Akinori Egashira

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

Deregulation of the akt pathway in human cancer

Akt (protein kinase B) is a serine/threonine kinase which is a central regulator of widely divergent cellular processes including proliferation, differentiation, migration, survival and metabolism. Akt is activated by a variety of stimuli, through growth factor receptors, in phosphatidylinositol 3-kinase (PI3K)-dependent manner. Akt is also negatively regulated by the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN). A disruption of normal Akt/PKB/PTEN signaling frequently occurs in many human cancers, which plays an important role in cancer development, progression and therapeutic resistance. Numerous studies have revealed the blockage of Akt signaling to result in apoptosis and growth inhibition of tumor cells. Therefore, this signaling pathway, including both upstream and downstream of Akt, has recently attracted considerable attention as a new target for effective cancer therapeutic strategies. In fact, many inhibitors of Akt pathway have been identified and clinical studies of some agents are ongoing. In this review, we describe Akt signaling pathway components and its cellular functions as well as the alterations in human cancers and the therapeutic approaches for targeting the Akt pathway in cancer.

Advances in esophageal cancer surgery in Japan: An analysis of 1000 consecutive patients treated at a single institute

BACKGROUND In Japan, most esophageal cancers are squamous cell carcinomas, and the results of esophagectomy have improved remarkably in recent years. The object of this study was to evaluate advances in operative therapy for esophageal cancer in Japan. METHOD We evaluated mortality, morbidity, and prognosis in 1000 consecutive patients who underwent esophagectomy for esophageal cancer at a single institution in Japan. The patients were divided into 3 groups according to the period when esophagectomy was performed: Group I (n = 197), 1964-1980; group II (n = 432), 1981-1993; and group III (n = 371), 1993-2006. RESULTS The incidence of squamous cell carcinoma was 94%. The morbidity rates were 62%, 38%, and 33 %, in groups I, II, and III, respectively (P < 0.01, groups I vs II and III), and the in-hospital mortality rates were 14.2%, 5.1%, and 2.4%, respectively (P < 0.01, between each group). The 5-year overall survival rate was 30% (14%, 27%, and 46% in groups I, II, and III, respectively; P < 0.0001). Multivariate analysis revealed age, gender, depth of invasion, node metastasis, distant metastasis, curability, extent of lymphadenectomy, resectability, and the period when the operation was performed as independent prognostic factors. CONCLUSION Generally, esophagectomy has been performed safely without critical complications; however, the prognosis has improved remarkably with advances in surgical techniques and treatment modalities.

MUTYH-Null Mice Are Susceptible to Spontaneous and Oxidative Stress–Induced Intestinal Tumorigenesis

MUTYH is a mammalian DNA glycosylase that initiates base excision repair by excising adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine, thereby preventing G:C to T:A transversion caused by oxidative stress. Recently, biallelic germ-line mutations of MUTYH have been found in patients predisposed to a recessive form of hereditary multiple colorectal adenoma and carcinoma with an increased incidence of G:C to T:A somatic mutations in the APC gene. In the present study, a systematic histologic examination revealed that more spontaneous tumors had developed in MUTYH-null mice (72 of 121; 59.5%) than in the wild type (38 of 109; 34.9%). The increased incidence of intestinal tumors in MUTYH-null mice (11 tumors in 10 of 121 mice) was statistically significant compared with the wild type (no intestinal tumors in 109 mice). Two adenomas and seven adenocarcinomas were observed in the small intestines, and two adenomas but no carcinomas were found in the colons. In MUTYH-null mice treated with KBrO(3), the occurrence of small intestinal tumors dramatically increased. The mean number of polyps induced in the small intestines of these mice was 61.88 (males, 72.75; females, 51.00), whereas it was 0.85 (males, 0.50; females, 1.00) in wild-type mice. The tumors developed predominantly in the duodenum and in the upper region of the (jejunum) small intestines. We conclude that MUTYH suppresses spontaneous tumorigenesis in mammals, thus providing experimental evidence for the association between biallelic germ-line MUTYH mutations and a recessive form of human hereditary colorectal adenoma and carcinoma.

Alcohol drinking, cigarette smoking, and the development of squamous cell carcinoma of the esophagus: molecular mechanisms of carcinogenesis

Esophageal cancer is the eighth most common incident cancer in the world and ranks sixth among all cancers in mortality. Esophageal cancers are classified into two histological types; esophageal squamous cell carcinoma (ESCC), and adenocarcinoma, and the incidences of these types show a striking variety of geographic distribution, possibly reflecting differences in exposure to specific environmental factors. Both alcohol consumption and cigarette smoking are major risk factors for the development of ESCC. Acetaldehyde is the most toxic ethanol metabolite in alcohol-associated carcinogenesis, while ethanol itself stimulates carcinogenesis by inhibiting DNA methylation and by interacting with retinoid metabolism. Cigarette smoke contains more than 60 carcinogens and there are strong links between some of these carcinogens and various smoking-induced cancers; these mechanisms are well established. Synergistic effects of cigarette smoking and alcohol consumption are also observed in carcinogenesis of the upper aerodigestive tract. Of note, intensive molecular biological studies have revealed the molecular mechanisms involved in the development of ESCC, including genetic and epigenetic alterations. However, a wide range of molecular changes is associated with ESCC, possibly because the esophagus is exposed to many kinds of carcinogens including alcohol and cigarette smoke, and it remains unclear which alterations are the most critical for esophageal carcinogenesis. This brief review summarizes the general mechanisms of alcohol- and smoking-induced carcinogenesis and then discusses the mechanisms of the development of ESCC, with special attention to alcohol consumption and cigarette smoking.

Trastuzumab and breast cancer: developments and current status

The emergence of trastuzumab has drastically changed therapy for breast cancer. Trastuzumab (Herceptin; Genentech) is a recombinant humanized monoclonal antibody that targets an epitope in the extracellular domain of the human epidermal growth factor receptor 2 (HER2) protein. HER2 is a member of a family of four transmembrane receptor tyrosine kinases that regulate cell growth, survival, and differentiation via multiple signal transduction pathways. Overexpression of HER2 or amplification of the HER2 gene occurs in 20%–30% of human breast cancers. Preclinical models have demonstrated that this antibody has significant antitumor activity as a single agent, and it also has a synergy with certain chemotherapeutic drugs. Phase II and III clinical trials performed in women with metastatic breast cancers that overexpress HER2 have shown trastuzumab to have clinical activity when used as monotherapy, while also improving survival when used as a first-line therapy in combination with chemotherapy. At present, clinical investigations are focusing attention on the efficacy of trastuzumab in both the adjuvant and neoadjuvant setting, as well as in the metastatic setting. In this review, we describe the developments and current status of trastuzumab-based treatment for breast cancer.

Overexpression of Hypoxia-Inducible Factor 1α and p53 Is a Marker for an Unfavorable Prognosis in Gastric Cancer

Purpose: To investigate the effects of the expression of hypoxia-inducible factor 1α (HIF-1α) and p53 on the prognosis of human gastric cancer, the clinicopathologic characteristics of the tumors and the clinical outcome were analyzed. Experimental Design: The expressions of HIF-1α and p53 proteins were studied by immunohistochemistry in 216 specimens of primary gastric cancer. Results: HIF-1α(+)/p53(+) tumors more frequently showed an undifferentiated type, an infiltrative growth appearance, and an invasive lymphatic involvement compared with HIF-1α(−)/p53(−) tumors. HIF-1α(+)/p53(+) tumors also had more lymph node metastasis compared with HIF-1α(−)/p53(−) tumors. When stratified for HIF-1α and p53 positivity, the patients who were p53-negative and HIF-1α-negative had the most favorable prognosis, whereas patients who were p53-positive and HIF-1α-positive had the worst prognosis (P = 0.0018). Using a multivariate Cox regression analysis, the depth of invasion, lymph node metastasis, and HIF-1α positivity were all found to be independent prognostic factors in patients with gastric cancer. Conclusion: Thus, HIF-1α is considered to be a useful independent prognostic factor in gastric cancer, and the combination of a HIF-1α protein overexpression with nonfunctional p53 tends to indicate a dismal prognosis.

Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes.

Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and aging. In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis. Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides. We have established MTH1(-/-) mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers. To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichia coli, was introduced into MTH1(-/-) mice. Interestingly, the net frequency of rpsL(-) forward mutants showed no apparent increase in MTH1(-/-) mice as compared to MTH1(+/+) mice. However, we found differences between these two genotypes in the class- and site-distributions of the rpsL(-) mutations recovered from the mice. Unlike MutT-deficient E. coli showing 1000-fold higher frequency of A:T-->C:G transversion than the wild type cells, an increase in frequency of A:T-->C:G transversion was not evident in MTH1 nullizygous mice. Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1(-/-) mice than in those of wild type mice. Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1(-/-) mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides. Consistent with this hypothesis, a significant increase in the frequency of G:C-->T:A transversions was observed with MTH1(-/-) MSH2(-/-) mice over MSH2(-/-) mice alone. These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxidized nucleotides.

Two modes of microsatellite instability in human cancer: differential connection of defective DNA mismatch repair to dinucleotide repeat instability

Microsatellite instability (MSI) is associated with defective DNA mismatch repair in various human malignancies. Using a unique fluorescent technique, we have observed two distinct modes of dinucleotide microsatellite alterations in human colorectal cancer. Type A alterations are defined as length changes of ≤6 bp. Type B changes are more drastic and involve modifications of ≥8 bp. We show here that defective mismatch repair is necessary and sufficient for Type A changes. These changes were observed in cell lines and in tumours from mismatch repair gene-knockout mice. No Type B instability was seen in these cells or tumours. In a panel of human colorectal tumours, both Type A MSI and Type B instability were observed. Both types of MSI were associated with hMSH2 or hMLH1 mismatch repair gene alterations. Intriguingly, p53 mutations, which are generally regarded as uncommon in human tumours of the MSI+ phenotype, were frequently associated with Type A instability, whereas none was found in tumours with Type B instability, reflecting the prevailing viewpoint. Inspection of published data reveals that the microsatellite instability that has been observed in various malignancies, including those associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), is predominantly Type B. Our findings indicate that Type B instability is not a simple reflection of a repair defect. We suggest that there are at least two qualitatively distinct modes of dinucleotide MSI in human colorectal cancer, and that different molecular mechanisms may underlie these modes of MSI. The relationship between MSI and defective mismatch repair may be more complex than hitherto suspected.

Concurrent genetic alterations in DNA polymerase proofreading and mismatch repair in human colorectal cancer

Genomic sequences encoding the 3′ exonuclease (proofreading) domains of both replicative DNA polymerases, pol delta and pol epsilon, were explored simultaneously in human colorectal carcinomas including six established cell lines. Three unequivocal sequence alterations, including one previously reported, were found, and all these were considered as dysfunctional mutations in light of the local amino-acid sequences. In particular, the F367S mutation found in the POLE gene encoding the pol epsilon catalytic subunit, which includes the proofreading domain, is the first found in human diseases. Surprisingly, the tumours carrying these proofreading domain mutations were all defective in DNA mismatch repair (MMR). In addition to the two cell lines with acknowledged MMR gene mutations, the third tumour was also demonstrated to harbour a distinct mutation in MLH1, and indeed exhibited a microsatellite-unstable phenotype. These findings suggest that, in concert with MMR deficiency, defective polymerase proofreading may also contribute to the mutator phenotype observed in human colorectal cancer. Our observations may suggest previously unrecognised complexities in the molecular abnormalities underlying the mutator phenotype in human neoplasms.