Date of Graduation
12-2015
Document Type
Dissertation (PhD)
Program Affiliation
Experimental Therapeutics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
George A. Calin
Committee Member
Anil K. Sood
Committee Member
Gabriel Lopez-Berestein
Committee Member
M. James You
Committee Member
Xiongbin Lu
Abstract
Long non-coding RNAs form the largest part of the mammalian non-coding transcriptome and control gene expression at various levels including chromatin modification, transcriptional and post-transcriptional processing. Although the underlying molecular mechanisms are not yet entirely understood, lncRNAs are implicated in initiation and progression of several cancers. CCAT2 is a lncRNA that spans the highly conserved 8q24 region associated with increased risk for various cancers. CCAT2 has been shown to play an important role in inducing chromosomal instability and supporting cell proliferation and cell cycle arrest. However, a causal role of CCAT2 in initiation of tumorigenesis and the importance of G/T SNP in CCAT2-induced phenotype still remains to be resolved. The purpose of this study was to elucidate the role of CCAT2 and its specific alleles (G/T) in regulation of cellular processes that drive tumorigenesis using a genetically engineered mouse model. We generated transgenic mice for each CCAT2 allele using random integration approach in C57Bl6/N background.
CCAT2(G/T) mice displayed spontaneous induction of widespread pancytopenias with splenomegaly and hepatomegaly. CCAT2(G/T) BM biopsies displayed severe myeloid and erythroid hyperplasia with enhanced proliferation and excessive apoptosis, along with extramedullary hematopoiesis in spleen and liver. Percentage of HSPCs was significantly reduced in BM of these mice, with increased presence of immature erythroid blasts and granulocyte-macrophage progenitors suggesting a block in differentiation. HSPCs of CCAT2(G/T) mice showed increased frequency of cytogenetic aberrations, including breaks and chromosomal fusions. However, these mice don’t develop AML, suggesting CCAT2 is critical in initiation of MDS. Microarray expression profiling of CCAT2(G/T) HSPCs revealed enrichment of pathways associated with epigenetic regulation, chromosomal instability and cell cycle regulation. We further identified significantly higher CCAT2 expression in the MDS patients as compared to healthy volunteers. Interestingly, patients with AML had significantly lower expression of CCAT2 as compared to patients with only MDS.
Based on these data, we conclude that CCAT2 plays an important role in regulation of normal hematopoiesis, and its deregulation can lead to MDS. CCAT2 lncRNA can be developed into a diagnostic and prognostic marker, as well as a novel intervention target for MDS therapy. CCAT2(G/T) mice can serve as a robust model for studying initiation of de novo MDS and as a pre-clinical model for evaluation of new therapies for low-risk MDS.
Keywords
Long non-coding RNAs, CCAT2, Myelodysplastic Syndrome, Transgenic mice