Growth inhibition of prostate cancer by an adenovirus expressing a novel tumor suppressor gene, pHyde

It has been estimated that there will be > 180,400 new cases of prostate cancer and 31,900 prostate cancer deaths in the United States this year. New therapeutic strategies against locally advanced prostate cancer are desperately needed. A novel gene (pHyde) was identified by an improved cDNA competition hybridization technique for Dunning rat prostate cancer cell lines. A recombinant replication-deficient E1/E3-deleted adenovirus type 5 containing a pHyde gene under the control of a truncated Rous sarcoma virus (RSV) promoter (AdRSVpHyde) was generated. In vitro, AdRSVpHyde significantly inhibited growth of human prostate cancer cell lines DU145 and LNCaP in culture. In vivo, a single injection of AdRSVpHyde (5 x 10(9) plaque-forming units) reduced DU145 tumors in nude mice remarkably compared with untreated control or viral control-treated DU145 tumors. Moreover, AdRSVpHyde induced apoptosis and stimulated p53 expression. These results together suggest that pHyde is a tumor suppressor gene that inhibits growth of prostate cancer and that this inhibition is at least in part due to the induction of apoptosis.     

Apoptosis induction in prostate cancer cells by a novel gene product, pHyde, involves caspase-3

A novel gene, pHyde, was recently cloned from Dunning rat prostate cancer cells. A recombinant adenovirus containing pHyde cDNA gene (AdpHyde) was generated to investigate the biological function of pHyde protein. AdpHyde inhibited the growth of human prostate cancer cells. Apoptosis was induced in AdpHyde transduced cells as demonstrated by DAPI (4', 6-diamino-2-phenylindole), TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling) staining, and flow cytometry assays. Apoptosis was also induced in human xenograft prostate cancer tumors growing in nude mice following treatment with AdpHyde. AdpHyde transduction resulted in a dose-dependent stimulation of caspase-3 activity in DU145 cells which was blocked by DEVD (succinyl-Asp-Glu-Val-Asp-aldehyde) and VAD (benzyloxycarbonyl - Val - Ala - Asp -fluoromethylketone), inhibitors specifically against caspase-3. Moreover, cancer cells that lacked expression of endogenous caspase-3 were not or barely inhibited by pHyde. These results taken together suggest that pHyde inhibits cancer growth by inducing apoptosis through a caspase-3 dependent pathway.     

Glioma pathogenesis-related protein 1 exerts tumor suppressor activities through proapoptotic reactive oxygen species-c-Jun-NH2 kinase signaling

Glioma pathogenesis-related protein 1 (GLIPR1), a novel p53 target gene, is down-regulated by methylation in prostate cancer and has p53-dependent and -independent proapoptotic activities in tumor cells. These properties suggest an important tumor suppressor role for GLIPR1, yet direct genetic evidence of a tumor suppressor function for GLIPR1 is lacking and the molecular mechanism(s), through which GLIPR1 exerts its tumor suppressor functions, has not been shown. Here, we report that the expression of GLIPR1 is significantly reduced in human prostate tumor tissues compared with adjacent normal prostate tissues and in multiple human cancer cell lines. Overexpression of GLIPR1 in cancer cells leads to suppression of colony growth and induction of apoptosis. Mice with an inactivated Glipr1 gene had significantly shorter tumor-free survival times than either Glipr1(+/+) or Glipr1(+/-) mice in both p53(+/+) and p53(+/-) genetic backgrounds, owing to their development of a unique array of malignant tumors. Mechanistic analysis indicated that GLIPR1 up-regulation increases the production of reactive oxygen species (ROS) leading to apoptosis through activation of the c-Jun-NH(2) kinase (JNK) signaling cascade. Thus, our results identify GLIPR1 as a proapoptotic tumor suppressor acting through the ROS-JNK pathway and support the therapeutic potential for this protein.     

Deletion at 13q21 is associated with aggressive prostate cancers

Previous cytogenetic and molecular genetic analyses suggest that the q21 band of chromosome 13 harbors a tumor suppressor gene(s) involved in prostatic carcinogenesis. The precise genetic location, however, has not been defined. In this study, we examined prostate cancer specimens and cell lines/xenograft for genetic deletions at 13q21, using the methods of tissue microdissection and duplex PCR. Deletions at 13q21 were detected in 13 of 147 (9%) prostate cancer samples. Deletion of the same region was also detected in the LNCaP cell line and the PC-82 xenograft of prostate cancer. The overlapping region of deletion in LNCaP and PC-82 spans 3.1 cM or 2.9 cR, which is equivalent to 1-3 Mb. The endothelin receptor B gene, a possible tumor suppressor gene at 13q21, was not located in the region of deletion. Among the 13 prostate neoplasms with deletion at 13q21, 5 were metastases, and 7 were poorly differentiated primary tumors. The only primary tumor that was not poorly differentiated but had deletion occurred in one of the youngest patients (49 years) at diagnosis. These results provide evidence that 13q21 may harbor an unidentified gene(s) whose inactivation occurs in some aggressive carcinomas of the prostate. In addition, this study provides a framework for the cloning and identification of the 13q21 gene(s).     

Loss of heterozygosity and tumor suppressor activity of Bin1 in prostate carcinoma

The genetic events underlying the development of prostate cancer are poorly defined. c-Myc is often activated in tumors that have progressed to metastatic status, so events that promote this process may be important. Bin1 is a nucleocytoplasmic adaptor protein with features of a tumor suppressor that was identified through its ability to interact with and inhibit malignant transformation by c-Myc. We investigated a role for Bin1 loss or inactivation in prostate cancer because the human Bin1 gene is located at chromosome 2q14 within a region that is frequently deleted in metastatic prostate cancer but where no tumor suppressor candidate has been located. A novel polymorphic microsatellite marker located within intron 5 of the human Bin1 gene was used to demonstrate loss of heterozygosity and coding alteration in 40% of informative cases of prostate neoplasia examined. RNA and immunohistochemical analyses indicated that Bin1 was expressed in most primary tumors, even at slightly elevated levels relative to benign tissues, but that it was frequently missing or inactivated by aberrant splicing in metastatic tumors and androgen-independent tumor cell lines. Ectopic expression of Bin1 suppressed the growth of prostate cancer lines in vitro. Our findings support the candidacy of Bin1 as the chromosome 2q prostate tumor suppressor gene.     

Alterations in the structural gene and the expression of p53 in rat liver tumors induced by aflatoxin B1.

Rat hepatocellular carcinomas (HCCs) induced by aflatoxin B1 (AFB) treatment were examined for changes in the p53 tumor suppressor gene and in p53 suppressor gene expression. A high proportion of HCCs (nine of 11 tumors in six of eight animals) exhibited new p53 restriction fragments, indicating genomic alterations of one of the p53 alleles. Each tumor with an altered p53 restriction-fragment pattern exhibited a new fragment in one of two size classes (3 kb or 7 kb with EcoRI digestion) that were missing portions of the 3' end of the p53 gene. These findings indicate that apparently similar genomic rearrangements or deletions occurred independently in AFB-induced tumors. When compared with nontumor liver tissue from the same animal, the tumors with p53 gene alterations showed dramatically reduced levels of p53 mRNA and protein and greatly increased levels of histone H2B and retinoblastoma tumor suppressor (Rb) mRNA. In two HCCs showing no evidence of p53 restriction-fragment alterations, mutant p53 protein was detected. Mutant protein was also detected in two liver samples containing an adenoma and altered foci. These data suggest that alterations of the p53 tumor suppressor gene are involved in the induction of rat HCC by AFB.     

KAI1 metastasis suppressor protein is down-regulated during the progression of human endometrial cancer.

PURPOSE: KAI1 is a metastasis suppressor gene located on human chromosome 11p11.2. It is a member of the structurally distinct family of cell surface glycoprotein, transmembrane 4 protein superfamily. KAI1 was initially isolated as a gene that suppressed metastasis of rat prostate tumor cells. Decreased KAI1 expression has been observed recently in various human cancers, including pancreatic, lung, hepatic, colorectal, breast, ovarian, esophageal, and cervical cancers. Frequent down-regulation of the KAI1 protein was also observed in endometrial cancer cell lines. The aim of this study was to determine whether this gene is altered in human endometrial carcinoma. In addition, its prognostic significance in this tumor was also evaluated. EXPERIMENTAL DESIGN: Tumor specimens from 18 cases with various degrees of endometrial hyperplasia, 97 primary endometrial carcinomas with various stages, and 28 metastatic lesions of this cancer were examined in this study. Using the method of immunohistochemistry, we characterized the KAI1 protein expression in the 143 endometrial tumors. Expression of KAI1 at RNA level was also examined in 35 of the 143 samples using a real-time quantitative PCR method. The data from immunohistochemical analysis were correlated with various clinicopathological factors. RESULTS: High levels of KAI1 protein expression were detected in almost all of the specimens with endometrial hyperplasia (17 of 18). In contrast, loss of KAI1 expression occurred in an increasing frequency (27.8-71.4%) from early stages of primary endometrial carcinomas to metastatic tumors (P < 0.001). In addition, more poorly differentiated tumors demonstrated significantly lower KAI1 expression as compared with the well-differentiated tumors (P < 0.001). It was also found that patients with KAI1-negative tumors had a lower survival rate than those with KAI1-decreased or positive tumors (P = 0.0042 and 0.0286, respectively). However, in multivariate analysis, the prognostic significance of KAI1 expression was inferior to tumor stage. CONCLUSION: These data suggest that KAI1 expression is down-regulated in advanced endometrial cancer. Clinically it may be a useful indicator of the tumor progression and may provide prognostic information on the outcome of this disease.     

FHITness and cancer

In early 1996, the Fragile Histidine Triad or FHIT gene (pronounced FIT) was cloned and shown to straddle the most fragile human chromosome site at chromosome band 3p14.2. The exceptionally large FHIT locus also encompasses a hereditary renal carcinoma associated chromosome translocation breakpoint and is very frequently altered by internal deletions in the most common human cancers. Germline alteration of one allele in familial cancer and deletions within the gene in sporadic cancers are hallmarks of tumor suppressor genes. Some of the DNA and RNA alterations exhibited by the FHIT gene in cancers showed features not previously encountered for known tumor suppressor genes, prompting a number of investigators to reject FHIT as a suppressor gene. However, evidence continues to accumulate, demonstrating that FHIT inactivation occurs in the majority of lung, gastric, cervical, and kidney carcinomas and that replacement of Fhit expression in such cancer cells suppresses their tumorigenicity.     

RTVP-1, a tumor suppressor inactivated by methylation in prostate cancer

We previously identified and characterized a novel p53-regulated gene in mouse prostate cancer cells that was homologous to a human gene that had been identified in brain cancers and termed RTVP-1 or GLIPR. In this report, we document that the human RTVP-1 gene is also regulated by p53 and induces apoptosis in human prostate cancer cell lines. We show that the expression of the human RTVP-1 gene is down-regulated in human prostate cancer specimens compared with normal human prostate tissue at the mRNA and protein levels. We further document epigenetic changes consistent with RTVP-1 being a tumor suppressor in human prostate cancer.     

New prognostic factors in human gastric carcinomas]

Correlation between the expression of growth factor/receptor systems or the alterations of tumor suppressor genes and biological malignancy of gastric cancer was described. Overexpression of many growth factors/receptors, such as EGF, TGF alpha, EGF receptor and ERBB2, and reduction of type I receptor for TGF beta may be linked with new prognostic factors of gastric carcinomas. The expression of cripto, a novel gene of EGF family, shows a tendency to correlate with tumor staging of well differentiated gastric adenocarcinomas. p53 gene abnormalities take place in 60% of gastric carcinomas including early stage carcinoma. Loss of heterozygosity on chromosomes 1q, 7p and 7q is frequently observed in advanced gastric carcinomas of well differentiated type. Molecules which regulate tumor invasion and metastasis such as nm23, tissue inhibitor of metalloproteinase (TIMP) and endogenous galactoside-binding lectin may provide for prognostic factors of gastric cancer.     

Down-regulation of fragile histidine triad expression in prostate carcinoma

BACKGROUND: The fragile histidine triad (FHIT) gene is a tumor suppressor gene that belongs to the histidine triad family of nucleoside binding proteins. The gene encompasses the common human chromosomal fragile site, the FRA3B locus at chromosome 3p14.2, and is expressed in most normal adult tissues and tumor cell lines. Numerous studies have indicated that the FHIT gene on chromosome 3p may play an important role in human neoplasia, although very few studies have investigated the FHIT gene in prostate carcinoma. METHODS: Using immunohistochemical analyses, the authors studied the expression of FHIT in prostate tumors from 84 radical prostatectomy specimens to determine whether there were any correlations between FHIT expression and various clinicopathologic characteristics. RESULTS: The percentages of cells stained with antibody to FHIT were significantly lower overall for tumor cells compared with normal cells (P = 0.0001). FHIT immunostaining intensity also was significantly lower for tumor cells compared with normal cells (P = 0.0001). A weak but statistically significant correlation (P = 0.045) was demonstrated with the presence of extraprostatic extension in the patient samples. No other significant correlation was seen between the percentage of cells stained for FHIT or FHIT immunostaining intensity and Gleason grade, tumor stage, tumor size, lymph node metastasis, surgical margins, vascular invasion, perineural invasion, or the presence of high-grade prostatic intraepithelial neoplasia. CONCLUSIONS: The data presented indicate a down-regulation of the FHIT tumor suppressor gene in prostate carcinoma and, thus, propose a potential target for therapeutic intervention.     

Protein phosphatase and TRAIL receptor genes as new candidate tumor genes on chromosome 8p in prostate cancer

BACKGROUND: Allelic losses on chromosome 8p are common in prostate carcinoma, but it is not known exactly how they contribute to cancer development and progression. MATERIALS AND METHODS: Expression of 12 genes located across chromosome 8p, including established tumor suppressor candidates (CSMD1, DLC1, NKX3.1), and others from a new microarray-based comparison was studied by quantitative RT-PCR in 45 M0 prostate carcinomas and 13 benign prostate tissues. RESULTS: Significantly reduced expression was observed for two protein phosphatase subunit genes (PPP2CB, PPP3CC) and two TRAIL decoy receptors (TNFRSF10C/DcR1, TNFRSF10D/DcR2), but not for the three established candidates nor for TRAIL death receptor genes. Low expression of PPP3CC and TNFRSF10C located at 8p21.3 was highly significantly associated with tumor recurrence. In addition to allele loss, down-regulation of TNFRSF10C and TNFRSF10D was found to be associated with hypermethylation, although bisulfite sequencing usually revealed it to be partial. CONCLUSION: Our data strongly support a recent proposal that a segment at 8p21.3 contains crucial prostate cancer tumor suppressors. In addition, they raise the paradoxical issue of why TRAIL decoy receptors rather than death receptors are down-regulated in aggressive prostate cancer.     

p53 mutation profiling of multiple esophageal carcinoma using laser capture microdissection to demonstrate field carcinogenesis

Inactivation of the p53 tumor suppressor gene is one of the most frequent genetic alterations observed in human esophageal carcinomas. In patients with esophageal carcinoma, one of the significant pathological features of the tumor is the presence of multiple lesions within the esophagus. However, the molecular mechanisms involved in the occurrence of multiple lesions have remained elusive. To characterize p53 alterations in multiple esophageal carcinomas and to study their roles in carcinogenesis, we performed p53 immunohistochemical and p53 mutation analyses using laser capture microdissection on surgically resected human esophageal carcinomas from 11 patients: 9 patients with multiple esophageal carcinomas, 1 with an intramural metastasis lesion within the esophagus and 1 with an intraepithelial carcinoma lesion contiguous to the main lesion. In each of the patients with multiple esophageal carcinomas, we examined samples from 1 main lesion and 1 representative concomitant lesion. Molecular analyses of samples from fresh-frozen normal tissues and tumor tissues of the main lesion (whole tumor) were also performed by the same method. p53 protein accumulation was observed in 16 (72.7%) of 22 lesions from the 11 cases. No p53 mutation was found in normal esophageal tissues. In the 9 cases of multiple esophageal carcinomas, point mutations were detected in the whole tumor in 1 (11.1%) case, in the microdissected tumor samples of main lesions in 3 (33.3%) cases and in the microdissected tumor samples of concomitant lesions in 3 (33.3%) cases. For the microdissected tumor samples, there was a 54.5% (12/22) concordance rate between the results of immunostaining and molecular analysis. In the 8 cases of whole tumors in which a p53 mutation was not observed, 2 cases revealed p53 mutation in the microdissected tumor samples of the main lesion. All 6 cases of multiple esophageal carcinomas that showed a p53 mutation in the microdissected tumor sample had a discordant p53 mutational status between the main and concomitant lesions. In contrast, both the intramural metastasis lesion and the intraepithelial carcinoma contiguous to the main lesion showed p53 mutational patterns identical to those of the main lesions. In conclusion, the analysis of microdissected DNA by laser capture microdissection is useful for characterizing the heterogeneity of the p53 gene mutation in multiple carcinoma lesions that cannot be accurately analyzed in whole esophageal tumor DNA. The finding of different p53 gene mutations among multiple esophageal carcinoma lesions suggest further evidence of multicentric or field carcinogenesis of the human esophagus.     

LOH of chromosome 12p correlates with Kras2 mutation in non-small cell lung cancer

Previous observation has shown that the wild-type Kras2 allele is a suppressor of lung cancer in mice. Here we report that loss of heterozygosity (LOH) of chromosome 12p was detected in approximately 50% of human lung adenocarcinomas and large cell carcinomas, and Kras2 mutations were detected at codon 12 in approximately 40% of adenocarcinomas and large cell carcinomas. Interestingly, all of the lung adenocarcinomas and large cell carcinomas containing a Kras2 mutation exhibited allelic loss of the wild-type Kras2 allele when a correlation between LOH of the region on chromosome 12p and Kras2 mutation was made. These results from human lung cancer tissues provide a strong evidence in support of our previous observation in mouse models that the wild-type Kras2 is a tumor suppressor of lung cancer.     

Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.

In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.12-14.2. The DICE1 gene is highly conserved in evolution and its mRNA is expressed in a wide variety of fetal and adult tissues. The DICE1 cDNA encodes a predicted protein of 887 amino acids corresponding to an 100 kD protein that shows 92.9% identity to the carboxy-terminal half of the mouse EGF repeat transmembrane protein DBI-1. The DBI-1 protein interferes with the mitogenic response to insulin-like growth factor 1 (IGF-I) and is presumably involved in anchorage-dependent growth. When compared to normal lung tissue expression of the DICE1 mRNA was reduced or undetectable in the majority of non-small cell lung carcinomas analysed. The location of the DICE1 gene in the region of allelic loss, its high evolutionary conservation and the downregulation of expression in carcinoma cells suggests that DICE1 is a candidate tumor suppressor gene in non-small cell lung carcinomas and possibly in other sporadic carcinomas.     

p53 Gene Mutations Associated with Anaplastic Transformation of Human Thyroid Carcinomas

Anaplastic carcinoma of the thyroid gland, which is one of the most aggressive, malignant tumors in humans, is considered to originate from preexisting differentiated thyroid cancer. To define the genetic alterations associated with such progression, we examined nine cases of anaplastic thyroid carcinoma for mutation in exons 4–9 of the p53 tumor suppressor gene. Preliminary screening for mutation by RNase protection analysis demonstrated that two out of nine anaplastic carcinomas contained sequence alterations in the p53 gene. Subsequent DNA sequencing identified the mutated nucleotides in these two cases; one was a nonsense mutation at codon 165, and the other was a single-base deletion at codon 176 resulting in the creation of a stop codon downstream due to frameshift. The fact that no mutations were detected in coexisting foci of papillary carcinomas from the same patients shows that these mutations of the p53 gene occurred after development of papillary carcinomas. These results suggest that p53 gene mutation triggers the progression from differentiated into anaplastic carcinoma in the human thyroid gland.