AbstractIn the current field of thyroidcancer, treatment methods are lacking. The usual treatment involves thyroidremoval consisting of surgery and iodine therapy. Thyroid cancer occurrence ispredicted to surpass colon cancer by 2030 (Nikiforov). Invasive surgeries and iodine therapy have limited survival rates anddecrease quality of life upon treatment. Thyroid cancer can be categorized intoWell differentiated thyroid cancer (WDTC), Poorly differentiated thyroid cancer(PDTC), and Anaplastic thyroid cancer (ATC). WDTC can be further divided intopapillary and follicular thyroid cancer.

            From preliminaryexperiments on PTC and FTC cell lines in Dr. MacDonald’s lab, we haveidentified certain genes upregulated in either papillary or follicular thyroidcancer. JAG2, a gene involved in theNotch signaling pathway, was highly upregulated in papillary thyroid cancer. Wehypothesize that the overexpression of JAG2drives papillary thyroid cancer progression. To evaluate JAG2’s role in PTC, we will use cellular and animal models as wellas cancer samples. Importantly, we will evaluate metastasis and progression ofpapillary thyroid cancer in our models. Introduction and Preliminary DataThyroid cancer consists of threedifferent cancer subtypes including WDTC, PDTC, and ATC. Approximately 95% ofthyroid cancer is categorized as WDTC.

The other 5% consists of PDTC and ATC.The most common type, WDTC, can be divided into papillary and follicularthyroid cancer depending on the respective mutations in the MAPK signalingpathway (Nikiforov). Papillary thyroid cancer isassociated with a BRAF mutation and metastasizes to local lymph nodes. PTC istypically more aggressive and leads to a much worse prognosis than FTC (Nikiforov). Follicular thyroid cancer is less common and arises frommutations in HRAS and metastasizes to bones and the lungs.

For our research, weare focusing on papillary thyroid cancer. We feel that papillary thyroidcancer, due to its dominance, will allow us to fully study and analyze itthrough our JAG2 models and cancersamples. Below, we provide a heatmap of the preliminary data illustrating JAG2’s importance in papillary thyroidcancer.             Fromthe past semester in the advanced cell biology lab under Dr. MacDonald, weexperimented with samples of cell lines of papillary and follicular thyroidcancer derived from mouse models with over expressed MAPK signaling. The BRAFcell line contained a PTEN deletion and a BRAFV600E mutation (PTC)while the HRAS cell line contained a PTEN deletion and a HRASG12Vmutation (FTC). We observed the difference in the transcriptional profile ofthe cell lines. After treatment, in figure 3, we also developed gene ontology,which groups differentially   expressedgenes by processes.

We put our DESeq data in a gene ontology program calledGOrilla. GOrilla helped us determined the important genes upregulated in eitherpapillary or follicular thyroid cancer. From GOrilla, we were able to find ourgene of interest being JAG2 and formour hypothesis dealing with JAG2overexpression in papillary thyroid cancer. JAG2 encodes for the JAG2 protein thatinteracts in the Notch signaling pathway. The Notch pathway is important forembryonic development and is highly conserved among eukaryotic organisms. Notchsignaling cascades can be turned on by the binding of JAG2 and other jaggedfamily proteins that function as the ligands to Notch receptors. Previousresearch has demonstrated that Notch signaling can be activated by the MAPKpathway and lead ultimately to deregulation of the pathway especially inpapillary thyroid cancer (Yamashita).

The Notch signaling works downstream ofthe MAPK pathway and has a number of recognized roles in other solid malignancies.Medulloblastoma (MB) is a malignant brain cancer that deals with children inearly development. The deregulation of Notch signaling cascades have been shownto result in MB samples.

MB tumors models importantly show the deregulation andoverexpression of JAG1 and JAG2 (Fiaschetti). JAG2 has even been connected with increasing levels of the oncogeneMYC. Researchers believe JAG2 levels have the ability to determines stages ofcancer and how aggressive the cancer might be. Similarly, high levels of JAG2 expression result in humanendometrial cancer (Sasnauskien?). Endometrial cancer begins at thelining of the uterus and spreads from there. It is also called uterine cancerand generally affects women after menopause. Interesting to note, uterinecancer is linked with high levels of survival and generally good prognoses.

Thedevelopment of normal cells into cancer cells most certainly correlates toNotch signaling, but to what extent is unknown. In endometrial cancer, theNotch pathway acts as an anti-oncogene and aids in protection of cells asopposed the other cancers. With that being said, the Notch pathway affectsnumerous cancers depending on the level of protein expression in thoserespective cancers. Notch and MAPK signaling have also been studied in breastcancer. Irregular Notch signaling leads to overexpression of various Notchproteins as well as JAG1 and JAG2.

Breast cancer tissues exhibited high levelsof these proteins as compared to normal tissues (Mittal). More research is needed to determine theentire mechanism involving the key players of receptors, ligands, anddownstream targets through breast cancer development. The role of Notchsignaling in thyroid cancer has yet to be investigated. This helps us see whatis not yet known with JAG2 and whyour work is valid. Also, we are optimistic that our research with our animalmodels will help the thyroid cancer field in creating better treatment andtherapies in this cancer.

Our experiments will target our two specific aims. Hypothesis and Specific AimsWe hypothesize that overexpression ofJAG2 drives papillary thyroid cancerprogression. Through testing this hypothesis, we hope to uncover JAG2’s role and importance in papillarythyroid cancer. To test this hypothesis, we will test JAG2 in vitro and in vivo on hopes of understanding why JAG2 is upregulated and how itsexpression 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 BRAFV600Emutation for our cellular model. Our cellular model will involve siRNAknockdowns to study cell morphology, motility, and metalloprotease assays. Ouranimal model experiment will be straightforward in testing mortality andpathology of mouse models.Specific Aim 2: Evaluate JAG2expression in different stages of cancer.

To test expression, we will usecancer 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 DesignThe first experiments for ourresearch will include various verifications and confirmations that the Notchpathway is activated by MAPK signaling and that JAG2 is expressed in Notch signaling. We will use BrafV600E/Pten-/- /TPO-Cre mice containing the mutationin BRAF and Pten deletion (Jolly).These were mice with Cre recombinase activation that developed papillarythyroid cancer (Jolly). WithBrafV600E/Pten-/- /TPO-Cre mice and wildtype thyrocytes, we willactivate the MAPK pathway with various growth and differentiation factors forassessment of the pathway.

The immunoprecipitations will tell us the proteininteractions in our cells. We will then run western blots with antibodies toMAPK 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 MAPKpathway.To test specific aim 1, we willinhibit and silence JAG2 in ourcellular and animal model to observe changes in metastasis of papillary thyroidcancer.

The first set of experiments will deal with cellular models. The BRAFcell lines from Dr. McDonald’s class will be used with the addition of normalthyroid cells derived from mice with no BRAF mutations. Most importantly, wewill incubate the derived BRAF cells with siRNA knockdowns of JAG2.

We will try differentconcentrations of siRNA and at various time points before moving on with anyexperiments to make sure the cells are not dead and we have JAG2 knockdowns in our cells. We willtest if JAG2 is present by western blots with antibodies to JAG2.To evaluate whether JAG2 is requiredfor EMT, we will use our cellular model. The normal thyroid cells derived frommice should exhibit epithelial cells as opposed to mesenchymal. Epithelialcells are flatter and rounder while mesenchymal cells are narrow and look likespindles.

Mesenchymal morphologies are present in tumor cells. They arerecruited to tumors and aid in the process of metastasis. Metastasisencompasses the spread of cancer from initial sites of origin to additionalsites like other organs. Mesenchymal cells should be much more prevalent in themutated cells as opposed to the normal cells. Light microscopy and staining forcadherins will allow us to capture photographs of the difference between theappearance of our cells. EMT cells express high levels of N-Cadherin as opposedthe E-Cadherin in epithelial cells. We will use immunofluorescent microscopy toevaluate the expression of cadherins indicative of EMT.

Additionally, we willuse time lapse microscopy to determine to motility rates of the respective celllines. The cells with high motility rates should be the mutation in BRAF cellsshowing the increased ability to metastasize as opposed to the wildtype thyrocytes and JAG2 knockdowns. This microscopy willallow us to observe live cells for longer periods of time and narrow down onthe differences between cancerous and normal cells. Assuming our siRNAknockdowns of JAG2 are sustainablefor long enough time points, we expect to see similar motility rates of JAG2 knockdowns and the wildtypethyrocytes as opposed to the mutated cells. Finally, we will assess theexpression of metallomatrix proteins like MMP6. Metallomatrix protein 6 degradesthe ECM and as well as cleaves receptors on the cell surface. MMP are important in metastasis as they destroy thesesurface receptors and trigger unwanted cell proliferation i.e.

cancer. We willrun western blots for MMP6 with our WT cells, BRAF cells, and BRAF JAG2 -/- cells. The western blots willshow what cell are expressing these metallomatrix proteins. These experimentswill illuminate whether metastasis occurs with JAG2 or without JAG2.With viable populations of mice andhigh survival rates, we will cross various mice resulting in four differentcell lines. The first line will be easy to acquire as it will be wildtype micemainly for controls. We will also use mice with a PTEN deletion and a BRAFV600Eas a control. The two lines of mice that will give us the information we arelooking for will be JAG2 knockoutswith no BRAF mutation and JAG2knockouts with the BRAF mutation.

We will first take time to make sure thesemice can survive for more than a few weeks. After completion of the preliminarychecks, we will first take cells out of the thyroid of each respective modeland grow them in cell cultures to determine if are controls working and our JAG2 knockouts are similar. These mousemodels will allow us to look at metastasis of papillary thyroid cancer throughsacrificing. We will study the tissue of these mice to look at metastasis. Wewill harvest tumors from the mouse models, and then do thin-tissue sectioningand staining with H to evaluate pathology. The sections will be labeledby the mouse model and organized in cohorts.

Additionally, staining will allowbetter visualization under the microscope. Pictures will be taken and analyzedthrough computer programs. Since these mice will have already been sacrificed,we will also take other samples of organs from their bodies to look formetastasis. We will specifically look at the lungs, lymph nodes, and even bonesamples to assess whether JAG2 isrequired for metastasis, organs will be harvested from the JAG2 knockout mice and evaluated for tumor presence.             Under oursecond aim, we will be comparing normal thyroid tissue to WDTC, PDTC, and ATCtissue. To test cancer progression, we will obtain our human samples from theCancer Genome Atlas. The human samples will allow us to make comparisons to ourcellular and animal models.

We will use a wide variety of samples with tissuesfrom all the stages of thyroid cancer. We will isolate mRNA in the respectivesamples and use RT-PCR for mRNA detection and quantification of JAG2. We will also run western blotswith antibodies to COL1A1 and LOX to compare levels in the normaltissue and the cancer tissue. COL1A1and LOX have shown upregulation andincreased mortality rates in PTC (Jolly).

We will see if the cells with the highestJAG2 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 ImplicationsWe expect that JAG2 upregulation in papillary cancer fuels its metastasis andprogression. First, the confirmation of MAPK pathway will show activation ofthe Notch signaling pathway.

JAG2influences the metastasis of papillary thyroid cancer through its upregulationand aggressive effects on thyroid cells. Normal thyrocytes will show no MMP6activity, but the BRAF mutation cells will indicate MMP6 activity leading tothe metastasis of PTC. On the other hand, the JAG2 knockdowns will indicate low MMP6 activity and decreasedmetastasis implicating possible treatments involving the inhibition of JAG2 that might help thyroid patients.

The pathology studies will show increased metastasis in organs of the mutatedmice while the JAG2 knockouts willshow decrease metastasis to other organs. The mouse model will then allowparallels with humans in the development of treatments involving knockouts ofthe JAG2 gene. The increased levelsof JAG2 expression in the cancersamples will illustrate that JAG2helps to drive the progression of papillary thyroid cancer. The samples withthe worse cancer subtypes will exhibit the highest levels of JAG2 expression.Ultimately, these results will uncover JAG2’sroles in papillary thyroid cancer and give important yet undiscoveredinformation to the medical and scientific fields.    Anticipated Problems or ComplicationsWe understand the potentialcomplications and variables could arise during our experiment that could alterour results.

We have to make sure our siRNA knockdowns are long enough and donot ultimately kill all the cells. We plan on having to adjust theconcentrations and conditions of our siRNA to successfully create knockdowns ofJAG2. We will account for thoseproblems if they occur. We might use a rat model if our mouse models cannotsurvive. Swine is another option as their anatomy and processes are highlyconserved with humans.

If the mice survival rates are very low and their organsdo not provide us with the information we need, rats might be an easier animalmodel to work with for metastasis studies. Immunoprecipitations and westernblots are not extremely complicated experiments, but take necessary time to dothem properly. We anticipate to have to read-do experiment and staining forvisualization of bands to ensure of results are accurate. It takes time andpractice to produce legible and sufficient readouts from gel electrophoresisand membrane blots that are trustable by the scientific field. We anticipatepreliminary complications with our antibodies that might alter our results, butwill make sure they are accurate by running controls to confirm the correctantibodies.

Another problem might arise in the with the COL1A1 and LOX blots. Wewill need to runs essential controls to JAG2 levels with the addition of theantibodies and without them as they could induce changes to the expressionlevels of JAG2. For example,extremely high levels of JAG2 mightinterrupt binding of the COL1A1 and LOX antibodies to their targets. Other approaches for papillarythyroid cancer research could involve the expression of chemokines andinflammatory cytokines. We would measure the expression through westernblotting expecting increased release of inflammatory cells to the sites ofcancer. With an animal model, we could then also use fluorescent microscopy tovisualize different tagged metastasis markers on the thyroid glands and otherorgans. We are opens to use and include other experiments as we see fit throughour research.     


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