Loss of PTEN has been shown to be associated with aggressive behavior of prostate cancer. It is less clear that loss of PTEN also increases the risk of cancer mortality. We investigated the association between PTEN expression and prostate cancer mortality, and the potential effect modification by IGF1R, a direct activator of the PI3K pathway.
Protein expression in tumor were evaluated using tumor tissues obtained from 805 participants of the Physicians’ Health and the Health Professionals Follow-up studies who were diagnosed with prostate cancer and underwent radical prostatectomy. Proportional hazard models were used to assess PTEN expression, and its interaction with IGF1R, in relation to lethal prostate cancer (cancer-specific death or distant metastases).
Low PTEN expression was associated with an increase risk of lethal prostate cancer (HR = 1.7, 95% CI: 0.98-3.2, P for trend = 0.04). The association was attenuated after adjustment for Gleason grade, tumor stage, and PSA at diagnosis. A significant negative interaction between PTEN and IGF1R was found (P for interaction = 0.03). Either reduction in PTEN or increase in IGF1R expression was sufficient to worsen prognosis. Models including PTEN and IGF1R expression offer additional predicting power to prostate cancer survival, comparing to those only including demographic and clinical factors.
Low PTEN protein expression significantly increases the risk of lethal prostate cancer, particularly when the IGF1R expression remains at normal level.
PTEN and IGF1R expression in tumor are promising candidates for independent prognostic factors to predict lethal prostate cancer.
Prostate cancer; PTEN; Tumor biomarker; Survival; Interaction
Cysteine- rich secretory protein 3 (CRISP3) prognostic significance in prostate cancer (PCA) has generated mixed result. Herein, we investigated and independently validated CRISP3 expression in relation to ERG and PTEN genomic aberrations and clinical outcome. CRISP3 protein expression was examined by immunohistochemistry using a cohort of patients with localized PCA (n = 215) and castration resistant PCA (CRPC) (n = 46). The Memorial Sloan Kettering (MSKCC) and Swedish cohorts were used for prognostic validation. Results showed, CRISP3 protein intensity to be significantly associated with neoplastic epithelium, being highest in CRPC vs. benign prostate tissue (p < 0.0001), but was not related to Gleason score (GS). CRISP3 mRNA was significantly associated with higher GS (p = 0.022 in MSKCC, p = 1.1e-4 in Swedish). Significant association between CRISP3 expression and clinical outcome was documented at the mRNA but not the protein expression levels. CRISP3 mRNA expression was related to biochemical recurrence in the MSKCC (p = 0.038) and lethal disease in the Swedish cohort (p = 0.0086) and retained its prognostic value in the subgroup of patients with GS 6 & 7. Furthermore, CRISP3 protein and mRNA expression was significantly associated with positive ERG status and with PTEN deletions. Functional biology analysis documented phenylalanine metabolism as the most significant pathway governing high CRISP3 and ERG expression in this subtype of PCA. In conclusion, the combined status of CRISP3, ERG and PTEN define a molecular subtype of PCA with poorest and lethal outcome. Assessing their combined value may be of added value in stratifying patients into different prognostic groups and identify those with poorest clinical outcome.
CRISP3; ERG; PTEN; Biological pathways; Prostate cancer; Poorest outcome; Molecular subtypes
A 51-year-old French Canadian man commenced screening for prostate cancer at the request of his family physician given his extensive family history of prostate cancer in five brothers, his father and two paternal uncles. His prostate specific antigen (PSA) level was 4.9ng/ml and a subsequent six-core biopsy revealed the presence of a prostate adenocarcinoma with a Gleason score of 7 (3+4). He was treated with a radical prostatectomy. Repeat PSA tests revealed a gradual rise in PSA levels despite androgen deprivation therapy with bicalutamide and goserelin over the course of 3 years. Genetic evaluation was undertaken in view of his personal and family history. The proband died a year later of widespread metastasis, at the age of 58.
Doppler ultrasound for the proband’s leg, abdominal and pelvic computed tomography (CT) scan with intravenous (IV) and oral contrast as well as chest CT with IV contrast for the assessment of metastatic prostate cancer; genetic counselling and mutation analysis for French Canadian founder mutations in the BRCA1 and BRCA2 genes; mutations specific analysis of the mother’s formalin-fixed, paraffin-embedded (FFPE) breast tissue blocks; examination of loss of heterozygosity at the BRCA2 gene locus; immunohistochemistry to determine the expression of the ERG nuclear oncoprotein in prostate tumours; genotyping with eight selected risk-associated single nucleotide polymorphisms (SNPs); genetic testing for the G84E variant in the HOXB13 gene
Early-onset and aggressive prostate cancer associated with a missense French Canadian BRCA2 founder mutation, c.5857G>T (p.Glu1953*).
Radical prostatectomy, hormone therapy with bicalutamide and goserelin, palliative chemotherapy initially with docetaxel plus prednisone then with mitoxantrone plus prednisone, as well as genetic counselling and testing for the proband and his family members.
Dynamin 2 (Dyn2) is essential for intracellular vesicle formation and trafficking, cytokinesis, and receptor endocytosis. In this study, we investigated the implication of Dyn2 as a prognostic marker and therapeutic target for progressive prostate cancer (PCA). We evaluated Dyn2 protein expression by immunohistochemistry in two cohorts: men with localized PCA treated by retropubic radical prostatectomy (n = 226), and men with advanced/castrate-resistant PCA (CRPC) treated by transurethral resection of prostate (TURP) (n = 253). The role of Dyn2 in cell invasiveness was assessed by in vitro and in vivo experiments using androgen-responsive and refractory PCA preclinical models. Dyn2 expression was significantly increased across advanced stages of PCA compared to benign prostate tissue (P < 0.0001). In the CRPC cohort, high Dyn2 was associated with higher Gleason score (P = 0.004) and marginally with cancer-specific mortality (P = 0.052). In preclinical models, Dyn2 gene silencing significantly reduced cell migration and invasion in vitro, as well as tumor size and lymph node metastases in vivo. In isolated PCA cells, Dyn2 was found to regulate focal adhesion turnover, which is critical for cell migration; this mechanism requires full Dyn2 compared to mutants deficient in GTPase activity. In conclusion, Dyn2 overexpression is associated with neoplastic prostate epithelium and is associated with poor prognosis. Inhibition of Dyn2 prevents cell invasiveness in androgen-responsive and -refractory PCA models, supporting the potential benefit of Dyn2 to serve as a therapeutic target for advanced PCA.
Cancer-specific mortality; castration resistance; dynamin; prostate cancer; therapeutic target
MicroRNAs are a class of small non-coding regulatory RNA molecules that regulate mRNAs post-transcriptionally. Recent evidence has shown that miRNAs target entire functionally related proteins such as protein complexes and biological pathways. However, characterizing the influence of miRNAs on genes whose encoded proteins are part of protein complexes has not been studied in the context of disease. We propose an entropy-based framework to identify miRNA-mediated dysregulation of functionally related proteins during prostate cancer progression. The proposed framework uses experimentally verified miRNA-target interactions, functionally related proteins and expression data to identify miRNA-influenced protein complexes in prostate cancer, and identify genes that are dysregulated as a result. The framework constructs correlation matrixes between functionally related proteins and miRNAs that have targets in the complex, and assesses the changes in the Shannon entropy of the modules across different stages of prostate cancer. Results reveal that SMAD4 and HDAC containing protein complexes are highly affected and disrupted by miRNAs, particularly miRNA-1 and miRNA-16. Using biological pathways to define functionally related proteins reveals that NF-kB-, RAS-, and Syndecan-mediated pathways are dysregulated due to miRNA-1- and miRNA-16-mediated regulation. These results suggest that miRNA-1 and miRNA-16 are important master regulators of miRNA-mediated regulation in prostate cancer. Moreover, results reveal that miRNAs with high-influence on the disrupted protein complexes are diagnostic and prognostic biomarker candidates for prostate cancer progression. The observation of miRNA-mediated protein complex regulation and miRNA-mediated pathway regulation, with partial experimental verification from previous studies, demonstrates that our framework is a promising approach for the identification of novel miRNAs and protein complexes related to disease progression.
Background. The prognostic significance of ERG expression in prostate cancer (PCA) has generated mixed results. We sought to investigate the prognostic significance of ERG expression in a localized cohort of men with PCA. Material and Methods. We investigated ERG protein expression in a cohort of 198 men with localized PCA. ERG expression was correlated with patients' clinical outcome and several pathological parameters, including Gleason score (GS), pathological stage, surgical margin, and extra-capsular extension. Results. ERG expression was detected in 86/198 (43.4%) patients exclusively in neoplastic epithelium. Overall, ERG mean expression intensity was 1.01 ± 1.27 versus 0.37 ± 0.83 in acinar PCA compared to foamy type PCA (P < 0.001). In HGPIN, ERG intensity levels were comparable to those in foamy type PCA (0.13 ± 0.56) but significantly lower than those in acinar PCA (P < 0.001). ERG expression was significantly associated with extra-prostatic extension and higher pathological stage and showed a trend toward seminal vesicle invasion. Herein, ERG expression was documented in 50/131 (38.1%) patients with pT2 versus 30/55 (54.5%) patients with pT3 (P = 0.04). ERG association with higher pathological stage was more pronounced in patients with GS > 7. Grouping patients into those with GS ≤ 7 versus >7, there was no significant association between ERG expression and GS. Similarly, no association was present in relation to either surgical margins or postsurgical serum PSA levels. Conclusion. We report significant association between ERG protein levels and extra-prostatic extension and higher pathological stage. ERG expression is not associated with adverse clinical outcome and is of limited prognostic value in localized PCA.
More than 1,300,000 prostate needle biopsies are performed annually in the U.S. with up to 16% incidence of isolated high-grade prostatic intraepithelial neoplasia (HGPIN). HGPIN has low predictive value for identifying prostate cancer (PCA) on subsequent needle biopsies in PSA screened populations. In contemporary series, PCA is detected in about 20% of repeat biopsies following a diagnosis of HGPIN. Further, discrete histological subtypes of HGPIN with clinical implication in management have not been characterized. The TMPRSS2-ERG gene fusion that has recently been described in PCA has also been demonstrated to occur in a subset of HGPIN. This may have significant clinical implications given that TMPRSS2-ERG fusion PCA is associated with a more aggressive clinical course.
In this study we assessed a series of HGPIN lesions and paired PCA for the presence of TMPRSS2-ERG gene fusion.
Fusion positive HGPIN was observed in 16% of the 143 number of lesions, and in all instances the matching cancer shared the same fusion pattern. 60% of TMPRSS2-ERG fusion PCA had fusion negative HGPIN.
Given the more aggressive nature of TMPRSS2-ERG PCA, the findings of this study raise the possibility that gene fusion positive HGPIN lesions are harbingers of more aggressive disease. To date, pathological, molecular and clinical parameters do not help stratify which men with HGPIN are at increased risk for a cancer diagnosis. Our results suggest that the detection of isolated TMPRSS2-ERG fusion HGPIN would improve the positive predictive value of finding TMPRSS2-ERG fusion PCA in subsequent biopsies.
There is a growing body of evidence associating microRNAs (miRNAs) with human diseases. MiRNAs are new key players in the disease paradigm demonstrating roles in several human diseases. The functional association between miRNAs and diseases remains largely unclear and far from complete. With the advent of high-throughput functional genomics techniques that infer genes and biological pathways dysregulted in diseases, it is now possible to infer functional association between diseases and biological molecules by integrating disparate biological information.
Here, we first used Lasso regression model to identify miRNAs associated with disease signature as a proof of concept. Then we proposed an integrated approach that uses disease-gene associations from microarray experiments and text mining, and miRNA-gene association from computational predictions and protein networks to build functional associations network between miRNAs and diseases. The findings of the proposed model were validated against gold standard datasets using ROC analysis and results were promising (AUC=0.81). Our protein network-based approach discovered 19 new functional associations between prostate cancer and miRNAs. The new 19 associations were validated using miRNA expression data and clinical profiles and showed to act as diagnostic and prognostic prostate biomarkers. The proposed integrated approach allowed us to reconstruct functional associations between miRNAs and human diseases and uncovered functional roles of newly discovered miRNAs.
Lasso regression was used to find associations between diseases and miRNAs using their gene signature. Defining miRNA gene signature by integrating the downstream effect of miRNAs demonstrated better performance than the miRNA signature alone. Integrating biological networks and multiple data to define miRNA and disease gene signature demonstrated high performance to uncover new functional associations between miRNAs and diseases.
miRNA; Protein interactions; Systems biology; Disease; Regression modeling
Neuroendocrine prostate cancer (NEPC), also referred to as anaplastic prostate cancer, is a lethal tumor that most commonly arises in late stages of prostate adenocarcinoma (PCA) with predilection to metastasize to visceral organs. In the current study, we explore for evidence that Aurora kinase A (AURKA) and N-myc (MYCN) gene abnormalities are harbingers of treatment-related NEPC (t-NEPC). We studied primary prostate tissue from 15 hormone naïve PCAs, 51 castration-resistant prostate cancers, and 15 metastatic tumors from 72 patients at different stages of disease progression to t-NEPC, some with multiple specimens. Histologic evaluation, immunohistochemistry, and fluorescence in situ hybridization were performed and correlated with clinical variables. AURKA amplification was identified in overall 65% of PCAs (hormone naïve and treated) from patients that developed t-NEPC and in 86% of metastases. Concurrent amplification of MYCN was present in 70% of primary PCAs, 69% of treated PCAs, and 83% of metastases. In contrast, in an unselected PCA cohort, AURKA and MYCN amplifications were identified in only 5% of 169 cases. When metastatic t-NEPC was compared to primary PCA from the same patients, there was 100% concordance of ERG rearrangement, 100% concordance of AURKA amplification, and 60% concordance of MYCN amplification. In tumors with mixed features, there was also 100% concordance of ERG rearrangement and 94% concordance of AURKA and MYCN co-amplification between areas of NEPC and adenocarcinoma. AURKA and MYCN amplifications may be prognostic and predictive biomarkers, as they are harbingers of tumors at risk of progressing to t-NEPC after hormonal therapy.
PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous-recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported.
Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN+/+; DU145-PTEN+/−; PC3-PTEN−/−) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression.
PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi amongst prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA-damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H2O2 and methyl-methanesulfonate; but not to cisplatin, camptothecin, or paclitaxel.
These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials.
PTEN; radiotherapy; chemotherapy; PARP; prostate cancer; RAD51
Gene alterations are a major component of the landscape of tumor genomes. To assess the significance of these alterations in the development of prostate cancer, it is necessary to identify these alterations and analyze them from systems biology perspective. Here, we present a new method (EigFusion) for predicting outlier genes with potential gene rearrangement. EigFusion demonstrated excellent performance in identifying outlier genes with potential rearrangement by testing it to synthetic and real data to evaluate performance. EigFusion was able to identify previously unrecognized genes such as FABP5 and KCNH8 and confirmed their association with primary and metastatic prostate samples while confirmed the metastatic specificity for other genes such as PAH, TOP2A, and SPINK1. We performed protein network based approaches to analyze the network context of potential rearranged genes. Functional gene rearrangement Modules are constructed by integrating functional protein networks. Rearranged genes showed to be highly connected to well-known altered genes in cancer such as AR, RB1, MYC, and BRCA1. Finally, using clinical outcome data of prostate cancer patients, potential rearranged genes demonstrated significant association with prostate cancer specific death.
While recurrent gene fusions involving ETS family transcription factors are common in prostate cancer, their products are considered “undruggable” by conventional approaches. Recently, rare “targetable” gene fusions (involving the ALK kinase), have been identified in 1–5% of lung cancers1, suggesting that similar rare gene fusions may occur in other common epithelial cancers including prostate cancer. Here we employed paired-end transcriptome sequencing to screen ETS rearrangement negative prostate cancers for targetable gene fusions and identified the SLC45A3-BRAF and ESRP1-RAF1 gene fusions. Expression of SLC45A3-BRAF or ESRP1-RAF1 in prostate cells induced a neoplastic phenotype that was sensitive to RAF and MEK inhibitors. Screening a large cohort of patients, we found that although rare (1–2%), recurrent rearrangements in the RAF pathway tend to occur in advanced prostate cancers, gastric cancers, and melanoma. Taken together, our results emphasize the importance of RAF rearrangements in cancer, suggest that RAF and MEK inhibitors may be useful in a subset of gene fusion harboring solid tumors, and demonstrate that sequencing of tumor transcriptomes and genomes may lead to the identification of rare targetable fusions across cancer types.
The actin cross-linking protein filamin A reduces migration, invasion, and metastasis of breast cancer cells.
The actin cross-linking protein filamin A (FLNa) functions as a scaffolding protein and couples cell cytoskeleton to extracellular matrix and integrin receptor signaling. In this study, we report that FLNa suppresses invasion of breast cancer cells and regulates focal adhesion (FA) turnover. Two large progression tissue microarrays from breast cancer patients revealed a significant decrease of FLNa levels in tissues from invasive breast cancer compared with benign disease and in lymph node–positive compared with lymph node–negative breast cancer. In breast cancer cells and orthotopic mouse breast cancer models, down-regulation of FLNa stimulated cancer cell migration, invasion, and metastasis formation. Time-lapse microscopy and biochemical assays after FLNa silencing and rescue with wild-type or mutant protein resistant to calpain cleavage revealed that FLNa regulates FA disassembly at the leading edge of motile cells. Moreover, FLNa down-regulation enhanced calpain activity through the mitogen-activated protein kinase–extracellular signal-regulated kinase cascade and stimulated the cleavage of FA proteins. These results document a regulation of FA dynamics by FLNa in breast cancer cells.
To elucidate the role of ETS gene fusions in castration-resistant prostate cancer (CRPC), we characterized the transcriptome of 54 CRPC tumor samples from men with locally advanced or metastatic disease. Trefoil factor 3 (TFF3) emerged as the most highly differentially regulated gene with respect to ERG rearrangement status and resistance to hormone ablation therapy. Conventional chromatin immunoprecipitation (ChIP)-polymerase chain reaction and ChIP followed by DNA sequencing (ChIP-seq) revealed direct binding of ERG to ETS binding sites in the TFF3 promoter in ERG-rearranged prostate cancer cell lines. These results were confirmed in ERG-rearranged hormone-naive prostate cancer (HNPC) and CRPC tissue samples. Functional studies demonstrated that ERG has an inhibitory effect on TFF3 expression in hormone-naive cancer but not in the castration-resistant state. In addition, we provide evidence suggesting an effect of androgen receptor signaling on ERG-regulated TFF3 expression. Furthermore, TFF3 overexpression enhances ERG-mediated cell invasion in CRPC prostate cancer cells. Taken together, our findings reveal a novel mechanism for enhanced tumor cell aggressiveness resulting from ERG rearrangement in the castration-resistant setting through TFF3 gene expression.
Checkpoint kinase 2 (CHEK2) is a protein involved in arresting cell cycle in response to DNA damage. To investigate whether it plays an important role in the development of prostate cancer (PRCA) in the Ashkenazi Jewish (AJ) population, we sequenced CHEK2 in 75 AJ individuals with prostate, breast, or no cancer (n = 25 each). We identified seven coding SNPs (five are novel) that changed the amino acid sequence, resulting in R3W, E394F, Y424H, S428F, D438Y, P509S, and P509L. We determined the frequency of each variant in 76 AJ families collected by members of the International Consortium for Prostate Cancer Genetics (ICPCG) where ≥ 2 men were affected by PRCA. Only one variant, Y424H in exon 11, was identified in more than two families. Exon 11 was then screened in nine additional AJ ICPCG families (a total of 85 families). The Y424H variant occurred in nine affected cases from four different families; however it did not completely segregate with the disease. We performed bioinformatics analysis, which showed that Y424H is a non-conservative missense substitution that falls at a position that is invariant in vertebrate CHEK2 orthologs. Both SIFT and Align-GVGD predict that Y424H is a loss of function mutation, however the frequency of Y424H was not significantly different between unselected AJ cases from Montreal/Memorial Sloan Kettering Cancer Centre (MSKCC) and AJ controls from Israel/MSKCC (OR 1.18, 95%CI: 0.34–4.61, p=.99). Moreover, functional assays using S. Cerevisae revealed that the Y424H substitution did not alter function of CHEK2 protein. Although we cannot rule out a subtle influence of the CHEK2 variants on PRCA risk, these results suggest that germline CHEK2 mutations have a minor role in PRCA susceptibility in AJ men.
checkpoint kinase 2; prostate cancer; single-nucleotide polymorphism; Ashkenazi Jewish; budding yeast
PALB2 has recently been identified as a breast cancer susceptibility gene. PALB2 mutations are rare causes of hereditary breast cancer but may be important in countries such as Finland where a founder mutation is present. We sought to estimate the contribution of PALB2 mutations to the burden of breast cancer in French Canadians from Quebec.
We screened all coding exons of PALB2 in a sample of 50 French-Canadian women diagnosed with either early-onset breast cancer or familial breast cancer at a single Montreal hospital. The genetic variants identified in this sample were then studied in 356 additional women with breast cancer diagnosed before age 50 and in 6,448 newborn controls.
We identified a single protein-truncating mutation in PALB2 (c.2323 C>T, resulting in Q775X) in 1 of the 50 high-risk women. This variant was present in 2 of 356 breast cancer cases and in none of 6,440 newborn French-Canadian controls (P = 0.003). We also identified two novel new non-synonymous single nucleotide polymorphisms in exon 4 of PALB2 (c.5038 A>G [I76V] and c.5156 G>T [G115V]). G115V was found in 1 of 356 cases and in 15 of 6,442 controls (P = 0.6). The I76V variant was not identified in either the extended case series or the controls.
We have identified a novel truncating mutation in PALB2. The mutation was found in approximately 0.5% of unselected French-Canadian women with early-onset breast cancer and appears to have a single origin. Although mutations are infrequent, PALB2 can be added to the list of breast cancer susceptibility genes for which founder mutations have been identified in the French-Canadian population.
The critical clinical question in prostate cancer research is: How do we develop means of distinguishing aggressive disease from indolent disease? Using a combination of proteomic and expression array data, we identified a set of 36 genes with concordant dysregulation of protein products that could be evaluated in situ by quantitative immunohistochemistry. Another five prostate cancer biomarkers were included using linear discriminant analysis, we determined that the optimal model used to predict prostate cancer progression consisted of 12 proteins. Using a separate patient population, transcriptional levels of the 12 genes encoding for these proteins predicted prostate-specific antigen failure in 79 men following surgery for clinically localized prostate cancer (P = .0015). This study demonstrates that cross-platform models can lead to predictive models with the possible advantage of being more robust through this selection process.
Metastasis; cancer; proteomics; prostate cancer; bioinformatics