Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Role of a novel transmembrane protein, MTTS1 in mitochondrial regulation and tumor suppression
(USC Thesis Other)
Role of a novel transmembrane protein, MTTS1 in mitochondrial regulation and tumor suppression
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
ROLE OF A NOVEL TRANSMEMBRANE PROTEIN, MTTS1 IN MITOCHONDRIAL
REGULATION AND TUMOR SUPPRESSION
by
Poornima Murali
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2012
Copyright 2012 Poornima Murali
ii
ACKNOWLEDGEMENTS
I would like to thank my mentor, Dr. Ebrahim Zandi, for his constant support and guidance in
research. I also thank Dr. Axel Schonthal and Dr. Joseph Landolph, for their valuable inputs and
suggestions, as members of the thesis committee.
I sincerely thank Dr.Murali Ganesan, Richa Aggarwal, Anita Ramanathan, Anketse Kassa and
Mario Pulido, for their support in various aspects of my project. In-vivo studies could not have
been possible without the help and guidance of Dr.Shili Xu and Youzhen Yan.
Last but not the least, I would like to owe my success to my parents, without whose motivation
and support, this achievement could not have been possible
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS……………………………………………………………………….ii
LIST OF FIGURES………………………………………………………………………………iv
LIST OF TABLES………………………………………………………………………………..v
ABSTRACT……………………………………………………………………………………...vi
CHAPTER ONE: INTRODUCTION ............................................................................................. 1
1.1 Components of ATP synthase ............................................................................................... 1
1.2 Warburg effect....................................................................................................................... 2
1.3 Mitochondrial respiration down-regulated in cancer ............................................................ 3
1.4 Mitochondrial DNA mutations in cancer .............................................................................. 4
1.5 Transmembrane proteins ....................................................................................................... 4
1.6 Functions of transmembrane proteins ................................................................................... 5
CHAPTER TWO: MATERIALS AND METHODS ..................................................................... 7
2.1 Cell culture Conditions.......................................................................................................... 7
2.2 Stable cell transfection .......................................................................................................... 7
2.3 Drug Selection ....................................................................................................................... 8
2.4 Mitochondrial abundance by qPCR ...................................................................................... 8
2.5 MTTS1 expression by RT-PCR ............................................................................................ 8
2.6 Mitochondrial respiration assay ............................................................................................ 9
2.7 In-vivo analysis in nude mice................................................................................................ 9
2.8 Immunohistochemistry using ATPase β ............................................................................. 10
CHAPTER THREE: RESULTS ................................................................................................... 11
3.1 Mitochondrial abundance by q PCR ................................................................................... 11
3.2 Stable cell transfection in HEK 293T cells ......................................................................... 14
3.3 Mitochondrial respiration decreases in shRNA-MTTS1cells ............................................. 15
3.4 In-vivo tumor studies .......................................................................................................... 18
3.5 Mitochondrial abundance in tumor tissues ......................................................................... 22
3.6 In-vivo tumor assay using PC3 cells ................................................................................... 23
CHAPTER FOUR: CONCLUSION AND DISCUSSIONS ........................................................ 24
REFERENCES………………………………………………………………………………......26
iv
LIST OF FIGURES
Figure 1: Rotary model of ATP synthase…………………………………………………………...2
Figure 2: Warburg effect showing alternate energy production by glycolysis……………………...3
Figure 3: q PCR and RT-PCR in head & neck cancer cell lines…………………………………...12
Figure 4: q PCR and RT-PCR in lung cell lines…………………………………………………...13
Figure 5: RT-PCR in HEK 293T cell lines………………………………………………………...14
Figure 6: Graph showing oxygen consumption in Complex I in HEK 293T cells………………...16
Figure 7: Graph showing oxygen consumption in Complex II in HEK 293T cells……………….17
Figure 8: Fluorescent Microscope images of HEK 293T cells injected in
immune-deficient mice for xenograft assay……………………………………………..19
Figure 9: Xenograft assay on immune-deficient mice……………………………………………..20
Figure 10: Graphical representation of tumor volume per day…………………………………….21
Figure 11: Tumors isolated from nude mice……………………………………………………….21
Figure 12: Immunohistochemistry with ATP synthase β viewed under microscope………….......25
Figure 13: In-vivo nude mice assay with PC3 cells………………………………………………..26
v
LIST OF TABLES
Table 1: List of primers used in q PCR and RT-PCR……………………………………………...9
Table 2: Respiratory Control Index (RCI) in Complex I and Complex II in
HEK 293T cells………………………………………………………………………....18
vi
ABSTRACT
A novel transmembrane protein was identified in Dr. Ebrahim Zandi’s lab as an IKK-β
interacting protein by mass spectrometry. Mass spectrometry and IP (Immunoprecipitation)
studies were performed to investigate functions of the newly identified protein. The study
showed the interaction of this protein with two subunits of F
1
components of mitochondrial ATP
synthase. The F
1
component of ATP synthase is the catalytic subunit that drives ATP synthesis.
Further analysis showed that this protein localizes to mitochondria and has a probable role in
regulating ATP synthesis by interacting with ATP synthase α and ATP synthase β. Research has
shown that mitochondrial dysfunction has a predominant role in cancer progression.
In this study, we characterized the role of this protein in regulating mitochondrial abundance and
its subsequent role in tumor regulation. Based on the finding of this study regarding the function
of the protein, we named it Mitochondrial Transmembrane Tumor Suppressor 1 (MTTS1).
MTTS1 levels of 4 out of 7 lung cancer cell lines correlated with their respective mitochondrial
abundance. A similar pattern was observed when 3 head and neck cancer cell lines were
screened. Stable cell transfection to overexpress and knockdown MTTS1 was performed in HEK
293T cells (human embryonic kidney cells). Levels of many mitochondrial genes, responsible for
cellular energy metabolism, were increased in HEK 293T cells overexpressing MTTS1. In-vivo
studies in mice using HEK 293T cells showed that there was a significant delay in tumor growth
in cells overexpressing MTTS1, suggesting that this protein can be a potential tumor suppressor.
1
CHAPTER ONE
INTRODUCTION
1.1 Components of ATP synthase
Mitochondria are cell organelles that participate in essential functions such as ATP production,
programmed cell death, and cell-specific functions like cholesterol metabolism, neurotransmitter
metabolism and detoxification of ammonia in urea cycle. Mitochondria are also responsible for
synthesis of hormones such as estrogen and testosterone, synthesis of steroids, regulation of
membrane potential, monitoring of cell differentiation, growth and development, and cell
signaling of neurons. Out of these, energy production by ATP is the most prominent function of
mitochondria carried out by the enzyme ATP synthase. ATP synthase is a large multi-protein
complex, located in the inner mitochondrial membrane that generates ATP from ADP and Pi.
The structure of ATP synthase comprises of two subunits namely F
1
and F
0
components. F
0
functions as a proton channel and F
1
functions as the catalytically active component comprising
of five subunits. The rotation of F
0
causes conformational changes in the subunits of F
1
, leading
to synthesis of ATP.
2
Fig 1: Rotary model of ATP synthase [1]
1.2 Warburg effect
The mitochondrion has its own genome, and it codes for 13 of the 87 proteins required for
oxidative phosphorylation as well as protein synthesis. A major cause of mitochondrial
abnormality is alterations in mtDNA (mitochondrial DNA). These can be germ line mutations, as
observed in Leber's hereditary optic neuropathy, diabetes mellitus, and Leigh's syndrome, or
somatic mutations, as observed in cancer [2]. The first relation between mitochondria and cancer
was established by Otto Warburg. He proposed that in all tumor cells, the glycolytic pathway is
stimulated, leading to high utilization of glucose. This is due to impaired mitochondria when
glycolysis compensates for impaired ATP production by mitochondria. This difference in energy
metabolism between a tumor and normal cell was the basis of Warburg’s theory.
3
Fig 2: Warburg effect showing alternate ATP production by glycolysis
1.3 Mitochondrial respiration down-regulated in cancer
Based on Warburg’s hypothesis, researchers elucidated how mitochondrial respiration was
regulated in cancer. Mitochondrial respiration was down-regulated and found to shift to
glycolysis in tumors with p53 mutations [3]. Several alterations in energy supplying pathways
were also observed in tumors. Phosphoinositide 3-kinase (PI3K) is involved in transmitting cell
survival signals from cell surface receptors. Activation of PI3K can stimulate glycolysis, cell
survival, and enhanced cell size in cancer cells. When mitochondria do not provide sufficient
ATP in tumors, HIF 1α induces upregulation of the glycolytic pathway under hypoxic conditions
[4]. Sometimes, Krebs cycle intermediates can accumulate during low ATP production and cause
signaling and upregulation of HIF1α [5]. OMM (Outer Mitochondrial Membrane)
permeabilization occurs during apoptosis in normal cells [6]. In cancer cells, a number of factors
associated with mitochondria, prevent permeabilization of OMM. Hexokinase is one such
4
enzyme that plays an important role in this prevention. Multiple researches prove that glycolytic
shift prevents tumor mitochondria from permeabilization [7].
1.4 Mitochondrial DNA mutations in cancer
Mutations were observed in 16S rRNA, ND2, and ATPase 6 genes in breast cancer [8]. Another
study confirmed somatic mutations observed in D-loop region in primary breast tumors. ND1,
ND4, ND5 and cytochrome C genes were also found to have mutations [9]. Studies in ovarian
cancer also identified somatic mutations in the D-loop, 12S rRNA, 16S rRNA, and cytochrome b
regions in mitochondrial genome [10]. Liang and his colleagues observed changes in mtDNA
copy number in 15 brain tumor sections [11]. Another group that studied 45 malignant glioma
cells found high mtDNA amplification in 87% of the cases. For over two decades now,
researchers have proved that thyroid tumors have high mitochondrial abundance [12]. Mutations
in genes coding for subunits of the respiratory chain have been found in a separate study that
screened 21 tumors specimens [13]. Similar mtDNA mutations in mitochondrial genome, with
special reference to D-Loop, Cyt-b, ND1, ND2, ATPase 6, COXI and COXII regions, were
observed in hepatic, pancreatic, gastric, and prostrate tumor samples.
1.5 Transmembrane proteins
Cells are bordered by biological membranes that act as barriers to prevent molecules from
leaking out or unwanted molecules from diffusing in. These membranes are composed of
proteins that float in a sea of lipids. The lipid components create the barrier for cells, and the
protein components act as channels and transport system.
5
Membrane proteins can be peripheral or integral proteins. Peripheral membrane proteins interact
indirectly with integral membrane proteins, or directly by lipid polar head groups to attach to the
membrane. Embedded in the phospholipid bilayer, are some segments of the integral membranes
proteins [14]. Most integral proteins span the entire phospholipid bilayer. These are called
transmembrane proteins and contain one or more membrane-spanning domains. A major type of
the membrane spanning domain is the alpha helical type, which comprises of over 27% of all
proteins in humans. The beta barrel is a less common type, found in outer membranes of
bacteria, mitochondria and chloroplasts.
Prominent cellular functions of these proteins include communication between cells, organelles
and cytosol, ion transport, transport of nutrients, and receptor for viruses. Transmembrane
protein activation also facilitates glucose metabolism and fatty acid production.
1.6 Functions of trans-membrane proteins
Transmembrane proteins are functionally diverse. Various transmembrane proteins have been
isolated and characterized. For example, TMEM 176A and TMEM-176B protein levels were
elevated in lymphoma and lung cancer cells [15]. TMEM-135 was found to be a regulatory
protein in mitochondrial genetic defects of fat metabolism [16].
Studies on TMEM 16A by Gatewood et al suggests that it plays an important role in generating
Ca
2+
activated Cl
-
currents in smooth muscles [17]. TMEM-237 was identified and characterized
as a protein that was involved in ciliogenesis [18]. TMEM-59 also called DCF1 has been showed
to mediate neural stem cell differentiation and APP (amyloid precursor protein) glycosylation
[19].
6
MTTS1 was identified in Dr. Ebrahim Zandi’s lab as an IKK-β interacting protein by mass
spectrometry. It is a 263AA protein with an approximate molecular weight of 30,041 Da. The
protein was identified as a result of abnormal aggregation, due to the boiling step prior to SDS-
PAGE in western blot. On isolating MTTS1, it was found to interact with three proteins of the
ATP synthase complex. Further experiments performed in Dr. Zandi’s lab proved that MTTS1
localized to mitochondria and regulates ATP synthesis. In this study, MTTS1 is shown to
regulate mitochondrial abundance and function as a tumor suppressor.
7
CHAPTER TWO
MATERIALS AND METHODS
2.1 Cell culture conditions
HEK 293T cells are human embryonic kidney cells that are transformed and express a large T
antigen on its surface. They were purchased from ATCC and grown in Dulbecco’s Modified
Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum and with 1% penicillin
and streptomycin at 37°C in a humidified 5% CO
2
atmosphere. The cells were trypsinized and
passaged at 70% confluence, in a 100mm x 200mm culture dish, every 3 to 4 days.
PC3 cells are prostate cancer cell lines, also purchased from ATCC. PC3 cells were grown in
Roswell Park Memorial Institute medium (RPMI), supplemented with 10% fetal bovine serum
and with 1% penicillin and streptomycin at 37°C in a humidified 5% CO
2
atmosphere. The cells
were trypsinized and passaged at 70% confluence, in a 100mm x 20mm culture dish, every 4 to 5
days.
2.2 Stable cell transfection
HEK 293T cells were transfected at 60% confluence using lipofectamine (Invitrogen, Carlsbad,
CA, USA) and PLUS reagent. For MTTS1 over-expression in 293 cells, 0.5μg of GFP-MTTS1
plasmid was incubated with 5μl of PLUS reagent and opti-MEM I. The complex was then mixed
with lipofectamine and incubated at room temperature for 20 to 30 minutes. After incubation,
spent cell media was replaced with opti-MEM I medium and the DNA-Reagent-lipofectamine
complex was added to the cells. The plates were incubated for 4 to 6 hours at 37°C in a CO
2
8
incubator. After incubation, fresh regular medium was added to the pre-existing transfection
mixture.
For stable knockdown of MTTS1 (shRNA-MTTS1), cells were transfected with Hu SH 29mer
shRNA p RFP-C-RS vector using oligofectamine (Invitrogen). 0.25μg of plasmid was
transfected using the same procedure as above.
2.3 Drug selection
HEK 293T cells that were transfected stably were selected by specific drugs. 0.5μg/ml of
geneticin-418 was used to selected cells overexpressing MTTS1. 0.25μg/ml of puromycin was
used to select cells that were transfected with shRNA. 10 days after transfection, cells were
sorted to obtain a pure population.
2.4 Mitochondrial abundance by qPCR
Total DNA was isolated from HEK 293T cells at 70% confluence using the DNeasy Tissue Kit
(Qiagen, France). 100ng of isolated DNA was used to quantify relative abundance of
mitochondria using qPCR. PCR kit was purchased from Quanta Biosciences. The copy number
ratios of mtND1 (a mitochondrial encoded NADH dehydrogenase 1) to beta actin (a nuclear
encoded gene) was used to compare the relative abundance of mitochondria in cells. Primers and
probes were added to the PCR mix in a 3:1 ratio. All PCR reactions were carried out in ABI
7200.
2.5 MTTS1 expression by RT-PCR
Total RNA was isolated according to the manufacturer’s protocol from HEK 293T cells at 70%
confluence using Qiagen RNeasy Mini kit. 100ng of isolated RNA was used for MTTS1 gene
9
expression by RT-PCR. The copy number ratio of MTTS1 to beta actin was used to calculate the
relative expression of MTTS1 in these cell lines. Reagents for RT-PCR were purchased from
Quanta Bioscience. Primers and probes were added in a 3:1 ratio.
FORWARD PRIMER (5’-3’) REVERSE PRIMER (5’-3’)
MTTS1 TTTCCAGCCCGTTTGTGAATCAGC CATTGAGGCAATCCGCACGATGAA
Beta Actin GGCACCCAGCACAATGAAG GCCGATCCACACGGAGTA
MtND1 CACCCAAGAACAGGGTTTGT TGGCCATGGGTATGTTGTTAA
Table 1: List of primers used for q PCR and RT-PCR
2.6 Mitochondrial respiration assay
Oxygen consumption was measured as an indicator of the mitochondrial respiratory activity.
Cells (3×10
6
) were suspended in 1 ml of respiration buffer (125mM KCl, 0.1% BSA, 20mM
HEPES, 2mM MgCl2, and 2.5mM KH2PO4; pH 7.2). For each experiment, equal numbers of
cells were pipetted into the chamber of an oxytherm electrode unit (Hansatech Instrument Ltd,
Norfolk, UK), which uses a Clark-type electrode to monitor the dissolved oxygen concentration
in the sealed chamber over time. The data were exported to a computerized chart recorder
(Oxygraph), which calculated the rate of O
2
consumption. The temperature was maintained at
37°C during the measurement.
2.7 In-vivo analysis in nude mice
HEK 293T cells, that overexpress MTTS1 and knockdown MTTS1, were each injected into
immune-deficient mice (Jackson Laboratories) to measure tumor growth. The animals were first
anaesthetized intra-peritoneally using Xylazine and Ketamine. 1*10
6
cells were suspended in
10
100μl PBS and injected subcutaneously on both sides of the mice. They were maintained in
special housing and monitored every day. Tumor volumes were measured by calipers using the
formula (D*d
2
)/2, where D is the longer diameter and d is the shorter diameter of tumor. The
animals were then sacrificed according to USC University guidelines by cervical dislocation.
2.8 Immunohistochemistry using ATPase β
Once tumors were isolated from mice, they were fixed in 10% formaldehyde. ATP synthase are
the prime producers of ATP, using the proton gradient generated by oxidative phosphorylation.
Tissue sections were analyzed by immunohistochemistry with anti-ATPase (BD Biosciences) to
detect mitochondrial content.
11
CHAPTER THREE
RESULTS
3.1 Mitochondrial abundance by q PCR
Since MTTS1 was found to interact with the F
1
component of ATP synthase, the study
investigated the possible relationship between mitochondrial abundance and MTTS1 expression
levels. Lung cancer cell lines and head & neck cancer cell lines were chosen for this study. To
determine mitochondrial abundance, genomic DNA was isolated from each of these cell lines.
The relative copy number ratios of mtND1 (mitochondrial NADH dehydrogenase 1) to beta actin
(nuclear encoded gene) was used to determine the mitochondrial abundance by q PCR. Fig 4A
shows the relative mitochondrial content in 4 lung cancer cell lines. Fig 3A shows the relative
mitochondrial content in 3 head & neck cancer cell lines. In order to establish a correlation
between mitochondrial content and MTTS1, RNA from the same cell lines were isolated. RT-
PCR was performed to determine the relative gene expression in the cell lines. The copy number
ratio of MTTS1 to beta actin was used to calculate the relative MTTS1 gene expression by real
time PCR. Fig 4B and 3B represent MTTS1 gene expression in lung and head & neck cancer
cells, respectively.
Comparing both sets of data, when MTTS1 levels increase, mitochondrial abundance also
increases. This is true for both lung cancer and head & neck cancer cells. Thus, there is an
evident correlation between MTTS1 gene expression and mitochondrial abundance in both cell
lines
12
A
B
Fig 3: q PCR and RT- PCR in Head & Neck cancer cell lines
A. q PCR data showing mitochondrial abundance in head & neck cancer cell lines.
B. RT-PCR data showing MTTS1 gene expression levels that correlate with mitochondrial
abundance in head & neck cancer cell lines
13
A
B
Fig 4: q PCR and RT- PCR in Lung cancer cell lines
A. q PCR data showing mitochondrial abundance in lung cancer cell lines.
B. RT-PCR data showing MTTS1 gene expression levels that correlate with mitochondrial
abundance in lung cancer cell lines
14
3.2 Stable cell transfection in HEK 293 T cells
To better understand the role of MTTS1, a stable cell transfection, to overexpress and
knockdown gene expression was performed in HEK 293T cells. Cells were transfected with
GFP-MTTS1 plasmid with a G418-resistant marker to overexpress MTTS1 and with Hu SH
29mer shRNA p RFP-C-RS vector with puromycin resistance to knockdown MTTS1. RT-PCR
was performed to determine levels of MTTS1 in transfected cells. The graph in Fig 5 shows the
copy number ratios of MTTS1 to beta actin (internal control). GFP-MTTS1 shows higher levels
of MTTS1 when compared to controls. shRNA-MTTS1 shows low levels of expression when
compared to controls.
Fig 5: RT-PCR in HEK 293 T cells
MTTS1 gene expression levels were analyzed in HEK 293T cells post transfection by RT-PCR
shRNA control shRNA-MTTS1 GFP Control GFP-MTTS1
MTTS1: Beta actin
15
3.3 Mitochondrial respiration decreases in shRNA- MTTS1cells
Mitochondrial respiration was analyzed by measuring oxygen consumption. Changes in oxygen
consumption in Complex I and Complex II were measured. 5mM glutamate and malate were
added to observe changes in Complex I. 5mM succinate was added to the cells to observe
changes in Complex II. This was followed by the addition of ADP, causing a sudden burst of
oxygen uptake. This process occurs as a result of ADP conversion into ATP. The actively
respiring state is known as “state 3” respiration, whereas the slower rate, is known as "state 4"
respiration. The ratio [state 3 rate]: [state 4 rate] is called the RCI (Respiratory control index).
RCI was calculated for each cell line. This indicates the tightness of coupling between
respiration and phosphorylation.
Data from Figure 6 and 7 show the rate of oxygen consumption in complex I and complex II
respectively. Oxygen consumption rate in complex I do not change with overexpression or
knocked down MTTS1. However, in complex II, the rate drops down in 293 shRNA-
MTTS1cells compared to control (Fig 7B). This means that mitochondrial respiration in complex
II is affected by knocking down MTTS1 in HEK 293T cells.
Cellular respiration analysis to measure the Oxygen Consumption Rate (OCR) (Seahorse
Bioscience, Billerica, MA) also proved that overexpressing MTTS1 caused an increase in OCR
in HEK 293T cells.
These data collectively emphasize that MTTS1 regulates mitochondrial respiration.
16
A
B
Fig 6: Graph showing oxygen consumption in Complex I in HEK 293 T cells
A. Rate of oxygen consumption in shRNA- MTTS1 and shRNA Control cells
B. Rate of oxygen consumption in GFP-MTTS1 and GFP Control cells
17
A
B
Fig 7: Graph showing oxygen consumption in Complex II in HEK 293 T cells
A. Rate of oxygen consumption in shRNA-MTTS1and shRNA Control cells
B. Rate of oxygen consumption in GFP-MTTS1 and GFP Control cells
18
From the data presented above, RCI (Respiratory Control Index) was calculated for each cell
line. 293 shRNA-MTTS1 exhibit low RCI ratio in both complex I and complex II suggesting low
oxygen consumption.
SAMPLE GFP
MTTS1
GFP Control shRNA-MTTS1 shRNA Control
RCI Complex I 1.670 1.948 1.542 1.835
RCI Complex II 1.648 1.851 1.54 2.13
Table 2: Respiratory Control Index (RCI) in Complex I and Complex II in HEK 293 T
cells
3.4 In-vivo tumor studies
Brandon et al., 2006, proved that cancer cells can result in defects in the mitochondrial genome,
resulting in deficient mitochondrial respiration and ATP generation. Since low levels of
mitochondria were observed in shRNA-MTTS1 cells, we hypothesized that MTTS1
overexpression could be important as a tumor suppressor. In-vivo xenograft assay on nude mice
was performed to observe the effect of MTTS1 on tumor growth. GFP-MTTS1 and shRNA-
MTTS1 cells were injected on either side of the mice. 12 days post injection, tumor growth was
first observed in mice injected with shRNA-MTTS1. No tumor growth was observed in mice
injected with 293 GFP-MTTS1 until 16 days post injection. This clearly suggests that when
MTTS1 is overexpressed, tumor growth is significantly delayed in immune-deficient mice.
Figure below show HEK 293T cells that overexpress MTTS1 (right) and cells transfected with
shRNA-MTTS1 (left), that were injected in mice for in-vivo analysis.
19
A B
Fig 8: Fluorescent Microscope images of HEK 293T cells injected in immune-deficient mice
for xenograft assay
A. HEK 293T cells transfected with Hu SH 29mer shRNA p RFP-C-RS vector with puromycin
resistance to knockdown MTTS1.
B. HEK 293T cells transfected with GFP-MTTS1 vector with G418 resistance to overexpress
MTTS1.
In-vivo analysis was carried out in 8 mice for each of the 4 groups of cell lines. To avoid bias, all
groups of cells were injected into both the right and left flanks of the mice. This is shown in Fig
9A where GFP-MTTS1 cells were injected on the right flank and in Fig 9B, on the left flank of
the mice. All cells were injected in the same way, creating 8 mice for each of the 4 cell lines.
From the data below, tumors from shRNA-MTTS1 grew much faster than those from GFP-
MTTS1. The difference in tumor size can also be seen in the figure below.
20
A B
C D
Figure 9: Xenograft assay on immune-deficient mice
A. shRNA-MTTS1 (right) showing higher tumor volume than GFP-MTTS1 (left)
B. shRNA-MTTS1 (left) showing higher tumor volume than 293 GFP MTTS1 (right)
C. shRNA Control (left) and GFP Control (right) showing no or low tumor volume
D. shRNA Control (right) and GFP Control (left) showing no or less tumor volume
A graphical representation of tumor volume measured each day is displayed below. From the
graph, tumor volume from shRNA-MTTS1 show higher tumor volumes than controls and tumors
from GFP-MTTS1 appear to be much lower than the controls. Besides tumor volume, the graph
also shows delayed onset of tumor in MTTS1 overexpressed cells.
21
Fig10:
Graphical representation of
tumor volume per day
Mice injected with shRNA-MTTS1
cells show high tumor volume
compared to GFP-MTTS1 and
controls.
Once tumors were isolated, differences in their color were observed (Fig 11). Tumors from GFP-
MTTS1 cells were white whereas tumors from shRNA-MTTS1 were red in color. Tumors from
controls were also white. Literature states that, a red tumor is a tumor with leaky vessels, while a
white tumor is a tumor with non-leaky vessels [24]. This suggests high vascularization in tumors
from shRNA-MTTS1 cells.
Fig 11: Tumors isolated from nude mice
Tumors from GFP-MTTS1 differ in size and color from those isolated from shRNA-MTTS1
Tumor from mice injected
with shRNA-MTTS1
Tumor from mice
injected with GFP-MTTS1
22
3.5 Mitochondrial abundance in tumor tissues
Tumors from mice were isolated and fixed in formalin for immunohistochemistry with ATPase
β. When tumor tissues from shRNA-MTTS1 and GFP-MTTS1 were analyzed using anti ATPase
β, tissues from shRNA-MTTS1 showed lesser mitochondria than GFP-MTTS1. This is evident
from the data below that showing shRNA-MTTS1 than GFP-MTTS1. The data thus prove that
MTTS1 regulate mitochondrial numbers in tumors.
A B
Fig 12: Immunohistochemistry with ATP synthase β viewed under microscope
A. Tumor sections from GFP-MTTS1 showing higher mitochondrial numbers
B. Tumor sections from shRNA-MTTS1 showing lower number of mitochondria
23
3.6 In-vivo tumor assay using PC3 cells
A xenograft experiment was also performed using PC3 cell lines. These are classical prostate
cancer cell lines. PC3 shRNA-MTTS1, PC3 GFP-MTTS1 and controls were injected using the
same procedure as HEK 293T cells. PC3 shRNA-MTTS1 cells were injected on the left flank
and GFP-MTTS1 on the right. From Fig 13, cells from shRNA-MTTS1 show higher tumor
volume. The onset of tumors from shRNA-MTTS1 was much faster (10 days post injection) than
GFP-MTTS1 (28 days post injection). The data correlate with results from HEK 293T cells.
MTTS1 suppresses tumor growth in PC3 cells and HEK 293T cells. Tumors from PC3 cells are
yet to be analyzed.
Fig 13: In-vivo nude mice assay with PC3 cells
Nude mice injected with PC3 GFP-MTTS1 and PC3 shRNA-MTTS1.
Difference in tumor growth shows GFP-MTTS1 cells suppress tumor.
PC3 shRNA-MTTS1
cells
PC3 GFP-MTTS1
cells
24
CHAPTER FOUR
CONCLUSION AND DISCUSSION
Previous research conducted on MTTS1 proves the association of MTTS1 with ATP synthase
and its role in regulating ATP synthase activity. In this study, more emphasis has been made to
identify the relation between MTTS1, mitochondria and cancer.
From the real-time PCR data, it is evident that MTTS1 expression correlates with, and regulates
mitochondrial numbers in cancer cells. When MTTS1 is overexpressed, mitochondrial numbers
also increase. This is also evident from immunohistochemistry with ATPase β. In cancer cells,
ATP synthesis occurs via glycolysis rather than oxidative phosphorylation. This is because most
cancer cells have few mitochondria, less mitochondrial content and thereby, highly glycolytic
[20, 21]. Increasing mitochondrial numbers or activity can be important in cancer when it can
synthesize ATP through oxidative phosphorylation as in normal cells. In-vivo nude mice assay
confirms the tumor suppressor function of MTTS1 in prostate cancer and HEK 293T cell lines.
MTTS1 can be a possible target for cancer by regulating mitochondria.
SDH (succinate dehydrogenase) complex also called complex II, is located in the inner
mitochondrial membrane and is involved in Citric acid cycle and Respiratory chain. It catalyzes
the oxidation of succinate. Previous studies and research indicate that mutations involved in SDH
gene causes paraganglioma [22]. Another study group observed increased oxidative stress
leading to tumorigenesis in Complex II gene mutation [23]. Complex II dysfunction is directly
linked to a number of diseases including cancer. Mitochondrial respiration measured in shRNA-
25
MTTS1 cells also show reduced oxygen consumption in complex II (Fig 7B). This correlation
may be important to further prove that MTTS1 play an important role as a tumor suppressor.
The literature states that a red tumor is a tumor with leaky vessels, while a white tumor is a
tumor with non-leaky vessels [24]. Leaky vessels are characteristic of cancer tumors [25, 26]. In
normal cells, the capillary endothelial cells that line the capillaries are smooth and tightly
packed. In cancerous tumors, they have gaps in between them. This causes the vessels to become
leaky. This correlation can be related to Fig 11, that show the difference in color of tumors
isolated from GFP-MTTS1 and shRNA-MTTS1 cells in mice. This can also imply that tumors
from GFP-MTTS1 are benign and those from shRNA-MTTS1 are malignant.
As mentioned, MTTS1 was identified as IKK-β interacting protein. However, the role of MTTS1
with IKK- β is unknown. Future directions of the project will be to establish their relation.
MTTS1 knock down in prostate cancer cell show early onset of tumor than GFP-MTTS1 in nude
mice. In-vivo analysis can be done with different cancer cell lines to observe if they have similar
effects as HEK 293T cells.
In sum, the novel transmembrane protein, MTTS1, regulates mitochondrial abundance in head &
neck cancer cell lines and lung cancer cell lines. Knockdown and overexpression of the gene
cause mitochondrial abundance to decrease and increase respectively, in HEK 293T cells. Low
oxygen consumption rates were observed in HEK 293T shRNA-MTTS1 cells. In-vivo
experiments in nude mice indicate that MTTS1, when overexpressed, play a role in tumor
suppression. Tumors isolated from MTTS1-GFP have shown high mitochondria by
immunohistochemistry. Hence, MTTS1 is important for mitochondrial regulation and tumor
suppression.
26
REFERENCES
24. Arbiser JL. Reversing the angiogenic switch with photodynamic therapy. J Invest Dermatol.
2003 Sep;121
22. Bayley JP, Devilee P, Taschner PE. The SDH mutation database: an online resource for
succinate dehydrogenase sequence variants involved in pheochromocytoma, paraganglioma and
mitochondrial complex II deficiency. BMC Med Genet. 2005 Nov 16;6:39
7. Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, Green DR.
Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and
apoptosis. Science. 2004 Feb 13;303(5660):1010-4
15. Cuajungco MP, Podevin W, Valluri VK, Bui Q, Nguyen VH, Taylor K. Abnormal
accumulation of human transmembrane (MTTS1)-176A and 176B proteins is associated with
cancer pathology. Acta Histochem. 2012 Jan 11
2. DiMauro S, Schon EA. Mitochondrial DNA mutations in human disease. Am J Med Genet.
2001 Spring;106(1):18-26. Review
16. Exil VJ, Silva Avila D, Benedetto A, Exil EA, Adams MR, Au C, Aschner M. Stressed-
induced MTTS1135 protein is part of a conserved genetic network involved in fat storage and
longevity regulation in Caenorhabditis elegans. PLoS One. 2010 Dec 3;5(12):e14228.
6. Gogvadze V, Orrenius S, Zhivotovsky B. Multiple pathways of cytochrome c release from
mitochondria in apoptosis. Biochim Biophys Acta. 2006 May-Jun;1757(5-6):639-47
5. Gottlieb E, Tomlinson IP. Mitochondrial tumour suppressors: a genetic and biochemical
update. Nat Rev Cancer. 2005 Nov;5(11):857-66.
24. Hobbs SK, Monsky WL, Yuan F, Roberts WG, Griffith L, Torchilin VP, Jain RK.
Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4607-12
27
18. Huang L, Szymanska K, Jensen VL, Janecke AR, Innes AM, Davis EE, Frosk P, Li C, Willer
JR, Chodirker BN, Greenberg CR, McLeod DR, Bernier FP, Chudley AE, Müller T, Shboul M,
Logan CV, Loucks CM, Beaulieu CL, Bowie RV, Bell SM, Adkins J, Zuniga FI, Ross KD,
Wang J, Ban MR, Becker C, Nürnberg P, Douglas S, Craft CM, Akimenko MA, Hegele RA,
Ober C, Utermann G, Bolz HJ, Bulman DE, Katsanis N, Blacque OE, Doherty D, Parboosingh
JS, Leroux MR, Johnson CA, Boycott KM. MTTS1237 is mutated in individuals with a Joubert
syndrome related disorder and expands the role of the MTTS1 family at the ciliary transition
zone. Am J Hum Genet. 2011 Dec 9;89
23. Ishii T, Yasuda K, Akatsuka A, Hino O, Hartman PS, Ishii N. A mutation in the SDHC gene
of complex II increases oxidative stress, resulting in apoptosis and tumorigenesis. Cancer Res.
2005 Jan 1;65(1)
1. Jiang W, Hermolin J, Fillingame RH. The preferred stoichiometry of c subunits in the rotary
motor sector of Escherichia coli ATP synthase is 10. Proc Natl Acad Sci U S A. 2001 Apr
24;98(9):4966-71
14. Lehninger A, Nelson DL, Cox MM. Principles of Biochemistry. 5
th
ed.W.H.Freeman; 2008
19. Li X, Feng R, Huang C, Wang H, Wang J, Zhang Z, Yan H, Wen T. MicroRNA-351
regulates MTTS1 59 (DCF1) expression and mediates neural stem cell morphogenesis. RNA
Biol. 2012 Mar 1;9
11. Liang BC. Evidence for association of mitochondrial DNA sequence amplification and
nuclear localization in human low-grade gliomas. Mutat Res. 1996 Jul 5;354(1):27-33.
10. Liu VW, Shi HH, Cheung AN, Chiu PM, Leung TW, Nagley P, Wong LC, Ngan HY. High
incidence of somatic mitochondrial DNA mutations in human ovarian carcinomas. Cancer Res.
2001 Aug 15;61
3. Matoba S, Kang JG, Patino WD, Wragg A, Boehm M, Gavrilova O, Hurley PJ, Bunz F,
Hwang PM. p53 regulates mitochondrial respiration. Science. 2006 Jun 16;312(5780):1650-3.
28
9. Parrella P, Xiao Y, Fliss M, Sanchez-Cespedes M, Mazzarelli P, Rinaldi M, Nicol T,
Gabrielson E, Cuomo C, Cohen D, Pandit S, Spencer M, Rabitti C, Fazio VM, Sidransky D.
Detection of mitochondrial DNA mutations in primary breast cancer and fine-needle aspirates.
Cancer Res. 2001 Oct 15;61(20):7623-6.
20. Pedersen PL. (1978). Prog. Exp. Tumor. Res. 22, 190-274
21. Pedersen PL. (1997). J. Bioenerg. Biomembrane 29, 301-302
12. Stefăneanu L, Taşcă C. An electron-microscopic study of human thyroid cancer.
Endocrinologie 1979 Oct-Dec;17(4):233-9. Review
8. Tan DJ, Bai RK, Wong LJ. Comprehensive scanning of somatic mitochondrial DNA
mutations in breast cancer. Cancer Res. 2002 Feb 15;62(4):972-6.
17. Thomas-Gatewood C, Neeb ZP, Bulley S, Adebiyi A, Bannister JP, Leo MD, Jaggar JH.
MTTS116A channels generate Ca² ⁺-activated Cl ⁻ currents in cerebral artery smooth muscle
cells. Am J Physiol Heart Circ Physiol. 2011 Nov;301 (5):H1819-27.
4. Wang GL, Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional
response to hypoxia. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4304-8.
13. Yeh JJ, Lunetta KL, van Orsouw NJ, Moore FD Jr, Mutter GL, Vijg J, Dahia PL, Eng C.
Somatic mitochondrial DNA (mtDNA) mutations in papillary thyroid Carcinomas and
differential mtDNA sequence variants in cases with thyroid tumours. Oncogene. 2000 Apr 13;19
(16):2060-6
26. Yuan F, Leunig M, Huang SK, Berk DA, Papahadjopoulos D, Jain RK. Microvascular
permeability and interstitial penetration of sterically stabilized (stealth) liposomes in a human
tumor xenograft. Cancer Res. 1994 Jul 1;54
Abstract (if available)
Abstract
A novel transmembrane protein was identified in Dr. Ebrahim Zandi’s lab as an IKK-β interacting protein by mass spectrometry. Mass spectrometry and IP (Immunoprecipitation) studies were performed to investigate functions of the newly identified protein. The study showed the interaction of this protein with two subunits of F1 components of mitochondrial ATP synthase. The F1 component of ATP synthase is the catalytic subunit that drives ATP synthesis. Further analysis showed that this protein localizes to mitochondria and has a probable role in regulating ATP synthesis by interacting with ATP synthase α and ATP synthase β. Research has shown that mitochondrial dysfunction has a predominant role in cancer progression. ❧ In this study, we characterized the role of this protein in regulating mitochondrial abundance and its subsequent role in tumor regulation. Based on the finding of this study regarding the function of the protein, we named it Mitochondrial Transmembrane Tumor Suppressor 1 (MTTS1). MTTS1 levels of 4 out of 7 lung cancer cell lines correlated with their respective mitochondrial abundance. A similar pattern was observed when 3 head and neck cancer cell lines were screened. Stable cell transfection to overexpress and knockdown MTTS1 was performed in HEK 293T cells (human embryonic kidney cells). Levels of many mitochondrial genes, responsible for cellular energy metabolism, were increased in HEK 293T cells overexpressing MTTS1. In-vivo studies in mice using HEK 293T cells showed that there was a significant delay in tumor growth in cells overexpressing MTTS1, suggesting that this protein can be a potential tumor suppressor.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Role of a novel transmembrane protein, TMEM56, in tumorigenic growth of human PC3 prostate cancer cell line
PDF
Studies on the expression and function of the human TMEM56 protein
PDF
Decreased levels of expression of transmembrane protein 56 (TMEM56) in breast cancer tissues
PDF
Studies on the role of a novel protein, TMEM 56 in tumorigenic growth for MCF-7 cells
PDF
Studies on the role of TMEM56 in tumorigenesis by using PTEN knockout mouse model
PDF
Design, synthesis and validation of Axl-targeted monoclonal antibody probe for microPET imaging of human lung cancer
PDF
Identify Werner protein molecular partners in S phase alt cell
PDF
Optimization of circulating tumor cells isolation for gene expression analysis
PDF
Corisol's role in breast-to-brain metastasis
PDF
Regulation of inflammation and angiogenesis by Kaposi’s sarcoma-associated herpesvirus
PDF
Regulation of acute KSHV infection by SIRT1
PDF
Hepatitis B virus X protein regulation of β-catenin and NANOG and co-regulatory role with YAP1 in HCC malignancy
PDF
The role of ErbB signaling in dendritic cells during inflammatory bowel disease
PDF
The role of GRP78 in the regulation of apoptosis and prostate cancer progression
PDF
Molecular targets for treatment of glioblastoma multiforme
PDF
Role of cancer-associated fibroblast secreted annexin A1 in generation and maintenance of prostate cancer stem cells
PDF
Cytotoxic effect of NEO212, a novel perillyl alcohol-temozolomide conjugate, on canine lymphoma
PDF
Targeting mitochondrion-nucleus PDH1 transfer to suppress self-renewal and epigenetic NANOG reprogramming of tumor-initiating cells
PDF
Cell surface translocation and therapeutic targeting of GRP78
PDF
MGMT in emergence of melanoma resistance to temozolomide
Asset Metadata
Creator
Murali, Poornima
(author)
Core Title
Role of a novel transmembrane protein, MTTS1 in mitochondrial regulation and tumor suppression
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
08/03/2014
Defense Date
06/15/2012
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
cancer,mitochondrial abundance,OAI-PMH Harvest,transmembrane protein,tumor supression
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Zandi, Ebrahim (
committee chair
), Landolph, Joseph R., Jr. (
committee member
), Schönthal, Axel H. (
committee member
)
Creator Email
muralipoornima@yahoo.in,poornimm@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-86121
Unique identifier
UC11288936
Identifier
usctheses-c3-86121 (legacy record id)
Legacy Identifier
etd-MuraliPoor-1139.pdf
Dmrecord
86121
Document Type
Thesis
Rights
Murali, Poornima
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
mitochondrial abundance
transmembrane protein
tumor supression