Joan J. Castellano

LincRNA-p21 Impacts Prognosis in Resected Non–Small Cell Lung Cancer Patients through Angiogenesis Regulation

Introduction: Long intergenic noncoding RNA‐p21 (lincRNA‐p21) is a long noncoding RNA transcriptionally activated by tumor protein p53 (TP53) and hypoxia inducible factor 1 alpha subunit (HIF1A). It is involved in the regulation of TP53‐dependent apoptosis and the Warburg effect. We have investigated the role of lincRNA‐p21 in NSCLC. Methods: LincRNA‐p21 expression was assessed in tumor and normal tissue from 128 patients with NSCLC and correlated with time to relapse and cancer‐specific survival (CSS). H23, H1299, and HCC‐44 cell lines were cultured in hypoxic conditions after silencing of lincRNA‐p21. The TaqMan human angiogenesis array was used to explore angiogenesis‐related gene expression. Levels of the protein vascular endothelial growth factor A were measured by enzyme‐linked immunosorbent assay in the cell supernatants. Angiogenic capability was measured by human umbilical vein endothelial cell tube formation assay. Microvascular density in tumor samples was analyzed by immunohistochemistry. Results: LincRNA‐p21 was down‐regulated in tumor tissue, but no association was observed with TP53 mutational status. High lincRNA‐p21 levels were associated with poor CSS in all patients (p = 0.032). When patients were classified according to histological subtypes, the impact of lincRNA‐p21 was confined to patients with adenocarcinoma in both time to relapse (p = 0.006) and CSS (p < 0.001). To explain the poor outcome of patients with high lincRNA‐p21 expression, we studied the role of lincRNA‐p21 in angiogenesis in vitro and observed a global downregulation in the expression of angiogenesis‐related genes when lincRNA‐p21 was inhibited. Moreover, supernatants from lincRNA‐p21–inhibited cells were significantly less angiogenic and had lower levels of secreted vascular endothelial growth factor A than controls did. Finally, tumor samples with high lincRNA‐p21 levels had higher microvascular density. Conclusions: Our findings suggest that lincRNA‐p21 affects outcome in patients with NSCLC adenocarcinoma through the regulation of angiogenesis.

Exosomal microRNAs isolated from plasma of mesenteric veins linked to liver metastases in resected patients with colon cancer

Before reaching a peripheral vein (PV), miRNAs released by the tumor are diluted and dispersed throughout the body or even retained in a specific organ. We hypothesized that blood drawn from the tumor-draining vein could provide more homogeneous information than blood drawn from the PV as that blood would contain all the biomarkers released by the tumor before they reach a potential metastatic site. We have profiled 754 miRNAs in 15 colon cancer plasma samples from the tumor-draining vein, the mesenteric vein (MV), identifying 13 microRNAs associated with relapse. The prognostic impact of these miRNAs were validated in 50 MV and 50 paired PV plasma samples of stage I-III colon cancer patients. Four miRNAs, let-7g, miR-15b, miR-155 and miR-328, were found overexpressed in MV compared to PV, and patients with high levels of those miRNAs in MV plasma had shorter time to relapse. Interestingly, in patients developing liver metastases, the exosomal cargo of miR-328 was much greater in MV than in PV plasma indicating a possible role of miR-328 in the development of liver metastases. Our results indicate that in colon cancer, the primary tumor releases high concentrations of miRNAs through the MV, and some of them are contained in tumor derived exosomes.

The expression level of BAALC-associated microRNA miR-3151 is an independent prognostic factor in younger patients with cytogenetic intermediate-risk acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (⩾60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P=0.0025), shorter leukemia-free survival (P=0.026) and higher cumulative incidence of relapse (P=0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P=0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML.

PiwiRNA-651 as marker of treatment response and survival in classical Hodgkin lymphoma.

PiwiRNAs, small non-coding RNAs processed by Piwi proteins, are involved in maintaining genome stability in germline cells. Recently, piwiRNA expression has been identified in some tumors. We have examined the potential reactivation of the Piwi/piwiRNA pathway in classical Hodgkin lymphoma (cHL). We found that Piwi proteins and three selected piwiRNAs, including piR-651, were expressed in cHL patients and cell lines, indicating that the Piwi/piwiRNA pathway is active in cHL. Interestingly, low levels of piR-651 were associated with lack of complete response to first-line treatment, as well as shorter disease-free and overall survival in a cohort of 94 cHL patients. At diagnosis, piR-651 was underexpressed in cHL serum samples compared to healthy controls, while after complete remission, piR-651 levels increased to levels similar to healthy controls. This is the first evidence that piwiRNAs are active in tumor and serum samples and impact prognosis in cHL.

Proteomic Analysis of Liquid Biopsy from Tumor-Draining Vein Indicates that High Expression of Exosomal ECM1 Is Associated with Relapse in Stage I-III Colon Cancer

BACKGROUND: The analysis of exosomes in blood obtained from the tumor-draining mesenteric vein (MV) can identify tumor biomarkers before they reach target organs and form the premetastatic niche where circulating tumor cells can anchor. Our group has recently shown that microRNAs in plasma from the MV—but not the peripheral vein (PV)—have been related to liver metastases in colon cancer (CC) patients. Here we examine the exosomal protein cargo in plasma from the MV and paired PV in 31 CC patients. PATIENTS AND METHODS: The study included patients who were initially diagnosed with stage I-III CC and 10 healthy controls. Exosomes from the MV and PV of all patients and controls were isolated by ultracentrifugation and confirmed by cryogenic transmission electron microscopy. High-throughput proteomic analysis by mass spectrometry was used to identify expression levels of exosomal proteins. Findings were confirmed by Western blot. RESULTS: Exosomal ECM1 protein was more highly expressed in patients than in controls and was 13.55 times higher in MV from relapsed than relapse-free patients. High exosomal ECM1 expression was associated with liver metastases. Patients with high exosomal ECM1 expression in MV—but not PV—plasma had shorter time to relapse than those with low ECM1 expression (P = .04). CONCLUSION: High levels of exosomal ECM1 protein can identify CC patients with a higher risk of relapse. The analysis of exosomes isolated from the tumor-draining MV is a promising method for the identification of biomarkers before they reach the target organ.

miR-328 mediates a metabolic shift in colon cancer cells by targeting SLC2A1/GLUT1

PurposeIncreasing evidence shows that altered metabolism is a critical hallmark in colon cancer. There is a strong need to explore the molecular mechanisms underlying cancer metabolism. Whether the aberrant expression of microRNAs contributes to cancer metabolism is not fully understood. miR-328 is a putative potential target of SLC2A1, but the regulating mechanism between them remains unknown. We have examined whether miR-328 directly regulates SLC2A1/GLUT1 expression in colon cancer cells.MethodsWe performed in silico bioinformatic analyses to identify miR-328-mediated molecular pathways and targets. We also performed luciferase assays and western blot analyses in LOVO and SW480 colon cancer cell lines. In addition, we assessed miR-328 expression in 47 paired tumor and normal tissue specimens from resected colon cancer patients.ResultsLuciferase reporter assays showed that miR-328 directly targeted SLC2A1 3′-untranslated region (UTR), with a significant decrease in luciferase activity in both LOVO and SW480 cell lines. These results were validated by western blot. miR-328 expression was significantly downregulated in tumor tissue compared with paired normal tissue.ConclusionsOur results show that miR-328 targets SLC2A1/GLUT1. We suggest that miR-328 may be involved in the orchestration of the Warburg effect in colon cancer cells. Furthermore, miR‐328 expression is reduced in colon cancer patients and thus inversely correlates with the classically reported upregulated SLC2A1/GLUT1 expression in tumors.

Prognostic Impact of miR-200 Family Members in Plasma and Exosomes from Tumor-Draining versus Peripheral Veins of Colon Cancer Patients

Objective: To evaluate the prognostic potential of expression levels of miR-200 family members (miR-200a, miR-200b, miR-200c, miR-429, miR-141) in plasma and exosomes from the tumor-draining vein (mesenteric vein [MV]) and peripheral vein (PV) of colon cancer (CC) patients. Methods: We analyzed the expression of miR-200 family members in matched samples of MV and PV plasma from 50 resected patients with CC and correlated our findings with overall survival (OS). We also examined the content of these microRNAs in MV and PV exosomes. Results: Expression levels were higher in MV than in PV (miR-200a, p < 0.001; miR-200b, p < 0.001; miR-429, p = 0.01; miR-200c, p = 0.05; miR-141, p = 0.05). Low levels of both miR-200c and miR-141 in MV plasma were associated with longer OS (p = 0.02). This association was maintained for the MV exosome cargo of miR-200c and miR-141 (p = 0.02). Conclusion: Our findings provide the first indication that expression levels of miR-200c and miR-141 in MV plasma can identify CC patients with poor prognosis. In addition, our results lend further support to the premise that tumor-draining veins constitute a better source of biomarkers than do PVs.

NKX2–1 expression as a prognostic marker in early-stage non-small-cell lung cancer

BackgroundNKX2–1, a key molecule in lung development, is highly expressed in non-small cell lung cancer (NSCLC), particularly in lung adenocarcinoma (ADK), where it is a diagnostic marker. Studies of the prognostic role of NKX2–1 in NSCLC have reported contradictory findings. Two microRNAs (miRNAs) have been associated with NKX2–1: miR-365, which targets NKX2–1; and miR-33a, which is downstream of NKX2–1. We have examined the effect of NKX2–1, miR-365 and miR-33a on survival in a cohort of early-stage NSCLC patients and in sub-groups of patients classified according to the mutational status of TP53, KRAS, and EGFR.MethodsmRNA and miRNA expression was determined using TaqMan assays in 110 early-stage NSCLC patients. TP53, KRAS, and EGFR mutations were assessed by Sanger sequencing.ResultsNKX2–1 expression was upregulated in never-smokers (P = 0.017), ADK (P < 0.0001) and patients with wild-type TP53 (P = 0.001). A negative correlation between NKX2–1 and miR-365 expression was found (ρ = −0.287; P = 0.003) but there was no correlation between NKX2–1 and miR-33a expression. Overall survival (OS) was longer in patients with high expression of NKX2–1 than in those with low expression (80.8 vs 61.2 months (P = 0.035), while a trend towards longer OS was observed in patients with low miR-365 levels (P = 0.07). The impact of NKX2–1 on OS and DFS was higher in patients with neither TP53 nor KRAS mutations. Higher expression of NKX2–1 was related to higher OS (77.6 vs 54 months; P = 0.017) and DFS (74.6 vs 57.7 months; P = 0.006) compared to low expression. The association between NKX2–1 and OS and DFS was strengthened when the analysis was limited to patients with stage I disease (P = 0.005 and P=0.003 respectively).ConclusionsNKX2–1 expression impacts prognosis in early-stage NSCLC patients, particularly in those with neither TP53 nor KRAS mutations.