Spatial transcriptomic interrogation of the tumour-stroma boundary in a 3D engineered model of ameloblastoma.

Stromal cells are key components of the tumour microenvironment (TME) and their incorporation into 3D engineered tumour-stroma models is essential for tumour mimicry. By engineering tumouroids with distinct tumour and stromal compartments, it has been possible to identify how gene expression of tumour cells is altered and influenced by the presence of different stromal cells. Ameloblastoma is a benign epithelial tumour of the jawbone. In engineered, multi-compartment tumouroids spatial transcriptomics revealed an upregulation of oncogenes in the ameloblastoma transcriptome where osteoblasts were present in the stromal compartment (bone stroma). Where a gingival fibroblast stroma was engineered, the ameloblastoma tumour transcriptome revealed increased matrix remodelling genes. This study provides evidence to show the stromal-specific effect on tumour behaviour and illustrates the importance of engineering biologically relevant stroma for engineered tumour models. Our novel results show that an engineered fibroblast stroma causes the upregulation of matrix remodelling genes in ameloblastoma which directly correlates to measured invasion in the model. In contrast the presence of a bone stroma increases the expression of oncogenes by ameloblastoma cells.

Prostate MR image quality of apparent diffusion coefficient maps versus fractional intracellular volume maps from VERDICT MRI using the PI-QUAL score and a dedicated Likert scale for artefacts.

PURPOSE: This study aimed to assess the image quality of apparent diffusion coefficient (ADC) maps derived from conventional diffusion-weighted MRI and fractional intracellular volume maps (FIC) from VERDICT MRI (Vascular, Extracellular, Restricted Diffusion for Cytometry in Tumours) in patients from the INNOVATE trial. The inter-reader agreement was also assessed. METHODS: Two readers analysed both ADC and FIC maps from 57 patients enrolled in the INNOVATE prospective trial. Image quality was assessed using the Prostate Imaging Quality (PI-QUAL) score and a subjective image quality Likert score (Likert-IQ). The image quality of FIC and ADC were compared using a Wilcoxon Signed Ranks test. The inter-reader agreement was assessed with Cohen's kappa. RESULTS: There was no statistically significant difference between the PI-QUAL score for FIC datasets compared to ADC datasets for either reader (p = 0.240 and p = 0.614). Using the Likert-IQ score, FIC image quality was higher compared to ADC (p = 0.021) as assessed by reader-1 but not for reader-2 (p = 0.663). The inter-reader agreement was 'fair' for PI-QUAL scoring of datasets with FIC maps at 0.27 (95% confidence interval; 0.08-0.46) and ADC datasets at 0.39 (95% confidence interval 0.22-0.57). For Likert scoring, the inter-reader agreement was also 'fair' for FIC maps at 0.38 (95% confidence interval; 0.10-0.65) and substantial for ADC maps at 0.62 (95% confidence interval; 0.39-0.86). CONCLUSION: Image quality was comparable for FIC and ADC. The inter-reader agreement was similar when using PIQUAL for both FIC and ADC datasets but higher for ADC maps compared to FIC maps using the image quality Likert score.

Spatial -omics technologies: the new enterprise in 3D breast cancer models.

The fields of tissue bioengineering, -omics, and spatial biology are advancing rapidly, each offering the opportunity for a paradigm shift in breast cancer research. However, to date, collaboration between these fields has not reached its full potential. In this review, we describe the most recently generated 3D breast cancer models regarding the biomaterials and technological platforms employed. Additionally, their biological evaluation is reported, highlighting their advantages and limitations. Specifically, we focus on the most up-to-date -omics and spatial biology techniques, which can generate a deeper understanding of the biological relevance of bioengineered 3D breast cancer in vitro models, thus paving the way towards truly clinically relevant microphysiological systems, improved drug development success rates, and personalised medicine approaches.

ST6GAL1-mediated aberrant sialylation promotes prostate cancer progression.

Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Accurate diagnosis of prostate cancer by combining Proclarix with magnetic resonance imaging.

OBJECTIVES: To assess of the clinical performance of Proclarix® (a novel Conformité Européenne [CE]-marked biomarker test aiding in the identification of clinically significant prostate cancer [csPCa]) alone or in combination with multiparametric magnetic resonance imaging (mpMRI) to predict csPCa (International Society of Urological Pathology Grade Group ≥2). PATIENTS AND METHODS: The study included blood samples from 721 men undergoing mpMRI followed by biopsy at University College London, London, and Vall d'Hebron University Hospital, Barcelona. Samples were tested blindly. The Proclarix-MRI model combining prostate volume, Proclarix and mpMRI results was trained using the UCL cohort (n = 159) and validated in the Vall d'Hebron cohort (n = 562). Its diagnostic performance was established in correlation to biopsy outcome and compared to available clinical parameters and risk calculators. RESULTS: Clinical performance of the Proclarix-MRI model in the validation cohort did not significantly differ from the training cohort and resulted in a sensitivity for csPCa of 90%, 90% negative predictive value and 66% positive predictive value. The Proclarix-MRI score's specificity (68%) was significantly (P < 0.001) better than the MRI-European Randomized study of Screening for Prostate Cancer risk score (51%), Proclarix (27%) or mpMRI (28%) alone. In addition, Proclarix by itself was found to be useful in the MRI Prostate Imaging-Reporting and Data System (PI-RADS) score 3 subgroup by outperforming prostate-specific antigen density in terms of specificity (25% vs 13%, P = 0.004) at 100% sensitivity. CONCLUSION: When combined with mpMRI and prostate volume, Proclarix reliably predicted csPCa and ruled out men with no or indolent cancer. A large reduction of two thirds of unneeded biopsies was achieved. Proclarix can further be used with high confidence to reliably detect csPCa in men with an indeterminate PI-RADS score 3 mpMRI. Despite these encouraging results, further validation is needed.

Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression.

Association between cannabis use with urological cancers: A population-based cohort study and a mendelian randomization study in the UK biobank.

BACKGROUND: Legislation of cannabis use has been approved in many European and North American countries. Its impact on urological cancers is unclear. This study was conducted to explore the association between cannabis use and the risk of urological cancers. METHODS: We identified 151,945 individuals with information on cannabis use in the UK Biobank from 2006 to 2010. Crude and age-standardized incidence ratios of different urological cancers were evaluated in the entire cohort and subgroups. Cox regression was performed for survival analysis. RESULTS: Previous use of cannabis was a significant protective factor for renal cell carcinoma (HR = 0.61, 95%CI:0.40-0.93, p = 0.021) and prostate cancer (HR = 0.82, 95%CI:0.73-0.93, p = 0.002) in multivariable analysis. The association between previous cannabis use and both renal cell carcinoma and bladder cancer was only observed in females (HRRCC  = 0.42, 95%CI:0.19-0.94, p = 0.034; HRBCa  = 0.43, 95%CI:0.21-0.86, p = 0.018) but not in men. There was no significant association between cannabis use and testicular cancer incidence. Mendelian randomization demonstrated a potential causal effect of cannabis use on a lower incidence of renal cell carcinoma. CONCLUSIONS: Previous use of cannabis was associated with a lower risk of bladder cancer, renal cell carcinoma, and prostate cancer. The inverse association between cannabis and both renal cell carcinoma and bladder cancer was only found in females but not in males.

Avoiding Unnecessary Biopsy after Multiparametric Prostate MRI with VERDICT Analysis: The INNOVATE Study.

Background In men suspected of having prostate cancer (PCa), up to 50% of men with positive multiparametric MRI (mpMRI) findings (Prostate Imaging Reporting and Data System [PI-RADS] or Likert score of 3 or higher) have no clinically significant (Gleason score ≤3+3, benign) biopsy findings. Vascular, Extracellular, and Restricted Diffusion for Cytometry in Tumor (VERDICT) MRI analysis could improve the stratification of positive mpMRI findings. Purpose To evaluate VERDICT MRI, mpMRI-derived apparent diffusion coefficient (ADC), and prostate-specific antigen density (PSAD) as determinants of clinically significant PCa (csPCa). Materials and Methods Between April 2016 and December 2019, men suspected of having PCa were prospectively recruited from two centers and underwent VERDICT MRI and mpMRI at one center before undergoing targeted biopsy. Biopsied lesion ADC, lesion-derived fractional intracellular volume (FIC), and PSAD were compared between men with csPCa and those without csPCa, using nonparametric tests subdivided by Likert scores. Area under the receiver operating characteristic curve (AUC) was calculated to test diagnostic performance. Results Among 303 biopsy-naive men, 165 study participants (mean age, 65 years ± 7 [SD]) underwent targeted biopsy; of these, 73 had csPCa. Median lesion FIC was higher in men with csPCa (FIC, 0.53) than in those without csPCa (FIC, 0.18) for Likert 3 (P = .002) and Likert 4 (0.60 vs 0.28, P < .001) lesions. Median lesion ADC was lower for Likert 4 lesions with csPCa (0.86 × 10-3 mm2/sec) compared with lesions without csPCa (1.12 × 10-3 mm2/sec, P = .03), but there was no evidence of a difference for Likert 3 lesions (0.97 × 10-3 mm2/sec vs 1.20 × 10-3 mm2/sec, P = .09). PSAD also showed no difference for Likert 3 (0.17 ng/mL2 vs 0.12 ng/mL2, P = .07) or Likert 4 (0.14 ng/mL2 vs 0.12 ng/mL2, P = .47) lesions. The diagnostic performance of FIC (AUC, 0.96; 95% CI: 0.93, 1.00) was higher (P = .02) than that of ADC (AUC, 0.85; 95% CI: 0.79, 0.91) and PSAD (AUC, 0.74; 95% CI: 0.66, 0.82) for the presence of csPCa in biopsied lesions. Conclusion Lesion fractional intracellular volume enabled better classification of clinically significant prostate cancer than did apparent diffusion coefficient and prostate-specific antigen density. Clinical trial registration no. NCT02689271 © RSNA, 2022 Online supplemental material is available for this article.

Histological analysis of fat grafting with platelet-rich plasma for diabetic foot ulcers-A randomised controlled trial.

Diabetic foot ulcers are often unresponsive to conventional therapy and are a leading cause of amputation. Animal studies have shown stem cells and growth factors can accelerate wound healing. Adipose-derived stem cells are found in fat grafts and mixing them with platelet-rich plasma (PRP) may improve graft survival. This study aimed to establish the histological changes when diabetic foot ulcers are treated with fat grafts and PRP. A three-armed RCT was undertaken of 18 diabetic foot ulcer patients: fat grafting; fat grafting with PRP; and routine podiatry care. Biopsies were obtained at week 0, 1, and 4, and underwent quantitative histology/immunohistochemistry (H&E, CD31, and Ki67). Treatment with fat and PRP increased mean microvessel density at 1 week to 1645 (SD 96) microvessels/mm2 (+32%-45% to other arms, P = .035). PRP appeared to increase vascularity surrounding fat grafts, and histology suggested PRP may enhance fat graft survival. There was no clinical difference between arms. This study demonstrates PRP with fat grafts increased neovascularisation and graft survival in diabetic foot ulcers. The histology was not, however, correlated with wound healing time. Future studies should consider using apoptosis markers and fluorescent labelling to ascertain if enhanced fat graft survival is due to proliferation or reduced apoptosis. Trial registration NCT03085550.

Exploring the Value of BRD9 as a Biomarker, Therapeutic Target and Co-Target in Prostate Cancer.

BACKGROUND AND AIMS: Despite recent advances in advanced prostate cancer treatments, clinical biomarkers or treatments for men with such cancers are imperfect. Targeted therapies have shown promise, but there remain fewer actionable targets in prostate cancer than in other cancers. This work aims to characterise BRD9, currently understudied in prostate cancer, and investigate its co-expression with other genes to assess its potential as a biomarker and therapeutic target in human prostate cancer. MATERIALS AND METHODS: Omics data from a total of 2053 prostate cancer patients across 11 independent datasets were accessed via Cancertool and cBioPortal. mRNA M.expression and co-expression, mutations, amplifications, and deletions were assessed with respect to key clinical parameters including survival, Gleason grade, stage, progression, and treatment. Network and pathway analysis was carried out using Genemania, and heatmaps were constructed using Morpheus. RESULTS: BRD9 is overexpressed in prostate cancer patients, especially those with metastatic disease. BRD9 expression did not differ in patients treated with second generation antiandrogens versus those who were not. BRD9 is co-expressed with many genes in the SWI/SNF and BET complexes, as well as those in common signalling pathways in prostate cancer. SUMMARY AND CONCLUSIONS: BRD9 has potential as a diagnostic and prognostic biomarker in prostate cancer. BRD9 also shows promise as a therapeutic target, particularly in advanced prostate cancer, and as a co-target alongside other genes in the SWI/SNF and BET complexes, and those in common prostate cancer signalling pathways. These promising results highlight the need for wider experimental inhibition and co-targeted inhibition of BRD9 in vitro and in vivo, to build on the limited inhibition data available.

Cellular senescence as a possible link between prostate diseases of the ageing male.

Senescent cells accumulate with age in all tissues. Although senescent cells undergo cell-cycle arrest, these cells remain metabolically active and their secretome - known as the senescence-associated secretory phenotype - is responsible for a systemic pro-inflammatory state, which contributes to an inflammatory microenvironment. Senescent cells can be found in the ageing prostate and the senescence-associated secretory phenotype and can be linked to BPH and prostate cancer. Indeed, a number of signalling pathways provide biological plausibility for the role of senescence in both BPH and prostate cancer, although proving causality is difficult. The theory of senescence as a mechanism for prostate disease has a number of clinical implications and could offer opportunities for targeting in the future.

Co-Targeting PIM Kinase and PI3K/mTOR in NSCLC.

PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.

Evaluation of PSA and PSA Density in a Multiparametric Magnetic Resonance Imaging-Directed Diagnostic Pathway for Suspected Prostate Cancer: The INNOVATE Trial.

Objectives: To assess the clinical outcomes of mpMRI before biopsy and evaluate the space remaining for novel biomarkers. Methods: The INNOVATE study was set up to evaluate the validity of novel fluidic biomarkers in men with suspected prostate cancer who undergo pre-biopsy mpMRI. We report the characteristics of this clinical cohort, the distribution of clinical serum biomarkers, PSA and PSA density (PSAD), and compare the mpMRI Likert scoring system to the Prostate Imaging-Reporting and Data System v2.1 (PI-RADS) in men undergoing biopsy. Results: 340 men underwent mpMRI to evaluate suspected prostate cancer. 193/340 (57%) men had subsequent MRI-targeted prostate biopsy. Clinically significant prostate cancer (csigPCa), i.e., overall Gleason ≥ 3 + 4 of any length OR maximum cancer core length (MCCL) ≥4 mm of any grade including any 3 + 3, was found in 96/195 (49%) of biopsied patients. Median PSA (and PSAD) was 4.7 (0.20), 8.0 (0.17), and 9.7 (0.31) ng/mL (ng/mL/mL) in mpMRI scored Likert 3,4,5 respectively for men with csigPCa on biopsy. The space for novel biomarkers was shown to be within the group of men with mpMRI scored Likert3 (178/340) and 4 (70/350), in whom an additional of 40% (70/178) men with mpMRI-scored Likert3, and 37% (26/70) Likert4 could have been spared biopsy. PSAD is already considered clinically in this cohort to risk stratify patients for biopsy, despite this 67% (55/82) of men with mpMRI-scored Likert3, and 55% (36/65) Likert4, who underwent prostate biopsy had a PSAD below a clinical threshold of 0.15 (or 0.12 for men aged <50 years). Different thresholds of PSA and PSAD were assessed in mpMRI-scored Likert4 to predict csigPCa on biopsy, to achieve false negative levels of ≤5% the proportion of patients whom who test as above the threshold were unsuitably high at 86 and 92% of patients for PSAD and PSA respectively. When PSA was re tested in a sub cohort of men repeated PSAD showed its poor reproducibility with 43% (41/95) of patients being reclassified. After PI-RADS rescoring of the biopsied lesions, 66% (54/82) of the Likert3 lesions received a different PI-RADS score. Conclusions: The addition of simple biochemical and radiological markers (Likert and PSAD) facilitate the streamlining of the mpMRI-diagnostic pathway for suspected prostate cancer but there remains scope for improvement, in the introduction of novel biomarkers for risk assessment in Likert3 and 4 patients, future application of novel biomarkers tested in a Likert cohort would also require re-optimization around Likert3/PI-RADS2, as well as reproducibility testing.

Author Correction: Co-targeting PIM and PI3K/mTOR using multikinase inhibitor AUM302 and a combination of AZD-1208 and BEZ235 in prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Genetic alterations in the 3q26.31-32 locus confer an aggressive prostate cancer phenotype.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A critical evaluation of visual proportion of Gleason 4 and maximum cancer core length quantified by histopathologists.

Gleason score 7 prostate cancer with a higher proportion of pattern 4 (G4) has been linked to genomic heterogeneity and poorer patient outcome. The current assessment of G4 proportion uses estimation by a pathologist, with a higher proportion of G4 more likely to trigger additional imaging and treatment over active surveillance. This estimation method has been shown to have inter-observer variability. Fifteen patients with Prostate Grade Group (GG) 2 (Gleason 3 + 4) and fifteen patients with GG3 (Gleason 4 + 3) disease were selected from the PROMIS study with 192 haematoxylin and eosin-stained slides scanned. Two experienced uropathologists assessed the maximum cancer core length (MCCL) and G4 proportion using the current standard method (visual estimation) followed by detailed digital manual annotation of each G4 area and measurement of MCCL (planimetric estimation) using freely available software by the same two experts. We aimed to compare visual estimation of G4 and MCCL to a pathologist-driven digital measurement. We show that the visual and digital MCCL measurement differs up to 2 mm in 76.6% (23/30) with a high degree of agreement between the two measurements; Visual gave a median MCCL of 10 ± 2.70 mm (IQR 4, range 5-15 mm) compared to digital of 9.88 ± 3.09 mm (IQR 3.82, range 5.01-15.7 mm) (p = 0.64) The visual method for assessing G4 proportion over-estimates in all patients, compared to digital measurements [median 11.2% (IQR 38.75, range 4.7-17.9%) vs 30.4% (IQR 18.37, range 12.9-50.76%)]. The discordance was higher as the amount of G4 increased (Bias 18.71, CI 33.87-48.75, r 0.7, p < 0.0001). Further work on assessing actual G4 burden calibrated to clinical outcomes might lead to the use of differing G4 thresholds of significance if the visual estimation is used or by incorporating semi-automated methods for G4 burden measurement.

Co-targeting PIM and PI3K/mTOR using multikinase inhibitor AUM302 and a combination of AZD-1208 and BEZ235 in prostate cancer.

PIM and PI3K/mTOR pathways are often dysregulated in prostate cancer, and may lead to decreased survival, increased metastasis and invasion. The pathways are heavily interconnected and act on a variety of common effectors that can lead to the development of resistance to drug inhibitors. Most current treatments exhibit issues with toxicity and resistance. We investigated the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, versus a combination of the PIM inhibitor, AZD-1208, and the PI3K/mTOR inhibitor BEZ235 (Dactolisib) to determine their impact on mRNA and phosphoprotein expression, as well as their functional efficacy. We have determined that around 20% of prostate cancer patients overexpress the direct targets of these drugs, and this cohort are more likely to have a high Gleason grade tumour (≥ Gleason 8). A co-targeted inhibition approach offered broader inhibition of genes and phosphoproteins in the PI3K/mTOR pathway, when compared to single kinase inhibition. The preclinical inhibitor AUM302, used at a lower dose, elicited a comparable or superior functional outcome compared with combined AZD-1208 + BEZ235, which have been investigated in clinical trials, and could help to reduce treatment toxicity in future trials. We believe that a co-targeting approach is a viable therapeutic strategy that should be developed further in pre-clinical studies.

Genetic alterations in the 3q26.31-32 locus confer an aggressive prostate cancer phenotype.

Large-scale genetic aberrations that underpin prostate cancer development and progression, such as copy-number alterations (CNAs), have been described but the consequences of specific changes in many identified loci is limited. Germline SNPs in the 3q26.31 locus are associated with aggressive prostate cancer, and is the location of NAALADL2, a gene overexpressed in aggressive disease. The closest gene to NAALADL2 is TBL1XR1, which is implicated in tumour development and progression. Using publicly-available cancer genomic data we report that NAALADL2 and TBL1XR1 gains/amplifications are more prevalent in aggressive sub-types of prostate cancer when compared to primary cohorts. In primary disease, gains/amplifications occurred in 15.99% (95% CI: 13.02-18.95) and 14.96% (95% CI: 12.08-17.84%) for NAALADL2 and TBL1XR1 respectively, increasing in frequency in higher Gleason grade and stage tumours. Gains/amplifications result in transcriptional changes and the development of a pro-proliferative and aggressive phenotype. These results support a pivotal role for copy-number gains in this genetic region.

Targeting the ERG oncogene with splice-switching oligonucleotides as a novel therapeutic strategy in prostate cancer.

BACKGROUND: The ERG oncogene, a member of the ETS family of transcription factor encoding genes, is a genetic driver of prostate cancer. It is activated through a fusion with the androgen-responsive TMPRSS2 promoter in 50% of cases. There is therefore significant interest in developing novel therapeutic agents that target ERG. We have taken an antisense approach and designed morpholino-based oligonucleotides that target ERG by inducing skipping of its constitutive exon 4. METHODS: We designed antisense morpholino oligonucleotides (splice-switching oligonucleotides, SSOs) that target both the 5' and 3' splice sites of ERG's exon 4. We tested their efficacy in terms of inducing exon 4 skipping in two ERG-positive cell lines, VCaP prostate cancer cells and MG63 osteosarcoma cells. We measured their effect on cell proliferation, migration and apoptosis. We also tested their effect on xenograft tumour growth in mice and on ERG protein expression in a human prostate cancer radical prostatectomy sample ex vivo. RESULTS: In VCaP cells, both SSOs were effective at inducing exon 4 skipping, which resulted in a reduction of overall ERG protein levels up to 96 h following a single transfection. SSO-induced ERG reduction decreased cell proliferation, cell migration and significantly increased apoptosis. We observed a concomitant reduction in protein levels for cyclin D1, c-Myc and the Wnt signalling pathway member ß-catenin as well as a marker of activated Wnt signalling, p-LRP6. We tested the 3' splice site SSO in MG63 xenografts in mice and observed a reduction in tumour growth. We also demonstrated that the 3' splice site SSO caused a reduction in ERG expression in a patient-derived prostate tumour tissue cultured ex vivo. CONCLUSIONS: We have successfully designed and tested morpholino-based SSOs that cause a marked reduction in ERG expression, resulting in decreased cell proliferation, a reduced migratory phenotype and increased apoptosis. Our initial tests on mouse xenografts and a human prostate cancer radical prostatectomy specimen indicate that SSOs can be effective for oncogene targeting in vivo. As such, this study encourages further in vivo therapeutic studies using SSOs targeting the ERG oncogene.