Non-canonical EphA2 activation underpins PTEN-mediated metastatic migration and poor clinical outcome in prostate cancer.

BACKGROUND: The key process of mesenchymal to amoeboid transition (MAT), which enables prostate cancer (PCa) transendothelial migration and subsequent development of metastases in red bone marrow stroma, is driven by phosphorylation of EphA2S897 by pAkt, which is induced by the omega-6 polyunsaturated fatty acid arachidonic acid. Here we investigate the influence of EphA2 signalling in PCa progression and long-term survival. METHODS: The mechanisms underpinning metastatic biopotential of altered EphA2 signalling in relation to PTEN status were assessed in vitro using canonical (EphA2D739N) and non-canonical (EphA2S897G) PC3-M mutants, interrogation of publicly available PTEN-stratified databases and clinical validation using a PCa TMA (n = 177) with long-term follow-up data. Spatial heterogeneity of EphA2 was assessed using a radical prostatectomy cohort (n = 67). RESULTS: Non-canonical EphA2 signalling via pEphA2S897 is required for PCa transendothelial invasion of bone marrow endothelium. High expression of EphA2 or pEphA2S897 in a PTENlow background is associated with poor overall survival. Expression of EphA2, pEphA2S897 and the associated MAT marker pMLC2 are spatially regulated with the highest levels found within lesion areas within 500 µm of the prostate margin. CONCLUSION: EphA2 MAT-related signalling confers transendothelial invasion. This is associated with a substantially worse prognosis in PTEN-deficient PCa.

Automated quantitative high-throughput multiplex immunofluorescence pipeline to evaluate OXPHOS defects in formalin-fixed human prostate tissue.

Advances in multiplex immunofluorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated high-throughput mIF workflow for quantitative single-cell level assessment of formalin fixed paraffin embedded tissue (FFPE), leveraging tyramide signal amplification on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign-adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fixed tissues, and their impact on long-term clinical outcomes.

Primary Mutational Landscape Linked with Pre-Docetaxel Lactate Dehydrogenase Levels Predicts Docetaxel Response in Metastatic Castrate-Resistant Prostate Cancer.

BACKGROUND: Docetaxel chemotherapy is a standard of care for metastatic castrate-resistant prostate cancer (mCRPC): 40-50% of patients achieve a biochemical response. However, there is a lack of response predictive biomarkers. OBJECTIVE: To assess lactate dehydrogenase (LDH) as a docetaxel response biomarker in mCRPC and to examine the association of LDH with genomic alterations in primary diagnostic biopsies. DESIGN, SETTING, AND PARTICIPANTS: Clinical and associated primary tumour-targeted next-generation sequencing data from matched training (n=150) and test (n=120) cohorts of progressive mCRPC patients receiving docetaxel therapy were analysed. Data were correlated with large-scale prostate cancer genomic datasets. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Prostate-specific antigen (PSA) response, radiographic response, biochemical progression-free survival (PFS), overall survival (OS), genomic analysis of primary biopsies, and genomic datasets (Memorial Sloan Kettering Cancer Center [MSKCC] and SU2C/PCF). RESULTS AND LIMITATIONS: Serum LDH ≥450U/l is a reliable prognostic biomarker (area under the curve: 0.757 [standard deviation 0.054, 95% confidence interval [CI] 0.650-0.864, p<0.001]) in progressive mCRPC, predicting PFS at 3 mo. Patients with LDH ≥450U/l were poorer PSA responders, with shorter PFS (213 vs 372 d, hazard ratio [HR] 1.876, 95% CI 1.289-2.7300) and OS (362 vs 563 d, HR 1.630, 95% CI 1.127-2.357). High LDH is an independent surrogate marker for survival following docetaxel and predicts a poor radiological response (p=0.043). Of the 14 patients with LDH ≥450U/l available for next-generation sequencing, nine (64.3%) were more likely to have DNA repair gene mutation(s) (BRCA1/2, ATM, CHEK2, Fanconi anaemia gene) in their primary biopsy. Cross correlation with MSKCC and SU2C/PCF databases revealed a positive correlation between LDHA, PARP1 (r=0.667, p<0.01), and other DNA repair genes. CONCLUSIONS: Genomic abnormalities of LDHA and DNA repair in primary biopsies link to high pretreatment LDH and poor response to docetaxel in mCRPC. PATIENT SUMMARY: The presence of mutations of the lactate dehydrogenase and DNA repair pathways are associated with aggressive prostate cancer and poor response to chemotherapy later in the disease.

Copper modulates zinc metalloproteinase-dependent ectodomain shedding of key signaling and adhesion proteins and promotes the invasion of prostate cancer epithelial cells.

A disintegrin and metalloproteinases (ADAMs) and matrix metalloproteinases (MMPs) are zinc metalloproteinases (ZMPs) that catalyze the "ectodomain shedding" of a range of cell surface proteins including signaling and adhesion molecules. These "sheddases" are associated with the invasion and metastasis of a range of cancers. Increased serum and tumor tissue levels of copper are also observed in several cancers, although little is known about how the metal might promote disease progression at the molecular level. In the current study, we investigated whether copper might regulate the ectodomain shedding of two key cell surface proteins implicated in the invasion and metastasis of prostate cancer, the Notch ligand Jagged1 and the adhesion molecule E-cadherin, and whether the metal was able to influence the invasion of the prostate cancer epithelial cell line PC3. Physiological copper concentrations stimulated the ZMP-mediated proteolysis of Jagged1 and E-cadherin in cell culture models, whereas other divalent metals had no effect. Copper-mediated Jagged1 proteolysis was also observed following the pretreatment of cells with cycloheximide and in a cell-free membrane system, indicating a posttranslational mechanism of sheddase activation. Finally, the concentrations of copper that stimulated ZMP-mediated protein shedding also enhanced PC3 invasion; an effect that could be negated using a sheddase inhibitor or copper chelators. Collectively, these data implicate copper as an important factor in promoting prostate cancer cell invasion and indicate that the selective posttranslational activation of ZMP-mediated protein shedding might play a role in this process.

SR-FTIR spectroscopy of renal epithelial carcinoma side population cells displaying stem cell-like characteristics.

It is hypothesized that cells with stem cell-like properties may be influential in carcinogenesis, possessing the ability to self-renew, produce differentiated daughter cells and resist environmental or therapeutic injury. This has led to a surge in interest in identifying and characterizing the tumour initiating or cancer stem cell (CSC) with the aim of discovering novel diagnostic and prognostic markers and of understanding the basic biology with the ultimate aim of generating new therapeutic approaches and biomarkers. However, a major hurdle to this process has been the lack of a truly specific cancer stem cell biomarker allied to the rarity of these cells. This has led to problems in characterising these CSCs by traditional '-omic' techniques. Using a renal carcinoma cell line model, we show that synchrotron radiation-Fourier transform infrared (SR-FTIR) spectroscopy is a suitable tool to measure discrete differences in the biochemistry of small numbers of single-cells. Using the chemometric techniques of Principal Component and Linear Discriminant Analysis (PCA and LDA) for multivariate reduction, biochemical differences between the cells from different sub-populations were evaluated. Results found lipid and phosphodiester vibrations to be particularly good discriminating markers in the spectra of these stem-like cells, relative to the more differentiated, proliferating cells that make up the majority of the cell population.

Hoechst 33342 side population identification is a conserved and unified mechanism in urological cancers.

Mutation within the adult human stem cell (SC) compartment has been proposed as a factor in the initiation and promotion of carcinogenesis. Isolation of these cancer stem cells (CSCs) has proven difficult, limiting their subsequent phenotypic, functional, and genetic characterization. We have used the Hoechst 33342 dye efflux technique to isolate an epithelial side population (SP) from genitourinary (GU) cancers, which is enriched for cells with SC traits. With informed consent, samples were taken from patients with primary tumors and undergoing surgery for prostatic (CaP), invasive bladder transitional cell (TCC), and renal cell carcinomas (RCC). Single cell epithelial suspensions were extracted from these and incubated with Hoechst 33342. Hoechst SP/non-SP profiles were then generated by flow cytometry using standardized protocols. SP/non-SP cell cycle status was established by Hoechst 33342 and Pyronin Y staining. Immunocytochemistry staining was performed for markers suggested as stem markers as well as lineage-specific markers. Functionality was determined using colony-forming assays and long-term monolayer culture. A characteristic verapamil-sensitive SP was isolated from all 3 urological malignancies and represented 0.57% +/- 0.11% (CaP), 0.52% +/- 0.49% (TCC), and 5.9% +/- 0.9% (RCC) of the total epithelial population. Cell cycle analysis showed that the SP had enhanced numbers of cells in G(0) as compared to the total cell population (CaP 12.4% +/- 3.2 vs. 3.8% +/- 1.0, RCC 23.2% +/- 3.4 vs. 1.8% +/- 0.9, and TCC 28.5% +/- 4.9 vs. 4% +/- 1.3). Immunocytochemistry demonstrated an increased expression of proliferative and putative stem markers within the SP fraction. Cultures confirmed significant enhancement of colony-forming ability and proliferative capacity of the SP fraction. A characteristic SP enriched for stem-like cells has been isolated from the 3 most common urological malignancies. This provides strong evidence that Hoechst 33342 efflux is a conserved and unified mechanism in GU cancer.

Molecular mechanisms of metastasis in prostate cancer.

Prostate cancer (PCa) preferentially metastasizes to the bone marrow stroma of the axial skeleton. This activity is the principal cause of PCa morbidity and mortality. The exact mechanism of PCa metastasis is currently unknown, although considerable progress has been made in determining the key players in this process. In this review, we present the current understanding of the molecular processes driving PCa metastasis to the bone.

Characterization of the Hoechst 33342 side population from normal and malignant human renal epithelial cells.

The fundamental changes which predispose for renal cell carcinoma (RCC) are poorly characterized. It is hypothesized that "cancer stem cells" may be influential in carcinogenesis, and the epithelial side population (SP) is enriched for stemlike cells in other epithelial cancers. In this study, we have isolated and characterized the SP and non-SP (NSP) populations from normal (NK) and malignant (RCC) human kidney tissue. NK specimens were taken from patients undergoing non-renal cancer surgery and paired malignant and macroscopically normal tissue samples were taken from patients undergoing surgery for RCC. The Hoechst 33342 dye efflux technique was used to isolate epithelial SP and NSP from normal and malignant human renal tissue. Cellular subpopulations were phenotyped for lineage, cell cycle, and putative stem cell markers, and functionally characterized using in vitro colony-forming and proliferation assays. The SP constituted 3.8 +/- 0.4 and 5.9 +/- 0.9% of epithelial cells in NK and RCC, respectively, of which 14.1 +/- 3.5 and 13.2 +/- 3.6% were shown to be in G(0). SP cells demonstrated greater proliferative potential in colony-forming efficiency, long-term culture, and spheroids assays and were shown to be maintained upon tissue culture passage. We have shown that the renal SP is enriched for quiescent cells, with a high proliferative capacity and stemlike properties. The population is, however, heterogeneous, confirming that the terms "SP cell" and "stem cell" cannot be used interchangeably.

Characterization of benign and malignant prostate epithelial Hoechst 33342 side populations.

BACKGROUND: The prostate epithelial stem cell has been proposed as the primary origin of neoplastic change in prostate cancer. However, the isolation and characterization of unexpanded prostate epithelial stem cells have proven problematic. METHODS: A prostate epithelial side population (SP) has been isolated utilizing a modified Hoechst 33342 dye efflux assay from both benign and malignant prostate tissue. CD45(-ve), integrin alpha2(+ve) Hoechst 33342 SP and NSP cells were isolated by FACS, immunophenotyped and functionally characterized in 3D culture. RESULTS: FACS analysis revealed a verapamil sensitive SP accounting for 0.93 +/- 0.12% and 0.57 +/- 0.11% of the total epithelial population from both benign and malignant prostates. The benign SP phenotype revealed a heterogeneous cell population consisting predominantly of small basal cells containing minimal cytoplasm. Conversely, the malignant SP was of undetermined acinar origin and with a complete loss of expression of the CDK2 inhibitor p21(WAF1/Cip1). In vitro androgen-enhanced 3D culture of the benign and malignant SP cells led to the production of spheroids which had acinus like morphology and expressed primitive and basal cell markers. Incorporation of the CD133 marker isolated a further SP sub-fraction accounting for 0.037 +/- 0.01% of epithelial cells. CONCLUSIONS: Our observations are consistent with the Hoechst 33342 dye efflux assay isolating a stem cell enriched population which can be further sub-fractionated by CD133 selection. Moreover, the loss of the CDK inhibitor in malignancy is consistent with the hypothesis that neoplastic change originates in the stem cell compartment.

Direct evidence of lipid translocation between adipocytes and prostate cancer cells with imaging FTIR microspectroscopy.

Various epidemiological studies show a positive correlation between high intake of dietary FAs and metastatic prostate cancer (CaP). Moreover, CaP metastasizes to the bone marrow, which harbors a rich source of lipids stored within adipocytes. Here, we use Fourier transform infrared (FTIR) microspectroscopy to study adipocyte biochemistry and to demonstrate that PC-3 cells uptake isotopically labeled FA [deuterated palmitic acid (D(31)-PA)] from an adipocyte. Using this vibrational spectroscopic technique, we detected subcellular locations in a single adipocyte enriched with D(31)-PA using the upsilon(as+s)(C-D)(2+3) (D(31)-PA): upsilon(as+s)(C-H)(2+3) (lipid hydrocarbon) signal. In addition, larger adipocytes were found to consist of a higher percentage of D(31)-PA of the total lipid found within the adipocyte. Following background subtraction, the upsilon(as)(C-D)(2+3) signal illuminated starved PC-3 cells cocultured with D(31)-PA-loaded adipocytes, indicating translocation of the labeled FA. This study demonstrates lipid-specific translocation between adipocytes and tumor cells and the use of FTIR microspectroscopy to characterize various biomolecular features of a single adipocyte without the requirement for cell isolation and lipid extraction.

A correlation of FTIR spectra derived from prostate cancer biopsies with gleason grade and tumour stage.

OBJECTIVES: We introduce biochemistry as a second dimension to Gleason grading, using Fourier transform infrared (FTIR) microspectroscopy. For the first time, we correlate FTIR spectra derived from prostate cancer (pCA) tissue with Gleason score and the clinical stage of the tumour at time of biopsy. METHODS: Serial sections from paraffin-embedded pCA tissue were collected. One was stained with hematoxylin and eosin and Gleason scored; FTIR spectra were collected from malignant locations using a second unstained section. FTIR spectra, representing different Gleason grades, were used to construct a diagnostic classifier for pCA using linear discriminant analysis (LDA). This model was blind tested using 383 IR spectra from 36 biopsies. RESULTS: Using a three-band Gleason criteria, we obtained sensitivity of > or =70% for the FTIR-LDA model to predict Gleason <7,=7, and >7, with specificities of > or =81%. Using a threshold of Gleason/FTIR-LDA score of > or =8, we obtained a sensitivity and specificity of 71% and 67%, respectively, for the correlation with metastatic tumours using the FTIR-LDA system and 85% and 63%, respectively, for the correlation of metastatic tumours using the Gleason system. CONCLUSIONS: There is a correlation between tissue architecture using Gleason score with tissue biochemistry using FTIR-LDA. Both systems are similar in their performance in predicting metastatic behaviour in tumours from individual patients.

AC133+ G0 cells from cord blood show a high incidence of long-term culture-initiating cells and a capacity for more than 100 million-fold amplification of colony-forming cells in vitro.

AC133+ cells may provide an alternative to CD34+ cells as a target for cell expansion and gene therapy protocols. We examined the differences in proliferative potential between cord blood selected for AC133 or CD34 in serum-free, stroma cell-free culture for up to 30 weeks. Because most hemopoietic stem cells reside within the G0/G1 phase of the cell cycle, we combined enrichment according to AC133 or CD34 expression with G0 position in the cell cycle to identify populations enriched for putative stem cells. Our results show that AC133+ G0 cells demonstrated a long-term culture-initiating cell incidence of 1 in 4.2 cells, had a colony-forming cell incidence of 1 in 2.8 cells, were capable of producing 660 million-fold expansion of nucleated cells and 120 million-fold expansion of colony-forming units-granulocyte-macrophage over a period of 30 weeks, and were consistently superior to CD34+ G0 cells according to these parameters. Furthermore, we have shown that AC133+CD34- cells have the ability to generate CD34+ cells in culture, which suggests that at least some AC133+ cells are ancestral to CD34+ cells. We conclude that AC133 isolation provides a better means of selection for primitive hemopoietic cells than CD34 and that, in combination with isolation according to G0 phase of the cell cycle, AC133 isolation identifies a highly enriched population of putative stem cells.

Differential inhibition of invasion and proliferation by bisphosphonates: anti-metastatic potential of Zoledronic acid in prostate cancer.

OBJECTIVES: To determine the mode of action of Zoledronic acid in the inhibition of metastasis in prostate cancer and the reduction of prostate cancer bone metastases. METHODS: Benign and malignant primary prostatic epithelial cells (PEC) and the PC-3 prostate cancer cell line were studied in co-culture using human bone marrow stroma in the presence of escalating doses of EDTA, Clodronate, Pamidronate and Zoledronic acid. PEC binding and colony growth in bone marrow stroma was measured using standardised quantitative techniques. PEC cellular invasion through Matrigel and an endothelial monolayer was measured either in invasion chambers or by the measurement of endothelial monolayer permeability to fluorescent dextran. Co-culture supernatants were assayed for specific cytokine levels. Bone marrow cellular toxicity was assessed using a standard Mix assay. RESULTS: Treatment of PEC with up to 100 microM bisphosphonate did not affect their ability to bind to bone marrow endothelium or stroma. Bone marrow endothelial permeability was reduced by 100 microM Zoledronic acid by 3.8% (p = 0.03856). Both Pamidronate (40% at 100 microM, p < or = 0.05) and Zoledronic acid inhibited PEC invasion, with Zoledronic acid being the most potent (40% at 10 microM, p < or = 0.05 rising to 91% at 100 microM, p < or = 0.001). Zoledronic acid inhibits malignant PEC proliferation in bone marrow stroma co-culture (26.5% at 10 microM rising to 66.5% at 40 microM). This was accompanied by changes within the cytokine milieu with a >800% rise in TIMP-2. CONCLUSION: Zoledronic acid is a potent inhibitor of PEC invasion across bone marrow endothelium and colony formation with the bone marrow stroma, affecting the MMP: TIMP-2 balance to favour MMP inhibition.

A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow.

Homing of bone marrow stromal cells (MSCs) to bone and bone marrow after transplantation, important for the correction of conditions such as metabolic storage disorders, can occur but with poor efficiency. Substantial improvements in engraftment will be required in order to derive a clinical benefit from MSC transplantation. Chemokines are the most important factors controlling cellular migration. Stromal-derived factor-1 (SDF-1) has been shown to be critical in promoting the migration of cells to the bone marrow, via its specific receptor CXCR4. The aim of our study was to investigate CXCR4 expression on MSCs and its role in mediating migration to bone marrow. We show that CXCR4, although present at the surface of a small subset of MSCs, is important for mediating specific migration of these cells to bone marrow.

The combined application of FTIR microspectroscopy and ToF-SIMS imaging in the study of prostate cancer.

At present. a prognosis for prostate cancer (CaP) is determined by its accurate assessment of disease grade and stage. Histopathological typing using the Gleason grading system is the most universally accepted approach for grading CaP and provides an indication as to the aggressiveness of the tumour at the time of presentation. However, this system is based upon a visual criterion of pattern recognition that is operator dependent and subject to intra- and inter-observer variability, which can result in inappropriate patient management. Thus, there is a need for a molecular based diagnostic technique to grade tissue samples in a reliable and reproducible manner. In this paper we report a prototype diagnostic classifier for Gleason graded CaP tissue, based upon the integration of FTIR microspectroscopy with linear discriminant analysis (LDA). Blind testing of this model demonstrates 80% agreement of FTIR-LDA grade to histology, for the specimens analysed. We also study the effects of connective tissue absorption upon the area ratio of peaks at A1030 cm(-1)/A1080(cm(-1) which we use as a criterion to biospectroscopically map and distinguish areas of benign from malignant tissue. In addition, imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been applied to study freeze-dried, freeze-fractured prostate cancer cells in vitro. Preliminary results demonstrate localisation of various species including K, Ca and Mg within the cytoplasm that are present at millimolar concentrations and vital to cell physiology. The soft ionisation technique employed also permits for molecular information to be obtained and this has been used to evaluate chemically, different fracture planes within the analysis area.

Novel method for the isolation and characterisation of the putative prostatic stem cell.

BACKGROUND: Prostate stem cells, responsible for the development, maturation, and function of the prostate, have been implicated in the aetiology of both benign prostate hyperplasia (BPH) and prostate cancer (CaP). However, research has been hampered by the lack of a definitive stem cell marker. We have adapted the protocol for differential Hoechst 33342 uptake by hemopoietic stem cells to enable isolation of putative stem cells from the prostate. METHODS: Prostate epithelial cells isolated from prostate tissue obtained from patients with BPH after transurethral resection of the prostate were stained with Hoechst 33342. The Hoechst 33342 Red/Blue flow cytometry profile was then determined. Hoechst 33342 and Pyronin Y staining was used to determined the cell cycle status. RESULTS: A verapamil-sensitive side population (SP) can be isolated from primary prostate tissue accounting for 1.38% +/- 0.07% of prostate epithelial cells. Cell cycle analysis of this SP population revealed that the majority of SP cells are in either G0 (12.38 +/- 0.31%) or G1 (63.19 +/- 2.13%). CONCLUSIONS: The Hoechst 33342 dye efflux protocol can be adapted for the isolation of a SP from primary prostate tissue.

Enhanced expression of vimentin in motile prostate cell lines and in poorly differentiated and metastatic prostate carcinoma.

BACKGROUND: The metastatic potential of a series of prostate cell lines was analysed by measuring motility and invasiveness, and further correlated to the expression of epithelial differentiation markers. METHODS: Invasion and motility were measured using in vitro assays. Immunohistochemistry of cell lines and tissues was used to identify expression of cytokeratins 8 and 1, 5, 10, 14, vimentin, prostate specific antigen, prostate specific membrane antigen, androgen receptor, desmoglein, E-cadherin, beta1 integrin, CD44, hmet, vinculin and actin. RESULTS: Expression of vimentin was the only marker to correlate with motility, no markers correlated to invasion. Lower vimentin expression was observed in cells with low motility (PNT2-C2) and high expression in cells with high motility (P4E6, PNT1a, PC-3). Vimentin expression was not detected in well differentiated tumours, moderately differentiated tumours contained vimentin positive cells (1/9 bone scan negative, 2/5 bone scan positive), but the majority of poorly differentiated cancers (4/11 bone scan negative, 9/14 bone scan positive) and bone metastases (7/8) had high vimentin expression in tumour cells. CONCLUSIONS: Motile prostate cancer cell lines express vimentin. In tissue sections, the presence of vimentin positive tumour cells correlated positively to poorly differentiated cancers and the presence of bone metastases.