Discussion

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Half Hairpin Analysis in Cancer Cells
Katie Stackpole Lucien Barnes Isabel
Vanderslice Andrew Borst
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Breast Cancer

• Second most common form of cancer in women,
  behind skin cancer
• Most common cause of death in Hispanic women

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Colon Cancer

• 72,007 men and 69,398 women were diagnosed with
  colorectal cancer.
• 26,781 men and 26,224 women died from colorectal
  cancer.
• Third most common type of cancer in men, and
  third leading cause of death from cancer.

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Cancer Proliferation Gene Discovery
Through Functional Genomics Michael R.
Schlabach,1 Ji Luo,1 Nicole L. Solimini,1
Guang Hu,1 Qikai Xu,1 Mamie Z. Li,1 Zhenming
Zhao,1 Agata Smogorzewska,1,2 Mathew E. Sowa,3
Xiaolu L. Ang,3 Thomas F. Westbrook,1 Anthony C.
Liang,1 Kenneth Chang,4 Jennifer A. Hackett,1 J.
Wade Harper,3 Gregory J. Hannon,4 Stephen J.
Elledge1 Retroviral short hairpin RNA
(shRNA)mediated genetic screens in mammalian
cells are powerful tools for discovering
loss-of-function phenotypes. We describe a highly
parallel multiplex methodology for screening
large pools of shRNAs using half-hairpin barcodes
for microarray deconvolution. We carried out
dropout screens for shRNAs that affect cell
proliferation and viability in cancer cells and
normal cells. We identified many shRNAs to be
antiproliferative that target core cellular
processes, such as the cell cycle and protein
translation, in all cells examined. Moreover, we
identified genes that are selectively required
for proliferation and survival in different cell
lines. Our platform enables rapid and
cost-effective genome-wide screens to identify
cancer proliferation and survival genes for
target discovery. Such efforts are complementary
to the Cancer Genome Atlas and provide an
alternative functional view of cancer cells.
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Key Terms

• DNA barcoding Uses a short genetic marker to
  identify species along the DNA
• shRNA RNA with a tight hairpin turn that can be
  used to silence gene expression via the RNAi
  pathway
• HHRNA 3 half of the shRNA stem, eliminates
  probe self-annealing during microarray
  hybridization
• Dropout Cell colonies that have shown lower
  proliferation after being introduced to shRNA
• Gene that was knocked down was necessary for
  cellular proliferation
• Knock-down The reduced expression of a gene.

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Roadmap of Lecture

• Central Goal Producing a high throughput screen
  to display what genes are not required for cancer
  cells
• Methods Retroviral transformation, HHRNA
  Production, HHRNA microarray hybridization, Cell
  viability assay
• Results Show cell viability when genes are
  knocked down, also shows validation of shRNA by
  using siRNA
• Discussion MDM2, PRPS2, and BUB1 shown to be
  required genes for cancer cells (with exceptions)

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Figure 1 Overview of the pool-based dropout
screen with barcode microarrays
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Figure 1 B Schematic of HH barcode hybridization
insertion region is unique in genome due to
inclusion of loop
190 bp HH DNA barcode
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1 C Comparison between HH amplicons and
full-hairpin PCR amplicons on an HH probe
microarray
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Retroviral transformation with MSCV
2nd generation Elledge-Hannon human and mouse
shRNA libraries
pMSCV -Puro-M1uI-shRNA library
- 1841 shRNAs targeting 1272 genes
cherry-picked, cloned as pools into
pMSCV-PM-PheS - Retroviruses produced by
transfection of MSCV-PM-shRNA, pCG-gag/pol, and
pVSV-G plasmids using TransIT-293 into 293T
cells. Retroviral supernatant was isolated -
Human cells were infected 3 times. Viruses
removed and uninfected cells selected against
using puromycin
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Controls and Verifications

• BUB1 (using siRNA and shRNAs to confirm knockdown
  because of similarity between processes)
• EIF 3510 and RBX1 (essential for viability in all
  cell lines)
• Included shRNA in screen and others from library

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Cell Viability Assay

• Cells infected in triplicate with retroviruses
• Replace media 24 hrs later
• On days 2, 6, or 9 post-infection using
  CellTiter-Glo reagent (glows in presence of ATP)
• Read on plate reader

Promega CellTiter-Glo reagent
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Comparisons to Other Techniques
siRNA
Main difference in new method using HHRNA
instead of shRNA eliminates probe self annealing,
allowing barcodes to be identified by 20 bp
hybridization to a plate rather than sequencing
of a 60bp barcode.
MUCH more high through-put than gene deletions
Analogous to a continually expressed RNAi BUT -
like a hybrid of microRNA and siRNA because it
comes from nucleus but cuts mRNA target
instead of simply sequestering it. - may
effect development - can test multiple genes at
once (as in this study)
microRNA
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Results
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What is?

• The meaning of log 2
• Most of the data in this paper uses log base 2 to
  normalize the results.
• How this works up-regulation by a factor of 2
  has an expression ratio of 2, and down-regulation
  by a factor of 2 has an expression ratio of ½.

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Discussion - What does this all mean?
Cats are awesome.regardless of what Dr. Young
says.
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Cancer Cell Line Similarity

• High degree of similarity for gene knock-down
  susceptibility between the two colon-cancer cell
  lines (DLD-1, HCT116).
• DLD-1 114 shRNAs (88 genes about 1.4 of
  genome)
• HCT116 202 shRNA (115 genes about 3.9 of
  genome)

IN ENGLISH The two colon cancer cell lines
required similar genes for successful
proliferation. Without these genes, the two lines
died.
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Normal Human Mammary Epithelial Cells vs.
Cancerous Cells

• 695 genes essential for HMEC survival
• Reflects ability of cells to respond to various
  stresses.
• The relatively few number of genes required for
  cancer cell proliferation demonstrates the
  ability for these cells to evade growth
  inhibitory cues.

These thousands of snowmen are analogous to the
uncontrolled proliferation of cancer cells.
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Genes selectively required for proliferation or
survival of cancer cells.
DLD-1 Colon Cancer HCT116 Colon
Cancer HCC1964 Breast Cancer HMEC Normal Human
Mammary Epithelial Cells
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Gene Knock-down Susceptibility

• Differs largely between cancer cells isolated
  from different tissue cultures.
• PRPS2 encodes nucleoside synthesis (dNTPs) in
  cells.
• Down-regulation of this gene proves to be
  inhibatory in DLD-1 (colon cancer) cells than in
  HCC1954 (breast cancer) cells

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MDM2 Gene Essentiality

• MDM2 E3 ubiquitin ligase for p53
• Large negative log2 ratio indicates that
  knocked-down gene is essential for growth
• Positive log2 ratios indicate knocking-down that
  gene helped with growth

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BUB1 Interesting

• BUB1 Spindle Checkpoint Kinase
• The spindle checkpoint blocks the entry of a cell
  undergoing mitosis into anaphase until all
  chromosomes are properly attached to the meiotic
  or mitotic spindle.
• It is a cell-cycle regulator

The spindle checkpoint stagnates mitosis until
all spindle microtubules are attached to the
chromosomal centromere at the metaphase plate.
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BUB1 more Necessary in Cancerous Breast Tissue.

• Enhanced sensitivity of HCC1954 (breast cancer)
  cells to BUB1 (spindle checkpoint kinase)
  knockdown.
• ENGLISH More breast cancer cells died when BUB1
  was knocked-down than in normal HMEC cells.

Both shRNA and siRNA knockdown of BUB1 reduce
HCC1954 cell viability but have little/no effect
on HMEC viability.
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BUB1 is more Necessary in Cancerous Breast Tissue.

• Again
• Large negative log2 ratio indicates that
  knocked-down gene is essential for growth
• Positive log2 ratios indicate knocking-down that
  gene helped with growth.
• Small negative log2 ratios (gt -1) indicate
  knocked-down gene wasnt very inhibitory for
  cellular proliferation.

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Enhanced Sensitivity to BUB1 Knockdown in breast
cancer tissue. Why?

• Breast cancer cells (HCC1954) are near-tetraploid
  in nature.
• BUB1 is putatively used to maintain genomic
  stability.
• Non-oncogene addiction
• Tendency of cancerous cells to become dependent
  on non-oncogenes for proliferation.

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In summary
It is now possible to easily identify specific
anti-proliferative genes for numerous cancer cell
lines.
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Genetic Cancer Genome Project

• Goal Generating cancer lethality signatures for
  different cancer types and thus identifying
  cancer typespecific lethal genes representing
  potential drug targets.

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