Jordi Canals

Background: When genes responsible for normal embryonic development are abnormally expressed in adults, it can lead to tumor development. This can suggest that the same mechanism that controls embryonic differentiation can also control tumor differentiation. We hypothesize that the malignant phenotype of lung cancer cells could acquire benign characteristics when in contact with an embryonic lung microenvironment. We cultured two lung cancer cell lines in embryonic lung mesenchyme-conditioned medium and evaluated morphological, functional and molecular changes. Methods: The human embryonic mesenchymal lung-conditioned medium (hEML-CM) was obtained by culturing lung cells from embryos in the pseudoglandular stage of development. The NSCLC cell lines A549 and H1299 we cultured in the hEML-CM and in a tumor-conditioned medium. Morphological changes were analyzed with optical and transmission electron microscopy. To evaluate the functional effect of conditioned medium in tumor cells, we analyzed cell proliferation, migration, colony formation capacity in 2D and 3D and in vivo tumor growth capacity. The expression of the pluripotency genes OSKM, the adenocarcinoma marker NKX2-1, the lung surfactant proteins SFTP, the myofibroblast marker MYH and DNMT3A/3B was analyzed with qRT-PCR and the presence of the myofibroblast markers vimentin and α-SMA with immunofluorescence. Transcriptomic analysis was performed using Affymetrix arrays. Results: The A549 and H1299 cells cultured in hEML-CM lost their epithelial morphology, acquired mesodermal characteristics, and decreased proliferation, migration, and colony formation capacity in 2D and 3D, as well as reduced its capacity to growth in vivo. The expression of OSKM, NKX2-1 and SFTP decreased, while that of DNMT3A/3B, vimentin, α-SMA and MYH increased. Distant matrix analysis based on transcriptomic profile showed that conditioned cells were closer to myoblast and human lung fibroblast than to normal epithelial immortalized lung cells. A total of 1631 for A549 and 866 for H1299 differentially expressed genes between control and conditioned cells were identified. Conclusions: To the best of our knowledge, this is the first study to report that stimuli from the embryonic lung can modulate the malignant phenotype of lung cancer cells, control their growth capacity and activate their differentiation into myofibroblasts. These findings could lead to new strategies for lung cancer management.

LincRNA-p21 is a long non-coding RNA involved in the p53 pathway and angiogenesis regulation that acts as prognostic marker in several tumors. In the present study, we aimed to analyze the clinical value of lincRNA-p21 in 177 resected stage I–III colorectal cancer (CRC) patients. Tumor and normal paired tissue and plasma samples from tumor-draining mesenteric veins and paired peripheral veins were analyzed. LincRNA-p21 expression was determined by RTqPCR and correlated with disease-free (DFS) and overall survival (OS). LincRNA-p21 was downregulated in tumor versus normal tissue (p = 0.0012). CRC patients with high lincRNA-p21 expression had shorter DFS (p = 0.0372) and shorter OS (p = 0.0465). Of note, the major prognostic impact was observed in the subset of rectal cancer patients where patients with high lincRNA-p21 levels had worse DFS (p = 0.0226) and OS (p = 0.0457). Interestingly, rectal cancer patients with high lincRNA-p21 benefited from post-operative chemoradiotherapy, as indicated by a longer OS in the group of high lincRNA-p21 patients receiving post-operative chemoradiotherapy (p = 0.04). Finally, patients with high lincRNA-p21 levels in mesenteric vein (MV) had shorter OS (p = 0.0329). LincRNA-p21 is a marker of advanced disease and worse outcome in CRC. Moreover, rectal cancer patients with high lincRNA-p21 levels could benefit from post-operative chemoradiotherapy, and plasmatic-lincRNA-p21 is a promising liquid biopsy biomarker.

Hypoxia-induced upregulation of lincRNA-p21 in tumor tissue was previously shown by our group to be related to poor prognosis in resected non-small cell lung cancer (NSCLC) patients. In the present study, we have evaluated the presence of lincRNA-p21 in extracellular vesicles (EVs) from NSCLC patients and assessed its potential as a prognostic biomarker. High EV lincRNA-p21 levels in blood from the tumor-draining vein were associated with shorter time to relapse and shorter overall survival. Moreover, the multivariate analysis identified high lincRNA-p21 levels as an independent prognostic marker. In addition, lincRNA-p21 was overexpressed in H23 and HCC44 NSCLC cell lines and their derived EVs under hypoxic conditions. Functional assays using human umbilical vein endothelial cells (HUVECs) showed that tumor-derived EVs enriched in lincRNA-p21 affected endothelial cells by promoting tube formation and enhancing tumor cell adhesion to endothelial cells. Additionally, the analysis of selected EV microRNAs related to angiogenesis and metastasis showed that the microRNAs correlated with EV lincRNA-p21 levels in both patients and cell lines. Finally, EV co-culture with HUVEC cells increased the expression of microRNAs and genes related to endothelial cell activation. In conclusion, EV lincRNA-p21 acts as a novel prognosis marker in resected NSCLC patients, promoting angiogenesis and metastasis.

HOTTIP, a long non-coding RNA located in the HOXA cluster, plays a role in the patterning of tissues with mesodermal components, including the lung. Overexpression of HOXA genes, including HOTTIP, has been associated with a more aggressive phenotype in several cancers. However, the prognostic impact of HOTTIP has not yet been explored in non-small-cell lung cancer (NSCLC). We have correlated HOTTIP expression with time to relapse (TTR) and overall survival (OS) in early-stage NSCLC patients. Ninety-nine early-stage NSCLC patients who underwent surgical resection in our center from June 2007 to November 2013 were included in the study. Mean age was 66; 77.8% were males; 73.7% had stage I disease; and 55.5% had adenocarcinoma. A validation data set comprised stage I-II patients from The Cancer Genome Atlas (TCGA) Research Network. HOTTIP was expressed in all tumor samples and was overexpressed in squamous cell carcinoma (p = 0.007) and in smokers (p = 0.018). Patients with high levels of HOTTIP had shorter TTR (78.3 vs 58 months; p = 0.048) and shorter OS (81.2 vs 61 months; p = 0.023) than those with low levels. In the multivariate analysis, HOTTIP emerged as an independent prognostic marker for TTR (OR: 2.05, 95%CI: 1–4.2; p = 0.05), and for OS (OR: 2.31, 95%CI: 1.04–5.1; p = 0.04). HOTTIP was validated as a prognostic marker for OS in the TCGA adenocarcinoma cohort (p = 0.025). Moreover, we identified a 1203-mRNA and a 61-miRNA signature that correlated with HOTTIP expression. The lncRNA HOTTIP can be considered a prognostic biomarker in early-stage NSCLC.

Since tumor-draining pulmonary vein blood (PV) is enriched in tumor-secreted products, we hypothesized that it would also be enriched in tumor-derived exosomes, which would be important in the metastasis process. We characterized exosomes from PV of 61 resected non-small cell lung cancer (NSCLC) patients to evaluate its potential as relapse biomarkers. Exosomes were characterized using transmission electron microscopy, western blot and nanoparticle tracking analysis and we examined time to relapse (TTR) and overall survival (OS). Differences between PV and peripheral vein were found. PV was enriched in smaller exosomes than the paired peripheral vein (p = 0.01). Moreover, PV exosome size mode was able to identify relapsed patients (Area under the curve [AUC] = 0.781; 95%CI: 0.6641–0.8978), in whom exosome size was smaller (p < 0.001). The combination of PV exosome size and N (lymph node involvement) showed an AUC of 0.89 (95%CI: 0.80–0.97). Moreover, smaller PV exosome size was associated with shorter TTR (28.3 months vs. not reached, p < 0.001) and OS (43.9 months vs. not reached, p = 0.009). Multivariate analyses identified PV exosome size and stage as independent prognostic markers for TTR and OS. PV exosome size is a promising relapse biomarker after surgery that can add valuable information to clinical variables.