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The API5-FGF2 complex functions in mRNA export and is a potential target for cancer therapeutics

SeonJeong Baek, Seoungmin Bong and Byung Il Lee

1 Research Institute, National Cancer center, Goyang, Gyeonggi 10408, Korea,

2 Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyang-si, Gyeonggi 10408, Republic of Korea

ABSTRACT

API5 (APoptosis Inhibitor 5) and nuclear FGF2 (Fibroblast Growth Factor 2) are upregulated in various human cancers and are correlated with poor prognosis. Although their physical interaction has been identified,

the function of the resulting complex in cancer is unknown. Here, we determined the crystal structure of the API5–FGF2 complex and identified critical residues driving the protein interaction. This provided

structural basis of nuclear localization of the FGF2 isoform lacking canonical nuclear localization signal and identified cryptic nuclear localization sequence in FGF2. The interaction between API5 and FGF2 was

important for mRNA export through both the TREX and eIF4E/LRPPRC mRNA export complexes, regulating the export of bulk mRNA and specific mRNAs containing eIF4E sensitivity elements, such as c-MYC

and cyclin D1. Interestingly, API5–FGF2 complex directly interacted with UAP56, a common factor of these two mRNA export complexes. Disruption of the API5–FGF2 interaction reduced cell proliferation and

increased drug sensitivity. These data reveal a previously unknown function for API5 and nuclear FGF2 and suggest a new therapeutic target for regulating the expression of oncogenes at the mRNA export level.

And in further studies, to discover chemicals that bind to the API5-FGF2 complex, we use thermal shift assay to prioritize Fragment-Based Drug Discovery (FBDD) library screening and select hits.

INTRODUCTION METHOD

- low-molecular weight (LMW) FGF2 CLONING & OVEREXPRESSION

isoform lacking the N-terminal extensions is ESCHERICHIA COLI EXPRESSION
SYSTEM (pET28b/pHis VECTOR)
usually secreted to function in autocrine

or paracrine FGF2 signaling by association

with heparan sulfate proteoglycans (HSPGs)

and FGF receptors (FGFRs).

PURIFICATION

- High-molecular-weight (HMW) FGF2

isoforms that possess N-terminal NLS

sequences are localized to the nucleus to

perform various FGFR-independent function.

Atlas of Genetics and Cytogenetics in Oncology and Haematology

Yang et al., 2009, Trends Mol Med

- Apoptosis inhibitor 5 (API5) is a nuclear

protein that inhibits apoptosis in human cells.

- API5 is important for cell cycle progression,

immune escape, metastasis, and the stem-

cell-like properties of cancer cells and that it CRYSTALLIZATION

promotes drug resistance in cancer.

- Interaction partners : Fibroblast growth

(Han et al 2012, J. Biol. Chem.) factor 2 (FGF2), Acinus, Influenza A virus
nucleoprotein, Estrogen receptor.

RESULTS Terai et al., 2013, Mol cancer Res.

STRUCTURE DETERMINATION,

REFINEMENT, AND ANALYSIS

HKL2000

Coot

Refmac

Phenix

The crystal structure of the API5–FGF2 complex reveals residues critical for the interaction

The interacting residues can be divided into segments of highly conserved basic (FGF2) or acidic

(API5) regions (FGF2-segment 1, 261KRTGQYKLGSKT272; API5-segment 1, 142QGEDIVR

148; API5-segment 2, 183LEDVTGEEF191; and API5-segment 3, 219EQADLEQTFNPSDPD

CVDR237) Interactome analysis with the API5–FGF2 complex suggests a functional link to mRNA export.
API5 and FGF2 interact directly withUAP56, a common factor of the TREX and eIF4E/

LRPPRC mRNA export machineries.

4 API5–FGF2 is involved in both NXF1- and CRM1-dependent mRNA export pathways

through direct interaction with UAP56.
2
Bulk mRNA export assay by

0
0 200 400 600 800 1000 1200 1400 1600 RNA-FISH
-2

API5–FGF2 complex functions in bulk
-4
ΔTm(°C) -6 mRNA export. Analysis of nuclear/cytosolic RNA

-8 The bulk mRNA export activity of API5 levels by RT-qPCR
depends largely on its interaction with
-10
FGF2.

Fragment-Based Drug Discovery (FBDD) conceptual diagram -12 API5–FGF2 complex regulates the

-14 export of 4E-SE containing REFERENCE
oncogenic mRNA
-16 Chemical numbers

Our study points to a model for the

function of the API5-FGF2 complex in Yang S.Y., Sales K.M., Fuller B, Seifalian A. M. and Winslet M. C.

the TREX and eIF4E/LRPPRCmRNA (2009) Apoptosis and colorectal cancer: implications for therapy.
Trends in Molecular Medicine
.
export pathways.

Han B.G., Kim K.H., Lee S.J., Jeong K.C., Cho J.W., Noh K.H., Kim

T.W., Kim S.J., Yoon H.J., Suh S.W., Lee S.H., and Lee B.I. (2012)

Helical Repeat Structure of Apoptosis Inhibitor 5 Reveals Protein
-Protein Interaction Modules. JOURNAL OF BIOLOGICAL

CHEMISTRY.

Atlas of Genetics and Cytogenetics in Oncology and
Haematology.

Terai H., Soejima K., Yasuda H., Nakayama S., Hamamoto J., Arai

Result from screening of chemicals that binds to the API5. D., Ishioka K., Ohgino K., Ikemura S., Sato T., Yoda S., Satomi R.,

we used thermal shift assay to prioritize Fragment-Based Drug Discovery (FBDD) library Naoki K., and Betsuyaku T. (2013) Activation of the FGF2-FGFR1
Autocrine Pathway: A Novel Mechanism of Acquired Resistance
screening (1600 chemicals) and the shifted Tm by compounds were presented by dot-plot. 7 hit to Gefitinib in NSCLC. Mol Cancer Res.

chemicals are highlighted on the plot chart. Show the chemical structure of 7 hits.