Abstract highly upregulated in papillary thyroid cancer. We

Abstract

In the current field of thyroid
cancer, treatment methods are lacking. The usual treatment involves thyroid
removal consisting of surgery and iodine therapy. Thyroid cancer occurrence is
predicted to surpass colon cancer by 2030 (Nikiforov). Invasive surgeries and iodine therapy have limited survival rates and
decrease quality of life upon treatment. Thyroid cancer can be categorized into
Well differentiated thyroid cancer (WDTC), Poorly differentiated thyroid cancer
(PDTC), and Anaplastic thyroid cancer (ATC). WDTC can be further divided into
papillary and follicular thyroid cancer.

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            From preliminary
experiments on PTC and FTC cell lines in Dr. MacDonald’s lab, we have
identified certain genes upregulated in either papillary or follicular thyroid
cancer. JAG2, a gene involved in the
Notch signaling pathway, was highly upregulated in papillary thyroid cancer. We
hypothesize that the overexpression of JAG2
drives papillary thyroid cancer progression. To evaluate JAG2’s role in PTC, we will use cellular and animal models as well
as cancer samples. Importantly, we will evaluate metastasis and progression of
papillary thyroid cancer in our models.

Introduction and Preliminary Data

Thyroid cancer consists of three
different cancer subtypes including WDTC, PDTC, and ATC. Approximately 95% of
thyroid cancer is categorized as WDTC. The other 5% consists of PDTC and ATC.
The most common type, WDTC, can be divided into papillary and follicular
thyroid cancer depending on the respective mutations in the MAPK signaling
pathway (Nikiforov). Papillary thyroid cancer is
associated with a BRAF mutation and metastasizes to local lymph nodes. PTC is
typically more aggressive and leads to a much worse prognosis than FTC (Nikiforov). Follicular thyroid cancer is less common and arises from
mutations in HRAS and metastasizes to bones and the lungs. For our research, we
are focusing on papillary thyroid cancer. We feel that papillary thyroid
cancer, due to its dominance, will allow us to fully study and analyze it
through our JAG2 models and cancer
samples. Below, we provide a heatmap of the preliminary data illustrating JAG2’s importance in papillary thyroid
cancer.

            From
the past semester in the advanced cell biology lab under Dr. MacDonald, we
experimented with samples of cell lines of papillary and follicular thyroid
cancer derived from mouse models with over expressed MAPK signaling. The BRAF
cell line contained a PTEN deletion and a BRAFV600E mutation (PTC)
while the HRAS cell line contained a PTEN deletion and a HRASG12V
mutation (FTC). We observed the difference in the transcriptional profile of
the cell lines. After treatment, in figure 3, we also developed gene ontology,
which groups differentially   expressed
genes by processes. We put our DESeq data in a gene ontology program called
GOrilla. GOrilla helped us determined the important genes upregulated in either
papillary or follicular thyroid cancer. From GOrilla, we were able to find our
gene of interest being JAG2 and form
our hypothesis dealing with JAG2
overexpression in papillary thyroid cancer.

JAG2 encodes for the JAG2 protein that
interacts in the Notch signaling pathway. The Notch pathway is important for
embryonic development and is highly conserved among eukaryotic organisms. Notch
signaling cascades can be turned on by the binding of JAG2 and other jagged
family proteins that function as the ligands to Notch receptors. Previous
research has demonstrated that Notch signaling can be activated by the MAPK
pathway and lead ultimately to deregulation of the pathway especially in
papillary thyroid cancer (Yamashita). The Notch signaling works downstream of
the MAPK pathway and has a number of recognized roles in other solid malignancies.
Medulloblastoma (MB) is a malignant brain cancer that deals with children in
early development. The deregulation of Notch signaling cascades have been shown
to result in MB samples. MB tumors models importantly show the deregulation and
overexpression of JAG1 and JAG2 (Fiaschetti). JAG2 has even been connected with increasing levels of the oncogene
MYC. Researchers believe JAG2 levels have the ability to determines stages of
cancer and how aggressive the cancer might be. Similarly, high levels of JAG2 expression result in human
endometrial cancer (Sasnauskien?). Endometrial cancer begins at the
lining of the uterus and spreads from there. It is also called uterine cancer
and generally affects women after menopause. Interesting to note, uterine
cancer is linked with high levels of survival and generally good prognoses. The
development of normal cells into cancer cells most certainly correlates to
Notch signaling, but to what extent is unknown. In endometrial cancer, the
Notch pathway acts as an anti-oncogene and aids in protection of cells as
opposed the other cancers. With that being said, the Notch pathway affects
numerous cancers depending on the level of protein expression in those
respective cancers. Notch and MAPK signaling have also been studied in breast
cancer. Irregular Notch signaling leads to overexpression of various Notch
proteins as well as JAG1 and JAG2. Breast cancer tissues exhibited high levels
of these proteins as compared to normal tissues (Mittal). More research is needed to determine the
entire mechanism involving the key players of receptors, ligands, and
downstream targets through breast cancer development. The role of Notch
signaling in thyroid cancer has yet to be investigated. This helps us see what
is not yet known with JAG2 and why
our work is valid. Also, we are optimistic that our research with our animal
models will help the thyroid cancer field in creating better treatment and
therapies in this cancer. Our experiments will target our two specific aims.

 

Hypothesis and Specific Aims

We hypothesize that overexpression of
JAG2 drives papillary thyroid cancer
progression. Through testing this hypothesis, we hope to uncover JAG2’s role and importance in papillary
thyroid cancer. To test this hypothesis, we will test JAG2 in vitro and in vivo on hopes of understanding why JAG2 is upregulated and how its
expression affects cell characteristics.

Specific Aim 1: Evaluate whether JAG2 is required for metastasis within papillary thyroid cancer.
We will use the BRAF cell line that contained a PTEN deletion and a BRAFV600E
mutation for our cellular model. Our cellular model will involve siRNA
knockdowns to study cell morphology, motility, and metalloprotease assays. Our
animal model experiment will be straightforward in testing mortality and
pathology of mouse models.

Specific Aim 2: Evaluate JAG2
expression in different stages of cancer. To test expression, we will use
cancer samples from the Cancer Genome Atlas. Various experiments like RT-PCR,
western blots, and tissue analysis will help us compare the expression of JAG2 in normal and cancer sample tissues.

 

 

 

 

 

Experimental Design

The first experiments for our
research will include various verifications and confirmations that the Notch
pathway is activated by MAPK signaling and that JAG2 is expressed in Notch signaling. We will use BrafV600E/Pten
-/- /TPO-Cre mice containing the mutation
in BRAF and Pten deletion (Jolly).
These were mice with Cre recombinase activation that developed papillary
thyroid cancer (Jolly). With
BrafV600E/Pten
-/- /TPO-Cre mice and wildtype thyrocytes, we will
activate the MAPK pathway with various growth and differentiation factors for
assessment of the pathway. The immunoprecipitations will tell us the protein
interactions in our cells. We will then run western blots with antibodies to
MAPK and ERK for confirmation of activation of the pathway. After confirmation,
we will run western blots with antibodies to Notch ligands and JAG2 protein.
This will confirm the Notch signaling is downstream and activated by the MAPK
pathway.

To test specific aim 1, we will
inhibit and silence JAG2 in our
cellular and animal model to observe changes in metastasis of papillary thyroid
cancer. The first set of experiments will deal with cellular models. The BRAF
cell lines from Dr. McDonald’s class will be used with the addition of normal
thyroid cells derived from mice with no BRAF mutations. Most importantly, we
will incubate the derived BRAF cells with siRNA knockdowns of JAG2. We will try different
concentrations of siRNA and at various time points before moving on with any
experiments to make sure the cells are not dead and we have JAG2 knockdowns in our cells. We will
test if JAG2 is present by western blots with antibodies to JAG2.

To evaluate whether JAG2 is required
for EMT, we will use our cellular model. The normal thyroid cells derived from
mice should exhibit epithelial cells as opposed to mesenchymal. Epithelial
cells are flatter and rounder while mesenchymal cells are narrow and look like
spindles. Mesenchymal morphologies are present in tumor cells. They are
recruited to tumors and aid in the process of metastasis. Metastasis
encompasses the spread of cancer from initial sites of origin to additional
sites like other organs. Mesenchymal cells should be much more prevalent in the
mutated cells as opposed to the normal cells. Light microscopy and staining for
cadherins will allow us to capture photographs of the difference between the
appearance of our cells. EMT cells express high levels of N-Cadherin as opposed
the E-Cadherin in epithelial cells. We will use immunofluorescent microscopy to
evaluate the expression of cadherins indicative of EMT. Additionally, we will
use time lapse microscopy to determine to motility rates of the respective cell
lines. The cells with high motility rates should be the mutation in BRAF cells
showing the increased ability to metastasize as opposed to the wildtype thyrocytes and JAG2 knockdowns. This microscopy will
allow us to observe live cells for longer periods of time and narrow down on
the differences between cancerous and normal cells. Assuming our siRNA
knockdowns of JAG2 are sustainable
for long enough time points, we expect to see similar motility rates of JAG2 knockdowns and the wildtype
thyrocytes as opposed to the mutated cells. Finally, we will assess the
expression of metallomatrix proteins like MMP6. Metallomatrix protein 6 degrades
the ECM and as well as cleaves receptors on the cell surface. MMP are important in metastasis as they destroy these
surface receptors and trigger unwanted cell proliferation i.e. cancer. We will
run western blots for MMP6 with our WT cells, BRAF cells, and BRAF JAG2 -/- cells. The western blots will
show what cell are expressing these metallomatrix proteins. These experiments
will illuminate whether metastasis occurs with JAG2 or without JAG2.

With viable populations of mice and
high survival rates, we will cross various mice resulting in four different
cell lines. The first line will be easy to acquire as it will be wildtype mice
mainly for controls. We will also use mice with a PTEN deletion and a BRAFV600E
as a control. The two lines of mice that will give us the information we are
looking for will be JAG2 knockouts
with no BRAF mutation and JAG2
knockouts with the BRAF mutation. We will first take time to make sure these
mice can survive for more than a few weeks. After completion of the preliminary
checks, we will first take cells out of the thyroid of each respective model
and grow them in cell cultures to determine if are controls working and our JAG2 knockouts are similar. These mouse
models will allow us to look at metastasis of papillary thyroid cancer through
sacrificing. We will study the tissue of these mice to look at metastasis. We
will harvest tumors from the mouse models, and then do thin-tissue sectioning
and staining with H to evaluate pathology. The sections will be labeled
by the mouse model and organized in cohorts. Additionally, staining will allow
better visualization under the microscope. Pictures will be taken and analyzed
through computer programs. Since these mice will have already been sacrificed,
we will also take other samples of organs from their bodies to look for
metastasis. We will specifically look at the lungs, lymph nodes, and even bone
samples to assess whether JAG2 is
required for metastasis, organs will be harvested from the JAG2 knockout mice and evaluated for tumor presence.

            Under our
second aim, we will be comparing normal thyroid tissue to WDTC, PDTC, and ATC
tissue. To test cancer progression, we will obtain our human samples from the
Cancer Genome Atlas. The human samples will allow us to make comparisons to our
cellular and animal models. We will use a wide variety of samples with tissues
from all the stages of thyroid cancer. We will isolate mRNA in the respective
samples and use RT-PCR for mRNA detection and quantification of JAG2. We will also run western blots
with antibodies to COL1A1 and LOX to compare levels in the normal
tissue and the cancer tissue. COL1A1
and LOX have shown upregulation and
increased mortality rates in PTC (Jolly). We will see if the cells with the highest
JAG2 expression also contain the highest levels of COL1A1 and LOX. Lastly,
we will also use similar tissue analysis through pathology from our first aim.

 

Interpretation and Implications

We expect that JAG2 upregulation in papillary cancer fuels its metastasis and
progression. First, the confirmation of MAPK pathway will show activation of
the Notch signaling pathway. JAG2
influences the metastasis of papillary thyroid cancer through its upregulation
and aggressive effects on thyroid cells. Normal thyrocytes will show no MMP6
activity, but the BRAF mutation cells will indicate MMP6 activity leading to
the metastasis of PTC. On the other hand, the JAG2 knockdowns will indicate low MMP6 activity and decreased
metastasis implicating possible treatments involving the inhibition of JAG2 that might help thyroid patients.
The pathology studies will show increased metastasis in organs of the mutated
mice while the JAG2 knockouts will
show decrease metastasis to other organs. The mouse model will then allow
parallels with humans in the development of treatments involving knockouts of
the JAG2 gene. The increased levels
of JAG2 expression in the cancer
samples will illustrate that JAG2
helps to drive the progression of papillary thyroid cancer. The samples with
the worse cancer subtypes will exhibit the highest levels of JAG2 expression.
Ultimately, these results will uncover JAG2’s
roles in papillary thyroid cancer and give important yet undiscovered
information to the medical and scientific fields.

 

 

 

Anticipated Problems or Complications

We understand the potential
complications and variables could arise during our experiment that could alter
our results. We have to make sure our siRNA knockdowns are long enough and do
not ultimately kill all the cells. We plan on having to adjust the
concentrations and conditions of our siRNA to successfully create knockdowns of
JAG2. We will account for those
problems if they occur. We might use a rat model if our mouse models cannot
survive. Swine is another option as their anatomy and processes are highly
conserved with humans. If the mice survival rates are very low and their organs
do not provide us with the information we need, rats might be an easier animal
model to work with for metastasis studies. Immunoprecipitations and western
blots are not extremely complicated experiments, but take necessary time to do
them properly. We anticipate to have to read-do experiment and staining for
visualization of bands to ensure of results are accurate. It takes time and
practice to produce legible and sufficient readouts from gel electrophoresis
and membrane blots that are trustable by the scientific field. We anticipate
preliminary complications with our antibodies that might alter our results, but
will make sure they are accurate by running controls to confirm the correct
antibodies. Another problem might arise in the with the COL1A1 and LOX blots. We
will need to runs essential controls to JAG2 levels with the addition of the
antibodies and without them as they could induce changes to the expression
levels of JAG2. For example,
extremely high levels of JAG2 might
interrupt binding of the COL1A1 and LOX antibodies to their targets.

Other approaches for papillary
thyroid cancer research could involve the expression of chemokines and
inflammatory cytokines. We would measure the expression through western
blotting expecting increased release of inflammatory cells to the sites of
cancer. With an animal model, we could then also use fluorescent microscopy to
visualize different tagged metastasis markers on the thyroid glands and other
organs. We are opens to use and include other experiments as we see fit through
our research.