Identification of sequences required for inhibition of oncogene-mediated transformation by pp32.

Oncogenic potential in prostate cancer is modulated in part by alternative use of genes of the pp32 family. This family includes the tumor suppressor pp32, expressed in normal tissue, and the pro-oncogenic genes pp32r1 and pp32r2 that are found principally in neoplastic cells. At the protein level, pp32, pp32r1, and pp32r2 are approximately 90% identical, yet they subsume opposite functions. In this study, we identify the region of pp32 associated with the ability to inhibit oncogene-mediated transformation in a rat embryo fibroblast system, an in vitro correlate of tumor-suppressive activity. Deletion and truncation analysis define a region spanning pp32 amino acids 150-174 as absolutely required for inhibition of transformed foci elicited by RAS and MYC. Comparison of pp32 with the pp32r1 sequence by moving averages of sequence identity reveals divergence over this region; pp32r2 also differs in this region through truncation after pp32 amino acid 131. The deletion experiments and the experiments of nature therefore converge to demonstrate that tumor-suppressive functions of pp32 reside in amino acids 150-174. Identification of this minimal tumor-suppressive region should help elaborate the pathways and mechanisms through which pp32 family members exert their functions.     

Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer

The regulation of intracellular Ca(2+) plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.     

Differentially Expressed Androgen-Regulated Genes in Androgen-Sensitive Tissues Reveal Potential Biomarkers of Early Prostate Cancer

Background

Several data favor androgen receptor implication in prostate cancer initiation through the induction of several gene activation programs. The aim of the study is to identify potential biomarkers for early diagnosis of prostate cancer (PCa) among androgen-regulated genes (ARG) and to evaluate comparative expression of these genes in normal prostate and normal prostate-related androgen-sensitive tissues that do not (or rarely) give rise to cancer.

Methods

ARG were selected in non-neoplastic adult human prostatic epithelial RWPE-1 cells stably expressing an exogenous human androgen receptor, using RNA-microarrays and validation by qRT-PCR. Expression of 48 preselected genes was quantified in tissue samples (seminal vesicles, prostate transitional zones and prostate cancers, benign prostatic hypertrophy obtained from surgical specimens) using TaqMan® low-density arrays. The diagnostic performances of these potential biomarkers were compared to that of genes known to be associated with PCa (i.e. PCA3 and DLX1).

Results and Discussion

By crossing expression studies in 26 matched PCa and normal prostate transitional zone samples, and 35 matched seminal vesicle and PCa samples, 14 genes were identified. Similarly, 9 genes were overexpressed in 15 benign prostatic hypertrophy samples, as compared to PCa samples. Overall, we selected 8 genes of interest to evaluate their diagnostic performances in comparison with that of PCA3 and DLX1. Among them, 3 genes: CRYAB, KCNMA1 and SDPR, were overexpressed in all 3 reference non-cancerous tissues. The areas under ROC curves of these genes reached those of PCA3 (0.91) and DLX1 (0.94).

Conclusions

We identified ARG with reduced expression in PCa and with significant diagnostic values for discriminating between cancerous and non-cancerous prostatic tissues, similar that of PCA3. Given their expression pattern, they could be considered as potentially protective against prostate cancer. Moreover, they could be complementary to known genes overexpressed in PCa and included along with them in multiplex diagnostic tools.     

Insulin Receptor Isoforms A and B as well as Insulin Receptor Substrates-1 and -2 Are Differentially Expressed in Prostate Cancer

Aims/Hypothesis

In different cancers types, insulin receptor isoform composition or insulin receptor substrate (IRS) isoforms are different to healthy tissue. This may be a molecular link to increased cancer risk in diabetes and obesity. Since this is yet unclear for prostate cancer, we investigated IR isoform composition and IRS balance in prostate cancer compared to benign and tumor adjacent benign prostate tissue and brought this into relation to cell proliferation.

Methods

We studied 23 benign prostate samples from radical cystectomy or benign prostatic hyperplasia surgery, 30 samples from benign tissue directly adjacent to prostate cancer foci and 35 cancer samples from different patients. RNA expression levels for insulin receptor isoforms A and B, IRS-1, IRS-2, and IGF-1 receptor were assessed by quantitative real-time RT-PCR. In addition, RNA- and protein expression of the cell cycle regulator p27^Kip1 was quantified by real-time RT-PCR and immunohistochemistry.

Results

Insulin receptor isoform A to B ratio was significantly higher in cancer as well as in tumor adjacent benign prostate tissue compared to purely benign prostates (p<0.05). IRS-1 to IRS-2 ratios were lower in malignant than in benign prostatic tissue (p<0.05). These altered ratios both in cancer and adjacent tissue were significantly associated with reduced p27^Kip1 content (p<0.02). Interestingly, IGF-1 receptor levels were significantly lower in patients with type 2 diabetes (p = 0.0019).

Conclusions/Interpretation

We found significant differences in the insulin signaling cascade between benign prostate tissue and prostate cancer. Histological benign tissue adjacent to cancer showed expression patterns similar to the malignancies. Our findings suggest a role of the insulin signaling pathway in prostate cancer and surrounding tissue and can hence be relevant for both novel diagnostic and therapeutic approaches in this malignancy.     

Markers of prostate region-specific epithelial identity define anatomical locations in the mouse prostate that are molecularly similar to human prostate cancers

Although the basic functions of the prostate gland are conserved among mammals, its morphology varies greatly among species. Comparative studies between mouse and human are important because mice are widely used to study prostate cancer, a disease that occurs in a region-restricted manner within the human prostate. An informatics-based approach was used to identify prostate-specific human genes as candidate markers of region-specific identity that might distinguish prostatic ducts prone to prostate cancer from ducts that rarely give rise to cancer. Subsequent analysis of normal and cancerous human prostates demonstrated that the genes microseminoprotein-beta (MSMB) and transglutaminase 4 (TGM4) were expressed in distinct groups of ducts in the normal human prostate, and only MSMB was detected in areas of prostate cancer. The mouse orthologs of MSMB and TGM4 were then used for expression studies in mice along with the mouse ventrally expressed gene spermine binding protein (SBP). All three genes were informative markers of region-specific epithelial identity with distinct expression patterns that collectively accounted for all ducts in the mouse prostate. Together with the human data, this suggested that MSMB expression defines an anatomical domain in the mouse prostate that is molecularly most similar to human prostate cancers. Computer-assisted serial section reconstruction was used to visualize the complete expression domains for MSMB, SBP, and TGM4 in the mouse prostate. This showed that MSMB is expressed in prostatic ducts that comprise 21% of the mouse dorso-lateral prostate. Finally, the expression of MSMB, SBP, and TGM4 was evaluated in a mouse prostate cancer model created by the prostate epithelium-specific deletion of the tumor suppressor PTEN. MSMB and TGM4 were rapidly and dramatically down-regulated in response to PTEN deletion suggesting that this model of prostate cancer includes a more rapid de-differentiation of the prostatic epithelium than is observed in organ-confined human prostate cancers.     

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.     

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer

Background: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. Methods: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. Results: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. Conclusions: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.     

Frequent 14-3-3 sigma promoter methylation in benign and malignant prostate lesions

14-3-3Sigma is a putative tumor suppressor gene involved in cell cycle regulation and apoptosis following DNA damage. 14-3-3Sigma loss of expression has been reported is several human cancers, including prostate adenocarcinoma and precursor lesions, and promoter hypermethylation has been proposed as the mechanism underlying gene silencing. Here, we investigate the frequency and extent of 14-3-3sigma promoter methylation in benign and cancerous prostate tissues. We examined tumor tissue from 121 patients with prostate carcinoma (PCa), 39 paired high-grade prostatic intraepithelial neoplasias (HGPIN), 29 patients with benign prostate hyperplasia (BPH), as well as four prostate cancer cell lines using quantitative methylation-specific PCR (QMSP). The percentage of methylated alleles (PMA) was calculated and correlated with clinical and pathological parameters. RT-PCR was performed in the cell lines to assess 14-3-3sigma mRNA expression. PCa, HGPIN, BPH, and cancer cell lines showed ubiquitous 14-3-3sigma promoter methylation. However, the PMA of HGPIN was significantly lower than that of PCa or BPH (P < 0.0001), while PCa and BPH did not significantly differ. The PMA did not correlate with any clinicopathological parameter. All prostate cancer cell lines expressed 14-3-3sigmamRNA. 14-3-3Sigma promoter methylation is a frequent event in prostate tissues and cancer cell lines. Furthermore, there is a progressive accumulation of neoplastic cells with 14-3-3sigma methylated alleles from HGPIN to PCa, suggesting a role for this epigenetic event in prostate carcinogenesis. However, other mechanisms besides promoter methylation might be required for effective 14-3-3sigma downregulation.     

Fibrils of Prostatic Acid Phosphatase Fragments Boost Infections with XMRV (Xenotropic Murine Leukemia Virus-Related Virus), a Human Retrovirus Associated with Prostate Cancer

The xenotropic murine leukemia virus-related virus (XMRV) has recently been detected in prostate cancer tissues and may play a role in tumorigenesis. It is currently unclear how this virus is transmitted and which factors promote its spread in the prostate. We show that amyloidogenic fragments known as semen-derived enhancer of virus infection (SEVI) originating from prostatic acid phosphatase greatly increase XMRV infections of primary prostatic epithelial and stromal cells. Hybrid simian/human immunodeficiency chimeric virus particles pseudotyped with XMRV envelope protein were used to demonstrate that the enhancing effect of SEVI, or of human semen itself, was at the level of viral attachment and entry. SEVI enhanced XMRV infectivity but did not bypass the requirement for the xenotropic and polytropic retrovirus receptor 1. Furthermore, XMRV RNA was detected in prostatic secretions of some men with prostate cancer. The fact that the precursor of SEVI is produced in abundance by the prostate indicates that XMRV replication occurs in an environment that provides a natural enhancer of viral infection, and this may play a role in the spread of this virus in the human population.Viruses are etiologic agents of various human cancers, including cervical carcinoma (caused by human papillomavirus), Kaposi''s sarcoma (caused by human herpesvirus 8), hepatocellular carcinoma (caused by hepatitis B virus and hepatitis C virus), and adult T-cell leukemia (caused by human T-cell leukemia virus type 1) (6). Genetic and epidemiologic evidence suggests that prostate cancer may also have an infectious etiology, although a causative agent has not been identified (4, 12). The gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV) is a candidate human tumor virus based on its association in human prostate tumors with a reduced-activity variant of the antiviral gene, RNASEL (also known as the hereditary prostate cancer 1 gene or HPC1) (17) and because it is a member of a viral family known to cause leukemias and lymphomas in different mammalian species (8). Interferon, through its effector RNase L, potently inhibits XMRV replication (5). XMRV integration sites in human prostate cancer tissues were mapped to cancer breakpoints, common fragile sites, micro-RNA genes, and cancer-related genes (11). Many of these genes are implicated directly or indirectly in prostate cancer and metabolic pathways that affect prostate cancer, including androgen signaling. XMRV has also been observed in prostate tissue from a nonfamilial prostate cancer patient and in an individual without prostate cancer (7). The possible role of XMRV in prostatic cancer raises questions about its ability to infect the prostate and the route of viral transmission.Recently, it has been shown that fragments of prostatic acid phosphatase (PAP), an abundant nonspecific protein phosphatase produced by the prostate (18) and secreted in semen in large quantities (about 2 mg/ml) (16), form amyloid fibrils that drastically enhance human immunodeficiency virus type 1 (HIV-1) infection (14). The fibrils of PAP_248-286, termed semen-derived enhancer of virus infection (SEVI), enhanced the infectious virus titer by several orders of magnitude by capturing HIV-1 virions and promoting their attachment to target cells. The ability of SEVI to promote the interaction between virions and the cell surface is independent of the viral glycoprotein and hence is not restricted to HIV-1, although subsequent fusion between the viral and cellular membranes still required gp120, CD4, and an appropriate coreceptor (14). A recent study indicates that the positive charges on SEVI (pI = 10.21) promote infectivity by neutralizing negative-charge repulsion between HIV particles and the cell surface (15).Because SEVI originates from the prostate (the organ from which XMRV infection was discovered [17]) and promotes viral attachment in a relatively nonspecific manner, we sought to determine its effect on XMRV infection. Here we demonstrate that XMRV infectivity is greatly enhanced by SEVI or human semen and that XMRV RNA is detectable in expressed prostatic secretions (EPS) from human tumor-bearing prostates.     

Identification of genes differentially expressed in benign prostatic hyperplasia.

Differences between benign prostatic hyperplasia (BPH) and normal prostate tissue at the level of mRNA expression provide an opportunity to identify candidate genes for this disease. A cDNA subtraction procedure was used to isolate differentially expressed genes in BPH. The subtraction was done by solution hybridization of BPH cDNA against excess normal prostate cDNA. We identified known, EST, and novel genes by sequence and database analysis of the subtracted cDNAs. Several of these cDNAs were used as probes in Northern blotting analysis to confirm over-expression of their corresponding mRNAs in BPH tissues. One highly upregulated sequence of interest shared identity with a known mRNA encoding human NELL2, a protein containing epidermal growth factor-like domains. NELL2 was not previously reported to be expressed in prostate and may code for a novel prostatic growth factor. In situ hybridization analysis of hyperplastic prostate specimens demonstrated that NELL2 mRNA expression is predominantly localized in basal cells of the epithelium. Disease-related changes in the levels of NELL2 may contribute to alterations in epithelial-stromal homeostasis in BPH. (J Histochem Cytochem 49:669-670, 2001)     

Localization of annexins I, II, IV and VII in whole prostate sections from radical prostatectomy patients

Annexins (ANXs) represent a family of calcium and phospholipid binding proteins that are involved in several physiological processes e.g. signal transduction, cellular differentiation and proliferation. Since they are known to be dysregulated in a variety of cancers we investigated the immunolocalization of ANXs in whole prostate sections containing benign prostatic epithelium (BPE), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN) and prostate cancer (PCa) in order to evaluate their possible role during tumorigenesis. Samples were obtained from 28 patients undergoing radical prostatectomy. Gross sections of whole prostates were examined immunohistochemically for the distribution of ANX I, II, IV and VII. In BPE all ANXs were localized to the cell membranes and the cytoplasm of all gland cells. In BPH the immunoreactivity of ANX I and II was restricted to the basal cells of glands and expression pattern of ANX IV and VII was similar to BPE. In PIN only basal cells expressed ANX II. In PCa ANX II immunoreactivity was absent and weak ANX I and ANX IV immunoreactivity was restricted to the cytoplasm of tumor cells. ANX VII immunoreactivity was seen in some but not all tumor cells. Since ANX IV and VII expression did not show significant changes in PCa compared to non-neoplastic tissue and PIN an essential role during prostate tumourigenesis seems unlikely. In contrast, as progression from PIN to PCa is characterized by a reduction of ANX I and II this suggests that downregulation of these proteins could represent an important event in prostate carcinogenesis.     

Prostate cancer vs hyperplasia: relationships with prostatic and adipose tissue fatty acid composition

The objective of the present study was to study whether adipose tissue and prostatic tissue fatty acid composition differentiates between prostate cancer and benign hyperplasia patients. In addition, the present investigation aimed at exploring the extent to which prostatic tissue fatty acid composition differentiates between prostate-confined cancer and extraprostatic disease including possible metastasis. The subjects were 71 male patients from the island of Crete. Half the patients (n=35) had been diagnosed with benign hyperplasia of the prostate, half with prostatic malignancy (n=36). Patients were examined at the outpatient clinic of the urology unit, University Hospital, Medical School, University of Crete. Relative to benign hyperplasia patients, cancer patients had elevated adipose tissue saturated and reduced monounsaturated fatty acid levels. Cancer patients had reduced prostate tissue stearic to oleic acid ratios and stearic acid levels as opposed to hyperplasia patients. The most pronounced difference between cancer patients and hyperplasia patients was a 3-fold elevated prostatic palmitoleic acid in the former group. Relative to benign hyperplasia patients, cancer patients had reduced prostate tissue arachidonic and docosahexaenoic acid levels. Finally, there was a significantly reduced omega-3/omega-6 polyunsaturated fatty acid ratio in the prostate cancer patient as opposed to the benign hyperplasia group. The pronounced elevations in prostatic tissue palmitoleic acid in cancer patients highlight a possible role of this fatty acid in neoplastic processes. The decreased arachidonic acid levels in cancer patients possibly stem from enhanced metabolism of arachidonic acid via lipoxygenase and cyclooxygenase pathways, and the formation of derivatives such as 5-HETE, 15-HETE, 12(S)-HETE and PGE(2).