MiR-151a: a robust endogenous control for normalizing small extracellular vesicle cargo in human cancer.

Small extracellular vesicles (sEVs) in the blood of cancer patients contain higher amounts of tumor markers than those identified as free-circulating. miRNAs have significant biomedical relevance due to their high stability and feasible detection. However, there is no reliable endogenous control available to measure sEVs-miRNA content, impairing the acquisition of standardized consistent measurements in cancer liquid biopsy. In this study, we identified three miRNAs from a panel of nine potential normalizers that emerged from a comprehensive analysis comparing the sEV-miRNA profile of six lung and ovarian human cancer cell lines in the absence of or under different conditions. Their relevance as normalizers was tested in 26 additional human cancer cell lines from nine different tumor types undergoing chemotherapy or radiotherapy treatment. The validation cohorts were comprised of 242 prospective plasma and ascitic fluid samples from three different human tumor types. Variability and normalization properties were tested in comparison to miR-16, the most used control to normalize free-circulating miRNAs in plasma. Our results indicate that miR-151a is consistently represented in small extracellular vesicles with minimal variability compared to miR-16, providing a novel normalizer to measure small extracellular vesicle miRNA content that will benefit liquid biopsy in cancer patients.

miR-124 as a Liquid Biopsy Prognostic Biomarker in Small Extracellular Vesicles from NSCLC Patients.

Despite advances in non-small cell lung cancer (NSCLC) research, this is still the most common cancer type that has been diagnosed up to date. microRNAs have emerged as useful clinical biomarkers in both tissue and liquid biopsy. However, there are no reliable predictive biomarkers for clinical use. We evaluated the preclinical use of seven candidate miRNAs previously identified by our group. We collected a total of 120 prospective samples from 88 NSCLC patients. miRNA levels were analyzed via qRT-PCR from tissue and blood samples. miR-124 gene target prediction was performed using RNA sequencing data from our group and interrogating data from 2952 NSCLC patients from two public databases. We found higher levels of all seven miRNAs in tissue compared to plasma samples, except for miR-124. Our findings indicate that levels of miR-124, both free-circulating and within exosomes, are increased throughout the progression of the disease, suggesting its potential as a marker of disease progression in both advanced and early stages. Our bioinformatics approach identified KPNA4 and SPOCK1 as potential miR-124 targets in NSCLC. miR-124 levels can be used to identify early-stage NSCLC patients at higher risk of relapse.

Prognostic Impact of Dihydropyrimidine Dehydrogenase Germline Variants in Unresectable Non-Small Cell Lung Cancer Patients Treated with Platin-Based Chemotherapy.

Platin-based chemotherapy is the standard treatment for patients with non-small cell lung cancer (NSCLC). However, resistance to this therapy is a major obstacle in successful treatment. In this study, we aimed to investigate the impact of several pharmacogenetic variants in patients with unresectable NSCLC treated with platin-based chemotherapy. Our results showed that DPYD variant carriers had significantly shorter progression-free survival and overall survival compared to DPYD wild-type patients, whereas DPD deficiency was not associated with a higher incidence of high-grade toxicity. For the first time, our study provides evidence that DPYD gene variants are associated with resistance to platin-based chemotherapy in NSCLC patients. Although further studies are needed to confirm these findings and explore the underlying mechanisms of this association, our results suggest that genetic testing of DPYD variants may be useful for identifying patients at a higher risk of platin-based chemotherapy resistance and might be helpful in guiding future personalized treatment strategies in NSCLC patients.

CDH6-activated αIIbß3 crosstalks with α2ß1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers.

Cadherin 6 (CDH6) is significantly overexpressed in advanced ovarian and renal cancers. However, the role of CDH6 in cancer metastasis is largely unclear. Here, we investigated the impact of CDH6 expression on integrin-mediated metastatic progression. CDH6 preferentially bound to αIIbß3 integrin, a platelet receptor scarcely expressed in cancer cells, and this interaction was mediated through the cadherin Arginine-glycine-aspartic acid (RGD) motif. Furthermore, CDH6 and CDH17 were found to interact with α2ß1 in αIIbß3low cells. Transient silencing of CDH6, ITGA2B, or ITGB3 genes caused a significant loss of proliferation, adhesion, invasion, and lung colonization through the downregulation of SRC, FAK, AKT, and ERK signaling. In ovarian and renal cancer cells, integrin αIIbß3 activation appears to be a prerequisite for proper α2ß1 activation. Interaction of αIIbß3 with CDH6, and subsequent αIIbß3 activation, promoted activation of α2ß1 and cell adhesion in ovarian and renal cancer cells. Additionally, monoclonal antibodies specific to the cadherin RGD motif and clinically approved αIIbß3 inhibitors could block pro-metastatic activity in ovarian and renal tumors. In summary, the interaction between CDH6 and αIIbß3 regulates α2ß1-mediated adhesion and invasion of ovarian and renal cancer metastatic cells and constitutes a therapeutic target of broad potential for treating metastatic progression.

Hypermethylation of Anti-oncogenic MicroRNA 7 is Increased in Emphysema Patients / La hipermetilación del microrna antioncogénico 7 se encuentra aumentada en pacientes con enfisema

INTRODUCTION: MicroRNA-7 (miR-7) has a suppressive role in lung cancer and alterations in its DNA methylation may contribute to tumorigenesis. As COPD patients with emphysema have a higher risk of lung cancer than other COPD phenotypes, we compared the miR-7 methylation status among smoker subjects and patients with various COPD phenotypes to identify its main determinants. METHODS: 30 smoker subjects without airflow limitation and 136 COPD patients without evidence of cancer were recruited in a prospective study. Clinical and functional characteristics were assessed and patients were classified into: frequent exacerbator, emphysema, chronic bronchitis and asthma COPD overlap (ACO). DNA collected from buccal epithelial samples was isolated and bisulfite modified. miR-7 methylation status was evaluated by quantitative methylation-specific polymerase chain reaction (qMSP). RESULTS: miR-7 Methylated levels were higher in COPD patients than in smokers without airflow limitation (23.7 ± 12.4 vs. 18.5 ± 8.8 %, p = 0.018). Among COPD patients, those with emphysema had higher values of methylated miR-7 (27.1 ± 10.2 %) than those with exacerbator (19.4 ± 9.9 %, p = 0.004), chronic bronchitis (17.3 ± 9.0 %, p = 0.002) or ACO phenotypes (16.0 ± 7.2 %, p = 0.010). After adjusting for clinical parameters, differences between emphysematous patients and those with other phenotypes were retained. In COPD patients, advanced age, mild-moderate airflow limitation, reduced diffusing capacity and increased functional residual capacity were identified as independent predictors of methylated miR-7 levels. CONCLUSION: The increase of miR-7 methylation levels experienced by COPD patients occurs mainly at the expense of the emphysema phenotype, which might contribute to explain the higher incidence of lung cancer in these patients

INTRODUCCIÓN: El microRNA-7 (miR-7) tiene un papel supresor en el cáncer de pulmón, y las alteraciones en la metilación de su DNA podrían contribuir a la tumorogénesis. Como los pacientes con EPOC y enfisema presentan un mayor riesgo de sufrir cáncer de pulmón frente a otros fenotipos de EPOC, comparamos la metilación de miR-7 entre los pacientes fumadores y los pacientes con varios fenotipos de EPOC para identificar sus factores determinantes principales. MÉTODOS: Se reclutaron para un estudio prospectivo 30 sujetos fumadores sin restricciones en el flujo aéreo y 136 pacientes con EPOC sin evidencia de cáncer. Se valoraron las características clínicas y funcionales y se clasificaron a los pacientes en: exacerbaciones frecuentes, enfisema, bronquitis crónica y solapamiento de asma y EPOC (ACO, por sus siglas en inglés). Se recogió ADN a partir de muestras de epitelio bucal, se aisló y se modificó con bisulfito. El estado de metilación del miR-7 se evaluó mediante la reacción cuantitativa en cadena de la polimerasa específica de la metilación (qMPS por sus siglas en inglés). RESULTADOS: Los niveles de metilación del miR-7 fueron más altos en los pacientes con EPOC que en los fumadores sin restricciones en el flujo aéreo (23,7 ± 12,4 frente a 18,5 ± 8,8%, p = 0,018). Entre los pacientes con EPOC, aquellos con enfisema presentaban valores más altos de miR-7 metilado (27,1 ± 10,2%) que aquellos con exacerbaciones (19,4 ± 9,9%, p = 0,004), bronquitis crónica (17,3 ± 9,0%, p = 0,002) o los fenotipos ACO (16,0 ± 7,2%, p = 0,010). Tras ajustar los resultados a los parámetros clínicos, las diferencias entre los pacientes enfisematosos y aquellos con otros fenotipos permanecieron. En los pacientes con EPOC, se identificaron como predictores independientes de los niveles de metilación del miR-7 a: la edad avanzada, limitación al flujo aéreo leve-moderada, capacidad de difusión reducida y capacidad residual funcional aumentada. CONCLUSIÓN: El aumento de los niveles de metilación del miR-7 que experimentan los pacientes con EPOC ocurre principalmente a expensas del fenotipo con enfisema, lo que podría contribuir a explicar la mayor incidencia de cáncer de pulmón en estos pacientes

Hypermethylation of Anti-oncogenic MicroRNA 7 is Increased in Emphysema Patients.

INTRODUCTION: MicroRNA-7 (miR-7) has a suppressive role in lung cancer and alterations in its DNA methylation may contribute to tumorigenesis. As COPD patients with emphysema have a higher risk of lung cancer than other COPD phenotypes, we compared the miR-7 methylation status among smoker subjects and patients with various COPD phenotypes to identify its main determinants. METHODS: 30 smoker subjects without airflow limitation and 136 COPD patients without evidence of cancer were recruited in a prospective study. Clinical and functional characteristics were assessed and patients were classified into: frequent exacerbator, emphysema, chronic bronchitis and asthma COPD overlap (ACO). DNA collected from buccal epithelial samples was isolated and bisulfite modified. miR-7 methylation status was evaluated by quantitative methylation-specific polymerase chain reaction (qMSP). RESULTS: miR-7 Methylated levels were higher in COPD patients than in smokers without airflow limitation (23.7±12.4 vs. 18.5±8.8%, p=0.018). Among COPD patients, those with emphysema had higher values of methylated miR-7 (27.1±10.2%) than those with exacerbator (19.4±9.9%, p=0.004), chronic bronchitis (17.3±9.0%, p=0.002) or ACO phenotypes (16.0±7.2%, p=0.010). After adjusting for clinical parameters, differences between emphysematous patients and those with other phenotypes were retained. In COPD patients, advanced age, mild-moderate airflow limitation, reduced diffusing capacity and increased functional residual capacity were identified as independent predictors of methylated miR-7 levels. CONCLUSION: The increase of miR-7 methylation levels experienced by COPD patients occurs mainly at the expense of the emphysema phenotype, which might contribute to explain the higher incidence of lung cancer in these patients.

Saa3 is a key mediator of the protumorigenic properties of cancer-associated fibroblasts in pancreatic tumors.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of abundant desmoplastic stroma primarily composed of cancer-associated fibroblasts (CAFs). It is generally accepted that CAFs stimulate tumor progression and might be implicated in drug resistance and immunosuppression. Here, we have compared the transcriptional profile of PDGFRα+ CAFs isolated from genetically engineered mouse PDAC tumors with that of normal pancreatic fibroblasts to identify genes potentially implicated in their protumorigenic properties. We report that the most differentially expressed gene, Saa3, a member of the serum amyloid A (SAA) apolipoprotein family, is a key mediator of the protumorigenic activity of PDGFRα+ CAFs. Whereas Saa3-competent CAFs stimulate the growth of tumor cells in an orthotopic model, Saa3-null CAFs inhibit tumor growth. Saa3 also plays a role in the cross talk between CAFs and tumor cells. Ablation of Saa3 in pancreatic tumor cells makes them insensitive to the inhibitory effect of Saa3-null CAFs. As a consequence, germline ablation of Saa3 does not prevent PDAC development in mice. The protumorigenic activity of Saa3 in CAFs is mediated by Mpp6, a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases (MAGUK). Finally, we interrogated whether these observations could be translated to a human scenario. Indeed, SAA1, the ortholog of murine Saa3, is overexpressed in human CAFs. Moreover, high levels of SAA1 in the stromal component correlate with worse survival. These findings support the concept that selective inhibition of SAA1 in CAFs may provide potential therapeutic benefit to PDAC patients.