Compartment-specific MicroRNA Expression Profiles

in Colorectal Cancer

Supervisors: Dr. TL Chan, Prof. SY LeungDepartment of Pathology,

The University of Hong Kong

Jackie Lau

Epithelial stem cells within human colon crypts

Confocal imaging of human colon cryptsPutative intestinal stem cell marker Musashi-1 (green) expression

Brittan & Wright (2004) Gut

Epithelial stem cell-pericryptal microenvironment (stem cell niche) interaction is crucial for homeostatic balance between epithelial cell self-renewal, cell division, cell differentiation and lineage determination

Blanpain et al. (2007) Cell Spradling et al. (2001) Nature

Modified from Spradling et al. (2001) Nature

Gene expression analysis of human colonic epithelial cell differentiation

Kosinski & Li et al. (2007) PNAS; Li et al. (2009) Gastroenterology Identified > 900 cDNA clones differentially expressed between top and basal crypts

in normal human colons Many involved in molecular pathways common to intestinal epithelial cell

development and neoplastic transformation

Modified from Kosinski & Li et al. (2007) PNAS

Resemblance between stem cell and cancer Immortality Self-renewal, clonal and heterogeneous properties Signals from surrounding microenvironment Common signaling pathways for regulation:

Wnt, Bone morphogenetic protein (BMP), Notch, Hedgehog

Colorectal Cancer (CRC) Tumor cells demonstrate “crypt-progenitor phenotype”, i.e.

phenotypic resemblance to basal crypt Deregulation of multiple signaling pathways mediating normal

intestinal epithelial development

Li, V.S. Profiling of gene expression changes in human colon crypt maturation and study on their dysregulation in tumorigenesis. PhD Thesis, The University of Hong Kong: Hong Kong, January 2008.

Project Aim

Based on the hypothesis that perturbation of normal colon maturation during tumorigenesis is cell type-dependent, the present study aims to investigate compartment-specific microRNA (miRNA) expression in normal human colons with tumor counterparts.

Experimental methodology I

Real-time quantitative reverse transcription (qRT)-PCRTaqMan® Human MicroRNA Array Set v2.0 Low Density Array (LDA)

Global expressions of 677 human miRNAs (miRBase v10) Duplicate quantification for each sample

Frozen colonic tissues Tumors Normal counterparts: microdissection-partitioned into

enrichments in whole mucosa, epithelium, stroma, top crypt and basal crypt

Crypt isolation from Stromal fractionProtocol modified by Dr. Helen Yan from our lab based on Strater et al. (1996) Gastroenterology; Whitehead et al. (1999) Gastroenterology; Hofmann et al. (2007) Gastroenterology

Microdissection into Samples enriched in: whole mucosa*, top crypt, basal crypt* * Mucularis mucosae and muscles removedKosinski & Li et al. (2007) PNAS; Li et al. (2009) Gastroenterology

Top Crypt harvested Basal Crypt harvested * Muscularis mucosa and muscles excluded

Experimental methodology II

Individual qRT-PCR validation of miRNAs differentially expressed across different compartments

TaqMan® MicroRNA Assay, Human Triplicate quantification for each sample

Formalin-fixed, paraffin-embedded (FFPE) tissues Tumors Normal counterparts: microdissection-partitioned into enrichments in whole mucosa, top crypt and basal crypt

Note: Concordance between qRT-PCR data from frozen tissues vs parallel FFPE samples in published reports and in-house data

Results

qRT-PCR detection for Purity of isolated Crypts and Stroma in Normal Colons

Relative quantification by Comparative Ct method Livak & Schmittgen (2001) Methods

Obtained “near-mesenchyme-free Crypts” &

“near-epithelium-free Stroma” fractions

Perreault & Beaulieu (1998) Exp. Cell Res.

Reproducibility between LDA Duplicates

15 20 25 30 35 40 4515

20

25

30

35

40

45

R² = 0.970221386323091

Replicate 1 Ct

Re

pli

ca

te 2

Ct

Ct values of all miRNAs on TaqMan® Human MicroRNA Array Set v2.0

Selection of Endogenous Control for Normalization

15

20

25

30

35 RNU6B

RNU48

RNU44

RNU43

RNU24

MammU6

Ct values of Endogenous Controls

Aver

age

Ct

Samples

6 TaqMan® MicroRNA Assay endogenous controls on each LDA set

RNU48 Lowest Ct variation across samples Ct values within reasonable range (21 – 25)

Statistical Analysis of LDA data

TM4: MeV (MultiExperiment Viewer) v4.4 Saeed et al. (2003) Biotechniques

Multiple statistical and computational algorithms identified distinct miRNA

expression patterns across compartments

Citical p-value = 0.01

75% amongst 29 miRNAs described to be associated with CRC show agreement in T:N expression ratio to published data

Sensitivity and reliability of high-throughput LDA detection

miRNA expression profilesdocumented in CRC

Lu et al. (2005) Nature

Bandres et al. (2006) Mol. Cancer

Schetter et al. (2009) JAMA

Aslam et al. (2009) Br. J. Surg.

In silico Target Prediction and Functional Enrichment Analysis

28 miRNAs D.E. between Top vs Basal crypts

Pathways involved in intestinal epithelial development along colon crypt axis

Candidate miRNAs for Individual qRT-PCR validation

Results collectively suggest D.E. miRNA listHigh concordance with published dataHigh involvement in intestinal epithelial development and CRC tumorigenesis

Selected 5 candidate miRNAs with robustness across various statistical and functional analyses

Formalin-fixed, paraffin-embedded (FFPE) tissues 42 Normal vs CRC Tumor pairs 16 Top vs Basal Crypt pairs (Normal)

Individual qRT-PCR validation of 5 candidate miRNAs

42 Normal vs Tumor pairs of FFPE Colonic tissues

miR-A miR-B miR-C miR-D miR-E

Individual qRT-PCR validation of 5 candidate miRNAs

16 Top vs Basal Crypt pairs (Normal)

* Log2 Fold Expression calibrated to Whole mucosa counterparts

miR-A miR-B miR-C miR-D miR-E

Conclusion

Protocols developed for isolation of compartments and high-throughput detection effective, as evident in data reproducibility and concordance with literature

Fundamental support for a hierarchy of miRNA expression along the colonic proliferation-differentiation axis

Foundation to identify candidate marker miRNAs that define colonic epithelial stem/progenitor cell niche

Based on resemblance between stem cell and cancer, such miRNA markers may constitute targets for mutation and malignant transformation, thus provide novel way to elucidate clonal origin of CRC

Current Work

Characterization of candidate miRNAs in CRC cell lines and a larger number of clinical samples

Functional Studies in CRC lines

Knockdown/Overexpression of candidate miRNAs

To elucidate biological roles of miRNAs

Monitoring phenotypic changes (cell proliferation, cell differentiation,

apoptosis) in transfected cells

Validate bioinformatically predicted mRNA targets

Acknowledgement

Department of Pathology, HKU

Dr. Chris TL Chan

Prof. SY Leung

Dr. Helen HN YanMiss Annie SY ChanMiss Grace ChengDr. Vivian Li

Department of Pathology,

St. Paul’s Hospital

Prof. ST Yuen

Department of Surgery, HKUProf. WL Law

Large intestine: Mucosa and Musculature in Humans

Adopted from Encyclopedia Britannica (http://www.britannica.com/eb/art-68639)

Why miRNA?

Gene expression profile – done Kosinski & Li et al. (2007) Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. PNAS, 104(39):15418-23.Li et al. (2009) Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms. Gastroenterology, 137:176-187.

Diagnostic and prognostic potential of miRNAs implicated in pathogenesis of CRC, either as oncogenes or tumor suppressors.

Potential to regulate multiple target genes or potentially an entire pathway; networks of several different miRNAs may act cooperatively in the translational regulation of individual mRNAs to elicit a biological phenotype

TaqMan® Human MicroRNA LDA

Fresh Tumors Flash freeze Cryosectioning

Fresh Normal counterparts Crypt isolation from Stromal fraction

Flash freeze Cryosectioning Microdissection into

Samples enriched in: whole mucosa*, top crypt, basal crypt* * Mucularis mucosae and muscles removed

Experimental methodology – Sample preparation