Immunohistochemical expression of prostate tumor overexpressed 1 in cystoprostatectomies with incidental and insignificant prostate cancer. Further evidence for field effect in prostatic carcinogenesis

Prostate tumor overexpressed 1 was recently identified as a novel gene and protein during a differential display screening for genes overexpressed in prostate cancer. It has been suggested that overexpression of prostate tumor overexpressed 1 can contribute to the proliferative status of prostate tumor cells and, thus, to their biologic behavior. Prostate tumor overexpressed 1 and Ki-67 were immunohistochemically evaluated in prostate cancer, high-grade prostatic intraepithelial neoplasia, and normal-looking epithelium in 20 cystoprostatectomies and 20 radical prostatectomies with pT2a Gleason score 6 prostate cancer. The aim was to see whether there were differences in marker expression between cystoprostatectomies and radical prostatectomies. The proportions of prostate tumor overexpressed 1– and Ki-67–positive cells in the cystoprostatectomies and radical prostatectomies increased from normal-looking epithelium through high-grade prostatic intraepithelial neoplasia, away from and adjacent to prostate cancer, to prostate cancer. Prostate tumor overexpressed 1 expression in prostate cancer in cystoprostatectomies was lower than in radical prostatectomies, the differences being significant; there were significant differences in Ki-67 indices. In conclusion, our findings related to prostate tumor overexpressed 1 expression in high-grade prostatic intraepithelial neoplasia, evaluated adjacent and away from prostate cancer, and in incidental and clinical cancers give further support to the concept of field effect in prostatic carcinogenesis as well as to differences in the process of prostatic carcinogenesis between cystoprostatectomies and radical prostatectomies.     

Overexpression of IGBFB2 is a marker for malignant transformation in prostate epithelium

Insulin-like growth factor binding protein 2 (IGFBP2) has been shown to be overexpressed in prostatic intraepithelial neoplasia (PIN) and invasive cancer and the serum level to parallel that of prostate-specific antigen (PSA) in prostate cancer. A combination of cDNA and tissue microarray results recently demonstrated overexpression of IGFBP2 in hormone refractory prostate cancer, indicating that the IGF system may be part of a key growth regulatory pathway in prostate cancer. The present study reexamines the immunohistochemical expression of IGFBP2 and its relationship to grade in tissue from 193 radical prostatectomy specimens from patients with localized prostate adenocarcinoma. We found a significant overexpression of IGFBP2 in all instances of PIN and in more than 90% of cancers regardless of the grade. An intense overexpression was noted in the neuroendocrine cells in normal glands as well as in cancer. The IGFBP2 expression was also analyzed in 18 cases of biopsy diagnosed prostate cancer. In all these cases, the glands interpreted as invasive cancer in hematoxylin-eosin stained sections overexpressed IGFBP2, without a significant correlation to grade. We conclude that overexpression of IGFBP2 is a powerful marker for malignant transformation in the prostate epithelium and suggest that optimized immunohistochemical detection of IGFBP2 expression may be an adjunct tool in the diagnosis of prostate cancer.     

Expression of group IIA secretory phospholipase A2 is elevated in prostatic intraepithelial neoplasia and adenocarcinoma

Phospholipase A2 (PLA2) enzymes release arachidonic acid from cellular phospholipids in a variety of mammalian tissues, including prostate. Group IIa secretory PLA2 (sPLA2) can generate arachidonate from cellular phospholipids. We examined the group IIa sPLA2 expression in benign prostatic tissues, prostatic intraepithelial neoplasia (PIN), and adenocarcinoma to determine whether sPLA2 expression is altered in the carcinogenesis of human prostatic cancer. Thirty-three of 74 total cases (45%) of benign prostatic tissue showed positive immunohistochemical staining for group IIA sPLA2, whereas 63 of 69 total cases (91%) of high-grade PINs and 70 of 78 total cases (90%) of adenocarcinomas gave positive results. Four of 10 cases of low-grade PIN showed positive immunoreactivity for sPLA2. The number of cells staining for sPLA2 was significantly less in benign epithelium (4%) and low-grade PIN (4%) compared to high-grade PIN (40%) or adenocarcinoma (38%) (P < 0.001). There was no significant difference between high-grade PIN and adenocarcinoma in the number of cells staining positively for sPLA2. The intensity of sPLA2 immunoreactivity was also different among benign prostatic tissue, low-grade PIN, high-grade PIN, and prostatic adenocarcinoma specimens. The malignant cells demonstrated more intense immunohistochemical staining (moderate to strong staining in 81% and 69% cases for high-grade PIN and adenocarcinoma, respectively) than benign glands (moderate staining in 11% of cases). No strong staining was observed in benign glands or low-grade PIN. Our data are consistent with the contention that group IIA sPLA2 expression is elevated in neoplastic prostatic tissue and support the hypothesis that dysregulation of sPLA2 may play a role in prostatic carcinogenesis.     

Skp2 overexpression is associated with loss of BRCA2 protein in human prostate cancer

BRCA2 (breast cancer 2, early onset) is a tumor suppressor gene that confers increased susceptibility for prostate cancer (PCa). Previous in vitro experiments demonstrated that Skp2, an E3 ubiquitin ligase aberrantly overexpressed in PCa, is involved in the proteolytic degradation of BRCA2 in PCa cells, suggesting that the BRCA2-Skp2 interaction may play a role in prostate tumorigenesis. Herein, we investigated BRCA2 and Skp2 expression during PCa development using a prostate TMA. Although luminal and basal benign prostate epithelium exhibited moderate to strong nuclear BRCA2 immunostaining, the intensity and number of positive nuclei decreased significantly in high-grade prostatic intraepithelial neoplasia and PCa. Decreased frequency and intensity of nuclear BRCA2 labeling were inversely correlated with Skp2 expression in high-grade prostatic intraepithelial neoplasia and PCa. To functionally assess the effects of BRCA2 and Skp2 expression on prostate malignant transformation, we overexpressed Skp2 in normal immortalized prostate cells. Skp2 overexpression reduced BRCA2 protein and promoted cell growth and migration. A similar phenotype was observed after reduction of BRCA2 protein levels using specific BRCA2 small-interfering RNA. Forced BRCA2 expression in Skp2-overexpressing stable transfectants inhibited the migratory and growth properties by >60%. These results show that loss of BRCA2 expression during prostate tumor development is strongly correlated with both migratory behavior and cancer growth and include Skp2 as a BRCA2 proteolytic partner in vivo.     

High-level expression of fatty acid synthase in human prostate cancer tissues is linked to activation and nuclear localization of Akt/PKB

Many human epithelial cancers, particularly those with a poor prognosis, express high levels of fatty acid synthase (FAS), a key metabolic enzyme linked to the synthesis of membrane phospholipids in cancer cells. In view of the recent finding that in the human prostate cancer cell line LNCaP, overexpression of FAS can be largely attributed to constitutive activation of the phosphatidylinositol-3 (PI3) kinase/Akt kinase pathway, the activation status of the Akt pathway, and whether this activation coincides with increased FAS expression, was examined in clinical prostate cancer tissues. Using well-preserved frozen prostatic needle biopsies and a sensitive Envision detection technique, S473-phosphorylated Akt (pAkt) was found in 11/23 low-grade prostatic intraepithelial neoplasia (PIN) lesions, in all (36/36) high-grade PINs, and in all (86/86) invasive carcinomas. Non-neoplastic tissues were negative. Interestingly, in low-grade PINs and low-grade carcinomas, pAkt was mainly cytoplasmic or membrane-bound and was associated with moderate elevation of FAS expression. In 24/36 high-grade PINs and 82/88 invasive carcinomas, pAkt was found at least partly in the nucleus. Greater nuclear pAkt staining, and higher FAS expression, correlated with a higher Gleason score. In the light of previous findings that pAkt plays a causative role in the overexpression of FAS in cancer cells in culture, these data strongly suggest that high-level expression of FAS in prostate cancer tissues is linked to phosphorylation and nuclear accumulation of Akt.     

Prostatic intraepithelial neoplasia

Prostatic intraepithelial neoplasia (PIN) is the histologic lesion most strongly associated with prostate cancer and has been postulated to be a pre-malignant lesion. However, much of the natural history of PIN remains unknown. A more fundamental understanding of the relationship between PIN and invasive tumors at the molecular level is critically needed and represents an important future challenge for investigators. The importance of their recognition are based on the correlation between their presence and the existence of prostatic adenocarcinomas. It seemed significant to us to point out the criteria of the diagnosis and the action to be taken in front of the discovery of such lesions on biopsies in prostate sufferers.     

Expression of the RNA component of human telomerase (hTR) in prostate cancer, prostatic intraepithelial neoplasia, and normal prostate tissue.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. While telomerase is undetectable in most normal somatic tissues, telomerase activation has been detected by a polymerase chain reaction (PCR)-based assay (TRAP) in many immortal cell lines and various cancers, including prostate cancers. To investigate the role of telomerase in prostate cancer at the cellular level, the expression of one of the ribonucleoprotein complexes, the RNA component of human telomerase (hTR), was studied in normal, preneoplastic, and cancerous prostate tissues using a non-radioactive in situ hybridization procedure. Nine human prostates resected at the time of radical prostatectomy were studied. In each case, archival paraffin-embedded samples from normal tissue, prostatic intraepithelial neoplasia (PIN) lesions, the putative precancerous lesion, and prostate carcinomas were selected for in situ hybridization. hTR mRNA expression was detected in carcinomatous glands of seven out of the nine cancers (75 per cent). Furthermore, in seven out of the eight cases showing PIN lesions, the epithelial cells of PIN foci also expressed hTR mRNA. By contrast, in normal tissue, epithelial cells were negative, whereas hTR mRNA expression was detected in the basal cells. The detection of hTR mRNA in PIN lesions clearly strengthens the link between PIN and carcinomatous glands and suggests that telomerase expression occurs early in prostate carcinogenesis. Furthermore, this study confirms previous experimental data suggesting that the basal cell layer is the stem cell compartment in prostate.     

Overexpression of Pim-1 during progression of prostatic adenocarcinoma

AIMS: Pim-1 is a serine/threonine kinase that has been shown to play an integral role in the development of a number of human cancers, such as haematolymphoid malignancies. Recently, evidence has shown Pim-1 to be important in prostatic carcinogenesis. In order to further our understanding of its role in prostate cancer, we investigated Pim-1 expression in normal, premalignant, and malignant prostate tissue. METHODS: Using immunohistochemistry, Pim-1 expression was analysed in prostate tissue from 120 radical prostatectomy specimens. In each case, Pim-1 staining was evaluated in benign prostatic epithelium, high grade prostatic intraepithelial neoplasia (PIN), and prostatic adenocarcinoma. The number of positively staining cells was estimated, and the intensity of staining was scored on a scale of 0 to 3+. RESULTS: Pim-1 immunoreactivity was identified in 120 cases (100%) of adenocarcinoma, 120 cases (100%) of high grade PIN, and 62 cases (52%) of benign glands. The number of cells staining in benign epithelium (mean 34%) was much lower than that in high grade PIN (mean 80%; p<0.0001) or adenocarcinoma (mean, 84%; p<0.0001). There was no significant difference between high grade PIN and adenocarcinoma in the percentage of cells staining positively for Pim-1 (p = 0.34). The staining intensity for Pim-1 was significantly lower in benign prostatic epithelium than in PIN and adenocarcinoma (p<0.001). There was no statistically significant correlation between the level of Pim-1 expression and Gleason score, patient age, tumour stage, lymph node metastasis, perineural invasion, vascular invasion, surgical margin status, extraprostatic extension, or seminal vesicle invasion. CONCLUSIONS: Pim-1 expression is elevated in PIN and prostatic adenocarcinoma compared with benign prostatic epithelium. This finding suggests that upregulation of Pim-1 may play a role in prostatic neoplasia.     

Age-related histopathological lesions in the Mongolian gerbil ventral prostate as a good model for studies of spontaneous hormone-related disorders

The Meriones unguiculatus (Mongolian) gerbil has demonstrated significant prostatic responses to hormonal treatments, and to drugs against human prostatic hyperplasia Spontaneous neoplasia develops in the older animals. Thirty gerbils (age 18 months) were divided into non-affected and prostatic lesion bearers and the prostate lesions were evaluated morphologically, immunohistochemically and quantitatively. The most frequent changes were in epithelial sites and, namely prostatic intraepithelial neoplasias, microinvasive carcinomas and adenocarcinomas. In the stromal compartment, cellular hyperplasia, when verified, was always associated with the sites of anomalous epithelium. Additionally, larger deposition of collagen fibrils, generating stromal fibrosis, was found in all the old gerbils analysed. The quantitative analysis showed that prostatic tissue proportions differed in altered areas, being specific for each lesion type. Isolated nuclear and nucleolar parameters were not effective in diagnosing the malign potential of lesions. However, the cellular proliferation and death indexes indicated larger cellular turnover in invasive lesions such as carcinomas. With these analyses, it could be verified that old gerbils present high propensity to develop spontaneous prostate changes and this may aid in a better understanding of the biological behaviour of human prostate cancer.     

Expression of KAI1 in paraffin-embedded normal, hyperplastic and neoplastic prostate and prostate carcinoma cell lines

Expression of KAI1, a tumor metastasis suppressor gene, was studied with different fixatives in frozen and paraffin-embedded sections of human and rat prostate carcinoma cell lines and human prostate lesions by Immunohisto-chemistry. Immunoreactivity of the membrane antigen in cell lines was associated with known expression levels in these lines and the fixative used. Formalin and paraformaldehyde helped maintain the immunoreactivity of cells. In human prostate, frozen sections revealed diffuse reactivity of the antigen in normal and neoplastic tissues while paraffin-embedded tissues usually showed focal reactivity, although more than 50% of cases with normal epithelium and adenocarcinomas were reactive. In some cases, pretreatment with trypsln enhanced immunoreactivity. Benign prostatic hyperplasia (BPH) showed the most intense diffuse immunoreactivity, which suggested enhanced expression. Prostatic intraepithelial neoplasia (PIN) also often expressed high levels of KAI1. Three of five metastases were reactive but two primaries and their metastases were not. Lymphocytes in primary carcinomas and lymphocytes and germinal center cells in lymph nodes were immunoreactive, while adjacent primary or metastatic prostate adenocarcinoma epithelium was not immunoreactive. Although paraffin-embedded human tissues were not optimal for determining levels of expression of KAI1, they did show immunoreactivity that could have prognostic value and showed the specific cytoplasmlc localization of the protein in cells.     

Decreased annexin I expression in prostatic adenocarcinoma and in high-grade prostatic intraepithelial neoplasia

AIMS: To analyse annexin I expression in prostatic carcinoma. Annexin I belongs to a family of structurally related calcium and phospholipid-binding proteins implicated in signal transduction, DNA replication, cell proliferation and apoptosis. The decreased expression of annexin I, II and VII proteins has been reported in different types of cancer. METHODS AND RESULTS: Using immunohistochemistry, we analysed annexin I expression in 77 cases of prostatic adenocarcinoma (Gleason score 6, N = 40; Gleason scores 7-8, N = 27; and Gleason scores 9-10, N = 10) and high-grade prostatic intraepithelial neoplasia (PIN, N = 50). Immunoreactivity of annexin I in tumour cells was evaluated as negative (< 5% of cells), focally positive (5-25% of cells) or positive (> 25% of cells). In contrast to positive staining in adjacent benign prostatic epithelium, annexin I expression was decreased (focally positive) in 76% of cases of high-grade PIN (P < 0.0001) and was decreased or absent in 81% of prostatic adenocarcinomas (P < 0.0001). Annexin I expression in all higher grade tumours (Gleason scores 7-10) was only focally positive or absent. CONCLUSIONS: Expression of annexin I inversely correlates with the increasing histological grade of prostatic adenocarcinoma. By showing a progressive loss of annexin I expression in high-grade PIN, intermediate-grade and high-grade cancer, our findings suggest that the loss of annexin I expression occurs early in prostatic tumorigenesis and becomes more prominent throughout tumour progression. The loss of expression of annexin I may serve as a useful marker of prostate cancer development and progression.     

Significance of apoptosis in prostatic intraepithelial neoplasia and carcinoma

This study was carried out to assess the significance of apoptosis in prostatic intraepithelial neoplasia (PIN) and prostate cancer. A total of 120 prostatic specimens were studied in the department of Pathology [corrected] JNMC, Aligarh. The rate of apoptosis in PIN and prostate cancer was examined by quantifying the number of apoptotic bodies per hundred cells (apoptotic index) on haematoxylin and eosin stained histological sections [corrected] A significant correlation was noted between increasing apoptotic indices and increasing Gleason grades within a cancer.     

Increased expression of delta-catenin/neural plakophilin-related armadillo protein is associated with the down-regulation and redistribution of E-cadherin and p120ctn in human prostate cancer

delta-Catenin, or neural plakophilin-related armadillo protein, is a unique armadillo domain-containing protein in that it is neural-specific and primarily expressed in the brain. However, our recent analysis of the human genome revealed a consistent association of delta-catenin messenger RNA sequences with malignant cells, although the significance of these findings was unclear. In this study, we report that a number of delta-catenin epitopes were expressed in human prostate cancer cells. Western blot and tissue microarray revealed a close association between increased delta-catenin expression and human primary prostatic adenocarcinomas. The analyses of 90 human prostate cancer and 90 benign prostate tissue samples demonstrated that an estimated 85% of prostatic adenocarcinomas showed enhanced delta-catenin immunoreactivity. delta-Catenin expression increased with prognostically significant increased Gleason scores. By analyzing the same tumor cell clusters using consecutive sections, we showed that an increased delta-catenin immunoreactivity was accompanied by the down-regulation and redistribution of E-cadherin and p120ctn, major cell junction proteins whose inactivation is frequently associated with cancer progression. Furthermore, overexpression of delta-catenin in tumorigenic CWR-R1 cells that are derived from human prostate cancer xenograft resulted in reduced immunoreactivity for E-cadherin and p120ctn at the cell-cell junction. This is the first study comparing overexpression of delta-catenin with the E-cadherin/catenin system in cancer and shows that delta-catenin may be intimately involved in regulating E-cadherin/p120ctn cell-cell adhesion in prostate cancer progression.     

Differential expression of cytokeratin mRNA and protein in normal prostate, prostatic intraepithelial neoplasia, and invasive carcinoma.

The expression of cytokeratin (CK) mRNA for CK5, -8, -14, -16, and -19 was investigated in normal prostate, prostatic intraepithelial neoplasia (PIN) lesions, and invasive carcinoma using in situ hybridization. Protein localization was carried out in adjacent sections using immunohistochemistry and correlated with mRNA expression. Snap-frozen human prostate samples including 22 examples of normal glands, 20 cases of PIN lesions, and 12 cases of invasive carcinoma were examined. CK5 and -14 mRNA and protein were prominently expressed only in the basal cells of normal glands and PIN lesions. CK14 mRNA was absent in the luminal cells of the most of the PIN lesions but was seen at a low level in some PIN lesions. CK14 protein was not detected in any PIN lesion, suggesting that, if the cell that makes up the PIN lesions is derived from a basal cell, CK14 translation is depressed although a low level of CK14 mRNA may persist. CK8 mRNA and protein were constitutively expressed in all epithelia of normal and abnormal prostate tissues. CK19 mRNA and protein were persistently expressed in both basal and luminal cells of the tubular portion of normal glands as well as PIN lesions, but were expressed heterogeneously in both basal and luminal cells of normal alveoli. CK16 mRNA was expressed in a similar pattern as CK19, but CK16 protein was not detected either in normal or in abnormal prostate tissues. In conclusion, the expression of CK19 in PIN lesions is similar to its tubular expression and would support an origin of PIN lesions from this structure rather than the alveolar portion of the glands. The similar cytokeratin expression between PIN lesions and invasive carcinoma further supports the concept that PIN is a precursor lesion of invasive carcinoma.     

Increased expression of FGF-8 isoforms and FGF receptors in human premalignant prostatic intraepithelial neoplasia lesions and prostate cancer.

SUMMARY: Fibroblast growth factor 8 (FGF-8) is implicated in growth of prostate cancer. Alternative splicing of the human FGF-8 gene potentially allows coding for four protein isoforms (a, b, e, and f). These isoforms differ in their binding to FGF receptors (FGFR) and in their mitogenic and transforming capacity in transfection assays. Here, we used RT-PCR and immunohistochemistry to study the expression of FGF-8 and FGFR isoforms in human prostate cancer (n = 31). Nonmalignant prostate specimens from cystoprostatectomies (n = 24) were examined as controls. Most prostate cancer samples and some control prostates also contained prostatic intraepithelial neoplasia (PIN) lesions. FGF-8a and e were expressed at significantly higher frequencies in prostate cancer (FGF-8a, 55%; FGF-8e, 45%) than in control samples (FGF-8a, 17%, p = 0.0052; FGF-8e, 8%, p = 0.0031). On the contrary, FGF-8b was found at an equal frequency in prostate cancer (55%) and in control prostates (50%). Furthermore, a combination of two or three FGF-8 isoforms (a, b, and/or e) was also expressed at a higher frequency in prostate cancer than in control samples (45% and 8%, respectively, p = 0.0031). Immunohistochemistry with an antibody recognizing all FGF-8 isoforms was more strongly immunoreactive in prostate cancer cells and PIN lesions than in normal-type epithelium. The receptor splicing variants FGFR1IIIc and FGFR2IIIc, which are activated by FGF-8, were found both in prostate cancer and control samples. Interestingly, immunoreactivity for FGFR1 and FGFR2 was much stronger in prostate cancer cells and PIN than in normal epithelium. These results demonstrate, for the first time, that FGF-8 isoforms and their receptors FGFR1IIIc and FGFR2IIIc are expressed at an increased level not only in prostate cancer but also in premalignant PIN lesions. These data suggest that FGF-8 may have an important autocrine role in the development of human prostate cancer. In addition to FGF-8b, the FGF-8 isoforms a and e may be involved in this process.