Nick Nemmers

Junior Seminar
Mentor: Dr. Speckhard
Thesis Introduction Second Draft 4/18/2016

Syndecan1 and its Effect on the Metastasis Signal

A great emphasis has been put on cancer research due to cancers extensive effect on
society. In 2012, there were 14 million new cases and 8.2 million cancer-related deaths
worldwide, which results in cancer being among the leading causes of death in the world. (2)
Cancer is a relevant issue in society and not easily solvable. Looking more specifically between
2010 and 2020, we expect the number of new cancer cases in the United States to go up about
24% in men to more than 1 million cases per year, and by about 21% in women to more than
900,000 cases per year. (1) This is a significant number of additional cases in the United States
alone, which adds additional risk for the possibility of tumor cells metastasizing. Metastasis is
when cancerous tumor cells move from the tumor to other parts of the body. In order to
metastasize, cancer cells must undergo a variety of steps before they can enter into the blood
stream or lymph system, which leads most of the cells to die over the course of the process, but
sometimes not all of them. (3) Cancer cells that spread to new parts of the body may not be
exactly the same as they started, which may make them harder to treat. (3) This process may be
detrimental to a cancer patient and adds difficulties to the treatment process, especially if the
primary tumor site is unknown because treatment is based on where the cancer originally started.
One potential step towards solving the problem of cancer is understanding this process of
metastasis, why it occurs, and if it is possible to stop it.
Like many other cell processes, the process of metastasis involves proteins and signaling.
There are many types of cells that have been seen to undergo metastasis originating from various
parts of the body such as the stomach, kidney, prostate, breast, colon, and many others. (3) It has
also been observed that the protein called T-cell lymphoma invasion and metastasis 1 (TIAM1) is
involved with some types of cancerous tumor cells and is important for cell-matrix interactions.
(12) TIAM1s deregulation has been implicated in invasive and metastatic forms of lung and
colorectal cancer and may be a predictor for poor patient outcome for renal cell, prostate, and

hepatocellular carcinomas. (12) Expression of the TIAM1 was easily detected in human giantcell lung carcinoma cell strains. (4) This and other similar studies showed that expression of
TIAM1 was evident in various types of cell strains and is associated with malignant grades of
human tissues. It was also determined that another protein found within the body, Syndecan1,
has been shown that it is a physiological partner of TIAM1 involved in cell migration and cellmatrix adhesion. (12) These proteins are but one combination of many that can cause cancer cells
to move, but understanding the interaction between these two proteins could lead to better
outcomes in the future.
Little is known about TIAM1 and Syndecan1, including the signaling and specificity
components of their binding interactions. The PDZ domain of the TIAM1 protein also serves a
significant purpose as the binding pocket for Syndecan1 and other proteins, but is also not
entirely known. If more information were known about how and why these two specific proteins
interact with one another to cause cell migration, then it may one day be possible to stop the
process of metastasis and contain the tumor. Containing a tumor within the body would then
allow for more effective cancer treatments and better results for cancer patients. Different
metastatic tumor nodules in the same patient may not be identical in terms of molecular changes
that are present. This raises the possibility that a drug might be effective in one part of a persons
tumor but not in another. (2) Stopping the spread of cancer and being able to contain it would
benefit cancer research greatly. But, before this is possible, the structure and functions of the
proteins involved in these cell processes must be understood to a great extent.
TIAM1 and Syndecan1 are the main proteins involved and it is important to have
information about them. TIAM1 is a protein made up of multiple domains, as shown in Figure 1,
and is a protein present in some types of cancer cells (12). Domains are a set of amino acids that
code for specific functions along the protein. Like most every protein, regulation and control are
both very important for TIAM1. Protein interactions is how TIAM1 obtains this control and
regulate Rac1 activity. (9) As a Rac1-specific guanine exchange factor (GEF), TIAM1 can
catalyze the transition of Rac1 from inactive GDP-bound state to active GTP-bound state, and
the active GTP-bound Rac1 is involved in many important cellular processes, such as gene
expression, apoptosis, and cell migration. (4) A particular domain of interest in TIAM1 and one
of the focus points of this research is the PDZ domain. This domain is the binding pocket and is

able to bind with proteins, such as Syndecan1. (10) With this binding function of the domain
comes the process of signaling. Proteins in cells are extensively involved in signaling, an
example being movement of the cell itself. The TIAM1 protein is thought to be the regulator,
depending on its binding ligand, when it comes to cell movement. This idea comes about because
it has been seen that Syndecan1 binding to the TIAM1 protein causes the cell to move. (9)
TIAM2 and
other
proteins are
not being
closely
looked at in

Figure 1. TIAM1 structure and a representation of its domains under investigation.

(Fuentes Proposal Correspondence, 10)

this case because Syndecan1 does not bind to them. TIAM1 and TIAM2 have different
structures, such as the binding pocket for ligands, and only allow certain ligands to interact them.
Syndecan1 is the binding protein that binds to the PDZ domain. Binding is a very specific
function for proteins and the C-terminus end must be the correct structure for it to have any
chance of binding. As stated, the structure of TIAM2 is different than that of TIAM1 and
Syndecan1 is not able to bind to it because it has a different specificity in terms of its binding
pocket. Syndecan1 is a cell surface proteoglycan that functions coordinately with integrins to
regulate cell-cell and cell-matrix adhesion. Moreover, its expression and co-localization are
correlated with E-cadherin, and their loss is associated with the EMT. (10) This concept is of
great interest and something that would be beneficial to learning more about the signaling
process of metastasis.
One way to learn more about binding properties between proteins is through Fluorescent
Resonance Energy Transfer (FRET). FRET is one experiment to look at protein interactions in
real-time and can produce valuable data that indicates properties such as the tightness of a
protein bond. The basis of FRET is that it uses proteins that are tagged with specific fluorescent
colors. In testing two different proteins that are each tagged with a different fluorescent color, the
spectrums of these colors have specific wavelengths that overlap slightly, one being an acceptor
excitation and the other being a donor emission as seen in Figure 2a. Only certain combinations
of colors are able to experience this phenomena because the wavelengths need to overlap. In

Figure 2a, the two excitation and emission spectrums of the proteins are represented on the left.
One protein is the donor and the other is the acceptor. In order for FRET to work, the spectrums
of each need to overlap. This allows for the phenomena of FRET to occur and the energy to be
transferred. Part b of the figure refers to the distance between the two interacting proteins. This
requirement is simple in that the proteins need to be extremely close in order to interact with one
another. Finally, part c refers to the correct orientation of the proteins. It is important to note also,
in parts b and c, the excitation and emissions. A single protein by itself should emit one color and
if there is FRET, a different color should be visible and fluoresce. This process is able to work in
real-time and give information about the binding of the two different fluorescent proteins.

Although
FRET has
not been
used before

Figure 2: FRET processes. a.) Spectrums from the two interacting proteins need to overlap in
order to experience FRET. b.) The proteins need to be close (several nanometers apart) in
order to interact and experience FRET. c.) The requirement of correct orientation of the
proteins is needed for FRET to occur. (Broussard, Rappaz, Webb, and Brown, 14)

to look at the TIAM1 and Syndecan1 interaction, other research methods dealing specifically
with TIAM1 and Syndecan1 have been carried out in the past. The most extensive work has been

done by Dr. Fuentes lab at the University of Iowa in recent years. One important part of their
research involved the PDZ domain and its specificity in terms of binding. Figure 3 pictured
below shows the TIAM1 protein found within humans. This was a result of Dr. Fuentes lab and
it is a good representation of the PDZ domain bound to a ligand, as well as unbound.

Figure 3: TIAM1 PDZ domain. (A.) Free and

peptide-bound structures of PDZ domain. (B.)
Interaction surface with labelled interactions.
(Fuentes Proposal Correspondence, 10)
The research done from this lab has preliminary results that support the already
established idea that TIAM1 and Syndecan1 have cancer implications. This inclination stemmed
from multiple different findings; the first of which relates to Figure 4. Figure 4 shows the PDZ
domain interactions with various binding partners. The data from this graph shows that
Syndecan1 is a binding partner with PDZ and thus TIAM. It is important because this data was
also taken in vitro, so as to have biological implications as well. It was determined that
Syndecan1 was a binding partner to the TIAM1 PDZ domain, as seen in the data from Figure 4.

Figure 4: PDZ Binding Domain Interactions. Syndecan1 () and other

binding partners are represented. Syndecan1 shows binding to the PDZ
domain of TIAM1 (left), but not to TIAM2 (right).
(Fuentes Proposal Correspondence, 10)

It is also determined from this table that Syndecan1 binds to TIAM1 and not to TIAM2,
which also indicates the role that specificity has when binding proteins. The Syndecan1 Cterminus is correct for TIAM1 and allows the binding to occur. Also, it means that Syndecan1
must have some effect on the signaling because TIAM1 is heavily reliant on regulation and
control through binding. The signal for cell movement is the main signal being looked at in this
particular case and, as mentioned before, Syndecan1 is involved in that process. This and several
other important concepts involving these two proteins was determined mainly through this
research and was a significant starting point for this current research.
Another important previous research done relating to this topic was done on FRET. The
research done discusses two important aspects of FRET. The first is that it performs research
using in vitro FRET, which can produce different results then in vivo. This allows for more
biological applications, and this is especially important for this research because the goal is to
look at proteins within the human body. The second important feature of this is that it uses
fluorescent proteins of blue and green. The particular fluorescent proteins used in this research
are blue and yellow (BFP and YFP), but there are similarities between this previous research.
Multiple experiments were run using FRET and different proteins in vitro that provide useful
information to be used when performing FRET for other research.
Finally, the research of previous Loras students will also be used in this research. Past
students have done very similar experiments. A student dealt with synthesizing the pET21A

vector and the DNA used in this research that is tagged with a specific fluorescence. (5)
Transformation and PCR are other important components for my research that were carried out
successfully by Loras students. Certain methods and preparations were modified by past
researchers to allow it to be carried out at Loras with the given resources available. DNA
manipulation is essentially my starting point for research and can be done efficiently using past
data regarding methods. In addition to the similarities in research, other results and methods can
also be references in these previous researches. One being that one student was able to
successfully transform some ligated DNA into competent cells prepared themselves. (13) Both
the ligation process and the difficulty of maintaining competent cells are common places of
difficulties. Overall, there is much useful information and techniques about bettering the current
research and optimizing the processes.
The goals of this research include several different parts. The first part being to
successfully modify strands of DNA into the specific and needed sequences. The final DNA
strands need to contain the proper fluorescent tags to be used later on in the research. Also, they
need to contain the specific primers, in this case Syndecan1, to be later transformed into
competent cells. Another goal is to transform the ligated DNA into competent cells to later be
used to test the interactions of proteins. The process of FRET is another goal of the research that
will ultimately give more data and insights on the binding of Syndecan1 and TIAM1. The
sensitivity of FRET is different from other techniques, such as NMR, and will allow us to
simulate biological processes to a good degree. Finally, an over-arching goal of this entire
research is to understand more about the proteins involved, as well as their processes. Some
information is known about how TIAM1 regulates, but the binding properties still have certain
parts to be determined yet. Testing specific protein interactions will allow for further insights as
to why the process of metastasis occurs. A potential goal for later research is then to find a way
to stop the metastasis signal and keep cancerous tumor cells from spreading.

References
1.) CDC-Expected New Cancer Cases and Deaths in 2020
http://www.cdc.gov/cancer/dcpc/research/articles/cancer_2020.htm (accessed Apr 17,
2016)
2.) Metastatic Cancer http://www.cancer.gov/research/areas/treatment and
http://www.cancer.gov/about-cancer/what-is-cancer/statistics (accessed Apr 17, 2016)

3.) What is Metastatic Cancer?

http://www.cancer.org/treatment/understandingyourdiagnosis/advancedcancer/advancedcancer-what-is-metastatic (Accessed Apr 17, 2016)
4.) Hou, M.; Tan, L.; Wang, X.; Zhu, Y. Antisense Tiam1 Down-Regulates the Invasiveness of
95D Cells in Vitro. Acta Biochemica et Biophysica Sinica 2004, 36 (8): 537-540.
5.) Pandey, S. Construction of pET21a Vector with Blue Fluorescent Protein Fragment
from pET30 and the Binding Partner CASPR4 of QMTIAM1. Loras College
Department of Molecular and Life Sciences.
6.) Lee, H.; Zheng, J. PDZ Domains and Their Binding Partners: Structure, Specificity, and
Modification. Cell Communication and Signaling 2010, 8, 8.
7.) Jares-Erijman, E.; Jovin, T. FRET Imaging. Nat Biotechnol 2003, 21, 1387-1395.
8.) Harris, B.; Lim, W. Mechanism and Role of PDZ Domains in Signaling Complex Assembly.
Journal of Cell Science 2001, 114, 3219-3231.
9.) Shepherd, T.; Klaus, S.; Liu, X.; Ramaswamy, S.; DeMali, K.; Fuentes, E. The Tiam1 PDZ
Domain Couples to Syndecan1 and Promotes Cell-Matrix Adhesion. Journal of
Molecular Biology 2010, 398, 730-746.
10.) Fuentes, E. Research Plan: Sections A-G, Principal Investigator: Fuentes, Ernesto. Research
Scholar Grant Application 2008-2009, 8.1-8.29
11.) Truong, K.; Ikura, M. The Use of FRET Imaging Microscopy to Detect Protein-Protein
Interactions and Protein Conformational Changes in Vivo. Current Opinion in Structural
Biology 2001, 11, 573-578.
12.) Shepherd, T.; Hard, R.; Murray, A.; Pei, D.; Fuentes, E. Distinct Ligand Specificity of the
Tiam1 and Tiam2 PDZ Domains. Biochemistry 2011, 50, 1296-1308.
13.) Obrien Thesis. Provided by Dr. Speckhard.
14.) Broussard, J.; Rappaz, B.; Webb, D.; Brown, C. Fluorescence Resonance Energy Transfer
Microscopy as Demonstrated by Measuring the Activation of the Serine/Threonine
Kinase Akt. Nature Protocols. 2013, 8, 265-281.