Celecoxib pre-treatment in human

colorectal adenocarcinoma patients is

associated with gene expression
alterations suggestive of diminished
cellular proliferation.
Authors:
James Todd Auman; Robert Church; Soo-Youn Lee; Mark A Watson; James W Fleshman; Howard L Mcleod
Journal:
European journal of cancer (Oxford, England : 1990) Volume: 44
ISO Abbreviation: Eur. J. Cancer Publication Date: 2008 Aug 

Presented by :
Ms. Nipaporn Thipmanee
Code 520531189

Adviser:
Asst. Prof. Dr. Sukon Prasitwattanasare
Celecoxib pre-treatment in human colorectal
adenocarcinoma…

Introduction

Materials and methods

Contents Results

Discussion

Conclusion

2
Introduction
Outlines
Affymetric
Microarray

• Celecoxib
Colorectal Cox-2
cancer

Gene
expression
profiling

3
Colorectal cancer

Colorectal cancer involves

• Colon
• Rectal
• Anal canal

Anatomy of colon and rectum 3rd most common cancers:

2 men & women
in the United States

4
Anal canal in rectum
Colorectal cancer [cont.]

Colorectal cancer : high mortality rate of the world

2008 new case In the United States

• Colon cancer -> 108,070
• Rectal cancer -> 40,740

3rd leading cancer diagnosis

2nd leading cause of cancer death.

Report in Cancer Statistics: Jemal et al; CA Cancer J Clin 2008

5
Colorectal cancer [cont.]
Colorectal cancer start

1
Polyp (Adenoma)
Definition : Begin tumor but not cancer
• Polyp begins as a small,
• It harmless growth in the wall of the colon

Polyp

2
Malignant Polyp
• These are cancer !!
• Abnormal cells grow
o in the lining of the large intestine(colon) or rectum
• Invade surrounding tissue & metastasize to other areas.

Malignant
Polyp 6
 Hereditary colorectal cancer syndromes

Scientists have discovered several genes

contributing to a susceptibility to two types of
colorectal cancer

FAP HNPCC
Hereditary
Familial nonpolyposis
adenomatous colon cancer) also
polyposis called Lynch
Syndrome

 FAP susceptibility gene with a specific

chromosomal locus (apc) that is mapped
within
5q15-q22{ Chromosome 5 th : ------- At locus query 15-22 }
 The occurrence within a family of many cases of CRC
without polyposis,
We are considered HNPCC by the pedigree in a
pattern consistent with an autosomal-dominant trait

http://www.genome.gov/10000466#al-2
 Hereditary colorectal cancer genes
Table. Show the Hereditary colorectal cancer genes

FAP = familial adenomatous polyposis; GI = gastrointestinal 8

Source :Vogelstein B, Kinzler KW: Cancer genes and the pathways they control. Nat Med 10 (8): 789-99, 2004. 
 Colorectal cancer [cont.]

Staging for Colorectal cancer

Stage I Cancer has grown through the inner layer of

the colon or rectum

Stage II Cancer has grown through or into the wall of

the colon or rectum: not reached the nearby lymph
nodes yet.

Stage III Cancer has metastasized to nearby organs

and lymph nodes near to the colon or rectum

Stage IV Cancer has metastasized to distant organs

spread to other parts of the body such as spread to
liver ,lung, or ovary and brain
Figure : Staging systems for colorectal cancer
(AJCC, American Joint Committee for Cancer;IUAC,
International Union Against Cancer.)

http://www.about.com/

10
Cox-2 (Celecoxib)

Nonsteroidal anti-inflammatory drugs

(NSAIDs)

Inhibit
Prostaglandin(PG)

cyclooxygenase-1(Cox-1) Cyclooxygenase-2(Cox-2)

Aspirin Celecoxib

Ibuprofen Rofecoxib

Etc. Etc. 11
Selective COX-2 inhibitor

(A) Nonselective inhibitors have access to the binding channels of both isoforms.
(B) The more voluminous residues in COX-1, Ile434, His513, and Ile532, obstruct access of the
bulky side chains of COX-2 inhibitors. 12
Cox-2 (Celecoxib)
Prostaglandin Biosynthesis

13
Celecoxib (cox-2 inhibitor)
Coxibs : Pharmacologic Characteristics

Celecoxib

14
Cox-2: Overexpressed in colon tissues

COX-2 protein staining in colon tumor Normal colon epithelium

15
Celecoxib (Cyclooxygenase-2)
Arachidonic Sphingomyelin Ceramide
Acid

COX
COX XX COX-1 Non-COX Targets:
inhibitor
inhibitor COX-2 P450s
PPAR/
PPAR,
Prostaglandins (PGE2)

Apoptosis

Proliferation

Angiogenesis

Free Radical Production

Carcinogen Activation

Immune Function
X 16
Carcinogenesis
Gene expression profiling

A genomic technique
• based on the cell expressed
• transcribed into messenger RNA (mRNA).
Gene
Measurement of the activity (the expression)
Expression of thousands of genes at once

Profiling Example for profile :

• distinguish between cells that are actively
dividing
• show how the cells react to a particular treatment

17
Microarray

What is a microarray?

• A tool for analyzing gene expression

• Consists small membrane or glass slide
• Glass slide contain samples (genes)

2 main types of microarray

 Affymetrix arrays – single channel
(background-green, foreground-red)

 cDNA arrays – two channel (red, green, yellow)

18
Affymetrix Array

GeneChips

Affymetrix : Prob

19
Affymetrix Array [cont.]

Collect microarray
data

Platform

• HG_ U95Av2 platform

• HG_U133Av2 platform

cDNA microarray method 20

Materials and method

21
Materials and methods

Human colorectal adenocarcinoma patients

ry
wi th prima
ts a
l ect patien nocarcinom Patients undergoing to
Se de
ctal a
colore
Washington University
School of Medicine's

22
Materials and methods[cont.]

Eligible Patients

After collect patients

Exclude patients

Take aspirin and NSAIDS

23
Materials and methods[cont.]

Random patients
to receive
celecoxib drug

Eligible
Patients 23 patients
(23 patients)

11 patients 12 patients
Receive celecoxib Not receive
drug celecoxib drug

21
Materials and methods[cont.]

Review Bords

11 12
patients patients

25
Materials and methods[cont.]

Celecoxib drug

Day 1st 2nd 7th

200 200
mg mg

400 mg

26
Materials and methods[cont.]

Microarray analysis

Gene annotation enrichment analysis

Separate groups from

difference genes
Hierarchical cluster

Identified difference genes

Volcano plot
Using GC-RMA algorithm

Normalization

Gene Expression Omnibus(GEO)

{GSE11237}
Microarray raw data
27
Materials and methods[cont.]

Microarray raw data

http://www.ncbi.nlm.nih.gov/geo/

GSE11237

Download
microarray data

Import microarray data into Genespring GX7.3

http://www.chem.agilent.com/en-US/Products/software/lifesciencesinformatics/genespringgx/Pages/default.aspx 28
Materials and methods[cont.]

Normalization

Processing of reduce unwanted variation across chips

May use normalization information from multiple chips

Pre-Normalisation Post-Normalisation
GeneChip Robust Multiarray Averaging:GC-RMA

• Improve form of RMA(Robust Multiarray Averaging )

• GC-RMA use the sequence-specific probe affinities of the GeneChip probes

29
Materials and methods[cont.]
Volcano Plot

Statistical measure
The Scatter plot
showing
differential
genes expression Absolute
fold change
(AFC > 1.5) Fisher's exact test
(p-value <0.05)

Example : Volcano plot

31
Materials and methods[cont.]

Hierarchical cluster

• Used for clustering the different of gene expression data

Hierarchical cluster often good for visualization

Outlines

Algorithm for hierarchical cluster

Distance measure

32
Materials and methods[cont.]
Algorithm for hierarchical cluster

Complete Linkage Clustering (or the farthest neighbor method)

Calculating distance between clusters

d(x, y)
X Y

Calculate distance by linkage function

1 n ( xi  x )( y i  y )
 
Pearson
D( X , Y )  max xX , yY d ( x, y ) Correlation d ( x, y ) 
n  1 i 1 x y
33
Materials and methods[cont.]

Hierarchical cluster

• Agglomerative method

1 Calculate all pair-wise distances

2 Join the closest pair

3 Calculate pair’s distance to all others

4 Repeat from 2 until all joined

34
Materials and methods[cont.]

Hierarchical cluster

35
Materials
Materialsand
andmethods
methods[cont.]
[cont.]

Hierarchical cluster
Hierarchical cluster

36
Materials and methods[cont.]

Hierarchical cluster

37
Materials and methods[cont.]

Hierarchical cluster

38
Materials and methods[cont.]

Hierarchical cluster

39
Materials and methods[cont.]

Hierarchical cluster

40
 Gene annotation enrichment analysis

Annotation coverage
The tool suite introduced in the
first version of DAVID
• Analyze genes

• In different biological
Analysis gene have enrichment process
in batch annotation and gene-GO
• Aspects their genes in
a single space
Highlight genes most relevant (chart report or pathway)
GO term

41
http://david.abcc.ncifcrf.gov/content.jsp?file=functional_annotation.html
Gene annotation enrichment analysis [cont.]

Using Fisher’s exact test

Functional
• Determine the proportion of those genes
Anannotation • Proportion value fall into each category
differs by group

Analysis Conclude:
Using fisher ‘s exact test for adopt to measure the
gene-enrichment in annotation terms

42
Gene annotation enrichment analysis [cont.]

Fisher’s exact test

The score for a GO term is the degree of independence between the two
properties:
A = {gene is in the list of significant genes}
B = {gene is found in the GO term}.

Testing the independence of two groups in the above contingency table corresponds
to Fisher’s exact test [Khatri and Draghici, 2005]

43
Gene annotation enrichment analysis

Fisher’s exact test (cont.)

Compute the significance of a gene

Text inset : Hypergeometric test
here

Define variables probability of getting exactly x by chance

(not what we want)
• N = genes are on
microarray
• Bio is a GO term
• M genes in Bio
• N −M genes not
in Bio
• K be the number of
significant genes

44
Results

45
 Results

Outlines

Gene annotation
Hierarchical enrichment
Volcano plot clustering analysis
Unsupervised N=190 genes of 190 genes
hierarchical clustering o 35 higher
expression
N=10,083 genes o 155 lower
expression
2 3 4
1

46
Results[cont.]

Volcano plot
Increase expression

Decrease expression

No difference
Excel File

Fig 2 A.
Volcano plot of gene expression changes between colorectal adenocarcinomas from patients pre-treated
and not pre-treated with celecoxib. 47
 Results[cont.] Black
to the
Results

Hierarchical clustering

• In sub-cluster A

8/ 9 tumour samples
** receiving no celecoxib pre-treatment

• In sub-cluster B
6/7 tumour sample
** receiving celecoxib pre-treatment

• In sub-cluster C
• 4 patients receiving celecoxib
• 3 patients receiving no drug.

Fig 2 B. Clustering using the 190 differentially expressed genes. 48

Results[cont.]
Black
to the
Results
Gene annotation enrichment analysis

Genes alter for celecoxib

pre-treatment for 7 days

23 genes,
involve in cellular
lipid metabolism

4 genes involve in
glutathione
metabolism

19 genes involve in
cell adhesnion

49
Discussion and Conclusion

50
Discussion
Agreement with
No obvious Low stringency cut-off
previous study
• Gender Celecoxib
• decrease expression of
• Achieve the goal of this
• Tumour site glutathione metabolism
pilot study
• Clinical stage Gene alteration (involve) • Identify gene expression
• Celecoxib • Lipid + glutathione alterations associate with
pre-treatment status metabolism celecoxib
• Cell adhesion

Usefulness Celecoxib & Time

for treat
• Pharmacology develop adjuvant • 7 days vs 12 months
chemotherapy by using celecoxib • 190 and over 1,000 genes
drug difference expression for time

Conclusion • But previous research reported

celecoxib increase risk for
respectively
• No genes alteration involve
cardiovascular side effect in transforming growth
• Recent phase II study : combine factor-β signaling
celecoxib and 5-fiuorourcil
chemotherapy
 mediate cytotoxicity
51
Conclusion
• Results suggest

Celecoxib reduces the proliferative capacity of colorectal adenocarcinomas

in short-term study

• Gene expression alterations celecoxib drug

Indicative decreased cellular lipid and glutathione metabolism

• Research team suggests

Should provide the impetus for larger studies in the future

Either using celecoxib treatment alone or in combination

with other chemotherapeutic regimens.
52
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