Transcript Microarray Shared Resource, UCSD

Locally Advanced Cervical Cancer in
Latinas: A Translational Approach
Dr. Francisco Pardo
David Chou
Cervical Cancer Overview
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Cancer of the Cervix is highly treatable when detected early
In recent years there has been a significant reduction in
incidence rate and mortality rate due to active and early
gynecological screening
More women with carcinoma of the cervix have been sexually
active before the age of 20
Human Papilloma viruses (HPV), a sexually transmitted virus,
has generally been associated with the disease.
Hispanic ethnic group has the highest incidence rate and
mortality rate for cervical cancer in California
Cervical Cancer: Population Disparity
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Hispanic women are 2-3 times more likely to have cervical
cancer than Caucasian women in California.
For example, data collected from clinics in San Francisco City
and San Mateo county all pointed to a higher incidence rate in
Hispanic women than other racial groups.
Cervical Cancer in Latinas:
A Translational Approach
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Cancer of the cervix is disproportionately
represented at higher stages in Hispanic subpopulations evaluated at UCSD
Besides socioeconomic and behavioral influences,
molecular and genetics factors need to be evaluated
in the Hispanic population in order to establish better
therapy regiments and control tumor progression
Using Affymetrix® GeneChip technology, we aim to
illustrate gene expression profiling of the locally
advanced cervical cancer patients
GeneChip Technology Overview
1.28 cm
Affymetrix® GeneChip HG-U133A
Affymetrix HG-U133A
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Analyzes the expression level of 18,400 transcripts and
variants, including 14,500 well-characterized human genes
25 mer oligonucleotides interrogate genes, sequences of
interest
Affymetrix has rules for probe selection based upon
hybridization properties, specificity, potential for crosshybridization
500,000 cells (probes) on an array
Typically a group of 11-20 probes (pairs) all for same gene
constitute a probe set
Project Flowchart
Patient Recruitment
Tissue Collection
Sample Preparation
Chip Hybridization/ Fluorescence Detection
(In Collaboration with the Microarray Shared Resource, UCSD)
Data Normalization/ Analysis
Tissue Collection
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30 Latina women with advanced cervical cancer will
participate in this pilot project.
Patients are giving necessary information on the
project and given consent forms to sign.
Confidentiality: Information obtained from patients
will be only identified by a code number.
Tissue samples are transported in liquid nitrogen
immediately after surgery. They are then stored at 70°C freezer before being processed.
Sample Preparation
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Tissues are sectioned <10 µm thick and staged on
glass slides.
Laser Captured Micro-dissection (LCM) are used to
encapsulate tumor cells to achieve homogenous cell
populations.
RNA extraction with the Qiagen MidiprepTM kit.
RNA samples stored at -70°C and then sent to the
UCSD Microarray Shared Resource for chip
hybridization
Laser Capture Micro-dissection (LCM)
PixCell® LCM System
LCM transfer of cancer cell clusters
Homogenous
poplution
Chip Hybridization
(Microarray Shared Resource, UCSD)
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Total RNA are cleaned up and reversed-transcribed
into cDNA. cDNA is then amplified to cRNA with
fluorescence tags.
cRNA is then fragmented and hybridized unto
Affymetrix HG-133A chip.
The cRNA fragments hybridize to corresponding
probes complementary to them.
Affymetrix GeneChip® Scanner detects fluorescence
level and reports intensity on an image file.
Chip Hybridization
(Microarray Shared Resource, UCSD)
Fluorescence Detection
Affymetrix
GeneChip®
Scanner 3000
Chip Image Produced by Fluorescence
Scanner
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Data Analysis
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Affymetrix® Microarray Suite (MAS5.0) software converts raw
image data created by the scanner into numerical values
(expression levels).
MAS5.0 applies unique algorithms to reduce background
noises and normalizes data sets.
MAS5.0 allows multiple array analysis and compare it to a preset baseline array.
MAS5.0 also allows online query to NetAffxTM to establish gene
identities and to search for relevant literatures on those genes.
We are also collaborating with the BioStastical Core in UCSD
to develop other analytical methods.
Pilot Project Status
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1.
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To date, the project has been reviewed and
approved by IRBs in both UCSD and SDSU. We are
currently recruiting patients and collecting samples.
For the samples collected, we are preparing tissue
slides for LCM.
Also, through a Mini-project involving HeLa cells:
We have established methods for RNA preprocessing and purification.
We have tested several analytical models for data
mining. Currently the Biostatics Core is comparing
preliminary data on HeLa using other algorithms.
Brief History: HeLa Cell Line
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HeLa line was first established in 1951, from a 31
year old woman from Baltimore named Henrietta
Lacks.
The cells taken from her cervix were malignant. They
were sent to John Hopkins and cultured.
HeLa became the first cell line to thrive and multiply
outside of the body, seemingly forever.
Henrietta Lacks eventually succumbed to cervical
cancer 8 months post surgery.
HeLa Project Experimental Methods
HeLa Cell Culture
Culture Irradiation
(0,2,5,10 Gy)
Sample Preparation
(RNA extraction)
Chip Hybridization/ Fluorescence Detection
(In Collaboration with the Microarray Shared Resource, UCSD)
Data Normalization/ Analysis
HeLa Project Results
2Gy
5Gy
10Gy
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Up37
regulat
ed
1
6
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DownRegul
ated
18
59
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10
3 radiation doses 2Gy, 5Gy,
and 10Gy compared to 0Gy
(no radiation)
At high radiation (5&10Gy),
Dkk1 expression is induced.
At lower doses (2&5Gy),
integrin β and RhoGDI
expression is decreased.
Result: Dkk1
DNA damage
(BCNU, cisplatin,H2O2,UV)
β-Catenin
dkk1
(cell adhesion molecule complex)
Apc
nucleus
cyclin D1
lrp
wnt
β-Catenin
Tcf
β-Catenin/Tcf
c-myc
Fibronectin
apoptosis
Dkk1 expression ultimately leads apoptosis and
down-regulation of cyclinD1.
re-entry
into cell
cycle
Result: Integrin β and RhoGDI
Growth factors could increase Rac GTP loading globally in the cell, but RhoGDI would prevent GTP-Rac binding to effectors in the cytoplasm. Integrins
would determine the specific local areas where Rac targets to membranes,
leading to the dissociation of Rho-GDI. Rac, RhoA, and Cdc42 are all part of
the Rho small G-protein family.
Rho and Cell Cycle Progression
Activation of RhoA inhibits the expression of
p21waf/cip, a regulator to the kinase activity of
cyclinD/CDK4 and cyclinE/CDK2.
Summary
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Dkk1 expression is increased at higher radiation
doses. Increased expression of Dkk1 ultimately
leads to decrease in cyclinD1 expression.
RhoGDI and Integrin β expressions are decreased at
lower radiation doses. Rho family G-proteins are free
to bind to down-stream effecter Pak due to the
decreased expression in integrin and RhoGDI.
Activation of Rho ultimately leads to increase in
cyclinD1 expression.
HeLa Project: Future Directions
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Validating the results and quantifying altered
expression by performing real-time quantitative RTPCR (qPCR), in particular the genes involving in cellcycle regulation (dkk1, integrin β, and rho-GDI).
Validating the results in the protein level through
Western Blotting analysis.
Establish probable relationships between regulated
genes through blocking antibodies and siRNA.
Conclusion
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To date, we have began recruiting patients
and collecting samples for analysis.
We have also started sample processing for
LCM.
Preliminary findings in HeLa project seem
promising to our overall Microarray
approach.
Future Directions
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Continue sample collection and prepare chip
hybridization en mass.
Developing new statistical algorithms to
complement MAS5.0
Biochemical studies for Dkk1, Integrin β, and
RhoGDI.
Acknowledgement
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Dr. Sandy Berstein, SDSU/UCSD Cancer
Partnership Program, SDSU
Joseph Aguillera and Dr. Yoo Hyuang, Radiation
Medicine Shared Resources, UCSD
Dr. Sonia Jain & Feng He, Biostatics Core, UCSD
Lutfunnessa Shireen, Microarray Shared Resources,
UCSD