Hideki Shimodaira

Randomized, Open-Label, Phase III Study Comparing Irinotecan With Paclitaxel in Patients With Advanced Gastric Cancer Without Severe Peritoneal Metastasis After Failure of Prior Combination Chemotherapy Using Fluoropyrimidine Plus Platinum: WJOG 4007 Trial

PURPOSE This phase III study compared treatment with weekly paclitaxel and biweekly irinotecan in patients with advanced gastric cancer refractory to treatment with fluoropyrimidine plus platinum. PATIENTS AND METHODS Patients were randomly assigned to receive either paclitaxel (80 mg/m(2) on days 1, 8, and 15, every 4 weeks) or irinotecan (150 mg/m(2) on days 1 and 15, every 4 weeks). Primary end point was overall survival (OS), and secondary end points were progression-free survival (PFS), response rate, adverse events, and proportion of patients who received third-line chemotherapy. RESULTS Of 223 patients, 219 were eligible for analysis. Median OS was 9.5 months in 108 patients allocated to the paclitaxel group and 8.4 months in 111 patients allocated to the irinotecan group (hazard ratio [HR], 1.13; 95% CI, 0.86 to 1.49; P = .38). Median PFS was 3.6 months in the paclitaxel group and 2.3 months in the irinotecan group (HR, 1.14; 95% CI, 0.88 to 1.49; P = .33). Response rate was 20.9% in the paclitaxel group and 13.6% in the irinotecan group (P = .24). Common grade 3 to 4 adverse events were neutropenia (paclitaxel group, 28.7%; irinotecan group, 39.1%), anemia (21.3%; 30.0%), and anorexia (7.4%; 17.3%). Treatment-related deaths occurred in two patients (1.8%) in the irinotecan group. Third-line chemotherapy was administered in 97 patients (89.8%) after paclitaxel treatment and in 80 patients (72.1%) after irinotecan treatment (P = .001). CONCLUSION No statistically significant difference was observed between paclitaxel and irinotecan for OS. Both are reasonable second-line treatment options for advanced gastric cancer.

Screening the p53 status of human cell lines using a yeast functional assay

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild‐type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53‐responsive GAL1 promoter in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature‐sensitive growth. p53 cDNA from eight cell lines showed p53‐dependent temperature‐sensitive growth, growing at 30°C but not at 37°C. Four temperature‐sensitive p53 mutations were isolated: CAT→CGT at codon 214 (H214R), TAC→TGC at codon 234 (Y234C), GTG→ATG at codon 272 (V272M), and GAG→AAG (E285K). Functionally wild‐type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization. Mol. Carcinog. 19:243–253, 1997.

Interaction of mismatch repair protein PMS2 and the p53-related transcription factor p73 in apoptosis response to cisplatin.

Mismatch repair (MMR) proteins contribute to genome integrity by correcting replication errors. In higher eukaryotes, MMR proteins also regulate the cellular response to DNA lesions such as oxidized, alkylated, or crosslinked bases. Previous studies have linked MMR proteins to the activation of apoptosis through p53-dependent and p53-independent mechanisms. MMR-deficient cells exhibit variable defects in the induction of p53 and its related p73, which are activators of apoptosis. However, the specific role of each MMR protein in the regulation of apoptosis has not been determined. Here, we describe an interaction between PMS2, an MMR protein, and p73. This interaction causes the stabilization of p73 and the redistribution of PMS2 to the nuclear compartment. Exposure to cisplatin enhances the association between PMS2 and p73. Moreover, stimulation of the p73 proapoptotic function by cisplatin requires PMS2. These results suggest that PMS2 contributes to genome integrity not only through DNA repair but also by enhancing DNA damage-induced apoptosis.

Functional analysis of human MLH1 mutations in Saccharomyces cerevisiae

Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.

Functional Analysis of Human MLH1 Variants Using Yeast and In vitro Mismatch Repair Assays

The functional characterization of nonsynonymous single nucleotide polymorphisms in human mismatch repair (MMR) genes has been critical to evaluate their pathogenicity for hereditary nonpolyposis colorectal cancer. We previously established an assay for detecting loss-of-function mutations in the MLH1 gene using a dominant mutator effect of human MLH1 expressed in Saccharomyces cerevisiae. The purpose of this study is to extend the functional analyses of nonsynonymous single nucleotide polymorphisms in the MLH1 gene both in quality and in quantity, and integrate the results to evaluate the variants for pathogenic significance. The 101 MLH1 variants, which covered most of the reported MLH1 nonsynonymous single nucleotide polymorphisms and consisted of one 3-bp deletion, 1 nonsense and 99 missense variants, were examined for the dominant mutator effect by three yeast assays and for the ability of the variant to repair a heteroduplex DNA with mismatch bases by in vitro MMR assay. There was diversity in the dominant mutator effects and the in vitro MMR activities among the variants. The majority of functionally inactive variants were located around the putative ATP-binding pocket of the NH(2)-terminal domain or the whole region of the COOH-terminal domain. Integrated functional evaluations contribute to a better prediction of the cancer risk in individuals or families carrying MLH1 variants and provide insights into the function-structure relationships in MLH1.

Romidepsin (FK228) and its analogs directly inhibit phosphatidylinositol 3‐kinase activity and potently induce apoptosis as histone deacetylase/phosphatidylinositol 3‐kinase dual inhibitors

Activation of phosphatidylinositol 3‐kinase (PI3K) signaling is involved in carcinogenesis and cancer progression. The PI3K inhibitors are considered candidate drugs for cancer treatment. Here, we describe a drug screening system for novel PI3K inhibitors using Saccharomyces cerevisiae strains with deleterious mutations in the ATP‐binding cassette transporter genes, because wild‐type S. cerevisiae uses drug efflux pumps for reducing intracellular drug concentrations. By screening the chemical library of the Screening Committee of Anticancer Drugs, we identified the histone deacetylase (HDAC) inhibitor romidepsin (FK228) and its novel analogs. In vitro PI3K activity assays confirmed that these compounds directly inhibit PI3K activity at μM‐range concentrations. FK‐A5 analog was the most potent inhibitor. Western blotting revealed that these compounds inhibit phosphorylation of protein kinase B and downstream signaling components. Molecular modeling of the PI3K–FK228 complex indicated that FK228 binds to the ATP‐binding pocket of PI3K. At μM‐range concentrations, FK228 and FK‐A5 show potent cytotoxicity, inducing apoptosis even in HDAC inhibitor‐resistant cells. Furthermore, HDAC/PI3K dual inhibition by FK228 and FK‐A5 at μM‐range concentrations potentiates the apoptosis induction, mimicking the effect of combining specific HDAC and PI3K inhibitors. In this study, we showed that FK228 and its analogs directly inhibit PI3K activity and induce apoptosis at μM‐range concentrations, similar to HDAC/PI3K dual inhibition. In future, optimizing the potency of FK228 and its analogs against PI3K may contribute to the development of novel HDAC/PI3K dual inhibitors for cancer treatment.

Dose escalation study of docetaxel and nedaplatin in patients with relapsed or refractory squamous cell carcinoma of the esophagus pretreated using cisplatin, 5-fluorouracil, and radiation

BackgroundDefinitive chemoradiation with cisplatin (CDDP) and 5-fluorouracil (5FU) has been playing an important role in the treatment of esophageal cancer, but some patients are not curable or have recurrent lesions. However, few chemotherapeutic regimens are available for such patients. Docetaxel and nedaplatin are active for esophageal cancer. We conducted a dose-escalation study of docetaxel and nedaplatin as second line-chemotherapy after definitive chemoradiation in patients with relapsed or refractory squamous cell carcinoma of the esophagus after chemoradiation.MethodsNedaplatin was administered on day 1 and docetaxel was administered on days 1 and 15, every 4 weeks. Dose escalation was based on the dose-limiting toxicity (DLT) observed during the first cycle.ResultsTwelve patients were enrolled. At a docetaxel dose of 30 mg/m2 and a nedaplatin dose of 80 mg/m2, one grade 4 neutropenia occurred and caused one treatment break longer than 2 weeks, but there were few DLTs. At doses of 35 and 80 mg/m2, respectively, two grade 4 neutropenias and one grade 2 thrombopenia occurred and caused three treatment breaks longer than 2 weeks. Therefore, the maximum tolerated dose was established at this dose level. Two grade 3 anorexias and one grade 3 nausea occurred, but other non-hematological toxicities were generally mild. Responses were seen in one-fourth of the 12 patients, including one complete remission.ConclusionThe recommended doses of docetaxel and nedaplatin were 30 and 80 mg/m2, respectively. This combination could be a potential second-line treatment for this target population.

Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients.

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.

Biochemical, biological and structural properties of romidepsin (FK228) and its analogs as novel HDAC/PI3K dual inhibitors

Romidepsin (FK228, depsipeptide) is a potent histone deacetylase (HDAC) inhibitor that has FDA approval for the treatment of cutaneous and peripheral T‐cell lymphomas. We have previously reported that FK228 and its analogs have an additional activity as phosphatidylinositol 3‐kinase (PI3K) inhibitors, and are defined as HDAC/PI3K dual inhibitors. Because a combination of an HDAC inhibitor and a PI3K inhibitor induces apoptosis in human cancer cells in a synergistic manner, development of an HDAC/PI3K dual inhibitor will provide an attractive novel drug for cancer therapy. Using structure‐based optimization of the analogs, FK‐A11 was identified as the most potent analog. FK‐A11 inhibited phosphorylation of AKT and accelerated histone acetylation at lower concentrations, resulting in stronger cytotoxic effects than FK228 and the other analogs in human cancer cells. In this study, we have characterized the biochemical, biological and structural properties of FK228 analogs as PI3K inhibitors. First, FK‐A11 is an ATP competitive PI3K inhibitor. Second, FK‐A11 is a pan‐p110 isoform inhibitor. Third, FK‐A11 selectively inhibits PI3K among 22 common cellular kinases. Fourth, conformational changes of FK228 analogs by reduction of an internal disulfide bond have no effect on PI3K inhibitory activity, unlike HDAC inhibitory activity. Finally, molecular modeling of PI3K‐FK228 analogs and analyses of the binding affinities identified the structure that defines potency for PI3K inhibitory activity. These results prove our concept that a series of FK228 analogs are HDAC/PI3K dual inhibitors. These findings should help in the development of FK228 analogs as novel HDAC/PI3K dual inhibitors.