Dissertation Final Copy

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EGF and SDF signalling and Akt-mediated resistance to apoptosis in
human MDA-MB-231 breast cancer cells.
By Matthew Cadwallen
Supervised by Dr. Phil Dash
March 2015
BSc Biological Sciences
School of Biological Sciences

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Contents
1. Abstract……………………………………………………………….3
2. Introduction…………………………………………….......………...3
2.1. Activation of Akt………………………………………………4
2.2. Downstream Activity of Phosphorylated Akt………………..5
2.3. Inhibiting the PI3K/Akt pathway…………………………….6
2.4. EGF and SDF…………………………………………………..7
3. Materials………………………………………………………………9
3.1. Chemicals and Reagents……………………………………….9
3.2. Cell Lines……………………………………………………….9
4. Methods………………………………………………………………..9
4.1. Cell Culture…………………………………………………….9
4.2. Treatment with EGF and SDF………………………………..10
4.3. Whole Cell Lysates…………………………………………….10
4.4. Bradford Assay………………………………………………...11
4.5. SDS-PAGE……………………………………………………..11
4.6. Western Blotting Reagents……………………………………12
4.7. Running Gels…………………………………………………...12
4.8. Gel Transfer……………………………………………………13
4.9. Blocking and Enhancing Chemilluminescence…….………...13
4.10. CellDesigner……………………………………………………14
4.11. Analysis of Images and Quantification……………………….14
5. Results………………………………………………………………….14
5.1. Activation of Akt by EGF and SDF…………………………...14
5.2. Processed Diagram of the EGF/EGFR/PI3K/Akt Signalling
Pathway Constructed Using CellDesigner…………………….16
5.3. The Role of SHC/Grb2/SOS1-mediated Activation of Ras in
EGFR Signalling………………………………………………..17
5.4. PI3K-mediated Activation of Akt……………………………...18
5.5. Processed Diagram of the SDF/CXCR4/JAK/STAT Pathway
Constructed Using CellDesigner……………………………….19
5.6. The Role of G-protein-recruitment of Scaffolding Proteins in
the Activation of the Ras/PI3K/Akt Pathway………………...20
6. Discussion………………………………………………………………20
7. Conclusion……………………………………………………………..22
8. Figures and Legends – CellDesigner………...……………………….23
9. References……………………………………………………………..29

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1. Abstract
Human breast cancer is the second most common cancer of humans, and is one of the more well-
studied cancers at present day because of this. It has become increasingly well understood that the
serine/threonine kinase and proto-oncogene known as Akt (PKB) plays a vital role in the majority of
human cells with regards to cell growth, proliferation and survival, and because of this it is prone to
mutations which often result in the uncontrolled activation of Akt, which can have tumorigenic
effects. Although Akt can be activated by several pathways, specific focus was placed on the PI3K
pathway and the receptors associated with its activation due to the fact that this signalling cascade
contains several oncoproteins, with genes that are known to be mutational hotspots in breast cancer.
Western Blotting of MDA-MB-231 cell lysates was carried out after exposure to Endothelial Growth
Factor (EGF) and Stromal Cell Derived Factor (SDF) for varied time intervals. This revealed that both
EGF and SDF caused activation of Akt. Interestingly, EGF activated Akt more sharply, but SDF was
able to activate Akt more quickly. In response to these observations, an investigation was
implemented using recognised bioinformatics tools in network biology to attempt to explain the above
results. It was found that EGF can play a role in activating PI3K through Grb2 dependent or
independent activation of Gab1, as well by activation of the Ras-GTP switch by EGFR activated
SHC/Grb2/SOS1 complex. SDF on the other hand, was found to activate PI3K by directly activating
the Ras-GTP switch in the membrane, although this mechanism is currently poorly understood. This
paper delivers a discussion regarding the possible targets for treatment in the inhibition of Akt action
if an oncogenic EGFR or CXCR4 receptor was found to be present in a patient. In addition, the
potential effects this would have on signalling molecules downstream of Akt and the possible
mutations influencing EGF/SDF signalling are discussed.
2. Introduction
Cancer remains one of the most significant and challenging illnesses to combat to date, due to the
numerous pathways involved in the development of very specific cancer diseases. It is often the case
that the cancer is unique in a highly specific way, for example a certain pathway may not be affected
in one type of cancer and then may be in another. One example of this is the Phosphatidylinositol 3-
kinase (PI3K)/Akt pathway, which plays a critical role in the regulation of several cancers, including
breast cancer (Osaki, Oshimura & Ito, 2004; Vivanco & Sawyers, 2002). The numerous effects it has
on cancerous cells means it remains very difficult to treat and resistance can quickly be evolved to
deal with any cytotoxic chemotherapy (Zhu et al, 2014). One large topic of interest is the role of Akt
(Protein Kinase B) in cancer cell survival and apoptosis resistance.

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2.1 Activation of Akt
The phosphorylation of Akt by serine/threonine kinases is primarily mediated by upstream signals
originating at the cell membrane. Stimulation of receptor tyrosine kinases by specific chemokines and
growth factors triggers a number of signalling cascades, some of which subsequently end in the
activation of Akt.
In summary, the essential steps that must take place for the activation of Akt involve a receptor-
mediated activation of PI3K activity, which facilitates the conversion of PIP2 to PIP3 as represented
in Figure 1. PIP3 is a membrane protein capable of binding to the Pleckstrin Homology (PH) domains
of PDK1 and Akt, resulting in the anchorage of Akt to the membrane where its kinase activity is
activated by PDK1-mediated phosphorylation of the Threonine 308 (Thr308) residue. In order to fully
activate Akt, phosphorylation of the Serine 473 (Ser473) residue must also take place. It is thought
that the rictor-MTOR complex plays a vital role in this process as well as insuring stabilisation of Akt
by binding Thr450, although the mechanisms for this are currently poorly understood (Sarbassov et
al, 2005). It is the activation of Akt that modulates the function of several molecules that are involved
in cell growth, proliferation and acceleration of the cell cycle itself (Fresno-Vara et al, 2004). The
PI3K/Akt pathway is normally a tightly regulated pathway that is important to normal cellular
Figure 1. Simplistic diagram of the mechanisms involved in the activation of Akt. Growth factor-
mediated activation results in the activation of the PI3K catalytic subunit (p110) when the
regulatory subunit (p85) binds to the growth factor receptor. In contrast, G Protein-coupled
receptor-mediated activation of PI3K involves the direct phosphorylation of GDP-Ras, generating
active GTP-Ras. GTP-Ras is capable of activating the PI3K catalytic subunit (Osaki, Oshimura &
Ito, 2004).

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function, however, once in a tumour environment it can express oncogenic potential. The overall
effect is an overexpression of proteins downstream of Akt resulting in anti-apoptotic effects.
2.2 Downstream Activity of Phosphorylated Akt
Figure 2 represents a summary of Akt downstream activity, such as the activation of anti-apoptotic
proteins like Bad, which when phosphorylated cannot bind and inhibit the survival factor Bcl-XL, so
proliferation is allowed to continue (Downward, 2004). Phosphorylated Akt in breast cancer can also
lead to the overexpression of the X-linked inhibitor of apoptosis (XIAP) gene. Increased expression of
Figure 2. Represents a basic overview on the mechanisms by which Akt is activated, as well as
a compilation of several downstream effectors of Akt, which may result in pro-survival and
anti-apoptotic effects as well encourage cell proliferation and differentiation through
Serine/Threonine phosphorylation activity.

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this gene is known to prevent apoptosis and be overexpressed in breast cancers. It was shown in 100
breast cancer patients, XIAP expression was significantly higher than that of a healthy patient, and
also correlated with the mutant TP53 gene (Xu et al, 2014). TP53 is also activated by Akt, with the
functioning tumour-suppressing P53 protein regulating cell proliferation. However, TP53 is the
largest mutation hotspot in breast cancer (30% of breast cancers possess a mutated TP53 gene) and
the protein product is dysfunctional and cannot regulate cell proliferation effectively (Bertheau et al,
2013). Just one more of the many ways in which Akt inhibits apoptosis and promotes survival of
tumour cells is by activating a known promoter of cell survival called Nuclear Factor kappa B (NF-
kB) indirectly. Akt phosphorylates IkB kinase which consequently degrades the NF-kB inhibitor and
allows it to promote cell survival (Downward, 2004). Mutations are capable of arising in several
molecules in the PI3K/Akt pathway, including Akt itself. The most common mutations occur in
molecules upstream of Akt, for example mutated version of PI3K is the most common in breast
cancers, but specifically a H1047R mutation in the catalytic subunit of PI3K (PIK3CA) can lead to
aberrant expression of these molecules and cause the overexpression of Akt (Garcia-Dios et al, 2013).
In association with mutations promoting increased expression of Akt, decreased expression in the
tumour suppressor gene Phosphatase and Tensin homologue (PTEN) is also very common. The
mutation involves a change to the sequence in the splice site at intron 4 and results in an absent exon
5, which is important for the tyrosine phosphatase activity of PTEN (Vranic et al, 2007). If PTEN
function is impaired, the pathway which activates Akt will be allowed to continue, increasing the
concentration of phosphorylated Akt. The overall effect of mutations such as this are present in
several cancers around the body, and correlate strongly with an increased aggressive tumour
behaviour and a derease in relapse-free survial. Akt therefore plays an outstanding role in the
uncontrolled proliferation of cancerous cells, and has become a central target for cancer therapy
(Garcia-Dios et al, 2013).
2.3 Inhibiting the PI3K/Akt Pathway
In terms of current treatment, a lot of focus has been placed on PI3K and upstream effectors of Akt
because they’re the oncogenes most often mutated and contributing to the aggressiveness of the breast
cancer. Wortmannin has been identified as a promising inhibitor of PI3K. It acts by permanently
inhibiting PI3K, which in turn has been proven to have antitumour effects, for example when studying
Wortmannin inhibition the 50% inhibitory concentration is approximately 2-4nm (Powis, 1994).
However, the problems associated with using Wortmannin as an effective treatment involve tackling
its poor stability in aqueous environments, but water-soluble equivalents are being developed for
clinical trials (Fresno-Vara, 2004).
In addition to both PI3K and Akt inhibitors, there have also been several attempts in targeting many
of the downstream substrates of Akt. It is important to remember that Akt has a number of molecules

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on which it has an effect, all of which play a role in either cell proliferation regulation (most notably
mammalian target of rapamycin or mTOR) or inhibition of apoptosis (BAD/procaspase-9 –
proapoptotic proteins that are deactivated when phosphorylated by Akt). The majority of these
molecules either have some form of treatment or have drugs in ongoing clinical trials, but because
there are more than one molecules that have the same effect, treating just one of these downstream
effectors will often not have a significant beneficial effect and have to be coupled with other drugs or
chemotherapy.
The inhibition of Akt itself though has proven in the past to be very difficult and generally
unsuccessful, with toxicity developing before little, if any, anti-tumour effects can be observed. One
prime example in the past was the use of Triciribine as an Akt inhibitor, which acts by preventing
DNA and protein synthesis (Hoffman et al, 1996). Triciribine yielded some success in Phase I trials,
leading to its testing in human Phase II trials. 14 patients metastatic for breast cancer were subject to
35mg/m2 per day by 24 hour infusion over a 5 day period, once every 6 weeks. This concentration
was increased by 5mg/m2 each time until toxicity was observed. The results presented brought about
the conclusion that triciribine was ineffective at all doses in patients metastatic to breast cancer due to
unaffected progression of metastasis accompanied by fatal side effects resulting from hyperlipidemia.
In spite of the toxicity, it is important to note that even in the presence of this inhibitor, Akt was
evidently relatively unaffected and activated. This has brought about several questions about how Akt
expression is much more challenging to inhibit in comparison to the success of PI3K and PDK1
inhibitors. The following suggests that there are several pathways that ultimately result in the
activation of Akt and its subsequent downstream anti-apoptotic effectors (Hoffman et al, 1996).
2.4 EGF and SDF
The chemokine, Stromal Cell Derived Factor (SDF) is known to result in the phosphorylation of Akt
when it binds to its membrane receptor, CXCR4. This of course triggers the anti-apoptotic and pro-
survival signals associated with the activation of Akt as well as those involved in the Nuclear Factor
kappa B (NF-kB) pathway. SDF binding can inhibit the production of Tumour Necrosis Factor (TNF)
which is normally induced by DNA damage resulting from radiation, contributing highly to
radiotherapy resistance (Wang et al, 1996). As well as this, NF-kB can upregulate CXCR4 expression
and therefore sensitise the cancer cell to SDF activation, promoting metastasis (Helbig et al. 2003).
Under normal circumstances, CXCR4 activation by SDF is vital for angiogenesis by mediating the
activation of Vascular Endothelial Growth Factor (VEGF) and inducing the recruitment of
Endothelial Progenitor Cells (EPCs) from the bone marrow. It is this involvement that, when
oncogenic, can induce neovascularisation and provide a transport medium for metastatic cancer cells.
In addition, as a target receptor for this chemokine, CXCR4 has been shown to be attracted to any
tissues which produce high levels of SDF (its ligand) through chemotactic mechanisms, which can aid

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the process of metastasis when oncogenic. As a result of this observation, it has become known that
the preferred metastatic sites of breast cancer such as the lung, bone marrow and lymph nodes happen
to produce peak levels of SDF. One of many functions of CXCR4 is to induce the polymerisation of
actin, which accounts for the ability of the breast cancer cells to migrate and possess invasive
properties when overexpressed due to mutation (Muller et al, 2001). In the absence of SDF however,
primary tumour growth and survival increases and there is no noticeable effect on apoptosis, even
though the rate of metastasis sharply decreased (Wendt et al, 2008). This suggest that there are several
other molecules involved that can encourage cell survival and avoid apoptosis in the tumour
environment. One notable example is Epidermal Growth Factor (EGF) and the pathways it activates.
EGF is a growth factor that promotes Akt phosphorylation through initial receptor tyrosine kinase
(EGFR) activity (Burgering & Coffer, 1995). Under normal function EGF binds EGFR and promotes
dimerization at the membrane, activating a number of signal transduction pathways that ultimately
result in cellular proliferation, survival and differentiation (Herbst, 2004). It is now known that there
is pathway crosstalk involved and this was the reason that led to the experimentation on their
activation of Akt.
These are two different mechanisms of activation that both result in the activation of Akt, and if they
are shown to activate Akt to varying extents, then this may help to confirm the initial hypothesis that
Akt can be activated from more than one pathway. We carried this out in MDA-MB-231 breast cancer
cell lines and attempted to then determine the chemical pathways involved for both SDF and EGF.
The reason for this is if there are any mutations detected in patients involving the PI3K/Akt pathways
for either SDF or EGF, mapping these pathways may help find a common factor that could potentially
be a target for therapy in these patients. Inhibiting the action of any mutated and overexpressing
proteins in these pathways may reduce the extent to which Akt is phosphorylated, reducing the anti-
apoptotic effects and pro-survival effects it has, and if this reduces the rate at which metastasis can
occur and then be coupled with chemo/radiotherapy, then treatment of the cancer could be very
positively affected.

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3. Materials
3.1 Chemicals and Reagents
Materials Supplier Catalogue Number
Dulbecco’s Modified Eagle
Medium (DNEM), Trypsin-
EDTA and Phosphate Buffer
Saline (PBS)
Life-technologies 31885-049, 25300-104 and
10010-056 respectively.
BSA Standards Thermo Scientific 23209
SDF and EGF Peprotech 300-28A and AF-100-15
respectively.
Fluorescent Rabbit Secondary
Antibody, membrane,
Ammonium persulphate
(APS), methanol and 100%
ethanol
Fisher Scientific 10348502, 15259894, 7727-54-
0, 67-56-1 and 64-17-5
respectively.
Akt (Ser) Antibody New England Bio-Labs 9271S
Protein Ladder Bio-rad Laboratories #161-0374
Protease Inhibitor Cocktail
(PIC)
Calbiochem, UK
3.2 Cell Lines
The cell line that was exclusively used in this study were invasive MDA-MB-231 breast cancer cells.
4. Methods
4.1 Cell Culture
All cell culturing took place within the tissue culture section of the lab, including the growth,
maintenance and the removal of the cells, with all cell lines operated on in a class II laminar flow
cabinet. For incubation, cell lines were stored in each of their 75cm tissue culture flasks in an
incubator that was kept at 37 degrees celcius, 5% carbon dioxide and a humidity of 95%. The
incubator, as well as all equipment used, were sterilised by spraying with ethanol before every use.
Cells were grown to approximately 90-100% confluence in the tissue culture flask, which contained
8ml of Dulbecco’s Modified Eagles Medium (DNEM). Upon reaching the above approximate
confluences, the old media was removed and safely disposed of, before washing with 2 or 3ml of
PBS. After the cells had been successfully washed, they were exposed to approximately 2ml of

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trypsin-EDTA in order to dissociate the cells from the flask surface and suspend them freely in the
media for transfer to another flask. Flasks containing the cells and 2ml of trypsin were moved to
another tissue culture flask where 6ml of new media was added (to neutralise the effects of trypsin) to
make it up to 8ml in total before incubation for another 48 hours. Once 90-100% confluence was
reached again, the 8 ml of media was again drained off and disposed of, cells were washed with PBS
and then 2 ml of trypsin was added, and then 6 ml of media after 3 minutes. 1 ml of media was taken
from this flask and placed into a new one, and made up to 8 ml with new media. In total, 8 new flasks
containing 1ml of approximately 10% cell confluence and 7 ml of media were made in preparation for
treatment with EGF and SDF. In all cases, flasks were labelled appropriately with the date and cell
type before incubation.
4.2 Treatment with EGF and SDF
Before the experiment took place, each of the 8 tissue culture flasks were labelled appropriately, 4
flasks were allocated to EGF and 4 to SDF, with a control for each. The flasks were labelled as EGF
10 minutes, EGF 20 minutes and EGF 30 minutes, and the same was carried out for the 4 SDF flasks.
EGF comes at a stock dilution of 10 µg/ml and SDF at 100 µg/ml, so in order to get the same
concentration the volume of SDF that needed to be added was worked out and divided by 10. The
following experiment took place in a sterile tissue culture hood, and PBS was placed on ice prior to
the experiment. 16 µl of EGF was added to its 3 respective flasks while the control received no EGF
treatment, and 1.6 µl of SDF was added to its 3 respective flasks while the control received no SDF
treatment. After each flask had successfully been exposed to EGF and SDF for the allocated amount
of time, all the media was swiftly removed and 5ml of ice cold PBS was added to the flasks to stop
the reaction. The flasks were left resting on ice and were ready to be prepared as whole cell lysates.
4.3 Whole Cell Lysates
Once all the samples had been exposed to their respective growth factor or chemokine for their
allocated amounts of time, the 5 ml of PBS added was removed from each of the 8 flasks, and then 1
ml of new PBS was added. A cell scraper was applied thoroughly in each tube to resuspend the cells
in the 1 ml of PBS, and once deemed to be fully resuspended, the 1 ml of PBS containing the
suspended, treated cells were pipetted into 8 eppendorf tubes and labelled in the same manner as the
flasks. The eppendorf tubes were then centrifuged at 1500 rpm for 10 minutes at 4 degrees Celcius.
This led to the formation of a pellet of cells at the bottom of each eppendorf tube, allowing the 1 ml of
PBS to be removed without removing any of the cells. 300 µl of RIPA buffer (50mM Tris-HCl pH 8.0
with 150mM of sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) and 3 µl of
Protease Inhibitor Cocktail (PIC) was then added to each eppendorf tube. The samples were
continuously resuspended through use of 21-guage shearing needles before being placed on ice again

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for 30 minutes. The eppendorf tubes were then centrifuged again for 15 minutes at 13,000 rpm at 4
degrees Celsius. The supernatant was then taken and collected into 8 new eppendorf tubes.
4.4 Bradford Assay
The Bradford Assay is a biochemical technique which requires the utilisation of a 96-well plate to
determine the protein concentration of a solution. The purpose of this, is by giving an indication of the
protein concentration of each of the 8 supernatants acquired, we can determine the volume to use in
each well for SDS-PAGE so that one sample is not higher or lower in protein concentration than any
other. The principle behind this experiment lies with the reagent Coomassie Brilliant Blue G dye,
which shifts from a brown colour (465nm) to a blue colour (595nm) when it comes into contact with
protein. More protein present will mean a deeper blue colour. The table shows the BSA standards
used in the Bradford Assay for this study.
Standard protein
Well
Row
µg/ml µl of 2mg/ml sample needed to make required
concentration
µl of dH20 needed =
300µl
A 25 3.75 296.25
B 125 18.75 281.25
C 250 37.5 262.5
D 500 75 225
E 750 112.5 187.5
F 1000 150 150
G 1500 225 75
H 2000 300 0
The first column consists of the control, in which 195 µl of Bradford reagent only. Each row in the
second and third columns contained the 196 µl of Bradford reagent as well as 5 µl of the BSA
standards in the fashion represented above. The remaining columns contained 195 µl of Bradfords
reagent as well as 5 µl of cell lysates in triplicate. Any air bubbles were removed and a precision
microplate reader was used to measure the 595 nm absorbance to get the protein concentration values.
These values were used to determine how much of each sample should be used in SDS-PAGE.
4.5 Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE allows separation of proteins by migration through sodium dodecyl sulphate-
polyacrylamide gel by molecular weight. The higher the weight of the protein the longer it will take to
travel through the pores developed in the gel during electrophoresis. For this study, a 10%
electrophoresis running gel was made up to 10 ml in total of the according quantities of 30%

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acrylamide, 1.5M Tris-HCl (pH 8.8), distilled water, 10% SDS, TEMED and 10% APS. The stacking
gel requires all the same reagent apart from a 1.0M Tris-HCl (pH 6.8) is used instead.
To do this successfully, all plates were sprayed with ethanol to ensure sterilisation and the plates were
placed with the tallest of the two at the back and the shortest at the front, where they were clamped
into place. When it was clear that the apparatus was not leaking, the aqueous gel was poured in
between the two plates to set, with air bubbles removed by adding a small amount of distilled water to
the top. When the gel is deemed to have ‘set’, the excess water on top was poured away and the
stacking gel was then prepared. Upon adding the aqueous stacking gel and ensuring that there were no
air bubbles present, a green comb was swiftly added to create the wells for the samples. After 20
minutes the stacking gel should be set, and the green comb was gently removed.
4.6 Western Blotting – Reagents
10x TBS (1L) 30g Tris-base, 80g Sodium chloride, 2g Potassium Chloride, made up
to 1L with double distilled water
1x TBS (1L) 100ml of 10x TBS, made up to 1L with double distilled water
TBST (1L) 1x TBS with 0.1% Tween 20, made up to 1L with double distilled
water
Transfer buffer (1L) 2.9g glycine, 2.8g Tris-base and 200ml of methanol, made up to 1L
with double distilled water
Blocking Solution 5% powdered skimmed milk with TBST
5% BSA Solution 5% BSA in TBST
4.7 Running Gels
Gel cast removed from gates and placed into the western blot machine. Prior to western blotting leaks
were checked for by the pouring of running buffer into the machine, including filling the gel wells.
Because only 1 gel was being run a buffer dam was employed to prevent leaking. Each well was
loaded with the calculated amounts acquired from the analysis of the results of the Bradford assay as
represented in the table below, but with 5 µl of precision protein ladder added to the first well.
Sample Volume added (µl)
EGF C 21.9
EGF 10 25.5
EGF 20 29.4
EGF 30 30.0
SDF C 20.9

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SDF 10 25.9
SDF 20 26.5
SDF 30 27.2
Once these were all added, the lid was placed on and the gels were allowed to run at 180V and 0.05A
for approximately 2 hours.
4.8 Gel Transfer
When SDS-PAGE was successfully completed, the gel was removed from the Western Blot machine
and the two glass plates were carefully removed to allow extraction of the gel. The stacking gel was
cut off and safely disposed of and the remaining gel was placed into 20 ml of transfer buffer for 20
minutes. A membrane was cut (9x6 cm) and placed in 20 ml of methanol for 3 minutes, this
membrane was not handled by anything other than ethanol sterilised tweezers. The membrane was
then transferred to 20 ml of transfer buffer for 10 minutes, and in that time 6 9x6 cm squares of filter
paper were cut out in preparation for the transfer machine, and were soaked in transfer buffer for 10
minutes. Once the transfer machine had been sterilised thoroughly, 3 pieces of filter paper were
placed in the machine, followed by the membrane and the gel, and then 3 more pieces of soaked filter
paper. The lid of the transfer machine was added and the transfer was allowed to run with at 0.04A at
250V for 1 hour and 35 minutes.
4.9 Blocking and Enhancing Chemiluminescence
After completion of the transferring process, the membrane was placed in blocking solution for 1
hour, while the gel and filter paper were properly disposed of. The membrane was then allowed to
wash in fresh TBST every 10 minutes for 30 minutes in total. The membrane was then placed into a
plastic pocket and allowed to be incubated at 4 degree Celsius overnight, shaken with 5 µl primary
Akt (Ser) antibody and blocking solution. Upon completion of this process, the membrane was
removed from the plastic pocket and the reagents were safely disposed of, and the membrane was
allowed to wash in TBST. The membrane was then placed into a new plastic pocket and allowed to be
incubated for 1 hour with 1.7 µl of the secondary rabbit antibody and blocking solution. The
membrane was then washed in TBST for the last time for 30 minutes. The membrane was then
exposed to ECL prime detection reagents 1 and 2 at a 1:1 ratio. This took place for 1 minute in the
absence of light before being carefully placed in the image quant 4000 machine, ensuring no air
bubbles were present when in the correct position for reading. Settings were set to precision exposure
and a high resolution, and the results of the Western Blots were observed.

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4.10 CellDesigner – Information Organiser for Research in Systems Biology
The 64-bit (Version 4.4) application is used in compliance with SBML, allows the storing of models
for analyses by other SBML-compliant applications. Was used for the storage of data on each
molecule in the processed diagrams produced in this study, as well as the organising of the diagram. It
is important to note that the diagrams produced are not completed exhausted and are open to be
updated in the future following further studies.
4.11 Analysis of Images and Quantification
Images acquired from the Western Blots were quantified using ImageJ software, Version 3.1.1. The
Western Blot display was altered to 8-bit and then each Western Blot was measured with the same
area. The results were represented as bar graphs constructed in Microsoft Excel (Version
15.0.4551.1011).
5. Results
5.1 Activation of Akt by EGF and SDF
Analysis of Akt activation by EGF and SDF in MDA-MB-231 cells took place when eight individual
samples including a control for each protein took place. Four wells were exposed to EGF with one
control (control was not subjected to EGF exposure) at ten minute intervals, meaning that the samples
ranged from one with a ten minute exposure and the maximum exposure time was thirty minutes. The
same format was carried out for SDF. After each allotted time allocation the reactions were halted by
placing each sample on ice, and then cell lysates were prepared separately to be ready for western blot
analysis with a rabbit Akt (Serine) primary antibody and detection was observed by analysing the
concentration of the presence of bound rabbit secondary antibody in the 10% gel electrophoresis.
EGF C EGF10 EGF20 EGF30 SDFC SDF10 SDF20 SDF30
Figure 3. Western Blot analysis representing the concentrations of phosphorylated Akt present
after exposure of Akt to EGF and SDF for ten, twenty and thirty minutes respectively.
60KdA

15
Figure 4. Bar graph constructed in Microsoft excel. Used values depicting the mean absorbance
values given by Fiji ImageJ, of phosphorylated Akt after exposure to EGF. Maximum absorbance
value (total phosphorylation and full black) used was 15 and the minimum absorbance value (no
phosphorylation and full white) used was 0 (Schneider, Rasband & Eliceiri, 2012).
2.086
6.521
12.618
13.537
EGF Control EGF 10 EGF 20 EGF 30
0
2
4
6
8
10
12
14
16
EGF Exposure Time (mins)
Absorbance
Graph to show the concentrations of phosphorylated Akt
after exposure to EGF for different amounts of time.
8.124
8.965
9.993
9.611
SDF Control SDF 10 SDF 20 SDF 30
0
2
4
6
8
10
12
SDF Exposure Time (mins)
Absorbance
Graph to show the concentrations of phosphorylated Akt
after exposure to SDF for different amounts of time.
Figure 5. Bar graph constructed in Microsoft excel. Used values depicting the mean absorbance
values given by Fiji ImageJ, of phosphorylated Akt after exposure to SDF. Maximum absorbance
value (total phosphorylation and full black) used was 12 and the minimum absorbance value (no
phosphorylation and full white) used was 0 (Schneider, Rasband & Eliceiri, 2012).

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The Western Blot analysis presented some interesting results. As expected, it was shown that both
SDF and EGF are fully capable of activating Akt by triggering signalling cascades that ultimately
result in the phosphorylation of Akt. However, as can be seen in figure 3 the extent to which each
molecule can activate Akt is varied. The overall conclusions drawn from Figure 4 were that both can
activate Akt, however EGF takes slightly longer to have a noticeable effect, and after thirty minutes
activates more Akt overall in comparison to SDF. With regards to SDF in Figure 5 however, the
results suggest that SDF activates Akt at a slightly faster rate than EGF, but concentration of
phosphorylated Akt plateaus and decreases after twenty minutes which is not the case with EGF. This
has led to the formation of the hypothesis that the two molecules activate different signalling
cascades, but both result in the activation of Akt to differing extents.
It is hypothesised that EGF-EGFR-mediated Akt phosphorylation is sharper because it activates more
signalling cascades that result in Akt activation, but takes longer to do so because the pathways
themselves may involve more molecules that need to be activated before Akt. This is in comparison to
SDF, with which it is hypothesised that it is activated more quickly than the EGFR signalling cascade
because the pathways involve less molecules that need to be activated before Akt. Furthermore, less
Akt was activated, and this is hypothesised to be because there are less signalling cascades that are
activated by SDF which result in Akt activation. This could be vital information when determining a
course of treatment when an MDA-MB-231 cell line is the underlying cause for the oncogenic effects
expressed in a patient with breast cancer, because treating a patient with inhibitors of EGFR for
example when the mutation lies within an SDF activated pathway would produce no beneficial
progress and submit the patient to potentially stressful toxicity.
5.2 Processed Diagram of The EGF/EGFR/PI3K/Akt signalling pathway constructed
using CellDesigner
In response to the results acquired in Figure 1, an investigation into the differing downstream
effectors of EGF and SDF took place through extensive textmining with the intention of mapping the
full pathway mediated by EGF and its complementary receptor which subsequently results in the
activation of Akt. The same was carried out with SDF to see if the two molecules shared any
downstream factors through pathway crosstalk. If any could be identified then the molecule in
question could provide a good target for therapy. Silencing a common factor could bring about a halt
in the SDF and EGF mediated activation of Akt and prevent overexpression of pro-apoptotic
downstream proteins.
Firstly, it is important to identify the numerous proteins involved after the initial activation of EGFR.
EGFR takes the form of the composition of the majority of Receptor Tyrosine Kinases (RTKs), with
one ligand-binding domain, as well as a cytoplasmic domain and a domain that mediates translocation
across the cell membrane, the transmembrane domain. In order to become active, the ligand-binding

17
domain is stimulated by EGF, and the molecule undergoes autophosphorylation of essential tyrosine
residues on the activation loop of the cytoplasmic domain. In order for this autophosphorylation to
occur, the transmembrane domains of two ligand-EGFR complexes dimerise (Schlessinger, 2002).
5.3 The Role of the SHC/Grb2/SOS1 mediated activation of Ras in EGFR Signalling
EGFR upon activation results in the autophosphorylation of several tyrosine residues that trigger
downstream binding of adaptor (scaffolding) proteins that facilitate the catalysis of downstream
signalling cascades, in spite of the fact that most, if not all of these adaptor proteins do not contain a
catalytic subunit themselves. Src homology domains are common in these adaptor proteins and enable
the binding of these proteins to EGFR tyrosine residues. EGFR overexpression arises due a mutation
that results in the constant or uncontrolled autophosphorylation of tyrosine residues 992, 1045, 1068,
1086, 1148, 1198 and 1173, (as represented in Figure 6) with either a single residue or a combination
of these residues resulting in the binding of specific scaffolding proteins to help mediate its function
of downstream signalling. Src homology 2 domain containing transforming protein (SHC) is just one
of these scaffolding proteins that is capable of forming complexes with EGFR by binding to specific
tyrosine residues. SHC becomes active once tyrosine 317 has been phosphorylated at the C-terminal
and this occurs upon the binding of SHC to EGFR predominantly on the 1148 tyrosine residue
(Pelicci et al, 1992). It was also later shown that Y239, Y240 and Y313 of SHC are also activated
shortly after cell exposure to EGF (Zheng et al, 2013). An oncogenic EGFR gene containing a
specific mutation that results in the 1148 tyrosine being substituted for phenylalanine has been shown
to decrease the extent to which SHC binds by as much as 60%, suggesting that Y1148 is a primary
binding residue for SHC. The remaining 40% of SHC that has successfully bound to EGFR is capable
to binding to another tyrosine residue found on EGFR. This was later determined to be tyrosine 1173,
which is also responsible for allowing the binding of Grb2, another scaffolding protein involved in
signalling pathways that generate similar phenotypic cellular responses by leading the activation of
Ras (Okabayashi et al, 1994; Batzer et al, 1994).
The significance of SHC has increased since its discovery in 1992, because it has recently been shown
to function not only once bound Grb2, but also independently. Furthermore, SHC has also been
proven to result in the activation of Akt and this can be seen in Figure 10, which shows that the initial
signalling of SHC results in Akt-mediated phosphorylation feedback of the Ser29 residue, which has
been linked with enhancing the ability of SHC to function independently of Grb2 (Zheng et al, 2013).
Grb2 itself plays a vital role in the activation of the PI3K/Akt pathway in response to EGFR
expression, for it is partially responsible for the Ras induced activation of the PI3K (p110 catalytic
subunit) pathway as well as facilitating the same activation in conjunction with the Grb2-associated
binding protein 1 (Gab1) (Batzer et al, 1994) as can be seen in Figures 8 and 7 respectively. Grb2
itself consists of an SH2 domain which facilitates the binding to the Y1173, Y1068 and Y1086 with

18
very high affinity to EGFR upon autophosphorylation, and two SH3 domains which bind to the
guanine-releasing factor SOS1 (Chardin et al. 1993; Egan et al, 1993). Figure 8 represents this
function, as well as showing the activation of Ras itself, which is located in the membrane of a cell.
Grb2 facilitates the translocation of SOS1 to the membrane so that it can undergo its function of
guanosine exchange, resulting in a decrease in the concentration of Ras-GDP and increasing the
concentration of the activated Ras bound to GTP (Batzer et al, 1994).
The only adaptor protein currently well understood that promotes the activation only of the PI3K/Akt
pathway is Gab1. The pathway depicted in Figure 7 represents Gab1 as a docking protein that can
function either with Grb2 or independently, although the function remains the same. This is in
contrast to the findings of Lock et al, who stated that the binding of Gab1 to Grb2 was essential for its
binding to EGFR (Lock et al, 2000). Functioning independently involves Gab1 binding to the ErbB-3
domain of the active EGFR receptor heterodimer, where it binds specifically to three of six PYXXM
motifs present. This subsequently results in the phosphorylation of Gab1 on Y446, Y472 and Y589
and activating its very own PYXXM motifs for the recruitment of PI3K to the membrane (Halgado-
Madruga et al, 1996). PI3K is known to possess its own PYXXM motifs in the SH2 domains of the
p85 regulatory subunit, facilitating the binding of PI3K to Gab1. Grb2-dependent activation of Gab1
requires an EGFR-Grb2 complex binding to the proline-rich region which is found on the C-terminal
end of the Gab1 SH2 domain. This also results in the activation of Gab1 and results in the subsequent
recruitment of PI3K to the membrane. In breast cancer MDA-MB-231 cell lines specifically, it has
been shown that a high level of EGFR-ErbB3 heterodimers associated with the EGFR oncogene
increases the amount of activated Gab1 present in the cell membrane (Kostenko et al, 2006).
5.4 PI3K-mediated activation of Akt
Through one, or a combination of the signalling cascades upstream of PI3K described above and
illustrated in Figure 6, phosphorylation of the catalytic subunit of PI3K is able to take place either by
binding to active Ras (Figure 8), or by activation by Gab1 (Figure 7). PI3K is located within the
plasma membrane, and its function when active is to phosphorylate PIP2 into PIP3 by mechanisms
described in Figure 9. The generation of PIP3 is responsible for the translocation of several molecules
that possess Pleckstrin Homology (PH) domains, including the likes of Akt and the enzyme that
catalyses its activation, PDK1 and PDK2, although the actual identity of PDK2 is yet to be
determined. This ambiguity arises from hypotheses that Ser473 – the phosphorylation site of PDK2 –
is either activated by autophosphorylation or potentially by Protein Kinase C (Toker & Newton, 2000;
Kawakami et al, 2004; Bayascas & Alessi, 2005). The action of PDK1 on the other hand has been
widely accepted. As illustrated in Figure 9, PDK1 catalyses the phosphorylation of the Thr308 residue
when Akt is bound to PIP3 in the cell membrane (Vivanco & Sawyers, 2002). Furthermore, Figure 9
identifies the specific roles of two tumour-suppressor genes, PTEN and PP2A. Functioning PTEN is

19
vital in the prevention of Akt phosphorylation by catalysing the reverse action of PI3K,
dephosphorylating PIP3 to PIP2 so Akt cannot translocate to the membrane to be activated
(Hlolbilkova et al, 2003). PP2A has a similar effect, but functions by dephosphorylating Akt itself on
the Thr308 and Ser473 residues. In addition, Baldacchino et al recently showed that PP2A was
deregulated in approximately 59.6% of basal breast tumours either by physical mutation of PP2A or
due to an overexpression of PP2A inhibitors. This was correlated to increased levels of
phosphorylated Akt (Baldacchino et al, 2014).
5.5 Processed Diagram of The SDF/CXCR4/JAK/STAT pathway constructed using
CellDesigner (Figure 11)
It was found that there are two main pathways involved in SDF/CXCR4-mediated activation of the
PI3K/Akt pathway, although it is important to note that specific residues involved in activation are
still quite poorly understood. However, the signalling pathways of the Janus kinases (JAK) such as
their vital role in the immune response, cell-growth, development and differentiation are ones that
have been studied extensively; only recently though has their role in breast cancer been scrutinised
(Wagner & Rui, 2008). Specifically, the role of JAK2 and its downstream signalling has been found
to have a ‘two-faced’ effect on the regulation of PI3K activity with the activation of different Signal
Transducers and Activators of Transcription (STATs) as shown in Figure 11 (Wagner & Schmidt,
2011). Firstly, when SDF binds CXCR4, phosphorylation of essential tyrosine residues occurs as well
as a conformational change in the cytoplasmic domain of the receptor, this promotes the chemotactic
translocation of JAK2 to the CXCR4 where it becomes transphosphorylated on its own essential
tyrosine residues. Through the mechanism of positive feedback, JAK2 is then able to create a docking
site for SH2 domain-containing proteins by further phosphorylating tyrosine residues on the CXCR4
receptor. STATs 3 and 5, significantly, are two of these proteins effected (Hosford & Miller, 2014).
Both STATs 3 and 5 are phosphorylated by JAK5 on unknown tyrosine residues which results in the
initiation of their activity. STAT3 has been linked with several pro-apoptotic effects by binding
directly to two small PI3K subunits p50a and p55a, and this results in the downregulation of PI3K and
minimises Akt phosphorylation (Abell & Watson, 2005) STAT3 activation requires the
phosphorylation of the Y157 residue on the CXCR4 intracellular loop 2 (Ahr et al, 2005). In contrast,
JAK2 has also been shown to mediate the activation of STAT5. A recent study revealed the anti-
apoptotic effects of STAT5, when a delay in mammary postlactation remodelling (normal breast
function) was noticed in spite of the pro-apoptotic activity of STAT3. This led to the formation of the
hypothesis that STAT5 was responsible for mediating AKT1 gene transcription and that this process
was in fact capable of eclipsing the pro-apoptotic effects of STAT3 (Creamer et al, 2010; Iavnilovitch
et al, 2002). Furthermore, it was also hypothesised that STAT5 plays a direct, stimulating role on
PI3K by binding to the SH2-containing domain of the p85 subunit and inducing phosphorylation of
the catalytic p110 subunit. However, this reaction requires further verification because the

20
mechanisms behind this are uncertain, and the phenotype observed could potentially be the result of a
mutation in gene coding for the PI3K p110 subunit, a known mutational hotspot (Sakamoto et al,
2007).
5.6 The Role of G-protein-recruitment of scaffolding proteins in the activation of the
Ras/PI3K/Akt pathway (Figure 11)
SDF/CXCR4 signalling has also been shown to result in the activation of Ras-GTP, resulting in the
translocation of Ras to the membrane where it is thought to activate the PI3K/Akt pathway. In
chemokine signalling, G-proteins Alpha, Beta and Gamma are present in a complex with CXCR4
while inactive. SDF ligand binding to the CXCR4 receptor triggers transphosphorylation of CXCR4
tyrosine residues and results in a conformational change of the receptor itself. This allows the three G
proteins to dissociate from each other as well as CXCR4 in an active state, although it is important to
note that G proteins Beta and Gamma remain in a complex while in the cell cytoplasm. Both G-Alpha
and the G-Beta-Gamma complex are capable of activating c-Src protein which in turn stimulates the
recruitment of the SHC/Grb2/SOS1 complex in essentially an identical fashion to that which is
represented in Figure 6 (Chang et al, 2002). This is represented in figure 11 with CXCR4 signalling,
and then end result is the guanine-nucleotide exchange factor SOS1 becomes active when in complex
with the Grb2/SHC scaffolding proteins and induces the formation of GTP-Ras from GDP-Ras and Pi
(Andrechek & Muller, 2000). Ras is then able to undergo its function represented in Figure 10,
activating the p110 catalytic subunit on PI3K and stimulating the downstream phosphorylation of Akt
represented in Figure 9.
6. Discussion
Upon observation of the processed diagrams produced to highlight the EGF/EGFR signalling pathway
(Figure 10) and the SDF/CXCR4 pathway, it is difficult to see any reason why one receptor would
potentially activate Akt at a faster rate than the other. This is because the literature suggested that both
signalling pathways are capable of activating PI3K through more than one distinct pathway, and both
are capable of activating Ras-GTP through the recruitment of scaffolding proteins. This could be
highly significant as a potential target for therapy, because blocking of the guanine-nucleotide
exchange factor SOS1, could lead to the prevention of the activation of Ras and decrease the extent to
which the PI3K/Akt pathways are activated. Furthermore, the Grb2 and SHC scaffolding proteins
were also shown to be involved in both pathways, and inhibition of these molecules could also lead to
a decrease in the activation of the Ras/PI3K/Akt pathway.
It was also difficult to understand why EGF/EGFR signalling was capable of activating Akt more
sharply, because the literature used to produce the diagrams suggested that the CXCR4 not only
facilitated the increase of PI3K activity by STAT5, but STAT5 also promoted the transcription of the
AKT1 gene itself. However, the extent to which these pathways actually result in the phosphorylation

21
of Akt still requires further research, and the reason why EGF activated Akt more strongly may then
be identified.
The processed diagrams produced were unable to reveal why SDF is capable of activating Akt faster
than EGF in MDA-MB-231 cells, it could be argued that an error in the Western Blots may have
occurred. However, results similar to Figure 3 were exhibited by Lee et al, who showed that Akt was
shown to be phosphorylated just 5 minutes after exposure of cells to SDF and then progressively
decrease after 20 minutes (Lee et al, 2004). It can be therefore be concluded that the SDF/CXCR4
pathway requires further research and better understanding in the context of breast cancer before a
processed diagram can be produced that is accurate and reliable.
As shown in Figure 3, it is clear to see that some Akt has been phosphorylated in the control sample
not subjected to SDF exposure. This could potentially just be a false result where perhaps the sample
was accidentally exposed to some SDF, but the more logical explanation is that a gain of function
mutation has taken place in a molecule upstream of Akt. It is common in MDA-MB-231 cell lines that
the catalytic subunit of PI3K (PI3KCA or P110) is mutated to gain function in breast cancer cells. For
example the H1047R mutation in exon 20 was identified as a mutational hotspot in a number of
studies and associated with an increase in PI3K activity, along with the E542K and E545K mutations
(Liang et al, 2006; Schneck et al, 2013). In addition, non-synonamous substitutions such as
c1264GA and c1663GA on exon 9 and c3140AG and c3140AT on exon 20 have been
linked also with increased PI3K activity (Harle et al, 2013). Presence of loss of function mutations on
PTEN are also known to exist prominently in breast cancer patients, with approximately 30%
metastatic breast cancer patients possessing a dysfunctional PTEN protein. This has been found to be
due to mutations in exons 1, 5, 7 and 9 in the PTEN gene, with most of these mutations resulting in a
truncated final PTEN. Loss of this molecule could also potentially give rise to activation of Akt even
in the absence of EGF or SDF and therefore potentially explains the anomalous result acquired.
(Pradella et al, 2014).
The processed diagrams produced in CellDesigner were intended to represent as many small molecule
interactions as was possible after exhaustive textmining. EGF/EGFR signalling pathways are
pathways that have been scrutinised for a particularly long time due to an early recognition for its role
in breast cancer, and this meant that it was possible to find research to a higher extent of detail. With
the high extent of understanding about the EGFR downstream signalling cascades, it was possible to
find detail down to the specific residues that are phosphorylated in order to mediate the resulting
activation of Akt. It can be argued therefore, that production of such processed diagrams may be more
beneficial than linear diagrams such as Figure 1, because the precise interactions between protein
domains and specific residues cannot be identified. On the other hand, SDF/CXCR4 pathways only
seem to have been studied in the context of breast cancer for a much shorter period of time. This

22
could perhaps be because chemokine signalling has long been studied in the context of other illnesses
such as the role of CXCR4 as a receptor that mediates the entry of HIV disease (Murakami &
Yamamoto, 2010). The fact that there was less literature available for the role of CXCR4 signalling in
breast cancer also limited the amount of detail that it was possible to go into. For example, research
available was only able to recognise the fact that STAT5-mediated the activation of Akt, but the stage
had not been reached where they could state confidently that STAT5 eclipsed the proapoptotic action
of STAT3 directly, due to the possibility that it could be due to the loss-of-function mutations of
PTEN or the presence of the overactive PI3KCA subunit described above.
7. Conclusion
This has allowed the formation of the conclusion that the compilation of all text and programs of
network biology use of processed diagrams are particularly useful if one is deciding to focus on an
already well-defined area of research. The development of a processed diagram highlighting all the
specific interactions may prove very useful to someone who is unsure on where to start in terms of
researching into a specific pathway. Analysis of a processed diagram can allow for the rapid
development of viable hypotheses for a laboratory experiment, and it can be argued that the fact that
most processed diagrams are not covered exhaustively allows for them to be continuously updated by
future experiments and provide an accurate representational summary of all the current literature in
the field., making it significantly easier to identify an experiment which may potentially add to the
growing network of research. For this reason, it is also important to note that no lab results can ever
be proven exclusively by a processed diagram, because it is quite possible that the results shown in
Figure 3 are indeed correct, but the mechanisms explaining why may just not yet have been
discovered.

23
8. Figures and Legends
Figure 6. A detailed processed diagram produced in CellDesigner representing the EGFR-
mediated recruitment of adaptor proteins and the residues phosphorylated that all result in PI3K
activation.

24
Figure 7. A processed diagram developed from existing literature and CellDesigner which
highlights the Gab1-mediated activation of PI3K in both the Grb2-dependent or independent
manner.

25
Figure 8. A processed diagram developed from existing literature and CellDesigner annotating
the role and coupling of the SHC-EGF-EGFR complex and the Grb2-SOS1 complex during the
EGF-EGFR-mediated activation of Ras-GDP in the membrane.

26
Figure 9. A processed diagram developed from existing literature and CellDesigner showing the
role PIP3 mediated translocation of PDK1, PDK2 and Akt to the membrane in Akt
phosphorylation that occurs when the processes described in figures 6, 7 and 8 take place. In
addition, the roles of tumour suppressor proteins PTEN and PP2A in the prevention of Akt
phosphorylation are also pin-pointed.

27
Figure 10. A processed diagram produced in CellDesigner depicting the full EGF-EGFR
signalling pathway that results in the activation of Akt. The pathway shows the catalytic effects of
the unique combinations of scaffolding proteins represented in more detail in Figure 6 when
bound to EGFR in the context of PI3K activation.

28
Figure 11. A processed diagram produced in CellDesigner depicting the pathways employed by
SDF/CXCR4 signalling complex to activate PI3K. Shows similarity to Figure 10 with the
induction of scaffolding proteins to activate Ras-GTP and highlights the unique ‘two-faced’ role
of Janus Kinase 5, for it is capable of activating pro-apoptotic STAT3 and anti-apoptotic STAT5
at the same time.

29
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