Function of STAT3 and SMG1 in maintaining Glioblastoma stem cells.
Glioblastoma multiforme (GBM) is the most common and most aggressive tumor of the
central nervous system, with a mean survival of only 14 months post diagnosis. Due to
the highly lethal nature of glioblastoma, new therapies are urgently needed. It is
thought that the major reason for poor prognosis of GBM is that a small population of
GBM stem cells (GSC) selectively survives therapy ... read moreand leads to tumor re-growth.
Therefore, in this thesis I have identified new potential therapeutic targets for GBM
stem cells and investigated the underlying mechanisms by which these targets regulate
the growth of GBM stem cells. The transcription factor STAT3 is required for the
self-renewal of several stem cell types including GSC. Interestingly, STAT3 inhibition
leads to an irreversible decrease in proliferation and neurosphere formation, as well as
loss of stem cell markers. The work presented in this thesis reports a novel epigenetic
mechanism for inhibiting self-renewal of GSC. by STAT3. Here, we show that STAT3
inhibition upregulates histone H3K27me2/3 demethylase JMJD3 (KDM6B), which can reverse
polycomb complex mediated repression of neural differentiation genes. To identify the
set of STAT3 regulated differentiation-specific genes, genome wide ChIP-sequencing and
microarray analysis were performed to determine changes in histone H3K27 methylation as
well as gene expression following STAT3 inhibition. STAT3 inhibition leads to reduced
promoter histone H3K27 trimethylation of neural differentiation genes, such as MYT1,
FGF21 and GDF15. While MYT1 gene expression requires the presence of an additional
growth factor in addition to STAT3 inhibition, FGF21 and GDF15 genes are expressed upon
STAT3 inhibition alone. In addition to genetic and epigenetic alterations,
microenvironment factors like tumor hypoxia play an important role in GSC
tumorigenicity. GSC preferentially reside in hypoxic niches in the tumor, which is
believed to be a major factor in maintaining GSC survival and tumor progression
following therapy. There have been no systematic attempts to identify and target
molecular mechanisms responsible for GSC survival under hypoxia. Since kinases are
druggable targets that play an important role in maintaining GSC growth and survival, we
conducted a functional RNAi kinome-wide screen across three GSC lines under normoxia
(21% oxygen) as well as hypoxia (1% oxygen). We found that only 25% of the kinase hits
in the screen were common to the three GSC lines screened, demonstrating a high level of
heterogeneity between these lines. Some of the common targets were previously known to
be involved in gliomagenesis, while some are novel targets in GBM. There were hits
unique to both hypoxia as well as normoxia, thereby emphasizing differential GBM stem
cell sensitivity to kinase inhibition in different microenvironments. The SMG1
(suppressor with morphological effects on genitalia) kinase was identified and validated
as a gene that can preferentially inhibit the growth of selected GBM stem cell lines
under hypoxia (1% oxygen). Strikingly, knockdown of SMG1 sensitized all the GBM stem
cell lines tested to temozolomide (TMZ), the frontline chemotherapeutic used to treat
GBM patients. Importantly, we found significant reduction in intracranial tumor growth
and prolonged survival in mice when SMG1 is knocked down in combination with TMZ
treatment, as compared to TMZ alone. We have used the Cell Collective modeling software
platform to simulate the mechanism of action of the SMG1 kinase network in GSC by
integrating our data with results in the literature. Based on the prediction of our
model, we found that SMG1 inhibits GSC growth through its well-characterized role in the
classical mRNA surveillance mechanism, nonsense mediated mRNA decay (NMD) pathway. Thus,
the kinase targets and the mechanisms identified here are therapeutically important for
developing inhibitors for treating GBM.
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Cellular & Molecular Physiology.
Advisor: Brent Cochran.
Committee: Daniel Jay, Peter Juo, and Keith Ligon.
Keywords: Physiology, and Cellular biology.read less