Munehiro Kishi

Heterogeneous Methylation and Deletion Patterns of the INK4a/ARF Locus Within Prostate Carcinomas

To elucidate the role of p53/p16(INK4a)/RB1 pathways in prostate carcinogenesis, we analyzed the p14(ARF), p16(INK4a), RB1, p21(Waf1), p27(Kip1), PTEN, p73, p53, and MDM2 gene status of multiple areas within 16 histologically heterogeneous prostate carcinomas using methylation-specific polymerase chain reaction, differential polymerase chain reaction, and immunohistochemistry. All focal areas examined had Gleason scores ranging from 1 to 5. Methylation of either PTEN or p73 was undetected in any sample, whereas expression of MDM2 seemed to be an independent event within small foci of 4 of 16 tumors. Loss of p14(ARF), p16(INK4a), RB1, and p27(Kip1) expression correlated with homozygous deletion or promoter hypermethylation. One carcinoma showed co-deletion of both p14(ARF) and p16(INK4a) in two of five areas examined; two areas within another tumor demonstrated concurrent hypermethylation of the promoter regions of the same genes. Focal hypermethylation of RB1, p21(Waf1), and p27(Kip1) was detected within two, two, and three tumors, respectively. These findings indicate that both genetic and epigenetic events occur independently in intratumor foci and further suggest hypermethylation-induced loss of gene function may be as critical as specific genetic mutations in prostate carcinogenesis.

Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas.

Frequent allelic losses on the long arm of chromosome 22 (22q) in gliomas indicate the presence of tumor suppressor gene (TSG) at this location. However, the target gene(s) residing in this chromosome are still unknown and their putative roles in the development of astrocytic tumors, especially in secondary glioblastoma, have not yet been defined. To compile a precise physical map for the region of common deletions in astrocytic tumors, we performed a high-density loss of heterozygosity (LOH) analysis using 31 polymorphic microsatellite markers spanning 22q in a series of grade II diffuse astrocytomas, anaplastic astrocytomas, primary glioblastomas, and secondary glioblastomas that had evolved from lower grade astrocytomas. LOH was found at one or more loci in 33% (12/36) of grade II diffuse astrocytomas, in 40% (4/10) of anaplastic astrocytomas, in 41% (26/64) of primary glioblastomas, and in 82% (23/28) of secondary glioblastomas. Characterization of the 22q deletions in primary glioblastomas identified two sites of minimally deleted regions at 22q12.3–13.2 and 22q13.31. Interestingly, 22 of 23 secondary glioblastomas affected shared a deletion in the same small (957 kb) region of 22q12.3, a region in which the human tissue inhibitor of metalloproteinases-3 (TIMP-3) is located. Investigation of the promoter methylation and expression of this gene indicated that frequent hypermethylation correlated with loss of TIMP-3 expression in secondary glioblastoma. This epigenetic change was significantly correlated to poor survival in eight patients with grade II diffuse astrocytoma. Our results suggest that a 957 kb locus, located at 22q12.3, may contain the putative TSG, TIMP-3, that appears to be relevant to progression to secondary glioblastoma and subsequently to the prognosis of grade II diffuse astrocytoma. In addition, the possibility of other putative TSGs on 22q12.3–13.2 and 22q13.31 that may also be involved in the development of primary glioblastomas cannot be ruled out.

DNA hypermethylation status of multiple genes in prostate adenocarcinomas

Multiple genetic mutations and epigenetic methylation are believed to be involved in prostate carcinogenesis, but it is not known whether these events are independent or correlated in some fashion. We therefore studied 32 prostate adenocarcinomas not only for deletions and/or mutations of multiple suspect genes, but also for aberrant DNA methylation using methylation‐specific PCR (MSP). Of those genes examined, p16INK4a, O6‐MGMT, and GST‐P were found to be the most frequently methylated (66%, 25% and 75% of cases, respectively), while methylations of p14ARF, RB1, p21waf1, and p27Kip1 were far less common (3%, 6%, 6% and 6% of cases, respectively). Methylation of O6‐MGMT and GST‐P genes was defective in about 19% of the cases and there were occasional simultaneous deletions and methylations of p14ARF and p16INK4a genes (13% and 3% of cases, respectively). In p16INK4a, methylation occurred in the promoter region in 9% of samples and in exon 2 in 66% of tumors. Hypermethylation of O6‐MGMT with concurrent p53 and ras gene mutations were found in 6% and 13% of specimens, respectively; among those tumors with high Gleason scores were 2 carcinomas showing hypermethylated O6‐MGMT with G‐to‐A transitions in K‐ras. Our results demonstrate that multiple genes of a subset common in prostate carcinomas are methylated and not infrequently show concurrent deletions. Further, there is a suggestion that specific combinations of hypermethylation and mutation correlate to tumor malignancy.

Contributions of mitogen‐activated protein kinase and nuclear factor kappa B to N‐(4‐hydroxyphenyl)retinamide–induced apoptosis in prostate cancer cells

The synthetic retinoid N‐(4‐hydroxyphenyl)retinamide (4‐HPR) has been shown to induce apoptosis in various types of tumors, including prostate cancer. We sought to examine the key mechanisms affecting the resistance to 4‐HPR–induced apoptosis in three human prostate cancer cell lines, PC‐3, DU145, and LNCaP. Concentrations of more than 40 μM 4‐HPR produced apoptosis to almost the same extent in all cell lines; however, only the LNCaP line remained highly sensitive to concentrations less than 10 μM. These differing sensitivities at low concentrations correlated well with the level of constitutive activation of nuclear factor kappa B (NFκB) in the individual cell lines. We found that NFκB activation inhibited c‐jun NH2‐terminal kinase and caspase 3 activation induced by 4‐HPR and that NFκB inhibition by the I kappa B alpha phosphorylation inhibitor compound Bay 117082 resulted in increasing sensitization of both PC‐3 and DU145 lines to apoptosis induced by 4‐HPR at low concentrations. Furthermore, we found that inhibition of extracellular signal–regulated kinase (ERK) enhanced the suppression of NFκB by 4‐HPR and also resulted in sensitization to apoptosis in the DU145 cell line, in which ERK is activated constitutively. It thus appears that mitogen‐activated protein kinase associated with the activity of NFκB plays an important role in the degree of resistance to 4‐HPR‐induced apoptosis in human prostate cancer cells.

The molecular mechanism of sensitization to Fas‐mediated apoptosis by 2‐methoxyestradiol in PC3 prostate cancer cells

It is widely known that death receptor Fas‐dependent apoptotic signals are associated with development of prostate cancer, but the key pathways involved in sensitivity to the apoptosis remain unclear. Here we investigated the molecular mechanism by which 2‐methoxyestradiol (2‐ME) effectively sensitizes a human prostate cancer cell line, PC3, to Fas‐mediated apoptosis. 2‐ME significantly inhibited nuclear factor‐κB (NF‐κB) activation and downregulated Fas‐associated death domain (FADD) protein interluekin‐1beta‐converting enzyme inhibitory protein (FLIP). Overexpression of the dominant negative mutant form of IκBα (d/n IκBα) or treatment with Iκ kinase‐specific inhibitor Bay117082 gave the same results, although the sensitizing effect was not as pronounced. A selective inhibitor of Akt phosphorylation, LY294002, accelerated formation of the death‐inducing signaling complex (DISC) not only by FLIP reduction but also by enhancement of recruitment of the FADD to Fas, thereby sensitizing PC3 cells to apoptosis similar to the case with 2‐ME stimulation. Moreover, we found that inhibition of 2‐ME–induced extracellular signal‐regulated kinase (ERK) activation by the upstream kinase inhibitor PD98059 significantly enhanced 2‐ME–mediated suppression of Akt activation, resulting in much greater sensitization to apoptosis. Taken together, the present findings indicate that 2‐ME suppresses NF‐κB/FLIP signaling and enhances DISC formation through inhibition of Akt, and that PC3 cells thereby are being sensitized to Fas‐mediated apoptosis and by a process closely associated with ERK.

Requirement of c-jun for testosterone-induced sensitization to N-(4-hydroxyphenyl)retinamide–induced apoptosis

Androgen stimulation strongly affects the sensitivity to anticancer drug–induced apoptosis in prostate cancer cells. We investigated the influence of androgen stimulation with testosterone on N‐(4‐hydroxyphenyl)retinamide (4‐HPR)–induced apoptosis in the androgen‐sensitive prostate cancer cell line LNCaP. Overexpression of a dominant negative form of mitogen‐activated protein kinase kinase 7, a specific kinase of c‐jun NH2‐terminal kinase (JNK), significantly inhibited 4‐HPR–induced JNK activation and apoptosis and canceled the hormone‐dependent sensitization. Testosterone activated extracellular signal‐regulated kinase (ERK), activating protein‐1, subsequently increased the expression of c‐jun. In addition, testosterone significantly enhanced in vivo phosphorylation of c‐jun by 4‐HPR as well as JNK activation. Transfection with an antisense oligonucleotide of c‐jun blocked 4‐HPR‐induced apoptosis and the testosterone‐induced sensitization, suggesting a major contribution of the JNK/c‐jun mediated pathway in androgen‐dependent sensitization. Interestingly, inhibition of testosterone‐induced activation by PD98059 also canceled an upregulation of c‐jun and increased apoptosis. These results suggested that modulation of JNK activation and expression of c‐jun through ERK might have been essentially involved in androgen‐mediated sensitization to 4‐HPR‐induced apoptosis in prostate cancer cells.

Loss of heterozygosity on chromosome 6q correlates with decreased thrombospondin‐2 expression in human salivary gland carcinomas

Since loss of heterozygosity (LOH) on the long arm of chromosome 6q is frequently observed in salivary gland carcinomas, we examined 28 salivary gland carcinomas using 24 microsat‐ellite markers mapping to 6q15–27 to identify the commonly deleted region that we felt might contain one or more tumor suppressor genes. LOH was detected in at least one locus in 10 of 28 tumors (35.7%). The most frequently deleted regions occurred between D6S1581 and D6S305 (LOH cluster region 1 (LCR1) and between D6S297 and D6S1590 (LCR2). LOH was observed in 60% of adenoid cystic carcinomas (ACC) and in 57.1% of mucoepider‐moid carcinomas (MEC), but was not observed in any locus in any other histological subtypes studied. The gene encoding for thrombospondin‐2 (TSP‐2) is located in LCR2 and 8 of 9 tumors demonstrating LOH in this region also showed significantly decreased TSP‐2 expression by immunohistochemistry. As TSP‐2 is a potent inhibitor of tumor growth and angiogenesis, we examined whether TSP‐2 expression correlated to microvascular angiogenesis in these tumors and discovered that microvessel counts were significantly higher in lesions with decreased TSP‐2 expression (P=0.02). Our results suggest that 6q LOH may be a significant event in salivary gland carcinogenesis, particularly in ACC and MEC, and that the correlated decrease of TSP‐2 expression also plays a critical role.

Phosphorylation of Fas-associated Death Domain Contributes to Enhancement of Etoposide-induced Apoptosis in Prostate Cancer Cells

Fas‐associated death domain (FADD) plays an important role as an adapter molecule in Fas (CD95/APO‐l)‐mediated apoptosis and contributes to anticancer drug‐induced cytotoxicity. We treated three human prostate cancer cell lines with etoposide, a toposiomerase II inhibitor with activity against various tumors including prostate cancer. We found that the overexpression of FADD sensitizes etoposide‐induced apoptosis through a rapid activation of c‐Jun NH2‐terminal kinase (JNK) and, subsequently, of caspase 3. In addition, phosphorylation of FADD at serine 194 coincided with this sensitization. Treatment with the caspase 3 inhibitor, N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (DEVD‐CHO), or overexpression of either mitogen‐activated protein kinase kinase (MKK) 7 or Bcl‐xL canceled FADD‐mediated sensitization to etoposide‐induced apoptosis. Moreover, treatment with the caspase 8 inhibitor, benzyloxy‐carbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐IETD‐fmk), or overexpression of viral FLICE/caspase‐8‐inhibitory protein (FLIP) from equine herpesvirus type 2 E8 also had an inhibitory effect, supporting a major involvement of a caspase 8‐dependent mitochondrial pathway. Interestingly, FADD was phosphorylated, and etoposide‐induced JNK/caspase activation and apoptosis were enhanced in the cells arrested at G2/M transition, but not in those overexpressing mutant FADD, in which 194 serine was replaced by alanine. Our results demonstrate that phosphorylated FADD‐dependent activation of the JNK/caspase pathway plays a pivotal role in sensitization to etoposide‐induced apoptosis in prostate cancer cells.

Genetic mapping of allelic loss on chromosome 6q within heterogeneous prostate carcinoma

A number of genetic events have been reported in prostate carcinogenesis, including frequent loss of heterozygosity (LOH) on chromosomes 8q, 10q, 16q and 18q, In samples of heterogeneous, multifocal prostate carcinomas, we focused on chromosome 6q using PCR‐based techniques with 15 microsatellite markers to identify the specific 6q deletion within tumors. LOH of one or more polymorphic markers was detected in 10 of 21 tumors (48%). Two of these 10 tumors demonstrated LOH in all cancerous foci at specific loci and 4 tumors showed deletion in one focus. Different deletion patterns were found in 3 tumors when different polymorphic markers were used. In 90% of tumors showing LOH in one or more foci, however, two common regions of LOH were identified; one at 1.81 cM on 6q15–16.3 between markers D6S1631 and D6S1056, and the other at 5.11 cM on 6q16–21 between markers D6S424 and D6S283. By RT‐PCR analysis, the TAK1 gene located at these loci did not correlate with LOH status, indicating that TAK1 is not a target gene in prostate carcinoma. The 6q deletion occurs heterogeneously and LOH was more frequent in tumors of higher pathological stages, implying that this alteration is a late event in prostate carcinogenesis. Because prostate carcinomas are genetically multicentric and of multifocal origin, it remains unclear whether the foci containing 6q deletions specifically expand within tumors or to what extent they contribute to the histological heterogeneity characteristic of the disease.

Androgen and the blocking of radiation-induced sensitization to Fas-mediated apoptosis through c-jun induction in prostate cancer cells.

Purpose: To clarify the key mechanism by which androgen makes prostate cancer cells highly resistant to Fas‐mediated apoptosis. Materials and methods: The role of c‐jun induction by 10 nM dihydrotestosterone (DHT) in 5 Gy radiation‐induced up‐regulation of Fas and sensitization to the apoptosis was studied by using the human prostate cancer cell line LNCaP. Results: On exposure to 5 Gy radiation, LNCaP cells demonstrated high sensitization to Fas‐mediated apoptosis through increased Fas expression, stabilized p53 expression and binding to p53 response elements within the promoter and first intronic region of the Fas gene. Following treatment with DHT, in vivo binding of p53 to its response elements was strongly inhibited. In addition, DHT significantly up‐regulated c‐jun expression through extracellular stress‐regulated kinase (ERK) activation, and transfection of an antisense oligonucleotide for c‐jun or ERK inhibition by PD98059 cancelled DHT‐mediated suppression of radiation‐induced transactivation of Fas gene and sensitization to Fas‐mediated apoptosis. Conclusions: Radiation‐induced Fas sensitization in prostate cancer cell was mediated through p53‐dependent transactivation of the Fas gene, which can be blocked by androgen stimulation mainly through induction of c‐jun.