Inhibition of endolysosomal two-pore channel 2 (TPC2) induces osteoblast differentiation and matrix mineralization while targeting autophagy Azadeh Montaseri, Michela Rossi, Giulia Battafarano, Anna Riccioli, Fioretta Palombi, Claudia Giampietri, Domenico Liguoro, Rita Mancini, Martina Meucci, Biagio Palmisano, Mara Riminucci, Marco Keller, Franz Bracher, Christian Grimm, Kaoru Umehara, Wanchai De-Eknamkul, Antonio Filippini, Andrea Del Fattore Journal of Endocrinological Investigation, 2026 Purpose Endolysosomal two-pore channels (TPCs) are non-selective cation channels that control the release of Ca 2+ and Na + from the endolysosomal lumen. TPCs also reportedly play a role in autophagy. Interestingly, autophagy regulates bone cell differentiation and function. This study aimed to provide an in-depth insight into TPC2’s action in the autophagy pathway to control osteoblast differentiation and function. Methods Primary human mesenchymal stem cells (hMSCs) and human osteoblast-like cells (Saos-2) were used to assess osteoblastogenesis and bone mineralization, respectively. MSCs were treated with different pharmacological TPC2 inhibitors including naringenin, tetrandrine, MT-8 and SG-094 during their differentiation process. Finally, formation of osteoblasts and in vitro bone mineralization were evaluated by alkaline phosphatase, alizarin red S and Von Kossa staining. Western blot analysis was performed to investigate the expression of autophagy-related molecules. Results The inhibition of TPC2 activity stimulates osteoblast differentiation from hMSCs and bone mineralization by Saos-2 cells. Interestingly, TPC2 inhibition reduces beclin-1 and LC3-II expression while that of the mammalian target of rapamycin (mTOR), the master regulator of autophagy, increases. Inhibition of mTOR activity by rapamycin reverses osteoblast differentiation induced by TPC2 inhibitor SG-094. Conclusion Inhibition of TPC2 channel activity increases osteoblast differentiation and bone mineralization in vitro and interferes with the completion of autophagy, upregulating phosphorylated mTOR.
miRNAs from Zebrafish Embryo Extracts Inhibit Breast Cancer Invasiveness and Migration by Modulating miR-218-5p/PI3K Pathway Noemi Monti, Daniele Antinori, Sara Proietti, Aurora Piombarolo, Alessandro Querqui, Guglielmo Lentini, Domenico Liguoro, Michele Aventaggiato, Marco Lucarelli, Andrea Pensotti, Alessandro Giuliani, Marco Tafani, Andrea Fuso, Mariano Bizzarri International Journal of Molecular Sciences, 2025 Herein, we demonstrate that soluble factors extracted from the distinct phases of the development of zebrafish embryos (ZFEs) exhibit a specific miRNA profile. We removed proteins and concentrated miRNAs in different phase-related samples, which we investigated further. We observed that ZFEs modulate miRNA expression in both normal and cancerous breast cells, significantly inhibiting the invasiveness and motility of triple-negative breast cancer cells. Namely, ZFEs reactivate the synthesis of miR-218-5p in cancerous cells, leading to the downregulation of PI3K, which consequently alters the distribution of phosphoinositides (such as PIP2/PIP3). Moreover, the silencing of miR-218-5p abolished the ZFE effects. Restoring a proper PIP2/PIP3 ratio is crucial for promoting the regression of the malignant phenotype. Phenotypic reversion follows the extensive cytoskeleton rearrangement and the re-emergence of E-cadherin/β-catenin complexes. In addition, ZFEs antagonize the Epithelial Mesenchymal Transition (EMT) by modulating several pathways, including the TCTP-p53 axis. Overall, these results show that embryo extracts enriched with fish miRNAs reactivate endogenous miR-218-5p in cancerous cells, which in turn downregulates critical pathways involved in tumor progression and metastasis.
Self-assembling nanoparticles for miRNA delivery towards precision medicine against melanoma Valeria Nele, Domenico Liguoro, Virginia Campani, Alessia Angelillo, Rachele Frigerio, Arianna Ortolano, Rita Mancini, Luigi Fattore, Giuseppe De Rosa, Gennaro Ciliberto Journal of Drug Delivery Science and Technology, 2024 Metastatic melanoma is a highly aggressive tumor with a poor prognosis. One of the therapeutic options for patients bearing BRAF V600E mutations is targeted therapy, which is based on the use of drugs able to inhibit the mitogen-activated protein kinase (MAPK) pathway. However, the long-term efficacy of targeted therapy is compromised by the onset of drug resistance. We previously identified a panel of oncosuppressor microRNAs (miRNAs) able to prevent the development of drug resistance to targeted therapy. We also developed self-assembling nanoparticles (SANP) as a promising and versatile nanomedicine platform for RNA-based personalized therapies. Here, we provide the proof-of-principle of a miRNA-based therapeutic strategy against BRAF-mutant melanomas. The role of the cationic lipid, ionizable lipid, cholesterol, and PEGylated lipid in SANP formulations was investigated to optimize miRNA encapsulation and delivery to melanoma cells. miRNA-loaded SANPs inhibited the release of soluble tumor-promoting factors and prevented the proliferation of metastatic melanoma cells. When used in combination with targeted therapy, miRNA-SANPs were able to potentiate its efficacy in a dose-response manner. These results pave the way for further studies on SANP as a platform for miRNA delivery to prevent the development of resistance to targeted therapy in metastatic melanoma.
The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors Domenico Liguoro, Rachele Frigerio, Arianna Ortolano, Andrea Sacconi, Mario Acunzo, Giulia Romano, Giovanni Nigita, Barbara Bellei, Gabriele Madonna, Mariaelena Capone, Paolo Antonio Ascierto, Rita Mancini, Gennaro Ciliberto, Luigi Fattore Cell Death and Disease, 2024 Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.
Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes through downregulation of intermediate filament nestin Vittorio Castaldo, Michele Minopoli, Francesca Di Modugno, Andrea Sacconi, Domenico Liguoro, Rachele Frigerio, Arianna Ortolano, Marta Di Martile, Luisa Gesualdi, Gabriele Madonna, Mariaelena Capone, Roberto Cirombella, Angiolina Catizone, Donatella Del Bufalo, Andrea Vecchione, Maria Vincenza Carriero, Paolo Antonio Ascierto, Rita Mancini, Luigi Fattore, Gennaro Ciliberto Journal of Experimental and Clinical Cancer Research, 2023 Background BRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488. Methods Invasion and migration have been determined by wound healing, transwell migration/invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by western blot, confocal immunofluorescence, immunohistochemical and flow cytometry analyses. Results We demonstrate that the two oncomiRs are responsible for the enhanced migratory and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients. Conclusions Altogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions. Graphical Abstract
ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation Luisa Gesualdi, Marika Berardini, Bianca Maria Scicchitano, Clotilde Castaldo, Mariano Bizzarri, Antonio Filippini, Anna Riccioli, Chiara Schiraldi, Francesca Ferranti, Domenico Liguoro, Rita Mancini, Giulia Ricci, Angela Catizone Biomedicines, 2023 c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype», while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium–mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.
Oncosuppressive miRNAs loaded in lipid nanoparticles potentiate targeted therapies in BRAF-mutant melanoma by inhibiting core escape pathways of resistance Luigi Fattore, Giordana Cafaro, Marta Di Martile, Virginia Campani, Andrea Sacconi, Domenico Liguoro, Emanuele Marra, Sara Bruschini, Daniela Stoppoloni, Roberto Cirombella, Francesca De Nicola, Matteo Pallocca, Ciro F. Ruggiero, Vittorio Castaldo, Angiolina Catizone, Donatella Del Bufalo, Giuseppe Viglietto, Andrea Vecchione, Giovanni Blandino, Luigi Aurisicchio, Maurizio Fanciulli, Paolo A. Ascierto, Giuseppe De Rosa, Rita Mancini, Gennaro Ciliberto Oncogene, 2023 BRAF-mutated melanoma relapsing after targeted therapies is an aggressive disease with unmet clinical need. Hence the need to identify novel combination therapies able to overcome drug resistance. miRNAs have emerged as orchestrators of non-genetic mechanisms adopted by melanoma cells to challenge therapies. In this context we previously identified a subset of oncosuppressor miRNAs downregulated in drug-resistant melanomas. Here we demonstrate that lipid nanoparticles co-encapsulating two of them, miR-199-5p and miR-204-5p, inhibit tumor growth both in vitro and in vivo in combination with target therapy and block the development of drug resistance. Mechanistically they act by directly reducing melanoma cell growth and also indirectly by hampering the recruitment and reprogramming of pro-tumoral macrophages. Molecularly, we demonstrate that the effects on macrophages are mediated by the dysregulation of a newly identified miR-204-5p-miR-199b-5p/CCL5 axis. Finally, we unveiled that M2 macrophages programs are molecular signatures of resistance and predict response to therapy in patients. Overall, these findings have strong translational implications to propose new combination therapies making use of RNA therapeutics for metastatic melanoma patients.
Radioresistance Mechanisms in Prostate Cancer Cell Lines Surviving Ultra-Hypo-Fractionated EBRT: Implications and Possible Clinical Applications Silvia Sideri, Francesco Petragnano, Roberto Maggio, Simonetta Petrungaro, Angela Catizone, Luisa Gesualdi, Viviana De Martino, Giulia Battafarano, Andrea Del Fattore, Domenico Liguoro, Paola De Cesaris, Antonio Filippini, Francesco Marampon, Anna Riccioli Cancers, 2022 The use of a higher dose per fraction to overcome the high radioresistance of prostate cancer cells has been unsuccessfully proposed. Herein, we present PC3 and DU-145, castration-resistant prostate cancer cell lines that survived a clinically used ultra-higher dose per fraction, namely, radioresistant PC3 and DU-145 cells (PC3RR and DU-145RR). Compared to PC3, PC3RR showed a higher level of aggressive behaviour, with enhanced clonogenic potential, DNA damage repair, migration ability and cancer stem cell features. Furthermore, compared to PC3, PC3RR more efficiently survived further radiation by increasing proliferation and down-regulating pro-apoptotic proteins. No significant changes of the above parameters were described in DU-145RR, suggesting that different prostate cancer cell lines that survive ultra-higher dose per fraction do not display the same grade of aggressive phenotype. Furthermore, both PC3RR and DU-145RR increased antioxidant enzymes and mesenchymal markers. Our data suggest that different molecular mechanisms could be potential targets for future treatments plans based on sequential strategies and synergistic effects of different modalities, possibly in a patient-tailored fashion. Moreover, PC3RR cells displayed an increase in specific markers involved in bone remodeling, indicating that radiotherapy selects a PC3 population capable of migrating to secondary metastatic sites. Finally, PC3RR cells showed a better sensitivity to Docetaxel as compared to native PC3 cells. This suggests that a subset of patients with castration-resistant metastatic disease could benefit from upfront Docetaxel treatment after the failure of radiotherapy.
Identification of a miRNA-based non-invasive predictive biomarker of response to target therapy in BRAF-mutant melanoma Ciro Francesco Ruggiero, Luigi Fattore, Irene Terrenato, Francesca Sperati, Valentina Salvati, Gabriele Madonna, Mariaelena Capone, Fabio Valenti, Simona Di Martino, Chiara Mandoj, Domenico Liguoro, Vittorio Castaldo, Giordana Cafaro, Ester Simeone, Vito Vanella, Michelangelo Russillo, Laura Conti, Giovanni Cuda, Diana Giannarelli, Paolo Antonio Ascierto, Rita Mancini, Gennaro Ciliberto Theranostics, 2022 Rationale: Metastatic melanoma is the most aggressive and dangerous form of skin cancer. The introduction of immunotherapy with Immune checkpoint Inhibitors (ICI) and of targeted therapy with BRAF and MEK inhibitors for BRAF mutated melanoma, has greatly improved the clinical outcome of these patients. Nevertheless, response to therapy remains highly variable and the development of drug resistance continues to be a daunting challenge. Within this context there is a need to develop diagnostic tools capable of predicting response or resistance to therapy in order to select the best therapeutic approach. Over the years, accumulating evidence brought to light the role of microRNAs (miRNAs) as disease biomarkers. Methods: In particular, the detection of miRNAs in whole blood or specific blood components such as serum or plasma, allows these molecules to be good candidates for diagnosis, prognosis and for monitoring response to anticancer therapy. In this paper, we evaluated circulating basal levels of 6 previously identified miRNAs in serum samples of 70 BRAF-mutant melanoma patients before starting targeted therapy. Results: Results show that the circulating levels of the oncosuppressor miR-579-3p and of the oncomiR miR-4488 are able to predict progression free survival (PFS) but not overall survival (OS). Most importantly, we observed that the best predictor of disease outcome is represented by the ratio of circulating miR-4488 vs. miR-579-3p (miRatio). Finally, the combination of the Lactate dehydrogenase (LDH) blood levels with the two circulating miRNAs alone or together did not produce any improvement in predicting PFS indicating that miR-579-3p and miR-4488 are independent predictors of PFS as compared to LDH. Conclusions: All together these data underscored the relevance of circulating miRNAs as suitable tools to predict therapy response in melanoma and maybe further developed as companion diagnostics in the clinic.
Reprogramming miRNAs global expression orchestrates development of drug resistance in BRAF mutated melanoma Luigi Fattore, Ciro Francesco Ruggiero, Maria Elena Pisanu, Domenico Liguoro, Andrea Cerri, Susan Costantini, Francesca Capone, Mario Acunzo, Giulia Romano, Giovanni Nigita, Domenico Mallardo, Concetta Ragone, Maria Vincenza Carriero, Alfredo Budillon, Gerardo Botti, Paolo Antonio Ascierto, Rita Mancini, Gennaro Ciliberto Cell Death and Differentiation, 2019
