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The interaction between mRNA and ribosomal RNA (rRNA) transcription in cancer remains unclear. RNAP I and II possess a common N-terminal tail (NTT), RNA polymerase subunit RPB6, which interacts with P62 of transcription factor (TF) IIH, and is a common target for the link between mRNA and rRNA transcription. The mRNAs and rRNAs affected by FUBP1-interacting repressor (FIR) were assessed via RNA sequencing and qRT-PCR analysis. An FIR, a c-myc transcriptional repressor, and its splicing form FIRΔexon2 were examined to interact with P62. Protein interaction was investigated via isothermal titration calorimetry measurements. FIR was found to contain a highly conserved region homologous to RPB6 that interacts with P62. FIRΔexon2 competed with FIR for P62 binding and coactivated transcription of mRNAs and rRNAs. Low-molecular-weight chemical compounds that bind to FIR and FIRΔexon2 were screened for cancer treatment. A low-molecular-weight chemical, BK697, which interacts with FIRΔexon2, inhibited tumor cell growth with rRNA suppression. In this study, a novel coactivation pathway for cancer-related mRNA and rRNA transcription through TFIIH/P62 by FIRΔexon2 was proposed. Direct evidence in X-ray crystallography is required in further studies to show the conformational difference between FIR and FIRΔexon2 that affects the P62-RBP6 interaction.

Anti-PUF60 autoantibodies are reportedly detected in the sera of patients with dermatomyositis and Sjögren's syndrome; however, little is known regarding its existence in the sera of cancer patients. FIR, a splicing variant of the PUF60 gene, is a transcriptional repressor of c-myc. In colorectal cancer, there is an overexpression of the dominant negative form of FIR, in which exon 2 is lacking (FIRΔexon2). Previously, large-scale SEREX (serological identification of antigens by recombinant cDNA expression cloning) screenings have identified anti-FIR autoantibodies in the sera of cancer patients. In the present study, we revealed the presence and significance of anti-FIR (FIR/FIRΔexon2) Abs in the sera of patients with esophageal squamous cell carcinoma (ESCC). Our results were validated by an amplified luminescence proximity homogeneous assay using sera of patients with various cancer types. We revealed that anti-FIRΔexon2 Ab had higher sensitivity than anti-FIR Ab. Receiver operating characteristic (ROC) analysis was applied for evaluating the use of anti-FIRΔexon2 Ab as candidate markers such as anti-p53 Ab and carcinoembryonic antigen, and the highest area under the ROC curve was observed in the combination of anti-FIRΔexon2 Ab and anti-p53 Ab. In summary, our results suggest the use of anti-FIRΔexon2 Ab in combination with the anti-p53 Ab as a predictive marker for ESCC. The area under the ROC curve was further increased in the advanced stage of ESCC. The value of anti-FIRΔexon2 autoantibody as novel clinical indicator against ESCC and as a companion diagnostic tool is discussed., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Anti-PUF60, poly(U)-binding-splicing factor, autoantibodies are reported to be detected in the sera of dermatomyositis and Sjogren's syndrome that occasionally associated with malignancies. PUF60 is identical with far-upstream element-binding protein-interacting repressor (FIR) that is a transcriptional repressor of c-myc gene. In colorectal cancers, a splicing variant of FIR that lacks exon2 (FIRΔexon2) is overexpressed as a dominant negative form of FIR. In this study, to reveal the presence and the significance of anti-FIRs (FIR/FIRΔexon2) antibodies in cancers were explored in the sera of colorectal and other cancer patients. Anti-FIRs antibodies were surely detected in the preoperative sera of 28 colorectal cancer patients (32.2% of positive rates), and the detection rate was significantly higher than that in healthy control sera (Mann-Whitney U test, p < 0.01). The level of anti-FIRs antibodies significantly decreased after the operation (p < 0.01). Anti-FIRs antibodies were detected in the sera of early-stage and/or recurrent colon cancer patients in which anti-p53 antibodies, CEA, and CA19-9 were not detected as well as in the sera of other cancer patients. Furthermore, the area under the curve of receiver operating characteristic for anti-FIRs antibodies was significantly larger (0.85) than that for anti-p53 antibodies or CA19-9. In conclusions, the combination of anti-FIRs antibodies with other clinically available tumor markers further improved the specificity and accuracy of cancer diagnosis.

Combination therapy of carbon-ion beam with the far upstream element-binding protein (FBP)-interacting repressor, FIR, which interferes with DNA damage repair proteins, was proposed as an approach for esophageal cancer treatment with low side effects regardless of TP53 status. In vivo therapeutic antitumor efficacy of replication-defective adenovirus (E1 and E3 deleted adenovirus serotype 5) encoding human FIR cDNA (Ad-FIR) was demonstrated in the tumor xenograft model of human esophageal squamous cancer cells, TE-2. Bleomycin (BLM) is an anticancer agent that introduces DNA breaks. The authors reported that Ad-FIR involved in the BLM-induced DNA damage repair response and thus applicable for other DNA damaging agents. To examine the effect of Ad-FIR on DNA damage repair, BLM, X-ray and carbon-ion irradiation were used as DNA damaging agents. The biological effects of high linear energy transfer (LET) radiotherapy used with carbon-ion irradiation are more expansive than low-LET conventional radiotherapy, such as X-rays or γ rays. High LET radiotherapy is suitable for the local control of tumors because of its high relative biological effectiveness. Ad-FIR enhanced BLM-induced DNA damage indicated by γH2AX in vitro. BLM treatment increased endogenous nuclear FIR expression in TE-2 cells, and P27Kip1 expression was suppressed by TP53 siRNA and BLM treatment. Further, Ad-FIRΔexon2, a dominant-negative form of FIR that lacks exon2 transcriptional repression domain, decreased Ku86 expression. The combination of Ad-FIR and BLM in TP53 siRNA increased DNA damage. Additionally, Ad-FIR showed synergistic cell toxicity with X-ray in vitro and significantly increased the antitumor efficacy of carbon-ion irradiation in the xenograft mouse model of TE-2 cells (P=0.03, Mann-Whitney's U-test) and was synergistic with the sensitization enhancement ratio (SER) value of 1.15. Therefore, Ad-FIR increased the cell-killing activity of the carbon-ion beam that avoids late-phase severe adverse effects independently of the TP53 status in vitro. Our findings indicated the feasibility of the combination of Ad-FIR with DNA damaging agents for future esophageal cancer treatment.

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR⁺/⁻) C57BL6 mice were generated. FIR complete knockout (FIR⁻/⁻) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR⁺/⁻ mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR⁺/⁻TP53⁻/⁻ generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR⁺/⁻TP53⁻/⁻ compared with that in FIR⁺/⁺TP53⁻/⁻ mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.

The far-upstream element-binding protein-interacting repressor (FIR) is a c-myc transcriptional suppressor. FIR is alternatively spliced to lack the transcriptional repression domain within exon 2 (FIRΔexon2) in colorectal cancers. FIR and FIRΔexon2 form homo- or heterodimers that complex with SAP155. SAP155, a subunit of the essential splicing factor 3b subcomplex in the spliceosome, is required for proper P27Kip1 pre-mRNA splicing, and P27Kip1 arrests cells at G1. In contrast, FIR was co-immunoprecipitated with Ku86 and DNA-PKcs. siRNA against Ku86/Ku70 decreased FIR and P27Kip1 expression, whereas siRNA against FIR decreased Ku86/XRCC5 and P27Kip1 expression. Thus the mechanical interaction of FIR/FIRΔexon2/SAP155 bridges c-myc and P27Kip1 expression, potentially integrates cell-cycle progression and c-myc transcription in cell. Bleomycin(BLM) is an anticancer agent that introduces DNA breaks. Because DNA breaks generate the recruitment of Ku86/Ku70 to bind to the broken DNA ends, the possible involvement of FIR and Ku86/Ku70 interaction in the BLM-induced DNA damage repair response was investigated in this study. First, BLM treatment reduced SAP155 expression and increased FIR and FIRΔexon2 mRNA expression as well as the ratio of FIRΔexon2:FIR in hepatoblastoma cells (HLE and HLF). Second, FIR or FIRΔexon2 adenovirus vectors (Ad-FIR or Ad-FIRΔexon2) increased Ku86/Ku70 and P27Kip1 expression in vitro. Third, BLM decreased P27Kip1 protein expression, whereas increased P27Kip1 and γH2AX expression with Ad-FIRΔexon2. Together, the interaction of FIR/SAP155 modulates FIR splicing and involves in cell-cycle control or cell fate via P27Kip1 and c-myc in BLM-induced DNA damage pathway. This novel function of FIR splicing will contribute to clinical studies of cancer management through elucidating the mechanical interaction of FIR/FIRΔexon2/SAP155 as a potential target for cancer treatment.