Induction by rapamycin and proliferation­promoting activity of Hspb1 in a Tsc2­deficient cell line.

Tuberous sclerosis complex (TSC) is an intractable inherited disease caused by a germline mutation in either the TSC complex subunit 1 (TSC1) or TSC2 tumor suppressor genes. Recent progress in the treatment of TSC with rapamycin has provided benefits to patients with TSC. However, the complete elimination of tumors is difficult to achieve as regrowth often occurs after a drug is suspended; thus, more efficient medication and novel therapeutic targets are required. To overcome tumor remnants in the treatment of TSC, the present study investigated rapamycin-responsive signaling pathways in Tsc2-deficient tumor cells, focusing on heat shock protein-related pathways. The expression levels of heat shock protein family B (small) member 1 (Hspb1; also known as HSP25/27) were increased by rapamycin treatment. The phosphorylation of Hspb1 was also increased. The knockdown of Hspb1 suppressed cell proliferation in the absence of rapamycin, and the overexpression of Hspb1 enhanced cell proliferation both in the presence and absence of rapamycin. Pathways associated with Hspb1 may present target candidates for treatment of TSC.

Combined use of irinotecan and p53 activator enhances growth inhibition of mesothelioma cells.

Malignant mesothelioma (MM) is an aggressive malignant neoplasm which rapidly invades pleural tissues and has a poor prognosis. Here, we explore enhancement of the effect of irinotecan [camptothecin-11 (CPT-11)] by the p53-dependent induction of carboxylesterase 2 (CES2), a CPT-11-activating enzyme, in MM. The level of CES2 mRNA was greatly increased on treatment with nutlin-3a. A combination of CPT-11 and nutlin-3a inhibited the growth of MM cells more effectively than either drug alone. Knocking down CES2 in MM cells reduced the effect of the drug combination, and its forced expression in MESO4 cells enhanced the growth inhibitory activity of CPT-11 in the absence of nutlin-3a. Enhancement of the growth inhibitory activity of CPT-11 by nutlin-3a suggests a possible new combinatorial MM chemotherapy regimen.

Genetics of synchronous uterine and ovarian endometrioid carcinoma: combined analyses of loss of heterozygosity, PTEN mutation, and microsatellite instability.

Synchronous development of carcinomas in the endometrium and ovaries is a fairly common phenomenon, but distinction of a single clonal tumor with metastasis from 2 independent primary tumors may present diagnostic problems. To determine clonality and the occurrence of progression, we microdissected multiple foci from 17 cases of synchronous endometrioid carcinomas and studied loss of heterozygosity (LOH), microsatellite instability (MI), and PTEN mutations. In 14 of the 17 cases, genetic alterations were either homogeneous or found in only some of the foci. LOH was detected for 10q (4 cases), 17p (2 cases), and 2p, 5q, 6q, 9p, 11q, 13q, and 16q (1 case each). Four cases had the MI phenotype with discordant MI patterns between both tumor sites, thus indicating a biclonal or triple clonal process. In 3 of 6 cases with PTEN mutations, identical mutations in both tumor sites indicated a single clonal neoplasm. Altogether, 14 synchronous tumors were genetically diagnosed as follows: single clonal tumor, characterized by concordant genetic alterations in both tumor sites, including identical LOH, identical PTEN mutations, and/or identical sporadic allelic instability patterns (4 cases); single clonal tumor with genetic progression, homogeneous LOH or identical PTEN mutations in both tumor sites and progressive LOH in ovarian metastatic foci (2 cases); and double (7 cases) or triple clonal tumors (1 case), determined by discordant PTEN mutations, heterogeneous LOH, and/or discordant MI patterns. Thus, 35% of synchronous tumors were monoclonal, 47% were polyclonal, and 18% were undetermined. The favorable prognosis of synchronous endometrioid carcinomas may be due to the occurrence of PTEN mutations in both independent and metastatic tumors, the MI-positive independent primary tumors, and the low frequency of LOH.

Frequent down-regulation of HIVEP2 in human breast cancer.

The HIVEP2 gene, located on 6q23-q24, belongs to a family of genes that encodes large zinc fingers containing transcription factor proteins. Although this gene has been implicated in the regulation of immune responses and cellular proliferation, its functions are largely unknown. In the present study, we investigated HIVEP2 gene abnormalities in microdissected breast cancer tissue. For real-time quantitational RT-PCR analysis of paired normal and tumor tissues, mRNA levels were down-regulated to a maximum of 96%. The overall median expression level in breast cancer (33 cases) was significantly lower than that in normal breast tissue (normalized median value of 4.49 versus 17.68; p < 0.0001). Through full-length 5'-RACE (rapid amplification of cDNA ends) analysis, we identified multiple exons in the 5'-untranslated regions with multiple transcriptional start sites, four of which were located in a large CpG island. No tissue- or cancer-specific usage patterns for the transcription start sites were identified by multiplex RT-PCR analysis. Only faint methylation was detected in the 5' region of the island in normal cells and breast cancer tissue, indicating physiological, aging and no tumor-specific methylation. Mutation screening showed only germline polymorphisms. Thus, down-regulation of the HIVEP2 genes frequently occurs and may be one of the genetic events responsible for breast cancer, and their transcription may be regulated by complex mechanisms involving interactions with other factors and/or by other genetic/epigenetic mechanisms.

Loss of heterozygosity in the clonal evolution of flat colorectal neoplasms.

In contrast to invasive colorectal carcinomas that develop in typical exophytic adenoma-carcinoma sequences, some invasive cancers may evolve from flat mucosal dysplastic lesions. Despite their relatively small size, these flat colorectal lesions are often associated with high-grade dysplasia and may show an aggressive clinical course. To delineate the genetic pathways in the clonal evolution of these tumors, multiple foci were microdissected from 13 cases and the allelic deletions of 15 chromosomal arms were analysed. Loss of heterozygosity (LOH) was detected most frequently on 17p (77%), followed by 18q (69%), and 5q (54%). In five cases with concomitant low-grade adenomas, only one case showed LOH in low-grade adenoma foci. In high-grade dysplasia with/without submucosal invasion, early and homogeneous LOH of one to several chromosomal arms was detected. Overall, homogeneous and thus early LOH were most frequently detected on 17p (seven of 10 cases with 17p LOH), followed by 3p (two of three cases with 3p LOH), and 5q (four of seven cases with 5q LOH). In addition to homogeneous LOH, the LOH patterns observed in different portions of dysplasias and invasive cancers in individual cases identified several different genetic patterns of tumour progression, either with linear or branching (divergent) trees. Positive immunostaining for p53 was detected in 10 of the 13 cases; of these, five cases were concomitant with 17p LOH in all of the microdissected foci, four cases were concomitant with 17p LOH in a majority of foci and, one case showed retention of 17p. Except for the flat configuration and early 17p LOH, genetic heterogeneity in the flat high-grade dysplastic foci was found to be similar to genetic chaos in the late dysplastic and preinvasive stages of exophytic adenoma. These findings suggest a potentially aggressive course for these neoplasms.