Wallace McKeehan Center for Cancer & Stem Cell Biology

Forty Years of FGF: Regulator of Cellular &Metabolic Homeostasis

F

IIII

III IIIH

SP

G

F

PLC-

Grb2

SHP2PKC

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sosRas

Raf

MEK

ERK1/2

Grb2

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AK

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The Klothos

Gordon Sato, The Mangrove Man

Gospodarowicz D, Jones KL, Sato G. 1974Purification of a growth factor for ovarian cells from bovine pituitary glands. PNAS 71:2295

Gospodarowicz D. 1975Purification of a fibroblast growth factor from bovine pituitary. JBC 250:2515

Maciag T, Mehlman T, Friesel R, Schreiber AB. 1984. Heparin binds endothelial cell growth factor, the principal endothelial cell mitogen in bovine brain. Science 31;225:932.

Gospodarowicz D, Cheng J, Lui GM, Baird A, Böhlen P. 1984. Isolation of brain fibroblast growth factor by heparin-Sepharose affinity chromatography:

identity with pituitary fibroblast growth factor. PNAS 81:6963.

1985-1986: Cloning of FGF1 and FGF2

1989-1993: Cloning and diversity of FGFR tyrosine kinases via splice variants

1993-2002: Structure of FGFR kinases and interaction with heparan sulfate

FGF22 FGF19 FGF21 FGF23

FGF11 FGF12

FGF16 FGF17 FGF18

FGF3 FGF5FGF1 FGF2 FGF4

FGF20

FGF6 FGF7 FGF8 FGF9 FGF10

Continued Discovery ThroughHomology & Genome Sequencing:

22 FGF Homologues

18 High Affinity FGFR Activators

Four TransmembraneTyrosine Kinase ReceptorsNumerous Splice Variants:

FGFR1-4

Blue: Positive ChargeRed: Negative Charge

White: Neutral

Structure, Mechanismof Assembly & Signaling

of the FGFR Complex

II

II

III

II

III

F

III III

FF

Pantoliano…Sisk Biochem. 1994

Spivak-Kroizman…Schlessinger 1994

DiGabriele…Hendrickson, Nature 1998

FII

III

II

III

F

Plotnikov…Mohammadi, Cell 1999

Kan…McKeehan, JBC 1996

McKeehan, Wang, Kan, PNAR 1998

Xu…McKeehan, JBC 1992Kan…McKeehan, Science 1993

F

II

III

II

III

F

III

F

Yayon…Ornitz, Cell 1991

II

III

II

III

F

F

F F

Venkataraman…Sasisekharan, PNAS 1996Moy…Powers, Biochem. 1997

+++++

IIII

F

IIIIII

F

II

IIF

III

II

+

Schlessinger….Mohammadi, Cell 2000

Pelligrini…..Blundell, Nature 2000

IIIIF

IIIIII

F

Kan…McKeehan, JBC 1996McKeehan, Wang, Kan, PNAR 1998

Conformational Model of Control and Activation (Derepression) of the FGFR Complex

Pre-existent unliganded symmetric complex of 2:2 FGFR-HS

Conformational maintenance of dependence on FGF for kinase derepression by transphorylation by heparan sulfate in the ectodomain

Conformational transmembrane communication between ecto and intracellular domains

How Can FGF-dependent Change Outside Be Transmitted Across the Membrane?

Next Generation: Are Intracellular Substrates/AdaptorsOrganized and Waiting for Conformational Derepression?

FIIII

III III

HS

PG

F

PLC-

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SHP2PKC

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sos Ras

Raf

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F F

Role of Heparan Sulfate

nIdoA/GlcA GlcN

Coreprotein

OCOO-

OX

OH OO

CH2OX

NY

OX OCOO- Linker

1. Matrix and Membrane: Reservoir, Stabilizer, FGF Delivery System

2. Integral Component of the FGFR Complex: Specificity for FGF Assembly of the oligomeric complex Negative restriction of the unliganded dimeric complex Stabilization of the derepressed, ligand-activated complex

2-O-, 2-N-, 3-O- and 6-O-sulfates,2-N-acetyl and epimerization

FGF

HS

HS proteoglycan

FGFR

Is There Structural Specificity in Heparan SulfateBeyond Variations in Charge Density?

Purification of an undersulfated 7,8-S-octasaccharide mixture

(7,8-S-OctaF7) by affinity chromatography with FGF7

7,8-S-OctaF7, which has anticoagulant activity and likely the motif containing a 3-O sulfate,specifically supports high affinity FGF7 binding and mitogenesis in cells expressing FGFR2IIIb.

It failed to support high affinity FGF1 binding and mitogenesis.

Hypothetical deduced structure of 7,8-S-OctaF7. The x, y or z could be a sulfate, preferably 6-O-sulfate with the other two as hydrogens (8-S), or x, y and z may all be hydrogens (7-S).

αKl1 αKl2N C

βKl1 βKl2N C

αKlotho (1014aa)βKlotho (1043aa)

The Klotho Co-factors: single pass transmembraneproteins involved in endocrine FGF activities

SS TM

Similarities to Heparan Sulfate (HS):Independent binding to eFGFs or FGFR

Direct participation in the FGFR complex with both FGF and FGFRDeterminants of specificity for eFGFs

Are HS and Klothos at play in the same FGFR complex?

Do HS or Klotho co-factors alter quality of the FGFR signal?

Does the protein core of HS or the intracellular domain ofHS or Klothos play a role in signaling?

Development and Adult Homeostasis

Modes of Function:Autocrine, Paracrine, Endocrine

Development: Autocrine and ParacrineControl: Changing, Short-lived Cell Autonomy,

Transcription rate-limiting

Adult Tissue Homeostasis: Largely ParacrineControl: Partition of FGF and FGFR between Cells,

Activity, not transcription rate-limitingAutocrine is Pathological

Intracrine (FGF, FGFR or FGF-FGFR)?

Is there tissue and target cell specificity among the 18 FGFsand the 4 tyrosine kinase receptors and variants?

Specificity set by:

1. Paracrine partition of FGF and the FGFR complex between cells.2. Endocrine partition of FGF and FGFR complex between organs.3. Cell-specific co-receptors HS and klothos.

Do different FGFR isotypes have exhibit different signaling endpoints in the same context?

FGF 7FGF10

FGR2b

FGFR3

FGF9

Epithelium

Stroma

Heparan sulfate

Subversion and Autocrine Switch of Canonical Matrix-controlled Short Range Paracrine FGF Signaling Results in Pathologies

Loss of R2bEctopicFGFR1

AutocrineSwitch

Cancer

Feng, Wang, Matsubara, Kan, McKeehan.Fibroblast growth factor receptor 2 limitsand receptor 1 accelerates tumorigenicityof prostate epithelial cells. Cancer Res. 1997

FGFRSHP2SHIP2ERK2IRS4FRS2PLCλ

RSK2FynFAKShcAP85αP85β

Paxillin

PTPN18CDK2 (Tyr15)

EmerinZRF1LAP2

SAP102

growth/tumor suppression, nuclear-cytosol interplay,

cell structural maintenance

FGFR2IIIb FGFR1IIIc

growth promotion,cell survival, adhesion,motility

Overlapping and Distinct TyrP Targets Between:

Luo et al. Novel phosphotyrosine targets of FGFR2IIIb signaling. Cell Signal 2009

Hepatocyte FGFR4

Hepatocyte Cyp7a

Cholesterol to Bile Acids

2000: First implication of FGF signaling in regulating metabolic circuits: Cholesterol/bile acid homeostasis

Time after partial hepatectomy (hr)0 24 48 72 96 120144168

1

2

3

4

5

0102030405060708090

0 24 48 72 96120144168

+/+-/-

+/+-/-

A B

Yu, Wang, Kan…McKeehan. Elevated cholesterol metabolism and bile acid synthesis in mice lacking membrane tyrosine kinase receptor FGFR4. JBC 2000

Early 2000:FGFR4 involved in cholesterol/bile acid metabolism. Yu et al. JBC 2000 Late 2000:Mutation in FGF23 involved in mineral metabolism. ADHR Consortium, Nat Genetics 2000

2002:FGF19 impacts metabolic rate and adiposity. Tomlinson et al., Endocrinol 2002

2005:FGF21 is a metabolic regulatorKharitonenkov et al., JCI 2005

2005:Klothos impact metabolism & FGF signaling

The FGF15/19-FGFR4 Axis:An enterohepatic regulatorof cholesterol/bile acid homeostasis

Inagaki et al., Cell Metab. 2005

Evolution of the Endocrine FGFS

D.D. Moore, Science 2007

Steroids

Steroidreceptors

Paracrine FGFsFGF7,9,10, others

heparan sulfates

FGFR2IIIbFGFR3

Cellularcompartmentalhomeostasis

FGF21 effectorshave expanded!

Hepatocyte FGFR4 Has Multiple EffectsBeyond Hepatic Cholesterol/Bile Acid Metabolism

1. Limits extent of toxic liver injury and fibrosis

2. Modulates hepatic lipid and glucose metabolism

3. Supports fatty liver in obesity or starvation

4. Reported as both hepatoma promoter and hepatoma suppressor

Which effects beyond bile acid metabolism occur directly within hepatocytes due to FGF19-FGFR4-bklotho signaling

is unclear.

Hepatocytes Hepatoma cells

R4 R4 R1

Resident hepatocyte FGFR4 limits hepatocarcinogenesis while ectopic hepatocyte FGFR1 accelerates it

Huang, et al. Ectopic activity of fibroblast growth factor receptor 1 in hepatocytesaccelerates hepatocarcinogenesis by driving proliferation and vascular endothelialgrowth factor-induced angiogenesis. Cancer Res 2006

Huang, et al. Resident hepatocyte fibroblast growth factor receptor 4 limitshepatocarcinogenesis. Mol Carcinog. 2008

WT R1TG WT R1TG

Ectopic FGFR1 in Hepatocytes

FGFR4 Knockout

Normal

liver

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1

1.5

2

2.5

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bklotho (KLB) is reduced in human & mouse hepatomas

-8

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6

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Normal LiverHepatoma

Human SIB-CleanEX DbGSE7307;2109

Restoration of KLB and FGFR4 to KLB- and FGFR4-deficientmouse hepatoma cells induces apoptosis

Without comparablechanges in FGFR4

Rel

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nKLB drops in liver after partial hepatectomy and recovers during restoration

Day after partial hepatectomy

0

2

4

6

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0-5 293 iR4cKLB

HEK293 KLB Constitutive(c) FGFR4 Induced(i) cKLB + iFGFR4

iR4cKLB

(R4)cKLB

iR4cKLB

F1

iR4cKLBF19

iR4F1

iR4F19

iR1cKLB

iR1cKLB

F1

iR1cKLBF19

KLB partners with FGFR4 to inhibit cell population growth via apoptosisApoptosis induced by the FGFR1/4-KLB pair is enhanced by either FGF1 or FGF19

FGF21 is specific for the FGFR1-KLB partner

KLB can confer growth controlling, anti-tumorigenic pro-apoptotic activity on both FGFR4 and FGFR1 signaling complexes.

This is in addition to the role of KLB in conferring high affinity of FGFR4 for endocrine FGF19 and FGFR1 for both FGF19 and FGF21.

Direction of pro-apoptotic signaling is likely through KLB-dependent abrogation of anti-apoptotic AKT and mTOR pathways

How might this KLB-dependent redirection occur?

KLB-dependent abrogation of anti-apoptotic AKT and mTOR pathways?

F

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III III

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cFF1

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Local

Canonical(c) FGF Paracrine/Autocrine

Signaling Endocrine(e) FGF Signaling

Cellular Homeostasis(Development & Adult)

Growth, migration, morphogenesisPromotion of Tumor Phenotype

Metabolic HomeostasisInhibition of growth, pro-cell death

Tumor Suppression

cF

DistalLocal

Local

Net KLB-directed anti-growth and anti-tumor effects is consistent with primary function of eFGFs in control of metabolic homeostasis.

Targeting hepatic FGFR4 will have serious effects on metabolic homeostasis, particularly bile acid metabolism and may have tumor-promoting effects.

Nicholes et al. A mouse model of hepatocellular carcinoma: ectopic expressionof fibroblast growth factor 19 in skeletal muscle of transgenic mice. Am J Pathol 2002

Desnoyers et al. Targeting FGF19 inhibits tumor growth in colon cancer xenograftand FGF19 transgenic hepatocellular carcinoma models. Oncogene 2008

French et al. Targeting FGFR4 inhibits hepatocellular carcinomain preclinical mouse models. PLoS One 2012

Mellor. Targeted inhibition of the FGF19-FGFR4 pathway in hepatocellularcarcinoma; translational safety considerations. Liver Int. 2014

FGF21

In contrast to bile acid-controlled diurnal ileal FGF19, FGF21 is significant under conditions of metabolic extremes as starvation and

obesity and other sources of organismic stress.

FGF21 binds and activates the FGFR1-KLB complex, but not FGFR4-KLB. FGFR1 is not expressed in hepatocytes, but is the major FGFR in adipocytes where KLB is also expressed. FGF19 binds and activates

both FGFR4- and FGFR1-KLB complexes. [Yang et al. Differential specificity of endocrine FGF19 and FGF21 to FGFR1 and FGFR4 in complex with KLB. PLoS

One 2012]

•What is the role of adipocyte FGFR1 during metabolic stress conditions where FGF21 is significant? •Is adipocyte FGFR1 an additional target of FGF19 and the major or sole target of FGF21? •Does FGFR1 account for the beneficial effects of FGF21 and FGF19 on obesity and diabetes?

Adipocyte-specific ablation of FGFR1 indicates that the adipocyte via FGFR1 is the (possibly the sole) FGFR target that accounts for

metabolic effects of FGF21 and the extra-hepatic effects of FGF19

•Yang et al. Control of lipid metabolism by adipocyte FGFR1-mediated adipohepatic communication during hepatic stress. Nutr Metab (Lond). 2012

•Adams & Yang et al. The breadth of FGF21s metabolic actions are governed by FGFR1 in adipose tissue. Mol. Metab. 2012

•Foltz et al. Treating diabetes and obesity with an FGF21-mimetic antibody activating the βKlotho/FGFR1c receptor complex. Sci Transl Med. 2012

Under normal conditions, adipocyte FGFR1 deficiency causes an increase in transcriptional activity of hepatic lipogenic genes without effect on adipocyte genes.

Under starvation conditions the FGFR1 deficiency indirectly causes an increase in hepatic steatosis concurrent with an increase in hepatic lipogenic geneswithout much effect on adipocyte gene expression.

Under starvation conditions the adipocyte FGFR1 deficiency causes concurrent elevation of triglyceride and NEFA without effect on glucose or ketone bodies. This occurs concurrent with an increase in adipocyte lipase activity.

Under starvation conditions that cause hepatic stress and steatosis, adipose FGFR1 concurrently imposes restrictions on adipocyte lipolysis and indirectly hepatic lipogenesis.

This serves to attenuate extent of compensatory hepatic steatosis that often occurs during hepatic stress.

Lipolysis and lipogenesis are normally tightly coupled to glucose and ketone body metabolism. Overall lipolysis and lipogenesis are tightly coupled and inversely related.

Uncoupling these normally tightly linked domains may mete out and extend lipid reserves for neural fuels (glucose and ketone bodies) during metabolic extremes and other conditions causing hepatic stress.

Adipocyte FGFR1-KLB is a target of FGF21 whose primary origin is hepatocytes and an additional target of FGF19 in addition to hepatocyte FGFR4.

We speculate this mechanism may underlie the beneficial effects of both endocrine FGFs under both metabolic extremes of starvation and obesity as well as other sources of hepatic stress.

[Yang et al. Control of lipid metabolism by adipocyteFGFR1-mediated adipohepatic communicationduring hepatic stress. Nutr Metab (Lond). 2012]

FGF21/FGFR1-mediated adipo-hepatic communication

Is adipocyte FGFR1 the sole direct mediator of beneficialeffects of FGF21 in obesity/diabetes (metabolic stress)?

Adipose FGFR1 deficiency abolishes weight loss, glucose and energy regulation by FGF21 in the obese

Foltz et al. Treating diabetes and obesity with an FGF21-mimetic antibodyactivating the βKlotho/FGFR1c receptor complex. Sci Transl Med. 2012

What about FGF19’s effects in the adipocytes?Adipose FGFR1 deficiency also abolishes weight loss, glucose

and energy regulation by FGF19 in the obese

Thus FGF19 targets adipocytes via FGFR1 with essentially identical effects to FGF21

IlealFGF19

FGFR4/KLB:Hepatic Cholesterol to Bile AcidsHepatic Lipogenesis

During normal feeding ileal FGF19 is likely the normal coordinator of adipo-hepatic communication in the lipid metabolism domain. FGF21 is a hepatokine reserved to instruct adipocytes under conditionsof metabolic extremes and other stressconditions sensed by the liver.

Effects of FGF19/FGF21 on glucosemetabolism are largely an indirect consequence of direct regulation of lipidmetabolism in both liver and adipocytesin both normal and extreme conditions.

Conclusion

Dr. Chundong YuProfessor BiologyXiamen University

Dr. Xinqiang HuangRegulus Therapeutics

Dr. Fen WangProfessor

Center DirectorIBT Texas A&M

US Public Health Service grantsJohn S. Dunn Research FoundationKomen Breast Cancer Foundation

Amgen & Eli Lilly

Dr. Yongde LuoAssistant ProfessorIBT Texas A&M

Chaofeng YangPostdoctoral

UT Southwestern

How could an extracellular co-factor (HS/klotho) alterdiverse intracellular signaling endpoints & phenotypes?

Transmembrane conformational transmission?

F

IIII

III III

HS

PG

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PLC-

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SHP2PKC

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sosRas

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s-k

loth

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m-k

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FGF15/19FGF21FGF23

GRB2 SHP-2

SNT1/FRS2

•Activated pathways remarkably similar

•Few P-Tyr substrate sites like other TKR

•Mostly SNT1/FRS2a multi-P adapter

Control cKLB iFGFR4 cKLB + iFGFR4

βKlotho (KLB) partners with FGFR4 or FGFR1 to inhibit cell population growth

KLB- and FGFR4-dependent apoptotic cell death is induced by either FGF19 or FGF1

4.5 9 10.5 13.4 21.4 14.5 Apoptosis (%)

cKLBiFGFR4

FGF19 (1)

cKLBiFGFR4

FGF19 (10)

cKLBiFGFR4

FGF19 (102)

cKLBiFGFR4

FGF19 (103)

PI

4.6 9 9.5 10.5 16.5 23.8

HEK293cKLB

iFGFR4

Annexin V

FGF1 (103)FGF1 (102)FGF1 (10)FGF1 (1)

Apoptosis (%)

PI

Luo et al. Metabolic regulator betaKlotho interacts with fibroblast growth factorreceptor 4 (FGFR4) to induce apoptosis and inhibit tumor cell proliferation 2010 JBC