Conference talk at BioSB 2015 in Lunteren, The Netherlands * Date: 20 May 2015 * Title: "Integrative network based analysis of mRNA and miRNA expression in vitamin D3-treated cancer cells"

1. Integrative network based analysis
of mRNA and miRNA expression in
vitamin D3-treated cancer cells
Martina Summer-Kutmon, PhD
Department of Bioinformatics (BiGCaT)
Maastricht Centre for Systems Biology (MaCSBio)
20 May 2015, BioSB 2015

2. Nutritional Systems Biology
● Understanding nutritional processes at a systems level
● Integrating the effects of nutritional compounds at the
gene expression level with information on the
regulatory level.
https://wellnessfx.files.wordpress.com/2011/06/picture-11.png

3. Nutrition and Epigenetics
www.int.laborundmore.de

4. Vitamin D3 metabolism
Deeb, KK, et al. "Vitamin D signalling pathways in cancer: potential for anticancer therapeutics." Nature Reviews Cancer (2007)

5. Vitamin D3-mediated regulation
Deeb, KK, et al. "Vitamin D signalling pathways in cancer: potential for anticancer therapeutics." Nature Reviews Cancer (2007)

6. Goal of this study
Pathway and network-based methods
Integrate mRNA and microRNA
expression data
Investigate regulatory action of
vitamin D in prostate cancer

7. Workflow

8. Multi-omics dataset
Human prostate cancer cell line
LNCaP -
Lymph node metastasis in Caucasian male
100 nM 1,25
dihydroxyvitamin D3
control group
(n=4)
VitD-treated group
(n=4)
Transcriptomics
Nimblegen-HG18-4plex
whole genome microarrays
GEO: GSE17461
MicroRNA-omics
Agilent Human microRNA v3
microarrays
GEO: GSE23814
RNA isolation
Wang, WL, et al. “Effects of 1alpha,25 dihydroxyvitamin D3 and testosterone on miRNA and mRNA expression in LNCaP cells." Molecular Cancer (2013)

9. Gene-level statistics
● Quality control and statistical analysis were
performed by Wang et al.
○ one-way ANOVA (p<0.05)
○ correction for multiple testing.
● Differentially expressed genes:
○ fold change cut-off at 1.5
○ p-value < 0.05
● Differentially expressed miRNAs:
○ fold change cut-off at 2.0
○ p-value < 0.05
Up-regulated Down-regulated
420 413
Up-regulated Down-regulated
9 0

10. VDR targets
Literature study (25 papers)
● 25 publications and books
● 178 human VDR targets
● 21 changed genes
CYP24A1 - degradation of vitamin D3 ↑
ORM1/ORM2 - acute phase plasma protein ↑
CDKN2D/2C - cell growth regulator ↓

11. Workflow

12. PathVisio
○ Open source pathway analysis toolbox
○ Data visualization and over-representation analysis
○ www.pathvisio.org
WikiPathways
○ Collaborative pathway database
○ 276 pathways in curated collection
○ www.wikipathways.org
Pathway analysis
Kelder, T, et al. "WikiPathways: building research communities on biological pathways." Nucleic Acids Res (2012)
Kutmon, M, et al. "PathVisio 3: An Extendable Pathway Analysis Toolbox." PLoS Comput Biol. (2015)

13. Pathway analysis
Transcriptomics
dataset
Pathway database
WikiPathways
Differentially
expressed genes
Calculates Z-Score
for each pathway
Overrepresentation
analysis
Ranked list of
pathways
Data visualization
on pathway
diagrams

14. Pathway analysis
Pathway Z-Score Category
DNA Replication 11.91 general
Cell Cycle 11.04 general
Histone Modifications 10.44 general
G1 to S cell cycle control 9.12 general
DNA damage response 5.40 general
ATM Signaling pathway 4.87 general
Fluoropyrimidine Activity 4.16 general
AhR signaling pathway 2.47 general
Pathway Z-Score Category
Retinoblastoma (RB) in Cancer 12.63 cancer
Gastric cancer network 1 10.44 cancer
Gastric cancer network 2 5.13 cancer
Integrated Pancreatic Cancer
Pathway
4.08 cancer
Integrated Cancer pathway 3.85 cancer
Integrated Breast Cancer Pathway 3.41 cancer
Signaling Pathways in
Glioblastoma
2.02 cancer
● Significantly altered pathways:
○ 8 general cell cycle related pathways
○ 7 cancer related pathways

15. Pathway analysis
● Most of the pathways are down-regulated
after vitamin D treatment
down
upCell Cycle Pathway Gastric Cancer Network 1

16. Pathway analysis
● Most of the pathways are down-regulated
after vitamin D treatment
down
upG1 to S Cell Cycle Control RB in Cancer

17. Workflow

18. Cytoscape
○ Network visualization and analysis tool
○ Extendable through apps
○ www.cytoscape.org
Network building
WikiPathways App
WikiPathways web
service client and
GPML file format
importer
Shannon, P et al. "Cytoscape: a software environment for integrated models of biomolecular interaction networks." Genome research (2003)

19. Network building WikiPathways App
Central genes:
TP53, CDKN1A and CDK2
linking 5 out of 8 pathways
Kutmon, M, et al. "WikiPathways App for Cytoscape: making biological pathways amenable to network analysis and visualization." F1000Research (2014)

20. Workflow

21. ● Problem:
Only ~50% of protein coding genes are in pathways
Network extension
changed genes in
complete dataset
changed genes in
all pathways
changed genes in altered
general pathways
833
420 up + 413 down
390
205 up + 185 down
73
14 up + 59 down
What about all the differentially
expressed genes that are not in
the altered pathways?

22. Network extension
● Identify known protein-protein interaction
partners of the genes in the selected pathways
Database First neighbours
STRING database
http://string-db.org/
443 changed genes
Database First neighbours
ENCODE
http://encodenets.gersteinlab.org/
67 changed genes
● Identify known transcription factor-target
interactions of the genes in the selected pathways
support from Georg Summer

23. VitD-extended network
● 583 out of 833
changed genes
(~70%)
● 238 up
● 345 down
up-regulated
down-regulated

24. Workflow

25. ● Connected sub-networks that are regulated
by Vitamin D treatment
● jActiveModules finds multiple active
networks with different scores
○ robust down-regulated module
○ 193 nodes
○ 41 from altered pathways
Active network modules jActiveModules App

26. Active network modules
Module in vitD-extended network
jActiveModules App

27. ● 195 genes
○ 192 changed genes, all down-regulated
○ 1 gene not changed (E2F4)
● Functional enrichment:
○ Cell cycle activity
○ DNA processing
○ Cytoskeleton organization
Active network modules jActiveModules App

28. Workflow

29. ● TargetScan + miRTarBase
○ 1,439 miRNAs → 25,886 miRNA-target
interactions
● 6 out of 9 DE miRNAs present
in vitD-microRNA network
VitD-microRNA network
● Extend biological network with regulatory
information
○ microRNAs, transcription factors, drugs, ...
CyTargetLinker App
Kutmon, M, et al. "CyTargetLinker: a cytoscape app to integrate regulatory interactions in network analysis." PloS one (2013)

30. VitD-microRNA network CyTargetLinker App
31 targets up-regulated (3 in pathways)
23 targets down-regulated (4 in pathways)
Targeted by multiple microRNAs:
CLSPN - cell cycle
FZD5 - receptor for Wnt proteins
CACNG4 - calcium channel

31. Conclusions
● Data integration
○ Multi-omics datasets
○ Pathway and interaction resources
● Cell cycle related and several cancer-related
pathways are down-regulated after vitamin
D treatment
● Possible regulatory mechanism of vitamin D
through microRNAs

32. Conclusion
● Combination of the network-based tools
PathVisio and Cytoscape
● Straightforward, in-depth and biological
meaningful
● Integration of different multi-omics data

33. Acknowledgments
I would like to thank:
Department of Bioinformatics, Maastricht University
Susan Coort
Kim de Nooijer
Claire Lemmens
Chris Evelo
Department of Cardiology, Maastricht University
Georg Summer
Dataset: Wei-Lin Wang et al.

34. Questions