Kazunori Otsuka

Understanding the function–structure and function–mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis

Inactivation of the tumor suppressor p53 by missense mutations is the most frequent genetic alteration in human cancers. The common missense mutations in the TP53 gene disrupt the ability of p53 to bind to DNA and consequently to transactivate downstream genes. However, it is still not fully understood how a large number of the remaining mutations affect p53 structure and function. Here, we used a comprehensive site-directed mutagenesis technique and a yeast-based functional assay to construct, express, and evaluate 2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the protein (5.9 substitutions per residue), and correlated p53 function with structure- and tumor-derived mutations. This high-resolution mutation analysis allows evaluation of previous predictions and hypotheses through interrelation of function, structure and mutation.

Lack of Correlation between p53-Dependent Transcriptional Activity and the Ability to Induce Apoptosis among 179 Mutant p53s

Tumor suppressor p53-dependent apoptosis is thought to be one of the most important tumor-suppressive functions in human tumorigenesis. However, whether the major mechanism underlying the p53-dependent apoptosis is transactivation dependent or independent remains unclear. Using 179 mutant p53s with diverse transcriptional activities for distinct p53-binding sequences in yeast, we evaluated both their sequence-specific transcriptional activities on six p53 target genes and their ability to induce apoptosis in Saos-2 cells. These mutant p53s also represented diversity in their ability to both transactivate target genes and induce apoptosis. We identified 17 mutant p53s with superior ability to induce apoptosis than wild-type p53 that tend to cluster at residues 121 or 290 to 292. There was no significant correlation between the two functional properties on any single target gene examined. Furthermore, the 17 mutant p53s were not classified in a specific cluster by hierarchical cluster analysis on their diverse transcriptional activities, indicating that these mutant p53s were not similar in the transcriptional activity of downstream genes. These results suggested that transactivation-dependent apoptosis does not always play a major role in p53-dependent apoptosis, indirectly supporting the importance role of the transactivation-independent mechanism.

Isolation of temperature-sensitive p53 mutations from a comprehensive missense mutation library.

Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. These mutants clustered in β-strands in the DNA-binding domain, particularly in one of the two β-sheets of the protein, and 15 residues (Thr155, Arg158, Met160, Ala161, Val172, His214, Ser215, Pro223, Thr231, Thr253, Ile254, Thr256, Ser269, Glu271, and Glu285) were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular β-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.

The relationship among p53 oligomer formation, structure and transcriptional activity using a comprehensive missense mutation library

Tumor suppressor p53 forms a homo-tetramer through its COOH-terminal oligomerization domain and acts as a sequence-specific transcription factor. We have analysed the interrelation among the transcriptional activities, the structure and the cancer-related mutations in the oligomerization domain by using a comprehensive missense mutation library. Here, we examined the ability of 184 mutant p53s in the domain to form an oligomer by expressing these mutant p53s in yeast, and compared the data with the previous information. We showed that specific residues in the α-helix and the β-strand of the oligomerization domain were critical for both oligomer formation and sequence-specific transactivation, and the activities were closely related. In particular, the α-helix was more sensitive to amino-acid substitutions than the β-strand. We found identity in the interrelation between the two activities, that is, monomer mutants were transcriptionally inactive whereas dimer and tetramer mutants retained their transcriptional activities. In TP53 mutation databases, a small number of mutations have been reported in this domain. Surprisingly, most do not encode p53s defective in functional properties. These results indicate that, although oligomer formation is essential for p53 transactivation function, the inactivation of oligomer formation and therefore the inactivation of transactivation may not be essential for tumor suppression by p53 because they do not lead to oncogenic proteins.

The screening of the second-site suppressor mutations of the common p53 mutants.

Second‐site suppressor (SSS) mutations in p53 found by random mutagenesis have shown to restore the inactivated function of some tumor‐derived p53. To screen novel SSS mutations against common mutant p53s, intragenic second‐site (SS) mutations were introduced into mutant p53 cDNA in a comprehensive manner by using a p53 missense mutation library. The resulting mutant p53s with background and SS mutations were assayed for their ability to restore the p53 transactivation function in both yeast and human cell systems. We identified 12 novel SSS mutations including H178Y against a common mutation G245S. Surprisingly, the G245S phenotype is rescued when coexpressed with p53 bearing the H178Y mutation. This result indicated that there is a possibility that intragenic suppressor mutations might restore the protein function in an intermolecular manner. The intermolecular mechanism may lead to novel strategies for restoring inactivated p53 function and tumor suppression in cancer treatment.

Phase I/II study of sequential therapy with irinotecan and S-1 for metastatic colorectal cancer

Background:Both irinotecan (CPT-11) and S-1 are active against colorectal cancer; however, as S-1 is a prodrug of 5-fluorouracil (5-FU), 5-FU and its metabolites might inhibit the antitumour effect of CPT-11. Therefore, we designed a sequential combination, in which CPT-11 infusion was given on day 1 and S-1 was given orally at 80 mg m−2 per day on days 3–16 every 3 weeks.Methods:Twelve patients entered the phase I study, and the recommended doses were determined as a CPT-11 dose of 150 mg m−2 and an S-1 dose of 80 mg m−2.Results:In all, 36 patients entered the phase II study, of whom 4 and 16 had complete and partial responses. The overall response rate was 55.6% (95% confidence interval, 38.1–72.1%), and median progression-free survival was 7.7 months (95% confidence interval, 4.8–12.6 months). Grade 3 neutropenia was the most common haematological toxicity and occurred in 6.5% of 215 treatment courses. Grade 3 non-haematological toxicities included anorexia (1.4%) and diarrhoea (0.9%). There was no grade 4 toxicity of any kind.Conclusion:Our results suggest that this regimen is convenient, safe and promising, compared with conventional regimens for patients with metastatic colorectal cancer.

A Curcumin Analog, GO-Y078, Effectively Inhibits Angiogenesis through Actin Disorganization

BACKGROUND The inhibition of angiogenesis is a theoretically ideal chemotherapy for cancer, but there remains room for improvement. Most inhibitors of angiogenesis approved to date target vascular endothelial growth factors (VEGFs); however, VEGFs are only one of the many classes of participant in tumor angiogenesis. Because tumor angiogenesis is orchestrated by many components, including growth factors, signal transducers, and effectors, its regulation exhibits redundancy. Curcumin can associate with many proteins, and it reportedly inhibits tumor angiogenesis. OBJECTIVE We investigated the ability of a new curcumin analog, GO-Y078, to inhibit tumor angiogenesis. RESULTS GO-Y078 inhibited human umbilical vascular endothelial cell sprouting. GO-Y078 also induced complete anoikis in vascular endothelial cells. Moreover, GO-Y078 suppressed the migration and invasion of vascular endothelial cells into extracellular matrix proteins. However, expression analysis revealed that GO-Y078 did not suppress molecules involved in VEGF signaling. Rather, GOY078 induced actin disorganization, dissociation of vinculin from actin, and destruction of focal adhesion, resulting in the inhibition of vascular endothelial cell mobility. GO-Y078 also suppressed in-vivo vasculogenesis in Xenopus laevis tadpoles. CONCLUSION Actin organization is a common effecter related to vascular endothelial cell mobility in angiogenesis. We demonstrated that GO-Y078 inhibits angiogenesis through actin disorganization.

Circulating tumor cell count as a biomarker of a specific gastric cancer subgroup characterized by bone metastasis and/or disseminated intravascular coagulation ‑ an early indicator of chemotherapeutic response

The incidence of gastric cancer coupled with multiple bone metastases, and/or disseminated intravascular coagulation (DIC), is characterized by the clinical presentation of rapid progression and a poor prognosis, and differs from typical gastric cancers. Circulating tumor cells (CTCs) are negligible in typical advanced gastric cancers, however, a considerable number of CTCs in the bloodstream may be detected in the subgroup demonstrating multiple bone metastases and/or DIC. The present study analyzed two cases, with the first case regarding a 51-year-old male who exhibited a CTC count of 275 cells/7.5 ml following an initial, ineffective, chemotherapy cycle. The patient underwent a second chemotherapy course that was effective, and the cell count was observed to reduced to 2 cells/7.5 ml. A decreased CTC count was first confirmed on day 16 following treatment. During the chemoresistant phase, the CTC count was observed to increase again. The second case presented by the current study describes a 59-year-old female who exhibited a CTC count of 235 cells/7.5 ml prior to chemotherapy. This subsequently decreased to 7 cells/7.5 ml following an effective course of chemotherapy. Notably, the CTC count increased alongside disease progression in this case. Within the rare subgroup of gastric cancer patients with multiple bone metastases and/or DIC, CTC count may serve as an early biomarker allowing the evaluation of therapeutic efficacy. However, due to the aggressive nature of this type of cancer, imaging analysis is not recommended as it may typically take several months to complete.

Analysis of the human APC mutation spectrum in a saccharomyces cerevisiae strain with a mismatch repair defect

Somatic APC mutations in colorectal tumors with an RER phenotype reflect excessive frameshift mutations, especially in simple repetition tracts within the coding sequence. Because this type of mutation is characteristic of cells with a deficient DNA MMR system, the APC mutation signature of RER tumors may be attributable to a defect in the MMR system. However, there is little experimental evidence to prove that the spectrum of mutations and the APC gene distribution are directly influenced by MMR system defects. We therefore examined the mutation spectrum of the MCR of the APC gene after transfection into both MMR‐proficient and MMR‐deficient yeast strains and compared it with a previously reported human APC mutation database. Small insertions or deletions in mono‐ or dinucleotide repeats were more common in the MMR‐deficient than in the MMR‐proficient strain (91.2% vs. 38.1%, Fishers exact test p < 0.0001). Furthermore, the 2 mutation hot spots, 4385–4394(AG)5 and 4661–4666(A)6, found in the yeast system corresponded with those in human tumors. Combining our data with those from human tumors, there appears to be hypermutable mutations in specific simple repetitive sequences within the MCR, which are more prevalent in MMR‐deficient cells and RER tumors than in MMR‐proficient cells and non‐RER tumors. We therefore consider that the differences in the spectra of RER and non‐RER tumors are attributable at least in part to the MMR system of the host cells.

18FDG-PET at 1-Month Intervals Is a Better Predictive Marker for GISTs That Are Difficult to Be Diagnosed Histopathologically: A Case Report

Imatinib mesylate is a tyrosine kinase inhibitor of c-KIT and PDGFRA. Imatinib mesylate is an effective drug that can be used as a first-choice agent for treatment of GISTs. Prior to treatment, molecular diagnosis of c-KIT or PDGFRA is necessary; however, in some types of GISTs, it is impossible to obtain a sufficient amount of specimen for diagnosis. An inoperable or marginally resectable GIST in a 79-year-old female was difficult to be diagnosed at a molecular pathological level, and hence, exploratory treatment was initiated using imatinib combined with 18FDG-PET evaluation at 1-month intervals. PET imaging indicated a positive response, and so we continued imatinib treatment in an NAC setting for 4 months. As a result, curative resection of the entire tumor was successfully performed with organ preservation and minimally invasive surgery. 18FDG-PET evaluation at 1-month intervals is beneficial for GISTs that are difficult to be diagnosed histopathologically.