Multiplexed Quantum Dot Labeling of Activated c-Met Signaling in Castration-Resistant Human Prostate Cancer

The potential application of multiplexed quantum dot labeling (MQDL) for cancer detection and prognosis and monitoring therapeutic responses has attracted the interests of bioengineers, pathologists and cancer biologists. Many published studies claim that MQDL is effective for cancer biomarker detection and useful in cancer diagnosis and prognosis, these studies have not been standardized against quantitative biochemical and molecular determinations. In the present study, we used a molecularly characterized human prostate cancer cell model exhibiting activated c-Met signaling with epithelial to mesenchymal transition (EMT) and lethal metastatic progression to bone and soft tissues as the gold standard, and compared the c-Met cell signaling network in this model, in clinical human prostate cancer tissue specimens and in a castration-resistant human prostate cancer xenograft model. We observed c-Met signaling network activation, manifested by increased phosphorylated c-Met in all three. The downstream survival signaling network was mediated by NF-κB and Mcl-1 and EMT was driven by receptor activator of NF-κB ligand (RANKL), at the single cell level in clinical prostate cancer specimens and the xenograft model. Results were confirmed by real-time RT-PCR and western blots in a human prostate cancer cell model. MQDL is a powerful tool for assessing biomarker expression and it offers molecular insights into cancer progression at both the cell and tissue level with high degree of sensitivity

Identification of Kinases Regulating Prostate Cancer Cell Growth Using an RNAi Phenotypic Screen

As prostate cancer progresses to castration-resistant disease, there is an increase in signal transduction activity. Most castration-resistant prostate tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes, despite the near absence of circulating androgen in these patients. The AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules. Thus, the elevated signaling activity that occurs during progression to castration resistance can affect prostate cancer cell growth either through the AR or independent of the AR. In order to identify signaling pathways that regulate prostate cancer cell growth, we screened a panel of shRNAs targeting 673 human kinases against LNCaP prostate cancer cells grown in the presence and absence of hormone. The screen identified multiple shRNA clones against known and novel gene targets that regulate prostate cancer cell growth. Based on the magnitude of effect on growth, we selected six kinases for further study: MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1. Knockdown of these kinases decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells. However, these kinases had different effects on basal or androgen-induced transcriptional activity of AR target genes. MAP3K11 knockdown most consistently altered transcription of AR target genes, suggesting that MAP3K11 affected its growth inhibitory effect by modulating the AR transcriptional program. Consistent with MAP3K11 acting on the AR, knockdown of MAP3K11 inhibited AR Ser 650 phosphorylation, further supporting stress kinase regulation of AR phosphorylation. This study demonstrates the applicability of lentiviral-based shRNA for conducting phenotypic screens and identifies MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1 as regulators of prostate cancer cell growth. The thorough evaluation of these kinase targets will pave the way for developing more effective treatments for castration-resistant prostate cancer

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1

Aberrant transforming growth factor–β (TGF-β) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-β signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-β signaling activity and that stromal cellconditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-β–induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment

WNT signaling regulates self-renewal and differentiation of prostate cancer cells with stem cell characteristics

Prostate cancer cells with stem cell characteristics were identified in human prostate cancer cell lines by their ability to form from single cells self-renewing prostaspheres in non-adherent cultures. Prostaspheres exhibited heterogeneous expression of proliferation, differentiation and stem cell-associated makers CD44, ABCG2 and CD133. Treatment with WNT inhibitors reduced both prostasphere size and self-renewal. In contrast, addition of Wnt3a caused increased prostasphere size and self-renewal, which was associated with a significant increase in nuclear Β-catenin, keratin 18, CD133 and CD44 expression. As a high proportion of LNCaP and C4-2B cancer cells express androgen receptor we determined the effect of the androgen receptor antagonist bicalutamide. Androgen receptor inhibition reduced prostasphere size and expression of PSA, but did not inhibit prostasphere formation. These effects are consistent with the androgen-independent self-renewal of cells with stem cell characteristics and the androgen-dependent proliferation of transit amplifying cells. As the canonical WNT signaling effector Β-catenin can also associate with the androgen receptor, we propose a model for tumour propagation involving a balance between WNT and androgen receptor activity. That would affect the self-renewal of a cancer cell with stem cell characteristics and drive transit amplifying cell proliferation and differentiation. In conclusion, we provide evidence that WNT activity regulates the self-renewal of prostate cancer cells with stem cell characteristics independently of androgen receptor activity. Inhibition of WNT signaling therefore has the potential to reduce the self-renewal of prostate cancer cells with stem cell characteristics and improve the therapeutic outcome.Peer reviewe

Spondin-2 (SPON2), a More Prostate-Cancer-Specific Diagnostic Biomarker

BACKGROUND: Prostate-specific antigen (PSA) screening, although common, has recently been called into question. To find prostate cancer (PCa) diagnostic biomarkers that can make up for the defects of PSA, we compared the secretomes of several benign and PCa cell lines, selected candidate molecules, and then confirmed their clinical value. METHODOLOGY/PRINCIPAL FINDINGS: We first identified extracellular proteins by two-dimensional gel electrophoresis (2-DE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification. We then validated the secreted proteins on a cellular level, and finally determined whether they could be used as PCa diagnostic biomarkers using prostate tissue and serum specimens of Chinese volunteers by immunohistostaining and sandwich ELISA. We obtained credible extracellular protein 2-DE graphs of prostate cell lines. The 5 spots that showed superior repeatability were selected for LC-MS/MS analysis, which identified seven candidate molecules. One of the candidate molecules, spondin-2 (SPON2), was only expressed in the conditioned media (CM) of androgen receptor (AR) positive PCa cell lines. Using tissue microarray by immunohistostaining, we found SPON2 to be over-expressed in PCa. SPON2 staining was more intense in Gleason score sum 7-8 and in PCa patients with metastasis. By receiver operator characteristic (ROC) curve analysis, we found that the serum SPON2 level was elevated in PCa patients, showing sensitivity and specificity suitable for diagnostic use. We also found that SPON2 could be used to identify PCa patients with serum PSA levels no higher than 10 ng/ml from healthy elderly men. CONCLUSION/SIGNIFICANCE: SPON2 is a new serum and histological diagnostic biomarker for PCa. It can avoid some of the problems of PSA testing and was here found to offer relatively high sensitivity and specificity relative to PSA

Prognostic significance of osteopontin expression in early-stage non-small-cell lung cancer

Osteopontin (OPN) is a multifunctional protein, which has recently been shown to be linked to tumorigenesis, progression and metastasis in different malignancies. Since non-small-cell lung cancer (NSCLC)'s prognosis remains bad, with few predictors of outcome, the purpose of this study was to evaluate if OPN might be involved in NSCLC's biology and therefore represent a prognostic marker and a target for new therapeutic trials. Immunohistochemistry was used to detect OPN expression, evaluated as percentage of neoplastic cells with cytoplasmic immunoreactivity, in a wide cohort of patients with stage I NSCLC (136 cases). The median value of this series (20% of positive cells) was used as the cutoff value to distinguish tumours with low (<20%) from tumours with high (⩾20%) OPN expression. A statistically significant correlation between high levels of OPN and shorter overall (P=0.034) and disease-free (P=0.011) survival in our patients was shown. Our results support the hypothesis that high OPN expression is a significantly unfavourable prognostic factor for the survival of patients with stage I NSCLC. This conclusion has notable importance in terms of the biological characterization of early-stage tumours and therapeutic opportunities

Optimization of the Balanced Steady State Free Precession (bSSFP) Pulse Sequence for Magnetic Resonance Imaging of the Mouse Prostate at 3T

INTRODUCTION: MRI can be used to non-invasively monitor tumour growth and response to treatment in mouse models of prostate cancer, particularly for longitudinal studies of orthotopically-implanted models. We have optimized the balanced steady-state free precession (bSSFP) pulse sequence for mouse prostate imaging. METHODS: Phase cycling, excitations, flip angle and receiver bandwidth parameters were optimized for signal to noise ratio and contrast to noise ratio of the prostate. The optimized bSSFP sequence was compared to T1- and T2-weighted spin echo sequences. RESULTS: SNR and CNR increased with flip angle. As bandwidth increased, SNR, CNR and artifacts such as chemical shift decreased. The final optimized sequence was 4 PC, 2 NEX, FA 50°, BW ±62.5 kHz and took 14-26 minutes with 200 µm isotropic resolution. The SNR efficiency of the bSSFP images was higher than for T1WSE and T2WSE. CNR was highest for T1WSE, followed closely by bSSFP, with the T2WSE having the lowest CNR. With the bSSFP images the whole body and organs of interest including renal, iliac, inguinal and popliteal lymph nodes were visible. CONCLUSION: We were able to obtain fast, high-resolution, high CNR images of the healthy mouse prostate with an optimized bSSFP sequence

Androgen and retinoic acid interaction in LNCaP cells, effects on cell proliferation and expression of retinoic acid receptors and epidermal growth factor receptor

BACKGROUND: Modulation of the expression of retinoic acid receptors (RAR) α and γ in adult rat prostate by testosterone (T) suggests that RAR signaling events might mediate some of the androgen effects on prostate cells. METHOD: In this study, we examined the interactions between T and retinoic acid (RA) in cell growth of human prostate carcinoma cells, LNCaP, and their relationship with the expression of RAR and epidermal growth factor receptor (EGF-R). RESULTS: Both T and RA, when administered alone, stimulated (3)H-thymidine incorporation in LNCaP cells in a dose-dependent manner; the effect of each agent was reciprocally attenuated by the other agent. Testosterone treatment of LNCaP cells also resulted in dose dependent, biphasic increases in RAR α and γ mRNAs; increases paralleled that of (3)H-thymidine incorporation and were attenuated by the presence of 100 nM RA. These results suggest a link between RAR signaling and the effect of T on LNCaP cell growth. Gel electrophoretic mobility shift assays revealed the presence of putative androgen responsive element (ARE) in the promoter region of RAR α gene, suggesting that a direct AR-DNA interaction might mediate the effects of T on RAR α gene. Furthermore, treatment of LNCaP cells with 20 nM T resulted in an increase in EGF-R. In contrast, EGF-R was suppressed by 100 nM RA that also suppressed the effect of T. CONCLUSIONS: Current results demonstrate interactions between T and RA in the expression of RARs and cell growth in LNCaP cells. The presence of putative ARE in the promoter of the RAR α gene suggests that AR-DNA interaction might mediate the effects of T on RAR α gene. The opposite effects of T and RA on the expression of RAR and EGF-R suggest that signal events of these receptors might be involved in the interaction between T and RA in the control of LNCaP cell growth

Increased transcriptional activity of prostate-specific antigen in the presence of TNP-470, an angiogenesis inhibitor

Prostate-specific antigen, PSA, is regarded as a reliable surrogate marker for androgen-independent prostate cancer (AIPC). Concern has been raised that investigational agents may affect PSA secretion without altering tumour growth or volume. In a phase I trial, several patients with AIPC had elevated serum PSA levels while receiving TNP-470 that reversed upon discontinuation. TNP-470 inhibits capillary growth in several angiogenesis models. These observations prompted us to determine if TNP-470, or its metabolite, AGM-1883, altered PSA secretion. Intracellular protein and transcriptional levels of PSA and androgen receptor were also determined. The highest TNP-470 concentration produced a 40.6% decrease in cell number; AGM-1883 had minimal effects on cell viability. PSA secretion per cell was induced 1.1- to 1.5-fold following TNP-470 exposure. The same trend was observed for AGM-1883. PSA and AR were transcriptionally up-regulated within 30 min after exposure to TNP-470. PSA transcription was increased 1.4-fold, while androgen receptor (AR) transcription was induced 1.2-fold. The increased PSA transcriptional activity accounts for the increased PSA secretion. Increased AR transcription was also reflected at the protein level. In conclusion, TNP-470 and AGM-1883 both up-regulated PSA making clinical utilization of this surrogate marker problematic. © 1999 Cancer Research Campaig