Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies

Characterization of the lncRNA Transcriptome in Multiple Myeloma

Multiple myeloma (MM) is an hematological neoplasm characterized by uncontrolled clonal proliferation of plasma cells in the bone marrow. MM is an incurable and very heterogeneous disease, whose clinical variability makes its management challenging, highlighting the need for biological features to improve the therapy and survival of MM patients. There are many studies about genetic and epigenetic alterations in MM; however, the magnitude of the heterogeneity of this neoplasm is still unknown. New approaches about the deregulation of specific long non-coding RNAs (lncRNAs) have been shown in MM; nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we have described the complete lncRNA transcriptome of MM patients in the context of B-cell differentiation. Thanks to our work, we identified 40,511 novel lncRNAs expressed in MM samples. We added to our group of novel lncRNAs all the coding genes and lncRNAs annotated before, detecting that 82% of the MM transcriptome corresponded to lncRNAs; and remarkably, 56% corresponded to the novel lncRNAs detected in MM patients. Furthermore, we identified that lncRNAs were more heterogeneously expressed than coding-genes. After a comparison between MM samples and their healthy counterpart, bone marrow plasma cells (BMPCs) from healthy donors, we identified a group of 10,351 overexpressed and 9,535 downregulated lncRNAs in MM patients. Transcriptional dynamics study of those deregulated lncRNAs in the context of normal B-cell differentiation revealed a group of 989 lncRNAs with specific expression in MM samples, among which 89 showed de novo epigenomic activation. From those 89 lncRNAs with specific expression and de novo gain active chromatin marks in MM, we selected SMILO for functional assays. Knockdown studies on SMILO (Specific Myeloma Intergenic LOng non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. These results showed the relevance of SMILO in the biology of MM cells, and that could be a potential therapeutic target. We also analyzed the expression of lncRNAs in the context of the clinical of MM patients, looking for a better stratification and survival of patients. We studied the expression of the 89 lncRNAs with specific expression and de novo gain active chromatin marks in MM, together with high risk genetic and clinical alterations. Results revealed that lncRNAs together with different genetic and clinical factors could better stratify MM patients into different risk groups for progression-free survival and overall survival. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease

Characterization of the lncRNA Transcriptome in Multiple Myeloma

Multiple myeloma (MM) is an hematological neoplasm characterized by uncontrolled clonal proliferation of plasma cells in the bone marrow. MM is an incurable and very heterogeneous disease, whose clinical variability makes its management challenging, highlighting the need for biological features to improve the therapy and survival of MM patients. There are many studies about genetic and epigenetic alterations in MM; however, the magnitude of the heterogeneity of this neoplasm is still unknown. New approaches about the deregulation of specific long non-coding RNAs (lncRNAs) have been shown in MM; nevertheless, the complete lncRNA transcriptome has not yet been elucidated. In this work, we have described the complete lncRNA transcriptome of MM patients in the context of B-cell differentiation. Thanks to our work, we identified 40,511 novel lncRNAs expressed in MM samples. We added to our group of novel lncRNAs all the coding genes and lncRNAs annotated before, detecting that 82% of the MM transcriptome corresponded to lncRNAs; and remarkably, 56% corresponded to the novel lncRNAs detected in MM patients. Furthermore, we identified that lncRNAs were more heterogeneously expressed than coding-genes. After a comparison between MM samples and their healthy counterpart, bone marrow plasma cells (BMPCs) from healthy donors, we identified a group of 10,351 overexpressed and 9,535 downregulated lncRNAs in MM patients. Transcriptional dynamics study of those deregulated lncRNAs in the context of normal B-cell differentiation revealed a group of 989 lncRNAs with specific expression in MM samples, among which 89 showed de novo epigenomic activation. From those 89 lncRNAs with specific expression and de novo gain active chromatin marks in MM, we selected SMILO for functional assays. Knockdown studies on SMILO (Specific Myeloma Intergenic LOng non-coding RNA), resulted in reduced proliferation and induction of apoptosis of MM cells, and activation of the interferon pathway. These results showed the relevance of SMILO in the biology of MM cells, and that could be a potential therapeutic target. We also analyzed the expression of lncRNAs in the context of the clinical of MM patients, looking for a better stratification and survival of patients. We studied the expression of the 89 lncRNAs with specific expression and de novo gain active chromatin marks in MM, together with high risk genetic and clinical alterations. Results revealed that lncRNAs together with different genetic and clinical factors could better stratify MM patients into different risk groups for progression-free survival and overall survival. In summary, our global analysis of the lncRNAs transcriptome reveals the presence of specific lncRNAs associated with the biological and clinical behavior of the disease

The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial–mesenchymal transition and invasive properties of prostate cancer

Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial-mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes

The oncoprotein SF2/ASF promotes non-small cell lung cancer survival by enhancing survivin expression

Abstract Purpose: SF2/ASF is a splicing factor recently described as an oncoprotein. In the present work, we examined the role of SF2/ASF in human non–small cell lung cancer (NSCLC) and analyzed the molecular mechanisms involved in SF2/ASF-related carcinogenesis. Experimental Design: SF2/ASF protein levels were analyzed in 81 NSCLC patients by immunohistochemistry. SF2/ASF downregulation cellular models were generated using small interfering RNAs, and the effects on proliferation and apoptosis were evaluated. Survivin and SF2/ASF expression in lung tumors was analyzed by Western blot and immunohistochemistry. Survival curves and log-rank test were used to identify the association between the expression of the proteins and time to progression. Results: Overexpression of SF2/ASF was found in most human primary NSCLC tumors. In vitro downregulation of SF2/ASF induced apoptosis in NSCLC cell lines. This effect was associated with a reduction in the expression of survivin, an antiapoptotic protein widely upregulated in cancer. In fact, SF2/ASF specifically bound survivin mRNA and enhanced its translation, via a mammalian target of rapamycin complex 1 (mTORC1) pathway-dependent mechanism, through the phosphorylation and inactivation of the translational repressor 4E-BP1. Moreover, SF2/ASF promoted the stability of survivin mRNA. A strong correlation was observed between the expression of SF2/ASF and survivin in tumor biopsies from NSCLC patients, supporting the concept that survivin expression levels are controlled by SF2/ASF. Furthermore, combined expression of these proteins was associated with prognosis. Conclusion: This study provides novel data on the mTORC1- and survivin-dependent mechanisms of SF2/ASF-related carcinogenic potential, and shows that SF2/ASF and survivin expression is involved in NSCLC progression

Development of a novel splice array platform and its application in the identification of alternative splice variants in lung cancer

Abstract Background Microarrays strategies, which allow for the characterization of thousands of alternative splice forms in a single test, can be applied to identify differential alternative splicing events. In this study, a novel splice array approach was developed, including the design of a high-density oligonucleotide array, a labeling procedure, and an algorithm to identify splice events. Results The array consisted of exon probes and thermodynamically balanced junction probes. Suboptimal probes were tagged and considered in the final analysis. An unbiased labeling protocol was developed using random primers. The algorithm used to distinguish changes in expression from changes in splicing was calibrated using internal non-spliced control sequences. The performance of this splice array was validated with artificial constructs for CDC6, VEGF, and PCBP4 isoforms. The platform was then applied to the analysis of differential splice forms in lung cancer samples compared to matched normal lung tissue. Overexpression of splice isoforms was identified for genes encoding CEACAM1, FHL-1, MLPH, and SUSD2. None of these splicing isoforms had been previously associated with lung cancer. Conclusions This methodology enables the detection of alternative splicing events in complex biological samples, providing a powerful tool to identify novel diagnostic and prognostic biomarkers for cancer and other pathologies

Nocturnal Hypoxemia and CT Determined Pulmonary Artery Enlargement in Smokers

Background: Pulmonary artery enlargement (PAE) detected using chest computed tomography (CT) is associated with poor outcomes in chronic obstructive pulmonary disease (COPD). It is unknown whether nocturnal hypoxemia occurring in smokers, with or without COPD, obstructive sleep apnoea (OSA) or their overlap, may be associated with PAE assessed by chest CT. Methods: We analysed data from two prospective cohort studies that enrolled 284 smokers in lung cancer screening programs and completing baseline home sleep studies and chest CT scans. Main pulmonary artery diameter (PAD) and the ratio of the PAD to that of the aorta (PA:Ao ratio) were measured. PAE was defined as a PAD ≥ 29 mm in men and ≥27 mm in women or as a PA:Ao ratio > 0.9. We evaluated the association of PAE with baseline characteristics using multivariate logistic models. Results: PAE prevalence was 27% as defined by PAD measurements and 11.6% by the PA:Ao ratio. A body mass index ≥ 30 kg/m2 (OR 2.01; 95%CI 1.06–3.78), lower % predicted of forced expiratory volume in one second (FEV1) (OR 1.03; 95%CI 1.02–1.05) and higher % of sleep time with O2 saturation < 90% (T90) (OR 1.02; 95%CI 1.00–1.03), were associated with PAE as determined by PAD. However, only T90 remained significantly associated with PAE as defined by the PA:Ao ratio (OR 1.02; 95%CI 1.01–1.03). In the subset group without OSA, only T90 remains associated with PAE, whether defined by PAD measurement (OR 1.02; 95%CI 1.01–1.03) or PA:Ao ratio (OR 1.04; 95%CI 1.01–1.07). Conclusions: In smokers with or without COPD, nocturnal hypoxemia was associated with PAE independently of OSA coexistence

Nocturnal Hypoxemia and CT Determined Pulmonary Artery Enlargement in Smokers

Background: Pulmonary artery enlargement (PAE) detected using chest computed tomography (CT) is associated with poor outcomes in chronic obstructive pulmonary disease (COPD). It is unknown whether nocturnal hypoxemia occurring in smokers, with or without COPD, obstructive sleep apnoea (OSA) or their overlap, may be associated with PAE assessed by chest CT. Methods: We analysed data from two prospective cohort studies that enrolled 284 smokers in lung cancer screening programs and completing baseline home sleep studies and chest CT scans. Main pulmonary artery diameter (PAD) and the ratio of the PAD to that of the aorta (PA:Ao ratio) were measured. PAE was defined as a PAD ≥ 29 mm in men and ≥27 mm in women or as a PA:Ao ratio &gt; 0.9. We evaluated the association of PAE with baseline characteristics using multivariate logistic models. Results: PAE prevalence was 27% as defined by PAD measurements and 11.6% by the PA:Ao ratio. A body mass index ≥ 30 kg/m2 (OR 2.01; 95%CI 1.06–3.78), lower % predicted of forced expiratory volume in one second (FEV1) (OR 1.03; 95%CI 1.02–1.05) and higher % of sleep time with O2 saturation &lt; 90% (T90) (OR 1.02; 95%CI 1.00–1.03), were associated with PAE as determined by PAD. However, only T90 remained significantly associated with PAE as defined by the PA:Ao ratio (OR 1.02; 95%CI 1.01–1.03). In the subset group without OSA, only T90 remains associated with PAE, whether defined by PAD measurement (OR 1.02; 95%CI 1.01–1.03) or PA:Ao ratio (OR 1.04; 95%CI 1.01–1.07). Conclusions: In smokers with or without COPD, nocturnal hypoxemia was associated with PAE independently of OSA coexistence