Characterization of the prostate cancer transcriptome and genome has identified chromosomal rearrangements and copy number gains/losses, including ETS gene fusions, PTEN loss and androgen receptor (AR) amplification, that drive prostate cancer development and progression to lethal, metastatic castrate resistant prostate cancer (CRPC)1. As less is known about the role of mutations2–4, here we sequenced the exomes of 50 lethal, heavily-pretreated metastatic CRPCs obtained at rapid autopsy (including three different foci from the same patient) and 11 treatment naïve, high-grade localized prostate cancers. We identified low overall mutation rates even in heavily treated CRPC (2.00/Mb) and confirmed the monoclonal origin of lethal CRPC. Integrating exome copy number analysis identified disruptions of CHD1, which define a subtype of ETS fusionnegative prostate cancer. Similarly, we demonstrate that ETS2, which is deleted in ~1/3 of CRPCs (commonly through TMPRSS2:ERG fusions), is also deregulated through mutation. Further, we identified recurrent mutations in multiple chromatin/histone modifying genes, including MLL2 (mutated in 8.6% of prostate cancers), and demonstrate interaction of the MLL complex with AR, which is required for AR-mediated signaling. We also identified novel recurrent mutations in the AR collaborating factor FOXA1, which is mutated in 5 of 147 (3.4%) prostate cancers (both untreated localized prostate cancer and CRPC), and showed that mutated FOXA1 represses androgen signaling and increases tumour growth. Proteins that physically interact with AR, such as the ERG gene fusion product, FOXA1, MLL2, UTX, and ASXL1 were found to be mutated in CRPC. In summary, we describe the mutational landscape of a heavily treated metastatic cancer, identify novel mechanisms of AR signaling deregulated in prostate cancer, and prioritize candidates for future study.
Patients with prostate cancer may present with metastatic or recurrent disease despite initial curative treatment. The propensity of metastatic prostate cancer to spread to the bone has limited repeated sampling of tumor deposits. Hence, considerably less is understood about this lethal metastatic disease, as it is not commonly studied. Here we explored whole-genome sequencing of plasma DNA to scan the tumor genomes of these patients non-invasively.
We wanted to make whole-genome analysis from plasma DNA amenable to clinical routine applications and developed an approach based on a benchtop high-throughput platform, that is, Illuminas MiSeq instrument. We performed whole-genome sequencing from plasma at a shallow sequencing depth to establish a genome-wide copy number profile of the tumor at low costs within 2 days. In parallel, we sequenced a panel of 55 high-interest genes and 38 introns with frequent fusion breakpoints such as the TMPRSS2-ERG fusion with high coverage. After intensive testing of our approach with samples from 25 individuals without cancer we analyzed 13 plasma samples derived from five patients with castration resistant (CRPC) and four patients with castration sensitive prostate cancer (CSPC).
The genome-wide profiling in the plasma of our patients revealed multiple copy number aberrations including those previously reported in prostate tumors, such as losses in 8p and gains in 8q. High-level copy number gains in the AR locus were observed in patients with CRPC but not with CSPC disease. We identified the TMPRSS2-ERG rearrangement associated 3-Mbp deletion on chromosome 21 and found corresponding fusion plasma fragments in these cases. In an index case multiregional sequencing of the primary tumor identified different copy number changes in each sector, suggesting multifocal disease. Our plasma analyses of this index case, performed 13 years after resection of the primary tumor, revealed novel chromosomal rearrangements, which were stable in serial plasma analyses over a 9-month period, which is consistent with the presence of one metastatic clone.
The genomic landscape of prostate cancer can be established by non-invasive means from plasma DNA. Our approach provides specific genomic signatures within 2 days which may therefore serve as 'liquid biopsy'.
Human cancers nearly ubiquitously harbor epigenetic alterations. While such alterations in epigenetic marks, including DNA methylation, are potentially heritable, they can also be dynamically altered. Given this potential for plasticity, the degree to which epigenetic changes can be subject to selection and act as drivers of neoplasia has been questioned. Here, we carried out genome-scale analyses of DNA methylation alterations in lethal metastatic prostate cancer and created DNA methylation “cityscape” plots to visualize these complex data. We show that somatic DNA methylation alterations, despite showing marked inter-individual heterogeneity among men with lethal metastatic prostate cancer, were maintained across all metastases within the same individual. The overall extent of maintenance in DNA methylation changes was comparable to that of genetic copy number alterations. Regions that were frequently hypermethylated across individuals were markedly enriched for cancer and development/differentiation related genes. Additionally, regions exhibiting high consistency of hypermethylation across metastases within individuals, even if variably hypermethylated across individuals, showed enrichment of cancer-related genes. Interestingly, whereas some regions showed intra-individual metastatic tumor heterogeneity in promoter methylation, such methylation alterations were generally not correlated with gene expression. This was despite a general tendency for promoter methylation patterns to be strongly correlated with gene expression, particularly at regions that were variably methylated across individuals. These findings suggest that DNA methylation alterations have the potential for producing selectable driver events in carcinogenesis and disease progression and highlight the possibility of targeting such epigenome alterations for development of longitudinal markers and therapeutic strategies.
By the age of 80, approximately 80% of men will manifest some cancerous cells within their prostate, indicating that prostate cancer constitutes a major health burden. While this disease is clinically insignificant in most men, it can become lethal in others. The most challenging task for clinicians is developing a patient-tailored treatment in the knowledge that this disease is highly heterogeneous and that relatively little adequate prognostic tools are available to distinguish aggressive from indolent disease. Next-generation sequencing allows a description of the cancer at an unprecedented level of detail and at different levels, going from whole genome or exome sequencing to transcriptome analysis and methylation-specific immunoprecipitation, followed by sequencing. Integration of all these data is leading to a better understanding of the initiation, progression and metastatic processes of prostate cancer. Ultimately, these insights will result in a better and more personalized treatment of patients suffering from prostate cancer. The present review summarizes current knowledge on copy number changes, gene fusions, single nucleotide mutations and polymorphisms, methylation, microRNAs and long non-coding RNAs obtained from high-throughput studies.
prostate cancer; next-generation sequencing; copy number changes; gene fusions; long non-coding RNAs; methylation; microRNAs; single nucleotide polymorphisms; single nucleotide variants
Vimentin was originally identified as an intermediate filament protein present only as an intracellular component in many cell types. However, this protein has now been detected on the surface of a number of different cancer cell types in a punctate distribution pattern. Increased vimentin expression has been indicated as an important step in epithelial-mesenchymal transition (EMT) required for the metastasis of prostate cancer. Here, using two vimentin-specific monoclonal antibodies (SC5 and V9 directed against the coil one rod domain and the C-terminus of the vimentin protein, respectively), we examined whether either of these domains would be displayed on the surface of three commonly studied prostate cancer cell lines isolated from different sites of metastases. Confocal analysis of LNCaP, PC3 and DU145 prostate cancer cell lines (derived from lymph node, bone or brain prostate metastases, respectively) demonstrated that both domains of vimentin are present on the surface of these metastatic cancer cell types. In addition, flow cytometric analysis revealed that vimentin expression was readily detected along with CD44 expression but only a small subpopulation of prostate cancer cells expressed vimentin and the putative stem cell marker CD133 along with CD44. Finally, Cowpea mosaic virus (CPMV) nanoparticles that target vimentin could bind and internalize into tested prostate cancer cell lines. These results demonstrate that at least two domains of vimentin are present on the surface of metastatic prostate cancer cells and suggest that vimentin could provide a useful target for nanoparticle- or antibody- cancer therapeutic agents directed against highly invasive cancer and/or stem cells.
vimentin; prostate cancer; metastases; stem cell; Cowpea mosaic virus
Prostate cancer, the majority of which is adenocarcinoma, is the most common epithelial cancer affecting a majority of elderly men in Western nations. Its manifestation, however, varies from clinically asymptomatic insidious neoplasms that progress slowly and do not threaten life to one that is highly aggressive with a propensity for metastatic spread and lethality if not treated in time. A number of somatic genetic and epigenetic alterations occur in prostate cancer cells. Some of these changes, such as loss of the tumor suppressors PTEN and p53, are linked to disease progression. Others, such as ETS gene fusions, appear to be linked more with early phases of the disease, such as invasion. Alterations in chromosome 8q24 in the region of MYC have also been linked to disease aggressiveness for many years. However, a number of recent studies in human tissues have indicated that MYC appears to be activated at the earliest phases of prostate cancer (e.g., in tumor-initiating cells) in prostatic intraepithelial neoplasia, a key precursor lesion to invasive prostatic adenocarcinoma. The initiation and early progression of prostate cancer can be recapitulated in genetically engineered mouse models, permitting a richer understanding of the cause and effects of loss of tumor suppressors and activation of MYC. The combination of studies using human tissues and mouse models paints an emerging molecular picture of prostate cancer development and early progression. This picture reveals that MYC contributes to disease initiation and progression by stimulating an embryonic stem cell–like signature characterized by an enrichment of genes involved in ribosome biogenesis and by repressing differentiation. These insights pave the way to potential novel therapeutic concepts based on MYC biology.
prostate cancer; tumor initiating cells; prostatic intraepithelial neoplasia
Red and processed meat may increase risk of advanced prostate cancer. Data on post-diagnostic diet and prostate cancer are sparse, but post-diagnostic intake of poultry with skin and eggs may increase risk of disease progression. Therefore, we prospectively examined total, unprocessed, and processed red meat, poultry, and eggs in relation to risk of lethal prostate cancer (e.g. men without cancer at baseline who developed distant organ metastases or died from prostate cancer during follow-up) among 27, 607 men followed from 1994–2008. We also performed a case-only survival analysis to examine post-diagnostic consumption of these foods and risk of lethal prostate cancer among the 3,127 men initially diagnosed with non-metastatic prostate cancer during follow-up. In the incidence analysis, we observed 199 events during 306,715 person-years. Men who consumed 2.5 or more eggs per week had an 81% increased risk of lethal prostate cancer compared to men who consumed less than 0.5 eggs per week (HR: 1.81; 95% confidence interval (CI): 1.13, 2.89; p-trend: 0.01). In the case-only survival analysis, we observed 123 events during 19,354 person-years. There were suggestive, but not statistically significant, positive associations between post-diagnostic poultry (HR ≥3.5 vs. <1.5 servings per week: 1.69; 95%CI: 0.96, 2.99; p-trend: 0.07) and post-diagnostic processed red meat (HR ≥3 vs. <0.5 servings per week: 1.45; 95%CI: 0.73, 2.87; p-trend: 0.08) and risk of progression of localized prostate cancer to lethal disease. In conclusion, consumption of eggs may increase risk of developing a lethal-form of prostate cancer among healthy men.
Eggs; red meat; poultry; prostate cancer; survival
Prostate cancer is widely observed to be biologically heterogeneous. Its heterogeneity is manifested histologically as multifocal prostate cancer, which is observed more frequently than unifocal prostate cancer. The clinical and prognostic significance of either focal cancer type is not fully established. To investigate prostate cancer heterogeneity, the genetic profiles of multifocal and unifocal prostate cancers were compared. Here, we report observations deduced from tumor-tumor comparison of copy number alteration data of both focal categories. Forty-one fresh frozen prostate cancer foci from 14 multifocal prostate cancers and eight unifocal prostate cancers were subjected to copy number variation analysis with the Affymetrix SNP 6.0 microarray tool. With the investigated cases, tumors obtained from a single prostate exhibited different genetic profiles of variable degrees. Further comparison identified no distinct genetic pattern or signatures specific to multifocal or unifocal prostate cancer. Our findings suggest that samples obtained from multiple sites of a single unifocal prostate cancer show as much genetic heterogeneity and variability as separate tumors obtained from a single multifocal prostate cancer.
prostate cancer; multifocal; unifocal; copy number variation; heterogeneity; affymetrix
Metastasis is characterized by spreading of neoplastic cells to an organ other than where they originated and is the predominant cause of death among cancer patients. This holds true for melanoma, whose incidence is increasing more rapidly than any other cancer and once disseminated has few therapeutic options. Here we performed whole exome sequencing of two sets of matched normal and metastatic tumor DNAs.
Using stringent criteria, we evaluated the similarities and differences between the lesions. We find that in both cases, 96% of the single nucleotide variants are shared between the two metastases indicating that clonal populations gave rise to the distant metastases. Analysis of copy number variation patterns of both metastatic sets revealed a trend similar to that seen with our single nucleotide variants. Analysis of pathway enrichment on tumor sets shows commonly mutated pathways enriched between individual sets of metastases and all metastases combined.
These data provide a proof-of-concept suggesting that individual metastases may have sufficient similarity for successful targeting of driver mutations.
The transition from androgen-dependent to castration-resistant prostate cancer (CRPC) is a lethal event of uncertain molecular etiology. Comparing gene expression in isogenic androgen-dependent and CRPC xenografts, we found a reproducible increase in N-cadherin expression, which was also elevated in primary and metastatic tumors of individuals with CRPC. Ectopic expression of N-cadherin in nonmetastatic, androgen-dependent prostate cancer models caused castration resistance, invasion and metastasis. Monoclonal antibodies against the ectodomain of N-cadherin reduced proliferation, adhesion and invasion of prostate cancer cells in vitro. In vivo, these antibodies slowed the growth of multiple established CRPC xenografts, blocked local invasion and metastasis and, at higher doses, led to complete regression. N-cadherin–specific antibodies markedly delayed the time to emergence of castration resistance, markedly affected tumor histology and angiogenesis, and reduced both AKT serine-threonine kinase activity and serum interleukin-8 (IL-8) secretion. These data indicate that N-cadherin is a major cause of both prostate cancer metastasis and castration resistance. Therapeutic targeting of this factor with monoclonal antibodies may have considerable clinical benefit.
Disseminated epithelial cells can be isolated from the bone marrow of a far greater fraction of prostate-cancer patients than the fraction of patients who progress to metastatic disease. To provide a better understanding of these cells, we have characterized their genomic alterations. We first present an array comparative genomic hybridization method capable of detecting genomic changes in the small number of disseminated cells (10-20) that can typically be obtained from bone-marrow aspirates of prostate-cancer patients. We show multiple regions of copy-number change, including alterations common in prostate cancer, such as 8p loss, 8q gain, and gain encompassing the androgen-receptor gene on Xq, in the disseminated cell pools from 11 metastatic patients. We found fewer and less striking genomic alterations in the 48 pools of disseminated cells from patients with organ-confined disease. However, we identify changes shared by these samples with their corresponding primary tumors and prostate-cancer alterations reported in the literature, evidence that these cells, like those in advanced disease, are disseminated tumor cells (DTCs). We also demonstrate that DTCs from patients with advanced and localized disease share several abnormalities, including losses containing cell-adhesion genes and alterations reported to associate with progressive disease. These shared alterations might confer the capability to disseminate or establish secondary disease. Overall, the spectrum of genomic deviations is evidence for metastatic capacity in advanced-disease DTCs and variation in that capacity in DTCs from localized disease. Our analysis lays the foundation for elucidation of the relationship between DTC genomic alterations and progressive prostate cancer.
Disseminated tumor cells; disseminated cells; prostate cancer; array CGH; genomic alterations
Expression of periostin is an indicator of epithelial-mesenchymal transition in cancer but a detailed analysis of periostin expression in prostate cancer has not been conducted so far.
Here, we evaluated periostin expression in prostate cancer cells and peritumoural stroma immunohistochemically in two independent prostate cancer cohorts, including a training cohort (n = 93) and a test cohort (n = 325). Metastatic prostate cancers (n = 20), hormone refractory prostate cancers (n = 19) and benign prostatic tissues (n = 38) were also analyzed.
In total, strong epithelial periostin expression was detectable in 142 of 418 (34.0%) of prostate carcinomas and in 11 of 38 benign prostate glands (28.9%). Increased periostin expression in carcinoma cells was significantly associated with high Gleason score (p < 0.01) and advanced tumour stage (p < 0.05) in the test cohort. Whereas periostin expression was weak or absent in the stroma around normal prostate glands, strong periostin expression in tumour stroma was found in most primary and metastatic prostate cancers. High stromal periostin expression was associated with higher Gleason scores (p < 0.001). There was a relationship between stromal periostin expression and shortened PSA relapse free survival times in the training cohort (p < 0.05).
Our data indicate that periostin up-regulation is related to increased tumour aggressiveness in prostate cancer and might be a promising target for therapeutical interventions in primary and metastatic prostate cancer.
To assess the feasibility of characterizing gene copy number alteration by fluorescence in situ hybridization of circulating tumor cells (CTC) isolated using the CellSearch system in patients with progressive castration resistant metastatic prostate cancer (CRPC).
We used probe combinations that included the androgen receptor (AR) and MYC genes for FISH analysis of CTC samples collected from 77 men with metastatic CRPC.
High-level chromosomal amplification of AR was detected in 37.5% of samples analyzed, and relative gain of MYC in 55.8%. No such abnormalities were detected in samples with CTC counts of less than 10, reflecting ascertainment difficulty in these lower count samples.
The CTC isolated from our patient cohort present a very similar molecular cytogenetic profile to that reported for late-stage tumors, and thus demonstrate that analysis of CTC can be a valuable, noninvasive surrogate for routine tumor profiling. Furthermore, we demonstrate that as many as 50% of these patients have substantial amplification of the AR locus, indicating that androgen signaling continues to play an important role in late-stage prostate cancer.
circulating tumor cells; prostate cancer; tumor markers; FISH; androgen receptor
Prostate cancer is the most commonly diagnosed and second most lethal malignancy in men, due mainly to a lack of effective treatment for the metastatic disease. A number of recent studies have shown that activation of the purine nucleoside receptor, adenosine A3 receptor (A3AR), attenuates proliferation of melanoma, colon, and prostate cancer cells. In the present study, we determined whether activation of the A3AR reduces the ability of prostate cancer cells to migrate in vitro and metastasize in vivo. Using severe combined immunodeficient mice, we show that proliferation and metastasis of AT6.1 rat prostate cancer cells were decreased by the administration of A3AR agonist N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide. In vitro studies show that activation of A3AR decreased high basal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity present in these cells, along with the expression of Rac1 and p47phox subunits of this enzyme. Inhibition of NADPH oxidase activity by the dominant-negative RacN17 or short interfering (si)RNA against p47phox reduced both the generation of reactive oxygen species and the invasion of these cells on Matrigel. In addition, we show that membrane association of p47phox and activation of NADPH oxidase is dependent on the activity of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase pathway. We also provide evidence that A3AR inhibits ERK1/2 activity in prostate cancer cells through inhibition of adenylyl cyclase and protein kinase A. We conclude that activation of the A3AR in prostate cancer cells reduces protein kinase A-mediated stimulation of ERK1/2, leading to reduced NADPH oxidase activity and cancer cell invasiveness.
Prostate carcinoma is among the most common types of cancer affecting hundreds of thousands people every year. Once the metastatic form of prostate carcinoma is documented, the majority of patients die from their tumors as opposed to other causes. The key to successful treatment is in the earliest possible diagnosis, as well as understanding the molecular mechanisms of metastatic progression. A number of recent studies have identified multiple biomarkers for metastatic progression. However, most of the studies consider only direct comparison between metastatic and non-metastatic classes of samples.
We propose an alternative concept of analysis that considers the entire multidimensional space of gene expression and identifies the partition of this space in which metastatic development is possible. To apply this concept in cancer gene expression studies we utilize a modification of high-dimension natural taxonomy algorithm FOREL. Our analysis of microarray data containing primary and metastatic cancer samples has revealed not only differentially expressed genes, but also relations between different groups of primary and metastatic cancer. Metastatic samples tend to occupy a distinct partition of gene expression space. Further pathway analysis suggests that this partition is delineated by a specific pattern of gene expression in cytoskeleton remodeling, cell adhesion and apoptosis/cell survival pathways. We compare our findings with both report of original analysis and recent studies in molecular mechanism of metastasis.
Our analysis indicates a single molecular mechanism of metastasis. The new approach does not contradict previously reported findings, but reveals important details unattainable with traditional methodology.
Reverse phase protein microarray technology was used to study key signaling pathways thought to be involved in the progression of benign epithelium to the lethal phenotype of prostate cancer. Specimens of androgen-stimulated localized prostate cancer (N=21) and androgen-deprivation therapy-recurrent local (N=4) or metastatic (N=11) prostate cancer were laser capture microdissected prior to analysis. The results showed significant increases in protein expression levels in malignant epithelial cells and patient-matched stromal tissue, which included higher levels of the apoptotic proteins Bax and Smac/Diablo and increased phosphorylation of Bcl2 (S70). The mitochondrial protein Smac/Diablo and the transcription regulatory protein STAT3 (Y705) correlated with Gleason sum and differed statistically in high Gleason grade (8-10) prostate cancers. Distinct metastasis-specific pathways were activated by caspase cleavage activation, ErbB2 phosphorylation, Bax total protein and Bcl-2 phosphorylation while phosphorylation of all three members of the MAPK family, ERK, p38 and SAP/JNK, were reduced significantly in metastatic lesions compared to primary cancers. This study, the most comprehensive pathway analysis ever performed for human prostate cancer, presents evidence of specific pathway biomarkers that may be useful for assessment of prognosis and stratification for therapy if validated in larger clinical study sets.
prostate cancer; signaling; proteomics; protein microarray; reverse phase array; metastasis
Coffee contains many biologically active compounds, including caffeine and phenolic acids, that have potent antioxidant activity and can affect glucose metabolism and sex hormone levels. Because of these biological activities, coffee may be associated with a reduced risk of prostate cancer.
We conducted a prospective analysis of 47 911 men in the Health Professionals Follow-up Study who reported intake of regular and decaffeinated coffee in 1986 and every 4 years thereafter. From 1986 to 2006, 5035 patients with prostate cancer were identified, including 642 patients with lethal prostate cancers, defined as fatal or metastatic. We used Cox proportional hazards models to assess the association between coffee and prostate cancer, adjusting for potential confounding by smoking, obesity, and other variables. All P values were from two-sided tests.
The average intake of coffee in 1986 was 1.9 cups per day. Men who consumed six or more cups per day had a lower adjusted relative risk for overall prostate cancer compared with nondrinkers (RR = 0.82, 95% confidence interval [CI] = 0.68 to 0.98, Ptrend = .10). The association was stronger for lethal prostate cancer (consumers of more than six cups of coffee per day: RR = 0.40, 95% CI = 0.22 to 0.75, Ptrend = .03). Coffee consumption was not associated with the risk of nonadvanced or low-grade cancers and was only weakly inversely associated with high-grade cancer. The inverse association with lethal cancer was similar for regular and decaffeinated coffee (each one cup per day increment: RR = 0.94, 95% CI = 0.88 to 1.01, P = .08 for regular coffee and RR = 0.91, 95% CI = 0.83 to 1.00, P = .05 for decaffeinated coffee). The age-adjusted incidence rates for men who had the highest (≥6 cups per day) and lowest (no coffee) coffee consumption were 425 and 519 total prostate cancers, respectively, per 100 000 person-years and 34 and 79 lethal prostate cancers, respectively, per 100 000 person-years.
We observed a strong inverse association between coffee consumption and risk of lethal prostate cancer. The association appears to be related to non-caffeine components of coffee.
Prostate cancer cell lines provide ideal in vitro systems for the identification and analysis of prostate tumor suppressors and oncogenes. A detailed characterization of the architecture of prostate cancer cell line genomes would facilitate the study of precise roles of various genes in prostate tumorigenesis in general. To contribute to such a characterization, we used the GeneChip 500K single nucleotide polymorphic (SNP) array for analysis of genotypes and relative DNA copy number changes across the genome of 11 cell lines derived from both normal and cancerous prostate tissues. For comparison purposes, we also examined the alterations observed in the cell lines in tumor/normal pairs of clinical samples from 72 patients. Along with genome-wide maps of DNA copy number changes and loss of heterozygosity for these cell lines, we report previously unreported homozygous deletions and recurrent amplifications in prostate cancers in this study. The homozygous deletions affected a number of biologically important genes, including PPP2R2A and BNIP3L identified in this study and CDKN2A/CDKN2B reported previously. Although most amplified genomic regions tended to be large, amplifications at 8q24.21 were of particular interest because the affected regions are relatively small, are found in multiple cell lines, are located near MYC, an oncogene strongly implicated in prostate tumorigenesis, and are known to harbor SNPs that are associated with inherited susceptibility for prostate cancer. The genomic alterations revealed in this study provide an important catalog of positional information relevant to efforts aimed at deciphering the molecular genetic basis of prostate cancer.
The intermediate filament protein Nestin identifies stem/progenitor cells in adult tissues, but the function of Nestin is poorly understood. We investigated Nestin expression and function in common lethal cancers. Nestin mRNA was detected in cell lines from small cell lung, and breast cancers, and particularly elevated in cell lines derived from prostate cancer metastases. Whereas the androgen-independent lines PC3, 22RV1, and DU145 all expressed Nestin transcripts under standard culture conditions, the androgen-dependent line LnCaP expressed Nestin only on androgen withdrawal. We confirmed associations of Nestin expression, androgen withdrawal, and metastatic potential by immunohistochemical analysis of samples from 254 prostate cancer patients. Cytoplasmic Nestin protein was readily identifiable in prostate cancer cells from 75% of patients with lethal androgen-independent disease, even in cancer sampled from the prostate itself. However, Nestin expression was undetectable in localized androgen-deprived tumors and in metastases without prior androgen deprivation. To address its function, we reduced Nestin levels with short hairpin RNAs, markedly inhibiting in vitro migration and invasion in prostate cancer cells but leaving cell growth intact. Nestin knockdown also diminished metastases 5-fold compared with controls despite uncompromised tumorigenicity at the site of inoculation. These results specify a function for Nestin in cell motility and identify a novel pathway for prostate cancer metastasis. Activity of this pathway may be selected by the extraprostatic environment or, as supported by our data, may originate within the prostate after androgen deprivation. Further dissection of this novel Nestin migration pathway may lead to strategies to prevent and neutralize metastatic spread.
To test the feasibility of using text mining to depict meaningfully the experience of pain in patients with metastatic prostate cancer, to identify novel pain phenotypes, and to propose methods for longitudinal visualization of pain status.
Materials and methods
Text from 4409 clinical encounters for 33 men enrolled in a 15-year longitudinal clinical/molecular autopsy study of metastatic prostate cancer (Project to ELIminate lethal CANcer) was subjected to natural language processing (NLP) using Unified Medical Language System-based terms. A four-tiered pain scale was developed, and logistic regression analysis identified factors that correlated with experience of severe pain during each month.
NLP identified 6387 pain and 13 827 drug mentions in the text. Graphical displays revealed the pain ‘landscape’ described in the textual records and confirmed dramatically increasing levels of pain in the last years of life in all but two patients, all of whom died from metastatic cancer. Severe pain was associated with receipt of opioids (OR=6.6, p<0.0001) and palliative radiation (OR=3.4, p=0.0002). Surprisingly, no severe or controlled pain was detected in two of 33 subjects’ clinical records. Additionally, the NLP algorithm proved generalizable in an evaluation using a separate data source (889 Informatics for Integrating Biology and the Bedside (i2b2) discharge summaries).
Patterns in the pain experience, undetectable without the use of NLP to mine the longitudinal clinical record, were consistent with clinical expectations, suggesting that meaningful NLP-based pain status monitoring is feasible. Findings in this initial cohort suggest that ‘outlier’ pain phenotypes useful for probing the molecular basis of cancer pain may exist.
The results are limited by a small cohort size and use of proprietary NLP software.
We have established the feasibility of tracking longitudinal patterns of pain by text mining of free text clinical records. These methods may be useful for monitoring pain management and identifying novel cancer phenotypes.
Natural Language Processing; Pain Management; Metastatic Prostate Cancer; Personalized Medicine; Individualized Medicine; Cancer Pain
Prostate cancer is a clinically heterogeneous disease, ranging from indolent asymptomatic disease to very aggressive metastatic and life threatening forms of the disease. Distant metastasis represents the major lethal cause of prostate cancer. The most critical clinical challenge in the management of the patients is identifying those individuals at risk of developing metastatic disease. To understand the molecular mechanisms of prostate cancer metastasis and identify markers with metastatic potential, we have analyzed protein expression in two syngeneic prostate cancer cells lines PC3-N2 and PC3-ML2 using isobaric tags for relative and absolute quantitation labeling and multi-dimensional protein identification technology liquid chromatography matrix assisted laser desorption ionization tandem mass spectrometry. PC3-N2 is lowly metastatic while PC3-ML2 highly metastatic. A total of 1,756 proteins were identified in the analyses with 130 proteins showing different expression levels (p<0.01) in the two cell lines. Out of these, 68 proteins were found to be significantly up-regulated while 62 are significantly down-regulated in PC3-ML2 cells compared with PC3-N2 cells. The upregulation of plectin and vimentin which were the most significantly differentially expressed were validated by Western blot and their functional relevance with respect to invasion and migration was determined by siRNA gene silencing. To our knowledge, this study is the first to demonstrate that up-regulation of vimentin and plectin expression positively correlates with the invasion and metastasis of androgen-independent PCA.
Breast and prostate cancer have a propensity to metastasize to bones and cause osteolysis and abnormal new bone formation. Metastases locally disrupt normal bone remodeling. Although metastases from prostate cancer have been classified as osteoblastic based on the radiographic appearance of the lesion, data gleaned from a rapid autopsy program indicate that the same prostate cancer patient may have evidence of both osteolytic and osteoblastic disease as shown by histologic examinations. Thus, bone metastases are heterogeneous, requiring combined treatment targeting on both osteolytic and osteoblastic lesions. While Samarium-153 (Sm-153) oxabifore treatment is widely used for the relief of pain in patients with osteoblastic metastatic bone lesions, Xgeva (Denosumab) is indicated for the prevention of skeletal-related events in patients with bone metastases from solid tumors. It is a fully human monoclonal antibody that has been designed to target receptor activator of nuclear factor-kB ligand (RANKL), a protein that acts as the primary signal to promote bone removal. In many bone loss conditions, RANKL overwhelms the body's natural defense against bone destruction. The main objectives of the current pilot study were to estimate the effectiveness of bone metastases treatment by a combination of Sm-153 oxabifore and Xgeva (Denosumab). Five patients (four female and one male, aged 35-64, mean age 50.8) with multiple skeletal metastases from prostatic carcinoma (1) and breast carcinoma (4) were studied. Their mean objective pain score according to visual analog scoring system on a 1-10 scoring system was 7.8 ± 0.5 (range 6-9). Sm-153 oxabifore was administered at the standard bone palliation dose of 37 MBq/kg body weight. Xgeva (Denosumab) was administered at a dosage of 120 mg every 4 weeks, with the monitoring of calcium level and administration of calcium, magnesium, and vitamin D. Whole body (WB) bone scan was performed before and 3 months after treatment in all patients. After Sm-153 oxabifore administration, pain relief occurred within 4.4 ± 1.25 days (range 2-9 days) and the objective pain score decreased to 0.2 ± 0.2 (range 0-1). There was statistically significant difference found, according to the pain score system, before and after treatment (P < 0.0001). WB bone scan showed that in one patient, there was significant reduction in the number and intensity of bone metastases, and in four patients, there was no evidence of bone metastases found. Based on our first experience, combined treatment of bone metastases with Sm-153 oxabifore and Denosumab is effective and safe.
Bone metastases; Sm-153 systemic radionuclide therapy; Xgeva (Denosumab)
Previous attempts to link prostate cancer progression to genetic alterations have been unsuccessful, and consequently, there is still no reliable predictor of prognosis for men with this disease. A recent study by Taylor et al. published in Cancer Cell, assesses copy number alterations, mutations and transcriptomes in 218 tumors and 12 prostate cancer cell lines and xenografts. Their analysis identifies frequencies of ERG alterations, 8p loss and 8q gain similar to previous findings. It also reveals novel genetic factors in prostate cancer progression, including the androgen receptor coactivator, NCOA2, which serves as an oncogene in about 11% of tumors, and a deletion at chromosome 3p14, which was associated with TMPRSS-ERG fusion. The copy number alteration data demonstrates six distinct subgroups of prostate cancer with considerable variation in time to biochemical relapse. Classification of prostate cancer into these genetic subgroups may help clinicians predict the likelihood of disease progression in newly diagnosed men, ultimately guiding treatment decisions and therapy development.
prostate cancer; pharmacogenetics; prognosis; copy number alterations; androgen receptor; TMPRSS-ERG; PI3K pathways; NCOA2
Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.
Adipocytokines may mediate the association between adiposity and lethal prostate cancer outcomes.
In the Physicians’ Health Study, we prospectively examined the association of prediagnostic plasma concentrations of adiponectin and leptin with risk of developing incident prostate cancer (654 case diagnosed 1982-2000 and 644 age-matched controls) and, among cases, risk of dying from prostate cancer by 2007.
Adiponectin concentrations were not associated with risk of overall prostate cancer. However, men with higher adiponectin concentrations had lower risk of developing high grade or lethal cancer (metastatic or fatal disease). The relative risk (95% confidence interval) comparing the highest to the lowest quintile (Q5 vs. Q1) was 0.25 (0.07-0.87; Ptrend=0.02) for lethal cancer. Among all the cases, higher adiponectin concentrations predicted lower prostate cancer-specific mortality (hazard ratio, HR Q5 vs. Q1=0.39; 0.17-0.85; Ptrend=0.02), independent of body mass index (BMI), plasma C-peptide (a marker of insulin secretion), leptin, clinical stage and tumor grade. This inverse association was apparent mainly among men whose BMI ≥25 kg/m2 (HR Q5 vs. Q1=0.10; 0.01-0.78; Ptrend=0.02), but not among men of normal weight (Ptrend=0.51). Although the correlation of leptin concentrations with BMI (r=0.58, P <0.001) was stronger than that of adiponectin (r=−0.17, P<0.001), leptin was unrelated to prostate cancer risk or mortality.
Higher prediagnostic adiponectin (but not leptin) concentrations predispose men to a lower risk of developing high grade prostate cancer and a lower risk of subsequently dying from the cancer, suggesting a mechanistic link between obesity and poor PCa outcome.
Adiponectin; obesity; prostate cancer; risk; survival