La figura de Alberto Sols fue durante décadas una referencia para los bioquímicos españoles. Los días 20 y 21 de febrero de 2017, la Fundación Ramón Areces dedicó un simposio internacional a su memoria, rindiendo homenaje a sus principales logros: las levaduras como modelo experimental, la enzimología y regulación metabólica y la patología molecular. El encuentro científico, que estuvo coordinado por Carlos Gancedo y Joan Guinovart, reunió a expertos internacionales para debatir sobre el legado de este científico español.

1. “Striking a Balance Between Cellular Growth
and Homeostasis”
M. Celeste Simon, Ph.D.
Madrid Symposium
February 20, 2017

2. Limited O2/Nutrient Availability in Solid Tumors

3. Tumor cells must balance:
expansion of biomass
viability in harsh microenvironment
evasion of the immune system

4. Chemical Biology of 2-Oxoglutarate Oxygenases
Loenarz, C et al. (2008) Nature Chem. Bio. 4:152
Thienpont, B et al. (2016) Nature 537:63
FIH Asparagine Hydroxylation
Histone, RNA, DNA Demethylation
Collagen Modification

5. Hypoxia Inducible Factor Pathways
Simon, MC (2016) New Engl. J. Med. 375:1687

6. Glycolysis, TCA Cycle, and Lipid Synthesis
Nakazawa M, B Keith, and MC Simon (2016) Nature Rev. Cancer 16:663

7. Overall Questions:
1) How does O2 and nutrient availability influence cancer
cell metabolism?
2) What are the metabolic interactions between multiple
cell types in the tumor microenvironment?
3) Can TME metabolic “rewiring” be therapeutically
targeted?
4) Can tumor metabolism teach us fundamental new
concepts about “normal metabolism”?

8. Clear cell renal cell carcinoma (ccRCC)
– Most common renal cancer
– 5 year survival
• >80% if localized/resected
• 23% if metastatic
– Hallmarks of disease:
• Constitutive HIF signaling
• Lipid Laden Cells

9. Metabolic profiling of ccRCC tumor and normal kidney
Urea cycle; arginine and proline metabolism
Nicotinamide (NAD) and Riboflavin (FAD) metabolism
Carnitine metabolism
Long chain fatty acid
Lysolipids
Li, B et al. (2014) Nature 513:251
Hakimi, A et al. (2016) Cancer Cell 29:104

10. Metabolic gene set analysis
of the
TCGA ccRCC RNAseq data
Steps:
1) RNAseq data of 480 ccRCC tumors and 69
normal kidney tissues (by TCGA)
2) Gene expression fold changes in tumor vs.
normal tissue (by Penn Bioinformatics Core)
3) 2753 metabolic genes, classified into 72
functional groups (bases on KEGG)
4) threshold p-adj value 0.1
Carbohydrate Storage
Gene
Gene expression changes in
tumor v.s. normal tissue
p-adj
G6PC -10.5772 6.22E-14
PCK1 -6.98445 6.58E-16
FBP1 -5.01838 1.86E-25
PCK2 -4.76531 1.69E-20
FBP2 -4.48907 0.0661758
GYG2 -2.11176 0.0870902
GYS1 1.87385 2.67E-08

11. 0
10
20
30
FBP1expression
(arbitraryunit)
ccRCC tissue
normal tissue
FBP1 staining in ccRCC tumor and normal kidney tissue

12. Central Carbon Metabolism

13. * *
* *
*
*
Metabolic changes in ccRCC tumor cells with
FBP1 re-expression

14. FBP1 inhibits ccRCC cell growth in vitro and in vivo
Cell growth
786-O
Xenograft

15. Nuclear FBP1 associates with HIF1 and HIF2
IP:
HIF1
Input
HIF1
FBP1
HIF1
FBP1
HIF2
FBP1
HIF2
FBP1
IP:
HIF2
Input

16. Both WT and G260R FBP1 suppress ccRCC growth
RCC10 (1 mM Glucose) 786-O (1 mM Glucose)

17. Conclusions
s
► FBP-1 is lost in 100% ccRCC (>1000 samples assayed).
► FBP-1 binds HIF’s at HREs, attenuating their activity .
►Fbp-1Δ/Δ mice to explore “metabolic zonation” in liver and kidney,
and generate ccRCC, HCC, and sarcoma GEMMs.
Summary

18. Marquardt JU. Cancer Cell. 2014; Shibata T. N Nat Rev Gastroenterol Hepatol. 2014 18
A snapshot of human HCC
 Chronic and multistep progression
 Inflammatory disease
 NAFLD is a metabolic predisposition
 Hepatocyte as one cell of origin
 Genetic heterogeneity
 Phenotypic heterogeneity and plasticity

19. Creatine
Mevalonate
Other Transport
Pentose Phosphate
Glycan Sulfate
Glycan Anchor
Nucleotide
Proton Transport
Complex III
Heparin Sulfate
Vitamin B6
Glycan Degradation
Glutamate
Complex IV
Pigment
Ion Transport
Glycolysis
Inositol Phosphate
NAD
Glycan
Purine
Complex I
Cholesterol
Membrane Lipid
Nucleotide Sugar
Small Molecule Transport
Proline
Krebs
Aminosugar
Pyrimidine
Sphingolipid
ATPase
ABC Transporter
Sugar
Hormone
Redox
Signalling
Methionine
CoA
Other
Neurotransmitter
Glutathione
Polyamine
Cofactor
Ubiquinone
Vitamin A
Anaerobic Glycolysis
Porphyrin
Multipurpose
Reactive Oxygen
Folate
Fatty Acid
B12
Sphingosine
Sulfate
Steroid
Amino Acid
Cysteine
Ketone Bodies
Complex II
BCAAs
Lysine
BH4
Propanoate
Carbohydrate Storage
Detox
Tyrosine
Xyulose
Bile Acid
Glycine
Tryptophan
Serine
Histidine
Glyoxylate
Urea
-5 -4 -3 -2 -1 0 1 2
Gene expression
(tumour /normal, Log2)
TCGA
Nucleotide Sugar
Pentose Phosphate
Mevalonate
Glycan Anchor
Cholesterol
Polyamine
Proton Transport
Complex III
Complex IV
Purine
CoA
Complex I
Inositol Phosphate
Sphingolipid
Other Transport
ATPase
Krebs
Glycolysis
Nucleotide
Glycan Degradation
Glycan
Aminosugar
Heparin Sulfate
Glycan Sulfate
Vitamin A
Anaerobic Glycolysis
Membrane Lipid
NAD
Vitamin B6
Cofactor
Redox
Methionine
Ubiquinone
Reactive Oxygen
Small Molecule Transport
Sugar
Ion Transport
Hormone
Pyrimidine
Porphyrin
B12
ABC Transporter
Signalling
Creatine
Pigment
Neurotransmitter
Glutathione
Multipurpose
Other
Amino Acid
Folate
Sphingosine
Fatty Acid
Glutamate
Proline
Steroid
Complex II
Carbohydrate Storage
Sulfate
BCAAs
Propanoate
Bile Acid
Serine
Detox
Ketone Bodies
BH4
Xyulose
Cysteine
Histidine
Glycine
Glyoxylate
Urea
Lysine
Tryptophan
Tyrosine
-5 -4 -3 -2 -1 0 1 2
Gene expression
(tumour /normal, Log2)
GSE14520
19
FBP1 in Hepatocellular Carcinoma

20. Normal Tumor
0
5
10
15
20
RNA-seqreadsofFBP1(Log2)
****
Normal Tumor
0
5
10
15
20
RNA-seqreadsofPCK1(Log2)
****
Normal Tumor
0
5
10
15
20
RNA-seqreadsofG6PC(Log2)
****
Underexpression of gluconeogenic genes in HCC
TCGA
Normal Tumor
0
5
10
15
RelativeexpressionofG6PC(Log2)
****
Normal Tumor
0
5
10
15
RelativeexpressionofFBP1(Log2)
****
Normal Tumor
0
5
10
15
RelativeexpressionofPCK1(Log2)
****
GSE14520
20
G6PC FBP1 PCK1
****p<0.0001

21. GSE14520
N
orm
al
Stage
I
Stage
II
Stage
III
0
5
10
15
20
RNA-seqreadsofFBP1(log2)
****
****
***
TCGA
N
orm
al
Stage
I
Stage
II
Stage
III
0
5
10
15
RelativeexpressionofFBP1(log2)
****
****
**

22. 22
H
Dox (ng/ml)
PLC/PRF/5-FBP1-V5
0 50 100 200 500 1000
GAPDH
FBP1
V5
0 2 4
0
5
10
15
20
Days
No.ofCells(x104)
PLC/PRF/5-FBP1-V5
No dox
Dox
1% serum/ 21%O2
FBP1 suppresses HCC cell proliferation
and xenograft tumor growth
H: human primary hepatocyte
Ctrl Dox
0.0
0.1
0.2
0.3
0.4
Tumorweight(g)
**
0 2 4
0
5
10
15
20
Days
No.ofCells(x104)
PLC/PRF/5-FBP1-V5
No dox
Dox
1% serum/ 1%O2
***
* p<0.05, **p<0.01, ***p<0.001
16 19 22 25 28 31 34 38
0
100
200
300
400
500
Days
TumorVolume(mm3)
PLC-PRF/5-FBP1-V5
Ctrl
Dox
Dox
**
- +
0
50
100
150
Coloniesperwell
*
Dox

23. DEN
Fbp1fl/fl
AAV-TBG-Cre/PBS
2 wk, male
4 wk 3-10 month
MRI
Pathological
analysis
DEN: diethyl nitrosamine
Role of FBP1 in HCC initiation: DEN model

24. 24
Fbp1
Gapdh
GFP Cre
AAV-mediated deletion of Fbp1 in livers of Fbp1fl/fl mice
****p<0.0001
GFP: Control group
Cre: Fbp1-deleted group
GFP Cre
0.0
0.5
1.0
Relativeexpression
Fbp1
****
0.000123362

25. 25
GFP Cre
0
5
10
15
20
25
OilRedO(areafraction)
****
GFP Cre
0
100
200
300
400
TGcontent(mg/gliver)
*
H&EOilRedO
GFP Cre
GS/DAPI
GFP Cre
FBP1-deficient mouse livers show NAFLD features
*p<0.05, ****p<0.0001
GFP Cre
0
2
4
6
LW/BWratio(%)
GFP Cre
0
20
40
60
80
ALT(mU/ml)
NAFLD: non-alcohol fatty liver disease
cv
cv
cv
cv

26. 26
DEN
Fbp1fl/fl
AAV-TBG-GFP/Cre
2 wk 6 wk 3-10 month
Analysis
DEN: diethyl nitrosamine
GFP Cre
0
50
100
ALT(mU/ml)
**
*p<0.05, **p<0.01, ***p<0.001
GFP Cre
H&E
GFP Cre
0
5
10
15
SurfacetumorNo.
***
GFP Cre
0
5
10
15
No.ofmacroscopictumors
(>0.5mm)
*
FBP1 deletion promotes DEN-induced HCC
tumorigenesis

27. 27GFP Cre
0
10
20
30OilRedO(areafraction) ***
*p<0.05, **p<0.01, ***p<0.001
GFP Cre
0
100
200
300
400
500
TGcontent(mg/gliver)
**
Ki67
GFP Cre
GFP Cre
0
50
100
150
No.ofKi67+cells/HPF
**
FBP1 deletion promotes DEN-induced HCC tumorigenesis
GFP Cre
OilRedO
GFP Cre
0
1
2
3
4
Relativeexpression
CD44
*
GFP Cre
0
10
20
30
40
50
Relativeexpression
Gpc3
*
GFP Cre
1
2
4
8
16
32
64
Relativeexpression(log2)
Afp
*

28. 28
N
orm
al
Stage
I
Stage
II
Stage
III
0
5
10
15
20
RNA-seqreadsofFBP1(log2)
****
****
***
DEN treatment downregulates Fbp1 expression in
mouse livers
*p<0.05, **p<0.01, ****p<0.0001
Ctrl DEN
0
50
100
ALT(mU/ml)
**
FBP1
GFP Cre
0.0
0.5
1.0
1.5
Relativeexpression
Fbp1
***
GFP Cre
0.0
0.5
1.0
1.5
2.0
2.5Relativeexpression
Pck1
GFP Cre
0
2
4
6
Relativeexpression
G6pc
N
orm
al
Stage
I
Stage
II
Stage
III
0
5
10
15
20
RNA-seqreads(Log2)
PCK1
****
N
orm
al
Stage
I
Stage
II
Stage
III
0
5
10
15
20
RNA-seqreads(Log2)
G6PC
****

29. Summary
• FBP1 is significantly downregulated during human HCC progression.
• FBP1 inhibits HCC cell proliferation and xenograft tumor growth.
• Liver-specific deletion of Fbp1 results in NAFLD features in mice.
• Deletion of Fbp1 promotes HCC tumorigenesis in the DEN model.
29
FBP1 NAFLD
DEN HCC
?
?
Mechanisms??
?
Walter Birchmeier. Nat Cell Biol, 2016

30. • Monitor Fbp1fl/fl mice for tumor development.
• RNAseq to identify differential genes/pathways.
• Metabolic profiling (lipidomics) to identify differential metabolite(s).
• IHC and/or flow cytometry analysis to characterize the differential
“immuno-microenvironment”: CD4, CD8 and Th17 cell populations….
• Modulating immuno-microenvironment by specific metabolite(s)?
• Compare HCC tumorigenesis between Fbp1fl/fl and Fbp1 fl/fl; Arntfl/fl
mice.
30
Current and Future directions

31. Urea cycle enzymes underexpressed in ccRCCs
Li, B et al. Nature (2014) 513:251
Dondeti, V et al. Cancer Res. (2012) 72:112
TCGA Network Nature (2013) 499:49
Gene
Gene Expression Change
(Tumor vs. Normal)
p-value
ARG2 -6.44696 1.023 E-24
ASS1 -3.22585 7.170 E-12
ORNT1 -1.9033 0.000790
ASL -1.28397 0.0695
CPS1 1.50423 1
OTC 5.99412 1

32. ARG2 and ASS1 expression lost in ccRCCs
Ochocki, J et al. (2016), unpublished

33. Urea Cycle

34. Metabolic profiling of primary ccRCCs
Li, B et al. (2014) Nature 513:251
Hakimi, A et al. (2016) Cancer Cell 29:104

35. ARG2/ASS1 re-expression reduces ccRCC cell growth

36. ARG2/ASS1 re-expression reduces tumor growth

37. Metabolomic profiling of ccRCC xenografts

38. Summary
• Multiple urea cycle enzymes are copy number lost
and underexpressed in ccRCC.
• ARG2 and ASS1 suppress ccRCC growth in vitro and
in vivo.
• ARG2 re-expression leads to decreased amino acid,
lipid, and nucleotide pools.
• ARG2 re-expression decreases mTORC1 activity.

39. Summary: But Why?
Multiple pathways implicated in urea cycle-dependent
tumor suppression of ccRCCs:
1. Decrease in amino acid pools reduces mTORC1 activity?
2. Decrease in aspartate causes reduced de novo nucleotide
synthesis?
3. Polyamine levels increase, which is detrimental to ccRCC cell
growth? Buildup of urea?
4. Depletion of pyridoxal phosphate, a critical biosynthetic cofactor?
5. Effects on nitric oxide homeostasis?
6. Impact on recruited immune cells?

40. Arginine metabolic tracing

41. ARG2 increases ODC and OAT metabolites,
decreasing Cofactor Pools
Pyridoxal Phosphate
(PLP):
• The active form of
Vitamin B6
• Required cofactor in
>140 reactions
• PLP-dependent
enzymes promote amino
acid, lipid, etc. synthesis
– eg. transaminations,
decarboxylations

42. ARG2 effects on Pyridoxal Phosphate

43. PDXK Rescues Growth in ARG2
Re-expressing ccRCC Cells in 3D

44. Summary
• Multiple urea cycle enzymes are copy number lost
and underexpressed in ccRCC.
• Urea cycle enzyme loss avoids a toxic buildup of
polyamines and urea in ccRCC cells.
• ARG2 loss protects pyridoxol phosphate abundance
needed for amino acid, lipid, etc. synthesis;
pyridoxol kinase a therapeutic target?
• Urea cycle enzyme loss affects NO levels, blood
vessels, and inflammatory cells.

45. Urea Cycle

46. ASS1/ASL/NOS Complex at Membranes
Erez A, et al. (2011) Nat. Medicine: 17:1619

47. Critical Questions
1) Do disparate oncogenic changes converge on more
common metabolic adaptations in tumors?
2) How can we “drug” missing enzymes in tumor cells?
3) Do changes in the abundance of asparagine, arginine,
and NO influence recruited immune cells?
4) Why do highly conserved metabolic enzymes have
both catalytic and structural properties?

48. The Simon Lab: 2016

49. Celeste Simon
Brian Keith
Sanika Khare
Nicole Anderson
Ankita Bansal
Peiwei Huang
Roy Lan
Kyoung Lee
Pearl Lee
Fuming Li
Nan Lin
Richard Maduka
Vivek Nimgaonkar
Danielle Sanchez
Michelle Spata
Hong Xie
Acknowledgement

50. Arginine Tracing in Normal Kidney Cells with
ARG2 Deletion

51. ARG2-/- HK2 cells sensitive to putrescine