Transporter Expression in the Gut and Tumors

Pascale Anderle, ISREC

Overview

Genomic profiling of transporters in Caco-2 cells and human intestine using a custom array

Genomic profiling of transporters along the mouse intestine using Affymetrix GeneChips

Identification of transporters responsible for chemo-sensitivity and chemo-resistance using a custom array

Goals:

Caco-2 cells: Differentiated cells vs. undifferentiated cells

Small intestinal and colonic tissues vs. Caco-2 cells

Evaluation of Transporter and Channel Genes in the Intestine

Reference:

5 days vs. 3 weeks

A-values7 9 11 13 15

-3

-

2

-

1

0

1

2

3

Differentiation of Caco-2 cells

M-v

alu

es

- 3

-

2

-

1

0

1

2

3

A-values7 9 11 13 15

5 days vs. 5 days

M-v

alu

es

- 3

-

2

-

1

0

1

2

3

A-values7 9 11 13 15

5 days vs. 1 week 5 days vs. 2 weeks

M-v

alu

es

- 3

-

2

-

1

0

1

2

3

A-values7 9 11 13 15

Time

Summary

Differentiation of Caco-2 cells:

During differentiation: Expression pattern changes Up and down regulation usually < 2 fold Significant changes between 5 days to 1 week and 1 week to 2 weeks No significant changes between 2 weeks and 3 weeks Genes in general related to ion household No major differences between flasks and filters (except GLUT3) Typical small intestinal transporters not especially up regulated in differentiated cells

Comparison Tissue vs. Caco-2 Cells:

Changes more pronounced between tissue and cell line than between undifferentiated and differentiated

cells Tissue vs. Caco-2 cells: More ratios > 2 fold No trend observed: undiff. cells to diff. cells = colon-like to small intestinal-like cells

Genomic Profiling of Membrane Transporters in the Intestine

Objective:

Identification of putative segment-specific and non-specific specific drug carriers

Study Design:

12 x Mu74Av2

12 x Mu74Bv2

12 x Mu74Cv2

Duodenum Jejunum Ileum Colon

Triplicates → 3 Pools of 10 mice

Accurate Annotation of Probe Sets

Mapping of single probes and attributing of annotation tags to each probe set (quality tags)

Assigning of flags to differentially regulated probe sets (F, G, 1)

G = 1

Gene B

F = 1

Gene C

1 = 1

Gene A

1 PS per Gene >2 PS per Gene>2 PS per Gene

A crucial step in classifying genes into different molecular functions is the use of a consistent and universal classification system and a precise annotation of Affymetrix probe sets.

Isrec Ontologizer Io

Selection of hierarchical level

P-value

P-value filterGene search

Classification of probe setsClassification of UniGenesClassification of RefSeqs

Quality filter

Ambiguity of results

Export

Regulation of Transporters along the Intestine

Fold Changes along the Intestine

Expression along the Crypt-Villus Axis

V C V C V C V C

D J I C

V C V C V C V C

D J I C

V C V C V C V C

D J I C

rel.

exp

res

sio

n l

evel

s [l

og

2]re

l. ex

pre

ssi

on

leve

ls [

log

2]re

l. ex

pre

ssio

n l

evel

s [l

og

2] Abca1

Slc34a2Slc2a1

Slc28a2Slc10a2Abcg8

Abcc1 Abcc3

Slc5a8

V C V C V C V C

D J I C

V C V C V C V C

D J I C

V C V C V C V C

D J I C

V C V C V C V C

D J I C

V C V C V C V C

D J I C

V C V C V C V C

D J I C

rel.

exp

res

sio

n l

evel

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og

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l. ex

pre

ssi

on

leve

ls [

log

2]re

l. ex

pre

ssio

n l

evel

s [l

og

2] Abca1

Slc34a2Slc2a1

Slc28a2Slc10a2Abcg8

Abcc1 Abcc3

Slc5a8

Summary

Majority of transporters not significantly expressed along the intestine

Carriers and in particular the classes “symporters” and “antiporters” change significantly

Transporter subclasses change along the whole intestine, most between ileum and colon

Similar number of transporters over-expressed in small and large intestine, but generally transporters are expressed at higher levels in the small intestine

Most differently regulated transporters are similarly expressed along the small intestine, yet various transporters are clearly segment-specific

Most transporters similarly expressed in humans and mice

Regulation of expression in whole tissue is a good indicator of the combined changed in epithelial crypt and villus cells

Identification of various new candidate carriers for drug delivery (mRNA protein)

Expression of Transporters in Cancers

Goal:

Study the role of transporters in cancer-sensitivity and cancer-resistance

Reference:

NCI Database

Scherf et al. 2000, Nature Genetics

SLCs: Chemo-sensitivity and -resistance

Sorted correlation coefficients

ABCB1

SLC29A1

Effects of regulation of transporters on dose-response curves

Summary

Important role of transporters in chemo-sensitivity and-resistance

ABCB1 and ABCB5 identified as resistance genes for various compounds

SLC29A1, nucleoside transporter, identified as sensitivity gene

Collection of information bits to perform follow-up studies

Acknowledgments I

UCSF/OSU

Wolfgang SadéeVera RakhmanovaYing HuangShoshana BrownAudrey PappZunyan Dai

Joe DeRisiAdam CarrollJingchun Zhu

Xenoport

Katie WoodfordNoa Zerangue

National Cancer Institute

John WeinsteinKimberly BusseyCatalin BarbacioruUma ShankavaramWilliam C. Reinhold

Acknowledgments II

ISREC

Jean-Pierre KraehenbuhlMartin Rumbo

Bioinformatics Core Facility

Thierry SengstagMauro Delorenzi

Nestlé

Muriel FiauxRobert MansourianDavid MutchMatthew-Alan RobertsGary Williamson

Swiss Institute of Bioinformatics

Philipp BucherChristian IseliViviane Praz