Prostatic Intraepithelial Neoplasia Cells Research Articles

Macrophages Cytokine Spp1 Increases Growth of Prostate Intraepithelial Neoplasia to Promote Prostate Tumor Progression.

Prostate cancer development and progression are associated with increased infiltrating macrophages. Prostate cancer is derived from prostatic intraepithelial neoplasia (PIN) lesions. However, the effects macrophages have on PIN progression remain unclear. Here, we showed that the recruited macrophages adjacent to PIN expressed M2 macrophage markers. In addition, high levels of Spp1 transcripts, also known as osteopontin, were identified in these macrophages. Extraneously added Spp1 accelerated PIN cell proliferation through activation of Akt and JNK in a 3D culture setting. We also showed that PIN cells expressed CD44, integrin αv, integrin β1, and integrin β3, all of which have been previously reported as receptors for Spp1. Finally, blockade of Akt and JNK activation through their specific inhibitor completely abolished macrophage Spp1-induced cell proliferation of PIN. Hence, our data revealed Spp1 as another macrophage cytokine/growth factor and its mediated mechanism to upregulate PIN cell growth, thus promoting prostate cancer development.

Macrophages expedite cell proliferation of prostate intraepithelial neoplasia through their downstream target ERK.

The risk factors for prostate cancer include a high-fat diet and obesity, both of which are associated with an altered cell environment including increased inflammation. It has been shown that chronic inflammation due to a high-fat diet or bacterial infection has the potential to accelerate prostate cancer as well as its precursor, prostatic intraepithelial neoplasia (PIN), development. However, the underlying mechanism of how chronic inflammation promotes prostate cancer development, especially PIN, remains unclear. In this study, we showed that more macrophages were present in PIN areas as compared to the normal areas of human prostate. When co-culturing PIN cells with macrophages in 3D, more PIN cells had nuclear localized cyclin D1, indicating that macrophages enhanced PIN cell proliferation. We identified ICAM-1 and CCL2 as chemoattractants expressed by PIN cells to recruit macrophages. Furthermore, we discovered that macrophage-secreted cytokines including C5a, CXCL1, and CCL2 were responsible for increased PIN cell proliferation. These three cytokines activated ERK and JNK signaling in PIN cells through a ligand-receptor interaction. However, only blockade of ERK abolished macrophage cytokines-induced cell proliferation of PIN. Overall, our results provide a mechanistic view on how macrophages activated through chronic inflammation can expedite PIN progression during prostate cancer development. The information from our work can facilitate a comprehensive understanding of prostate cancer development, which is required for improvement of current strategies for prostate cancer therapy.

Apoptotic and predictive factors by Bax, Caspases 3/9, Bcl-2, p53 and Ki-67 in prostate cancer after 12\u2009Gy single-dose

Radio-induced apoptosis is mediated by the activation of tumor protein p53, Bax and caspases. The purpose of this study was to investigate the early activation of this pathway in men receiving in vivo irradiation immediately before radical prostatectomy for locally advanced prostate cancer. We also investigated cell proliferation index (Ki-67), proto-oncogene (p53) and anti-apoptotic protein (Bcl-2) levels as potential predictive factors. We selected a homogeneous sample of 20 patients with locally advanced prostate cancer and candidate to radical prostatectomy. To assess the apoptotic pathways, Bax, is studied through immunofluorescence assay, before and after 12 Gy single dose intraoperative radiotherapy (IORT) to the prostate, on bioptic samples and on surgical specimens. Moreover, before and after IORT, Bcl-2, p53, and Ki-67 were also detected through immunohistochemistry. A count of positive Bax spots for immunofluorescence was performed on tumor cells, prostatic intraepithelial neoplasia (PIN), and healthy tissue areas before and after IORT. We also analyzed Caspases 3 and 9 expressions after IORT. Before IORT, Bcl-2 mean value in neoplastic cells was 2.23% ± 1.95, mean Ki-67 in neoplastic area was 4.5% ± 3.8, and p53 was 22.5% ± 6.8. After IORT, Bcl-2 mean value in neoplastic cells was 8.85 ± 8.92%, Ki-67 in neoplastic area was 7.8 ± 6.09%, and p53 was 24.9 ± 26.4%. After the irradiation, healthy areas expressed significantly lower levels of Bax (2.81 ± 1.69%) with respect to neoplastic cells (p < 0.0001), while in PIN areas, Bax positive cells were significantly more present than in neoplastic areas (p = 0.0001). At statistical analysis, it was observed that cancer cells with Ki-67 ≥ 8% had a trend toward greater expression of Bax (p = 0.0641). We observed an increase of Bcl-2 expression after IORT in neoplastic areas (p = 0.0041). Biopsy specimens with p53 ≥ 18% and Ki-67 ≥ 8% had worse post-operative staging with extracapsular invasion (p = 0.04 for both parameters) and nodal positivity (p = 0.04 for p53 and p = 0.0001 at pathology for ki-67). No correlation between IORT and Caspases activation was noted. In conclusion, after 12 Gy IORT, Bax was overexpressed in tumor and PIN cells. Pre-operative Ki-67 and p53 definition could be used in future studies to predict patients with worse pathological stage, while Bcl-2 activation after IORT might be a predictive factor for loco-regional failure.

Abstract LB-160: Human telomerase RNA component (hTR/TERC) is consistently overexpressed in prostate cancer, required for cell proliferation and is positively regulated by MYC

Abstract Introduction: Telomerase activity (TA) is increased in most human cancers (80 - 90%) as a mechanism to maintain telomere function, yet the complex regulatory mechanisms driving TA in different cancer types remain obscure. Telomerase consists of at least two essential elements, a catalytic component (TERT) and an RNA subunit (hTR or TERC). Early data showed that TERC was constitutively expressed at similar levels in different tissues and cancer and that TERT mRNA levels best correlated with enzymatic activity across normal tissues and cancer cells. However, accumulating evidence suggests a more important role for the regulation of TERC in telomere maintenance, as well as possible telomerase activity-independent functions. MYC (c-Myc) is overexpressed in most prostate cancers and is known to regulate TERT mRNA levels in a number of cell types and hence to drive TA. However, there are no prior studies of MYC regulation of TERC. We performed a comprehensive study of TERC expression in cancer cell lines and prostatic tissue, across normal, high grade prostatic intraepithelial neoplasia (PIN), primary carcinomas (PCa), and castrate resistant metastatic disease. We further explored MYC regulation of TERC in Pca and examined cell proliferation in TERC-depleted Pca cell lines. Experiments and results: Using a novel chromogenic In Situ Hybridization (CISH) RNA assay in FFPE tissues, validated in cell lines by RT-PCR, we found consistent TERC overexpression in nuclei of PIN (p&amp;lt;0.001, N = 26 patients) and Pca cells (p&amp;lt;0.001, N = 66 patients), that was confirmed by qRT-PCR (n = 25) and RNAseq (n = 12) in tumor/benign frozen tissue pairs. There was no association with Gleason score or pathologic stage, and a weak correlation between TERC and TERT levels by RT-PCR. There was a correlation between TERC CISH and MYC protein expression in prostate tissue (Spearman 0.4540, p&amp;lt;0.0001). Further, we show: i) that forced reductions of MYC resulted in decreased TERC levels in 8 cancer cell lines (prostate, lung, breast, and colorectal); ii) MYC occupies the TERC locus by chromatin immunoprecipitation (ChIP); iii) there is a decrease in human TERC promoter activity with MYC silencing; iv) increased Terc levels in PIN and carcinoma in 2 different mouse models of prostate-restricted overexpression of MYC; and v) knockdown of TERC by siRNA resulted in reduced growth and decreased Ki-67 in TERC depleted human prostate cancer cells. Conclusions: We report consistent overexpression of TERC in PIN and PCa and show the first evidence of MYC regulation of TERC. These studies suggest an important role for MYC-regulated TERC overexpression in human prostate cancer development and progression. Further studies are required to better understand the functional roles of TERC (e.g. TA mediated vs. other activity) in carcinogenesis of the prostate and other organ sites and its regulation by MYC. Citation Format: Javier A. Baena Del Valle, Qizhi Zheng, Michael Rubenstein, Alan K. Meeker, Christopher M. Heaphy, Gretchen Hubbard, Charles J. Bieberich, Srinivasan Yegnasubramanian, David Esopi, Sarah J. Wheelan, Angelo M. De Marzo. Human telomerase RNA component (hTR/TERC) is consistently overexpressed in prostate cancer, required for cell proliferation and is positively regulated by MYC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-160.

Abstract A151: Identification of antibody fragments specific for high-grade prostatic intraepithelial neoplasia cells via SELEX.

Abstract The purpose of this study is to develop a minimally invasive molecular tool for the early detection and specific treatment of prostate cancer. A human non-immune single-chain Fragment variable (scFv) library with 10∧9 diversity has been displayed by a yeast host surface with the ability to bind to whole cell targets. This protein library was cloned into plasmids and transformed into the yeast strain Saccharomyces cerevisiae. Surface expression of the scFv library was achieved by expressing the scFv under control of a galactose promoter preceding incubation with the target cell line. A molecular recognition element (MRE) specific for HGPIN cells will be selected for through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX). From an initial scFv library with 10∧9 diversity, seven rounds of selection will be completed in order to enrich the antibody fragment library that binds only to the prostate cancer cell line HGPIN and not those that bind to normal and cancerous cells of the prostate epithelium. This involves screening a library of random molecules for their ability to bind to a target; non-binding molecules are removed and binding molecules are amplified. Amplified molecules are subjected to negative targets, and molecules that don't bind to these are amplified, thus completing a round of selection. Stringency is increased through each round of selection by shortening the yeast + prostate cell incubation period and decreasing the number of target cells used in incubation. After multiple rounds, one or a few strong binding, highly specific molecules are selected for. Diversity of the library is decreased with each round of selection. This is followed by amplification of the remaining yeast. Sequencing is performed throughout the selection process in order to monitor both the decrease in diversity and the emergence of the most selective sequences. The obtained MRE will be useful for specific diagnosis in the pre-malignant stages of prostate cancer. It may also be conjugated to drugs for targeted therapeutic applications. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A151. Citation Format: Cyrus J. Hajiran, Letha J. Sooter. Identification of antibody fragments specific for high-grade prostatic intraepithelial neoplasia cells via SELEX. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A151.

Primary Cilia Are Lost in Preinvasive and Invasive Prostate Cancer

Prostate cancer is the second most commonly diagnosed cancer in men worldwide. Little is known about the role of primary cilia in preinvasive and invasive prostate cancer. However, reduced cilia expression has been observed in human cancers including pancreatic cancer, renal cell carcinoma, breast cancer, cholangiocarcinoma, and melanoma. The aim of this study was to characterize primary cilia expression in preinvasive and invasive human prostate cancer, and to investigate the correlation between primary cilia and the Wnt signaling pathway. Human prostate tissues representative of stages of prostate cancer formation (normal prostate, prostatic intraepithelial neoplasia (PIN), and invasive prostate cancer (including perineural invasion)) were stained for ciliary proteins. The frequency of primary cilia was determined. A decrease in the percentage of ciliated cells in PIN, invasive cancer and perineural invasion lesions was observed when compared to normal. Cilia lengths were also measured to indirectly test functionality. Cilia were shorter in PIN, cancer, and perineural invasion lesions, suggesting dysfunction. Primary cilia have been shown to suppress the Wnt pathway. Increased Wnt signaling has been implicated in prostate cancer. Therefore, we investigated a correlation between loss of primary cilia and increased Wnt signaling in normal prostate and in preinvasive and invasive prostate cancer. To investigate Wnt signaling in our cohort, serial tissue sections were stained for β-catenin as a measure of Wnt signaling. Nuclear β-catenin was analyzed and Wnt signaling was found to be higher in un-ciliated cells in the normal prostate, PIN, a subset of invasive cancers, and perineural invasion. Our results suggest that cilia normally function to suppress the Wnt signaling pathway in epithelial cells and that cilia loss may play a role in increased Wnt signaling in some prostate cancers. These results suggest that cilia are dysfunctional in human prostate cancer, and increase Wnt signaling occurs in a subset of cancers.

Abstract 3260: Primary cilia are lost early in prostate cancer progression

Abstract Prostate cancer is the most prominently diagnosed cancer in men, aside from non-melanoma skin cancer, and the second leading cause of cancer related deaths in men in the U.S. Little is known about the role of primary cilia in prostate cancer progression, so our goal is to elucidate this role. Primary cilia are microtubule-based organelles present on many mammalian cell types and help the cell sense the extracellular environment. Various signaling pathways important in development involve primary cilia, such as the Hedgehog (Hh) and Wingless/Int (Wnt) pathways, and these pathways are known to be misregulated in prostate cancer. Dysfunction of primary cilia is the cause of numerous ciliopathies, such as polycystic kidney disease and Bardet-Biedl syndrome, and has been linked to cancers, like basal cell carcinoma and medulloblastoma. We hypothesize that primary cilia suppress prostate tumorigenesis, and cilia loss promotes prostate cancer by altering cell signaling pathways like Hh and canonical Wnt. To test this hypothesis, we looked in human prostate cancers at cilia, Hh and canonical Wnt signaling. Human prostate tissue from prostate cancer patients was stained for ciliary proteins (acetylated and gamma tubulin). The expressions of these proteins were analyzed using confocal microscopy and compared between adjacent normal, hyperplastic, prostatic intraepithelial neoplasia (PIN), malignant and perineural invasion areas of each patient's tissue. As a control, normal prostate tissue from bladder cancer patients who underwent prostatectomies as part of treatment was used. A marked decrease in the percentage of ciliated cells in cancer and PIN was observed compared to normal prostate tissue. Higher grade cancer (Gleason sum α7) also had fewer cilia than lower grade (Gleason sum = 6). We are currently investigating Hh and canonical Wnt signaling activity in serial sections of the tissue used to analyze primary cilia. The expressions of Beta-Catenin, a protein used to measure canonical Wnt signaling activity, and Gli1, a transcription factor used as a readout of Hh activity, are being analyzed based on expression levels and localization within the cell, and correlated to primary cilia data. Thus far, our results suggest a role of primary cilia in promoting prostate cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3260. doi:1538-7445.AM2012-3260

Abstract 2695: Olfactomedin 4 gene is associated with progression of prostate cancer: Evidence from an OLFM4 knockout mouse model

Abstract The olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein whose physiological and pathological functions are largely unknown. We have previously reported that expression of OLFM4 is down regulated during the progression of human prostate cancer, and that reduced expression is due to gene deletions of OLFM4 (36.7%) and/or hyper-methlytion of CpG sites in the promoter region. We also demonstrated that ectopic expression of OLFM4 in several human prostate cancer cell lines inhibits proliferation, invasion and metastatic potential. In this study, we further investigated the potential role of OLFM4 in the carcinogenesis of prostate cancer utilizing an OLFM4 knock out mouse model. Histopathological analysis revealed that homozygous OLFM4 mutant mice develop prostatic epithelial lesions in an age-dependent manner. The prostatic epithelial hyperplasia was seen at 6 months of age, prostatic intraepithelial neoplasia (PIN) was found at 10-12 months of age, and prostate adenoma was observed from 18-24 months of ages in the both anterior prostate (AP) and dorsal-lateral prostate (DLP) of OLFM4-null mice. The frequency of PIN lesions at the AP and DLP was 0 % (0/20) at 3-6 months, 38% (5/13) at 10-12 months and 44% (11/25) at 18-24 months. The frequency of prostatic adenoma was 28% (7/25), and 8% (2/25) of OLFM4-null mice developed lung metastases at 18-24 months of age. To determine whether OLFM4 affects proliferation or apoptosis of prostatic epithelial cells in the OLFM4-null mice, we carried out Ki-67 immunohistochemical staining and TUNEL assays. The ratio of Ki-67 positive cells was significantly higher at 3-6 months (p=0.04180), 10-12 months (P=0.002) and 18-20 months (p=0.0038) compared to age-matched controls, OLFM4 wild type mice, or heterozygous mice, thus indicating an increase in proliferation in OLFM4-null prostate tissues. There was no difference in the number of apoptotic cells in the control versus null mice. Since we have previously found that OLFM4 blocks CXCL12 induced AKT activity in the human prostate cancer cell PC-3, and inhibits cell invasion, we studied the phosphorylation of AKT and downstream signaling pathway components by Western blot analysis of prostate samples from 3-4 month old mice. Compared with OLFM4 wild type mice, pAKT, pGSK3β, β-catenin, cyclin D1 and c-Myc were increased in the OLFM4-null mice tissue even in the absence of morphological abnormalities. Elevated β-catenin protein in OLFM4-null mice prostate epithelial cells was also observed in PIN and tumor cells at 18 months of age, and positive staining with Cyclin D1 in OLFM4-null tumor was also detected. Taken together, the data show OLFM4-null prostatic epithelial cells exhibit higher levels of AKT and β-catenin, suggesting this is the mechanism leading to their enhanced cellular proliferation and carcinogenetic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2695. doi:1538-7445.AM2012-2695

Sodium/Glucose Co-transporter 1 Expression Increases in Human Diseased Prostate

Sodium/glucose co-transporter 1 (SGLT1) is an active glucose transporter that takes up glucose into cells independent of the extracellular concentration of glucose. This transporter plays a critical role in maintaining glucose homeostasis at both physiological and pathological levels. The expression level of SGLT1 in normal and diseased human prostatic tissue has not been determined. We produced two rabbit polyclonal antibodies against human SGLT1, one each for immunohistochemical and Western blot analyses, and characterized the expression of SGLT1 in human prostate tissues: normal prostate (n=3), benign prostatic hyperplasia (BPH) (n=53), prostatic intraepithelial neoplasia (PIN) (n=9), and prostate cancer (PCa) (n=44). In normal prostate tissue, SGLT1 was weakly expressed exclusively in the epithelium. The transporter was significantly increased in the basal cells and stromal cells of BPH, increased in the epithelial cells of PIN, and frequently overexpressed in stromal cells and universally overexpressed in the tumor cells of PCa. The pattern of expression was shown as membranous/ cytoplasmic staining in low-grade cancer cells and nuclear envelope staining in high-grade cancer cells. The SGLT1-positive stromal cells of BPH and PCa tissues were negative for tenascin, a marker of reactive stromal cells. We concluded that SGLT1 is up-regulated in BPH and PCa, and SGLT1 may serve as a potential therapeutic target for treating these prostate disorders.

Pathological significance and predictive value for biochemical recurrence of c‐Fes expression in prostate cancer

c-Fes is a proto-oncogene encoded non-receptor protein-tyrosine kinase (PTK). However, genetic studies have indicated that it has anti-tumorigenic effects in certain cancers. The pathological and clinical significance of c-Fes in prostate cancer are unknown. Expression of c-Fes was evaluated in normal glands, prostatic intraepithelial neoplasia (PIN), cancer cells in tissues of knock-in mouse adenocarcinoma prostate (KIMAP) model, and prostate cancer patients free of metastasis. Expression of c-Fes was analyzed by immunohistochemistry, and quantified by using the immunoreactivity score (IRS) (staining intensity × percentage of positive cells). Relationships between c-Fes expression and pT stage, Gleason's score (GS), and biochemical recurrence in patients who underwent radical surgery were also investigated. In KIMAP, the percentage in normal glands, PIN and cancer cells positive for c-Fes expression were 0 (0/7), 25.0 (2/8), and 100% (7/7), respectively. In human tissues, c-Fes expression was also significantly higher in cancer cells than in normal cells and PIN, and it correlated with pT stage (P < 0.001) and GS (P = 0.047). Multivariate analysis showed that c-Fes expression was an independent predictor of poor outcome poor prognosis (hazard ratio = 3.21, 95% confidence interval = 1.11-9.37, P = 0.032). The results suggested that c-Fes expression is a useful predictor of biochemical recurrence after radical surgery. The results also suggested that c-Fes is a potentially useful therapeutic target in prostate cancer and a predictor of biochemical recurrence after radical prostatectomy.

Abstract 1436: Interleukin-17A stimulates expression of chemokines and cytokines in prostatic epithelial cells via nuclear factor-kappaB and extracellular signal-regulated kinase pathways

Abstract T helper 17 (Th17) cells secret interleukin-17A (IL-17A) and interleukin-17F. Th17 cells and IL-17A are increased in prostate tumors. The role of IL-17A in prostate cancer is not clear. The objective of this in-vitro study was to investigate the effects of IL-17A on prostatic epithelial cells. Cell growth assay, Western blot, and real-time quantitative reverse transcription- polymerase chain reaction (qRT-PCR) analysis were used to measure the effects of IL-17A on the immortalized normal human prostatic epithelial cell lines (RWPE-1 and pRNS-1-1), human high-grade prostatic intraepithelial neoplasia (PIN) cell line, human prostate cancer LNCaP cell line, and mouse prostate cancer TRAMP-C1 cell line. We found that recombinant human IL-17A (20 ng/ml) did not affect cell growth rate in any of the prostatic cell lines studied. IL-17A did not significantly activate Nuclear Factor-kappaB (NF-kappaB) or Extracellular signal-Regulated Kinase (ERK) signaling pathway in RWPE-1, pRNS-1-1, or PIN cells. IL-17A activated NF-kappaB and/or ERK signaling pathways in LNCaP and TRAMP-C1 cells. When IL-17RC (receptor of IL-17A) was overexpressed in PIN and LNCaP cells, activation of NF-kappaB or ERK signaling pathway by IL-17A was significantly enhanced. IL-17A modestly induced mRNA expression of chemokines (CXCL1 and CXCL2) in RWPE-1, pRNS-1-1, and PIN cells. IL-17A induced mRNA expression of chemokines (CXCL1, CXCL2, CCL2, CCL5, CCL7, and CCL20) and cytokine IL-6 in LNCaP cells. In mouse TRAMP-C1 cells, IL-17A induced mRNA expression of chemokines (CXCL1, CXCL2, CXCL5, CCL2, and CCL5) and cytokine IL-6. When IL-17RC was overexpressed in PIN and LNCaP cells, chemokine expression was significantly enhanced. Our findings suggest that although IL-17A has no effects on cell growth in-vitro, it may modulate the stroma-epithelial interaction in tumor microenvironment in-vivo via stimulation of expression of chemokines and cytokines. Acknowledgement: DoD W81XWH-05-1-0567, NIH/NCRR 2P20 RR020152-06, and LCRC Fund. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1436.

MYC Overexpression Induces Prostatic Intraepithelial Neoplasia and Loss of Nkx3.1 in Mouse Luminal Epithelial Cells

Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate[1]. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina—features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These “Super-Lo-MYC” mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a “paradoxical” increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.

PTEN inhibits BMI1 function independently of its phosphatase activity

BackgroundPTEN is the second most mutated tumor suppressor gene other than p53. It suppresses tumorigenesis by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate (PIP3) to phosphatidylinositol (4,5)-biphosphate (PIP2), thereby directly inhibiting phosphatidylinositol 3 kinase (PI3K)-mediated tumorigenic activities. Consistent with this model of action, cytosolic PTEN is recruited to the plasma membrane to dephosphorylate PIP3. While nuclear PTEN has been shown to suppress tumorigenesis by governing genome integrity, additional mechanisms may also contribute to nuclear PTEN-mediated tumor suppression. The nuclear protein BMI1 promotes stem cell self-renewal and tumorigenesis and PTEN inhibits these events, suggesting that PTEN may suppress BMI1 function.ResultsWe investigated whether PTEN inhibits BMI1 function during prostate tumorigenesis. PTEN binds to BMI1 exclusively in the nucleus. This interaction does not require PTEN's phosphatase activity, as phosphatase-deficient PTEN mutants, PTEN/C124S (CS), PTEN/G129E (GE), and a C-terminal PTEN fragment (C-PTEN) excluding the catalytic domain, all associate with BMI1. Furthermore, the residues 186-286 of C-PTEN are sufficient for binding to BMI1. This interaction reduces BMI1's function. BMI1 enhances hTERT activity and reduces p16INK4A and p14ARF expression. These effects were attenuated by PTEN, PTEN(CS), PTEN(GE), and C-PTEN. Furthermore, knockdown of PTEN in DU145 cells increased hTERT promoter activity, which was reversed when BMI1 was concomitantly knocked-down, indicating that PTEN reduces hTERT promoter activity via inhibiting BMI1 function. Conversely, BMI1 reduces PTEN's ability to inhibit AKT activation, which can be attributed to its interaction with PTEN in the nucleus, making PTEN unavailable to dephosphorylate membrane-bound PIP3. Furthermore, BMI1 appears to co-localize with PTEN more frequently in clinical prostate tissue samples from patients diagnosed with PIN (prostatic intraepithelial neoplasia) and carcinoma compared to normal prostate epithelium. While PTEN co-localized with BMI1 in 2.4% of normal prostate epithelial cells, co-localization was observed in 37.6% and 18.5% of cells in PIN and carcinoma, respectively. Collectively, we demonstrate that PTEN inhibits BMI1 function via binding to BMI1 in a phosphatase independent manner.ConclusionWe demonstrate that nuclear PTEN reduces BMI1 function independently of its phosphatase activity. It was recently observed that nuclear PTEN also suppresses tumorigenesis. Our results, therefore, provide a plausible mechanism by which nuclear PTEN prevents tumorigenesis.

Expression and prognostic role of syndecan-2 in prostate cancer

Syndecans are a four-member family of transmembrane heparan sulphate proteoglycans that have different functions in cell signalling, adhesion, cytoskeleton organization, migration, proliferation, and angiogenesis. Several studies investigated the role of syndecan-2 (SDC2) in different carcinomas; however, only one being focused on SDC2 in prostate cancer. SDC2 expression and relationship with established prognostic features were assessed in a cohort of 86 patients treated with radical prostatectomy for clinically localized prostate adenocarcinoma. SDC2 expression was present in the majority of prostate cancers and absent in only 11.6% of cases. SDC2 expression was also recorded in cells of prostatic intraepithelial neoplasia, whereas normal prostatic epithelial tissue and stroma did not express SDC2. SDC2 overexpression in prostate cancer was significantly associated with established features indicative of worse prognosis such as higher preoperative PSA (P=0.011), higher Gleason score (P<0.001), positive surgical margins (P<0.003), and extraprostatic extension of disease (P<0.003). Moreover, expression of SDC2 was also associated with biochemical disease progression on univariate analysis (P<0.001). Study results supported the potential role of SDC2 in prostatic carcinogenesis and cancer progression. Moreover, SDC2 could serve as an additional prognostic marker that might help in further stratifying the risk of disease progression in patients with prostate cancer.

Precursor of prostate‐specific antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: a study of 90 cases

To assess the expression of the precursor of prostate-specific antigen (pro-PSA), a distinct molecular form of serum-free PSA that includes native and truncated forms, in benign epithelium, high-grade prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma. We immunohistochemically evaluated 90 formalin-fixed, paraffin-embedded prostate needle biopsies using monoclonal antibodies against [-2] pro-PSA, native [-5/-7] pro-PSA, prostate-specific membrane antigen (PSMA), PSA and racemase. Staining intensity was recorded using a scale of 0-3 (0, no staining; 3, highest staining). The percentage of immunoreactive cells in benign epithelium, high-grade PIN and adenocarcinoma was estimated in increments of 10%. All cases had [-5/-7] pro-PSA immunoreactivity. There was weak focal perinuclear cytoplasmic immunoreactivity for [-5/-7] pro-PSA in 62% (range 0-90%) of benign epithelial cells, whereas there was strong diffuse cytoplasmic staining in 83% (range 10-90%) of high-grade PIN and 87% (range 40-90%) of cancer cells. Almost all (99%) cases were immunoreactive for [-2] pro-PSA. The median (range) proportion of cells expressing [-2] pro-PSA was lower in benign epithelium, at 17 (0-80)%, than in high-grade PIN, at 55 (0-90)% (P < 0.001) and adenocarcinoma, at 55 (0-100)% (P < 0.001). The intensity of immunoreactivity for both isoforms increased from benign to neoplastic (high-grade PIN and adenocarcinoma) epithelium. A total of 31% of high-grade PIN and 11% of cancer cases with negative racemase staining showed strong staining for [-5/-7] pro-PSA. The expression of [-5/-7] pro-PSA in benign and neoplastic cells might be used in combination with high molecular weight keratin, p63, and racemase to distinguish benign epithelium from high-grade PIN and adenocarcinoma, particularly when racemase staining is negative. Both isoforms are sensitive markers for prostatic epithelium, making them possible candidates for investigating carcinoma with an unknown primary, particularly in cases in which PSA staining is negative and the level of suspicion is high.

Ribosome‐inactivating proteins isolated from dietary bitter melon induce apoptosis and inhibit histone deacetylase‐1 selectively in premalignant and malignant prostate cancer cells

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS-PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome-inactivating proteins (RIPs), alpha-momorcharin and beta-momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC-3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase-1 (HDAC-1) activity and promotes histone-3 and -4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of beta-catenin and decreased levels of c-Myc and Cyclin-D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC-1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.

A prostatic intraepithelial neoplasia-dependent p27Kip1 checkpoint induces senescence and inhibits cell proliferation and cancer progression: Majumder PK, Grisanzio C, O'Connell F, Barry M, Brito JM, Xu Q, Guney I, Berger R, Herman P, Bikoff R, Fedele G, Baek W-K, Wang S, Ellwood-Yen K, Wu H, Sawyers CL, Signoretti S, Hahn WC, Loda M, Sellers WR, Department of Medical Oncology, Dana-Farber

Transgenic expression of activated AKT1 in the murine prostate induces prostatic intraepithelial neoplasia (PIN) that does not progress to invasive prostate cancer (CaP). In luminal epithelial cells of Akt-driven PIN, we show the concomitant induction of p27(Kip1) and senescence. Genetic ablation of p27(Kip1) led to down-regulation of senescence markers and progression to cancer. In humans, p27(Kip1) and senescence markers were elevated in PIN not associated with CaP but were decreased or absent, respectively, in cancer-associated PIN and in CaP. Importantly, p27(Kip1) up-regulation in mouse and human in situ lesions did not depend upon mTOR or Akt activation but was instead specifically associated with alterations in cell polarity, architecture, and adhesion molecules. These data suggest that a p27(Kip1)-driven checkpoint limits progression of PIN to CaP.

A Prostatic Intraepithelial Neoplasia-Dependent p27 Kip1 Checkpoint Induces Senescence and Inhibits Cell Proliferation and Cancer Progression

Transgenic expression of activated AKT1 in the murine prostate induces prostatic intraepithelial neoplasia (PIN) that does not progress to invasive prostate cancer (CaP). In luminal epithelial cells of Akt-driven PIN, we show the concomitant induction of p27(Kip1) and senescence. Genetic ablation of p27(Kip1) led to downregulation of senescence markers and progression to cancer. In humans, p27(Kip1) and senescence markers were elevated in PIN not associated with CaP but were decreased or absent, respectively, in cancer-associated PIN and in CaP. Importantly, p27(Kip1) upregulation in mouse and human in situ lesions did not depend upon mTOR or Akt activation but was instead specifically associated with alterations in cell polarity, architecture, and adhesion molecules. These data suggest that a p27(Kip1)-driven checkpoint limits progression of PIN to CaP.

Decreased NKX3.1 protein expression in focal prostatic atrophy, prostatic intraepithelial neoplasia, and adenocarcinoma: Association with Gleason score and chromosome 8p deletion: Bethel CR, Faith D, Li X, Guan B, Hicks JL, Lan F, Jenkins RB, Bieberich CJ, De Marzo AM, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Baltimore, MD

NKX3.1 is a homeobox gene located at chromosome 8p21.2, and one copy is frequently deleted in prostate carcinoma. Prior studies of NKX3.1 mRNA and protein in human prostate cancer and prostatic intraepithelial neoplasia (PIN) have been conflicting, and expression in focal prostate atrophy lesions has not been investigated. Immunohistochemical staining for NKX3.1 on human tissue microarrays was decreased in most focal atrophy and PIN lesions. In carcinoma, staining was inversely correlated with Gleason grade. Fluorescence in situ hybridization showed that no cases of atrophy had loss or gain of 8p, 8 centromere, or 8q24 (C-MYC), and only 12% of high-grade PIN lesions harbored loss of 8p. By contrast, NKX3.1 staining in carcinoma was correlated with 8p loss and allelic loss was inversely related to Gleason pattern. Quantitative reverse transcription-PCR for NKX3.1 mRNA using microdissected atrophy revealed a concordance with protein in 5 of 7 cases. In carcinoma, mRNA levels were decreased in 6 of 12 cases but mRNA levels correlated with protein levels in only 4 of 12 cases, indicating translational or post-translational control. In summary, NKX3.1 protein is reduced in focal atrophy and PIN but is not related to 8p allelic loss in these lesions. Therefore, whereas genetic disruption of NKX3.1 in mice leads to PIN, nongenetic mechanisms reduce NKX3.1 protein levels early in human prostate carcinogenesis, which may facilitate both proliferation and DNA damage in atrophic and PIN cells. Monoallelic deletions on chromosome 8p are associated with more advanced invasive and aggressive disease.

Glutathione S-transferase: differential expression of α, μ, and π isoenzymes in benign prostate, prostatic intraepithelial neoplasia, and prostatic adenocarcinoma

Glutathione S-transferases (GST) comprise a family of enzymes which are critical for inactivation of toxins and carcinogens. We examined the cellular expression of multiple subclasses of GST immunohistochemically in 25 radical prostatectomy specimens with clinically localized prostate cancer. Gleason scores ranged from 5 to 9, and pathologic stages varied from pT2a to pT3b (all N0M0). Antibodies were directed against GST Ya, Yc, and Yk (alpha subclass), Yb1 (micro subclass), and YPr (pi subclass). The percentage of positive cells and intensity of staining was assessed for benign epithelium, high-grade prostatic intraepithelial neoplasia (PIN), and adenocarcinoma. GSTalpha (Ya) was detected in 30% of cells (mean) in benign acini, 4.9% of cells in high-grade PIN, and 4.5% of cells in adenocarcinoma. The corresponding results for alpha (Yk), micro (Yb1), and pi (Yp) were 12.7%, 10.9%, and 3.5%; 8.7%, 5.2%, and 0.6%; and 66.7,% 0%, and 0%, respectively. GST Yc (alpha subclass) displayed the lowest level of expression, with diffuse weak staining in scattered benign secretory cells and only single cells (<1%) in high-grade PIN and carcinoma. These results demonstrate consistent reduction or loss of expression of all subclasses of GST with progression of prostatic neoplasia from benign epithelium to high-grade PIN and carcinoma. We hypothesize that carcinogenesis in the prostate results from impaired cellular handling of mutagenic agents owing to reduction or loss of expression of multiple GST and other detoxifying and antimutagenesis agents.