Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a dominantly inherited multisystemic disease caused by CTG tandem repeat expansions in the DMPK 3' untranslated region. These expanded repeats are transcribed and produce toxic CUG RNAs that sequester and inhibit activities of the MBNL family of developmental RNA processing factors. Although myotonic dystrophy is classified as a muscular dystrophy, the brain is also severely affected by an unusual cohort of symptoms, including hypersomnia, executive dysfunction, as well as early onsets of tau/MAPT pathology and cerebral atrophy. To address the molecular and cellular events that lead to these pathological outcomes, we recently generated a mouse Dmpk CTG expansion knock-in model and identified choroid plexus epithelial cells as particularly affected by the expression of toxic CUG expansion RNAs. To determine if toxic CUG RNAs perturb choroid plexus functions, alternative splicing analysis was performed on lateral and hindbrain choroid plexi from Dmpk CTG knock-in mice. Choroid plexus transcriptome-wide changes were evaluated in Mbnl2 knockout mice, a developmental-onset model of myotonic dystrophy brain dysfunction. To determine if transcriptome changes also occurred in the human disease, we obtained post-mortem choroid plexus for RNA-seq from neurologically unaffected (two females, three males; ages 50-70 years) and myotonic dystrophy type 1 (one female, three males; ages 50-70 years) donors. To test that choroid plexus transcriptome alterations resulted in altered CSF composition, we obtained CSF via lumbar puncture from patients with myotonic dystrophy type 1 (five females, five males; ages 35-55 years) and non-myotonic dystrophy patients (three females, four males; ages 26-51 years), and western blot and osmolarity analyses were used to test CSF alterations predicted by choroid plexus transcriptome analysis. We determined that CUG RNA induced toxicity was more robust in the lateral choroid plexus of Dmpk CTG knock-in mice due to comparatively higher Dmpk and lower Mbnl RNA levels. Impaired transitions to adult splicing patterns during choroid plexus development were identified in Mbnl2 knockout mice, including mis-splicing previously found in Dmpk CTG knock-in mice. Whole transcriptome analysis of myotonic dystrophy type 1 choroid plexus revealed disease-associated RNA expression and mis-splicing events. Based on these RNA changes, predicted alterations in ion homeostasis, secretory output and CSF composition were confirmed by analysis of myotonic dystrophy type 1 CSF. Our results implicate choroid plexus spliceopathy and concomitant alterations in CSF homeostasis as an unappreciated contributor to myotonic dystrophy type 1 CNS pathogenesis.

Suppression of SIN3A by miR-183 Promotes Breast Cancer Metastasis.

Recent work has established that SWI-independent-3 (SIN3) chromatin modification complexes play key roles in cancer progression. We previously demonstrated that knockdown of SIN3A expression promotes human breast cancer cell invasion and metastasis; however, the levels of SIN3A in patient breast carcinoma are not known. We therefore examined SIN3A mRNA and protein in patient tissues and determined that SIN3A expression is lower in breast carcinoma relative to normal breast. Given the 3'-untranslated region (UTR) of SIN3A has several conserved binding sites for oncogenic miRNA, we hypothesized that SIN3A is targeted by miRNA and found that ectopic miR-183 results in decreased SIN3A in breast carcinoma cell lines. Functionally, we demonstrate that miR-183 promotes breast cancer cell migration and invasion in a SIN3A-dependent manner and ectopic miR-183 promotes metastasis in vivo. Patients with breast cancer with high levels of miR-183 and low levels of SIN3A have the shortest overall survival. Given the critical link between metastasis and survival in patients with breast cancer, it is of utmost importance to identify clinically relevant genes involved in metastasis. Here, we report for the first time the aberrant expression of the putative metastasis suppressing gene SIN3A in human breast cancers and propose a mechanism of SIN3A suppression by miR-183. IMPLICATIONS: SIN3A expression is decreased in metastatic breast cancer in part due to miR-183.

Collagen modifying enzyme P4HA1 is overexpressed and plays a role in lung adenocarcinoma.

Lung cancer is the leading cause of cancer-related deaths globally and is histologically defined as either small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC), with the latter accounting for 80% of all lung cancers. The 5-year overall survival rate for lung cancer patients is low as it is often discovered at advanced stages when potential cure by surgical resection is no longer an option. To identify a biomarker and target for lung cancer, we performed analysis of multiple datasets of lung cancer gene expression data. Our analyses indicated that the collagen-modifying enzyme Prolyl 4-Hydroxylase Subunit Alpha 1 (P4HA1) is overexpressed in NSCLC. Furthermore, our investigation found that overexpression of enzymes involved in this pathway predicts poor outcome for patients with lung adenocarcinoma. Our functional studies using knockdown strategies in lung cancer cell lines in vitro indicated that P4HA1 is critical for lung cancer growth, migration, and invasion. Additionally, diethyl pythiDC (PythiDC), a small molecule inhibitor, decreased the malignant phenotypes of lung cancer cells. Moreover, we found that miR-124 regulates and targets P4HA1 in lung cancer cells. Thus, our study suggests that collagen-modifying enzymes play an important role in lung cancer aggressiveness. Furthermore, our studies showed that P4HA1 is required for lung cancer cell growth and invasion, suggesting its potential as a valid therapeutic target in lung adenocarcinoma.

miR-31 Displays Subtype Specificity in Lung Cancer.

miRNA rarely possess pan-oncogenic or tumor-suppressive properties. Most miRNAs function under tissue-specific contexts, acting as either tumor suppressors in one tissue, promoting oncogenesis in another, or having no apparent role in the regulation of processes associated with the hallmarks of cancer. What has been less clear is the role of miRNAs within cell types of the same tissue and the ability within each cell type to contribute to oncogenesis. In this study, we characterize the role of one such tissue-specific miRNA, miR-31, recently identified as the most oncogenic miRNA in lung adenocarcinoma, across the histologic spectrum of human lung cancer. Compared with normal lung tissue, miR-31 was overexpressed in patient lung adenocarcinoma, squamous cell carcinoma, and large-cell neuroendocrine carcinoma, but not small-cell carcinoma or carcinoids. miR-31 promoted tumor growth in mice of xenografted human adenocarcinoma and squamous cell carcinoma cell lines, but not in large- or small-cell carcinoma lines. While miR-31 did not promote primary tumor growth of large- and small-cell carcinoma, it did promote spontaneous metastasis. Mechanistically, miR-31 altered distinct cellular signaling programs within each histologic subtype, resulting in distinct phenotypic differences. This is the first report distinguishing diverse functional roles for this miRNA across the spectrum of lung cancers and suggests that miR-31 has broad clinical value in human lung malignancy. SIGNIFICANCE: These findings demonstrate the oncogenic properties of miR-31 in specific subtypes of lung cancer and highlight it as a potential therapeutic target in these subtypes. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/1942/F1.large.jpg.

Perfusion and Inflation of the Mouse Lung for Tumor Histology.

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform necropsies rapidly and efficiently from studies without sacrificing the quality of the tissues procured. The goal of this protocol is to present a method to rapidly perfuse, inflate, and fix mouse lungs for downstream histological analysis. This method does not standardize lung inflation; thus, it does not require any special procedures or equipment and instead simply instills fixative directly through the trachea following perfusion through the heart. This allows for sufficient estimation of tumor size, histology, and scoring. This also allows for the collection of frozen tissue prior to lung tissue fixation. This method is limited in that it does not allow for later morphometric quantification of the lung; however, it is more than sufficient for lung tumor analysis from genetically engineered mouse models (GEMMs), syngeneic models, as well as xenograft tumor and metastasis studies.

Tobacco SABP2-interacting protein SIP428 is a SIR2 type deacetylase.

Salicylic acid is widely studied for its role in biotic stress signaling in plants. Several SA-binding proteins, including SABP2 (salicylic acid-binding protein 2) has been identified and characterized for their role in plant disease resistance. SABP2 is a 29 kDA tobacco protein that binds to salicylic acid with high affinity. It is a methylesterase enzyme that catalyzes the conversion of methyl salicylate into salicylic acid required for inducing a robust systemic acquired resistance (SAR) in plants. Methyl salicylic acid is one of the several mobile SAR signals identified in plants. SABP2-interacting protein 428 (SIP428) was identified in a yeast two-hybrid screen using tobacco SABP2 as a bait. In silico analysis shows that SIP428 possesses the SIR2 (silent information regulatory 2)-like conserved motifs. SIR2 enzymes are orthologs of sirtuin proteins that catalyze the NAD+-dependent deacetylation of Nε lysine-acetylated proteins. The recombinant SIP428 expressed in E. coli exhibits SIR2-like deacetylase activity. SIP428 shows homology to Arabidopsis AtSRT2 (67% identity), which is implicated in SA-mediated basal defenses. Immunoblot analysis using anti-acetylated lysine antibodies showed that the recombinant SIP428 is lysine acetylated. The expression of SIP428 transcripts was moderately downregulated upon infection by TMV. In the presence of SIP428, the esterase activity of SABP2 increased modestly. The interaction of SIP428 with SABP2, it's regulation upon pathogen infection, and similarity with AtSRT2 suggests that SIP428 is likely to play a role in stress signaling in plants.