Hyperactive HRAS dysregulates energetic metabolism in fibroblasts from patients with Costello syndrome via enhanced production of reactive oxidizing species.

Germline-activating mutations in HRAS cause Costello syndrome (CS), a cancer prone multisystem disorder characterized by reduced postnatal growth. In CS, poor weight gain and growth are not caused by low caloric intake. Here, we show that constitutive plasma membrane translocation and activation of the GLUT4 glucose transporter, via reactive oxygen species-dependent AMP-activated protein kinase α and p38 hyperactivation, occurs in primary fibroblasts of CS patients, resulting in accelerated glycolysis and increased fatty acid synthesis and storage as lipid droplets. An accelerated autophagic flux was also identified as contributing to the increased energetic expenditure in CS. Concomitant inhibition of p38 and PI3K signaling by wortmannin was able to rescue both the dysregulated glucose intake and accelerated autophagic flux. Our findings provide a mechanistic link between upregulated HRAS function, defective growth and increased resting energetic expenditure in CS, and document that targeting p38 and PI3K signaling is able to revert this metabolic dysfunction.

Assessing the Impact of Polyethylene Nano/Microplastic Exposure on Human Vaginal Keratinocytes.

The global rise of single-use throw-away plastic products has elicited a massive increase in the nano/microplastics (N/MPLs) exposure burden in humans. Recently, it has been demonstrated that disposable period products may release N/MPLs with usage, which represents a potential threat to women's health which has not been scientifically addressed yet. By using polyethyl ene (PE) particles (200 nm to 9 µm), we showed that acute exposure to a high concentration of N/MPLs induced cell toxicity in vaginal keratinocytes after effective cellular uptake, as viability and apoptosis data suggest, along with transmission electron microscopy (TEM) observations. The internalised N/MPLs altered the expression of junctional and adherence proteins and the organisation of the actin cortex, influencing the level of genes involved in oxidative stress signalling pathways and that of miRNAs related to epithelial barrier function. When the exposure to PE N/MPLs was discontinued or became chronic, cells were able to recover from the negative effects on viability and differentiation/proliferation gene expression in a few days. However, in all cases, PE N/MPL exposure prompted a sustained alteration of DNA methyltransferase and DNA demethylase expression, which might impact epigenetic regulation processes, leading to accelerated cell ageing and inflammation, or the occurrence of malignant transformation.

Translational Implications for Radiosensitizing Strategies in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence that includes FP-RMS, harboring the fusion oncoprotein PAX3/7-FOXO1 and FN-RMS, often mutant in the RAS pathway. Risk stratifications of RMS patients determine different prognostic groups and related therapeutic treatment. Current multimodal therapeutic strategies involve surgery, chemotherapy (CHT) and radiotherapy (RT), but despite the deeper knowledge of response mechanisms underpinning CHT treatment and the technological improvements that characterize RT, local failures and recurrence frequently occur. This review sums up the RMS classification and the management of RMS patients, with special attention to RT treatment and possible radiosensitizing strategies for RMS tumors. Indeed, RMS radioresistance is a clinical problem and further studies aimed at dissecting radioresistant molecular mechanisms are needed to identify specific targets to hit, thus improving RT-induced cytotoxicity.

MS-275 (Entinostat) Promotes Radio-Sensitivity in PAX3-FOXO1 Rhabdomyosarcoma Cells.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Clinically relevant radioresistant rhabdomyosarcoma cell lines: functional, molecular and immune-related characterization.

BACKGROUND: The probability of local tumor control after radiotherapy (RT) remains still miserably poor in pediatric rhabdomyosarcoma (RMS). Thus, understanding the molecular mechanisms responsible of tumor relapse is essential to identify personalized RT-based strategies. Contrary to what has been done so far, a correct characterization of cellular radioresistance should be performed comparing radioresistant and radiosensitive cells with the same isogenic background. METHODS: Clinically relevant radioresistant (RR) embryonal (RD) and alveolar (RH30) RMS cell lines have been developed by irradiating them with clinical-like hypo-fractionated schedule. RMS-RR cells were compared to parental isogenic counterpart (RMS-PR) and studied following the radiobiological concept of the "6Rs", which stand for repair, redistribution, repopulation, reoxygenation, intrinsic radioresistance and radio-immuno-biology. RESULTS: RMS-RR cell lines, characterized by a more aggressive and in vitro pro-metastatic phenotype, showed a higher ability to i) detoxify from reactive oxygen species; ii) repair DNA damage by differently activating non-homologous end joining and homologous recombination pathways; iii) counteract RT-induced G2/M cell cycle arrest by re-starting growth and repopulating after irradiation; iv) express cancer stem-like profile. Bioinformatic analyses, performed to assess the role of 41 cytokines after RT exposure and their network interactions, suggested TGF-ß, MIF, CCL2, CXCL5, CXCL8 and CXCL12 as master regulators of cancer immune escape in RMS tumors. CONCLUSIONS: These results suggest that RMS could sustain intrinsic and acquire radioresistance by different mechanisms and indicate potential targets for future combined radiosensitizing strategies.

Deep Sequencing the microRNA profile in rhabdomyosarcoma reveals down-regulation of miR-378 family members.

BACKGROUND: Rhabdomyosarcoma (RMS) is a highly malignant tumour accounting for nearly half of soft tissue sarcomas in children. MicroRNAs (miRNAs) represent a class of short, non-coding, regulatory RNAs which play a critical role in different cellular processes. Altered miRNA levels have been reported in human cancers, including RMS. METHODS: Using deep sequencing technology, a total of 685 miRNAs were investigated in a group of alveolar RMSs (ARMSs), embryonal RMSs (ERMSs) as well as in normal skeletal muscle (NSM). Q-PCR, MTT, cytofluorimetry, migration assay, western blot and immunofluorescence experiments were carried out to determine the role of miR-378a-3p in cancer cell growth, apoptosis, migration and differentiation. Bioinformatics pipelines were used for miRNA target prediction and clustering analysis. RESULTS: Ninety-seven miRNAs were significantly deregulated in ARMS and ERMS when compared to NSM. MiR-378 family members were dramatically decreased in RMS tumour tissue and cell lines. Interestingly, members of the miR-378 family presented as a possible target the insulin-like growth factor receptor 1 (IGF1R), a key signalling molecule in RMS. MiR-378a-3p over-expression in an RMS-derived cell line suppressed IGF1R expression and affected phosphorylated-Akt protein levels. Ectopic expression of miR-378a-3p caused significant changes in apoptosis, cell migration, cytoskeleton organization as well as a modulation of the muscular markers MyoD1, MyoR, desmin and MyHC. In addition, DNA demethylation by 5-aza-2'-deoxycytidine (5-aza-dC) was able to up-regulate miR-378a-3p levels with a concomitant induction of apoptosis, decrease in cell viability and cell cycle arrest in G2-phase. Cells treated with 5-aza-dC clearly changed their morphology and expressed moderate levels of MyHC. CONCLUSIONS: MiR-378a-3p may function as a tumour suppressor in RMS and the restoration of its expression would be of therapeutic benefit in RMS. Furthermore, the role of epigenetic modifications in RMS deserves further investigations.

Anti­oncogenic and pro­myogenic action of the MKK6/p38/AKT axis induced by targeting MEK/ERK in embryonal rhabdomyosarcoma.

Insights into the molecular and cellular biology of embryonal rhabdomyosarcoma (ERMS), an aggressive paediatric tumour, are required in order to identify new targets for novel treatments that may benefit patients with this disease. The present study examined the functional effects of MKK3 and MKK6, two upstream kinases of p38, and found that the ectopic expression of MKK6 led to rapid p38 activation and the myogenic differentiation of ERMS cells, whereas MKK3 failed to induce differentiation, while maintaining the proliferation state. Myogenin and myosin heavy chain were induced in MKK6­overexpressing ERMS cells and were inhibited by the p38 inhibitor, SB203580. The expression of Myc and ERK­PO4 increased under the effect of SB203580, whereas it decreased in MKK6­overexpressing cells. AKT activation was part of the myogenic program triggered by MKK6 overexpression alone. To the best of our knowledge, the present study demonstrates, for the first time, that the endogenous MKK6 pathway may be recovered by MEK/ERK inhibition (U0126 and trametinib) and that it concomitantly induces the reversal of the oncogenic pattern and the induction of the myogenic differentiation of ERMS cell lines. The effects of MEK/ERK inhibitors markedly increase the potential clinical applications in ERMS, particularly on account of the MEK inhibitor­induced early MKK6/p38 axis activation and of their anti­oncogenic effects. The findings presented herein lend further support to the antitumour effects of MKK6; MKK6 may thus represent a novel target for advanced personalised treatments against ERMS.

Radioresistance in rhabdomyosarcomas: Much more than a question of dose.

Management of rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, frequently accounting the genitourinary tract is complex and requires a multimodal therapy. In particular, as a consequence of the advancement in dose conformity technology, radiation therapy (RT) has now become the standard therapeutic option for patients with RMS. In the clinical practice, dose and timing of RT are adjusted on the basis of patients' risk stratification to reduce late toxicity and side effects on normal tissues. However, despite the substantial improvement in cure rates, local failure and recurrence frequently occur. In this review, we summarize the general principles of the treatment of RMS, focusing on RT, and the main molecular pathways and specific proteins involved into radioresistance in RMS tumors. Specifically, we focused on DNA damage/repair, reactive oxygen species, cancer stem cells, and epigenetic modifications that have been reported in the context of RMS neoplasia in both in vitro and in vivo studies. The precise elucidation of the radioresistance-related molecular mechanisms is of pivotal importance to set up new more effective and tolerable combined therapeutic approaches that can radiosensitize cancer cells to finally ameliorate the overall survival of patients with RMS, especially for the most aggressive subtypes.

MET Inhibition Sensitizes Rhabdomyosarcoma Cells to NOTCH Signaling Suppression.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth in vitro and in vivo. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.

The botanical drug PBI-05204, a supercritical CO2 extract of Nerium oleander, sensitizes alveolar and embryonal rhabdomyosarcoma to radiotherapy in vitro and in vivo.

Treatment of rhabdomyosarcoma (RMS), the most common a soft tissue sarcoma in childhood, provides intensive multimodal therapy, with radiotherapy (RT) playing a critical role for local tumor control. However, since RMS efficiently activates mechanisms of resistance to therapies, despite improvements, the prognosis remains still largely unsatisfactory, mainly in RMS expressing chimeric oncoproteins PAX3/PAX7-FOXO1, and fusion-positive (FP)-RMS. Cardiac glycosides (CGs), plant-derived steroid-like compounds with a selective inhibitory activity of the Na+/K+-ATPase pump (NKA), have shown antitumor and radio-sensitizing properties. Herein, the therapeutic properties of PBI-05204, an extract from Nerium oleander containing the CG oleandrin already studied in phase I and II clinical trials for cancer patients, were investigated, in vitro and in vivo, against FN- and FP-RMS cancer models. PBI-05204 induced growth arrest in a concentration dependent manner, with FP-RMS being more sensitive than FN-RMS, by differently regulating cell cycle regulators and commonly upregulating cell cycle inhibitors p21Waf1/Cip1 and p27Cip1/Kip1. Furthermore, PBI-05204 concomitantly induced cell death on both RMS types and senescence in FN-RMS. Notably, PBI-05204 counteracted in vitro migration and invasion abilities and suppressed the formation of spheroids enriched in CD133+ cancer stem cells (CSCs). PBI-05204 sensitized both cell types to RT by improving the ability of RT to induce G2 growth arrest and counteracting the RT-induced activation of both Non-Homologous End-Joining and homologous recombination DSBs repair pathways. Finally, the antitumor and radio-sensitizing proprieties of PBI-05204 were confirmed in vivo. Notably, both in vitro and in vivo evidence confirmed the higher sensitivity to PBI-05204 of FP-RMS. Thus, PBI-05204 represents a valid radio-sensitizing agent for the treatment of RMS, including the intrinsically radio-resistant FP-RMS.

Altered Expression of Candidate Genes in Mayer-Rokitansky-Küster-Hauser Syndrome May Influence Vaginal Keratinocytes Biology: A Focus on Protein Kinase X.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a rare and complex disease defined by congenital aplasia of the vagina and uterus in 46,XX women, often associated with kidney and urinary tract anomalies. The aetiopathogenesis of MRKH syndrome is still largely unknown. Herein, we investigated the role of selected candidate genes in the aetiopathogenesis of MRKH syndrome, with a focus on PRKX, which encodes for protein kinase X. Through RT-qPCR analyses performed on vaginal dimple samples from patients, and principal component analysis (PCA), we highlighted a phenotype-related expression pattern of PRKX, MUC1, HOXC8 and GREB1L in MRKH patients. By using an in vitro approach, we proved that PRKX ectopic overexpression in a cell model of vaginal keratinocytes promotes cell motility through epithelial-to-mesenchymal transition (EMT) activation, a fundamental process in urogenital tract morphogenesis. Moreover, our findings showed that PRKX upregulation in vaginal keratinocytes is able to affect transcriptional levels of HOX genes, implicated in urinary and genital tract development. Our study identified the dysregulation of PRKX expression as a possible molecular cause for MRKH syndrome. Moreover, we propose the specific role of PRKX in vaginal keratinocyte biology as one of the possible mechanisms underlying this complex disease.

Calcineurin Gamma Catalytic Subunit PPP3CC Inhibition by miR-200c-3p Affects Apoptosis in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) outpaces all the other forms of the female reproductive system malignancies. MicroRNAs have emerged as promising predictive biomarkers to therapeutic treatments as their expression might characterize the tumor stage or grade. In EOC, miR-200c is considered a master regulator of oncogenes or tumor suppressors. To investigate novel miR-200c-3p target genes involved in EOC tumorigenesis, we evaluated the association between this miRNA and the mRNA expression of several potential target genes by RNA-seq data of both 46 EOC cell lines from Cancer Cell line Encyclopedia (CCLE) and 456 EOC patient bio-specimens from The Cancer Genome Atlas (TCGA). Both analyses showed a significant anticorrelation between miR-200c-3p and the protein phosphatase 3 catalytic subunit γ of calcineurin (PPP3CC) levels involved in the apoptosis pathway. Quantitative mRNA expression analysis in patient biopsies confirmed the inverse correlation between miR-200c-3p and PPP3CC levels. In vitro regulation of PPP3CC expression through miR-200c-3p and RNA interference technology led to a concomitant modulation of BCL2- and p-AKT-related pathways, suggesting the tumor suppressive role of PPP3CC in EOC. Our results suggest that inhibition of high expression of miR-200c-3p in EOC might lead to overexpression of the tumor suppressor PPP3CC and subsequent induction of apoptosis in EOC patients.

OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence.

Ovarian cancer (OC) is the most aggressive gynecological tumor worldwide and, notwithstanding the increment in conventional treatments, many resistance mechanisms arise, this leading to cure failure and patient death. So, the use of novel adjuvant drugs able to counteract these pathways is urgently needed to improve patient overall survival. A growing interest is focused on epigenetic drugs for cancer therapy, such as Bromodomain and Extra-Terminal motif inhibitors (BETi). Here, we investigate the antitumor effects of OTX015, a novel BETi, as a single agent or in combination with ionizing radiation (IR) in OC cellular models. OTX015 treatment significantly reduced tumor cell proliferation by triggering cell cycle arrest and apoptosis that were linked to nucleolar stress and DNA damage. OTX015 impaired migration capacity and potentiated IR effects by reducing the expression of different drivers of cancer resistance mechanisms, including GNL3 gene, whose expression was found to be significantly higher in OC biopsies than in normal ovarian tissues. Gene specific knocking down and computational network analysis confirmed the centrality of GNL3 in OTX015-mediated OC antitumor effects. Altogether, our findings suggest OTX015 as an effective option to improve therapeutic strategies and overcome the development of resistant cancer cells in patients with OC.

DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Recently, we demonstrated the overexpression of both DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B) in RMS tumour biopsies and cell lines compared to normal skeletal muscle. Radiotherapy may often fail due to the abnormal expression of some molecules able to drive resistance mechanisms. The aim of this study was to analyse the involvement of DNMT3A and DNMT3B in radioresistance in RMS. RNA interference experiments against DNMT3A/3B were performed in embryonal RMS cells, upon ionizing radiation (IR) exposure and the effects of the combined treatment on RMS cells were analysed. DNMT3A and DNMT3B knocking down increased the sensitivity of RMS cells to IR, as indicated by the drastic decrease of colony formation ability. Interestingly, DNMT3A/3B act in two different ways: DNMT3A silencing triggers the cellular senescence program by up-regulating p16 and p21, whilst DNMT3B depletion induces significant DNA damage and impairs the DNA repair machinery (ATM, DNA-PKcs and Rad51 reduction). Our findings demonstrate for the first time that DNMT3A and DNMT3B overexpression may contribute to radiotherapy failure, and their inhibition might be a promising radiosensitizing strategy, mainly in the treatment of patients with metastatic or recurrent RMS tumours.

MiR-200c-3p maintains stemness and proliferative potential in adipose-derived stem cells by counteracting senescence mechanisms.

Adipose-derived mesenchymal stem cells (ASCs) are promising therapeutic tools in regenerative medicine because they possess self-renewal, differentiation and immunomodulatory capacities. After isolation, ASCs are passaged multiple times in vitro passages to obtain a sufficient amount of cells for clinical applications. During this time-consuming procedure, ASCs become senescent and less proliferative, compromising their clinical efficacy. Here, we sought to investigate how in vitro passages impact ASC proliferation/senescence and expression of immune regulatory proteins. MicroRNAs are pivotal regulators of ASC physiology. Particularly, miR-200c is known to maintain pluripotency and targets the immune checkpoint Programmed death-ligand 1 (PD-L1). We therefore investigated its involvement in these critical characteristics of ASCs during in vitro passages. We found that when transiently expressed, miR-200c-3p promotes proliferation, maintains stemness, and contrasts senescence in late passaged ASCs. Additionally, this miRNA modulates PD-L1 and Indoleamine 2,3-Dioxygenase (IDO1) expression, thus most likely interfering with the immunoregulatory capacity of ASCs. Based on our results, we suggest that expression of miR-200c-3p may prime ASC towards a self-renewing phenotype by improving their in vitro expansion. Contrarily, its inhibition is associated with senescence, reduced proliferation and induction of immune regulators. Our data underline the potential use of miR-200c-3p as a switch for ASCs reprogramming and their clinical application.

BET inhibition therapy counteracts cancer cell survival, clonogenic potential and radioresistance mechanisms in rhabdomyosarcoma cells.

The antitumour effects of OTX015, a first-in-class BET inhibitor (BETi), were investigated as a single agent or in combination with ionizing radiation (IR) in preclinical in vitro models of rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma. Herein, we demonstrated the upregulation of BET Bromodomain gene expression in RMS tumour biopsies and cell lines compared to normal skeletal muscle. In vitro experiments showed that OTX015 significantly reduced RMS cell proliferation by altering cell cycle modulators and apoptotic related proteins due to the accumulation of DNA breaks that cells are unable to repair. Interestingly, OTX015 also impaired migration capacity and tumour-sphere architecture by downregulating pro-stemness genes and was able to potentiate ionizing radiation effects by reducing the expression of different drivers of tumour dissemination and resistance mechanisms, including the GNL3 gene, that we correlated for the first time with the RMS phenotype. In conclusion, our research sheds further light on the molecular events of OTX015 action against RMS cells and indicates this novel BETi as an effective option to improve therapeutic strategies and overcome the development of resistant cancer cells in patients with RMS.

MiR-200c sensitizes Olaparib-resistant ovarian cancer cells by targeting Neuropilin 1.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecological malignancy and the second leading cause of cancer-related death in women. Treatment with PARP inhibitors (PARPi), such as Olaparib, has been recently introduced for OC patients, but resistance may occur and underlying mechanisms are still poorly understood. The aim of this study is to identify target genes within the tumor cells that might cause resistance to Olaparib. We focused on Neuropilin 1 (NRP1), a transmembrane receptor expressed in OC and correlated with poor survival, which has been also proposed as a key molecule in OC multidrug resistance. METHODS: Using three OC cell lines (UWB, UWB-BRCA and SKOV3) as model systems, we evaluated the biological and molecular effects of Olaparib on OC cell growth, cell cycle, DNA damage and apoptosis/autophagy induction, through MTT and colony forming assays, flow cytometry, immunofluorescence and Western blot analyses. We evaluated NRP1 expression in OC specimens and cell lines by Western blot and qRT-PCR, and used RNA interference to selectively inhibit NRP1. To identify miR-200c as a regulator of NRP1, we used miRNA target prediction algorithms and Pearsons' correlation analysis in biopsies from OC patients. Then, we used a stable transfection approach to overexpress miR-200c in Olaparib-resistant cells. RESULTS: We observed that NRP1 is expressed at high levels in resistant cells (SKOV3) and is upmodulated in partially sensitive cells (UWB-BRCA) upon prolonged Olaparib treatment, leading to poor drug response. Our results show that the selective inhibition of NRP1 is able to overcome Olaparib resistance in SKOV3 cells. Moreover, we demonstrated that miR-200c can target NRP1 in OC cells, causing its downmodulation, and that miR-200c overexpression is a valid approach to restore Olaparib sensitivity in OC resistant cells. CONCLUSIONS: These data demonstrate that miR-200c significantly enhanced the anti-cancer efficacy of Olaparib in drug-resistant OC cells. Thus, the combination of Olaparib with miRNA-based therapy may represent a promising treatment for drug resistant OC, and our data may help in designing novel precision medicine trials for optimizing the clinical use of PARPi.

NRF2 orchestrates the redox regulation induced by radiation therapy, sustaining embryonal and alveolar rhabdomyosarcoma cells radioresistance.

PURPOSE: Tumor cells generally exhibit higher levels of reactive oxygen species (ROS), however, when stressed, tumor cells can undergo a process of 'Redox Resetting' to acquire a new redox balance with stronger antioxidant systems that enable cancer cells to become resistant to radiation therapy (RT). Here, we describe how RT affects the oxidant/antioxidant balance in human embryonal (RD) and alveolar (RH30) rhabdomyosarcoma (RMS) cell lines, investigating on the molecular mechanisms involved. METHODS: Radiations were delivered using an x-6 MV photon linear accelerator and their effects were assessed by vitality and clonogenic assays. The expression of specific antioxidant-enzymes, such as Superoxide Dismutases (SODs), Catalase (CAT) and Glutathione Peroxidases 4 (GPx4), miRNAs (miR-22, -126, -210, -375, -146a, -34a) and the transcription factor NRF2 was analyzed by quantitative polymerase chain reaction (q-PCR) and western blotting. RNA interference experiments were performed to evaluate the role of NRF2. RESULTS: Doses of RT higher than 2 Gy significantly affected RMS clonogenic ability by increasing ROS production. RMS rapidly and efficiently brought back ROS levels by up-regulating the gene expression of antioxidant enzymes, miRNAs as well as of NRF2. Silencing of NRF2 restrained the RMS ability to counteract RT-induced ROS accumulation, antioxidant enzyme and miRNA expression and was able to increase the abundance of γ-H2AX, a biomarker of DNA damage, in RT-treated cells. CONCLUSIONS: Taken together, our data suggest the strategic role of oxidant/antioxidant balance in restraining the therapeutic efficiency of RT in RMS treatment and identify NRF2 as a new potential molecular target whose inhibition might represent a novel radiosensitizing therapeutic strategy for RMS clinical management.

PARP inhibitors affect growth, survival and radiation susceptibility of human alveolar and embryonal rhabdomyosarcoma cell lines.

PURPOSE: PARP inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent childhood soft-tissue sarcoma. The cellular and molecular effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthesized PARP1/2/3 inhibitor, were assessed in alveolar and embryonal RMS cells both as single-agent and in combination with ionizing radiation (IR). METHODS: Cell viability was monitored by trypan blue exclusion dye assays. Cell cycle progression and apoptosis were measured by flow cytometry, and alterations of specific molecular markers were investigated by, Real Time PCR, Western blotting and immunofluorescence experiments. Irradiations were carried out at a dose rate of 2 Gy (190 UM/min) or 4 Gy (380 UM/min). Radiosensitivity was assessed by using clonogenic assays. RESULTS: Olaparib and AZD2461 dose-dependently reduced growth of both RH30 and RD cells by arresting growth at G2/M phase and by modulating the expression, activation and subcellular localization of specific cell cycle regulators. Downregulation of phospho-AKT levels and accumulation of γH2AX, a specific marker of DNA damage, were significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to apoptosis-related cell death. Both PARPi significantly enhanced the effects of IR by accumulating DNA damage, increasing G2 arrest and drastically reducing the clonogenic capacity of RMS-cotreated cells. CONCLUSIONS: This study suggests that the combined exposure to PARPi and IR might display a role in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.

HDAC4 and HDAC6 sustain DNA double strand break repair and stem-like phenotype by promoting radioresistance in glioblastoma cells.

The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis- and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM.