Eiwa Ishida

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.

High Expression of a New Marker PCA-1 in Human Prostate Carcinoma

Purpose: Identifying the genetic factors involved in prostate carcinogenesis is critical. Novel cancer-specific markers aid in early detection, in differentiating between cancer and nonmalignant disorders, and in monitoring clinical of prostate disease. We therefore examined differential gene displays in an attempt to identify genes that may be involved in prostate carcinogenesis. Experimental Design: Applying fluorescent differential display analysis to human prostate carcinomas, we have identified and cloned several cDNA transcripts. Antisera were raised against synthetic peptides and used in Western blot and immunohistochemical analyses. The mRNAs were also analyzed by real-time reverse transcription-PCR. For functional analysis, we assessed methylmethane sulfonate (MMS)–induced toxicity in COS-7 cells after cDNA transfection. Results: We identified a gene, designated prostate cancer antigen-1 (pca-1), which shows high mRNA expression in prostate carcinoma. Database analysis of the deduced amino acid sequence of PCA-1 indicated high similarity to Escherichia coli AlkB, a DNA alkylation damage repair enzyme. By immunohistochemical analysis, PCA-1 was expressed in a high number of both prostate carcinoma samples and in the atypical cells within high-grade prostatic intraepithelial neoplasias but not in benign prostatic hyperplasia or normal adjacent tissues. PCA-1-transfected COS-7 cells further showed resistance against MMS-induced cell death. Conclusions: These findings suggest that PCA-1 could be a useful diagnostic marker. Furthermore, because this human counterpart of AlkB exhibits a protective function against alkylation damage in mammalian cells, PCA-1 may also serve as a therapeutic target molecule for prostate cancer.

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.

Phosphorylation status of fas-associated death domain-containing protein (FADD) is associated with prostate cancer progression

It has recently been demonstrated that phosphorylation of FADD at serine 194 plays an important role in the induction of apoptosis by anti‐cancer drugs in human prostate cancer cells. The present study has assessed whether this phosphorylation status is valuable as a marker for human prostate cancer progression, and has investigated its biological role in cell growth. Immunohistochemical studies revealed much higher phosphorylation of FADD at serine 194 in normal epithelial cells than in cancer cells, although FADD was found to be highly expressed to the same extent in both cases. The positivity for phosphorylated FADD was significantly lower for patients with a Gleason score greater than or equal to 7, a positive surgical margin, extracapsular or seminal vesicle invasion. In addition, a relationship was also apparent in cancer cells refractory to neoadjuvant hormonal therapy. Interestingly, in Gleason score 3 + 4 tumours, the positivity for FADD phosphorylation was statistically increased by neoadjuvant hormonal therapy, resulting in a reduced percentage of cases with a positive surgical margin and extracapsular invasion. In vitro data showed different functions of phosphorylated and non‐phosphorylated FADD: in normal epithelial cells, overexpression of a phosphorylation‐mimicking mutant FADD (S194E) caused G2/M cell‐cycle arrest, while a non‐phosphorylation‐mimicking mutant (S194A) had no effect, whereas S194A overexpression resulted in cell cycle progression and enhanced colony‐forming activity in cancer cells, but S194E FADD was without influence. These results clearly demonstrate that transition from phosphorylated FADD to the non‐phosphorylated form might be associated with carcinogenesis and that induction of FADD phosphorylation could therefore be a target for chemohormonal therapy of human prostate cancer. Moreover, assessment of FADD phosphorylation may be useful as a new biomarker to predict cancer progression. Copyright

Molecular pathology of prostate cancer

The incidence of prostate cancer has increased in Japan recently and is developing into a life‐threatening disease for many Japanese men. This is a result of several convergent factors including the adoption of a Western lifestyle, the widespread use of prostate‐specific antigen (PSA) testing, and an increased population of advanced years in Japanese men. Although there is much information to date relating to molecular events underlying the etiology of prostate cancer, it is still unclear as to how and when these genetic alterations occur in each step of tumorigenesis. One fruitful area of investigation has been in the analysis of chromosomal abnormalities commonly observed in prostate cancer. However, no single candidate gene has been definitely identified in cancer initiation and/or progression; in addition, less research has been devoted to understanding the molecular events that underlie tumor histogenesis in terms of likely precursor lesions, such as prostatic intraepithelial neoplasia (PIN). This article reviews the current knowledge of the molecular pathology of prostate cancer, including its histogenesis, genetic and epigenetic alterations, and hereditary factors.

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.

Frequent HRK inactivation associated with low apoptotic index in secondary glioblastomas

To detect and identify the genetic alterations and methylation status of the HRK gene in human glioblastomas, we analyzed a cohort of astrocytic tumors for hypermethylation, loss of heterozygosity on 12q13.1, and gene expression. Our study examined a series of 36 diffuse low-grade astrocytomas, 32 anaplastic astrocytomas, 64 primary glioblastomas, and 28 secondary glioblastomas that had evolved from either 24 low-grade diffuse astrocytomas or 4 anaplastic astrocytomas. The region around the HRK transcription start site was methylated in 19% of diffuse astrocytomas, in 22% of anaplastic astrocytomas, in 27% of primary glioblastomas, and in 43% of secondary glioblastomas. HRK expression was significantly reduced in 61% of secondary glioblastomas as compared to other types of tumors, and aberrant methylation was closely associated with loss of expression. Reverse transcription-PCR analysis also demonstrated a clear agreement between reduced HRK protein levels and low or absent HRK transcripts. Lack of HRK immunoreactivity was significantly correlated with a low apoptotic index, whereas a strong association between methylation status and apoptosis was found only in secondary glioblastomas. Abnormal methylation of HRK was detected in astrocytic tumors concurrent with methylation of multiple genes, including p16INK4a and p14ARF. Interestingly, these epigenetic changes in secondary glioblastoma were further associated with wild-type p53. Our findings suggest that HRK is inactivated mainly by aberrant DNA methylation in astrocytic tumors and that reduced HRK expression contributes to the loss of apoptotic control in high-grade tumors. Reduced expression of HRK may serve as one important molecular mechanism in progression to secondary glioblastoma.

FADD phosphorylation is critical for cell cycle regulation in breast cancer cells

Anti-oestrogen therapy is effective for control of hormone receptor-positive breast cancers, although the detailed molecular mechanisms, including signal transduction, remain unclear. We demonstrated here that long-term tamoxifen treatment causes G2/M cell cycle arrest through c-jun N-terminal kinase (JNK) activation, which is dependent on phosphorylation of Fas-associated death domain-containing protein (FADD) at 194 serine in an oestrogen (ER) receptor-positive breast cancer cell line, MCF-7. Expression of a dominant negative mutant form of MKK7, a kinase upstream of JNK, or mutant FADD (S194A) in MCF-7 cells suppressed the cytotoxicity of long-term tamoxifen treatment. Of great interest, similar signallings could be evoked by paclitaxel, even in an ER-negative cell line, MDA-MB-231. In addition, immunohistochemical analysis using human breast cancer specimens showed a close correlation between phosphorylated JNK and FADD expression, both being significantly reduced in cases with metastatic potential. We conclude that JNK-mediated phosphorylation of FADD plays an important role in the negative regulation of cell growth and metastasis, independent of the ER status of a breast cancer, so that JNK/FADD signals might be promising targets for cancer therapy.