Role of microRNAs in Chemoresistamce

1
Role of microRNAs in Chemoresistamce
Paul Blower
MicroRNA in Development and Cancer Mathematical
Biosciences Institute April 13, 2007
2
Overview

• Chemogenomic methods applied to NCI-60 to study
  chemoresistance
• Measurement of microRNA expression in the NCI-60
• Role of microRNAs in chemoresistance/ sensitivity

3
Conceptual Framework
4
NCI Cancer Screening

• Compounds tested against 60 tumor cell lines
• Breast (5) Leukemia (6) Ovarian (7)
• CNS (6) Lung (9) Prostate (2)
• Colon (7) Melanoma (10) Kidney (8)
• Compounds tested for growth inhibition of tumor
  cell lines, determine GI50
• Since 1990, gt100,000 compounds screened, data for
  43,000 compounds publicly available

Shoemaker, Nat. Rev. Cancer, 2006, 6, 813-23
http//dtp.nci.nih.gov/docs/cancer/cancer_data.htm
l
5
Compound-Gene Correlations
60 cell lines
60 cell lines
22,000 genes
x

22,000 genes
43,000 compounds
43,000 compounds
AT'
Activity (A)
Targets (T)

• Matrix of Pearson correlation coefficients
• Each row of A and T is normalized by mean and
  standard deviation,
• Matrices are multiplied to obtain AT'
• Each entry is divided by n - 1, where n (60) is
  the number of cell lines
• Optionally, the rows and columns of the product
  matrix are arranged in cluster order

6
Gene-Compound Correlations

• Breast
• CNS
• Colon
• Leukemia
• Lung
• Melanoma
• Ovarian
• Prostate
• Renal

across NCI60 cell lines, r -0.87
7
Conceptual Framework
60 Cell Lines
60 Cell Lines
43,000 Cmpds
Database S (Molecular Structure Features)
Database A (Activity Patterns)
Database T (Molecular Targets)
43,000 Cmpds
27,000 Features
22,000 Genes
22,000 Genes
SAT (Feature Gene Correlation)
27,000 Features
8
Chemogenomic Studies

• Multiple drug resistance gene (ABCB1) SAR of
  ellipticines
• Cystine/glutamate transport system
  glutathione-mediated chemoresistance
• Genes involved in growth factor signaling

9
Molecular Profiling of NCI-60
DNA
Compound screening
Protein
RNA
Weinstein, Mol. Cancer Ther., 2006 5(11) 2601-5
10
MicroRNA Expression in NCI-60

• Measured expression levels of microRNAs in the
  NCI-60 using custom microarray
• Custom pin-spotted microRNA microarray (OSU
  Comprehensive Cancer Center, version 3.0)
• Contains 627 human microRNA probes, spotted in
  duplicate, corresponding to 321 mature microRNAs
• Data deposited in
• ArrayExpress, accession number E-MEXP-1029
  (http//www.ebi.ac.uk/arrayexpress)
• NCIs CellMiner database (http//discover.nci.nih.
  gov)

11
Experimental Design

• 60 cell lines run in three batches, 20 samples
  4 controls per batch, 72 samples total
• Arranged cells in cluster order and divided into
  3 groups
• For each batch, a balanced random selection was
  taken from each group
• One cell line selected as control from each batch
  based on tissue diversity and moderate doubling
  time

Scherf et al. Nat. Genet. 2000, 236-44.
12
Quality Assessment
Quantile plots of microarray batches
Batch 25th 50th 75th A 1.3
5.5 8.7 B 4.3 6.1
8.6 C 4.1 6.2 8.8
microRNA expression (log2)
Percentile
13
Estimated Bias Curves

• Generated for 5 replicates of the 3 control cell
  lines
• For each probe-cell, true expression is average
  of 5 replicates
• For each batch, paired true and observed
  expression
• bias observed true

14
Quality Assessment

• Plots of bias versus true expression
• At expression levels where log2(exp) 8, all
  three batches show low variability and low bias
• Selected 279 probes for which log2(exp) 8 in at
  least 10 of the cell lines

The y-axis is the difference between true and
observed expression levels, and the x-axis is the
true expression level
15
Quality Assessment

• Clustered 72 samples based on expression levels
  of 279 selected microRNA probes
• For each of the 3 control sets, all 5 replicates
  clustered together with no intervening cell lines
• Mean pair-wise correlation coefficient within
  replicate sets

Complete linkage clustering with correlation
metric
16
Clustering of NCI-60 Cell Lines
Complete linkage clustering of cell lines using a
correlation metric based on expression levels
microRNA - 279 probes with log2(exp) 8 in at
least 10 of the cell lines
mRNA 1000 U133A probes with the largest
inter-quartile range
17
Heatmap of microRNA Expression
Heat map of 279 microRNAs. Complete linkage
clustering was performed using the correlation
metric. The color coding represents the
standardized expression level in each cell line
according to color bar
18
MicroRNAs in Chemoresistance

• Study affects of altered microRNA levels on
  compound potency in selected cell lines
• Manipulate microRNA levels by transfecting cells
  with microRNA precursors or antisense inhibitors
• Experimental design
• microRNAs
• cell lines
• compounds tested

19
MicroRNAs in Chemoresistance
Selection of microRNAs for Experimental Studies
Esquela-Kerscher Slack, Nat. Rev. Cancer
2006, 259269
20
NCI-60 Expression for mir-21

• Breast
• CNS
• Colon
• Lung
• Leukemia
• Melanoma
• Ovarian
• Prostate
• Renal

SNB-19
A549
log2(exp) difference from mean
OVCAR-3
CCRF-CEM
21
Manipulation of Cellular microRNA Levels
Measured by RT-PCR values are ?CT using U6 as
internal standard
Fold change is estimated increase in microRNA
level between inhibitor and precursor calculated
as 2(I-P)
22
Western Blots of microRNA Targets
let-7i
mir-16
mir-21
P I C
P I C
P I C
RAS BCL2 PTEN MYC ACTIN

• A549 cells transfected with microRNA precursor,
  inhibitor and control
• Transfection with mir-16 precursor reduces K-RAS
  2A expression to 50 of control

23
Structures of Tested Compounds
24
Compound Dosing Experiments

• Compare effects of microRNA levels on compound
  potency in selected cell lines
• Cells transfected with microRNA inhibitor,
  precursor or control
• After 24 hours, transfected cells were treated
  with compounds in a dilution series
• 48 hours after treatment, total protein measured
  with sulforhodamine B assay

purchased from Ambion, Inc., Austin TX
25
Compound Dosing Experiments
Plate Layout
medium precursor inhibitor
control blank
High Compound concentration Low No compound
26
Effect of miR-21 Level on Compound Potency in A549
Dose-response curves comparing inhibition and
forced expression of mir-21 with control
27
Effect of miR-21 Level on Compound Potency in A549
Relative GI50 values (control 1)






Compound for which both t-test and slope test
have p lt 0.01 and P-I fold difference gt 2
28
Effect of miRNA Level on Compound Potency in A549
Values are ratio of compound GI50 for microRNA
precursor / inhibitor
Colors indicate compound-microRNAs for which
both t-test and slope test have p lt 0.01
29
Effect of miR-21 Level on Compound Potency
Values are ratio of compound GI50 for microRNA
precursor / inhibitor
Colors indicate compound-microRNAs for which
both t-test and slope test have p lt 0.01
30
Effect of miRNA Level on Compound Potency
P/I gt 1.5-fold
Plot of p-value vs P/I Ratio
P/I gt 2-fold
t-test, p lt 0.01
31
Summary

• Measured microRNA expression levels in NCI-60
• At higher expression levels microarray data show
  low variability and low bias
• Cell line groupings based on microRNA expression
  consistent with tissue type and with cell line
  clustering based on mRNA
• MicroRNA targets may be cell specific
• Could not confirm published targets of let7i
  RAS, Myc mir16 - Bcl2 mir21 PTEN)
• K-RAS 2A is target of mir-16
• Mir-21 level had the opposite effect on potency
  of doxorubicin in different cell lines

32
Summary

• Confirmed role for microRNAs in chemoresistance/
  sensitivity
• Mir-21 level had a significant effect on compound
  potency in 36 (10/28) of compound-cells tested
• Altering microRNA level can have opposite effects
  on different drugs
• MicroRNA function in chemoresistance is generally
  consistent in across cell lines some exceptions

33
Acknowledgement
MBI / Statistics Joseph Verducci Shili Lin Jin
Zhou Shuying Sun Pharmacology Wolfgang
Sadee Ji-Hyun Chung Zunyan Dai MVIMG Carlo
Croce Chang-Gong Liu
Pharmacy Tom Schmittgen Jong-Kook Park NCI John
Weinstein William Reinhold Philip Lorenzi