Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...

Dana-Farber/Harvard Cancer Center 

CURE 

Continuing Umbrella of Research Experiences 

Scientific Presentations 

Summer 2009

Launched in 2002, the Continuing Umbrella of Research 

Experiences (CURE) at Dana-Farber/Harvard Cancer Center 

is an important building block in research training initiatives. 

This program is designed to provide underrepresented 

minority high school and college students with a stimulating 

and rewarding hands-on research experience that encourages 

students to pursue education and training in the biomedical 

sciences and careers in basic, clinical and population sciences 

cancer research.

CURE Scientific Presentations 

Dana Building, Room 1620 

(Smith Family Room) 

Wednesday, August 19, 2009 

1:00–4:00 p.m. 

Thursday, August 20, 2009 

1:30–5:00 p.m. 

Friday, August 21, 2009 

1:00–4:00 p.m.

Incidence and Predictors of a Late Prostate Cancer Recurrence Among Men Who 

Have No Evidence of Disease at Five Years Following a Radical Prostatectomy 

Abiola Ahove 

Mentor: Paul Nguyen, MD 

Scientific Advisor: Vince D’Amico, MD, PhD 

Dana-Farber Cancer Institute/Brigham and Women’s Hospital 

Background Information: Prostate cancer is the second-leading cause of cancer 

death in American men. A prostate-specific antigen (PSA) level >=0.2 ng/mL following 

radical prostatectomy is called a PSA failure and is considered to be a prostate 

cancer recurrence. Objective: In this study, we examined the incidence and predictors 

of developing a late PSA failure among men who have achieved at least five years 

of recurrence-free survival following a radical prostatectomy. Methods: The clinical 

records of 508 men who underwent radical prostatectomy for prostate cancer from 

1985-2000 at Brigham and Women’s Hospital and who achieved at least five years of 

recurrence-free survival (i.e. all PSAs

Production of ß 2 

M and HLA-A2 for MHC Tetramers Formation 

Judith Alonzo 

Mentor: Karen S. Anderson MD, PhD 

Scientific Advisors: Jaewon Choi, MS; Jun Zhou, PhD; and Jessica Wong, BA 

Dana-Farber Cancer Institute 

Background: The major histocompatibility complex (MHC) is a genomic region 

that plays a crucial role in the immune system of most vertebrates. MHC tetramers 

are proteins that monitor T-cells in the blood and bind to T-cell receptors. They are 

folded by binding ß 2 

-microglobulin (ß 2 

M) with HLA-A2 and antigenic peptide. 

MHC tetramers require refolding individual sub-units of high purity, quantity, and 

quality. The goal of this project is to produce high yields of ß 2 

M and A0201 to fold 

MHC tetramers with the desired peptide. Methods: Three A2 DNA constructs were 

tested, HLA-A2Nohis1, A2his+4, and A*0201 and one ß 2 

M. Culture conditions 

were optimized to obtain higher yields of both, ß 2 

M and HLA-A. Proteins were 

expressed in E. coli bacteria. Two methods were employed for ß 2 

M and HLA-A production 

to fold MHC tetramers, sonication and B-PER Reagent. Both techniques 

disrupt the cell membranes and release cell contents, including proteins. Refolding 

reaction and biotinylation of each protein follows. Purity is monitored on 4-20% 

SDS-PAGE gel. Results: HLA-A*0201 was found to work better than the other 

DNA constructs, giving higher yields (390.80mg for 1L culture) and more pure 

samples; similar results have been obtained for ß 2 

M but at lower yields (13.46mg for 

250mL culture). Currently, sonication has proven to be more suitable for large-scale 

protein preparation than B-PER reagent. Conclusion: A0201 was obtained in high 

yields and purity, as well as ß 2 

M. The sonication method is more efficient for Largescale 

protein production while B-PER reagent is more convenient for smaller sample 

sizes. No final conclusions can be made at this time since folding and biotinylation 

processes are ongoing.

Medical and Psychological Outcomes of Sibling Donors for Patients Undergoing 

Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) at a Single Institution 

Bethel Belai 

Mentor: Leslie Lehmann, MD 


Bone marrow transplant (BM) and cytokine mobilized peripheral blood stem cell 

(PBSC) transplantation can be curative for patients with malignant and nonmalignant 

hematologic diseases. Siblings are most often used as donor matches for such 

patients. In cases where siblings are not donor matches, unrelated donors are used 

as donor matches for these same patients. Much psychological assessment has been 

done regarding unrelated donors post-donation, but none involving sibling donors 

specifically. This exploratory study aims to assess the medical and psychological outcomes 

of sibling donors following a bone marrow harvest performed at Dana-Farber 

Cancer Institute from January1998 through December 2008. Donor outcome was 

assessed at a time, 3-9 months following donation. The first part of the study is the 

medical element, compiled by a list of an estimated 150 sibling donors within the 

past 10 years, who are between 5-19 years of age. The second part is the psychological 

element which examines the donors mental state pre and post-donation. Assessments 

were performed in person, or by phone by a member of the HSCT team who did 

not perform the actual harvest. Donors were asked questions regarding any medical 

or psychological issues related to the process of serving as a donor (guardian was 

present for questioning unless donor was under 18 years of age and without guardian 

consent). Data will be gathered based on the outcome of these assessments as 

documented in the donor medical record.

The extracellular matrix influences mesenchymal stem cell 

activation toward a myofibroblast-like phenotype 

Melissa Bryll 

Mentor: Raghu Kalluri, PhD 

Scientific Advisor: Michael Duncan, PhD 

Beth Israel Deaconess Medical Center/Harvard Medical School 

Numerous studies suggest that mesenchymal stem cells (MSCs) have the potential 

to serve a therapeutic role in a variety of disease states including cancer. However, 

recent reports indicate that MSCs in the context of the tumour microenvironment 

may serve as a source of carcinoma-associated fibroblasts (CAFs). The extracellular 

matrix is an important component of the tumor stroma and regulates cell adhesion, 

survival, and proliferation. This study examines the influence of the extracellular matrix 

(ECM) on TGFß1- induced transition of MSCs to CAFs. We propose that distinct 

ECM components within the tumour microenvironment can modulate MSC 

activation into fibroblasts through cell-matrix adhesion events. We have conducted 

cell adhesion assays on matrix-coated plates to determine which matrix components 

effectively promote cell adhesion during MSC activation. Subsequently, immunoblotting 

and gene expression profiling for MSC and CAF markers of TGFß1 treated 

cells cultured on the ECM molecules will emphasize the extent to which the matrix 

influences MSC activation. We hypothesize fibronectin will have a significant effect, 

as it is a common component of the tumour stroma and MSCs readily adhere to it. 

Determining a role of the reactive stroma in MSC activation will further enhance our 

understanding of MSC utilization and its potential limitations in cancer therapy.

Targeting Glioma Growth 

Cody Cameron 

Mentor: Khalid Shah, PhD 

Scientific Advisor: Tugba Bagci, PhD 

Massachusetts General Hospital 

Glioma is a form of brain cancer which results in very sudden yet detrimental effects 

to the patient. Like most cancers, gliomas result from the deregulation of certain 

components of the cell cycle, such as proliferation. While normal cells regulate their 

proliferation and undergo apoptosis when damaged, glioma cells undergo uncontrolled 

proliferation, thereby creating neoplasms. Among other pathways, the PI3 

Kinase-Akt pathway is commonly mutated in gliomas resulting in uncontrolled 

proliferation. Over activation of PI3 Kinase leads to increased phosphorylation and 

thereby activation of Akt, which then leads to increased cell growth. The inhibition 

of activity along the PI3 Kinase pathway can achieved with a new drug, PI-103. The 

overall goal of the project is to test the efficacy of PI-103 alone and in combination 

with stem cell based pro-apoptotic therapies in both malignant and invasive glioma 

cells in vitro as well as in mouse models of glioma. My part in the project is to test the 

effect of PI-103 on the PI3 Kinase pathway components in different glioma cells in 

culture. Specifically, I aim to demonstrate the changes in the phosphorylation of Akt 

levels in response to different doses of PI-103 treatment in six established and two 

primary human glioma cell lines. My results will determine whether this drug will 

inhibit the activation of the PI3-kinase-Akt pathway in human glial cells. This should 

set up a clear-cut plan for in vivo experiments that will follow in culture studies and 

will be subsequently performed in the laboratory.

Identification of novel genes that influence virulence of vibrio cholerae 

Cindy Capitolin 

Mentor: Paula Watnick, MD, PhD 

Scientific Advisor: Alexandra Purdy, PhD 

Children’s Hospital Boston 

Cholera, a fatal disease characterized by severe diarrhea, is caused by a Gram-negative 

bacterium called Vibrio cholerae which is acquired via ingestion of contaminated food 

or water. Although cholera is practically non-existent in the US, it is a major concern 

in the developing world, particularly in countries with poor sanitation. Identification 

of the genes required for intestinal survival of the Vibrio cholerae bacterium will help 

us to understand the pathogenesis of this disease and develop new ways of treating 

cholera. 

The toxin-co regulated pilus and cholera toxin, which are responsible for the intense 

diarrhea that is seen in cholera, are already known to be essential for virulence of V. 

cholerae. We hypothesize that there are additional genes that contribute to the virulence 

of Vibrio cholerae. The purpose of this study is to identify these genes. 

The Watnick lab has developed Drosophila melanogaster as a model organism for the 

study of cholera. Several virulence-defective mutants were identified in a primary 

screen. In my study, survival assays were set up in Drosophila and the virulence defect 

of 8 transposon insertion mutants was confirmed. Colonization assays were then 

set up in order to determine the ability of each specific mutant strain to survive in 

and colonize the Drosophila intestine. We also isolated, amplified and sequenced the 

transposon insertion sites of each of these mutant strains. 

Thus far we have identified the transposon insertion sites of four mutant strains. 

These include flagellar synthesis genes, a phosphate transport system, and an uncharacterized 

ABC transport system. We anticipate that at the end of this project we 

will discover and characterize other genes that are relevant to the virulence of Vibrio 

cholerae.

Determination of Elafin’s role in cellular proliferation 

in the ovarian cancer microenvironment 

Amber Channer 

Mentor: Ronny Drapkin, MD, PhD 

Scientific Advisor: Adam Clauss, PhD 


Ovarian cancer is a major cause of cancer-related mortality in women worldwide. 

Unfortunately, the lack of efficient screening methods results in the majority of patients 

being diagnosed with late stage ovarian cancer, when a surgical cure is highly 

unlikely. Although most patients with advanced stage disease initially respond to 

chemotherapy, the majority ultimately relapse with chemo-resistant tumors. Therefore, 

there is a pressing need to develop serum biomarkers for early detection. We 

have previously described two potential biomarkers: HE4 and Elafin. Both are located 

in the WAP locus on chromosome 20q13, a region frequently amplified in 

ovarian cancers. The FDA recently approved the HE4 ELISA test, in conjunction 

with CA125, to monitor patients with ovarian cancer for recurrence. Elafin is a secreted 

serine protease inhibitor, which has shown to specifically inhibit Elastase, a 

serine protease. We know that the protease function of Elastase is important to release 

TGF-ß and thereby active it. TGF-ß (Transforming Growth Factor) is a secreted 

protein that mainly functions to inhibit epithelial proliferation, tumor development 

and morphogenesis. During oncogenesis, TGF-ß is silenced and cannot control the 

cell cycle, thus enabling cells to grow uncontrollably. This work aims to characterize 

the role Elafin plays in the ovarian cancer microenvironment. We hypothesize that 

by inhibiting Elastase, Elafin prevents the release of active TGF-ß. To analyze we will 

transform a panel of ovarian cell-lines with either wild type Elafin, which has been 

previously shown to increase cellular proliferation, or two mutated forms of Elafin 

(M25K and M25G). The mutation of methionine to a lysine or a glycine generates 

a protein that no longer functions as a serine protease inhibitor. By comparing the 

proliferation effect of the wild type Elafin versus the mutated counterpart, we will be 

able to tell whether the serine protease inhibitor function of Elafin is necessary for its 

role in increasing cellular proliferation.

Co-crystallization of PIP2 and the FERM domain of focal adhesion kinase 

Douglas Chapski 

Mentor: Daniel Lietha, PhD 

Scientific Advisor: Michael Eck, MD, PhD 


Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays an essential 

role in focal adhesions and cell migration. FAK’s overexpression in diseases such as 

glioblastomas and ovarian cancers suggests that it is a good target for cancer therapies. 

However, the mechanism of FAK regulation downstream of integrin signaling 

is not fully understood. Previous research indicates that phosphatidylinositol 

4,5-bisphosphate (PIP2) activates FAK by binding to the F2 lobe of the FERM domain. 

Crystallographic studies show that existing crystal packing of FERM domain 

crystals does not allow PIP2 to be soaked into the hypothesized binding region of the 

F2 lobe. Co-crystallization was attempted as an alternative method for solving the 

FERM+PIP2 structure, but crystals were too small for analysis. Future optimization 

of co-crystallization conditions and diffraction studies will give us insight on how 

PIP2 binds to the FERM domain, and thus how FAK gets activated. With the new 

crystal structure, we will be able to construct a more detailed activation pathway for 

FAK.

The Effect of PolyIC on Cultured Lymph Node Fibroblastic Reticular Cells 

Pamela Cote 

Mentor: Shannon J. Turley, PhD 


Fibroblastic reticular cells (FRCs) in the lymph nodes (LNs) have been characterized 

as a subset of lymph node stromal cells (LNSCs) with a CD45-, gp38+, CD140a+, 

CD31- surface phenotype. These cells have also been found to express MHC Class I 

and II, costimulatory molecules (PD-L1, CD80) and adhesion molecules (VCAM-1, 

ICAM-1). Expression of these molecules may allow LNSCs to regulate T cell responses 

in the LNs and shape immunological tolerance. FRCs are important cells 

because they also secrete survival factors for memory and resting T cells in the LNs 

and they secrete chemokines which are necessary for the recruitment of Dendritic 

cells (DCs) and naïve T cells to the LNs. It has been established that certain viruses 

such as the Ebola virus and persistent lymphocytic choriomeningitis (LCMV) infect 

FRCs in the LNs which may result in immunological dysfunction and chronic infection. 

The purpose of this study is to ascertain whether exposure to PolyIC causes 

LNSCs to acquire immunostimulatory potential using flow cytometery. This study 

will allow us to determine if viral pathogens or viral products can cause FRCs to 

undergo phenotypic changes. Therefore this will allow us to establish whether FRCs 

continue to promote self tolerance induction or auto- reactivity of T cells during a 

viral infection and to ultimately recognize what important implications these cells 

have on immunological disorders.

Conjugating Aptamers to Nanorods Allowing for Chemotherapeutic Drug Delivery 

Elise Digga 

Mentor: Omid Farokhzad, MA, MD 

Scientific Advisor: Xiao Zeyu, PhD 

Brigham and Women’s Hospital 

In recent in vivo experiments, nanorods have been increasingly employed as carriers, 

capable of transporting chemotherapeutic drugs to targeted cancer cells. Their ability 

to convey the drugs is due to RNA sequences called aptamers. The entire complex 

consists of an aptamer attached to strands of nucleic acids, which in turn are conjugated 

to the nanorods. The drugs are stored between the strands of nucleic acids and 

released when the aptamers bind to membrane receptors specific to the malignant 

cells. We are currently trying to find which RNA sequence can bind to which cancer 

cells, using the cell lines PC3 (PSMA- prostate cancer cell), LNCaP (PSMA+ prostate 

cancer cell), BPH (prostate normal cell). By using FACS (fluorescence-activated cell 

sorter) we found an RNA sequence that binds to LNCaP, but not BPH (as expected) 

or PC3 due to the membrane receptor specific to LNCaP. We can cut down this 

sequence further to find smaller segments that will conjugate with LNCaP and by 

doing so will further impact drug transport studies in the future.

Role of SCCRO/DCUN1D1 in Prostate Cancer 

Thao Xuan Do 

Mentor: Luiz Zerbini, PhD 

Beth Israel Deaconess Medical Center 

Squamous Cell Carcinoma Related Oncogene (SCCRO)/DCUN1D1 has been 

known to be amplified and over-expressed in up to 40% of lung, head and neck, 

and cervical squamous cell carcinomas, suggesting that it plays an oncogenic role 

in these cancers. However, the role of SCCRO/DCUN1D1 in prostate cancer is 

unknown. Therefore we target SCCRO/DCUN1D1 in prostate cancer cell line. By 

using Western Blot and PCR, we found that SCCRO/DCUN1D1 is upregulated 

at both the mRNA and protein level in primary and metastatic prostate cancer cell 

lines such as CW19, CW22, VCaP, LNCaP, DUCaP, DU145, PC3, CL1 and PREC. 

Knockdown of this gene in prostate cancer cell lines in vitro using RNA interference 

technique resulted in a decrease in proliferation rate by MTT cell proliferation assay 

using MTT (yellow tetrazolium salt). Suppression of SCCRO/DCUN1D1 also inhibited 

the migration and invasion characteristics of prostate cancer cell lines in vitro 

by migration/ invasion assay using BD BioCoat Matrigel Invasion Chambers. With 

all of the above results, we strongly believe that SCCRO/DCUN1D1 is required for 

the development of prostate cancer tumor.

The Growth of Malignant Plasma Cells in Zebrafish Embryos 

Jessica Francois 

Mentor: Robert I. Handin, MD 

Scientific Advisor: Nikhil Munshi, MD 

Brigham and Women’s Hospital/Dana-Farber Cancer Institute 

Multiple myeloma is a malignant disorder of plasma cells, which reside in the bone 

marrow and are the antibody-producing cells of the immune system. Our laboratory 

uses zebrafish embryos to study the growth of both murine and human myeloma 

cell lines and primary myeloma cells purified from patient bone marrow aspirates. 

Our objective was to determine whether zebrafish could be used to study growth 

characteristics of human myeloma cells. Zebrafish are an increasingly popular animal 

model as they are easy to breed and manipulate. Zebrafish embryos are transparent 

and tumor cells grow rapidly after injection. We injected 50 nl fluid containing 

1,000-2,000 myeloma cells suspended in matrigel into the pervitelline space of 

anesthetized, dechorionated zebrafish embryos 48 hours post fertilization. In some 

cases, unlabelled myeloma cells were injected into Tg(Fli1:GFP) fish, which express 

GFP in endothelial cells, to assess tumor-induced angiogenesis. The angiogenic response 

to tumor injection was also assessed by alkaline phosphatase staining of fixed 

embryos. Twenty four to forty eight hours after injection, myeloma cells induced an 

angiogenic response in the host with proliferation of vessels from the sub-intestinal 

venous (SIV) system toward the tumor. Within 2-3 days, visible growth of myeloma 

cells was observed. In some fish, myeloma cells grew out of the yolk sac and into 

vertebral bodies. In contrast, plasma cells from normal individuals or individuals 

with monoclonal gammopathy of uncertain significance (MGUS), a benign precursor 

to myeloma, did not induce angiogenesis nor form tumors. We conclude that 

zebrafish can be used to study the growth characteristics of human myeloma cells and 

may provide a convenient model to identify novel or combinations of drugs that will 

inhibit myeloma cell growth.

Targeting Communication Disparities with Click to Connect 

Charline Gay 

Mentor: Vish Viswanath, PhD 

Scientific Advisors: Sara Minsky, BA and Emily Zobel Kontos, AB, ScM 


Despite improvements in overall health of Americans, some racial/ethnic minority 

populations and lower socioeconomic position (SEP) individuals experience health 

disparities that may be attributed to inequalities in communication. The Internet has 

emerged as a powerful source of health information with growing penetration. Yet, 

limited attention has been paid to the utility and usability of websites for low literacy 

and low SEP individuals. In our approach to improving this communications gap, 

we developed an intervention (Click to Connect) focused on improving access and 

ability to use the Internet for low literacy and low SEP individuals. The components 

of the intervention include a low literacy web portal designed specifically for our 

audience, computer use/Internet training, and technical support. The goal of my 

project is to analyze and report the results of the intervention’s process data, which 

will aid in the assessment of the operation of the program, program strengths and 

weaknesses, and will allow for mid-course corrections. We hypothesize that participants 

will increase their usage of the Internet to seek health information over time 

as they acquire computer and Internet literacy skills. Each participant was asked to 

complete an online survey once a month to assess their Internet and computer use, 

their experience with the system, and a training evaluation questionnaire. In this 

survey, we focused on evaluating the effectiveness of the intervention in terms of 

the acquisition of computer and Internet literacy skills, and the effectiveness of the 

training component of the intervention among participants over the course of the 

intervention period. Qualitative and quantitative analysis was then conducted on the 

web portal survey results and the training evaluation.

High level baseline in language fmri 

Carl Glaser 

Mentor: Alexandra Golby, MD 

Scientific Advisor: Laura Rigolo 


Functional Magnetic Resonance (fMRI) imaging of the brain has been widely used 

in research for years but is just recently becoming prominent clinically for presurgical 

planning of tumor resection. The use of fMRI to identify and map functional areas 

of the brain, those used in motor, language functions etc, has specifically gained support 

in the clinical field because of its non invasive nature. This surge in fMRI’s use 

clinically has sent researchers looking for ways to increase its effectiveness. This has 

resulted in investigation into the format used to administer fMRI. Currently one 

test used to identify areas of language activation consists of a task involving antonym 

generation and a baseline scan. The baseline must be obtained in order to show the 

change in neural activity. The baseline involves subjects fixating on a crosshair. Because 

of this, increased activation between scan occurs not only in the area involved 

in language function, it also occurs in areas such as those of working memory, attention, 

execution, and vision. Therefore the areas of increased activation during a 

language task vs. the standard baseline are not all correlated to the use of language 

functions. This provides the user with a wide range of data that isn’t language related. 

This experiment will attempt to remedy this fact by introducing a high-level 

baseline. This task consists of the subjects identifying whether a series of letters are 

uppercase or lowercase. When compared to the traditional baseline it is expected that 

the high level baseline will show increases in neurological activation in similar areas 

to the language task, excluding the language activation itself. This would then allow 

comparison of the language task and the high level baseline, removing all areas of 

similar activation from the language task results, leaving only the desired language 

activation.

Determining the Mechanisms of DNA Double-Strand Break in 

Heterochromatin & Euchromatin 

Raquel L. Graham 

Mentor: Peter O’Donovan, PhD 

Scientific Advisor: David Livingston, MD 


Our genetic material DNA resides in a chromatin structure composed of DNA and 

histone proteins. There are two forms of Chromatin—heterochromatin and euchromatin. 

Heterochromatin is tightly compacted and transcriptionaly inactive, whereas 

euchromatin is less compacted and transcriptionaly active. Chromatin impacts all 

DNA Metabolic processes, including DNA damage and DNA repair. The DNA double-strand 

break [DSB] is the most toxic of various types of DNA damage because 

both DNA strands are damaged. There are two major categories to repair DNA DSB: 

homologous recombination and non-homologous endjoining. Homologous recombination 

is error free and non-homologous endjoining is error prone. It has been 

suggested that there are differences in DNA repair between heterochromatin and euchromatin. 

The purpose of this study is to find out whether or not there are differences 

in mechanisms of DNA DSB repair of heterochromatin and euchromatin. To test 

this hypothesis, we have developed a system that allows us to generate a single DNA 

DSB in a cell. The DNA DSB will be either in heterochromatin or euchromatin. We 

will be using an enzyme called ISce-I and female mouse embryonic fibro-blast cells 

[MEFs]. A restriction site will be placed in the X Chromosome of the MEFs because 

the female has two X Chromosomes, active X (euchromatin) and inactive X (heterochromatin). 

Then ISce-I will be introduced into the nucleus to generate a DNA DSB 

in the X Chromosome. After DNA DSB repair, genomic DNA will be extracted from 

cells, PCR amplified, and sequenced for the region around DSB. The sequence will 

tell what repair mechanism was used for DSB, i.e. homologous recombination or 

non-homologous endjoining. Therefore we will be able to determine whether different 

repair mechanisms were used between heterochromatin vs. euchromatin.

Early Detection of Morphological Changes After 

Massive Small Bowel Resection in Rats 

Winta Haile 

Mentor: Stanley Ashley, MD 

Scientific Advisor: Anita Balakrishnan, MD 


Although small bowel resection incites adaptive morphological changes in remnant 

intestine, such changes have only been identified a week beyond resection. We aimed 

to determine whether these changes would occur at two days post-resection. 

Sprague Dawley rats were subjected to either 80% resection (n=4)or transection and 

reanastomosis (n=4) of the small bowel. Two days later, rats were sacrificed and sections 

of remaining jejunum harvested, fixed in formalin and embedded in paraffin. 

7um thick sections were stained with haematoxylin and eosin and used for measurement 

of villous height and width, crypt depth, enterocytes per crypt, enterocytes per 

villus, and smooth muscle thickness. 

I anticipate changes in morphology following resection in response to small bowel 

length, such as increases in villous height, crypt depth, number of enterocytes per 

crypt and villus, and smooth muscle thickness. If we find reproducible morphological 

changes in response to resection, we can use this model to identify potential specific 

genetic pathways regulating the adaptive response. Identifying early morphological 

changes and the regulating factors will allow improved treatments for patients with 

insufficient adaptation in short bowel syndrome.

Patient Acceptability of the Electronic Self Report 

Assessment Cancer Program (ESRA-C) Version 2 to Enhance 

Communication Between Patients and Clinicians 

Tanya Harrison 

Mentor: Donna Berry, PhD, RN, AOCN, FAAN 

Scientific Advisor: Barbara Halpenny, MA 


Assessing cancer symptoms and quality of life issues is essential for excellent clinical 

care in cancer patients. The Electronic Self Report Assessment-Cancer program 

(ESRA-C) version 1 was demonstrated to be an efficacious and acceptable method to 

cancer patients for reporting symptom and quality of life information to clinicians. 

ESRA-C version 2 is a further developed program that contains additional symptom 

questionnaires and has been used in a multi-site sample in a randomized clinical trial 

at two comprehensive cancer centers. The objective of this analysis is to determine 

if ESRA-C version 2 is as equally acceptable to patients as version 1 and whether 

acceptability differed in various demographics. We evaluated the average score of 

the five acceptability questions with response options ranging from one (not at all acceptable) 

to six (very much acceptable) among patients that have completed ESRA-C 

version 2. We also assessed demographics such as age, gender, and computer literacy. 

Results showed that 40% of the sample (n=65) are female, 40% are age 60 or older, 

and only 74% of the participants use a computer often or very often. There were no 

differences in acceptability of the program between men and women, patients under 

or over 60 years old, and frequent or non-frequent computer users. Although specific 

measures of acceptability varied between the two groups, the average acceptability 

score was lower in version 2. We conclude that ESRA-C version 2 is not as equally 

acceptable to patients as version 1 due to the amount of time it takes for the participants 

to complete the report.

Investigating the role of Esrrb and its regulation 

of satellite cell potential in vitro 

Keyauna Hoffman 

Mentor: Glen Rowe, PhD 


Estrogen related receptor beta (Esrrb) encodes for a protein that is involved in many 

biological processes including placental and inner ear development. Esrrb has also 

been reported to play a role in embryonic cell biology, their self renewal, and the 

potential to differentiate. However, little is known at present of the role of Esrrb in 

the regulation of other cell systems such as muscle biology and satellite cell’s ability 

to differentiate into myoblasts. We therefore wanted to determine if Esrrb played a 

role in the regulation of satellite cell’s potential in vitro. Satellite cells are located on 

the surface of myofibers and in response to muscle injury, they provide the necessary 

skeletal muscle growth and repair. Preliminary data from our lab has shown that elongated 

myotubes in culture treated with Esrrb agonist, transform into circular colonies, 

which morphologically resemble satellite cells. We therefore performed qPCR to 

determine if these circular colonies expressed markers of satellite cells or if they were 

simply defused myotubes. We observed a significant change in both markers of satellite 

cells and terminally differentiated myotubes, suggesting that the cells treated with 

the Esrrb agonist were indeed changing their differentiation program. In summary, 

our experiments suggest that Esrrb could potentionally be a regulatory factor in the 

satellite differentiation program. We therefore hope to determine whether these dedifferentiated 

myotubes can differentiate again into myotubes. In addition, we hope to 

perform both gain and loss of function experiments with Esrrb to determine whether 

it is either necessary or sufficient to regulate satellite cell differentiation. There are still 

many outstanding questions about Esrrb’s contribution to the adult myogenic program 

and how it could contribute to advances in stem cell based therapies. However, 

these questions are currently the focus of other ongoing studies.

Interstitial Cystitis Antiproliferative Factor Modulates MDM2, 

Leading to P53 Stability 

Nichole Jones 

Mentor: Jayoung Kim, PhD 


Objectives: Interstitial cystitis (IC) Antiproliferative Factor (APF) has been reported 

as a sialoglycopeptide urinary biomarker of IC, a chronic syndrome of unknown 

etiology with variable symptoms including pelvic and or perineal pain, urinary frequency, 

and urgency. We previously reported that APF suppressed the proliferation 

rate of normal bladder epithelial cells through a mechanism that involves increased 

levels of p53 and activated signal pathways (Kim et al. FEBS Lett. 581:3795, 2007; 

Kim et al. BJU Int. 103(4):514, 2009). The goal of present study is to understand 

the process whereby p53 expression level elicits cell growth arrest in response to APF. 

Methods: We performed MTT-based proliferation assay to measure APF effects on 

growth of T24 human bladder cells. We engineered the expression levels of p53 and 

the murine double minute 2 E3 ubiquitin ligase (MDM2) by transient transfection 

with silencing RNAs or by the use of overexpression constructs. Various biochemical 

experiments including Immunofluorescence Staining, western blot analysis and immunoprecipitation 

were used. Results: APF treatment significantly decreased protein 

level of MDM2 within 2 hours. Time course experiments showed that p53 level was 

enhanced when MDM2 expression was suppressed. Inhibition of proteasomal activity 

enhanced the APF-induced p53 increase. MDM2 bound to p53 and triggered 

ubiquitination and degradation of p53. Furthermore, APF attenuated the association 

of p53/MDM2 and delayed p53 degradation. Conclusions: We demonstrate that 

MDM2, a negative regulator of p53 expression, is functionally attenuated by APF 

in bladder epithelial cells. These data suggest that targeting MDM2 or p53/MDM2 

complex formation may be relevant in the development of novel therapeutic approaches 

to IC.

Exploring the Effects of Estrogen on Apoptosis 

Walker Keenan 

Mentor: Shannon Bailey, PhD 

Scientific Advisor: Myles Brown, MD 


The risk of a woman developing breast cancer over the course of her lifetime is 13% 

and 1 in 4 diagnosed cancers in 2008 was breast cancer. Anti-estrogen therapy, which 

prevents estrogen receptor (ER) activity, works well in ER positive breast cancer patients. 

Previously, our lab has determined all the binding sites for the estrogen receptor 

and the pro-apoptotic transcription factor p53, in the human genome. We discovered 

that both of these proteins share a subset of binding sites in the MCF7 breast 

cancer cell line. It has been determined that some of the genes near these binding 

sites are known to initiate apoptosis. We found that a number of these genes are differently 

regulated by either p53 or ER, where genes down-regulated by estrogen are 

up-regulated by p53 stimuli. We therefore hypothesized that estrogen inhibits apoptosis. 

To determine if ER can block p53-mediated apoptosis, we will treat the breast 

cancer cell lines, MCF7 and T47D, with apoptosis inducing drugs in the absence or 

presence of ß-estradiol (E2), an estrogen receptor agonist. By determining the effects 

of E2 on p53-mediated apoptosis, we may be able to provide a new paradigm for the 

discovery of more effective breast cancer treatments.

Repression Of Transcription Factor 7-Like 2 Increases Risk Of Type 2 Diabetes 

Tsega Meshesha 

Mentor: Melissa K. Thomas, MD, PhD 


Diabetes mellitus is a disorder characterized by hyperglycemia that can occur through 

mechanisms such as impaired insulin secretion, insulin resistance in peripheral tissues 

and increased glucose output by liver. Genome-wide association studies have identified 

Transcription factor 7-like 2 (TCF7L2), a key element of the Wnt signaling 

pathway, as a genetic variant linked to a higher risk of developing type 2 diabetes. 

However, the mechanisms by which TCF7L2 is implicated in this disease are unknown. 

The objective of this study is to determine whether TCF7L2 deficiency alters 

pancreatic beta cell mass, function or insulin production. To address this question, 

we are investigating the phenotype of TCF7L2 heterozygous knockout as compared 

to wild-type control mice. The mice were genotyped by extracting DNA from tail 

biopsies. The extracted DNA was analyzed by polymerase chain reaction (PCR). To 

determine whether TCF7L2 deficiency alters insulin production and pancreatic architecture, 

pancreatic tissues from TCF7L2 heterozygote and wild-type mice were 

immunostained for TCF7L2, insulin, and glucagon. It is expected that if TCF7L2 

regulates pancreatic endocrine cell mass or hormone production, the immunostaining 

will reveal a change in beta and/or alpha cell mass or pancreatic hormone expression 

patterns when pancreatic tissue of TCF7L2 heterozygotes is compared to controls. 

This study may elucidate how TCF7L2 influences and/or regulates glucose levels.

Untitled 

Michel Moravia 

Mentor: Michael F. Gurish, PhD 

Scientific Advisor: Tatiana G. Jones, MD, PhD 

Brigham and Women’s Hospital/Harvard Medical School 

Mast cells (MC) are found throughout the body near blood vessels and mucosal 

interfaces with the environment where they are thought to play a role in mast cell 

progenitor homing in innate immunity. The MC homes to the lungs as a committed 

progenitor (MCp). A recent investigation of MCp homing uncovered a role for T-bet, 

a transcription factor protein that regulates T cell development, as T-bet deficient 

mice had reduced numbers of MCp in both the intestine and the lungs (Alcaide et 

al, 2007). Surprisingly, the study found that T-bet expression in dendritic cells (DC) 

was critical to the homing of MCp to the intestine. We are extending this finding by 

investigating whether T-bet+ DC are also required for MCp homing to the lungs. To 

evaluate this, I will culture bone marrow-derived DC (BMDC) from both wild type 

C57BL/6 mice and T-bet knockout mice. These DC will be transferred to T-bet deficient 

mice to evaluate whether T-bet+ DC restores MCp homing to the lung. Two 

weeks after transfer, I will perform a limiting dilution assay to quantitatively assess 

the total numbers of MCps present in the lungs of the respective mice. This work has 

the potential to provide new checkpoints for applied research and drug discoveries. 

The use of VIA and HPV testing was favored in most studies and successful results 

were attained. VIA fit the criteria of being efficient compared to cytology, affordable, 

sensitive and not much training required although is not very specific. Colposcopy 

use requires a lot of training and experience in order to get the right results. HPV 

vaccination is costly according to most of the countries economic status, in addition, 

the 3 doses is problematic since in most studies many patients were lost in follow up 

visits.

Cervical cancer prevention in developing countries 

Vivian Nassali 

Mentor: Annekathyrn Goodman, MD 

Scientific Advisors: R. Sankaranarayanan, MD and Paul D. Blumenthal, MD 


Introduction: Cervical cancer is the second most common cancer cause of death in 

women in undeveloped countries. The sexually transmitted viral infection, Human 

Papillomavirus (HPV), has been identified as the leading etiologic agent of malignant 

transformation of the epithelium of the lower genital tract. Specific “high risk” 

genital subtypes HPV 16 and 18 are highly linked with cervical cancer. It has a long 

pre-invasive phase called dysplasia or squamous intraepithelial lesion (SIL) that can 

be detected by a pelvic exam and a Pap smear. Early intervention of pre-invasive 

and invasive cancer could eradicate this cancer. Other screening interventions for 

early detection that may fit in resource poor environments have been developed. 

Some of the alternative strategies include; HPV testing, and Visual Inspection with 

Acetic acid, (VIA), HPV vaccination. Methods: We reviewed 13 of the world’s recent 

literature. Search terms such as “HPV high risk” “cervical Cancer” “Developing 

Countries” were used in search programs. We reviewed methods that are effective, 

acceptable, and sensitive, cost efficient as well as rapid tests that promote screen and 

treat approaches in some developing areas in Africa, Thailand, rural China and India. 

Results: The following interventions were used: VIA, HPV DNA testing, colposcopy 

use, as well as HPV vaccination. A total of about 55271 women took part in 

these different studies, their ages ranging from 24-65 years. VIA, HPV testing, cytology 

and vaccination showed sensitivity ranging from 67-78%,66-100,53.4%,91.6% 

and specificity ranging from 48-86%, 61-96%, 93%,99.7 respectively. 99.4 % acceptability 

by women whom where screened by VIA, method would reduce cancer 

by 25-30%. HPV testing requires an expert and cytology can detect CIN, CIN2 

CIN3 but requires infrastructure and a lab. HPV vaccination would reduce cancer 

by 40%. Conclusion: The use of VIA and HPV testing was favored in most studies 

and successful results were attained. VIA fit the criteria of being efficient compared 

to cytology, affordable, sensitive and not much training required although is not very 

specific. Colposcopy use requires a lot of training and experience in order to get the 

right results. HPV vaccination is costly according to most of the countries economic 

status, in addition, the 3 doses is problematic since in most studies many patients 

were lost in follow up visits.

Massively Parallel Amplification of cDNAs 

Ugo Nduaguba 

Mentors: David E. Hill, PhD and Marc Vidal, PhD 

Scientific Advisors: Lila Ghamsari, Elizabeth Mello, Kourosh Salehi-Ashtiani 


Primer, cloning, and sequencing costs are major obstacles in carrying out genome 

wide amplification and cloning of an organism’s protein coding regions. Although 

the advent of “next generation” sequencing technology platforms has led to decreased 

cost and increased throughput of DNA sequencing, the power of these methodologies 

cannot be fully utilized due to the lack of PCR technologies that match the 

throughput at which these new sequencing platforms operate cost effectively. 

In situ microarray synthesis of primers provides an avenue for inexpensive synthesis of 

primers. However, the use of such array-synthesized primers in PCR, typically generated 

as a complex mixture of several thousand primers, remains problematic. One of 

the challenges in carrying out such complex amplification reactions is that because 

primers are used in pools, the concentration with respect to each specific primer 

pair can be considerably lower than what they would be in a conventional PCR, 

altering the primer hybridization kinetics significantly. In order to develop a PCR 

protocol capable of generating hundreds of amplicons simultaneously, we have used 

a set of 500 primer pairs, targeting metabolic protein coding regions of Chlamydomonas 

reinhardtii, to determine the required amplification parameters. To compensate 

for the lower primer concentration, we increase the primer pool concentration by 

tenfold and extend the annealing time by ~15 to 30 fold (~1,000 - 2,000 seconds) 

for each PCR cycle. Using these modified parameters along with reverse transcribed 

Chlamydomonas RNA as template; we obtain gene-specific amplification products in 

reactions containing the 500 primer pairs. Cloning and sequencing of the obtained 

products verifies correct amplification of the intended targets.

Identifying Potential HLA-A*0201-restricted, 

Immunogenic Epitopes from Myosin VI 

Osamede Obanor 

Mentor: Simo Arredouani, PhD 

Scientific Advisors: Wen Yue, MD; Bin Lu, PhD; and Martin G. Sanda, MD 


Background: Myosin VI previously found to be overexpressed in prostate tumors 

and play a role in tumorigenesis, could potentially be a target for immunotherapy 

geared towards prostate cancers. Hypothesis: By analyzing and testing Myosin VI’s 

HLA-A2.1 epitopes, novel epitopes will be identified that can elicit a cytotoxic lymphocyte 

immune response to prostate tumors. Preliminary data: Comprehensive 

in-silico analysis was conducted that identified 57 novel, human prostate cancer-restricted, 

tumor-associated antigens (TAA). Fifteen of these TAA, including Myosin 

VI, were ascertained as showing prostate cancer-associated expression by Q-RTPCR 

of human specimens. Aims & Methods: in-silico analysis using multiple prediction 

algorithms was performed in order to identify novel peptides of Myosin VI that bind 

to human HLA-A2.1 receptors. Potential binders were then run through an in vitro 

HLA-A2.1 stabilization assay to determine whether they bind to the HLA-A2.1 receptor. 

HLA-A2.1 transgenic (HHD) mice were injected with the 5 peptides that 

showed significant binding, along with a constant helper peptide to test for their 

immunogenicity. Spleens were harvested from the immunized mice 12 days later; 

splenocytes were prepared and subjected to an ELISPOT testing that determined if 

an immune response was elicited. Results: 10 peptides from Myosin VI were chosen 

to go through HLA-A2.1 stabilization assay, and 5 of the 10 showed significant binding 

to HLA-A2.1 receptors. When the 5 peptides were injected in transgenic mice, 2 

showed the ability to elicit an immune response. Conclusions: 2 Myosin VI peptides 

were able to bind to HLA-A2.1 receptors and elicit an immune response, making 

them potential targets for immunotherapy.

The Role of TPBG in Melanoma Metastasis 

Barbara Owens 

Mentor: Lawrence N. Kwong, PhD 

Scientific Advisor: Lynda Chin, MD 


Metastasis is the spread of cancer from its primary site to other places in the body 

(e.g., brain, liver). These cancer cells utilize the lymphatic and blood vessels as their 

means of transport to distant normal organs where they grow. In the process of trying 

to identify genes that play a role in regulating metastasis, we tested 50 genes expected 

to be tumor suppressor genes (TSG). Based on preliminary data using a 96-well Boyden 

chamber format, 10 significantly scoring genes were selected to be further tested. 

I then focused on TPBG (trophoblast glycoprotein) for validation, as results from this 

round of testing showed that loss of TPBG enhanced invasion through matrigel by 

approximately 1.8 fold using mouse melanoma cells. To evaluate and validate TPBG 

as a TSG, we would knock down and overexpress TPBG in both mouse and human 

cells. Methods of testing TPBG will include additional invasion assays, overexpressing 

TPBG to find a suppressive effect, and use of realtime RT PCR and western blots 

to determine the basal level of TPBG in cell lines with known invasive properties. 

This will allow us to further analyze and determine the effect of TPBG on invasion in 

vitro. This is an interesting discovery because the role of TPBG in melanoma has not 

been studied previously, and knowing which genes have this effect on cancer cells will 

help in the diagnosis and treatment of melanoma.

Glioblastoma Multiforme and MGMT promoter Methylation 

Lydia Price 

Mentor: Clark Chen, MD, PhD 

Scientific Advisor: Kimberly Ng 


Glioblastoma Multiforme (GBM) is the most common type of brain cancer. While 

the current standard of care for GBM patients involves surgical resection followed by 

radiation and temozolomide (TMZ) chemotherapy, this therapeutic strategy is only 

palliative, and most patients with this advanced brain disease die within two years after 

diagnosis. By examining 24 GBM cell lines; we hoped to determine a correlation 

between O-6-methylguanine-DNA Methyltransferase (MGMT) promoter methylation 

and MGMT expression level. MGMT is a protein that allows cells to repair 

the damage to their DNA in a single step, hereby permitting the cells to continue 

proliferation. In many cases, high levels of MGMT has been linked to resistance to 

radiation and chemotherapy. Thus, cells that contain low levels of MGMT may be 

highly sensitive to these forms of treatment. The data was collected by western blot 

and clonogenic assay analysis. The results of these experiments are currently pending, 

and further information will be available following data collection.

Role of selectins in the pathogenesis of multiple myeloma 

Phong Quang 

Mentor: Irene Ghobrial, MD 

Dana-Farber Cancer Institute/Harvard Medical School 

Introduction: Multiple-Myeloma (MM) is characterized by the disseminated involvement 

of the bone-marrow (BM), and its progression involves a continuous circulation 

of the MM cells (MMCs) in the peripheral blood and homing back to the 

BM. Selectins are adhesion molecules involved in the primary interaction of lymphocytes 

with the endothelial cells (ECs) of blood vessels. In this study we studied 

the role selectins in the pathogenesis of MM. Methods: We have characterized the 

expression of E, L and P-selectins and their ligands on MM cell lines, patient sample 

and plasma cells from normal subjects (NPCs). We have tested the effect of blockade 

of each of the selectins and selectin-ligands on the interaction of MMCs with ECs. 

Moreover, we tested the effect of a pan selectin inhibitor (GMI-1070) on MMCs 

adhesion to ECs, and trans-well (through filter) and trans-endothelial SDF1-induced 

migration in vitro, and characterized its effect on cytoskeletal signaling induced by 

the interaction of MMCs and ECs. Moreover, we have tested the effect of the inhibitor 

on homing of MMCs to the BM in mice using in vivo flow cytometry to detect 

the number of circulating cells, and in vivo confocal microscopy to directly visualize 

the interaction. Results: All MM cell lines and patient samples had low expression 

of all selectins and high expression of L and P, but not E, selectin ligands. While 

NPCs showed low expression of all selectins and ligands. Blockade of L and P-selectin 

ligands reduced the interaction of MMCs with ECs in vitro, while blockade of 

E-selectin ligand or any of selectins did not show any effect. The pan-selectin inhibitor 

reduced the interaction of MMCs with ECs in vitro, did not alter their SDF1- 

induced migration through filter, but reduced significantly the migration through 

ECs. The inhibitor inhibited the activation of FAK and ERK induced by interaction 

of MMCs and ECs. Moreover, the selectin inhibitor extending the circulation time of 

MM cells in mice, and reduced the homing of MMCs. Conclusion: We found that 

the overexpressed P selectin ligands in MMCs play a major role in their homing to 

the BM. Moreover, the pan-selectin inhibitor (GMI-1070) prevented the homing of 

MMCs to the BM. This provides a basis for testing the effect of the inhibitor on MM 

tumor progression and initiation.

High-throughput Methods for Human Interactome Network Mapping 

Nancy Rivera 

Mentors: David E. Hill, PhD and Marc Vidal, PhD 

Scientific Advisors: Matija Dreze and Pascal Braun, PhD 


Protein-protein interactions (PPI) are of central importance to the functioning of 

cells. Thus, studying PPI is essential to improve our understanding of basic biology, 

organism development and diseases. Advancements in technology have allowed researchers 

to progress from studying one unique PPI at a time to global investigations 

of hundreds to thousands of PPI at a time. These advancements will eventually lead 

to a better understanding of disease processes and more rapid development of therapeutics, 

particularly for cancer. In order to evaluate the quality of PPI datasets, we 

are developing a high throughput validation system based on testing each interaction 

pair using multiple, distinct assays having empirically determined assay sensitivities 

and specifities. To calibrate each of the assays with respect to their inherent false 

negative and false positive rates, two reference sets of interaction pairs have been 

assembled. The positive reference set (PRS) is comprised of 100 well-documented 

pairs of interacting human proteins while a random reference set (RRS) contains 100 

pairs in which each partner is randomly chosen from a collection of ~15,000 individual 

proteins. The two reference sets are then tested in all assays to establish assay 

parameters. Any given interaction pair from a PPI dataset is then tested in each of 

the distinct assays and the results are combined to derive a “confidence score” for each 

interaction pair. Affinity purification assay using Glutathione S-transferase (GST) 

pulldowns followed by western blotting has been traditionally used for confirming 

PPI but overall assay sensitivity and specificity has not been established. We now are 

defining the assay parameters for GST pull-downs by first applying it to the PRS/ 

RRS sets. GST pull-down results for the PRS/RRS compare favorably to those seen 

for other assays, albeit with some interesting differences that will be discussed.

Tumor-derived Neuropilin-2 acts as a reservoir for 

VEGF-A and VEGF-C and increases angiogenesis and 

lymphangiogenesis in a breast cancer model 

Jeans Santana 

Mentor: Diane Renee Bielenberg, PhD 

Scientific Advisor: Meetu Seth 

Children’s Hospital Boston/Harvard Medical School 

Background: Vascular endothelial growth factor (VEGF-A), an angiogenic factor, 

binds to VEGF receptor-2 (VEGFR-2) and promotes the proliferation and migration 

of endothelial cells (EC). Neuropilins (NRP1, NRP2), co-receptors for VEGF- 

A, enhance VEGF activity but lack kinase activity. Neuropilin-2 (NRP2) is expressed 

on EC and tumor cells and binds VEGF-A and VEGF-C (lymphangiogenic protein). 

Tumor cells lack VEGFR-2 and therefore cannot directly signal upon VEGF binding. 

Our goal is to understand the contribution of tumor-derived NRP2 toward tumor 

progression. We hypothesize that NRP2 will act as a reservoir for VEGF-A and 

VEGF-C thus increasing angiogenesis and lymphangiogenesis in breast carcinoma 

models. Methods: Breast carcinoma cells, MCF7MFP1, were transfected with NRP2 

in vitro. RT-PCR, western blot, and ELISA were used to determine the expression of 

endogenous VEGF ligands and receptors in these breast cancer cells. Binding assays 

were used to determine whether NRP2 enhanced the binding of VEGF-A or VEGF- 

C to the tumor cells. The tumorigenicity and metastatic potential of MCF7MFP- 

1NRP2 and MCF7MFP1Mock were compared by injecting the cells orthotopically 

into immunodeficient mice. Results: Immunoblotting confirmed the overexpression 

of NRP2 in MCF7MFP1NRP2 cells. RT-PCR, western blot, and ELISA showed 

expression of VEGF-A and VEGF-C in tumor cell lysates and conditioned medias. 

Expected Results: It is anticipated that these studies will demonstrate that tumor 

cells overexpressing NRP2 are able to bind more VEGF-A and VEGF-C than untransfected 

cells. These results may suggest that tumors expressing NRP2 will sequester 

VEGF-A and VEGF-C ligands in the tumor microenvironment and stimulate 

(lymph)angiogenesis.

The Potential Role of Pituitary Tumor Transforming Gene 1 in 

Breast Cancer Development and Progression 

Bhaumika Shah 

Mentor: Towia Libermann, PhD 


Breast cancer is the most common cause of cancer in women and the second most 

common cause of cancer death in women in the U.S. While Pituitary Tumor-Transforming 

Gene 1 (PTTG1) has been recognized as an oncogene in the context of 

several types of tumors and a single nucleotide polymorphism in the PTTG1 gene 

has been linked to breast cancer development, no further studies into the potential 

role of PTTG1 in breast cancer have been pursued. Our preliminary analysis of 

genomic data sets link PTTG1 overexpression to recurrence and poor outcome in 

breast cancer patients and provide the basis for our innovative study that PTTG1 

is a novel breast cancer oncogene that plays an essential role in breast cancer development, 

progression and aggressiveness. We analyzed a variety of publicly available 

breast cancer microarray datasets for correlation between high expression of PTTG1 

and survival. Kaplan Meier curve analysis demonstrated that high PTTG1 expression 

strongly correlated with poor survival. The purpose of this study is to determine the 

potential role of PTTG1 in breast cancer development and progression as it relates to 

poor outcome. The potential biological relevance of the PTTG1 gene in breast cancer 

will be assessed by evaluating its mRNA (real time PCR) and protein expression levels 

(Western Blot) in breast cancer tissue and cell lines. We will also verify if PTTG1 promotes 

breast cancer survival or if the blockage of PTTG1 gene expression can induce 

cell death in breast cancer cells through RNA interference. The preliminary results 

display that the PTTG1 is upregulated at both mRNA and protein levels in breast 

cancer cell lines. For mRNA levels, about 60% of breast cancer tissue samples were 

upregulated from the PTTG1. There were high expression levels for both the PTTG1 

mRNA and protein levels in the MDA453 cell line. siRNA will be introduced into 

these breast cancer cell lines to verify the effect of PTTG1 blockage on breast cancer 

proliferation and apoptosis. We anticipate that PTTG1 overexpression is required for 

breast cancer progression and metastasis.

Genetic Characterization of a New Leukemia Cell Line 

Bethlehem Solomon 

Mentor: David Sweetser, MD, PhD 


Cancer derives from sequential mutations in a cell. This is especially documented in 

leukemia. Our lab is studying a specific type of leukemia known as acute myeloid leukemia 

(AML). The most common type of AML has a balanced translocation between 

chromosome 8 and 21, which creates a fusion gene called AML1-ETO. Although 

this step creates a preleukemic state, it needs other mutations to become a full-blown 

leukemia. The most common additional mutation seen with AML1-ETO is a partial 

deletion on the long arm of chromosome 9, del(9q). Our lab has studied leukemia 

cells from patients with t(8;21) and del(9q). However, there currently is no cell line 

that grows in culture with t(8;21) and del(9q). It would be helpful to be able to study 

these cells in a culture to examine their biochemistry and drug sensitivity. We obtained 

an immortalized cell line that started with umbilical cord blood samples that 

had AML1-ETO introduced into them and from initial analysis appeared to have 

subsequently spontaneously deleted a portion of chromosome 9. Thus, the purpose 

of this project is to verify and precisely map the chromosomal deletion in this cell 

line. 

We hypothesize that there will be a similar del(9q) deletion and similar gene expression 

patterns in this cell line as compared to patient AML samples. PCR were used 

to allelotype this cell line to compare it to a control cell line in order to precisely map 

the deletion. We anticipate to map a large commonly deleted region (CDR) in these 

cells that compares to the CDR in the AML samples. More precise mapping and 

gene expression analysis by quantitative RT-PCR is also underway. We project that 

the outcome of this project will be validation of a new cell line that will be useful in 

evaluating new drugs that might be used to treat leukemia.

The Role of PTEN and SHIP1 in Efferocytosis of 

Apoptotic Neutrophils By Macrophages 

Yunwei Sun 

Mentors: Hongbo R. Luo, PhD 

Scientific Advisor: Subhanjan Mondal, PhD 


Neutrophils are the most abundant and shortest-lived cells of the immune system 

Substantial daily turnover of neutrophils occurs through spontaneous apoptosis followed 

by clearance by macrophages. Defects in the clearance mechanism cause increased 

necrotic cells in circulation, which may lead to generation of self antibodies 

and may cause autoimmune diseases such as systemic lupus erythmatorus (SLE). 

Phosphatidylinositol 3-kinase (PI3K) is important for efferocytosis,the engulfment 

of apoptotic cells by phagocytes. Inhibition of PI3K reduces efferocytosis efficiency. 

Phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3), a product of PI3K, localizes 

in the phagocytic cup during efferocytosis. After engulfment, PtdIns(3,4,5)P3 is 

converted to PtdIns(3,4)P2 by phosphatase SHIP1 or to PtdIns(4,5)P2 by PTEN. 

The focus of this study is to determine whether PTEN and SHIP1 localize to the 

phagocytic cup in macrophagesduring engulfment of neutrophils. This will lead to a 

greater understanding of the roles of PTEN/SHIP1 in efferocytosis. 

A phagocytosis assay with immunostaining was performed by labeling apoptotic 

neutrophils (isolated from mice) with snarf-1 and by transfecting RAW 264.7 macrophages 

with GFP conjugated PTEN or SHIP1. Western blots were also used to 

analyze PTEN and SHIP1 protein levels. 

It was found that PTEN is not enriched in the phagocytic cup and is globally distributed 

throughout macrophages. Therefore, it can be inferred that the little amount 

of PTEN is enough for the converting PtdIns(3,4,5)P3 to PtdIns(4,5)P2. Ship1 is 

found to be enriched at the phagocytic cup indicating that it is involved in the conversion 

of PtdIns(3,4,5)P3 to PtdIns(3,4)P2.

Optimizing HA Immunopurification Strategies Using Click Chemistry 

Maria N. Tomar, BS 

Mentor: Jarrod A. Marto, PhD 

Scientific Advisors: Scott B. Ficarro, PhD and Guillaume Adelmant, PhD 


Insight into many cellular processes requires an understanding of protein-protein 

interaction networks. These networks can be established by purification of protein 

complexes followed by their component analysis by mass spectrometry. Tandem affinity 

purification (TAP), which relies on antibodies (Abs) to affinity tags (i.e. flag and 

HA), is a valuable technique to purify protein complexes from cells and organisms, 

yielding greater purity than using only one affinity label. Although this combination 

has been successfully used by many laboratories, the elution from the HA Ab poses 

a challenge for MS based experiments due to the high concentration of HA peptide 

required for elution. Although protein precipitation or MWCO membranes can be 

used to remove HA peptide prior to MS analysis, they typically lead to sample losses 

and are therefore not desirable. To circumvent this problem, we are synthesizing two 

HA peptide analogues and their corresponding scavenger resins that should allow a 

quantitative removal of the HA peptide using click chemistry, an approach that exploits 

the fast, selective reactions that occur between azide and alkyne or phosphine 

functional groups. So far, alkyne and azide functionalized HA peptides have been 

synthesized, along with an intermediate required to produce the azide capture resin. 

Synthesis of other reagents is underway, and when complete, we will assess removal 

of HA peptide from protein extracts.

Patient-Provider Communication Regarding 

Self-Reported Insomnia in Cancer Care 

Bianca Valcarce 

Mentor: Donna Berry, PhD, RN, AOCN, FAAN 

Scientific Advisor: Barbara Halpenny, MA 


Sleep problems experienced by patients with cancer are often caused by the anxiety 

and stress of a cancer diagnosis, plus multiple bio-physiological factors. The disturbance 

in sleep patterns often develops into serious insomnia, requiring attention and 

intervention from the oncology provider. Insomnia has been associated with cognitive 

dysfunction, changes in ability to work, a decline in quality of life, as well as 

alterations to bodily functions. The symptom should be a concern of clinicians and 

should be openly and routinely communicated in cancer care. The purpose of this 

analysis is to illustrate the nature of the discussions between patients and their clinicians 

regarding patient-reported insomnia during the course of treatment. A secondary 

analysis of data collected in the larger Electronic Self Report Assessment- Cancer 

(ESRA-C) trial will be performed. In particular, patient self-report of insomnia will 

be evaluated and corresponding audio-recordings of clinic visits will be content-analyzed. 

Out of the 590 patients who had their clinic visits audio-recording during 

the ESRA-C study, 120 reported serious insomnia. Only 81 patients, however, had 

the insomnia issues discussed during the clinic visit. Content-analysis of these audio 

recordings is ongoing. We expect that the nature of the conversations will vary from 

case to case, including clinician abrupt initiation of another topic without addressing 

the issue of insomnia, pharmacologic and non-pharmacologic interventions.

CDK8 overexpression as a possible regulator of ß-catenin 

localization in colon cancer 

Lise Wagnac 

Mentor: William Hahn, MD, PhD 

Scientific Advisor: Xiaoxing Wang, PhD 

Broad Institute/Dana-Farber Cancer Institute 

Colon cancer is the third most common cancer in the United States and also the 

second leading cause of cancer deaths. Despite the vast efforts that have been made 

to make colon cancer more understandable, only a few molecularly targeted therapies 

have entered clinical practice. A lack of therapeutic approaches exist that target 

specific molecular pathways responsible for the development of tumors adds to the 

difficulty of treating colon cancer. In the case of colon cancer, aberrant activation 

of Wnt/ß-catenin signaling and successive up-regulation of ß-catenin transcriptional 

response is a critical event for its development. Upon activation of Wnt ligand/receptor 

complex, ß-catenin proteins become stabilized and enter the nucleus. By several 

research groups, it has been demonstrated that if ß-catenin is aberrantly activated, 

it remains in the nucleus and functions as an oncogene. Recent work has identified 

Cyclin-Dependent Kinase 8 (CDK8) as a regulator of ß-catenin signaling in colon 

cancer. However, the mechanism of regulation is not entirely clear. Since nuclear 

accumulation of ß-catenin is critical for its biological function, we propose to determine 

whether CDK8 overexpression affects ß-catenin subcellular localization. We 

will investigate localization of ß-catenin in colon cancer cell lines with various level 

of CDK8 expression using both immunofluorescence and biochemical fractionation 

approaches. To assess if the localization of ß-catenin is specifically a result of CDK8 

expression, we will introduce shRNA against CDK8 and CDK8 kinase mutants into 

the cells and evaluate if the ß-catenin localization is altered. We hypothesize that 

ß-catenin will accumulate under CDK8 overexpression and prove that CDK8 does 

have a correlation with ß-catenin localization. This will aid us in understanding the 

biology of ß-catenin in signaling pathway in colon cancer and lead to new ideas for 

potential cancer therapeutic targets.

Validation of an anti-Dkk1 monoclonal antibody for 

the treatment of bone disease in multiple myeloma 

Cienna Wesley 

Mentor: Noopur Raje, MD 

Scientific Advisors: Samantha Pozzi, MD and Sonia Vallet, MD 


Multiple Myeloma (MM) is a cancer of the bone marrow caused by a proliferation of 

malignant plasma cells. MM plasma cells interfere with bone remodeling, acting on 

osteoblasts (OB) (bone producing cells) and the osteoclasts (OC) (bone resorption 

cells) creating an imbalance in their number and function resulting in the development 

of osteolytic lesions, typical of the disease. Increased serum concentration of 

Dkk1, an inhibitor of the Wnt pathway essential for osteoblastogenesis, has been 

observed in MM patients. It correlates with osteolytic lesions and inhibits the activity 

of OB in MM patients. It is our hypothesis that targeting this serum protein will 

enable us to restore bone homeostasis in MM and thus prevent bone disease. We will 

test this hypothesis by using a novel anti-Dkk1 monoclonal antibody with the goal 

of decreasing bone disease in MM. 

We have previously identified its effects on OB. Here I will focus on effects of anti- 

DKK1 on OC. OC were generated from bone marrow mononuclear cells derived 

from MM patients. After 2-3 weeks of culture, cells were fixed and stained for Tartrate 

Resistant Acid Phosphatase (TRAP) characteristic of OC and counted. Our 

data demonstrates a reduction of OC number in the treated samples compared with 

control. We will next test whether OC function is impacted as well. These experiments 

will highlight the effects of anti-DKK1 on the OC in MM.

Nerve Regeneration II 

Further Studies on the Mechanisms of Mst3b Function 

Sherry Wu 

Mentor: Nina Irwin, PhD 


Axon outgrowth is necessary for wiring the brain during development and repairing 

nerve damage following injury. I have previously shown that two proteins that 

are each part of protein complexes and are critical for axon outgrowth, Mst3b and 

RGS12, bind in response to growth factors to form a large protein complex. This 

summer, to learn more about their roles in a signal transduction pathway leading 

to axon outgrowth, I will complete experiments in which RNAi and mutated proteins 

will be used to analyze the mechanisms by which Mst3b and RGS12 regulate 

outgrowth. In addition, Nina Irwin has recently shown that Mst3b can be cleaved 

by caspase. I will examine the effects of caspase cleavage on the activity of Mst3b in 

COS-7 cells by transfection with plasmids expressing wild-type, cleaved, or uncleavable 

Mst3b; and immunoprecipitating Mst3b from cell lysates to measure differences 

in kinase activity. I will study the role of caspase cleavage in the regulation of axon 

growth in response to growth factor by transfecting embryonic day 18 (E18) neurons 

with plasmids expressing wild-type, cleaved, or uncleavable Mst3b; and co-expressing 

Mst3b siRNA to block endogenous Mst3b expression. Interestingly, caspase cleavage 

removes the nuclear export signal in Mst3b, but retains the nuclear import signal. 

Thus, I hypothesize that cleaved Mst3b will be found in the nucleus. I will test this 

hypothesis by a localization assay using fluorescent microscopy of E18 neurons transfected 

with plasmids expressing wild-type, cleaved, or uncleavable EGFP/Mst3b. In 

summary, I expect that following the addition of growth factors, caspase cleavage allows 

nuclear localization of the Mst3b and RGS12 protein complex, which I predict 

to be necessary for axon outgrowth to occur.

Alumni Notable Achievements 

This past year a CURE student: 

• Completed their second year at Chicago Medical School. 

• Presented a scientific poster at the Annual Biomedical Research Conference for 

Minority Students. 

• Won the 2008 Annual Biomedical Research Conference for Minority Students 

award in Cell Biology Oral presentation. 

• Was a Tufts Summer Scholar. 

• Enrolled in the MED Prep program at UNC Chapel Hill. 

• Presented research at the National McNair Scholars Research Conference. 

• Participated in Project Success 2009. 

• Was employed in the Vidal lab at Dana-Farber Cancer Institute. 

• Won the Harvard Vanguard Scholarship. 

• Received the Gardener’s Award for Research in Science. 

• Obtained a scholarship from Genzyme. 

• Was awarded 1st Place in the Massachusetts State Science and Engineering Fair 

2007 and was a finalist in the Intel International Science and Engineering Fair 

(ISEF) 2007. 

• Was inducted as a member of National Honors Society 2008. 

• Received the Gateway to Research Scholarship from the American Foundation 

for Pharmaceutical Education. 

• Traveled to Brazil to conduct cancer research. 

• Attended one of these conferences: the National Conference for Undergraduate 

Researchers, American Association of Cancer Research Conference, or the 

Biomedical Science Careers Program. 

• Presented a poster or an oral presentation at the New England Science 

Symposium. 

• Finished their first year at Boston Medical School.

Dana-Farber/Harvard Cancer Center’s Continuing Umbrella of Research Experiences 

(CURE) wishes to acknowledge and thank the CURE Advisory Committee, 

mentors, scientific advisors, lecturers, and supporters for expanding the academic 

and career horizons of our students. 

Many thanks to the following organizations: 

• Amgen 

• Biogen/Idec 

• The Friends of Dana-Farber 

• National Cancer Institute 

• University of Massachusetts Bridges to Baccalaureate program 

• Dana-Farber Cancer Institute’s Workforce Development 

A special thanks to: 

• Tracy Callahan 

• Hachung Chung 

• Nicole Cohen 

• Kirsten Fertuck 

• Girija Goyal 

• Carla Hawkins 

• Petra Loesch 

• Iqbal Massodi 

• Narie Storer 

• Venus Swearingen 

• Michael Verzi 

• Janghee Woo 

• Adrienne Yanez

© 

NCI 

CCC 

DANA-FARBER/HARVARD CANCER CENTER 

A Comprehensive Cancer Center 

Designated by the National Cancer Institute

Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...

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