Altered ovarian cancer metabolism increases neuronal n-acetylaspartate to promote tumor growth

149 Pages·2016·4.98 MB·English

by  Behrouz Zand

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The University of Texas MD Anderson Cancer
The University of Texas MD Anderson Cancer
Center UTHealth Graduate School of
Center UTHealth Graduate School of
Biomedical Sciences Dissertations and Theses
Biomedical Sciences
(Open Access)
8-2013
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Behrouz Zand
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Zand, Behrouz, "Altered ovarian cancer metabolism increases neuronal n-acetylaspartate to promote
tumor growth" (2013). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of
Biomedical Sciences Dissertations and Theses (Open Access). 378.
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Altered ovarian cancer metabolism increases neuronal n-acetylaspartate to
promote tumor growth
Behrouz Zand, M.D.

APPROVED:

Anil K. Sood, M.D.

Menashe Bar Eli, M.D.

Gary Gallick, Ph.D.

Eric Wagner, Ph.D.

Peiying Ying, Ph.D.

APPROVED:

Dean, the University of Texas- Health Science Center at Houston
Graduate School of Biomedical Science
Altered ovarian cancer metabolism increases the neuronal n-acetylaspartate
to promote tumor growth

Thesis
Presented to the Faculty of
The University of Texas
Health Science Center at Houston
And
The University of Texas
M.D. Anderson Cancer Center
Graduate School of Biomedical Sciences
In Partial Fulfillment
Of the Requirements
For the Degree of MASTER OF SCIENCE
By
Behrouz Zand, M.D.
August 2013
© Copyright 2013
All Rights Reserved

Dedication
To my parents, who have always taught me that higher education is one of the most
important virtues to strive for in one’s life. To my wife Sara, who has been nothing short of
100% supportive of my work and endeavors rain or shine. To my son Aria, who has
changed my life more than I would have ever imagined before.

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Altered ovarian cancer metabolism increases the neuronal n-acetylaspartate to
promote tumor growth
Publication No._____
Behrouz Zand, M.D.
Supervisory Professor: Anil K. Sood, M.D.
Background: Altered metabolism is a well-established trait in many cancers, and is
an emerging hallmark of cancer. Recent resurgence of cancer metabolism studies
has identified dysregulated metabolic pathways that produce novel oncometabolites
in various cancers. However, large scale studies of dysregualted high grade serous
epithelial ovarian cancers (HGSOC) are unknown.
Materials and Methods: Following IRB approval, metabolic profiling of 101
HGSOC patients and 15 normal ovaries were obtained using GC/LC mass
spectrometry from 2 U.S. academic centers to identify highly up-regulated
metabolites. Samples from a cohort of 135 and 208 patients from a single
institution were evaluated for gene expression and protein expression of NAT8L,
respectively. Gene expression of NAT8L and clinical outcomes were further
investigated from publicly available databases from the cancer genomics atlas
(TCGA) using www.cbioportal.org, and two previously published melanoma gene
expression profiles. Reverse Phase Protein Array (RPPA) and gene expression
array were evaluated in HeyA8 ovarian cancer cell lines to investigate the protein
and gene expression changes associated with NAT8L siRNA. In vitro and in vivo
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experiments of NAT8L siRNA were investigated to evaluate its effects on cancer
proliferation, apoptosis, cell cycle, and invasion/migration.
Results: A total of 313 metabolites were identified between these two groups, of
which 172 were significantly altered (p<0.05) between HGSOC and normal ovary
tissues. NAA was one of the most significant alterations in HGSOC compared to
the normal ovary with a greater than 28 fold elevation in ovarian cancer compared
to the normal ovary (p=2.30E-11). NAA levels in HGSOC were strongly correlated
with its biosynthetic enzyme NAT8L gene expression levels (r=0.52, p<0.0001), and
not with its degradation enzyme ASPA (r= -0.11, 0=0.30). Patients with higher
levels of NAA had worse overall survival (1295 days) compared to patients with low
NAA levels (not reached) (p=0.038). Two separate HGSOC gene expression
cohorts revealed that high expression of NAT8L is associated with worse median
overall survival in HGSOC (35 months, 40 months) compared to low NAT8L gene
expression (45 months, 52 months) (p=0.03, p=0.005). High NAT8L protein
expression was associated with poor overall survival in ovarian cancer with 3.86
years overall survival compared to 9.09 years with low NAT8L expression
(p<0.001). Furthermore, high NAT8L gene expression was found to have
significantly worse overall survival in invasive breast, lung squamous, colon, uterine,
melanoma and kidney renal cell cancers.
HeyA8 and A2780 cell lines showed that NAT8L siRNA significantly
increased total apoptosis compared to control (NT) siRNA by 38.53% (p<0.001) and
37.85% (p<0.001), respectively. HeyA8 cells treated with paclitaxel and NAT8L
siRNA had a 29.83% increase in apoptosis compared to paclitaxel and NT siRNA
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treated cells (p<0.001). HEYA8 and A2780 cells treated with NAT8L siRNA had a
significantly decreased cell proliferation by 23.65% (p<0.001) and 19.13%
(p<0.001), respectively. HEYA8 cells treated with NAT8L siRNA had an 8.4%
increase in the number of cells with G1 phase (p<0.001), and an 11.65% decrease
in the number cells in the S phase (p<0.001). Knockdown of NAT8L in HEYA8 cells
significantly decreased migration and invasion by 91% and 92%, respectively
(p<0.001). In HEYA8 orthotopic ovarian cancer mouse models, DOPC NAT8L
siRNA had significantly decreased tumor burden by 69.17% (p<0.01), respectively.
Furthermore, NAT8L siRNA + paclitaxel had significantly less tumor burden
compared to NT siRNA (p<0.001), paclitaxel + NT siRNA (p=0.004), and NAT8L
siRNA alone (p=0.032). We observed similar effects in A2780, SKOV3 orthotopic
ovarian cancer mouse models and orthotopic A375-SM melanoma mouse model.
Genomic analysis of HEYA8 cells transfected with NAT8L siRNA compared
to NT siRNA showed 1961 significantly different gene expression data (p<0.001).
Hierarchical cluster analysis of RPPA from NAT8L siRNA and NT siRNA had 171
significantly different protein expression data (p<0.05). Computational network
analysis (NetWalker) showed significant decreases in a large number of genes
involved in mitosis and the M phase of the cell cycle, regulation of catabolic
processes, and regulation of cell death being altered.
Conclusion: HGSOC metabolic profiling revealed highly altered metabolism
compared to the normal ovary. NAA is one of the most up-regulated metabolites in
HGSOC. High levels of NAA are associated with worse overall survival in HGSOC.
Furthermore, high expression of its biosynthetic gene (NAT8L) is associated with
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worse overall survival in HGSOC, invasive breast, lung squamous, colon, uterine,
melanoma and renal cell cancers. Inhibiting NAA production decreases tumor
growth, and tilts the cancer cell to a more catabolic steady state. Therefore, our
data indicate that targeting cancer’s NAA production maybe an effective therapeutic
approach.

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Table of Contents
Approvals…………………………………………………………………………………….i
Title…………………………………………………………………………………………...ii
Dedication…………………………………………………………………………………..iii
Abstract………………………………………………………………………………..…....iv
Table of Contents…………………………………………………………………………viii
List of Figures………………………………………………………………………………ix
List of Tables…………………………………………………………………………….…xii
Background and Introduction……………………………………………………………...1
Hypotheses and Specific Aims…………………………………………………………..27
Methods…………………………………………………………………………………….28
Results……………………………………………………………………………………...43
Discussion………………………………………………………………………………….95
Bibliography…………………………………………………………………………..…..104
Vita………………………………………………………………………………………...135

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