Integrin alpha9 emerges as a key therapeutic target to reduce metastasis in rhabdomyosarcoma and neuroblastoma.

The majority of current cancer therapies are aimed at reducing tumour growth, but there is lack of viable pharmacological options to reduce the formation of metastasis. This is a paradox, since more than 90% of cancer deaths are attributable to metastatic progression. Integrin alpha9 (ITGA9) has been previously described as playing an essential role in metastasis; however, little is known about the mechanism that links this protein to this process, being one of the less studied integrins. We have now deciphered the importance of ITGA9 in metastasis and provide evidence demonstrating its essentiality for metastatic dissemination in rhabdomyosarcoma and neuroblastoma. However, the most translational advance of this study is to reveal, for the first time, the possibility of reducing metastasis by pharmacological inhibition of ITGA9 with a synthetic peptide simulating a key interaction domain of ADAM proteins, in experimental metastasis models, not only in childhood cancers but also in a breast cancer model.

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program.

BACKGROUND: Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. METHODS: Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. RESULTS: Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. CONCLUSIONS: We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Dickkopf-1 Inhibition Reactivates Wnt/ß-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo.

The Wnt/ß-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/ß-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to ß-catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression.

Functional high-throughput screening reveals miR-323a-5p and miR-342-5p as new tumor-suppressive microRNA for neuroblastoma.

Current therapies for most non-infectious diseases are directed at or affect functionality of the human translated genome, barely 2% of all genetic information. By contrast, the therapeutic potential of targeting the transcriptome, ~ 70% of the genome, remains largely unexplored. RNA therapeutics is an emerging field that widens the range of druggable targets and includes elements such as microRNA. Here, we sought to screen for microRNA with tumor-suppressive functions in neuroblastoma, an aggressive pediatric tumor of the sympathetic nervous system that requires the development of new therapies. We found miR-323a-5p and miR-342-5p to be capable of reducing cell proliferation in multiple neuroblastoma cell lines in vitro and in vivo, thereby providing a proof of concept for miRNA-based therapies for neuroblastoma. Furthermore, the combined inhibition of the direct identified targets such as CCND1, CHAF1A, INCENP and BCL-XL could reveal new vulnerabilities of high-risk neuroblastoma.

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Circulating tumor cells in peripheral blood or disseminated to bone marrow, a concept commonly referred to as minimal residual disease (MRD), are thought to be key to the prediction of metastasis and treatment efficacy. To date, two MRD markers, MYOD and MYOGENIN, have been tested; however, MRD detection continues to be challenging mainly owing to the closeness of the detection limit and the discordance of both markers in some samples. Therefore, the addition of a third marker could be useful for more accurate MRD assessment. The PAX3 gene is expressed during embryo development in all myogenic precursor cells in the dermomyotome. As RMS cells are thought to originate from these muscle precursor cells, they are expected to be positive for PAX3. In this study, PAX3 expression was characterized in cancer cell lines and tumors, showing wide expression in RMS. Detection sensitivities by quantitative polymerase chain reaction (qPCR) of the previously proposed markers, MYOD and MYOGENIN, were similar to that of PAX3, thereby indicating the feasibility of its detection. Interestingly, the flow cytometry experiments supported the usefulness of this technique in the quantification of MRD in RMS using PAX3 as a marker. These results indicate that flow cytometry, albeit in some cases slightly less sensitive, can be considered a good approach for MRD assessment in RMS and more consistent than qPCR, especially owing to its greater specificity. Furthermore, fluorescence-activated cell sorting permits the recovery of cells, thereby providing material for further characterization of circulating or disseminated cancer cells.

In vivo cisplatin-resistant neuroblastoma metastatic model reveals tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as an independent prognostic factor of survival in neuroblastoma.

Neuroblastoma is the most common solid extracranial tumour in children. Despite major advances in available therapies, children with drug-resistant and/or recurrent neuroblastoma have a dismal outlook with 5-year survival rates of less than 20%. Therefore, tackling relapsed tumour biology by developing and characterising clinically relevant models is a priority in finding targetable vulnerability in neuroblastoma. Using matched cisplatin-sensitive KellyLuc and resistant KellyCis83Luc cell lines, we developed a cisplatin-resistant metastatic MYCN-amplified neuroblastoma model. The average number of metastases per mouse was significantly higher in the KellyCis83Luc group than in the KellyLuc group. The vast majority of sites were confirmed as having lymph node metastasis. Their stiffness characteristics of lymph node metastasis values were within the range reported for the patient samples. Targeted transcriptomic profiling of immuno-oncology genes identified tumour necrosis factor receptor superfamily member 4 (TNFRSF4) as a significantly dysregulated MYCN-independent gene. Importantly, differential TNFRSF4 expression was identified in tumour cells rather than lymphocytes. Low TNFRSF4 expression correlated with poor prognostic indicators in neuroblastoma, such as age at diagnosis, stage, and risk stratification and significantly associated with reduced probability of both event-free and overall survival in neuroblastoma. Therefore, TNFRSF4 Low expression is an independent prognostic factor of survival in neuroblastoma.

TRIB3 silencing promotes the downregulation of Akt pathway and PAX3-FOXO1 in high-risk rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), such as other childhood tumors, has witnessed treatment advancements in recent years. However, high-risk patients continue to face poor survival rates, often attributed to the presence of the PAX3/7-FOXO1 fusion proteins, which has been associated with metastasis and treatment resistance. Despite efforts to directly target these chimeric proteins, clinical success remains elusive. In this study, the main aim was to address this challenge by investigating regulators of FOXO1. Specifically, we focused on TRIB3, a potential regulator of the fusion protein in RMS. Our findings revealed a prominent TRIB3 expression in RMS tumors, highlighting its correlation with the presence of fusion protein. By conducting TRIB3 genetic inhibition experiments, we observed an impairment on cell proliferation. Notably, the knockdown of TRIB3 led to a decrease in PAX3-FOXO1 and its target genes at protein level, accompanied by a reduction in the activity of the Akt signaling pathway. Additionally, inducible silencing of TRIB3 significantly delayed tumor growth and improved overall survival in vivo. Based on our analysis, we propose that TRIB3 holds therapeutic potential for treating the most aggressive subtype of RMS. The findings herein reported contribute to our understanding of the underlying molecular mechanisms driving RMS progression and provide novel insights into the potential use of TRIB3 as a therapeutic intervention for high-risk RMS patients.

microRNAs as pharmacological targets in cancer.

The survival rate of cancer patients has increased considerably in the last 20 years owing to significant efforts made in prevention, early detection protocols, combined chemotherapy regimens, targeted therapies, refined radiotherapy and cancer vaccines. However, metastasis and acquired resistance to current therapies represent two major challenges for achieving long-term cure. Therefore, new treatment strategies must be developed. One promising alternative is epigenetic-based therapies, of which miRNAs are at the forefront. MicroRNAs are endogenous small non-coding RNAs, often deregulated in cancer, which regulate gene expression by specific binding to the 3'-UTR of target genes. They are excellent candidates for therapy since miRNAs can regulate multiple targets of the same or different pathways, thereby minimizing the risk of resistance development or compensatory mechanisms. In this review, the mechanisms that lead to miRNA deregulation in cancer, their feasibility as therapeutic tools and the different strategies for the pharmacological manipulation of miRNAs in preclinical animal models are discussed.

Prevalence and Impact of Asthma and Allergy on Daily Life, Health Outcomes and Use of Healthcare Services in Children: A Population-Based Study.

INTRODUCTION: Studies on the prevalence of asthma and allergies often lack representation of the pediatric population, and their impact has not been examined using children without these diseases as a reference group. This study aimed to determine the prevalence of asthma and allergies in children under 14 years old in Spain and their impact on health-related quality of life, activities, healthcare utilization, and environmental and household exposure risk factors. METHODS: Data came from a Spanish population-based representative survey of children aged <14 years (N=6297). A sample of controls (1:4) from the same survey was matched using propensity score matching. Logistic regression models and population-attributable fractions were calculated to determine the impact of asthma and allergy. RESULTS: The population prevalence of asthma was 5.7% (95% CI: 5.0%, 6.4%), and of allergy was 11.4% (95% CI: 10.5%, 12.4. In children with lower percentiles of health-related quality of life (≤20th), 32.3% (95% CI, 13.6%, 47.0%) was attributed to asthma and 27.7% (95% CI: 13.0%, 40.0%) to allergy. Forty-four percent of restrictions in usual activity were attributed to asthma (OR: 2.0, p-value: <0.001), and 47.9% to allergy (OR: 2.1, p-value: <0.001). 62.3% of all hospital admissions were attributed to asthma (OR: 2.8, p-value: <0.001), and 36.8% (OR: 2.5, p-value: <0.001) of all specialist consults to allergy. CONCLUSIONS: The high prevalence of atopic disease and its impact on daily life and healthcare utilization call for an integrated healthcare system focused on children and caregivers' needs with continuity of care across education and healthcare settings.

Targeting the Hedgehog Pathway in Rhabdomyosarcoma.

Aberrant activation of the Hedgehog (Hh) signalling pathway is known to play an oncogenic role in a wide range of cancers; in the particular case of rhabdomyosarcoma, this pathway has been demonstrated to be an important player for both oncogenesis and cancer progression. In this review, after a brief description of the pathway and the characteristics of its molecular components, we describe, in detail, the main activation mechanisms that have been found in cancer, including ligand-dependent, ligand-independent and non-canonical activation. In this context, the most studied inhibitors, i.e., SMO inhibitors, have shown encouraging results for the treatment of basal cell carcinoma and medulloblastoma, both tumour types often associated with mutations that lead to the activation of the pathway. Conversely, SMO inhibitors have not fulfilled expectations in tumours-among them sarcomas-mostly associated with ligand-dependent Hh pathway activation. Despite the controversy existing regarding the results obtained with SMO inhibitors in these types of tumours, several compounds have been (or are currently being) evaluated in sarcoma patients. Finally, we discuss some of the reasons that could explain why, in some cases, encouraging preclinical data turned into disappointing results in the clinical setting.

Analysis of Cancer Genomic Amplifications Identifies Druggable Collateral Dependencies within the Amplicon.

The identification of novel therapeutic targets for specific cancer molecular subtypes is crucial for the development of precision oncology. In the last few years, CRISPR/Cas9 screens have accelerated the discovery and validation of new targets associated with different tumor types, mutations, and fusions. However, there are still many cancer vulnerabilities associated with specific molecular features that remain to be explored. Here, we used data from CRISPR/Cas9 screens in 954 cancer cell lines to identify gene dependencies associated with 16 common cancer genomic amplifications. We found that high-copy-number genomic amplifications generate multiple collateral dependencies within the amplified region in most cases. Further, to prioritize candidate targets for each chromosomal region amplified, we integrated gene dependency parameters with both druggability data and subcellular location. Finally, analysis of the relationship between gene expression and gene dependency led to the identification of genes, the expression of which may constitute predictive biomarkers of dependency. In conclusion, our study provides a set of druggable targets specific for each amplification, opening the possibility to specifically target amplified tumors on this basis.

Single-cell profiling of alveolar rhabdomyosarcoma reveals RAS pathway inhibitors as cell-fate hijackers with therapeutic relevance.

Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.

uPA deficiency exacerbates muscular dystrophy in MDX mice.

Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.

Notch, wnt, and hedgehog pathways in rhabdomyosarcoma: from single pathways to an integrated network.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Regarding histopathological criteria, RMS can be divided into 2 main subtypes: embryonal and alveolar. These subtypes differ considerably in their clinical phenotype and molecular features. Abnormal regulation or mutation of signalling pathways that regulate normal embryonic development such as Notch, Hedgehog, and Wnt is a recurrent feature in tumorigenesis. Herein, the general features of each of the three pathways, their implication in cancer and particularly in RMS are reviewed. Finally, the cross-talking among these three pathways and the possibility of better understanding of the horizontal communication among them, leading to the development of more potent therapeutic approaches, are discussed.

Locally Grown Crops and Immunocastration in Fattening Heavy Pigs: Effects on Performance and Welfare.

This study aimed to explore dietary replacement soybean meal (SBM) with local pea seeds (PS-L) and the effects of surgically castrated (CM) or immunocastration (IM) in heavy male pigs, on growth performance, feeding behaviour, and tail and ear lesions. Four treatments were arranged factorially (2 × 2), with two sexes and two dietary treatments (96 pigs in eight pens). The inclusion of PS-L was 25%, 30%, and 40% during three phases (40−80 kg, 80−110 kg, and 110−140 kg, respectively). No difference in average daily feed intake (ADFI) and body-weight (BW) between PS-L and SBM could be demonstrated (p > 0.05), but PS-L diet decreased the average daily gain (ADG) at 110−140 kg of BW (p < 0.001) and increased feed conversion rate (FCR) (p < 0.05). The ADG was higher (p < 0.01) in IM than CM in all fattening periods, and the FCR in IM was lower (p < 0.05) than in barrows. IM pigs had lower dressing percentage than CM (p < 0.01). Pigs fed a PS-L diet ate faster but increased their passive behaviour compared with those fed SBM. In conclusion, the PS-L diet did not reduce BW and improved passive behaviour, and IM grew more efficiently, regardless of diet.

MEK and MCL-1 sequential inhibition synergize to enhance rhabdomyosarcoma treatment.

Targeted agents have emerged as promising molecules for cancer treatment, but most of them fail to achieve complete tumor regression or attain durable remissions due to tumor adaptations. We used dynamic BH3 profiling to identify targeted agents effectiveness and anti-apoptotic adaptations upon targeted treatment in rhabdomyosarcoma. We focused on studying the use of BH3 mimetics to specifically inhibit pro-survival BCL-2 family proteins, overwhelm resistance to therapy and prevent relapse. We observed that the MEK1/2 inhibitor trametinib rapidly depleted the pro-apoptotic protein NOXA, thus increasing MCL-1 availability. Indeed, we found that the MCL-1 inhibitor S63845 synergistically enhanced trametinib cytotoxicity in rhabdomyosarcoma cells in vitro and in vivo. In conclusion, our findings indicate that the combination of a BH3 mimetic targeting MCL-1 with trametinib improves efficiency on rhabdomyosarcoma by blocking tumor adaptation to treatment.

Methodological advances in the discovery of novel neuroblastoma therapeutics.

INTRODUCTION: Neuroblastoma is a cancer of the sympathetic nervous system that causes up to 15% of cancer-related deaths among children. Among the ~1,000 newly diagnosed cases per year in Europe, more than half are classified as high-risk, with a 5-year survival rate <50%. Current multimodal treatments have improved survival among these patients, but relapsed and refractory tumors remain a major therapeutic challenge. A number of new methodologies are paving the way for the development of more effective and safer therapies to ultimately improve outcomes for high-risk patients. AREAS COVERED: The authors provide a critical review on methodological advances aimed at providing new therapeutic opportunities for neuroblastoma patients, including preclinical models of human disease, generation of omics data to discover new therapeutic targets, and artificial intelligence-based technologies to implement personalized treatments. EXPERT OPINION: While survival of childhood cancer has improved over the past decades, progress has been uneven. Still, survival is dismal for some cancers, including high-risk neuroblastoma. Embracing new technologies (e.g. molecular profiling of tumors, 3D in vitro models, etc.), international collaborative efforts and the incorporation of new therapies (e.g. RNA-based therapies, epigenetic therapies, immunotherapy) will ultimately lead to more effective and safer therapies for these subgroups of neuroblastoma patients.

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.

Dickkopf Proteins and Their Role in Cancer: A Family of Wnt Antagonists with a Dual Role.

The Wnt signaling pathway regulates crucial aspects such as cell fate determination, cell polarity and organogenesis during embryonic development. Wnt pathway deregulation is a hallmark of several cancers such as lung, gastric and liver cancer, and has been reported to be altered in others. Despite the general agreement reached by the scientific community on the oncogenic potential of the central components of the pathway, the role of the antagonist proteins remains less clear. Deregulation of the pathway may be caused by overexpression or downregulation of a wide range of antagonist proteins. Although there is growing information related to function and regulation of Dickkopf (DKK) proteins, their pharmacological potential as cancer therapeutics still has not been fully developed. This review provides an update on the role of DKK proteins in cancer and possible potential as therapeutic targets for the treatment of cancer; available compounds in pre-clinical or clinical trials are also reviewed.