Lineage of drug discovery research on fluorinated pyrimidines: chronicle of the achievements accomplished by Professor Setsuro Fujii.

Prof. Setsuro Fujii achieved significant results in the field of drug discovery research in Japan. He developed nine well-known drugs: FT, UFT, S-1 and FTD/TPI are anticancer drugs, while cetraxate hydrochloride, camostat mesilate, nafamostat mesilate, gabexate mesilate and pravastatin sodium are therapeutic drugs for various other diseases. He delivered hope to patients with various diseases across the world to improve their condition. Even now, drug discovery research based on Dr. Fujii's ideas is continuing.

CD44v3,8-10 is essential for Slug-dependent vimentin gene expression to acquire TGF-ß1-induced tumor cell motility.

CD44 is a widely expressed polymorphic adhesion molecule that has pleiotropic functions in development and tumor progression. Its mRNA undergoes alternative splicing to generate multiple variant (CD44v) isoforms, although the function of each CD44v isoform is not fully elucidated. Here, we show that CD44v plays an important role in the induction of vimentin expression upon transforming growth factor-ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT). Among multiple CD44v isoforms expressed in NUGC3 gastric cancer cells, CD44v8-10 and CD44v3,8-10 are involved in the acquisition of migratory and invasive properties associated with TGF-ß1-induced EMT, and only CD44v3,8-10 induces the transcription of vimentin mediated by the EMT transcription factor Slug. In primary tumor specimens obtained from patients with gastric cancer, CD44-containing variant exon 9 (CD44v9) expression and EMT features [E-cadherin(-)vimentin(+)] were significantly correlated, and EMT features in the cells expressing CD44v9 were associated with tumor invasion depth, lymph node metastasis, and pStage, which indicate invasive and metastatic properties, and poor prognosis. These results indicate that certain CD44v isoforms promote tumor cell motility and metastasis in gastric cancer in association with EMT features, and CD44v3,8-10 may contribute to these clinical characteristics.

The balance of forces generated by kinesins controls spindle polarity and chromosomal heterogeneity in tetraploid cells.

Chromosomal instability, one of the most prominent features of tumour cells, causes aneuploidy. Tetraploidy is thought to be an intermediate on the path to aneuploidy, but the mechanistic relationship between the two states is poorly understood. Here, we show that spindle polarity (e.g. bipolarity or multipolarity) in tetraploid cells depends on the level of functional phosphorylated Eg5, a mitotic kinesin, localised to the spindle. Multipolar spindles are formed in cells with high levels of phosphorylated Eg5. This process is suppressed by inhibition of Eg5 or expression of a non-phosphorylatable Eg5 mutant, as well as by changing the balance between opposing forces required for centrosome separation. Tetraploid cells with high levels of functional Eg5 give rise to a heterogeneous aneuploid population through multipolar division, whereas cells with low levels of functional Eg5 continue to undergo bipolar division and remain tetraploid. Furthermore, Eg5 protein levels correlate with ploidy status in tumour specimens. We provide a novel explanation for the tetraploid intermediate model, i.e. spindle polarity and subsequent tetraploid cell behaviour are determined by the balance of forces generated by mitotic kinesins at the spindle.

The evolution of surgical treatment for gastrointestinal cancers.

INTRODUCTION: According to the latest Japanese nationwide estimates, over a million Japanese people are newly diagnosed with cancer each year. Since gastrointestinal cancers account for more than 40% of all cancer-related deaths, it is imperative to formulate effective strategies to control them. MATERIALS AND METHODS, AND RESULTS: Basic drug discovery research Our research has revealed that the abnormal expression of regulators of chromosomal stability is a cause of cancers and identified an effective compound against cancers with chromosomal instability. We revealed the molecular mechanism of peritoneal dissemination of cancer cells via the CXCR4/CXCL12 axis to CAR-like cells and identified an MEK inhibitor effective against these tumors. Residual tumor cells after chemotherapy in colorectal cancer are LGR5-positive cancer stem cells and their ability to eliminate reactive oxygen species is elevated. The development of surgical procedures and devices In cases of gastric tube reconstruction for esophageal cancer, we determined the anastomotic line for evaluating the blood flow using ICG angiography and measuring the tissue O2 metabolism. We established a novel gastric reconstruction method (book-binding technique) for gastric cancer and a new rectal reconstruction method focusing on the intra-intestinal pressure resistance for rectal cancer. We established a novel tissue fusion method, which allows contact-free local heating and retains tissue viability with very little damage, and developed an understanding of the collagen-related processes that underpin laser-induced tissue fusion. Strategy to prevent carcinogenesis We succeeded in cleaving hepatitis B virus DNA integrated into the nucleus of hepatocytes using genome editing tools. The development of HCC from non-alcoholic steatohepatitis (NASH) may be prevented by metabolic surgery. CONCLUSION: We believe that these efforts will help to significantly improve the gastrointestinal cancer treatment and survival.

Clinical significance of programmed cell death-ligand 1 expression and the immune microenvironment at the invasive front of colorectal cancers with high microsatellite instability.

Immunotherapy is reportedly effective in colorectal cancers (CRCs) with high microsatellite instability (MSI-H); however, the specific cell types that respond to immune checkpoint therapy are unclear. Herein, we aimed to examine the expression of programmed cell death-ligand 1 (PD-L1) and related proteins in MSI-H and microsatellite-stable (MSS) CRCs to investigate the immune microenvironment at the tumor's invasive front. The MSI status was retrospectively assessed in 499 patients undergoing surgical resection of primary CRC; of these, 48 were classified as MSI-H. Propensity score matching was performed, and tissues from 36 and 37 patients with MSI-H and MSS CRCs, respectively, were immunohistochemically evaluated for PD-L1, PD-1, CD8 and CD68. PD-L1 expression was evaluated separately for tumor cells (PD-L1 [T]) and tumor-infiltrating myeloid cells in the stroma (PD-L1 [I]). PD-L1 (T) was positive in only 5.4% and 36.1% of MSS and MSI-H CRCs, while PD-L1 (I) was positive in 27% and 72.2% of these CRCs, respectively. The PD-L1 (T) and PD-L1 (I) expression levels in MSI-H CRCs significantly correlated with poor differentiation, lymphatic invasion and vascular invasion (p < 0.05), and with early-stage adenocarcinoma and high budding grade (p < 0.05), respectively. Significantly more PD-L1 (I), CD8-positive cells and CD68-positive macrophages were present at the invasive front than in the central tumor in MSI-H CRCs (p < 0.005). PD-L1 was expressed on both tumor cells and CD68/CD163-positive (M2) macrophages at the invasive front of MSI-H CRCs. In conclusion, PD-L1-positive tumor cells and M2-type tumor-associated macrophages may contribute to tumor invasion and immune escape at the invasive front.

DNA replication stress and cancer chemotherapy.

DNA replication is one of the fundamental biological processes in which dysregulation can cause genome instability. This instability is one of the hallmarks of cancer and confers genetic diversity during tumorigenesis. Numerous experimental and clinical studies have indicated that most tumors have experienced and overcome the stresses caused by the perturbation of DNA replication, which is also referred to as DNA replication stress (DRS). When we consider therapeutic approaches for tumors, it is important to exploit the differences in DRS between tumor and normal cells. In this review, we introduce the current understanding of DRS in tumors and discuss the underlying mechanism of cancer therapy from the aspect of DRS.

Gastric Cancer Patients with High PLK1 Expression and DNA Aneuploidy Correlate with Poor Prognosis.

Gastric cancer is the fourth most common cancer worldwide. Although it is important to identify patients at high risk for a poor outcome, factors correlating with prognosis in gastric cancer are largely unknown. Here, we focus on the correlations among expression of Polo-like kinase 1 (PLK1), DNA ploidy, and clinical outcome in gastric cancer patients. Gastric cancer specimens were analyzed from 207 consecutive patients. Patients were classified into two groups according to tumor PLK1 expression and DNA content, and an analysis of their clinical outcomes was carried out. Prognoses of patients with PLK1-high tumors were worse than those of patients with PLK1-low tumors, but the differences were not statistically significant. In cell lines, overexpression of PLK1 induced centrosome amplification and multipolar spindles, potentially leading to DNA aneuploidy. Indeed, high expression of PLK1 was also associated with DNA aneuploidy in clinical gastric cancer specimens. Patients with both high PLK1 expression and DNA aneuploidy had poor recurrence-free survival, whereas PLK1 expression and DNA ploidy status alone were not significantly associated with outcome. Here, we provide clinical evidence that high expression of PLK1 could have detrimental effects in tumors with DNA aneuploidy, which may increase the risk of recurrence in gastric cancer patients.

[Antitumor Molecular Mechanism of Trifluridine and Tipiracil Hydrochloride (TAS-102: TFTD)].

Treatment options for patients with metastatic colorectal cancer (mCRC), who are refractory to standard chemotherapy, are limited. In a global multicenter randomized double-blind phase III study (RECOURSE study), TAS-102 (TFTD) administration significantly improved overall survival rate with favorable safety profile in mCRC patients refractory to standard chemotherapy (HR=0.68, p<0.001). TFTD was approved initially in Japan in March 2014 and is currently under review by health authorities in the United States and Europe. TFTD is expected to play an important role in salvage-line treatment for patients with mCRC. In this review, we present the history of its clinical development and the experimental data that elucidate the underlying molecular mechanism of action of TFTD and its key component, trifluridine.

[Evolving 5-Fluorouracil Therapy to Achieve Enhanced Efficacy-Past and Current Efforts of Researchers].

5-fluorouracil(5-FU)therapy has advanced greatly over the past 50 years, achieving enhanced therapeutic effects and reduced adverse effects. By taking advantage of the metabolism of 5-FU, researchers have made efforts to develop prodrugs, combination drug products, and combination therapy regimens via biochemical modulation(BCM)with alteration of the drug metabolism. Examples include the advent of the prodrug tegafur(FT), followed by tegafur-uracil(UFT)and tegafurgimeracil- potassium oxonate(S-1)as combined products based on BCM. In the current standard treatment for gastrointestinal cancers, anticancer 5-FU derivatives serve as a platform for combination regimens with other cytotoxic agents or molecular- targeted drugs. To provide further improvements in anticancer therapy outcomes, novel molecular-targeted agents, immune checkpoint inhibitors, and other drugs are being developed, but 5-FU remains an attractive target that shows further potential for increased efficacy. In the future, the evolution of anticancer therapy with 5-FU derivatives is expected to continue via a variety of approaches.

[The Molecular Aspect of the Antitumor Effect of Oxaliplatin in Combination with 5-FU].

Platinum-based chemotherapeutic drugs as a component of combination chemotherapy are widely used in the treatment of cancer. In particular, oxaliplatin(L-OHP), one such platinum-based chemotherapeutic drug, has a synergistic effect in combination with 5-FU and Leucovorin for the treatment of advanced colorectal cancer. However, the underlying molecular mechanism of this synergistic effect has not been fully clarified yet. In this review, we summarize several updates about the in vitro action of oxaliplatin in human tumor cells and discuss the underlying mechanism of its synergistic effect with 5-FU.

Contribution of Aurora-A and -B expression to DNA aneuploidy in gastric cancers.

PURPOSE: DNA aneuploidy, which is characterized by cells containing an abnormal number of chromosomes, is closely associated with carcinogenesis and malignant progression. Aneuploidy occurs during cell division when the chromosomes do not separate properly. Aurora kinases (Aurora-A, -B, and -C) contribute to accurate cell division, and are candidate molecular targets for mitosis-specific anticancer drugs. METHODS: We determined the expression of Aurora-A and -B in 110 gastric cancer specimens by performing an immunohistochemical analysis. We also determined the DNA content, TP53 gene mutations, and microsatellite instability in the same samples. RESULTS: We found the nuclear expression of Aurora-A and -B to increase in tumor tissue in comparison to that in normal epithelial tissue. A high Aurora-B expression significantly correlated with aneuploidy and TP53 mutations, but not with microsatellite instability. In contrast, the Aurora-A expression did not correlate with either aneuploidy or microsatellite instability. In addition, the expression of Aurora-A or -B was not significantly associated with the clinical outcomes or prognosis. CONCLUSIONS: Our results suggest that an overexpression of Aurora-B, but not of Aurora-A, might contribute to DNA aneuploidy in gastric cancers by promoting chromosomal instability.

The anti-tumor effect of trifluridine via induction of aberrant mitosis is unaffected by mutations modulating p53 activity.

The fluorinated thymidine analog trifluridine (FTD) is a chemotherapeutic drug commonly used to treat cancer; however, the mechanism by which FTD induces cytotoxicity is not fully understood. In addition, the effect of gain-of-function (GOF) missense mutations of the TP53 gene (encoding p53), which promote cancer progression and chemotherapeutic drug resistance, on the chemotherapeutic efficacy of FTD is unclear. Here, we revealed the mechanisms by which FTD-induced aberrant mitosis and contributed to cytotoxicity in both p53-null and p53-GOF missense mutant cells. In p53-null mutant cells, FTD-induced DNA double-stranded breaks, single-stranded DNA accumulation, and the associated DNA damage responses during the G2 phase. Nevertheless, FTD-induced DNA damage and the related responses were not sufficient to trigger strict G2/M checkpoint arrest. Thus, these features were carried over into mitosis, resulting in chromosome breaks and bridges, and subsequent cytokinesis failure. Improper mitotic exit eventually led to cell apoptosis, caused by the accumulation of extensive DNA damage and the presence of micronuclei encapsulated in the disrupted nuclear envelope. Upon FTD treatment, the behavior of the p53-GOF-missense mutant, isogenic cell lines, generated by CRISPR/Cas9 genome editing, was similar to that of p53-null mutant cells. Thus, our data suggest that FTD treatment overrode the effect on gene expression induced by p53-GOF mutants and exerted its anti-tumor activity in a manner that was independent of the p53 function.

CtIP- and ATR-dependent FANCJ phosphorylation in response to DNA strand breaks mediated by DNA replication.

FANCJ, also called BACH1/BRIP1, is a 5'-3' DEAH helicase, whose mutations are known as a risk factor for Fanconi anemia and also breast and ovarian cancer. FANCJ is thought to contribute to DNA double-strand break (DSB) repair and S-phase checkpoint through binding to multiple partner proteins, such as BRCA1 and TopBP1, but its molecular regulation remains unclear. We focused on DNA damage-induced phosphorylation of FANCJ and found that reagents that cause DSB or replication fork stalling induce FANCJ hyperphosphorylation. In particular, camptothecin (CPT) induced rapid and efficient FANCJ hyperphosphorylation that was largely dependent on TopBP1 and ATM-Rad3 related (ATR) kinase. Furthermore, DNA end resection that exposes single-strand DNA at the DSB site was required for hyperphosphorylation. Interestingly, upon CPT treatment, a dramatic increase in the FANCJ-TopBP1 complex was observed, and this increase was not alleviated even when ATR-dependent hyperphosphorylation was suppressed. These results suggest that FANCJ function may be modulated by hyperphosphorylation in a DNA end resection- and ATR-dependent manner and by FANCJ-TopBP1 complex formation in response to replication-coupled DSBs.

A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules.

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a +TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure.

FANCJ expression predicts the response to 5-fluorouracil-based chemotherapy in MLH1-proficient colorectal cancer.

PURPOSE: Fanconi anemia protein, FANCJ, directly interacts with MLH1, a key protein involved in DNA mismatch repair. Deficient mismatch repair, or microsatellite instability, is a potent marker for the ineffectiveness of 5-fluorouracil (5-FU) in colorectal cancer (CRC). We investigated the significance of FANCJ expression in CRC, focusing on the effects of 5-FU-based adjuvant chemotherapy. METHODS: Clinicopathologic features and immunohistochemical expression of FANCJ and MLH1 were studied in 219 patients with CRC. We also analyzed 5-FU sensitivity in CRC cell lines with varying levels of FANCJ expression. RESULTS: FANCJ expression was elevated in tumor tissues compared with normal epithelial tissue. High expression of FANCJ was significantly associated with 5-FU resistance measured by the SDI test (P < 0.05) and poor recurrence-free survival (RFS) (P < 0.05). Among patients with stage II/III tumors who received 5-FU, patients with tumors exhibiting high FANCJ expression had significantly worse RFS than did patients with tumors exhibiting low FANCJ expression (P < 0.01). Among patients who did not receive adjuvant chemotherapy, FANCJ expression was not correlated with RFS (P = 0.76). High FANCJ expression was correlated with 5-FU resistance in tumors with normal MLH1 expression (P < 0.05) but not in tumors not expressing MLH1 (P = 0.9). In vitro, FANCJ overexpression was correlated with 5-FU resistance in MLH1-proficient HCT116 3-6 cells but not in MLH1-deficient HCT116 cells. CONCLUSIONS: FANCJ could be a useful biomarker to predict the response to 5-FU and prognosis of CRC, particularly in tumors with normal MLH1 expression.

High expression of BUBR1 is one of the factors for inducing DNA aneuploidy and progression in gastric cancer.

Gastric cancers show high frequency of DNA aneuploidy, a phenotype of chromosomal instability. It is suggested that the abnormal spindle assembly checkpoint is involved in DNA aneuploidy, but the underlying mechanism is still unclear. We studied the mechanism by assessing the expression of BUBR1 in gastric cancer. The DNA ploidy patterns of 116 gastric cancer samples obtained from the Department of Surgery and Science at Kyushu University Hospital were analyzed. Of those, DNA aneuploidy was seen in 70 (60.3%) cases of gastric cancer. The expression of BUBR1 was studied by immunohistochemistry in 181 gastric cancer samples and by real-time RT-PCR in several gastric cancer cell lines. Ninety-one (50.3%) cases had high expression of BUBR1 and those cases correlated significantly with DNA aneuploidy (P < 0.05). Also high expression of BUBR1 cases had significant correlation with deep invasion, lymph node metastasis, liver metastasis, and poor prognosis. In gastric cancer cell lines, high expression of BUBR1 had a significant relationship with DNA aneuploidy (P < 0.05). Then, gastric cancer cell lines MKN-28 and SNU-1 were transfected with full-length BUBR1 to observe the significance of the change in BUBR1 expression. Enforced expression of BUBR1 resulted in changes to the ploidy pattern and high Ki-67 expression. Collectively, our clinical and in vitro data indicate that high expression of BUBR1 may be one of causative factors for the induction of DNA aneuploidy and progression of gastric cancer.

DNA Replication Stress Induced by Trifluridine Determines Tumor Cell Fate According to p53 Status.

DNA replication stress (DRS) is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analogue-type chemotherapeutic drugs introduce DNA damage and exacerbate DRS in tumor cells. However, the mechanisms underlying the antitumor effect of these drugs are not fully understood. Here, we show that the fluorinated thymidine analogue trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases by acting both as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand, which caused DRS. In cells, FTD decreased the thymidine triphosphate level in the dNTP pool and increased the FTD triphosphate level, resulting in the activation of DRS-induced cellular responses during S-phase. In addition, replication protein A-coated single-stranded DNA associated with FancD2 and accumulated after tumor cells completed S-phase. Finally, FTD activated the p53-p21 pathway and suppressed tumor cell growth by inducing cellular senescence via mitosis skipping. In contrast, tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results demonstrate that DRS induced by a nucleoside analogue-type chemotherapeutic drug suppresses tumor growth irrespective of p53 status by directing tumor cell fate toward cellular senescence or apoptotic cell death according to p53 status. IMPLICATIONS: Chemotherapeutic drugs that increase DRS during S-phase but allow tumor cells to complete S-phase may have significant antitumor activity even when functional p53 is lost.

Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level.

Trifluridine (FTD), a tri-fluorinated thymidine analogue, is a key component of the oral antitumor drug FTD/TPI (also known as TAS-102), which is used to treat refractory metastatic colorectal cancer. Thymidine kinase 1 (TK1) is thought to be important for the incorporation of FTD into DNA, resulting in DNA dysfunction and cytotoxicity. However, it remains unknown whether TK1 is essential for FTD incorporation into DNA and whether this event is affected by the expression level of TK1 because TK1-specific-deficient human cancer cell lines have not been established. Here, we generated TK1-knock-out human colorectal cancer cells using the CRISPR/Cas9 genome editing system and validated the specificity of TK1 knock-out by measuring expression of AFMID, which is encoded on the same locus as TK1. Using TK1-knock-out cells, we confirmed that TK1 is essential for cellular sensitivity to FTD. Furthermore, we demonstrated a correlation between the TK1 expression level and cytotoxicity of FTD using cells with inducible TK1 expression, which were generated from TK1-knock-out cells. Based on our finding that the TK1 expression level correlates with sensitivity to FTD, we suggest that FTD/TPI might efficiently treat cancers with high TK1 expression.

MDC1 methylation mediated by lysine methyltransferases EHMT1 and EHMT2 regulates active ATM accumulation flanking DNA damage sites.

Chromatin dynamics mediated by post-translational modifications play a crucial role in cellular response to genotoxic stress for the maintenance of genome integrity. MDC1 is a pivotal chromatin adaptor in DNA damage response (DDR) and its methylation is essential to recruit repair factors at DNA double-strand break (DSB) sites, yet their precise molecular mechanisms remain elusive. Here we identified euchromatic histone-lysine N-methyltransferase 1 (EHMT1) and EHMT2 as novel regulators of MDC1, which is required for the accumulation of DDR factors e.g. 53BP1 and RAP80, at the DSB sites. MDC1 interacts mainly with EHMT1, which is facilitated by DNA damage-initiated ATM signalling, and EHMT2 dominantly modulates methylation of MDC1 lysine 45. This regulatory modification promotes the interaction between MDC1 and ATM to expand activated ATM on damaged chromatin and dysfunctional telomere. These findings identify EHMT1 and EHMT2 as DDR components, with implications for genome-integrity maintenance through proper dynamic methylation of MDC1.

Changes in HER2 Expression and Amplification Status Following Preoperative Chemotherapy for Gastric Cancer.

BACKGROUND: It is essential to establish a strategy for second-line treatment for human epidermal growth factor receptor 2 (HER2)-positive gastric cancer; however, HER2 expression status after chemotherapy treatment is not routinely determined. MATERIALS AND METHODS: We analyzed 25 cases of gastric cancer that received preoperative chemotherapy and selected the six pre-treatment samples that were HER2-positive. Pre- and post-treatment tumor samples were examined for HER2 expression, and for HER2, epidermal growth factor receptor (EGFR), and hepatocyte growth factor receptor (MET) gene amplification. RESULTS: Three patients had been treated with trastuzumab plus chemotherapy, and three patients with cytotoxic chemotherapy alone. Only one case that had an initial HER2 score of 3+ and had received trastuzumab plus chemotherapy remained HER2-positive after treatment. Decrease or loss of HER2 expression and amplification was observed in the other five patients. Amplification of EGFR or MET was not observed in any pre- or post-treatment specimens. CONCLUSION: Our data suggest that trastuzumab plus chemotherapy or chemotherapy alone may induce loss of HER2 positivity.