whether trabectedin could reduce the in vitro and in vivo invasiveness of metastatic ACC cell models through the modulation of MMP2 expression/activity, the MMP2 and its inhibitors TIMP1 and TIMP2 gene expression were first evaluated in vehicle- treated and trabectedin-treated cells. Results demonstrated a low, no significant reduction in MMP2 gene expression (MUC-1: 44Ct=0.815; TVBF-7: 44Ct=0.261. Measuring the gene expression of the tissue inhibitors of MMP2, namely TIMP-1 and TIMP-2 and a low, no significant increase in TIMP-2 gene expression (MUC-1: 44Ct =- 0.85; TVBF-7: 44Ct =- 0.03) was observed, while a significant increase in gene expression was, however, observed for TIMP1 in both cell models (MUC-1: 44Ct =- 2.00; TVBF-7: 44Ct =- 1.75).
MMP2 mRNA was translated into its protein and actively secreted in the medium, as reported in Fig. 5A. Interestingly, the Western blot results obtained detecting MMP2 in the conditioned medium of MUC-1 and TVBF-7 cells demonstrated a significant reduction of MMP2 protein after trabectedin treatment (Fig. 5B). The secreted MMP2 was functionally active as reported in the zymography assay performed using gels containing gelatin, an MMP2 substrate (Fig. 5C). Indeed, the signal detected at the expected MMP2 molecular weight was reduced in the conditioned medium obtained from trabectedin- treated cells (trabectedin-treated MUC-1 (mean ± S.D.) -45.3 ± 11.5% vs untreated, P=0.0012; trabectedin-treated TVBF-7 cells: - 33.8 ± 13.9% vs untreated, P=0.0065). These results strengthen the involvement of MMP2 in the progression and invasiveness of ACC.
Discussion
Trabectedin is an anticancer drug approved for the pharmacological management of STS and OC (Larsen et al. 2016). We have previously demonstrated the tumoral
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cytotoxic effect of trabectedin in different experimental cell models of ACC, both of primary and metastatic origin, with a long-lasting effect maintained after the discontinuation of drug treatment (Abate et al. 2020). One of the aims of this study was to extend the observation about the cytotoxicity activity also in the recently established ACC cell line, named
TVBF-7 cells, a new cellular model derived from a lymph node metastasis in a patient with disease progression after EDP-M. In this new ACC cell model, trabectedin was shown to be very active, being able to induce cytotoxicity at low nanomolar concentrations. Besides the cytotoxic effect, trabectedin also affected the cell proliferation rate in each
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ACC experimental cell model, obtained from both primary and metastatic disease, and more importantly, these effects were observed at concentrations reachable in humans at the doses already approved for clinical use (https://www. micromedexsolutions.com/home/dispatch). Due to the high heterogeneity of the ACC, especially in the advanced stages of the disease (Gara et al. 2018), the availability of different experimental cell models characterized by peculiar molecular characteristics is mandatory. On this line, compared to the past, an increasing number of ACC cell lines with peculiar biological characteristics are now available (Sigala et al. 2022b). The results obtained in the in vitro experiments were often endowed with the limit of reproducibility in in vivo models. As a matter of fact, in our recent experience, we showed that cabazitaxel was active in vitro, but this activity was not confirmed in ACC patients enrolled in a prospective phase II trial (Fragni et al. 2019, Laganà et al. 2020). As a further step, therefore, we evaluated the antineoplastic effect of trabectedin in zebrafish xenografts of each ACC experimental cell model in order to evaluate the efficacy of trabectedin in a more physiological and complex in vivo environment. The zebrafish embryos model, although much more simple when compared with mammalian animal models, is now recognized as a good model for the translation process of the data obtained with cell cultures (Marques et al. 2009, Jung et al. 2012, Tonon et al. 2016, Bootorabi et al. 2017). The result relating to the amount of drug absorbed by the embryo indicates a concentration consistent with those used in in vitro assays, which, as indicated earlier, are comparable to that obtainable in the plasma of trabectedin- treated patients following administration according to the dosage schedules for approved indications (https://www. micromedexsolutions.com/home/dispatch). Data relating to the reduction of the xenograft area confirmed the data obtained with the in vitro cell cultures.
Based on these in vitro and in vivo results, we wanted to investigate whether trabectedin could interfere with the invasion/metastatic ability of ACC cells. The metastatic cell lines MUC-1 and TVBF-7 cells showed to be able to form metastases in the embryos. No metastases were found in the embryo injected with NCI-H295R and a low invasive ability was revealed in in vitro invasion assay. In our experimental settings, NCI-H295R cells displayed a lower invasive capability compared to results reported elsewhere (Ferruzzi et al. 2005). This apparent discrepancy in in vitro results could be due to both different cell culture conditions or due to technical issues such as, for example, kits from different manufacturers. Both MUC-1 and TVBF-7 cells proved to be useful models for studying the trabectedin-induced effect on the metastasizing process, and a higher incidence of metastasis-positive embryos was observed for the MUC-1 cell line in comparison to TVBF- 7. The usefulness of these two cellular models is reinforced by the observation that they were able to metastasize in different body areas of the embryo. Taken together, these observations showed that these two lines, although both deriving from metastatic disease, possess divergent peculiarities, in accordance with the known heterogeneity of ACC (Gara et al. 2018).
The role of matrix metalloproteinases (MMPs) in the processes of invasion and metastasis of tumor cells is well-known (Abdel-Hamid et al. 2021). Volante et al. identified MMP2 as a useful marker for the immunohistochemical diagnosis of ACC. Furthermore, a positive correlation between high MMP2 expression and disease aggressiveness was found (Volante et al. 2006). On this basis, we analyzed the effect of trabectedin on MMP2, and our results support the hypothesis of the involvement of this drug on proteinase secretion and activity in ACC. Certainly, we cannot exclude an effect of trabectedin on other proteases that may contribute to the
inhibition of the invasion process induced by the drug. Indeed, in different cell models, MMP2 and other MMPs have been identified as direct ß-catenin transcriptional targets and their expression was found to be regulated by the activating status of Wnt/B-catenin signaling pathway (Nguyen et al. 2019). We previously linked the effect of trabectedin in NCI-H295R cells with an inhibitory effect on the Wnt/ß-catenin pathway (Abate et al. 2020). Furthermore, we demonstrated the cytotoxic activity of trabectedin in both Wnt/ß-catenin pathway WT and mutated ACC cells. The effect of trabectedin on MMP2 observed in the present study, therefore, could have potentially involved the Wnt/ß-catenin pathway, but this interesting hypothesis needs confirmation in a well- designed preclinical study. The cancer cell’s capability to invade and migrate is a complex process involving a number of different actors in a precise timeline, although the precise timing and intracellular pathways involved in complex and peculiar for each cancer (Fares et al. 2020). Accordingly, the metastasizing process in ACC can be due to different molecular mechanisms (for an extensive discussion refer to Lalli & Luconi 2018). Results reported here strongly indicated the involvement of the MMP2, although other pathways targeted by trabectedin in our ACC cell models cannot be excluded. The demonstration of the efficacy of trabectedin in different ACC cell models, characterized by peculiar molecular characteristics, strengthens the evidence that trabectedin may be effective to impair cell viability and the process of invasion/metastasis in an extended group of ACC cells, mimicking the heterogeneity of the disease in clinics. Although these data are preliminary, it is possible to hypothesize that trabectedin could be effective in an advanced and metastatic setting of ACC. Results of the present work together with those previously published (Abate et al. 2020) could provide the preclinical rationale for an evaluation of trabectedin efficacy in ACC in an appropriate clinical trial.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ ERC-22-0273.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
@ 2022 the author(s) Published by Bioscientifica Ltd. Printed in Great Britain
Funding
Trabectedin was kindly provided by Pharma Mar S.A. (Madrid, Spain). This work was supported by AIRC project IG23009 (PI AB) and by University of Brescia local grants.
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Received in final form 20 November 2022 Accepted 29 November 2022 Accepted Manuscript published online 30 November 2022
@ 2022 the author(s) Published by Bioscientifica Ltd. Printed in Great Britain