Mitotic quiescence, but not unique "stemness," marks the phenotype of bone metastasis-initiating cells in prostate cancer

This study aimed to identify subpopulations of prostate cancer cells that are responsible for the initiation of bone metastases. Using rapidly dividing human prostate cancer cell lines, we identified mitotically quiescent subpopulations (<1%), which we compared with the rapidly dividing populations for patterns of gene expression and for their ability to migrate to the skeletons of athymic mice. The study used 2-photon microscopy to map the presence/distribution of fluorescently labeled, quiescent cells and luciferase expression to determine the presence of growing bone metastases. We showed that the mitotically quiescent cells were very significantly more tumorigenic in forming bone metastases than fast-growing cells (55 vs. 15%) and had a unique gene expression profile. The quiescent cells were not uniquely stem cell like, with no expression of CD133 but had the same level expression of other putative prostate stem cell markers (CD44 and integrins α2/β1), when compared to the rapidly proliferating population. In addition, mitotic quiescence was associated with very high levels of C-X-C chemokine receptor type 4 (CXCR4) production. Inhibition of CXCR4 activity altered the homing of quiescent tumor cells to bone. Our studies suggest that mitotic dormancy is a unique phenotype that facilitates tumor cell colonization of the skeleton in prostate cancer.—Wang, N., Docherty, F., Brown, H. K., Reeves, K., Fowles, A., Lawson, M., Ottewell, P. D., Holen, I., Croucher, P. I., Eaton, C. L. Mitotic quiescence, but not unique “stemness,” marks the phenotype of bone metastasis-initiating cells in prostate cancer

iTRAQ Identification of Candidate Serum Biomarkers Associated with Metastatic Progression of Human Prostate Cancer

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer ‘BPH’, (ii) localised cancer with no evidence of progression, ‘non-progressing’ (iii) localised cancer with evidence of biochemical progression, ‘progressing’, and (iv) bone metastasis at presentation ‘metastatic’. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and ‘panels’ of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation

The frequency of osteolytic bone metastasis is determined by conditions of the soil, not the number of seeds; evidence from in vivo models of breast and prostate cancer

Background While both preclinical and clinical studies suggest that the frequency of growing skeletal metastases is elevated in individuals with higher bone turnover, it is unclear whether this is a result of increased numbers of tumour cells arriving in active sites or of higher numbers of tumour cells being induced to divide by the bone micro-environment. Here we have investigated how the differences in bone turnover affect seeding of tumour cells and/or development of overt osteolytic bone metastasis using in vivo models of hormone-independent breast and prostate cancer. Methods Cohorts of 6 (young) and 16 (mature)-week old BALB/c nude mice were culled 1, 7 and 21 days after received intracardiac injection of luciferase expressing human prostate (PC3) or breast cancer (MDA-MB-231) cell lines labelled with a fluorescent cell membrane dye (Vybrant DiD). The presence of growing bone metastases was determined by bioluminescence using an in vivo imaging system (IVIS) and followed by anatomical confirmation of tumour metastatic sites post mortem, while the presence of individual fluorescently labelled tumour cells was evaluated using two-photon microscopy ex vivo. The bone remodelling activities were compared between young and mature naïve mice (both male and female) using micro-CT analysis, ELISA and bone histomorphometry. Results Both prostate and breast cancer cells generated higher numbers of overt skeletal lesions in young mice (~80%) than in mature mice (~20%). Although mature mice presented with fewer overt bone metastases, the number of tumour cells arriving/colonizing in the tibias was comparable between young and mature animals. Young naïve mice had lower bone volume but higher bone formation and resorption activities compared to mature animals. Conclusions Our studies suggest that higher frequencies of growing osteolytic skeletal metastases in these models are linked to increased bone turnover and not to the initial number of tumour cells entering the bone microenvironment

Human bone marrow stromal cells protect prostate cancer cells from TRAIL-induced apoptosis.

Tumor-derived OPG has recently been shown to protect prostate cancer cells from apoptosis. This study has confirmed that bone marrow stromal cell-derived OPG also suppresses cytokine-induced apoptosis in this tumor type, suggesting that it may be the presence of bone-derived OPG that is responsible for the observed preference of these cells in colonizing the skeleton. INTRODUCTION - Metastasis to the skeleton occurs in around 70% of patients with advanced prostate cancer (CaP), suggesting that the bone microenvironment may provide factors that favor the growth and survival of prostate cancer cells. Osteoprotegerin (OPG) is a molecule involved in bone remodeling, where it acts as an inhibitor of osteoclastogenesis, but it is also a decoy receptor for TRAIL/Apo 2L, a member of the TNF family of pro-apoptotic cytokines. The aim of this study was to determine whether OPG produced by human bone marrow stromal cells could protect prostate cancer cells from TRAIL-induced apoptosis. MATERIALS AND METHODS - Human bone marrow stromal cell cultures were generated from bone biopsies taken from newly diagnosed untreated CaP patients with (M1) or without (M0) bony metastasis. The stromal origin of these cells was confirmed by Western blot analysis using antibodies raised to stromal and epithelial markers. Media were conditioned over the cultures of these cells for 4 days, and levels of OPG were determined using an ELISA. The human prostate cancer cell line PC3 was challenged with TRAIL (50 ng/ml) in fresh media or in media supplemented with 50% conditioned media, and apoptosis was assessed using DAPI stain. The effects of specific removal of OPG activity by immunoprecipitation or by co-treatment of cultures with an alternative ligand for OPG (RANKL) were also tested. RESULTS AND CONCLUSIONS - The presence of stromal cell conditioned media in PC3 culture significantly reduced TRAIL-induced apoptosis. All stromal cell lines isolated were shown to express OPG and to release this protein into the conditioned media. Immunoprecipitation of OPG and co-treatment of cultures with sRANKL reversed the protective effects of the conditioned media. These data suggest that at least part of the survival advantage gained by CaP cells in colonizing bone may be caused by the production of OPG by tumor-associated stromal cells.</p

Evaluation of the frequency of putative prostate cancer stem cells in primary and metastatic prostate cancer

Tumour cells with a stem cell-like phenotype have recently been identified in prostate tumors and it has been suggested that this population may be responsible for the diversity of cell types within tumors and also for the initiation of metastases. These cells carry a number of defined markers: they are cd133 and cd44+ve and express high levels of alpha2beta1 integrin. In this study we have, for the first time, assessed matched primary and bone marrow biopsies from prostate cancer patients for the distribution of cells carrying these and a number of other putative stem cell markers

iTRAQ-facilitated Proteomic analysis of human prostate cancer cells identifies proteins associated with progression

The unpredictable behavior of prostate cancer presents a major clinical challenge during patient management. In order to gain an insight into the molecular mechanisms associated with prostate cancer progression, we employed the shot-gun proteomic approach of isobaric tags for relative and absolute quantitation (iTRAQ), followed by 2D-LC-MS/MS, using the poorly metastatic LNCaP cell line and its highly metastatic variant LNCaP-LN3 cell line as a model. A total number of 280 unique proteins were identified (≥95% confidence), and relative expression data was obtained for 176 of these. Ten proteins were found to be significantly up-regulated (≥1.50 fold), while 4 proteins were significantly down-regulated (≥−1.50 fold), in LNCaP-LN3 cells. Differential expression of brain creatine kinase (CKBB), soluble catechol- O-methyltransferase (S-COMT), tumor rejection antigen (gp96), and glucose regulated protein, 78 kDa (grp78), was confirmed by Western blotting or independent 2D-PAGE analysis. Additionally, iTRAQ analysis identified absence of the lactate dehydrogenase-B (LDH-B) subunit in LNCaP-LN3 cells, confirming our published data. The clinical relevance of gp96 was assessed by immunohistochemistry using prostate tissues from benign ( n = 95), malignant ( n = 66), and metastatic cases ( n = 3). Benign epithelium showed absent/weak gp96 expression in the basal cells, in contrast to the moderate/strong expression seen in malignant epithelium. Furthermore, there was a statistically significant difference in the intensity of gp96 expression between benign and malignant cases ( p < 0.0005, Mann–Whitney U). Our study is the first to report the application of iTRAQ technology and its potential for the global proteomic profiling of prostate cancer cells, including the identification of absent protein expression

Osteoprotegerin (OPG) expression by breast cancer cells in vitro and breast tumours in vivo - a role in tumour cell survival?

In addition to its role in bone turnover, osteoprotegerin (OPG) has been reported to bind to and inhibit Tumour necrosis factor-related apoptosis inducing ligand (TRAIL). TRAIL is produced in tumours by invading monocytes, inducing apoptosis in neoplastic cells sensitive to this cytokine. OPG production by tumour cells would therefore be a novel mechanism whereby cancer cells evade host defences and gain a growth advantage. In this study we show that OPG produced by breast cancer cells enhances tumour cell survival by inhibiting TRAIL-induced apoptosis. OPG expression by breast cancer cells (MDA-MB 436/231) grown in vitro was examined using PCR and ELISA, and the sensitivity of these cells to TRAIL was determined. The effects of OPG on TRAIL induced apoptosis was investigated by exposing MDA-MB 436 cells to TRAIL, in the presence or absence of OPG, followed by assessment of nuclear morphology. We found that the levels of OPG produced were sufficient to inhibit TRAIL-induced apoptosis, suggesting that OPG may play a role in tumour cell survival. We also examined the expression pattern of OPG in a selection of breast tumours (n=400) by immunohistochemistry, and related OPG expression to the clinico-pathological data for each tumour. OPG expression was found to be negatively correlated with increasing tumour grade. To our knowledge these results are the first to demonstrate that OPG can act as an endocrine survival factor for breast cancer cells, as well as reporting the expression patterns of OPG in a large cohort of human breast tumours

Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells identifies candidate biomarkers of progression: an exploratory study.

BACKGROUND: Due to the heterogeneity in the biological behavior of prostate cancer, biomarkers that can reliably distinguish indolent from aggressive disease are urgently needed to inform treatment choices. METHODS: We employed 8-plex isobaric Tags for Relative and Absolute Quantitation (iTRAQ), to profile the proteomes of two distinct panels of isogenic prostate cancer cells with varying growth and metastatic potentials, in order to identify novel biomarkers associated with progression. The LNCaP, LNCaP-Pro5, and LNCaP-LN3 panel of cells represent a model of androgen-responsive prostate cancer, while the PC-3, PC-3M, and PC-3M-LN4 panel represent a model of androgen-insensitive disease. RESULTS: Of the 245 unique proteins identified and quantified (>or=95% confidence; >or=2 peptides/protein), 17 showed significant differential expression (>or=+/-1.5), in at least one of the variant LNCaP cells relative to parental cells. Similarly, comparisons within the PC-3 panel identified 45 proteins to show significant differential expression in at least one of the variant PC-3 cells compared with parental cells. Differential expression of selected candidates was verified by Western blotting or immunocytochemistry, and corresponding mRNA expression was determined by quantitative real-time PCR (qRT-PCR). Immunostaining of prostate tissue microarrays for ERp5, one of the candidates identified, showed a significant higher immunoexpression in pre-malignant lesions compared with non-malignant epithelium (P < 0.0001, Mann-Whitney U-test), and in high Gleason grade (4-5) versus low grade (2-3) cancers (P < 0.05). CONCLUSIONS: Our study provides proof of principle for the application of an 8-plex iTRAQ approach to uncover clinically relevant candidate biomarkers for prostate cancer progression