“Targeting Thymidylate Synthase in Cancer Therapy”

F.G. Berger

Department of Biological Sciences

Center for Colon Cancer Research

University of South Carolina

Thymidylate synthase (TS)

Catalyzes the conversion of dUMP into dTMP

Figure adapted from Lehninger

C

N

5-Fluorouracil (FUra) 5-Fluoro-2’-deoxyuridine (FdUrd)

Thymidylate Synthase Inhibitors:

Longley et al. Nature Reviews Cancer 3:330 (2003)

Control TNFα

FUra FUra

FdUrd FdUrd

RTX

Role of post-translational processes in TS function:

● Degradation of the TS polypeptide

● SUMO modification of TS

● Nuclear localization

E. Chu and C.Allegra. 1999. Adv Enzyme Regul. 36:143-63.             

Intracellular stability of human TS

HCT15

HCT15/200

Kitchens, M. E. et al. 1999. J. Biol. Chem. 274:12544-12547

Polysome profiles for TS mRNA

● TS is degraded intracellularly by the 26S proteasome.

● TS is not ubiquitinylated.

● Abrogation of the ubiquitin ligation pathway does not alter TS degradation.

● Conversion of all Lys residues to Arg does not stabilize the TS polypeptide.

Therefore, the TS polypeptide is degraded in a ubiquitin-independent manner.

Role of the N-terminus in TS degradation

The N-terminal region of human TS destabilizes E. coli TS

The N-terminal region of human TS destabilizes GFP

t1/2 ≤ 12 h

t1/2 ≥ 36 h

Role of the penultimate residue in TS degradation.

N-terminal processing of the TS polypeptide:

● Methionine excision● N-α-acetylation

STABILITY PHENOTYPE

PENULTIMATE RESIDUE

MET EXCISION

PREDICTED N-TERMINUS

% LIKELIHOOD*

Unstable(Half-life <14 h)

P (WT) + P 100G + G 100V + V 90

Stable(Half-life >36 h)

A + Ac-A 74C + Ac-C 10D - Ac-M 67T + Ac-T 23S + Ac-S 85

F, H, I, K, L, M, N, Q, R, W, Y

- Ac-M 11

* From Meinnel et al. 2005. Biochimie 87, 701-712.

Human Pro2 - Val3 - Ala4 - Gly5----

Mouse Ac-Met1 - Leu2 - Val3 - Val4 - Gly5----

Rat Ac-Met1 - Leu2 - Val3 - Glu4 - Gly5----

2D Western blots of human TS

2D Western blots of TS/GFP fusions

2 10

PVAGSELPR

Bait:

hTS-(1-313) Gal4-BD

PreyHuman Placental c-DNA library

fused to the C-terminus of Gal4-AD

Yeast two-hybrid screen

Bait Plasmids Identified

● UBC9 is a conjugating enzyme required for protein sumoylation, and has been implicated in regulating several critical cellular pathways.

● SUMO is activated for conjugation by the E1 enzyme AOS/UBA2, transferred to the E2 conjugation enzyme UBC9, and finally conjugated to target proteins.

● Known target proteins of sumoylation include p53, MDM2, PML, RanGAP1, IκB, androgen receptor, and c-Jun.

● Modification of these proteins by sumoylation changes their subcellular localization, function, and/or stability.

● Sumoylation is reversible, and there are at least seven mammalian SUMO-specific proteases, which are designated the SENP family proteins.

In vitro TS sumoylation occurs, and requires all components of sumoylation pathway

TS +

SUM

O-1TS

+ m

SUMO-1

TS +

E1

TS +

E2

No Pro

tein

TS o

nly

WB: Anti-hTS AbTS-SUMO1

TS

Formation of Inhibitory Ternary Complex blocks TS Sumoylation

- + CH 2

THF only

+ CH 2

THF +

FdUMP

+ FdU

MP only

Sumoy

lated

TS + CH 2

THF + FdU

MP

TS +

CH 2TH

F + Fd

UMP

TS on

ly

* *

* TS-SUMO1

TS

* - inhibitory ternary complex

_____TS + SUMO1_____

Consensus SUMO ligation sites in TS Ψ-K-X-E - Ψ is bulky hydrophobic residue, X is any residue

Site A (K284)Site B (K292) Site C (K308)

281 313

● ●

Human TS: …ILRKVEKIDDFKAEDFQIEGYNPHPTIKMEMAV

Mouse TS: T V

Rat TS: T V

K308 is the site of TS sumoylation

1 2 1 2 1 2 1 2 1 2 1 2 1 2

WTSite AK284R

Site BK292A

Site CK308R

K284RK292R

K284RK292RK308R

Noplasmid

TnT-Coupled in vitro Translated human TS

hTS-SUMO1

hTS

1 – in vitro synthesized TS2 – in vitro synthesized TS + SUMO1

Fang, Deyu and Kerppola (2004) Proc. Natl. Acad. Sci. USA 101, 14782-14787

Fluorescence complementation assay

TS

K308

CC

SUMO1+

TS

SUMO1

YN

Bissoon-Haqqani, S. et al. J. Histochem. Cytochem. 2006;54:19-29

Confocal microscopy of HeLa-55 cells stained by immunofluorescence for thymidylate synthase (TS)

Ligands inhibit TS accumulation in the nucleus

Such ligand-mediated inhibition of nuclear TS accumulation is not a consequence of DNA damage

TS localization to the nucleus is also modulated by residue K308, but does not require sumoylation at this site

“Working” Model for nuclear import of TS:

(1.) C-terminal end (including K308) of TS is required for its binding to the nuclear periphery.

(2.) This is abrogated by either ligands or the K308A mutation.

(3.) TS goes through a sumoylation/desumoylation cycle as it enters the nucleus.

(4.) This determines the enzyme’s intra-nuclear locale.

Ligands affect exposure of C-terminal region;

Sumoylation affects intra-nuclear locale, but not entry.

5-yr Survival

p-value Risk Ratio p-value

Nuclear TS Low High

70%51% 0.026 1.46 (1.13-1.89) 0.004

Cytoplasmic TS Low High

70%58% 0.038 1.32 (1.02-1.70) 0.030

N/C Ratio Low High

65%45% 0.010 1.61 (1.09-2.37) 0.012

M. Gustavson et al. AACR Conference on Molecular Diagnostics in Cancer Therapeutic Development (Chicago, IL., Sept. 2006)

High nuclear TS levels in colorectal tumors are associated with poorer survival:

Could the cytotoxicity of TS inhibitors derive from abrogation of the enzyme’s nuclear accumulation,

and subsequent effects on DNA repair?

Karen Barbour Marj Peña Kenn White Yang Yang XingSandra Melo