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Page 6 of 17 Matsushita et al. Hepatoma Res 2018;4:61 I http://dx.doi.org/10.20517/2394-5079.2018.81

RESULTS
Mechanism in carcinogenesis of FIR/FIRΔexon2/PUF60 as a target for cancer diagnosis and
therapy
Previous studies revealed that FUBP1, FIR (PUF60)/FIRΔexon2, SAP155, and SAP130 were over expressed in
[18]
[38]
hepatocellular carcinoma (HCC) tissue . Additionally, FIR/FIRΔexon2 mRNA levels were increased in HCC .
Recent studies have been revealed regarding direct protein interactions between UHM family and ULM family
[Supplementary Figure 1] [39-41] . SAP155/SAP145/SAP130 subunits consist of SF3B complex and UHM of FIR/
PUF60 directly binds to ULM of SAP155 (SF3B1) [Supplementary Figure 1A-C] [32,41,42] . Further, FUBP1, FIR
(PUF60)/FIRΔexon2, SAP155, and SAP130 were over expressed in hepatitis C virus (HCV)-related HCC tissue
[18]
and FIR (PUF60)/FIRΔexon2 reflects DNA damage [Supplementary Figure 1D] . Bleomycin-induced DNA
damage decreased SAP155 and significantly increased FIR/FIRΔexon2 mRNA expression as well as the
[18]
FIRΔexon2: FIR ratio in hepatoblastoma (HLE and HLF) cells . Therefore, FUBP1/FIR (PUF60)/FIRΔexon2
proteins, mRNAs and/or autoantibodies against these peptides are highly possible biomarker candidates
for hepatoma diagnosis. Anti-FIR/FIRΔexon2 autoantibodies were detected in several gastrointestinal
cancers [27,28] . Anti-FIR/FIRΔexon2 autoantibodies in the sera of HCC patients are now under investigation.
Given FIRΔexon2 is a dominant negative regulator of FIR/PUF60, FIRΔexon2 inhibition is an advantageous
target for cell growth suppression [Figure 2A]. Inhibition of SF3B (SAP155) by siRNA or SSA resulted in c-Myc
[1]
overexpression possibly due to the FIR downregulation [Figure 2B] . Knockdown of SAP155 or FIR was used
to investigate their reciprocal influence on each other and on c-Myc transcription, pre-mRNA splicing, and
[31]
[31]
protein expression . FIR and FIRΔexon2 were co-immunoprecipitated with SAP155 . UHM of FIR/PUF60
at carboxyl-terminus directly binds to W (Typ) D (Asp)-domain of SF3B1 (SAP155) as ULM [42,43] . The tight
FIR/FIRΔexon2-SAP155 interaction disables established FIR and SAP155 functions disturbing the synthesis
of normally spliced FIR mRNA. FIRΔexon2 potently forms a heterodimer with FIR and thus FIRΔexon2
interferes with FIR to bind to FUSE [Supplementary Figure 1D]. These results strongly suggest that FIRΔexon2
antagonized FIR in c-Myc transcriptional suppression and simultaneously interferes with SF3B in splicing
during tumor progression. Therefore, both common and discriminating recognition elements in the UHM-
ULM binding interface provide a rationale for a structural basis for specific UHM-ULM interactions and a
[40]
platform of intermolecular interactions governing disease-related AS in eukaryotic cells . For instance, SF3B1
(SAP155)/FIR/PUF60 complex is a target of cancer therapy. In these scenarios, low molecular weight artificial
chemical, BK697, was synthesized by in silico screening that targets FIRΔexon2 in this study [Figure 2B]. Small
molecular chemical compounds against FIRΔexon2 were screened among 23,275 chemicals of NPDepo by
Dr Hiroyuki Osada and his colleagues (RIKEN, Wako, Saitama, Japan) to develop cancer therapy [Figure 1].
Nine small molecular chemicals were identified by NPDepo screening against FIR and four chemicals against
FIRΔexon2 as candidates of interacting chemicals [Figure 1].

The interaction of FIRΔexon2 and WD-like domain of FBW7 and in silico screening of small
molecular chemical compounds against FIR/FIRΔexon2 for cancer therapy
[44]
FBW7 frequently is mutated in hematopoietic tumors . FBW7 is a member of the Skp1-Cull-F-box
type ubiquitin ligase complex and is involved in degradation of various growth-related proteins, Notch1,
[44]
c-Myc, c-Jun, and cyclin E via the proteasome system , indicating FBW7 is a tumor suppressor in cancer
[45]
development and progression . Remarkably, three-dimensional structure analysis revealed the hypothetical
inhibitory mechanism of FBW7 function by FIR/FIRΔexon2 [Figure 3]. The binding structure between
SAP155 (SF3B1) and one of the splicing factors containing UHM, SPF45, was already clarified by X-ray crystal
analysis (PDB code: #2PEH) [Figure 3A and B]. In the 2PEH structure, the crystal unit cell contains two
SPF45 recombinant proteins (a.a. 301-401) and two SAP155 partial peptides (a.a. 333-342). SPF45 has an amino
sequence of LNGRYFGGRVVKA [Figure 3A] and similar sequences are commonly seen at the C-terminal
domains of FIR and U2AF65 [Figure 3B]. According to the crystal structure, 2PEH, SPF45 makes a strong
interaction with a WD part of SAP155 at the domain of the above-mentioned conserved sequence.