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Studies on the role of TMEM56 in tumorigenesis by using PTEN knockout mouse model

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Studies on the role of TMEM56 in tumorigenesis by using PTEN knockout mouse model

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STUDIES ON THE ROLE OF TMEM56 IN
TUMORIGENESIS BY USING PTEN KNOCKOUT
MOUSE MODEL

by

Jiabo Xu

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)
May 2016

Copyright 2016 Jiabo Xu

ii

TABLE OF CONTENTS
LIST OF FIGURES ....................................................................................................................... iii
LIST OF TABLES ......................................................................................................................... iv
ABSTRACT .................................................................................................................................... v
CHATPER 1 INTRODUCTION .................................................................................................... 1
1.1 PTEN ..................................................................................................................................... 1
1.2 Membrane Proteins ............................................................................................................... 4
1.3 Transmembrane (TMEM) Proteins and TMEM56 ............................................................... 5
CHAPTER 2 HYPOTHESIS .......................................................................................................... 9
CHAPTER 3 METHODS ............................................................................................................. 10
3.1 Construction of Mouse Models ........................................................................................... 10
3.2 Mice Maintenance ............................................................................................................... 11
3.3 Genotyping .......................................................................................................................... 11
3.4 Harvest of Mouse Livers ..................................................................................................... 12
CHAPTER 4 RESULTS ............................................................................................................... 13
4.1 Analysis of PTEN liver-specific Deletion Mouse Models ................................................. 13
4.2 Analysis of Double knockout of TMEM56 and PTEN Mice ............................................. 18
4.2.1 Comparison of the mice sacrificed at 12 weeks of age ................................................ 18
4.2.2 Comparison of the mice sacrificed at 40 weeks of age ................................................ 23
CHAPTER 5 DISCUSSIONS AND CONCLUSIONS ................................................................ 28
REFERENCES ............................................................................................................................. 31

iii

LIST OF FIGURES
Figure 1: The Structure of PTEN Protein ............................................................................... 2
Figure 2: Model for the Inhibition of PKB/Akt Signaling by PTEN ...................................... 3
Figure 3: TMEM56 ................................................................................................................. 6
Figure 4: TMEM56 Expression Level in Different Tissues. .................................................. 8
Figure 5: A. TMEM56 Expression Level in Different Cell Lines. B. In-vivo Xenograft
Assays. ............................................................................................................................ 8
Figure 6: Construction of Mouse Models ............................................................................. 10
Figure 7: Example of the Genotyping Results. ..................................................................... 14
Figure 8: Liver Size of WT and PTEN KO Mice Sacrificed at 40 Weeks of Age ............... 16
Figure 9: Liver Appearances of WT and PTEN KO Mice Sacrificed at 40 Weeks of Age
....................................................................................................................................... 17
Figure 10: Histological Analyses of Liver Sections from WT Mice and PTEN KO Mice
Sacrificed at 40 Weeks of Age. ..................................................................................... 18
Figure 11: Liver Size of Different Genotypes of Mice Sacrificed at 12 Weeks of Age ....... 19
Figure 12: Liver Appearances of Different Genotypes of Mice Sacrificed at 12 Weeks of
Age. ............................................................................................................................... 20
Figure 13: Histological Analyses of Liver Sections from Different Genotypes of Mice
Sacrificed at 12 Weeks of Age ...................................................................................... 22
Figure 14: Liver Size of Different Genotypes of Mice Sacrificed at 40 Weeks of Age ....... 24
Figure 15: Liver Appearances of Different Genotypes of Mice Sacrificed at 40 Weeks of
Age ................................................................................................................................ 25
Figure 16: Histological Analyses of Liver Sections from Different Genotypes of Mice
Sacrificed at 40 Weeks of Age ...................................................................................... 27

iv

LIST OF TABLES
Table 1: The Forward and Reverse Primers Used for the PCR. ................................................. 12
Table 2: The Sizes of the Genes. ................................................................................................. 13

v

ABSTRACT
A novel transmembrane protein, TMEM56, was first identified as an IKK-β interacting protein
by using mass spectrometry in Dr. Ebrahim Zandi’s laboratory. Previous studies in the laboratory
have shown that liver has the highest TMEM56 expression level among different tissues in mice.
Further analysis showed that knocking down TMEM56 level in HEPG2 cells resulted in slower
cell proliferation and smaller tumors in xenograft mouse models. However, knocking out
TMEM56 did not result in tumor growth in any organ of the mice.
In this study, we investigated the role of TMEM56 in tumorigenesis by combining TMEM56
knockout with liver-specific knockout of a known tumor suppresser, PTEN. In-vivo studies from
other groups have shown that deletion of PTEN in liver resulted in hepatomegaly, fatty liver,
increased glycogen synthesis and hepatocellular carcinomas. We first generated four different
mice genotypes, AlbCre
-/-
Pten
+/+
TM
+/+
(WT), AlbCre
-/-
Pten
+/+
TM
-/-
(TMEM56 KO),
AlbCre
+/-
Pten
loxP/loxP
TM
+/+
(PTEN KO) and AlbCre
+/-
Pten
loxP/loxP
TM
-/-
(PTEN&TMEM56 KO).
We compared the liver size, liver morphological and histological features of mice sacrificed at
12 and 40 weeks of age. PTEN KO and PTEN&TMEM56 KO mice were subjected to similar
degree of injury, indicating that knocking out TMEM56 did not have any effect on early or late
stage of tumorigenesis in liver. In summary, these studies suggest that TMEM56 does not
inhibit or further activate the tumor suppressor function of PTEN/PKB/Akt pathway. Further
studies are needed to detective whether TMEM56 has a role in tumorigenesis.

1

Chapter 1
Introduction
1.1 PTEN
PTEN (also known as MMAC1 and TEP1) stands for “Phosphatase and tensin homolog deleted
on chromosome 10”. Pten is a tumor suppressor gene which was identified in 1997 by two
independent groups (1, 2). PTEN is one of the most commonly lost tumor suppressors in human
cancer (3). Many studies have focused on the function of PTEN protein in tumorigenesis and
have demonstrated a significant rate of mutations of the Pten gene in gliomas cancer(4), prostate
cancer(5), breast cancer (6) and liver cancer (7). Specifically, in human liver cancers, the
expression of the Pten gene, which encodes the protein PTEN, is suppressed in almost half of all
liver cancers, and about 45% of all liver cancers have mutant PTEN. In addition, germline
mutations of Pten were also found in Cowden disease (CD), Lhermitte-Duclos disease (LDD)
and Bannayan-Zonana syndrome (BZS) (8).
The PTEN protein (Figure 1) is a 403 amino-acid protein, and a member of the large PTP
(protein tyrosine phosphatase) family (3). The N-terminal region of PTEN contains a
phosphatase domain (residues 7-185) with a phosphatase motif (HCSSGSSR, residues 123-130)
implicated in its tumor suppressor activity. The C-terminal region contains a C2 domain
(residues 186-351) and a C terminal tail (residues 352-403). C2 domain allows for the binding of
PTEN to phospholipids. The protein kinase CK2 phosphorylation sites are important for the
stability and activity of PTEN, and are contained in the tail region. A PDZ domain is also within
the tail region, which allows PTEN to bind membrane-associated guanylate kinase inverted
(MAGI) proteins to enhance efficiency of PTEN signaling. (3, 9, 10)

2

Figure 1: The Structure of PTEN Protein (9).
PTEN exerts its role as a tumor suppressor by negatively regulating the PKB/Akt signaling
pathway (11) (Figure 2). The PKB/Akt pathway is implicated in protection from apoptosis in
response to several growth factors and cytokines (12). Activated PI3K phosphorylates
phosphatidylinositol 4,5-bisphosphate [PIP2] to form phosphatidylinositol 3,4,5-trisphosphate
[PIP3] on the inner cell membrane, which propagates activation signals to downstream
molecules. The major function of PTEN is to dephosphorylate PIP
3
(13) and convert PIP3 back
to PIP2, so that the PKB/Akt pathway is turned off and the cell will undergo apoptosis (14).
Studies showed that deletions at chromosome 10q23 will cause the loss of function of the pten
gene (2), which will cause the PKB/Akt pathway be derepressed and stimulate cell growth and
survival (11, 15).

3

Figure 2: Model for the Inhibition of PKB/Akt Signaling by PTEN (16)
Besides its function to regulate the metabolic pathway, PTEN also plays an important role in cell
motility, especially in controlling the directionality of chemotaxis (17). Scientists also found that
PTEN can suppress tumor-induced angiogenesis within brain tumors (18). Moreover, studies
have demonstrated that in the nucleus, PTEN also plays an important role to maintain
chromosomal stability and DNA repair (19).
In vivo studies showed that Pten deletion in liver results in hepatomegaly, fatty liver, increased
glycogen synthesis and hepatocellular carcinomas. A hepatocyte-specific null mutation of pten in
mice, using the AlbCre-loxP system, has been generated to study the function of PTEN (20). In
this model, the progression of the fatty liver phenotype appeared to be age-dependent. At 1
month of age, the mutant hepatocytes appeared swollen with minimal lipid deposition, starting
from the area surrounding the central terminal hepatic venule. By the age of 3 months, the

4

mutant livers became bigger and revealed pale color. The ratio of liver weight to body weight in
the mutant animals also increased significantly. A substantial amount of lipid accumulation in
the mutant hepatocytes was clearly evident and further progressed to entire liver by 6 months,
with no apparent histological changes in other tissues (20). About 47% of AlbCrePten
loxP/loxP
livers develop liver cell adenomas by 10 months of age. All of AlbCrePten
loxP/loxP
livers showed
adenomas and 66% had hepatocellular carcinomas by the age of 19 months (7).
The Knudson two-hit or multi hit hypothesis states that cancer requires accumulation of a
number of gene mutations (21, 22). The Pten liver knockout model described above has been
used in several studies to test the Knudson hypothesis. For example, combined deletion of
Glucose-regulated protein 78 (GRP78) and Pten in mouse liver accelerated significantly tumor
development (23).
1.2 Membrane Proteins
Membrane proteins are proteins that interact with cell membranes. Based on the nature of the
membrane-protein interactions, membrane proteins can be categorized into two groups: integral
(intrinsic) membrane proteins and peripheral (extrinsic) membrane proteins (24). Most integral
membrane proteins are embedded in the phospholipid bilayer. Some integral membrane proteins
are anchored to the membrane by covalent bonds. Peripheral membrane proteins are not
embedded in the membrane. They attach to integral membrane proteins or directly interact with
lipid polar head groups (24).
Since membrane proteins are associated with lipid bilayer in different ways, the functions of
membrane proteins are different (16). Integral membrane proteins can function as channels to
convey large molecules and ions across the membrane. Some membrane receptor proteins, such
as enzyme-linked receptors, ion channel-linked receptors and G protein-coupled receptors, can

5

transfer signals between the cell’s internal and external environments. They can also act as
enzymes and cell adhesion molecules (25).
1.3 Transmembrane (TMEM) Proteins and TMEM56
A transmembrane protein is a type of integral membrane protein that can span entirely a
biological membrane(24). Transmembrane proteins function in various biological processes. For
example, TMEM16A and TMEM16B in the anoctamin family can act as calcium-activated
chloride channels (26). And abnormal accumulation of human transmembrane TMEM176A and
176B proteins has been associated with cancer pathology (27). Another TMEM protein,
TMEM237, was found mutated in individuals with a Joubert Syndrome Related Disorder,
expanding the role of the TMEM at the ciliary transition zone (28).
TMEM56 (Figure 3) was first discovered in Dr. Ebrahim Zandi’s laboratory by mass
spectrometry and immunoprecipitation as an IKK-β interacting protein. It has 263 amino acids,
an approximate molecular weight of 30KDa, and six α-helix transmembrane domains. The gene
that codes for TMEM56 is located on chromosome 1 (1p21 3).

6

A. Amino Acid Sequence of TMEM56

B. Predicted Structure of TMEM56

Figure 3: TMEM56. A. The amino acid sequence of TMEM 56; B. Predicted Structure of
TMEM56
http://www.uniprot.org/uniprot/Q96MV1

7

Previous studies of TMEM56 in Dr. Zandi’s laboratory have shown that liver has the highest
TMEM56 expression level among different tissues of mice (Figure 4). Reducing the expression
of TMEM56 in two different stable HEPG2 cell line resulted in much slower cell proliferation.
The in vivo xenograft assays also showed that reducing TMEM56 expression resulted in
formation of much smaller tumors. These data indicate that knocking down the level of
TMEM56 can affect tumor growth (Figure 5). However, there were no remarkable pathological
differences between TMEM56 knockout mice and normal mice (Zandi, unpublished data). This
indicates that TMEM56 by itself is not sufficient for tumorigenesis. However, the in vitro data
with HEPG2 cells indicated that it is possible that TMEM56 would either accelerate or suppress
cancer. The goal of this study is to examine if TMEM56 can alter the function of PTEN tumor
suppressor in mouse liver.

8

Figure 4: TMEM56 Expression Level in Different Tissues.
A

B

Figure 5: A. TMEM56 Expression Level in Different Cell Lines. B. In-vivo Xenograft Assays.

9

Chapter 2
Hypothesis
According to the previous studies in Dr. Zandi’s laboratory, knocking down TMEM56 in the
mouse did not cause tumor growth in any organ. However, knocking down TMEM56 levels in
HEPG2 cells resulted in slower cell proliferation and smaller tumors in xenograft mouse model.
As HEPG2 is a cancer cell line and had accumulated several mutations, to determine whether
TMEM56 plays a role in the process of carcinogenesis in combination with a known tumor
suppressor (PTEN), we hypothesized that knocking out TMEM56 in mice with null mutations in
Pten will alter the tumorigenic function of PTEN. The reason we chose PTEN and liver to test
this hypothesis is because liver has the highest TMEM56 expression level, and the Pten liver
knockout mouse model is a well-established cancer model, which has been used for similar
studies (23, 29, 30).

10

Chapter 3
Methods
3.1 Construction of Mouse Models
Targeted deletion of Pten has been reported previously (20). AlbCre
+/-
Pten
loxP/loxP
TM
+/+
mice on a
C57BL/6J background were kindly provided by Dr. Bangyan Stiles’ laboratory.
AlbCre
+/-
Pten
loxP/loxP
TM
+/+
mice were crossed with AlbCre
-/-
Pten
+/+
TM
-/-
mice on a C57BL/6J
background to generate AlbCre
+/-
Pten
loxP/+
TM
+/-
mice. The method to generate the
AlbCre
+/-
Pten
loxP/loxP
TM
-/-
mice is as shown in figure 6. Male animals of C57BL/6J background
were used for all experiments.

Figure 6: Construction of Mouse Models

11

3.2 Mice Maintenance
All protocols for animal use were reviewed and approved by the USC Institutional Animal Care
and Use Committee. The conditions are: The rodents room temperature ranged from 70-72℉; The
humidity ranged from 30%-70% RH and the room lights were 12/12 with a 6:00AM “on” and
6:00PM “off”.
3.3 Genotyping
Mouse tail biopsies were lysed in 150 µl lysis buffer (150 µl DirectPCR Lysis Reagent, 50 µg/ml
Proteinase K). Tubes were incubated in 55℃ blocks overnight and then shook to make sure no
tissue clumps were observed. Crude lysates were incubated at 85℃ for 45min on heat blocks
before spinning the hair down. The DNA extracts were then subjected to PCR analysis.
Three genes that needed to be identified were AlbCre, Pten and TM. For PCR 1.5 µl of DNA
lysate of each sample was used. The forward and reverse primers used for the PCR are listed in
Table 1. For each reaction tube, 0.5 µl of the forward and reverse primers were used. 0.8% agarose
gel was used to conduct the agarose gel electrophoresis (AGE).

12

A. Primer for Cre
Cre84-F 5’-GCG GTC TGG CAG TAA AAA CTA TC-3’
Cre85-R 5’-GTC AAA CAG CAT TGC TGT CAC TT-3’
B. Primer for PTEN
PTEN6637-F 5’-TCC CAG AGT TCA TAC CAG GA-3’
PTEN7319-R 5’-AAT CTG TGC ATG AAG GGA AC-3’
C. Primer for TMEM56
L5exn5tm-F 5’-GTA GTA TGC AGT TAG TCC TG-3’
L3ecn6tm-R 5’-GTG ATC CAA CGT ATG TGA AC-3’
Table 1: The Forward and Reverse Primers Used for the PCR.
3.4 Harvest of Mouse Livers
Mice were sacrificed in batches by the age of 12 weeks and 40 weeks. The whole livers were
harvested from the chests of mice and were weighed. The length of the harvested liver was
measured and pictures were taken. A 4-5mm wide edge of each left liver’s lateral lobe was cut and
fixed in formalin. Hematoxylin and Eosin (H&E) Staining was performed by USC/Norris
Comprehensive Cancer Center Translational Pathology Core Facility.

13

Chapter 4
Results
4.1 Analysis of PTEN liver-specific Deletion Mouse Models
The sizes of the genes are shown in Table 2. The genotypes of AlbCre
+/-
and Pten
loxP/loxP
, the
PTEN liver-specific deletion mouse model, as well as the AlbCre
+/-
Pten
loxP/+
TM
+/+
,
AlbCre
+/-
Pten
loxP/+
TM
-/-
, the double knockout of PTEN and TMEM56 mouse model were
confirmed by PCR and agarose gel. An example of the genotyping results was shown in Figure
7.

Cre84-F+Cre85-R
PTEN6637-F+
PTEN7319-R L5exn5tm-F+L3exn6tm-R
Cre +/+ 100bp Pten +/+ 500bp TM +/+ 600bp
Cre +/- 100bp
Pten
loxP/+ 500bp+650bp TM+/- 600bp
Cre -/- ---
Pten
loxP/loxP 650bp TM-/- ---
Table 2: The Sizes of the Genes.

14

A

B

Figure 7: Example of the Genotyping Results.

15

Liver-specific PTEN deletion will cause hepatomegaly, fatty liver, increased glycogen synthesis
and hepatocellular carcinomas (7, 20). To confirm the phenotype of PTEN specific deletion in
liver, we compared the morphological and histological differences between livers of
AlbCre
+/-
Pten
loxP/loxP
TM
+/+
(PTEN KO) mice and AlbCre
-/-
Pten
+/+
TM
+/+
(WT) mice. For this
comparison we used male mice, which were sacrificed at 40 weeks of age. Each group had five
mice.
Length of liver and ratio of liver weight to body weight were analyzed in WT and PTEN KO
mice at the age of 40 weeks (Figure 8). The liver length and the ratio of liver weight to body
weight in the mutant animals were both significantly increased, indicating that the mutant liver
was significantly larger than the wild type livers as previously has been reported (7).

16

A

B

Figure 8: Liver Size of WT and PTEN KO Mice Sacrificed at 40 Weeks of Age: A. Ratio of liver
weight to body weight. B. Liver Length.
In addition to the size difference, the mutant livers also had a different appearance from WT
livers (Figure 9). The mutant liver was pale in color and showed marked hepatomegaly. Tumors
had already developed on the left lobe of mutant liver in all five animals.
N=5

N=5

N=5

N=5

17

Figure 9: Liver Appearances of WT and PTEN KO Mice Sacrificed at 40 Weeks of Age (black
arrows denote examples of tumors).
Histological analysis demonstrated significant morphological changes of mutant livers (Figure
10). H&E staining results showed that mutant hepatocytes extended remarkably. The
cytoplasmic vacuoles became larger and pushed the nucleus against the cell membrane. The
histological section of aged mutant liver showed a lipid accumulation that resembled adipocytes
in fat tissues. We also observed an accumulation of cells in the periductal region of the PTEN
KO liver.

18

Figure 10: Histological Analyses of Liver Sections from WT Mice and PTEN KO Mice
Sacrificed at 40 Weeks of Age. A. Lower (×4) Magnification; B. Higher (×10) Magnification.
Together, these results indicated that PTEN KO liver was undergoing steatohepatitis and
developing hepatocellular carcinomas.
4.2 Analysis of Double knockout of TMEM56 and PTEN Mice
4.2.1 Comparison of the mice sacrificed at 12 weeks of age
Firstly, we compared the liver size, liver morphology, and the histology of four different mouse
genotypes at the age of 12 weeks to determine whether TMEM56 had any impact on the early
stage of the tumorigenesis. The histograms showed that knocking out PTEN significantly

19

increased both the ratio of liver weight to body weight and the liver length, but there was no
remarkable difference between TM
+/+
and TM
-/-
livers in liver weight ratio and liver length. This
result indicates that knocking out TMEM56 in mice does not have an effect on the liver size at
12 weeks of age. (Figure 11)
A

B

Figure 11: Liver Size of Different Genotypes of Mice Sacrificed at 12 Weeks of Age. A. Ratio
of Liver Weight to Body Weight; B. Liver Length.
N=5
N=5

N=5
N=5

N=5
N=5

N=5
N=5

20

The livers of PTEN liver-specific knockout mice didn’t show any tumorigenesis, but the fatty
accumulation was obvious. The difference between TM
+/+
liver and TM
-/-
liver was unremarkable.
The livers of 12-week-old PTEN KO and PTEN&TMEM56 KO mice were both enlarged and
light-colored. The PTEN KO and PTEN&TMEM56 KO livers were subjected to similar degree
of injury, which meant that knocking out of TMEM56 did not affect the early stage of
tumorigenesis in liver-specific PTEN knockout (Figure 12).

Figure 12: Liver Appearances of Different Genotypes of Mice Sacrificed at 12 Weeks of Age.

21

The histological analysis of liver sections from PTEN KO

and PTEN&TMEM56 KO

mice
showed similar histological features as that of sections from the PTEN knockout liver. The
cytoplasmic vacuoles became bigger and the lipid accumulation had started. Some nuclei were
still centrally located. No carcinoma foci could be seen in the sections. By comparing the PTEN
KO and PTEN&TMEM56 KO liver, we found that the levels of lipid accumulation and
hepatocytes injury were similar. The results showed that TM
-/-
liver didn’t have any remarkable
difference from TM
+/+
liver histologically. (Figure 13)

22

A

B

Figure 13: Histological Analyses of Liver Sections from Different Genotypes of Mice
Sacrificed at 12 Weeks of Age. A. Lower (×4) Magnification; B. Higher (×10) Magnification.

23

4.2.2 Comparison of the mice sacrificed at 40 weeks of age
We then studied the mice that were sacrificed at 40 weeks of age. Firstly, we compared the liver
size among four different groups of mice (Figure 14). Specifically deleting PTEN in the liver can
cause a remarkable increase in the liver size, both in liver weight and liver length. But TMEM56
knockout mice didn’t show much difference in liver size compared to wild type animals. There
was no significant difference between AlbCre
-/-
Pten
+/+
TM
+/+
(WT) and AlbCre
-/-
Pten
+/+
TM
-/-

(TMEM56 KO) mice either in the ratio of liver weight to body weight or the liver length. By
comparing the differences between the liver size of AlbCre
+/-
Pten
loxP/loxP
TM
+/+
(PTEN KO) mice
and AlbCre
+/-
Pten
loxP/loxP
TM
-/-
(PTEN&TMEM56 KO) mice, we found that knocking out
TMEM56 can only increase less then 1% in the ratio of liver weight to body weight and about
0.2cm in the liver length.

24

A

B

Figure 14: Liver Size of Different Genotypes of Mice Sacrificed at 40 Weeks of Age. A. Ratio
of Liver Weight to Body Weight. B. Liver Length.

N=5

N=5
N=5

N=5

N=5
N=5

N=5
N=5

25

The appearances of different livers are shown in Figure 15. Livers of WT and TMEM56 KO
mice were normal and looked similar. There were no remarkable pathological changes on
TMEM56 KO livers. As we discussed before, livers of the PTEN liver-specific knockout mice
had fatty accumulation and developed hepatocellular carcinomas. Similar to PTEN KO liver,
PTEN&TMEM56 KO liver also developed tumors at the age of 40 weeks. We counted the
average number of the tumors in the livers of PTEN KO

and PTEN&TMEM56 KO

and found
that the average number of the tumors on PTEN KO liver was 10 and the average number of the
tumors on PTEN&TMEM56 KO liver was 12, which was approximately same as that of PTEN
KO liver (Figure 15).

Figure 15: Liver Appearances of Different Genotypes of Mice Sacrificed at 40 Weeks of Age
(black arrows denote examples of tumors).

26

The H&E staining (Figure 16) comparison of the liver sections from WT and TMEM56 KO

mice
didn’t show any cytological changes in hepatocytes. The histological differences between
TMEM56 KO mice and PTEN&TMEM56 KO mice were remarkable as we discussed before.
Nevertheless, there were no remarkable differences between the liver sections from PTEN KO

and PTEN&TMEM56 KO mice. Together the data show that knocking out TMEM56 does not
cause a histological difference between PTEN KO mice and PTEN&TMEM56 KO mice.

27

A

B

Figure 16: Histological Analyses of Liver Sections from Different Genotypes of Mice
Sacrificed at 40 Weeks of Age. A. Lower (×4) Magnification; B. Higher (×10) Magnification.

28

Chapter 5
Discussions and Conclusions
TMEM56 is a transmembrane protein that was first identified in Dr. Ebrahim Zandi’s laboratory
as an IKK-β interacting protein by using immunoprecipitation and mass spectrometry. Prior
studies in Dr. Zandi’s laboratory showed a potential role of TMEM56 in tumorigenesis.
We used the PTEN liver-specific deletion mouse model to determine whether TMEM56 has a
role in tumorigenesis in vivo. In vivo studies have shown that PTEN deletion in liver results in
hepatomegaly, fatty liver, increased glycogen synthesis and hepatocellular carcinomas in mouse
models. About 47% of AlbCrePten
loxP/loxP
livers developed liver cell adenomas by 10 months of
age. All of AlbCrePten
loxP/loxP
livers showed adenomas and 66% had hepatocellular carcinomas
by the age of 19 months (7). Pten liver knockout mouse model is a well-established cancer
model, which has been used for similar studies, for example, combined deletion of
Glucose-regulated protein 78 (GRP78) and Pten in mouse liver accelerated tumor development
significantly (23).
In this study, we generated PTEN&TMEM56 KO mice by crossing AlbCre
+/-
Pten
loxP/loxP
TM
+/+
mice with AlbCre
-/-
Pten
+/+
TM
-/-
mice on a C57BL/6J background. First, we used mice that were
sacrificed at 12 weeks of age to examine if TMEM56 could affect early stage of tumorigenesis in
the liver. At 12 weeks of age, PTEN KO liver did not show any tumorigenesis, but the fat
accumulation could be observed both by morphology and histology. PTEN&TMEM56 KO livers
had the same features and degree of injury as PTEN KO livers, indicating that knocking out
TMEM56 did not have any additional effect on early stage of tumorigenesis in liver caused by
PTEN deletion.

29

Then we compared the liver size, liver morphology and histology features from different
genotypes of mice sacrificed at 40 weeks of age. PTEN knockout livers showed larger size and
fatty accumulation compared to WT. PTEN&TMEM56 KO livers did not show any significant
morphological and histological difference from PTEN KO livers. These results indicate that
knocking out TMEM56 in mice did not affect the hepatocellular carcinomas more than what is
caused by PTEN deletion.
PTEN exerts its role as a tumor suppressor by negatively regulating the PKB/Akt pathway and
stimulating the cell growth. However, the pathway through which TMEM56 functions is
unknown. The reason why TMEM56 does not affect hepatocellular carcinomas more than what
is caused by PTEN deletion may be that TMEM56 affects a pathway other than the PKB/Akt.
Therefore, knocking out TMEM56 in PTEN knockout livers did not cause any difference in
tumorigenesis.
Future studies can focus on investigating why knocking down TMEM56 in HEPG2 cells can
cause slower cell proliferation and smaller tumors in the xenograft mouse model and get
TMEM56 does not seem to have any affect on hepatocellular carcinomas. The fact that knocking
out TMEM56 did not have any effect on tumorigenesis in mouse models could be the result of
protein redundancy. The whole body TMEM56 knockout may induce some other pathways that
can compensate for the lack of TMEM56 in mice. HEPG2 cells may have lost such
compensatory pathways. Therefore, knocking down TMEM56 in HEPG2 cells can result in
slower cell proliferation and smaller tumor growth. Further investigation of the role of TMEM56
in HEPG2 cells may provide some insight into function of TMEM56.
Previous studies in Dr. Zandi’s laboratory indicated that TMEM56 might be a potential oncogene
or tumor suppressor gene. This study, however, indicates that in mice, TMEM56 by itself does
not function as either an oncogene, or tumor suppressor. Furthermore, in a liver-specific PTEN

30

deletion system, TMEM56 did not show any effect on hepatocellular carcinogenesis. Further
studies are needed to investigate the function of TMEM56 in tumorigenesis.

31

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advanced cancers. Nature genetics. 1997;15(4):356-62.
2. Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, et al. PTEN, a putative protein
tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science (New
York, NY). 1997;275(5308):1943-7.
3. Leslie NR, Downes CP. PTEN function: how normal cells control it and tumour cells lose it.
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Abstract (if available)

Abstract A novel transmembrane protein, TMEM56, was first identified as an IKK-β interacting protein by using mass spectrometry in Dr. Ebrahim Zandi’s laboratory. Previous studies in the laboratory have shown that liver has the highest TMEM56 expression level among different tissues in mice. Further analysis showed that knocking down TMEM56 level in HEPG2 cells resulted in slower cell proliferation and smaller tumors in xenograft mouse models. However, knocking out TMEM56 did not result in tumor growth in any organ of the mice. ❧ In this study, we investigated the role of TMEM56 in tumorigenesis by combining TMEM56 knockout with liver-specific knockout of a known tumor suppresser, PTEN. In-vivo studies from other groups have shown that deletion of PTEN in liver resulted in hepatomegaly, fatty liver, increased glycogen synthesis and hepatocellular carcinomas. We first generated four different mice genotypes, AlbCre⁻⁄⁻Pten⁺⁄⁺TM⁺⁄⁺ (WT), AlbCre⁻⁄⁻Pten⁺⁄⁺TM⁻⁄⁻ (TMEM56 KO), AlbCre⁺⁄⁻Ptenˡᵒˣᴾ⁄ˡᵒˣᴾTM⁺⁄⁺ (PTEN KO) and AlbCre⁺⁄⁻Ptenˡᵒˣᴾ⁄ˡᵒˣᴾTM⁻⁄⁻ (PTEN&TMEM56 KO). We compared the liver size, liver morphological and histological features of mice sacrificed at 12 and 40 weeks of age. PTEN KO and PTEN&TMEM56 KO mice were subjected to similar degree of injury, indicating that knocking out TMEM56 did not have any effect on early or late stage of tumorigenesis in liver. In summary, these studies suggest that TMEM56 does not inhibit or further activate the tumor suppressor function of PTEN/PKB/Akt pathway. Further studies are needed to detective whether TMEM56 has a role in tumorigenesis.

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Asset Metadata

Creator Xu, Jiabo (author)

Core Title Studies on the role of TMEM56 in tumorigenesis by using PTEN knockout mouse model

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Publication Date 04/20/2016

Defense Date 03/22/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag hepatocellular carcinomas,mouse model,OAI-PMH Harvest,PTEN,TMEM56

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Zandi, Ebrahim (committee chair), Hsieh, Chih-Lin (committee member), Landolph, Joseph R., Jr. (committee member)

Creator Email jiaboxu@usc.edu,xujiabo123456@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-235549

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Legacy Identifier etd-XuJiabo-4322.pdf

Dmrecord 235549

Document Type Thesis

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Rights Xu, Jiabo

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...

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