1
Identification and functional analysis of fusion gene in breast cancer through
comprehensive analysis of genomic structure
by Pang Qing Yuan
Supervisor: Koichiro Inaki Genome Institute of Singapore
Cancer Biology and Pharmacology 2
Submitted to the School of Biological Sciences in partial fulfillment of the requirements for Final Year Project
NANYANG TECHNOLOGICAL UNIVERSITY
April 2010
2
DECLARATION I declare that, in accordance with School requirements this thesis is under 6000 words in length; all presented work was performed within the official project time frame as stated below; all presented work was performed by me, unless otherwise specified; all relevant work experience gained before the Final Year Project is stated below; the input by my supervisor, or delegated supervisor, into this thesis was limited to reviewing of up to two hard copy drafts; this thesis is my own work, unless otherwise referenced, as defined by the NTU policy on plagiarism and I have read the NTU Honour Code and Pledge; the included Abstract, Introduction, Results, Discussion and Conclusions sections will be submitted to SafeAssignment no later than 24 hours after hardcopy submission. Final Year Project start date: 11th January 2010 Final Year Project submission date: 23rd April 2009 Total number of weeks: 16 weeks Pre-Final Year Project experience May-Dec 2008, Research attachment at Genome Institute of Singapore, Cancer Biology and Pharmacology 2 under Dr Koichiro Inaki’s supervision. May-July 2009, Research attachment at Genome Institute of Singapore, Cancer Biology and Pharmacology 2 under Dr Koichiro Inaki’s supervision. Student’s signature: Date:
3
TABLE OF CONTENTS
ACKNOWLEDGEMENTS - 5
ABRREVIATIONS - 6
ABSTRACT - 7
INTRODUCTION - 8
MATERIAL AND METHODS - 10
Functional prediction of fusion gene protein products - 10
Plasmid constructs - 10
Cell culture - 10
Transfection and transient colony formation assay - 10
Scattering assay - 11
Generation of over-expressing stable cell lines - 11
Western Blot - 12
Co-immunoprecipitation - 13
RESULTS - 14
Functional prediction of RPS6KB1:TMEM49 fusion gene
products
- 14
Transient over-expression of HA tagged S6K fusion constructs - 15
S6K fusion constructs when cultured in RPMI-1640
supplemented with 3% FBS showed significant increased colony
size but not when cultured in RPMI-1640 supplemented with
10% FBS
- 16
S6K fusion construct E4-E12 displayed scattered colonies when
cultured in RPMI-1640 supplemented with 10% FBS
- 18
Over-expression of proposed p70-binding partners fused with
FLAG tags in T47D cell lines followed by immuno-precipitation
- 19
DISCUSSION - 22
Functional possibilities of S6K fusion constructs - 22
Over-expression of S6K fusion constructs could confer a cell
growth advantage when cells are cultured in low serum
conditions.
- 23
Scattered colonies observed for over-expressing E4:E12 T47D
cells suggest a migratory phenotype
- 23
4
Higher expression profile of E4-E12 was observed in cells co-
transfected with p85 and p70 as compared to other FLAG
constructs
- 24
Future work 24
CONCLUSIONS 26
REFERENCES 27
5
ACKNOWLEDGEMENTS
I would like to express my gratitude towards Dr Koichiro Inaki for his guidance
and tutelage throughout my duration on this project. The experience in the lab
has allowed me to pick up many techniques and also the process thought
which are essential in being a good scientist. I would also like to extend my
gratitude to Dr Leena Ukil, Edward Chee Yu Wing, Shakeela Banu and
Tareen Ho Yuet Fern for their help and guidance when I am in doubt. Lastly, I
would like to thank Jarius Ng Jia Jin and Natalie See and other lab members
for assisting me in one way or another.
6
ABBREVIATIONS
AKT v-akt murine thymoma viral oncogene homolog 3, protein
kinase B γ
CMV Cytomegalovirus
DMEM Dulbecco Modified Eagle Medium
EDTA Ethylenediaminetetraacetic acid
EGTA Ethylene glycol tetraacetic acid
ETS Homo sapiens ETS variant 1
FISH Fluorescent in situ hybridization
FLAG FLAG octapeptide
G418 Geneticin
HA Hemagglutanin
IRS-1 Insulin receptor substrate 1
mAb Monoclonal antibody
MCF7 Human breast adenocarcinoma cell line, Michigan Cancer
Foundation-7
mTOR Mammalian target of rapamycin
PET Paired end ditag
PPP2CA Protein phosphotase 2 catalytic subunit
qPCR Quantitative polymerase chain reaction
RACE Rapid amplification of cDNA ends
RPMI Roswell Park Memorial Institute
RPS6 Ribosomal protein S6
RPS6KB1 Ribosomal protein S6 Kinase
S6K S6 Kinase
SMART Simple modular architecture research tool
T47D Human ductal breast epithelial tumour cell line
TMEM49 Transmembrane protein 49
TMPRSS2 Transmembrane protease, serine 2
VMP1 Vacuolar membrane protein 1
PI3K Phosphatidylinositol 3-kinase
RPTOR regulatory associated protein of mTOR
7
ABSTRACT
Identification and functional analysis of fusion gene in breast cancer through comprehensive analysis of genomic structure
by
Pang Qing Yuan
Chromosomal abnormalities are common hallmark features in cancer
and they give rise to fusion genes through various mechanical process. In
order to identify these novel fusion genes, PET technology was employed in
this study. From gPET analysis in the frequently amplified locus of 17q23,
multiple gene fusions between RPS6K1 and TMEM49 were identified. This
particular gene fusion was found to be expressed in MCF7 cell lines as well
as 22 out of 70 clinical samples. In our study, we have focused on 6 fusion
structures, which were identified previously by Inaki et al, unpublished data.
We show that by over-expressing these 6 fusion constructs, E4:E12 in
particular displayed significant increase in colony size in low serum conditions,
which suggests that it could confer a cell growth advantage. In addition, we
show that this particular S6K fusion construct displayed scattered colonies
suggesting a migratory phenotype associated with increased p70-S6K activity.
Taken together, these findings suggest that these S6K fusion products could
affect counterparts within the PI3K/Akt/mTOR pathway and contribute towards
oncogenic phenotype. This study also suggests several possibilities into
understanding the functional role of S6K fusion gene and its contribution in
breast cancer.
8
INTRODUCTION
Breast cancer is one of the most common cancers that afflict women
around the world. An average of 1000 women are being diagnosed with
breast cancer annually in Singapore (Singapore Cancer Society). Therefore in
recent years, it has been of increasing importance to discover more
biomarkers as well as anti-cancer drugs that will allow early intervention and
better prognosis.
Chromosomal abnormalities are one of the common features in cancer
cells. These chromosomal abnormalities result in fusion genes that could
eventually cascade towards the progression of cancer. As normal somatic
chromosomes are stable in nature, fusion gene identification serves as an
ideal target in the diagnosis of cancer as well as development of anti-cancer
drugs. Gene fusions could result from translocations, deletions and tandem
duplications of chromosomal structure (Prensner and Chinnaiyan 2009;
Rabbitts et al., 2009). If certain functional gene fusions expressions were to
be up regulated by strong promoters or enhancers, it could result in increased
proliferation of cells and affect the progression of cancer. A classic example
would be Bcr-Abl fusion gene which is formed due to t(9;22)(q34.1;q11.2) and
is observed in 90% of chronic myeloid leukemia.
Tomlins et al (2005) have reported the presence of genomic
rearrangements that resulted in the fusion of TMPRSS2 to ETS family
members. The strong promoter elements of TMPRSS2 mediates the over-
expression of ETS family members which are implicated in 23 out of 29 cases
of prostate cancer. Other studies (Soda et al., 2007; Tomlins et al., 2007)
have reported various gene fusions in different carcinomas. Therefore, by
investigating the transcriptional and functional consequence of these gene
fusions, it would allow us to gain a better understanding of these structural
abnormalities contributing to effective therapy intervention.
Comprehensive analysis of the breast cancer genome structure using
PET (paired end ditag) (Ng et al., 2006; Ruan et al., 2007; Fullwood et al.,
2009) technology developed by Genome Institute of Singapore allowed us to
identify abnormal genetic structures, which produced novel gene fusions
(Inaki et al., unpublished data). From data screening using gPET (genomic
PET) browser, 2687 genetic abnormalities were discovered. Out of the 2687
9
genetic abnormalities, 160 were predicted to produce fusion genes. RT-PCR
validation was conducted for the predicted genes, and 77 out of the 160
(48.1%) were expressed (Sinclair et al., 2003). Among the fusion gene
transcripts, RPS6KB1-TMEM49 fusion gene expressed in MCF-7 cell line was
further analyzed (Inaki et al., unpublished data). This fusion gene is caused by
tandem duplication event within 17q23, which is a high frequently amplified
locus in breast cancer (Sinclair, Rowley et al. 2003). Thus 5’ part of RPS6KB1
is fused with 3’ part of TMEM49. RPS6KB1 is a kinase involved in
PI3K/Akt/mTOR pathway, which is highly activated in the cancers where over-
expression of this gene is known to confer cell growth advantage. Over-
expression of this gene is also reported to promote epithelial to mesenchymal
phenotype in ovarian cell lines. (Mahalingam and Templeton 1996; Dennis et
al., 1998; Pullen et al., 1998) TMEM49/VMP1 is a cell membrane associated
protein reported to be involved in organelle trafficking and triggering
autophagy. Its reduced expression has also been reported to decrease
invasive capability in tumour cells. (Ropolo et al., 2007; Calvo-Garrido et al.,
2008; Sauermann et al., 2008)
In order to identify cancer cell lines and clinical specimens that express
these RPS6KB1-TMEM49 fusion transcripts, RT-PCR was conducted.
Although the expression of the fusion gene was only found in MCF-7 among
the 8 cell lines tested, it showed recurrent expression (22 out of 70) in clinical
samples. In the expressed tumors, 10 multiple fusion points were determined.
After validation of the expression of these fusion transcripts, investigations of
the functional implications and their putative role in the contribution to the
oncogenic phenotype were followed.
10
MATERIALS AND METHODS
Functional prediction of fusion gene protein products
mRNA sequences of functional RPS6KB1 and TMEM49 were obtained from
NCBI. The various exons combinations of the RPS6KB1 and TMEM49 were
created and predicted using Simple Modular Architecture Research Tool
(http://smart.embl-heidelberg.de/).
Plasmid constructs
RPS6, PPP2CA, S6K-p70 and S6K-p85 were constructed by RT-PCR using
primers targeting full-length coding sequence PCR enzyme used was
Phusion® Flash High-Fidelity PCR Master Mix (Finnzymes, Finland). To
construct IRS-1 and mTOR full-length cDNA were first amplified by PCR and
ligated into pSC-B-amp/kan (Stratagene, Agilent Technologies), followed by
PCR targeting full-length coding sequence. As the IRS-1 and mTOR are of
length 3.8-kb and 7.7-kb respectively, sequencing primers (1st Base,
Singapore) were designed in order to validate the sequence. All PCR
constructs were subjected to NotI/XbaI restriction endonuclease digestion
(New England Biolabs, Canada) before ligation into p3XFLAG-CMV
expression vector (Sigma-Aldrich).
Cell culture
T47D cells were propagated at 37°C and 5% CO2 in humidified atmosphere in
RPMI 1640 (BSF, A*STAR) supplemented with 10% heat-inactivated FBS
(Invitrogen). MCF7 cells were propagated with D-MEM (BSF, A*STAR)
supplemented with 10% heat-inactivated FBS.
Transfection and colony formation assay
T47D cells were plated to 90% confluency on a 15 cm2 tissue culture plate
before trypsinization using 0.25% Trypsin-EDTA (Gibco®, Invitrogen). 2x105
cells/well were then plated in triplicate sets into 6 well plates and left into the
incubator for 2 days. Transfection was carried out once the T47D cells
reaches 80% confluency. T47D were transfected with modified pcDNA3.1(-)
(Invitrogen) vectors where inserted constructs are fused with HA tag at C-
11
terminus namely Mock (empty vector), E1:E7, E1:E8, E4:E12, E1:E11,
E2:E11 and E4:E11. 250μL of Opti-MEM® I Reduced Serum Media
(Invitrogen) were added to respective labeled 4 ml polycarbonate tubes.
Following which, 4 μg (equivalent of Mock) of various fusion constructs were
added respectively. In the meantime, 10μL of Lipofectamine™ 2000
(Invitrogen) was added into another set of labeled tubes with 250μL of Opti-
MEM® I Reduced Serum Media (Invitrogen) followed by incubation at room
temperature for 5 minutes before combining the fractions together and adding
it drop-wise into the respective wells of the 6 well plates after 30 minutes
incubation. Transfected cells were then left in the incubator for 24 hours. The
transfected cells were then trypsinized and re-plated into 1x105 cells/well in
triplicate samples. Cells were then left in the incubator for 24 hours. After 24
hours, RPMI-1640 (BSF, A*STAR) supplemented with 3% or 10% FBS was
added to the respective plates. 400 μg/mL of G418 (Gibco®) was added to the
media as means of selection. Fresh selection media was changed every week
and growth was observed. After 3-5 weeks, cells were fixed with methanol
and stained using crystal violet.
Scattering Assay
The scattering assay was performed as described in both Pon et al and
Zhou et al (Zhou and Wong 2006; Pon et al. 2008), colony morphologies were
defined as normal or scattered. After the colony formation assay as
mentioned above, pictures were taken under 10X objective lenses using
image capture (Nikon, Japan). Scattered colonies were judged by a typical
change in morphology, characterized by cell-cell dissociation and the
acquisition of a migratory fibroblast-like phenotype. Photos were randomized
and tagged before they are shown to participants. Scattering activity was
measured in a total number of scattered colonies from 50 colonies for 2 wells
in a single batch for each S6K construct. Participants were recruited to judge
the scattering activity and their scores were recorded and tabulated.
Generation of over-expressing stable cell lines
T47D cells were plated to 90% confluency on a 15 cm2 tissue culture
plate before trypsinization using 0.25% Trypsin-EDTA (Gibco®, Invitrogen).
12
1x106 cells/well were then plated in triplicate sets into 10 cm2 dishes and left
into the incubator for 2 days. Transfection was carried out once the T47D cells
reaches 80% confluency. T47D were transfected with modified pcDNA3.1(-)
(Invitrogen) vectors where inserted constructs are fused with HA tag at C-
terminus namely Mock (empty vector) and E4:E12. 1.25mL of Opti-MEM® I
Reduced Serum Media (Invitrogen) were added to respective labeled 14 ml
polycarbonate tubes. Following which, 20 μg (equivalent of Mock) of fusion
construct was added respectively. In the meantime, 50μL of Lipofectamine™
2000 (Invitrogen) was added into another set of labeled tubes with 1.25mL of
Opti-MEM® I Reduced Serum Media (Invitrogen) followed by incubation at
room temperature for 5 minutes before combining the fractions together and
adding it drop-wise into the respective wells of the 10 cm2 dishes after 30
minutes incubation. Transfected cells were then left in the incubator for 24
hours before RPMI-1640 (BSF, A*STAR) supplemented with 10% FBS and
400 μg/mL of G418 (Gibco®) as mean of selection. Fresh selection media was
changed every week and growth was observed over a period of 2 weeks.
Stable over-expressing cells were harvested after 2 weeks to determine the
level of protein expression.
Western Blot
Transfected cells were lysed in 1X cell lysis buffer (20mM Tris HCl, pH
7.5, 150mM NaCl, 1mM Na2EDTA, 1mM EGTA, 1% Triton, 2.5mM Sodium
pyrophosphate, 1mM β-glycerophosphate, 1mM Na2VO4, 1μg/ml Leupeptin)
(Cell Signaling Technology®) with 1X Complete Proteinase inhibitor (Roche
Applied Science). 15 μL of total cell lysate from each sample was separated
by 15% Tris-Glycine SDS-PAGE and transferred to 0.2 μm nitrocellulose
membrane. Membranes were blocked at room temperature for an hour in 5%
Milk-TBST (Tris-Buffered Saline Tween-20). The membranes were then
incubated overnight with following antibodies overnight: anti HA High Affinity
Rat IgG (Roche Applied Science), Phospho-AKT Thr308 Rabbit mAb (Cell
Signaling Technology®, 9275), AKT Rabbit mAb (Cell Signaling Technology®)
Phospho-S6 Ribosomal Protein Rabbit mAb (Cell Signaling Technology®), S6
Ribosomal Protein (5G10) Mouse mAb (Cell Signaling Technology®, 2217).
Horse-radish peroxidase (HRP) conjugated secondary antibodies – goat anti-
13
rat, goat anti-rabbit, goat anti-mouse (BioRad) - were then used to detect the
primary antibodies. Protein of interest was then visualized using Enhanced
Chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech,
GE Healthcare).
Co-immunoprecipitation
Pre-clearing of lysate was conducted using 100μL of diluted lysate
added with 50μL of Rec. Protein A-Sepharose 4B beads (Zymed Laboratories,
Invitrogen) for 1 hour at 4°C with rotary shaking. Pre-cleared lysate was then
incubated with anti-FLAG®-M2 mouse mAb (Sigma-Aldrich) for 2 hours at 4°C
with rotary shaking. After which, Rec Protein A-Sepharose 4B beads (Zymed
Laboratories, Invitrogen) was added and incubated for an hour at 4°C with
rotary shaking. The mixture was precipitated and wash 5 times before elution
in 1X SDS sample buffer with β-mercaptanol. The eluted mixture was then
subjected to western blot.
14
RESULTS
Functional prediction of RPS6KB1:TMEM49 fusion gene products
Previously, work has been done to validate the presence of fusion
transcripts in MCF7 cell line and clinical samples. In order to predict whether
these fusion transcripts could give rise functional novel protein products, the
sequences of the validated transcripts were subjected to analysis using
SMART. All fusion gene constructs were found to contain no functional kinase
domain. E1:E7 (Fig 1A) and E1:E8 (Fig 1B) were predicted to contain 4 trans-
membrane domains, while E2:E11 (Fig 1C) and E4:E12 (Fig 1D) contained 1
trans-membrane domain, On the other hand, E1:E11 (Fig 1E) and E4:E11
(Fig 1F) were predicted to be truncated protein products due to a frame shift
in the fusion point.
However, all constructs still retain the N terminal region of the
RPS6KB1 protein which is reported to bind to C terminal region. This
interaction results in the kinase activity inhibition (Dennis et al. 1998). The N
terminus is also reported to contain a conserved TOS motif, which is
important in the binding with mTOR/RPTOR to facilitate the phosphorylation
of the T389 residue required in p70 S6K activation. (Schalm and Blenis, 2002;
Nojima et al., 2003)
Figure 1: Various RPS6KB1:TMEM49 fusion gene construct protein domains predictions. (A) E1:E7 and (B) E1:E8 predicted with 4 transmembrane domains, (C) E2:E11 and (D) E4:E12 predicted with 1 transmembrane domain, (E) E1:E11 and (F) E4:E11 were predicted to be truncated proteins.
15
Transient over-expression of HA tagged S6K fusion constructs
6 fusion constructs with various fusion points fused with HA tags were
previously constructed during my attachment. These fusions constructs
consisted of various combinations of fusions between exons of RPS6KB1 and
TMEM49, which were eventually termed like E1:E11 where the former refers
to the exon of RPS6KB1 and the latter being the exon of TMEM49. As both
E1:E11 and E4:E11 were predicted to be truncated products due to out of
frame fusion, the constructs were designed to be in coding frame so as to
express the following truncated products. In order to understand the
expression profile of the various constructs, it was transfected and transiently
over-expressed in T47D (Figure 2A) as well as MCF7 cell lines. (Figure 2B).
Figure 2: Transient over-expression of S6K fusion constructs in T47D and MCF7 cell lines. (A) Over-expression of HA-tagged S6K fusion constructs in T47D cell lines. (B) Over-expression of HA-tagged S6K fusion constructs in MCF7 cell lines.
From the results, expression profiles of the S6K constructs were
identical in both cell lines. Low expression profiles were observed for E1:E7
16
(24.5 kDa), E1:E8 (29.3 kDa) and E1:E11 (9.95 kDa) in both cell lines.
However, E4:E11 (18.7 kDa) was not expressed at all even after it was
designed to be in coding frame. Strong expression profiles were seen for
E2:E11 (16.7 kDa) and E4:E12 (19.7 kDa) constructs. Expected protein
weight was calculated based on the expression sequence of the various
sequences. Some of the over-expressed proteins were detected at higher
molecular weights, which could be a result of post-translational modifications.
We next sought to determine whether these fusion constructs could confer a
cell growth advantage in low S6K expressing T47D cell lines.
S6K fusion constructs when cultured in RPMI-1640 supplemented with
3% FBS showed significant increased colony size but not when
cultured in RPMI-1640 supplemented with 10% FBS
In order to understand if the S6K constructs could confer cell growth
advantage, colony formation assay was performed to find out if there was any
significant colony size phenotype in cancer cell growth by over-expression of
the constructs in T47D cell line which are low RPS6KB1 expressing cells.
Transfected cells with the respective various constructs were observed for
changes in cell morphology, significant colony morphological changes in
comparison to negative control (Mock). When the cells were cultured in 10%
FBS serum, there was significant colony size reduction for 4 of 6 the
constructs only in the 3rd batch (Figure 3C). However, when it was cultured in
3% FBS, there was marked significant increase in colony size observed
(Figure 4). However, this was only observed in the first batch but not in the
subsequent batches. Therefore there is a need to repeat the experiment in
triplicates in order to observe any significant colony size differences.
17
Figure 3: Colony formation assay cultured in RPMI-1640 supplemented with 10% FBS over a period of 5 weeks. Biological replicates were done and fixed after 5 weeks. (A) Batch 1 was conducted in duplicate sets (B) Batch 2 was conducted in duplicate sets (C) Batch 3 was conducted in triplicate sets, Student’s T-test P<0.05 (D) Western Blot of the over-expressing S6K constructs.
18
Figure 4: Colony formation assay cultured in RPMI-1640 supplemented with 3% FBS over a period of 5 weeks. Experiment was conducted in duplicate sets, Student’s T-test P<0.05, P<0.005, P<0.0005 vs control.
We next sought to determine whether there are other phenotypes
associated with the over-expression of these S6K fusion constructs.
S6K fusion construct E4-E12 displayed scattered colonies when
cultured in RPMI-1640 supplemented with 10% FBS
As reported in Pon et al. and Zhou et al. (Zhou and Wong 2006; Pon,
Zhou et al. 2008), over-expression of constitutive active p70 S6K in ovarian
cancer cell lines promotes epithelial to mesenchymal transition (EMT), which
is seen as scattered colonies when cells were cultured in media.
In order to understand if S6K fusion constructs could enhance the over-
expression of p70-S6K, a scattering assay was conducted. Scattered colonies
was observed for E4:E12 (53.5%) when it was cultured in RPMI-1640
supplemented with 10% FBS as scored by participants (Figure 5A). Even
when it was cultured in RPMi-1640 supplemented with 3% FBS (Figure 6), the
scattered colonies was even more evident for E4:E12 construct (77.5%).
19
Figure 5: Scattered colonies cultured in RPMI-1640 supplemented with 10% FBS were scored. (A) Percentage of scattered colonies. Columns, means of three experiments done in duplicates; bars, standard deviation. Student’s T-Test P<0.0005 vs control (B) Representative photos of over-expressing Mock and E4:E12 transfected T47D cells.
Figure 6: Scattered colonies cultured in RPMI-1640 supplemented with 3% FBS were scored. (A) Percentage of scattered colonies. Columns, means of single experiment done in duplicates; bars, standard deviation. Student’s T-Test P<0.05 vs control (B) Representative photos of over-expressing Mock and E4:E12 transfected T47D cells.
Next, we sought to understand how E4:E12 construct could contribute
to such a phenotype observed. Therefore, we proposed that E4:E12 could
possibly bind to various known p70-S6K binding partners to give rise to the
phenotype observed. We would also like to study whether stable over-
expressing E4:E12 cells could possess migratory or invasive properties.
Over-expression of proposed p70-binding partners fused with FLAG
tags in T47D cell lines followed by immuno-precipitation
Proposed partners in the PI3K/Akt/mTOR pathway that may interact
with S6K fusion construct E4:E12 are the 2 isofoms of RPS6KB1 namely p85-
20
S6K and p70-S6K. Other proposed partners included RPS6, PPP2CA, mTOR
and IRS-1 (Ferrari et al. 1993; Nojima et al. 2003; Zhang et al. 2008).
However as mTOR and IRS require more time to generate since they are
rather large fragments. Thus we were able to generate only p85-S6K, p70-
S6K, RPS6 and PPP2CA, which are fused with FLAG tags. We sought to
determine the binding interactions between these proposed partners and
E4:E12 via co-transfection and transient over-expresion (Figure 7).
Figure 7: Expression profile of co-transfected T47D cells. Left: co-transfected Mock-HA cells with proposed binding FLAG tag partners. Right: co-transfected S6K E4:E12-HA with proposed binding FLAG tag partners
Proposed FLAG tagged partners p85-S6K (85 kDa), p70-S6K (70 kDa),
PPP2CA (35.6 kDa) and RPS6 (28.7 kDa) displayed high expression profiles
while E4:E12 displayed a low expression profile across the board. However,
T47D cells that are co-transfected with p85-S6K and E4:E12 displayed slightly
higher expression level as compared to other FLAG tagged constructs.
21
Figure 8: Co-immunoprecipitation using anti-FLAG-m2 mouse IgG and detection using anti-HA rat mAb with anti-rat goat antibody.
Immuno-precipitation was conducted to determine if the protein
products could interact with each other. However, as the antibody used for the
pull-down was anti-FLAG mouse mAb and detected was using anti-HA rat as
primary antibody while anti-rat IgG goat antibody as secondary antibody
resulted in species cross-reactivity and no clear result could be detected
(Figure 8). Anti-HA rabbit antibody will be used in the subsequent experiments
in order to detect the presence of HA tagged E4:E12 in the immuno-
precipitated samples.
22
DISCUSSION
Functional possibilities of S6K fusion constructs
The N terminal region of p85/p70-S6K has been extensively studied
and reported to be involved in the regulation of activity by binding to the C
terminus (Mahalingam and Templeton 1996; Dennis et al. 1998; Pullen et al.
1998). Since all fusion constructs still retained their N terminus despite lacking
a functional kinase domain, they could possibly bind to functional p85/p70-
S6K C terminus in order to relieve the inhibition and bypass the first step of
activation. Thus the fusion constructs would exert a dominant negative effect
and eventually enhance the activity of the functional p85-S6K or p70-S6K.
Recent studies have also identified a conserved TOS motif in both N
terminus and C terminus region, which is required for binding to
mTOR/RPTOR in order to facilitate the phosphorylation of T389 (Schalm et al.
2002; Nojima et al, 2003). As described in Dennis and Pullen et al, the
phosphorylation of this residue allows the S6K protein to adopt a open
confirmation thereby allowing PDK1 to bind and activate it through
phosphorylation of the T229 in the kinase domain. This also suggests that a
fusion construct could possibly bind to mTOR/RPTOR to enhance functional
p85-S6K/ p70-S6K activation.
Functional p70-S6K is also reported to be involved in the regulation of
IRS-1 via negative feedback mechanisms (Zhang et al., 2008). A dominant
negative mutant S6K as described in the same paper prevented the
degradation of the IRS-1 when stimulated by tumour necrosis factor alpha
(TNF-α). This dominant negative mutant described (Addgene, Plasmid 8985:
pRK7-HA-S6K1-KR) only possessed a functional N terminus that is encoded
by exons 1 to 5 and its protein profile is similar to that of our fusion constructs.
In essence, these studies suggest that all fusion constructs could
possibly sustain the activation of PI3K/Akt/mTOR pathway and eventually
result in the constitutive activation of all functional counterparts within this
pathway. Thus in theory, phenotypes that are associated with the over-
expression of constitutive active S6K mutants should manifest with the over-
expression of these fusion constructs should the hypothesis be true.
23
Over-expression of S6K fusion constructs could confer a cell growth
advantage when cells are cultured in low serum conditions.
In low serum conditions (3% FBS), over-expression of S6K fusion
constructs E1:E8, E4:E12, E1:E11 and E2:E11 showed a marked significant
increase in colony size as compared to control. However, this was not the
same case when the cells were cultured in ample serum conditions (10%
FBS). Surprisingly, we observed a larger number of colonies of about two-fold
difference when cells were cultured in ample serum conditions as compared
to low serum conditions. This could be possibly relate to the fact that these
cells aggregate together in low serum conditions in order to survive better
thus resulting in lower numbers of colonies but with increased size. However,
differences in colony size in relation to serum conditions were only significant
(Student’s T-test, p<0.05) for control (Mock) and fusion constructs E1:E7 and
E2:E11. This phenotype observed was also only documented between
samples from a single biological set. It also coincided with preliminary
screening data conducted by other previous students during their stint (not
presented). Nonetheless, biological replicates are still required in order to
reach a definitive conclusion. If this phenotype was to be validated true, then it
would suggest that the fusion constructs have a part to play in the over-
activation of the functional counterparts within the PI3K/Akt/mTOR pathway.
Scattered colonies observed for over-expressing E4:E12 T47D cells
suggest a migratory phenotype.
Over-expression of E4:E12 in T47D resulted in an average of 53.5%
scattered colonies as scored by participants. This scattered colony phenotype
suggests that over-expression of this construct may be involved in intrinsic
pathways that contributed to such an observation.
In order to be able to investigate further, we have to look at pathways
and effectors implicated in the actin cell remodeling. Activating missense
mutations of PI3K reported in breast and ovarian cancers resulted in the
oncogenic phenotype, which promotes cell survival and increased cell
migration (Qian et al., 2004; Levine et al., 2005; Meng et al., 2006). All 3
effectors PI3K, mTOR and p70-S6K are required in the induction of actin
filament remodeling as reported in Qian et al. (2004). Since cell migration is
24
closely related to invasive capability of a tumour cell. It would be interesting to
understand the biological mechanisms in which fusion construct E4:E12 could
give rise to this phenotype observed.
Higher expression profile of E4-E12 was observed in cells co-
transfected with p85 and p70 as compared to other FLAG constructs.
Increased expression of E4:E12 was observed from the western blot
(refer to Figure 7) for T47D cells which were transiently transfected with
functional p85 and p70 S6K. The result suggest 2 possibilities where the first
would be over-expression of p85/p70 S6K may give rise to increased protein
synthesis thus explaining the increased protein expression profile of fusion
construct E4:E12. While the second possibility would be that with over-
expression of the fusion construct E4-E12, IRS-1 is prevented from
degradation thus sustaining the activation of endogenous S6K eventually
resulting in a constant loop activation of increased protein synthesis.
In order to understand whether this over-expression could be due to
which possibility mentioned. Another western blot has to be performed using
antibodies targeted at IRS-1, PI3K phospho-RPS6, RPS6, Akt and phospho-
Akt. If protein expression levels are high across the board, then the second
possibility as mentioned would make a more convincing conclusion. However,
if protein levels are high only for phospho-RPS6 and RPS6 then the first
possibility mentioned would be considered as a plausible conclusion.
Future work
Currently, IRS-1 and mTOR FLAG tagged constructs are still in the
process of being cloned and generated. These constructs upon completion
will be used for transient co-transfection and subjected to immuno-
precipitation in order to find out if E4:E12 fusion construct could interact with
these 2 proposed binding partners. This will allow us to obtain a better
understanding of the mechanisms in which this dominant negative S6K
protein could contribute to the cancer cell progression.
To gain a better conclusive finding on whether over-expression of
E4:E12 could contribute to cell proliferation and migration phenotype similar to
that of PI3K active mutants as reported. Functional PI3K constructs could be
25
generated and over-expressed in T47D cells. If these 2 populations of cells
displayed a similar phenotype, then it would suggest that fusion construct
E4:E12 work via prevention of IRS-1 degradation and eventually leading to
constant loop activation of the PI3K/Akt/mTOR pathway as described in the
first section. Having said so, an over-expressing IRS-1 cell line could also be
generated in the meantime to understand if both IRS-1 over-expression and
PI3K over-expression would give rise to the same phenotype observed.
Other studies such as scratch or wound assay have to be conducted in
order to understand if the cell migratory phenotype is observed in stable over-
expressing E4:E12 cells. Invasion assay will proceed once the migratory
phenotype is validated.
26
CONCLUSIONS
In this study, we have identified that over-expression of fusion
construct E4:E12 could confer a cell growth advantage. We have also
identified a scattering phenotype, which suggests that the over-expression
confers a migratory phenotype. These results suggests a possibility that the
fusion construct may act similarly as a dominant negative mutant of S6K and
could possibly give rise to the phenotypes observed through enhanced activity
of functional p85/p70-S6K within the PI3K/Akt/mTOR pathway. Extensive
studies still has to be conducted in order to seek more insight into the exact
molecular mechanisms of this fusion construct and its functional role in the
oncogenic phenotype.
27
REFERENCES Calvo-Garrido, J., S. Carilla-Latorre, et al. (2008). "Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion, and development." Mol Biol Cell 19(8): 3442-3453. Dennis, P. B., N. Pullen, et al. (1998). "Phosphorylation sites in the autoinhibitory domain participate in p70(s6k) activation loop phosphorylation." J Biol Chem 273(24): 14845-14852. Ferrari, S., R. B. Pearson, et al. (1993). "The immunosuppressant rapamycin induces inactivation of p70s6k through dephosphorylation of a novel set of sites." J Biol Chem 268(22): 16091-16094. Fullwood, M. J., C. L. Wei, et al. (2009). "Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses." Genome Res 19(4): 521-532. Levine DA, et al. (2005). “Frequent mutation of the PIK3CA gene in ovarian and breast cancers.” Clin Cancer Res 11:2875–2878 Mahalingam, M. and D. J. Templeton (1996). "Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains." Mol Cell Biol 16(1): 405-413. Meng, Q., Xia, C., et al. (2006). “Role of PI3K and AKT specific isoforms in ovarian cancer cell migration, invasion and proliferation through the p70S6K1 pathway.” Cell. Signal. 18, 2262–2271 Ng, P., J. J. S. Tan, et al. (2006). "Multiplex sequencing of paired-end ditags (MS-PET): a strategy for the ultra-high-throughput analysis of transcriptomes and genomes." Nucl. Acids Res. 34(12): e84-. Nojima, H., C. Tokunaga, et al. (2003). "The mammalian target of rapamycin (mTOR) partner, raptor, binds the mTOR substrates p70 S6 kinase and 4E-BP1 through their TOR signaling (TOS) motif." J Biol Chem 278(18): 15461-15464. Pon, Y. L., H. Y. Zhou, et al. (2008). "p70 S6 kinase promotes epithelial to mesenchymal transition through snail induction in ovarian cancer cells." Cancer Res 68(16): 6524-6532. Prensner, J. R. and A. M. Chinnaiyan (2009). "Oncogenic gene fusions in epithelial carcinomas." Current Opinion in Genetics & Development 19(1): 82-91. Pullen, N., P. B. Dennis, et al. (1998). "Phosphorylation and activation of p70s6k by PDK1." Science 279(5351): 707-710.
28
Rabbitts, T. H. (2009). "Commonality but Diversity in Cancer Gene Fusions." Cell 137(3): 391-395. Ropolo, A., D. Grasso, et al. (2007). "The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells." J Biol Chem 282(51): 37124-37133. Ruan, Y., H. S. Ooi, et al. (2007). "Fusion transcripts and transcribed retrotransposed loci discovered through comprehensive transcriptome analysis using Paired-End diTags (PETs)." Genome Res 17(6): 828-838. Sauermann, M., O. Sahin, et al. (2008). "Reduced expression of vacuole membrane protein 1 affects the invasion capacity of tumor cells." Oncogene 27(9): 1320-1326. Schalm, S. S. and J. Blenis (2002). "Identification of a conserved motif required for mTOR signaling." Curr Biol 12(8): 632-639. Sinclair, C. S., M. Rowley, et al. (2003). "The 17q23 amplicon and breast cancer." Breast Cancer Res Treat 78(3): 313-322. Singapore Cancer Society. (Retrieved on 09 April 2010) http://www.singaporecancersociety.org.sg/lac-fcb-how-common-is-breast-cancer.shtml Soda, M., Y. L. Choi, et al. (2007). "Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer." Nature 448(7153): 561-566. Tomlins, S. A., B. Laxman, et al. (2007). "Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer." Nature 448(7153): 595-599. Qian, Y., Corum L. et al. (2004). “PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration.” Am. J. Physiol. Cell Physiol. 286: C153–C163 Zhang, J., Z. Gao, et al. (2008). "S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2." J Biol Chem 283(51): 35375-35382. Zhou, H. Y. and A. S. Wong (2006). "Activation of p70S6K induces expression of matrix metalloproteinase 9 associated with hepatocyte growth factor-mediated invasion in human ovarian cancer cells." Endocrinology 147(5): 2557-2566.