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Decreased levels of expression of transmembrane protein 56 (TMEM56) in breast cancer tissues
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Decreased levels of expression of transmembrane protein 56 (TMEM56) in breast cancer tissues
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Content
DECREASED LEVELS OF EXPRESSION OF
TRANSMEMBRANE PROTEIN 56 (TMEM56) IN BREAST
CANCER TISSUES
By
Fikir Mesfin
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 2014
Copyright 2014 Fikir Mesfin
ii
TABLE OF CONTENTS
LIST OF FIGURES ........................................................................................... iii
LIST OF TABLES .............................................................................................. iv
ABSTRACT ........................................................................................................ v
INTRODUCTION ...............................................................................................1
1.1 Breast cancer ........................................................................................1
1.2 Mitochondria and cancer ...................................................................... 2
1.3 The ATP synthase ................................................................................4
1.4 Membrane proteins and their functions .................................................. 6
1.5 Transmembrane (TMEM) protein family ............................................... 7
HYPOTHESIS ................................................................................................... 10
MATERIALS AND METHODS ....................................................................... 11
3.1 Breast cancer tissue sections................................................................. 11
3.2 Laser Capture Microdissection ............................................................ 12
3.3 RNA extraction of FFPE tissues ........................................................... 13
3.4 Real time RT-PCR ............................................................................... 13
3.5 Immunohistochemistry using ATPase β ...................................................... 14
3.6 Analysis of ATPase β stained tissues ..................................................... 14
RESULTS .......................................................................................................... 15
4.1 Laser Capture Microdissection of breast cancer tissues .......................... 15
4.2 ATPase β staining ................................................................................ 18
DISCUSSION AND CONCLUSION ................................................................ 21
REFERENCES .................................................................................................. 25
iii
LIST OF FIGURES
Figure 1: Mitochondrial ATP synthase .................................................................. 5
Figure 2: TMEM56 protein sequence and predicted structure .................................9
Figure 3: Breast Tissue Section ............................................................................ 12
Figure 4: Breast Tissue Section pre and post microdissection ................................ 15
Figure 5: TMEM56 Expression in twelve breast cancer tissues .............................. 16
Figure 6: Analysis of TMEM56 Expression breast cancer tissues .......................... 16
Figure 7:TMEM56 Expression level in Stage I cancer tissues ................................ 17
Figure 8: Analysis of TMEM56 Expression level in Stage I breast cancer tissues.... 18
Figure 9: Breast Cancer tissue SP-12-7163 after ATPase β staining ....................... 18
Figure 10: Total ATPase β level in breast cancer tissues ....................................... 19
Figure 11: TMEM56 Expression Level and total ATPase β level of breast cancer
tissues ................................................................................................................ 20
iv
LIST OF TABLES
Table 1: Patient Breast Cancer Tissues ................................................................. 11
Table 2: Forward and Reverse primers for Real time RT PCR .............................. 13
v
ABSTRACT
Membrane proteins are indispensable members of the cell membrane. They anchor
the cell, regulate its communication with the environment and degrade damaged cell
membrane. Transmembrane proteins (TMEM) are a group of novel proteins that
have been found to have key roles in differentiation and regulation of the cell [28].
TMEM56 is a new protein that was first identified in Dr. Ebrahim Zandi’s laboratory
as an IKK- β interacting protein by immunoprecipitation and mass spectrometry [25].
Studies done in the lab have shown that TMEM56 has a potential tumor suppressor
role. Therefore, we hypothesize that TMEM56 expression will be suppressed in
cancer cells compared to normal cell and its expression level will be lower in cancer
region.
This study examines the TMEM56 expression level in normal and cancer regions of
twelve breast cancer tissues. Laser capture Microdissection was used to isolate the
cancer cells and the normal cells. Real time RT-PCR was performed on RNA
samples that were extracted from isolated region. The data shows that 78% of the
stage I cancer samples analyzed had lower TMEM56 expression level in the cancer
region compared to the normal region. This is consistent with our hypothesis that the
cancer cells will have lower expression level of TMEM56 as a result of its potential
tumor suppressor role. Further studies can illustrate the mechanism by which
TMEM56 possibly suppressor tumor growth.
1
CHAPTER I
INTRODUCTION
1.1 Breast Cancer
Breast cancer is the second most common cancer and is one of the leading
causes of death in women in America. According to the American Cancer Society,
over 200,000 new breast cancer cases were estimated for 2013 and over 39,000
patients were expected to die from it [2].
Breast cancer is a disease caused by abnormal growth of the cells of the breast
[2]. The female breast tissue consists of lobules (milk producing glands), ducts
(transport milk from the lobules to the nipple) and stroma, which consists of
connective and adipose tissue. The majority of breast cancers are a type of
adenocarcinoma, as almost all breast cancers originate in the epithelial cell lining of
the lobules and ducts [2].
Breast cancer is staged based on the tumor size, how many lymph nodes are
involved, and how far it has spread in the body. The most common staging system is
the TNM staging system. The letter ‘T’ followed by a number from 0 to 4 describes
the tumor size. The higher the ‘T’ number the larger the tumor. The letter ‘N’
followed by a number from 0 to 3 indicates if the tumor has spread to nearby lymph
nodes and if so to how many. The letter ‘M’ followed by a 0 or 1 indicates whether
the cancer has spread to distant organs [2].
2
1.2 Mitochondria and Cancer
The relationship between mitochondria and cancer was established in the
early decades of the twentieth century when the Nobel Laureate, Otto Warburg,
described that cancer cells showed a higher rate of glycolysis compared to normal
cells in the presence or absence of oxygen [3]. Warburg observed that cancer cells
challenge Pasteur’s Effect, as they do not show inhibition of fermentation in the
presence of oxygen [5]. Busk et.al showed that glycolysis accounts for 60% of ATP
production in most cancer cells [6]. One explanation put forward for this
phenomenon is the hypoxic tumor environment that limits the use of the oxidative
phosphorylative pathway [7]. In addition, a key regulatory enzyme of glycolysis
(phosphofructokinase -1; PFK-1) is inhibited by ATP. Therefore, a reduction in the
production of ATP due to the inhibition of oxidative phosphorylation can reduce the
inhibition of PFK-1 and enhance glycolysis [7]. Most tumors also show an increased
activity of Hypoxia Induced Factors (HIF). HIF is a family of transcription factors
that are maintained at low levels in normoxia due to continuous degradation.
However, in hypoxia, HIF degradation is inhibited, and this regulates two major
enzymes- pyruvate dehydrogenase (PDH) and lactate dehydrogenase (LDH). These
enzymes determine the fate of pyruvate, its reduction in the cytosol to lactate or its
oxidation in the mitochondria. HIF induces LDH levels and reduces the activity of
PDH, thereby decreasing pyruvate’s translocation and oxidation in the mitochondria
[7].
The mitochondrion is associated with cancer at the genetic level as well. The
mitochondrial DNA is about 16Kb in size. It contains the genes for all the
3
mitochondrial rRNAs and tRNAs and some of the proteins involved in the electron
transport chain. The mtDNA does not have histones, making it more susceptible to
mutagens. The direct effect of somatic mtDNA mutation on tumorigenesis remains
uncertain. However, in some cancers, mutation in somatic mtDNA has been shown
to correlate with a low respiratory rate and decreased activity of complexes I and III
[8-10]. In addition, studies have shown that non-oncogenic genetic changes that
alter mitochondrial metabolism affect tumor growth [4]. It has also been shown that
mtDNA mutations that produce excess reactive oxygen species (ROS) increase the
metastatic potential of certain tumor cells [11]. Mutations of nuclear-encoded
enzymes such as succinate dehydrogenase (SDH), which play a vital role in
metabolism, have been shown to predispose patients to certain cancers [12].
The mitochondria also have a role in intrinsic cellular apoptosis. The
mitochondrial membrane houses pro- and anti-apoptotic proteins. Upon activation
by pro-apoptotic proteins, a protein complex forms a pore in the mitochondrial
membrane, leading to the loss of proton gradients and the release of mitochondrial
proteins such as cytochrome c into the cytosol. Cancer cells often have gene
mutations that suppress pro-apoptotic pathways and induce anti-apoptotic pathways
[14]. These mutations include loss of function of pro-apoptotic proteins such as p53,
and gain of function of anti-apoptotic proteins such as Bcl-2, both of which are key
players in mitochondrial mediated intrinsic apoptosis [14].
4
1.3 The ATP synthase
The mitochondrial ATP synthase is made up of two components, F
o
and F
1
components. The F
o
component is an intergral part of the membrane, while the F
1
component, associated with the F
o
component, protrudes to the mitochondrial
matrix. The F
o
-ATPase is involved in the translocation of protons, while the F
1
-
ATPase is involved in ATP synthesis. The F
o
component has three subunits-one ‘a’
subunit, two ‘b’ subunits and 8-15 ‘c’ subunits depending on the organism. On the
other hand, the F
1
component has 9 subunits; 3 α, 3 β, γ, δ, ε. The three β subunits
have identical polypeptide chain but have different conformation at a given time.
This difference accounts for their differential binding of ADP and ATP. At a given
time, a β subunit starts in the β-ADP form (the conformation that binds ADP) and
binds ADP and Pi. The subunit then changes conformation to that of β-ATP (the
conformation that binds ATP tightly), resulting in the synthesis of ATP. Finally, the
conformation of the subunit then shifts to β-empty (a conformation that has a low
affinity for ATP) which leads to the release of ATP [15]. The conformational change
is brought about by the passage of protons though the F
o
pore which cause a rotation
in the ‘c’ subunit of F
o
and the γ subunit of F
1
. The γ subunit is associated with one β
subunit at a given time, and it brings about conformational change of the β subunit
[15]. In this way, the proton gradient is coupled with ATP synthesis by ATP
synthase in the mitochondria.
Although F-ATPase (the ATPase found in mitochondrial membrane) is a
well-known, well-studied ATPase, there are other ATPases that play important roles
in the proper functioning of the cell. The P-ATPase family, of which Na+/K+
5
ATPase is a member of, are cation transporting complexes that are reversibly
phosphorylated by ATP [16]. V-type ATPases, yet another class of ATPase, are
proton pumps that are found in organelles such as the lysosomes, endosomes, the
Golgi complex and plant vacuole and are responsible for the acidification of these
organelles.
Recent studies have shown that various subunits of the F
1
F
o
-ATPase are also
found on the surface of certain cells [17, 18]. These surface F
1
F
o
-ATPase subunits
have been named ecto-F
1
F
o
-ATPase [19, 20]. The β subunit of F
1
F
o
-ATPase has been
found to be expressed on the surface of the cell as a receptor for various ligands [19].
The majority of the cells that have been found to express ecto- F
1
F
o
-ATPase β
subunit are cancer cell lines, such as leukemia cell lines [17, 18, 21].
Figure 1:
Mitochondrial ATP
synthase [16]
6
1.4 Membrane Proteins and their functions
Membrane proteins are a major class of proteins and are structurally and
functionally diverse. Based on membrane-protein interactions, membrane proteins
can be classified into integral and peripheral proteins [1]. Integral proteins have a
stretch of hydrophobic amino acids that are embedded in the membrane, thereby
anchoring the protein. Integral proteins can also be covalently linked to hydrocarbon
groups that are embedded in the membrane [1]. Peripheral proteins on the other
hand do not have a domain that is embedded in the membrane. They are held at the
membrane by interaction with membrane polar head or by interaction with integral
proteins [1].
The hydrophobic chain of integral proteins usually spans the entire
membrane. The most common structure of this membrane spanning chain is an α-
helix. The amino acids making up this α-helix are pre-dominantly hydrophobic and a
given protein can have several α-helices spanning the membrane [1].
Membrane proteins have numerous functions. Some membrane proteins are
channels for the passage of large or hydrophilic molecules in and out of the cell.
Others are receptors for ligands, while some maintain the integrity of the cell
structure by anchoring structural proteins such as beta actin. Certain enzymes such
as phospholipases, that play an important role in the degradation of damaged cell
membranes by hydrolyzing the polar head group of phospholipids, are peripheral
membrane proteins [1]. Further still, the protein complexes that are involved in the
oxidation of NADH and FADH
2
in the oxidative phosphorylation pathway, as well
7
as the F
o
subunit of the ATP synthase, are all integral proteins of the mitochondrial
inner membrane.
1.5 Transmembrane (TMEM) protein family
The first of the TMEM family of proteins was first identified from bronchial
epithelial cells, Xenopus oocytes, and murine eyes [22]. One of the well-studied
members of this family of proteins is TMEM16A. This protein is differentially
spliced in different organs and organisms, and it acts as a calcium-dependent chloride
channel (CaCC) [22, 23]. The amino acid sequence and structure-predicting
programs indicate that TMEM16A has eight transmembrane domains, with both the
carboxyl and amino termini directed into the intracellular medium [23]. Another
TMEM protein is TMEM237. This protein has four transmembrane domains.
Similar to TMEM16A, the predicted structure shows that it has both the COOH and
NH
2
termini protruding into the intracellular medium [24]. Studies have shown that
TMEM237 plays a role in cilium biogenesis and that this protein is mutated in
patients with Joubert Syndrome related disorders [24].
TMEM56 was first identified as an IKK- β interacting protein by
immunoprecipitation and mass spectrometry in Dr. Ebrahim Zandi’s laboratory. It is
a 263 amino acid protein and its estimated molecular weight is approximately
30kDa. It is an integral membrane protein, and it has six alpha helix transmembrane
domains. The gene that codes for TMEM56 is located on chromosome 1 (1p21.3).
8
Previous studies conducted in the laboratory using immunopreciptation and
mass spectrometry have shown that TMEM56 has a strong association with ATP
synthase. In addition, it has been shown that Flag tagged TMEM56 co-localized
with ATP synthase using immunostaining with anti-flag and anti-ATPase α
antibodies [25]. Furthermore, in-vivo xenograft assay using nude mice and stable
HEK 293T cells has shown that mice injected with TMEM56 knockdown stable
293T cells had larger tumors than mice that were injected with TMEM56
overexpressed 293T stable cells [26].
9
Human Transmembrane protein 56 (TMEM56)
A. Amino acid sequence of TMEM56
1 MEINTKLLIS VTCISFFTFQ LLFYFVSYWF SAKVSPGFNS LSFKKKIEWN SRVVSTCHSL
61 VVGIFGLYIF LFDEATKADP LWGGPSLANV NIAIASGYLI SDLSIIILYW KVIGDKFFIM
121 HHCASLYAYY LVLKNGVLAY IGNFRLLAEL SSPFVNQRWF FEALKYPKFS KAIVINGILM
181 TVVFFIVRIA SMLPHYGFMY SVYGTEPYIR LGVLIQLSWV ISCVVLDVMN VMWMIKISKG
241 CIKVISHIRQ EKAKNSLQNG KLD
B. Predicted Structure (Unitprot KB database)
Figure 2:
A. The amino acid sequence of TMEM56 from NCBI (NP_001186608.1)
http://www.ncbi.nlm.nih.gov/protein/NP_001186608.1
B. Predicted Structure of TMEM56 from UnitprotKB database (Q96MV1)
http://www.uniprot.org/uniprot/Q96MV1
10
CHAPTER II
HYPOTHESIS
As mentioned earlier, previous studies conducted in Dr. Zandi’s laboratory
have shown that TMEM56 has a strong association with ATP synthase [25].
Furthermore, in vivo xenograft assay has shown that TMEM56 knock-down cells
have greater tumor growth rate [26]. As a result, it was hypothesized that TMEM56
might have a tumor suppressor role. Consequently, this study was designed to look
at TMEM56 expression level in breast cancer tissues.
We hypothesize that because of the potenital tumor suppressor role of TMEM56,
cancer cells will suppress the expression of TMEM56 and normal cells will have
higher TMEM56 expression. In addition, in connection with the strong association
between TMEM56 and ATPase, we hypothesize that the TMEM56 expression level
will correspond with ATPase expression.
11
CHAPTER III
METHODS AND MATERIALS
3.1 Breast Cancer Tissue sections
The breast cancer tissues used for this study were obtained from patient
biopsy samples (acquired from Dr. Wafaa Elatre, M.D. - Department of Pathology,
USC). The biopsy blocks were formalin fixed and paraffin embedded. The blocks
were cut to 5 µm thickness and were stained with Hematoxylin and Eosin (H&E) for
better visibility and contrast. For use in microdissection, the tissues had to be
prepared on plain non-charged glass slides in order to efficiently pick up laser-cut
tissue sections. The tissue samples were prepared in the Translational Pathology
Core of Norris Comprehensive Cancer Center. The cancers were staged using the
TNM staging system.
Cancer (5uM per block)
Path # Block Tissue Type of Cancer Stage
SP-12-8952 D12 Breast Ductal pT1N0MX Stage IA
SP-11-7116 C5 Breast Ductal and Lobular pT1cN0MX Stage IA
SP-12-4881 F6 Breast Ductal pT1cN0MX Stage IA
SP-11-4330 C5 Breast Ductal pT1N0MX Stage IA
SP-12-7206 D2 Breast Ductal pT1cN0MX Stage IA
SP-12-7163 D5 Breast Ductal pT1bN0 Stage IA
SP-12-3933 A7 Breast Ductal pT2NX
SP-11-9600 C8 Breast Ductal pT1cN0MX Stage IA
SP-12-6727 F4 Breast Ductal pTisT2N1bMX Stage IIB
SP-12-2489 A13 Breast Ductal pT3N1MX Stage IIIA
SP-12-6720 F9 Breast Ductal pTisT1aN0MX Stage IA
SP-12-2214 D17 Breast Ductal pT1c(m)N0MX Stage IA
Table 1: Patient Breast Cancer Tissues (obtained from Dr. Wafaa Elatre, M.D. – Dept.
of Pathology, USC)
12
3.2 Laser Capture Microdissection
Laser Capture Microdissection is a technique used to isolate a cell or group of
cells from a tissue section. The instrument used is the PixCell II LCM system from
Arcturus Biosciences (Grand Island, NY). The method requires particular caps that
are coated with a polymer for the capture of laser-cut tissues (Arcturus Capsure
Macro LCM caps – life technologies). The instrument has a laser source and a stage
hand that holds the caps in place. The caps are placed on the desired tissue section.
When the laser is shot through the caps, the polymer melts onto the tissue section.
Then, when the caps are lifted, the cut tissue section lifts along with the cap. For
each sample a cancer region and a corresponding normal region was cut and the
tissue sections were then processed for RNA extraction.
Figure 3: Breast Tissue sections
A. Cancer region (SP-12-8952)
B. Normal region (SP-12-8952)
B
A
13
3.3 RNA Extraction of Formalin Fixed Paraffin Embedded tissues
The caps with the dissected tissues attached are placed on a 0.5 mL
Eppendorf tubes. The RNA was extracted from the breast cancer tissues using
Qiagen RNeasy FFPE kit. Because of the abundance of RNase in tissues and the
environment the slides come in contact with during storage, the samples obtained
from microdissection had to be processed differently than regular RNA extraction
from cells or frozen tissues. The extraction method includes an extra protease
inhibition step, where the samples are incubated with proteinase K overnight at
55ºC.
3.4 Real Time RT PCR
The concentration of total RNA extracted from FFPE microdissected tissue
samples is very small. On average, 40-50 ng of total RNA was used for the real time
RT PCR. Beta actin was used as an internal control. Reagents for RT-PCR were
purchased from Quanta Biosciences (Gaithersburg, MD). The forward and reverse
primers used for the real time RT PCR are listed in table (Table 2). 250 ηM of the
forward and reverse primers was used for each reaction tube.
Forward Primer Reverse Primer
TMEM56 TTTCCAGCCCGTTTGTGAATCAGC CATTGAGGCAATCCGCACGATGAA
B-actin GCACCCAGCACAATGAAG GCCGATCCACACGGAGTA
Table 2: Forward and reverse primers for Real time RT PCR
14
3.5 Immunohistochemitry using ATPase β
Fresh tissue slides were cut again from the breast cancer sample blocks. The
tissue sections where then stained with anti-ATPase β antibody (BD Biosciences-San
Jose, CA). The antibody stains the total ATPase β subunit. The staining was
performed in the USC immunohistochemistry department of the clinical laboratories.
3.6 Analysis of ATPase β stained Tissues
A digital slide was made from the ATPase β stained tissues using an Aperio
ScanScope. The scanned tissues where then analyzed using the image analysis
feature of Aperio ScanScope. The positive pixel intensity of the cancer regions was
compared with that one of the normal regions for each tissue sample to examine the
difference in total ATPase β level.
15
CHAPTER IV
RESULTS
4.1 Laser Capture Microdissection of Breast Cancer Tissues
For each of the twelve samples, a cancer region and a corresponding normal
region was isolated using laser microdissection. The isolated sections were then
processed to extract total RNA. The total RNA was then used to perform real time
RT PCR using TMEM56 primers. Beta actin was used as an internal control.
A B
C D
E F
Figure 4:
A. Breast Cancer Tissue
Sample (SP-12-8952)
cancer region pre-
dissection
B. Breast Cancer Tissue
Sample (SP-12-8952)
cancer region post-
dissection
C. Breast Cancer Tissue
Sample (SP-11-9600)
cancer region pre-
dissection
D. Breast Cancer Tissue
Sample (SP-11-9600)
cancer region post-
dissection
E. Breast Cancer Tissue
Sample (SP-11-9600)
normal region pre-
dissection
F. Breast Cancer Tissue
Sample (SP-11-9600)
normal region post-
dissection
16
Out of the twelve samples analyzed, eight of the breast tissues (8952, 7116,
4881, 4330, 9600, 2214, and 6720, 6727) had less TMEM56 mRNA level in the
cancer region compared to the corresponding normal regions. Three of the samples
(7206, 2489, and 3933) had less TMEM56 level in the normal regions compared to
0.001
0.01
0.1
1
10
100
1000
10000
100000
8952 7116 4881 4330 7206 7163 9600 2214 6720 6727 2489 3933
Relative Expression (Log Scale)
TMEM56 Expression Level
Normal
Cancer
Stage I
SIII
SII
NA
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
Less in Cancer Tissues Less in Normal
Tissues
Difference is
insignificant
TMEM56 Expression Level
Figure 5: TMEM56 Expression in twelve breast cancer tissues
Figure 6:
Analysis of
TMEM56
expression
level in
twelve breast
cancer
tissues
17
the cancer regions. The mRNA level difference was insignificant in one of the
samples (7163).
When the stages of the breast cancer tissues were interpreted using the
American Cancer Society TNM staging system, out of the twelve samples we were
able to obtain, there was only one stage II and one stage III cancer tissues. The stage
II (6727) showed lower TMEM56 level in the cancer region while the Stage III
(2489) breast cancer tissues showed higher TMEM56 level in the cancer region
compared to the normal region. Out of the stage I breast cancer tissues, about 78% (7
out of 9) showed a lower TMEM56 level in the cancer regions than in the
corresponding normal regions.
0.001
0.01
0.1
1
10
100
1000
10000
100000
8952 7116 4881 4330 7206 7163 9600 2214 6720
Relative Expression (Log Scale)
TMEM56 Expression Level in Stage I Breast Cancer
Tissues
Normal
Cancer
Figure 7: TMEM56 expression level in stage I Breast cancer tissues
18
4.2 ATPase β staining
Fresh tissues slides were cut from eleven of the breast cancer tissue blocks.
The slides were then stained with anti-ATPase β antibody. Digital slides were created
from the stained tissue slides using Aperio ScanScope.
The scanned slides where then analyzed using the Aperio ScanScope’s
positive pixel analysis. On the stained slides, the positive signal (ATPase β) is brown
and the negative signal (counter staining) is blue. For analysis equal areas were
selected from the cancer and corresponding normal regions of each tissue sample.
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
Less in Cancer
Tissues
Less in Normal
Tissues
Difference is
insignificant
TMEM56 Expression Level
A B
Figure 9: Breast
Cancer tissue SP-12-
7163 after ATPase β
staining.
A. Normal region
B. Cancer region
Figure 8: Analysis of TMEM56 expression level in stage I breast cancer tissues
19
The positive signal intensity of these equivalent regions was then analyzed using the
Aperio software.
The results obtained in the immunohistochemistry (ATPase β staining) of the
breast cancer tissues show that the cancer regions have higher ATPase β level than
the corresponding normal regions.
As shown above, not all the cancer regions have lower TMEM56 level as
predicted in the hypothesis (Figure 5). However, because of the strong relation
shown between TMEM56 and ATP synthase by immunoprecipitation and
immunostaining [25, 26], a comparison was done to determine whether the
expression level of TMEM56 in the breast cancer tissues corresponds with the total
ATPase β level from immunohistochemistry (Figure 9).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
8952 7116 4330 7206 7163 9600 2214 6727 2489 3933
Ration of Average Positive Pixel
Intensity
ATP5b Level
Normal
Cancer
Stage I
SII
SIII NA
Figure 10: Total ATPase β level in eleven breast cancer tissues
20
As seen in Figure 10, all the cancer tissues have higher ATPase β level
compared to the normal regions. However, the ATPase β level seen from
immunohistochemistry does not correspond with the TMEM56 level seen from real
time RT-PCR for each tissue sample (Figure 11).
0
0.5
1
1.5
2
2.5
3
8952 7116 4330 7206 7163 9600 2214 6727 2489 3933
Normal - TMEM Exp
Cancer - TMEM Exp
Normal - ATP5b Level
Cancer - ATP5b Level
Figure 11: TMEM56 Expression level and total ATPase β level of breast cancer tissues
21
CHAPTER V
DISCUSSION AND CONCLUSIONS
TMEM56 is a transmembrane protein that was first identified in Dr. Ebrahim
Zandi’s laboratory through immunoprecitation and mass spectrometry as an IKK- β
interacting protein. Previous studies in the lab using immunostaining,
immunoprecipation and mass spectrometry have shown that TMEM56 has a strong
association with ATP synthase. Furthermore, studies done in the laboratory of in
vivo xenograft using nude mice and TMEM56 overexpressed and knock-down HEK
293T stable cells have shown that mice that were injected with TMEM56 knock-
down stable cells had a higher rate of growth and formed larger tumors indicating a
potential tumor suppressor role for TMEM56.
Therefore, this study was designed to further study the potential tumor
suppressor role of TMEM56 by examining its expression level in breast cancer
tissues. As a result of the potential tumor suppressor role of TMEM56, it was
hypothesized that the cancer regions of the breast cancer tissues will suppress the
expression of TMEM56, and they will have a lower TMEM56 level compared to the
normal regions.
The real time RT-PCR result of the laser microdissected cancer and normal
regions have shown that 67% of the tissue samples have lower TMEM56 level in the
cancer regions compared to the corresponding normal regions. As mentioned earlier,
out of the twelve breast cancer tissues that we were able to obtain, nine tissue
samples were stage I, one was stage II, and one was stage III. The stage of one tissue
22
sample (SP-12-3933) could not be determined because the ‘N’ number in the TNM
staging system was not available. Among the stage I cancer tissues analyzed, seven
out of the nine (78%) had lower TMEM56 level in the cancer regions compared to
the corresponding normal regions. One sample (7206) had higher TMEM56
expression in the cancer region compared to the normal region. However, the
TMEM56 expression level in the normal region for this sample is lower compared to
other samples. Therefore, the increased expression of TMEM56 in the cancer region
compared to the normal region in this sample will be physiologically insignificant.
The data has shown that the majority of the tissues have lower TMEM56 level in the
cancer regions than in the normal regions, supporting the hypothesis that TMEM56
might have a tumor suppressor role.
One of the challenges in cancer treatment is the absence of a specific gene or
protein that is consistently overexpressed or down-regulated in all the patients
diagnosed with a particular cancer. The majority of the time, it is a combination or a
cascade of genes that are altered that leads to the abnormal cell growth. Though one
gene is overexpressed in one group of cancer patients, its expression can be normal in
another group of patients. In breast cancer treatment, for example, the breast cancer
cells have to be identified as ER+/-, PR+/- or HER2 +/- for their expression of
estrogen, progesterone, and human epidermal growth factor receptors respectively.
Depending on the expression of these receptors on the breast cancer cells, the
treatment regimen varies. Therefore, though the expression level of TMEM56 is not
consistent across all the breast cancer tissues analyzed in this study, the majority of
23
the samples have shown lower expression levels that it definitely warrants further
study to elucidate its specific role in breast cancer and its treatment.
The immunohistochemistry result using ATPase β have shown that total
ATPase β expression is higher in the cancer regions than the corresponding normal
regions. However, the data shows that the total ATPase β level does not seem to
have a positive or negative correspondence with the TMEM56 expression level.
Studies have shown that cancer cells express more ecto-ATPase subunits than
normal cells [27]. Ecto-ATPase subunits are the cell surface ATPase subunits. As
mentioned above, studies done in Dr. Zandi’s laboratory have shown a strong
association between TMEM56 and ATP synthase. In addition, immunostaining
performed in the laboratory has consistently shown the co-localization of TMEM56
with ATP synthase, but no co-localization has been seen with mitochondrial marker.
Therefore, it is possible that TMEM56 has a role in the specific expression or
translocation of ATPase to the cell surface. Though the exact distribution is not
known, over 95% of the ATP synthase is located in the mitochondria, and only a
small percent of the ATP synthase subunits are on the cell surface. The
immunohistochemistry result compares the total ATPase level of the cancer region
and corresponding normal region. The positive signal from ecto-ATPase is a small
fraction of the signal seen from the total ATPase and if TMEM56 is only associated
with the expression or translocation of cell surface ATPase and not that of
mitochondrial ATPase, this can be a possible explanation for the lack of observed
correspondence between TMEM56 level and ATPase level.
24
In conclusion, the data has shown 78% of the stage I cancer cells have lower
TMEM56 expression level compared to the normal region consistent with our
hypothesis. Further studies can illustrate the specific role of TMEM56 in cancer, one
of the novel members of the transmembrane family of proteins.
25
REFERENCES
1. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New
York: W. H. Freeman; 2000. Section 3.4, Membrane Proteins. Available from:
http://www.ncbi.nlm.nih.gov/books/NBK21570/
2. American Cancer Society. Breast Cancer.
http://www.cancer.org/acs/groups/cid/documents/webcontent/003090-pdf.pdf
3. Warburg O. On the origin of cancer cells. Science. 1956;123:309–14
4. Chen Y., Cairns R., Papandreou I., Koong A., Denko N. C. (2009). Oxygen
consumption can regulate the growth of tumors, a new perspective on the Warburg
effect. PLoS ONE 4:e7033 10.1371/journal.pone.0007033
5. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to
current concepts of cancer metabolism. Nat Rev Cancer 69: 325–337
6. Busk M. et al. Aerobic glycolysis in cancers: implications for the usability of oxygen-
responsive genes and fluorodeoxyglucose-PET as markers of tissue hypoxia. Int J
Cancer. 2008;8(12):2726–2734. doi: 10.1002/ijc.23449.
7. Frezza C, Gottlieb E. Mitochondria in cancer: not just innocent bystanders. Semin
Cancer Biol. 2009;19(1):4–11. doi: 10.1016/j.semcancer.2008.11.008.
8. G. Gasparre, A.M. Porcelli, E. Bonora, L.F. Pennisi, M. Toller, L. Iommarini et al.
Disruptive mitochondrial DNA mutations in complex I subunits are markers of
oncocytic phenotype in thyroid tumors. Proc Natl Acad Sci USA, 104 (21) (2007),
pp. 9001–9006
9. G. Gasparre, E. Hervouet, E. de Laplanche, J. Demont, L.F. Pennisi, M.
Colombel et al. Clonal expansion of mutated mitochondrial DNA is associated with
26
tumor formation and complex I deficiency in the benign renal oncocytoma. Hum
Mol Genet, 17 (7) (2008), pp. 986–995
10. E. Bonora, A.M. Porcelli, G. Gasparre, A. Biondi, A. Ghelli, V. Carelli et al.
Defective oxidative phosphorylation in thyroid oncocytic carcinoma is associated
with pathogenic mitochondrial DNA mutations affecting complexes I and III.
Cancer Res, 66 (12) (2006), pp. 6087–6096
11. K. Ishikawa, K. Takenaga, M. Akimoto, N. Koshikawa, A. Yamaguchi, H.
Imanishi et al. ROS-generating mitochondrial DNA mutations can regulate tumor
cell metastasis. Science, 320 (5876) (2008), pp. 661–664
12. B.E. Baysal, R.E. Ferrell, J.E. Willett-Brozick, E.C. Lawrence, D. Myssiorek, A.
Bosch et al. Mutations in SDHD, a mitochondrial complex II gene, in hereditary
paraganglioma. Science, 287 (5454) (2000), pp. 848–851
13. I.P. Tomlinson, N.A. Alam, A.J. Rowan, E. Barclay, E.E. Jaeger, D. Kelsell et al.
Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin
leiomyomata and papillary renal cell cancer. Nat Genet, 30 (4) (2002), pp. 406–410
14. Scatena R, Bottoniand P, Giardina B, Mitochondria and Cancer: A Growing Role
in Apoptosis, Cancer Cell Metabolism and Dedifferentiation. Adv Exp Med
Biol. 2012;942:287-308. doi: 10.1007/978-94-007-2869-1_13.
15. Lehninger A, Nelson DL, Cox MM. Principles of Biochemistry. 6
th
edition. W.H.
Freeman; 2012
16. Jiang W, Hermolin J, Fillingame RH. The preferred stoichiometry of c subunits in
the rotary motor sector ofEscherichia coli ATP synthase is 10. Proc Natl Acad Sci U S
A. 2001; 98:4966–4971.
27
17. Das B, Mondragon MO, Sadeghian M, Hatcher VB, Norin AJ (1994) A novel ligand
in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+
transporting ATP synthase on the surface of tumor cell lines. J Exp Med 180: 273–
281
18. von Haller PD, Donohoe S, Goodlett DR, Aebersold R, Watts JD. Mass
spectrometric characterization of proteins extracted from Jurkat T cell detergent-
resistant membrane domains.Proteomics. 2001;1:1010–1021. doi: 10.1002/1615-
9861(200108)1:8<1010::AID-PROT1010>3.0.CO;2-L.
19. Moser TL, Stack MS, Asplin I, Enghild JJ, Hojrup P, et al. (1999)Angiostatin binds
ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci
USA 96: 2811–2816
20. Moser TL, Kenan DJ, Ashley TA, Roy JA, Goodman MD, et al. (2001) Endothelial
cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by
angiostatin. Proc Natl Acad Sci USA 98: 6656–6661
21. Zhao Wen-Li, Wang Jian, Tao Yan-Fang, Feng Xing, et. al. Inhibition of the ecto-
beta subunit of F1F0-ATPase inhibits proliferation and induces apoptosis in acute
myeloid leukemia cell lines. J Exp Clin Cancer Res. 2012; 31(1): 92.
22. Fuller CM, Time for TMEM? The Journal of Physiology,590, 5931-5932. December
1, 2012
23. Ferrera L, Caputo A, Galietta LJ (2010) TMEM16A protein: a new identity for
Ca(2+)-dependent Cl( −) channels. Physiology (Bethesda) 25: 357–363
24. Huang L, Szymanska K, Jensen VL, Janecke AR, Innes AM, et al.
(2011) TMEM237 Is Mutated in Individuals with a Joubert Syndrome Related
28
Disorder and Expands the Role of the TMEM Family at the Ciliary Transition
Zone. Am J Hum Genet 89: 713–730
25. Hsiao-Fan WEI. Thesis: Studies on the expression and function of the human
TMEM56 protein. USC master thesis. 2011
26. Poornima Murali. Role of a novel transmembrane protein, MTTS1 in mitochondrial
regulation and tumor suppressor. USC master thesis. 2012
27. Pan J, Sun LC, Tao YF, Zhou Z, et al. ATP synthase ecto- α-subunit: a novel
therapeutic target for breast cancer. Journal of Translational Medicine 2011, 9:211
28. Li X, Feng R, Huang C, Wang H, Wang J, Zang Z. MicroRNA-351 regulates
MTTS1 59 (DCF1) expression and mediates neural stem cell morphogenesis. RNA
Biol. 2012 Mar1; 9
Abstract (if available)
Abstract
Membrane proteins are indispensable members of the cell membrane. They anchor the cell, regulate its communication with the environment and degrade damaged cell membrane. Transmembrane proteins (TMEM) are a group of novel proteins that have been found to have key roles in differentiation and regulation of the cell [28]. TMEM56 is a new protein that was first identified in Dr. Ebrahim Zandi’s laboratory as an IKK-β interacting protein by immunoprecipitation and mass spectrometry [25]. Studies done in the lab have shown that TMEM56 has a potential tumor suppressor role. Therefore, we hypothesize that TMEM56 expression will be suppressed in cancer cells compared to normal cell and its expression level will be lower in cancer region. ❧ This study examines the TMEM56 expression level in normal and cancer regions of twelve breast cancer tissues. Laser capture Microdissection was used to isolate the cancer cells and the normal cells. Real time RT-PCR was performed on RNA samples that were extracted from isolated region. The data shows that 78% of the stage I cancer samples analyzed had lower TMEM56 expression level in the cancer region compared to the normal region. This is consistent with our hypothesis that the cancer cells will have lower expression level of TMEM56 as a result of its potential tumor suppressor role. Further studies can illustrate the mechanism by which TMEM56 possibly suppressor tumor growth.
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Creator
Mesfin, Fikir
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Core Title
Decreased levels of expression of transmembrane protein 56 (TMEM56) in breast cancer tissues
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
08/11/2014
Defense Date
06/11/2014
Publisher
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Tag
ATP synthase,breast cancer,laser capture microdissection,OAI-PMH Harvest,transmembrane protein 56
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Zandi, Ebrahim (
committee chair
), Hsieh, Chih-Lin (
committee member
), Landolph, Joseph R., Jr. (
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fmesfin@usc.edu,fmmesfin@gmail.com
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ATP synthase
breast cancer
laser capture microdissection
transmembrane protein 56