MiR-223 Exclusively Impairs In Vitro Tumor Growth through IGF1R Modulation in Rhabdomyosarcoma of Adolescents and Young Adults.

Adolescents and young adults (AYA) with rhabdomyosarcoma (RMS) form a subgroup of patients whose optimal clinical management and best possible access to care remain a challenge and whose survival rates lag behind that of children diagnosed with histologically similar tumors. A better understanding of tumor biology that differentiates children (PEDS-) from AYA-RMS could provide critical information and drive new initiatives to improve their final outcome. We investigated the functional role of miRNAs implicated in AYA-RMS development, as they have the potential to lead to discovery of new targets pathways for a more tailored treatment in these age groups of young RMS patients. MiR-223 and miR-486 were observed de-regulated in nine RMS tissues compared to their normal counterparts, yet only miR-223 replacement impaired proliferation and aggressiveness of AYA-RMS cell lines, while inducing apoptosis and determining cell cycle arrest. Interestingly, IGF1R resulted in the direct target of miR-223 in AYA-RMS cells, as demonstrated by IGF1R silencing. Our results highlight an exclusive functional role of miR-223 in AYA-RMS development and aggressiveness.

BMP7 promotes cardiomyocyte regeneration in zebrafish and adult mice.

Zebrafish have a lifelong cardiac regenerative ability after damage, whereas mammals lose this capacity during early postnatal development. This study investigated whether the declining expression of growth factors during postnatal mammalian development contributes to the decrease of cardiomyocyte regenerative potential. Besides confirming the proliferative ability of neuregulin 1 (NRG1), interleukin (IL)1b, receptor activator of nuclear factor kappa-Β ligand (RANKL), insulin growth factor (IGF)2, and IL6, we identified other potential pro-regenerative factors, with BMP7 exhibiting the most pronounced efficacy. Bmp7 knockdown in neonatal mouse cardiomyocytes and loss-of-function in adult zebrafish during cardiac regeneration reduced cardiomyocyte proliferation, indicating that Bmp7 is crucial in the regenerative stages of mouse and zebrafish hearts. Conversely, bmp7 overexpression in regenerating zebrafish or administration at post-mitotic juvenile and adult mouse stages, in vitro and in vivo following myocardial infarction, enhanced cardiomyocyte cycling. Mechanistically, BMP7 stimulated proliferation through BMPR1A/ACVR1 and ACVR2A/BMPR2 receptors and downstream SMAD5, ERK, and AKT signaling. Overall, BMP7 administration is a promising strategy for heart regeneration.

The metastatic niche formation: focus on extracellular vesicle-mediated dialogue between lung cancer cells and the microenvironment.

Lung cancer is the deadliest cancer in the world, with the majority of patients presenting with advanced or metastatic disease at first diagnosis. The lungs are also one of the most common sites of metastasis from lung cancer and other tumors. Understanding the mechanisms that regulate metastasis formation from primary lung cancer and in the lungs is therefore fundamental unmet clinical need. One of the first steps during the establishment of lung cancer metastases includes the formation of the pre-metastatic niche (PMN) at distant organs, which may occur even during the early phases of cancer development. The PMN is established through intricate cross-talk between primary tumor-secreted factors and stromal components at distant sites. Mechanisms controlling primary tumor escape and seeding of distant organs rely on specific properties of tumor cells but are also tightly regulated by interactions with stromal cells at the metastatic niche that finally dictate the success of metastasis establishment. Here, we summarize the mechanisms underlying pre-metastatic niche formation starting from how lung primary tumor cells modulate distant sites through the release of several factors, focusing on Extracellular Vesicles (EVs). In this context, we highlight the role of lung cancer-derived EVs in the modulation of tumor immune escape. Then, we illustrate the complexity of Circulating Tumor Cells (CTCs) that represent the seeds of metastasis and how interactions with stromal and immune cells can help their metastatic dissemination. Finally, we evaluate the contribution of EVs in dictating metastasis development at the PMN through stimulation of proliferation and control of disseminated tumor cell dormancy. Overall, we present an overview of different steps in the lung cancer metastatic cascade, focusing on the EV-mediated interactions between tumor cells and stromal/immune cells.

Extracellular vesicles from subjects with COPD modulate cancer initiating cells phenotype through HIF-1α shuttling.

Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer development. COPD induces activation of hypoxia-induced signaling, causing remodeling of surrounding microenvironmental cells also modulating the release and cargo of their extracellular vesicles (EVs). We aimed to evaluate the potential role of circulating EVs from COPD subjects in lung cancer onset. Plasma-EVs were isolated by ultracentrifugation from heavy smoker volunteers with (COPD-EVs) or without (heavy smoker-EVs, HS-EV) COPD and characterized following MISEV guidelines. Immortalized human bronchial epithelial cells (CDK4, hTERT-HBEC3-KT), genetically modified with different oncogenic alterations commonly found in lung cancer (sh-p53, KRASV12), were used to test plasma-EVs pro-tumorigenic activity in vitro. COPD-EVs mainly derived from immune and endothelial cells. COPD-EVs selectively increased the subset of CD133+CXCR4+ metastasis initiating cells (MICs) in HBEC-sh-p53-KRASV12high cells and stimulated 3D growth, migration/invasion, and acquisition of mesenchymal traits. These effects were not observed in HBEC cells bearing single oncogenic mutation (sh-p53 or KRASV12). Mechanistically, hypoxia-inducible factor 1-alpha (HIF-1α) transferred from COPD-EVs triggers CXCR4 pathway activation that in turn mediates MICs expansion and acquisition of pro-tumorigenic effects. Indeed, HIF-1α inhibition or CXCR4 silencing prevented the acquisition of malignant traits induced by COPD-EVs alone. Hypoxia recapitulates the effects observed with COPD-EVs in HBEC-sh-p53-KRASV12high cells. Notably, higher levels of HIF-1α were observed in EVs from COPD subjects who subsequently developed cancer compared to those who remained cancer-free. Our findings support a role of COPD-EVs to promote the expansion of MICs in premalignant epithelial cells through HIF-1α-CXCR4 axis activation thereby potentially sustaining lung cancer progression.

NRG1/ERBB3/ERBB2 Axis Triggers Anchorage-Independent Growth of Basal-like/Triple-Negative Breast Cancer Cells.

ERBB3, also known as HER3, is a tyrosine kinase transmembrane receptor of the ERBB family. Upon binding to neuregulin 1 (NRG1), ERBB3 preferentially dimerizes with HER2 (ERBB2), in turn inducing aggressive features in several cancer types. The analysis of a dataset of breast cancer patients unveiled that higher ERBB3 mRNA expression correlates with shorter relapse-free survival in basal-like breast cancers, despite low ERBB3 expression in this breast cancer subtype. Administration of neuregulin 1 beta (NRG1ß) significantly affected neither cellular proliferation nor the basal migratory ability of basal-like/triple-negative quasi-normal MCF10A breast cells, cultured in mono-layer conditions. Furthermore, no significant regulation in cell morphology or in the expression of basal/myoepithelial and luminal markers was observed upon stimulation with NRG1ß. In non-adherent conditions, NRG1ß administration to MCF10A cells did not significantly influence cell survival; however, it robustly induced cell growth as spheroids (3D growth). Intriguingly, a remarkable upregulation of ERBB3 and ERBB2 protein abundance was observed in 3D compared to 2D cell cultures, and NRG1ß-induced 3D cell growth was efficiently prevented by the anti-HER2 monoclonal antibody pertuzumab. Similar results were obtained by the analysis of basal-like/triple-negative breast cancer cellular models, MDA-MB-468 and MDA-MB-231 cells, in which NRG1ß induced anchorage-independent cell growth that in turn was prevented or reduced by the simultaneous administration of anti-HER2 neutralizing antibodies. Finally, the ability of pertuzumab in suppressing NRG1ß-induced 3D growth was also evaluated and confirmed in MCF10A engineered with HER2-overexpression. We suggest that the NRG1/ERBB3/ERBB2 pathway promotes the anchorage-independent growth of basal-like breast cancer cells. Importantly, we provide evidence that ERBB2 neutralization, in particular by pertuzumab, robustly inhibits this process. Our results pave the way towards the development of novel anticancer strategies for basal-like breast cancer patients based on the interception of the NRG1/ERBB3/ERBB2 signaling axis.

Circulating CD81-expressing extracellular vesicles as biomarkers of response for immune-checkpoint inhibitors in advanced NSCLC.

PD-L1 in tumor cells is the only used biomarker for anti PD1/PD-L1 immune-checkpoints inhibitors (ICI) in Non Small Cell Lung Cancer (NSCLC) patients. However, this parameter is inaccurate to predict response, especially in patients with low tumor PD-L1. Here, we evaluated circulating EVs as possible biomarkers for ICI in advanced NSCLC patients with low tumoral PD-L1. EVs were isolated from plasma of 64 PD-L1 low, ICI-treated NSCLC patients, classified either as responders (R; complete or partial response by RECIST 1.1) or non-responders (NR). EVs were characterized following MISEV guidelines and by flow cytometry. T cells from healthy donors were triggered in vitro using patients' EVs. Unsupervised statistical approach was applied to correlate EVs' and patients' features to clinical response. R-EVs showed higher levels of tetraspanins (CD9, CD81, CD63) than NR-EVs, significantly associated to better overall response rate (ORR). In multivariable analysis CD81-EVs correlated with ORR. Unsupervised analysis revealed a cluster of variables on EVs, including tetraspanins, significantly associated with ORR and improved survival. R-EVs expressed more costimulatory molecules than NR-EVs although both increased T cell proliferation and partially, activation. Tetraspanins levels on EVs could represent promising biomarkers for ICI response in NSCLC.

Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through transfer of miR-126 and miR-320.

BACKGROUND: Extracellular vesicles (EVs) containing specific subsets of functional biomolecules are released by all cell types and analysis of circulating EVs can provide diagnostic and prognostic information. To date, little is known regarding the role of EVs both as biomarkers and potential key players in human lung cancer. METHODS: Plasma EVs were isolated from 40 cancer-free heavy-smokers classified according to a validated 24-microRNA signature classifier (MSC) at high (MSCpos-EVs) or low (MSCneg-EVs) risk to develop lung cancer. EVs origin and functional properties were investigated using in vitro 3D cultures and in vivo models. The prognostic value of miRNAs inside EVs was assessed in training and in validation cohorts of 54 and 48 lung cancer patients, respectively. RESULTS: Different membrane composition, biological cargo and pro-tumorigenic activity were observed in MSCpos vs MSCneg-EVs. Mechanistically, in vitro and in vivo results showed that miR-126 and miR-320 from MSCpos-EVs increased pro-angiogenic phenotype of endothelial cells and M2 polarization of macrophage, respectively. MSCpos-EVs prompted 3D proliferation of non-tumorigenic epithelial cells through c-Myc transfer. Moreover, hypoxia was shown to stimulate the secretion of EVs containing c-Myc from fibroblasts, miR-126-EVs from endothelial cells and miR-320-EVs from granulocytes. Lung cancer patients with higher levels of mir-320 into EVs displayed a significantly shorter overall survival in training [HR2.96] and validation sets [HR2.68]. CONCLUSION: Overall our data provide a new perspective on the pro-tumorigenic role of circulating EVs in high risk smokers and highlight the significance of miR-320-EVs as a new prognostic biomarker in lung cancer patients.

Cotargeting of miR-126-3p and miR-221-3p inhibits PIK3R2 and PTEN, reducing lung cancer growth and metastasis by blocking AKT and CXCR4 signalling.

Lung cancer is the leading cause of cancer-related death worldwide. Late diagnosis and metastatic dissemination contribute to its low survival rate. Since microRNA (miRNA) deregulation triggers lung carcinogenesis, miRNAs might represent an interesting therapeutic tool for lung cancer management. We identified seven miRNAs, including miR-126-3p and miR-221-3p, that are deregulated in tumours compared with normal tissues in a series of 38 non-small-cell lung cancer patients. A negative correlation between these two miRNAs was associated with poor patient survival. Concomitant miR-126-3p replacement and miR-221-3p inhibition, but not modulation of either miRNA alone, reduced lung cancer cell viability by inhibiting AKT signalling. PIK3R2 and PTEN were validated as direct targets of miR-126-3p and miR-221-3p, respectively. Simultaneous miRNA modulation reduced metastatic dissemination of lung cancer cells both in vitro and in vivo through CXCR4 inhibition. Systemic delivery of a combination of miR-126-3p mimic and miR-221-3p inhibitor encapsulated in lipid nanoparticles reduced lung cancer patient-derived xenograft growth through blockade of the PIK3R2-AKT pathway. Our findings reveal that cotargeting miR-126-3p and miR-221-3p to hamper both tumour growth and metastasis could be a new therapeutic approach for lung cancer.

Glucocorticoid Receptor Modulates EGFR Feedback upon Acquisition of Resistance to Monoclonal Antibodies.

Evidences of a crosstalk between Epidermal Growth Factor Receptor (EGFR) and Glucocorticoid Receptor (GR) has been reported, ranging from the modulation of receptor levels or GR mediated transcriptional repression of EGFR target genes, with modifications of epigenetic markers. The present study focuses on the involvement of EGFR positive and negative feedback genes in the establishment of cetuximab (CTX) resistance in metastatic Colorectal Cancer (CRC) patients. We evaluated the expression profile of the EGFR ligands TGFA and HBEGF, along with the pro-inflammatory cytokines IL-1B and IL-8, which were previously reported to be negatively associated with monoclonal antibody response, both in mice and patient specimens. Among EGFR negative feedback loops, we focused on ERRFI1, DUSP1, LRIG3, and LRIG1. We observed that EGFR positive feedback genes are increased in CTX-resistant cells, whereas negative feedback genes are reduced. Next, we tested the expression of these genes in CTX-resistant cells upon GR modulation. We unveiled that GR activation leads to an increase in ERRFI1, DUSP1, and LRIG1, which were shown to restrict EGFR activity, along with a decrease in the EGFR activators (TGFA and IL-8). Finally, in a cohort of xenopatients, stratified for response to cetuximab, we observed an inverse association between the expression level of LRIG1 and CRC progression upon CTX treatment. Our model implies that combining GR modulation to EGFR inhibition may yield an effective treatment strategy in halting cancer progression.