Osamu Tetsu

Beta-catenin regulates expression of cyclin D1 in colon carcinoma cells.

Mutations in the adenomatous polyposis coli (APC) tumour-suppressor gene occur in most human colon cancers. Loss of functional APC protein results in the accumulation of β-catenin. Mutant forms of β-catenin have been discovered in colon cancersthat retain wild-type APC genes,, and also in melanomas, medulloblastomas, prostate cancer and gastric and hepatocellular, carcinomas. The accumulation of β-catenin activates genes that are responsive to transcription factors of the TCF/LEF family, with which β-catenin interacts. Here we show that β-catenin activates transcription from the cyclin D1 promoter, and that sequences within the promoter that are related to consensus TCF/LEF-binding sites are necessary for activation. The oncoprotein p21ras further activates transcription of the cyclin D1 gene, through sites within the promoter that bind the transcriptional regulators Ets or CREB. Cells expressing mutant β-catenin produce high levels of cyclin D1 messenger RNA and protein constitutively. Furthermore, expression of a dominant-negative form of TCF in colon-cancer cells strongly inhibits expression of cyclin D1 without affecting expression of cyclin D2, cyclin E, or cyclin-dependent kinases 2, 4 or 6. This dominant-negative TCF causes cells to arrest in the G1 phase of the cell cycle; this phenotype can be rescued by expression of cyclin D1 under the cytomegalovirus promoter. Abnormal levels of β-catenin may therefore contribute to neoplastic transformation by causing accumulation of cyclin D1.

Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome.

Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder involving characteristic craniofacial features, cardiac defects, ectodermal abnormalities, and developmental delay. We demonstrate that heterogeneous de novo missense mutations in three genes within the mitogen-activated protein kinase (MAPK) pathway cause CFC syndrome. The majority of cases (18 out of 23) are caused by mutations in BRAF, a gene frequently mutated in cancer. Of the 11 mutations identified, two result in amino acid substitutions that occur in tumors, but most are unique and suggest previously unknown mechanisms of B-Raf activation. Furthermore, three of five individuals without BRAF mutations had missense mutations in either MEK1 or MEK2, downstream effectors of B-Raf. Our findings highlight the involvement of the MAPK pathway in human development and will provide a molecular diagnosis of CFC syndrome.

Proliferation of cancer cells despite CDK2 inhibition.

We have investigated the contribution of CDK4 and CDK2 inhibition to G1 arrest in colon cancers following inhibition of the MEK/MAP kinase pathway. CDK4 inhibition is sufficient to cause arrest, but inhibition of CDK2 by p27 Kip1 redistribution or ectopic expression has no effect on proliferation. Likewise, inhibition of CDK2 through expression of dominant-negative (DN) CDK2 or antisense oligonucleotides did not prevent cell proliferation in these cells. We therefore tested whether CDK2 activity is dispensable in other cells. Surprisingly, osteosarcomas and Rb-negative cervical cancers continued to proliferate after depletion of CDK2 through antisense oligonucleotides or small interfering (si) RNA. Here we report of sustained cell proliferation in the absence of CDK2, and we suggest that CDK2 is not a suitable target for cancer therapy.

A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer cell proliferation

Cyclin D1 regulates G1 progression. Its transcriptional regulation is well understood. However, the mechanism underlying cyclin D1 ubiquitination and its subsequent degradation is not yet clear. We report that cyclin D1 undergoes increased degradation in the cytoplasm during S phase in a variety of cancer cells. This is mediated by phosphorylation at Thr286 through the activity of the Ras/Raf/MEK/ERK cascade and the F-box protein FBXW8, which is an E3 ligase. The majority of FBXW8 is expressed in the cytoplasm during G1 and S phase. In contrast, cyclin D1 accumulates in the nucleus during G1 phase and exits into the cytoplasm in S phase. Increased cyclin D1 degradation is linked to association with FBXW8 in the cytoplasm, and enhanced phosphorylation of cyclin D1 through sustained ERK1/2 signaling. Depletion of FBXW8 caused a significant accumulation of cyclin D1, as well as sequestration of CDK1 in the cytoplasm. This resulted in a severe reduction of cell proliferation. These effects could be rescued by constitutive nuclear expression of cyclin D1-T286A. Thus, FBXW8 plays an essential role in cancer cell proliferation through proteolysis of cyclin D1. It may present new opportunities to develop therapies targeting destruction of cyclin D1 or its regulator E3 ligase selectively.

The Role of mel-18, a Mammalian Polycomb Group Gene, during IL-7–Dependent Proliferation of Lymphocyte Precursors

mel-18 is a mammalian homolog of Drosophila melanogaster Polycomb group genes. Mice lacking the mel-18 gene show a posterior transformation of the axial skeleton, severe combined immunodeficiency, and a food-passing disturbance in the lower intestine due to hypertrophy of the smooth muscle layer. In this study, the severe combined immunodeficiency observed in mel-18 mutant mice is correlated with the impaired mitotic response of lymphocyte precursors upon interleukin-7 stimulation. Strikingly, the axial skeleton and lymphoid phenotypes are identical in both mel-18 and bmi-1 mutants, indicating that the Mel-18 and Bmi-1 gene products might act in the same genetic cascade. These results suggest that mammalian Polycomb group gene products are involved in cell cycle progression in the immune system.

Genetic Profiling Reveals Cross-Contamination and Misidentification of 6 Adenoid Cystic Carcinoma Cell Lines: ACC2, ACC3, ACCM, ACCNS, ACCS and CAC2

Adenoid cystic carcinoma (ACC) is the second most common malignant neoplasm of the salivary glands. Most patients survive more than 5 years after surgery and postoperative radiation therapy. The 10 year survival rate, however, drops to 40%, due to locoregional recurrences and distant metastases. Improving long-term survival in ACC requires the development of more effective systemic therapies based on a better understanding of the biologic behavior of ACC. Much preclinical research in this field involves the use of cultured cells and, to date, several ACC cell lines have been established. Authentication of these cell lines, however, has not been reported. We performed DNA fingerprint analysis on six ACC cell lines using short tandem repeat (STR) examinations and found that all six cell lines had been contaminated with other cells. ACC2, ACC3, and ACCM were determined to be cervical cancer cells (HeLa cells), whereas the ACCS cell line was composed of T24 urinary bladder cancer cells. ACCNS and CAC2 cells were contaminated with cells derived from non-human mammalian species: the cells labeled ACCNS were mouse cells and the CAC2 cells were rat cells. These observations suggest that future studies using ACC cell lines should include cell line authentication to avoid the use of contaminated or non-human cells.

mel-18 negatively regulates cell cycle progression upon B cell antigen receptor stimulation through a cascade leading to c-myc/cdc25

mel-18 is a mammalian Polycomb group gene encoding a transcriptional repressor with tumor suppressive activity. Overexpression of mel-18 in mice results in cell cycle arrest of B cells upon B cell receptor stimulation with downregulation of c-myc. This phenotype is rescued in mel-18/c-myc double-transgenic mice, suggesting that c-myc locates downstream of mel-18. In mel-18 transgenic mice, the downregulation of cyclins D2 and E; CDK4, -6, and -7; and CDC25A causes the impairment in the activities of cyclin-dependent kinases, resulting in hypophosphorylation of the retinoblastoma protein. In contrast, the upregulation of c-Myc, CDC25, and CDC2/CDK2 kinase activities results in the augmentation of B cell proliferation in mel-18-deficient mice. We therefore propose that mel-18 negatively regulates the cell cycle through a c-myc/cdc25 cascade.

EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function

Significance Lung cancer is the leading cause of cancer death worldwide. About 10% harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with EGFR inhibitors, only 5% of patients achieve tumor reduction >90%, even though all of those treated have EGFR mutations. Our study addressed this discrepant response by investigating the mechanism of innate drug resistance, i.e. resistance inherent in the tumor cells even before treatment begins and not acquired over its course. Because overcoming innate resistance will increase the primary response, our findings may provide an opportunity to develop new therapies to reduce the probability of emergent resistance to EGFR inhibitors. Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.

Short Communication Mammalian Polycomb group genes are categorized as a new type of early response gene induced by B-cell receptor cross-linking

Polycomb group (PcG) genes were initially described in Drosophila melanogaster as regulators of the homeobox gene. Four mammalian homologues, mel-18, bmi-1, M33 and rae-28, are analyzed in this study. They not only regulate mammalian homeotic genes by analogy with their Drosophila counterparts, but also have some influence on the growth and differentiation of B lymphocytes. Here we report that these four mammalian PcG genes are rapidly induced after antigen-receptor cross-linking in B cells. Thus we would like to propose that mammalian PcG genes can be categorized as a new type of immediate early gene.

A case of spontaneous intramural hematoma of the esophagus

SummaryThe authors experienced a case of spontaneous intramural hematoma of the esophagus (SIHE). This 44-year-old Japanese woman was admitted to our hospital bacause of chest pain accompanied hy minimal hematemesis. Endoscopy revealed an elevated intraluminal bleeding bulge. Barium esophagograms showed a smooth and giant elevated intraluminal lesion. CT and MRI also revealed thickening of the esophageal wall. Fasting and intravenous hyperalimentation were prescribed on admission. The conditions improved and she became asymptomatic on the fifth day of hospitalization. Subsequent examinations by esophagography and endoscopy showed that the elevated lesion had disappeared and that the inflamed mucosal lesion had improved. The prognosis of cases of SIHE is excellent under conservative therapy, but close follow-up care is necessary.