Sithara Raju Ponny

Background: Anaplastic thyroid carcinoma (ATC) accounts for only 3% of thyroid cancers, yet strikingly, it accounts for almost 40% of thyroid cancer deaths. Currently, no effective therapies exist. In an effort to identify ATC-specific therapeutic targets, we analyzed global gene expression data from multiple studies to identify ATC-specific dysregulated genes. Methods: The National Center for Biotechnology Information Gene Expression Omnibus database was searched for high-throughput gene expression microarray studies from human ATC tissue along with normal thyroid and/or papillary thyroid cancer (PTC) tissue. Gene expression levels in ATC were compared with normal thyroid or PTC using seven separate comparisons, and an ATC-specific gene set common in all seven comparisons was identified. We investigated these genes for their biological functions and pathways. Results: There were three studies meeting inclusion criteria, (including 32 ATC patients, 69 PTC, and 75 normal). There were 259 upregulated genes and 286 downregulated genes in ATC with at least two-fold change in all seven comparisons. Using a five-fold filter, 36 genes were upregulated in ATC, while 40 genes were downregulated. Of the 10 top globally upregulated genes in ATC, 4/10 ( MMP1 , ANLN , CEP55 , and TFPI2 ) are known to play a role in ATC progression; however, 6/10 genes ( TMEM158 , CXCL5 , E2F7 , DLGAP5 , MME , and ASPM ) had not been specifically implicated in ATC. Similarly, 3/10 ( SFTA3 , LMO3 , and C2orf40 ) of the most globally downregulated genes were novel in this context, while 7/10 genes ( SLC26A7 , TG , TSHR , DUOX2 , CDH1 , PDE8B , and FOXE1 ) have been previously identified in ATC. We experimentally validated a significant correlation for seven transcription factors ( KLF16 , SP3 , ETV6 , FOXC1 , SP1 , EGFR1 , and MAFK ) with the ATC-specific genes using microarray analysis of ATC cell lines. Ontology clustering of globally altered genes revealed that “mitotic cell cycle” is highly enriched in the globally upregulated gene set (44% of top upregulated genes, p -value <10 −30 ). Conclusions: By focusing on globally altered genes, we have identified a set of consistently altered biological processes and pathways in ATC. Our data are consistent with an important role for M-phase cell cycle genes in ATC, and may provide direction for future studies to identify novel therapeutic targets for this disease.

Background: Myxomatous valve degeneration (MVD) involves the progressive thickening and degeneration of the heart valves, leading to valve prolapse, regurgitant blood flow, and impaired cardiac function. Leukocytes composed primarily of macrophages have recently been detected in myxomatous valves, but the timing of the presence and the contributions of these cells in MVD progression are not known. Methods: We examined MVD progression, macrophages, and the valve microenvironment in the context of Marfan syndrome (MFS) using mitral valves from MFS mice ( Fbn1 C1039G/+ ), gene-edited MFS pigs ( FBN1 Glu433AsnfsX98/+ ), and patients with MFS. Additional histological and transcriptomic evaluation was performed by using nonsyndromic human and canine myxomatous valves, respectively. Macrophage ontogeny was determined using MFS mice transplanted with mTomato+ bone marrow or MFS mice harboring RFP (red fluorescent protein)–tagged C-C chemokine receptor type 2 (CCR2) monocytes. Mice deficient in recruited macrophages ( Fbn1 C1039G/+ ;Ccr2 RFP/RFP ) were generated to determine the requirements of recruited macrophages to MVD progression. Results: MFS mice recapitulated histopathological features of myxomatous valve disease by 2 months of age, including mitral valve thickening, increased leaflet cellularity, and extracellular matrix abnormalities characterized by proteoglycan accumulation and collagen fragmentation. Diseased mitral valves of MFS mice concurrently exhibited a marked increase of infiltrating (MHCII+, CCR2+) and resident macrophages (CD206+, CCR2–), along with increased chemokine activity and inflammatory extracellular matrix modification. Likewise, mitral valve specimens obtained from gene-edited MFS pigs and human patients with MFS exhibited increased monocytes and macrophages (CD14+, CD64+, CD68+, CD163+) detected by immunofluorescence. In addition, comparative transcriptomic evaluation of both genetic (MFS mice) and acquired forms of MVD (humans and dogs) unveiled a shared upregulated inflammatory response in diseased valves. Remarkably, the deficiency of monocytes was protective against MVD progression, resulting in a significant reduction of MHCII macrophages, minimal leaflet thickening, and preserved mitral valve integrity. Conclusions: All together, our results suggest sterile inflammation as a novel paradigm to disease progression, and we identify, for the first time, monocytes as a viable candidate for targeted therapy in MVD.

Aberrant alternative splicing of mRNAs results in dysregulated gene expression in multiple neurological disorders. Here, we show that hundreds of mRNAs are incorrectly expressed and spliced in white blood cells and brain tissues of individuals with fragile X syndrome (FXS). Surprisingly, the FMR1 (Fragile X Messenger Ribonucleoprotein 1) gene is transcribed in >70% of the FXS tissues. In all FMR1 -expressing FXS tissues, FMR1 RNA itself is mis-spliced in a CGG expansion–dependent manner to generate the little-known FMR1 -217 RNA isoform, which is comprised of FMR1 exon 1 and a pseudo-exon in intron 1. FMR1 -217 is also expressed in FXS premutation carrier–derived skin fibroblasts and brain tissues. We show that in cells aberrantly expressing mis-spliced FMR1 , antisense oligonucleotide (ASO) treatment reduces FMR1 -217, rescues full-length FMR1 RNA, and restores FMRP (Fragile X Messenger RibonucleoProtein) to normal levels. Notably, FMR1 gene reactivation in transcriptionally silent FXS cells using 5-aza-2′-deoxycytidine (5-AzadC), which prevents DNA methylation, increases FMR1 -217 RNA levels but not FMRP. ASO treatment of cells prior to 5-AzadC application rescues full-length FMR1 expression and restores FMRP. These findings indicate that misregulated RNA-processing events in blood could serve as potent biomarkers for FXS and that in those individuals expressing FMR1-217 , ASO treatment may offer a therapeutic approach to mitigate the disorder.

The HER2 + subtype of human breast cancer is associated with the malignant transformation of luminal ductal cells of the mammary epithelium. The sequence analysis of tumor DNA identifies loss of function mutations and deletions of the MAP2 K4 and MAP2 K7 genes that encode direct activators of the JUN NH 2 -terminal kinase (JNK). We report that in vitro studies of human mammary epithelial cells with CRISPR-induced mutations in the MAPK and MAP2K components of the JNK pathway caused no change in growth in 2D culture, but these mutations promoted epithelial cell proliferation in 3D culture. Analysis of gene expression signatures in 3D culture demonstrated similar changes caused by HER2 activation and JNK pathway loss. The mechanism of signal transduction cross-talk may be mediated, in part, by JNK-suppressed expression of integrin α6β4 that binds HER2 and amplifies HER2 signaling. These data suggest that HER2 activation and JNK pathway loss may synergize to promote breast cancer. To test this hypothesis, we performed in vivo studies using a mouse model of HER2 + breast cancer with Cre/loxP -mediated ablation of genes encoding JNK ( Mapk8 and Mapk9 ) and the MAP2K ( Map2 k4 and Map2 k7 ) that activate JNK in mammary epithelial cells. Kaplan–Meier analysis of tumor development demonstrated that JNK pathway deficiency promotes HER2 + -driven breast cancer. Collectively, these data identify JNK pathway genes as potential suppressors for HER2 + breast cancer.