Lecture by Prof. Lorenz Poellinger in the course: "Tumour Hypoxia: From Biology to Therapy III". For the complete e-Course see http://www.myhaikuclass.com/MaastroClinic/metoxia

1. Epigenetic Mechanisms Regulating the Cellular
!
Response to Hypoxia!
Lorenz Poellinger
!
Dept. Cell Mol. Biol., Karolinska Institute, Stockholm, Sweden!

2. Cellular Adaptation to Hypoxia
-mediated via hypoxia-inducible factors (HIFs)
-Three known members, HIF-1α, HIF-2α, and HIF-3α
-bHLH /PAS transcription factors
-Dimerization partner Arnt
-Complexes bind to hypoxia responsive elements (HREs)
-HIF protein stabilization at low oxygen concentrations
(HIF-1α, HIF-2α, and HIF-3α)
-Derepression of HIF function at low oxygen concentrations
(HIF-1α, and HIF-2α, but not HIF-3α)

3. Different HIF Functions in Tumor Cells
(neuroblastoma, breast cancer cells):
- HIF-1: mediator of acute responses to hypoxia
- HIF-2: mediator of long-term (chronic) responses to hypoxia

4. Differential Utilization of HIFs in Response to Oxygen Shortage
HIF-1α 1% O2
HIF-2α 1% O2
HIF activity HIF-2α 5% O2
4 72
Time (h)

5. Inhibitory PAS domain protein - IPAS
IPAS
IPAS/HIF-1α
HRE

6. Application of IPAS antisense induces
angiogenesis in the cornea!
V! S!
R! AS!
Cornea angiogenesis study!
5 days after implantation of micro pellet containing !
2.5 µg of oligonucleotide (arrows)!
V: vehicle R: randomized oligonucleotide!
S: sense oligonucleotide AS: antisense oligonucleotide !

8. IPAS is a splicing variant of the HIF-3α locus!
bHLH! PAS domain! TAD!
HIF-3α! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11! 12! 13!14!15!
1a! 1! 2! 3! 4a!4! 5! 6! 7! 8! 9! 10! 11! 12! 13! 14!15! 16!
genome!
-14! -87!
ATG!ATG! STOP!STOP!
IPAS! 1a! 2! 3! 4! 5! 6! 16!
4a!

9. Oxygen concentration-dependent expression
of the IPAS-specific splicing product of the
HIF-3α locus in the mouse heart!

10. Different HIF Functions in Tumor Cells
- HIF-1: mediator of acute responses to hypoxia
- HIF-2: mediator of long-term (chronic) responses to hypoxia
- HIF-3: (negative) modulator of the hypoxic response
(IPAS splice variant: potent dominant negative regulator)
(specific splice variants: cornea epithelium; inducible in
many adult tissues, embryonic forms)

11. IPAS-/- mice!
(collaboration with Dr. Masayuki Yamamoto, Univ. Tsukuba, Japan)!
- IPAS-GFP expression in heart, lung, kidney and endothelium!
- IPAS-/- mice: very pronounced Cardiac RV Hypertension!
(block of negative regulation of endothelin-1 expression during!
development and postnatally).!

12. Human Neuroblastoma (childhood tumors derived from
the sympathetic nervous system)
Hypoxic human neuroblastoma cells
-induce genes traditionally linked to a hypoxic phenotype
-(VEGF, GLUT1, GLUT3, HK2, ENO etc.)
-lose neuronal/neuroendocrine marker gene expression
-(GAP-43, SCG-10, NPY, NF, dHAND, HASH-1 etc. are downregulated)
-become neural-crest like
-(increased expression of vimentin, ID2, Notch-1, HES-1, c-kit)

13. -Hypoxia drives neuroblastoma cells towards an immature,
stem cell-like phenotype.
-By histopathological criteria, hypoxic breast cancer cells become
dedifferentiated in vivo. These cells express cytokeratin 19, a
stem cell marker.
-In both neuroblastoma and breast cancer, low differentiation
stage correlates with poor prognosis.

14. In neuroblastoma, high HIF-2α protein levels in
restricted tumor areas are associated with:
- high VEGF production
- tumor vascularization
- poor prognosis
- unfavorable outcome
A small, distinct population of cells determines
outcome
Holmquist-Mengelbier et al., Cancer Cell 2006

15. A Molecular Model for Hypoxia-induced Dedifferentiation of Neuroblastoma Cells
Id2!
Normoxia !
dHAND!
Hash1!
E2-2!
E2-2!
Neuronal!
marker genes !
E-box ! E-box !
Tumor growth ! Id1, Id2!
Notch1/3!
Notch pathway blocks!
Hash1 expression!
Hes1, Hey1!
Hypoxia!
E2-2!
HIFs! dHand!
Hash1!
!
Hypoxia Id2!
E2-2!
Hash1!
dHAND!
Id2!
Id1!
Neuronal!
marker genes !
E-box ! E-box !

16. Notch Signaling Pathway
Notch ligand
Notch receptor
cleavage by
γ-secretase
Sel-10
NICD
+
Hes-1
CSL
Hey-2
Proteasome
Inhibition of differentiation

17. Regulation of the cell differentiation status by hypoxia
(stem cells, endothelial cells, cancer cells):
- Hypoxia and HIFs are important for stem cell maintenance
- Certain tumor cells de-differentiate under hypoxic conditions
(correlation with increased tumor aggressiveness)
- Many of the mechanisms regulating the cell differentiation status in hypoxia
seem to be integrated with the Notch signaling pathway
(e.g. integration between the Notch and hypoxia signaling pathways in angiogenesis
and EMT)

18. Integration between the hypoxia and Notch signaling pathways

19. Activation of Notch in a Coculture System
CSL
binding site
Hey-2
ATG
-293
-118
HRE
ATG
PGK1
-274
-95
293T -/+Jagged-1
Test of species specific primers
C2C12 Fl Notch-1
Hey-2 promoter
PGK1 promoter

20. HIF-1α interacts with Notch
on the Hey-2 promoter
Chromatin IP
N
N
H
H
N
N
H
H
Ligand:
-
+
-
+
Ligand:
-
+
-
+
α-HIF-1α
α-Notch 1 ICD
Hey-2
PGK1
α-goat IgG
input

21. Epithelial Mesenchymal Transition (EMT), Metastasis!
Hypoxia Snail E-cadherin EMT !

22. Hypoxia-induced downregulation of E-cadherin
requires Notch signaling!

23. Metastasis!
Hypoxia Snail E-cadherin EMT !
Does Notch-mediated EMT and E-cadherin
downregulation proceed via Snail ?!

24. Hypoxia-dependent upregulation of Snail1
requires Notch signaling!
- - - - + DAPT
24h 48h 72h 72h
Normoxia Hypoxia
Snail1!
Direct transcriptional control ?!

25. !
Notch ICD binds to the Snail promoter
Snail
ttcacttcctctgggaagtcaccccgacc ATG
CSL-binding site
ChIP
Input IgG NICD
2.5
Snail-promoter Snail-promoter∆CSL
Fold activation
2
1.5
1
Normoxia Hypoxia
0.5
0 -
Ligand + +
Notch 1 ICD
α-HIF-1α
Snail
α-IgG
Input

26. LOX (lysyl oxidase)!
LOX
Erler et al., Nature 2006
Hypoxia Notch Snail E-cadherin EMT !
LOX
- LOX is upregulated by hypoxia
- LOX stabilizes Snail protein via! GSK3
negative regulation of GSK3β
PPPP
Snail Snail

27. Is Notch involved in LOX regulation ?!
Notch potentiates HIF-1α-binding to the
hypoxia-induced LOX LOX promoter is
expression! potentiated by Notch!
LOX
ATG
HRE
Normoxia Hypoxia
3T3-B + + -
3T3-J - - +
α-HIF-1α
α-IgG
LOX
Input

28. Metastasis!
Notch
LOX
Hypoxia Notch Snail E-cadherin EMT !

29. Metastasis!
Notch
LOX
Hypoxia Notch Snail E-cadherin EMT !

30. Category
Late response
Continuous response
Model Gene
CA9
PGK1
Gene-specific roles of HIF/HRE in chromatin dynamics
• Establishment of hypoxia-dependent NFR in promoter regions
(e.g. CA9)
• HIF binding to HRE in NFR to enhance transcription
(e.g. PGK1)

31. Chromatin remodeling
during hypoxia-responsive transcription
Inducible NFR
I
Normoxia
HRE
Stabilization
Activation
Hypoxia
Remodeling
CBP
HRE
Degradation
Reoxygenation

32. Conserved core hypoxia gene expression signatures common to seven cancer cell lines
Jmjd1a is a JmjC domain-containing
histone H3K9 demethylase.
Lendahl U, Lee KL, Yang H, and Poellinger L.
Nat Rev Genet. (2009)

33. The Epigenetic landscape of Cells in Hypoxia?
Silence
HYPOXIA
Active
H3K9
Me H3K9
A
Active
Silence
H3K9 Me H3K9

34. G9a / GLP (Eu-HMTase 1) vs Jmjd1a (Jhdm2a, Kdm3a)
Cystein Rich Ankyrin
哺乳類のヒストン・リジン・
Poly E Region SET domain
Repeat メチル化酵素
Repressor
G9a
G9a 1,172 a.a.
Dimerization
Poly ED
GLP
GLP
1,296 a.a.
Adds me1 or me2
G9a / GLP function as a heterodimer
Cooperate with Dnmts to silence genes
K79
K4
K9
K27
K36
H3
H4
K20
Removes me1 or me2
Zinc Finger-like JmjC-domain
Jmjd1a
Jmjd1a
Activator
1,323 a.a.