A genome-wide CRISPR/Cas9 screen identifies DNA-PK as a sensitiser to 177Lutetium-DOTA-octreotate radionuclide therapy.

Peptide receptor radionuclide therapy (PRRT) using 177Lutetium-DOTA-octreotate (LuTate) for neuroendocrine tumours (NET) is now an approved treatment available in many countries, though primary or secondary resistance continue to limit its effectiveness or durability. We hypothesised that a genome-wide CRISPR/Cas9 screen would identify key mediators of response to LuTate and gene targets that might offer opportunities for novel combination therapies for NET patients. Methods: We utilised a genome-wide CRISPR-Cas9 screen in LuTate-treated cells to identify genes that impact on the sensitivity or resistance of cells to LuTate. Hits were validated through single-gene knockout. LuTate-resistant cells were assessed to confirm LuTate uptake and retention, and persistence of somatostatin receptor 2 (SSTR2) expression. Gene knockouts conferring LuTate sensitivity were further characterised by pharmacological sensitisation using specific inhibitors and in vivo analysis of the efficacy of these inhibitors in combination with LuTate. Results: The CRISPR-Cas9 screen identified several potential targets for both resistance and sensitivity to PRRT. Two gene knockouts which conferred LuTate resistance in vitro, ARRB2 and MVP, have potential mechanisms related to LuTate binding and retention, and modulation of DNA-damage repair (DDR) pathways, respectively. The screen showed that sensitivity to LuTate treatment in vitro can be conferred by the loss of a variety of genes involved in DDR pathways, with loss of genes involved in Non-Homologous End-Joining (NHEJ) being the most lethal. Loss of the key NHEJ gene, PRKDC (DNA-PK), either by gene loss or inhibition by two different inhibitors, resulted in significantly reduced cell survival upon exposure of cells to LuTate. In SSTR2-positive xenograft-bearing mice, the combination of nedisertib (a DNA-PK specific inhibitor) and LuTate produced a more robust control of tumour growth and increased survival compared to LuTate alone. Conclusions: DDR pathways are critical for sensing and repairing radiation-induced DNA damage, and our study shows that regulation of DDR pathways may be involved in both resistance and sensitivity to PRRT. Additionally, the use of a DNA-PK inhibitor in combination with LuTate PRRT significantly improves the efficacy of the treatment in pre-clinical models, providing further evidence for the clinical efficacy of this combination.

E6AP Promotes a Metastatic Phenotype in Prostate Cancer.

Although primary prostate cancer is largely curable, progression to metastatic disease is associated with very poor prognosis. E6AP is an E3 ubiquitin ligase and a transcriptional co-factor involved in normal prostate development. E6AP drives prostate cancer when overexpressed. Our study exposed a role for E6AP in the promotion of metastatic phenotype in prostate cells. We revealed that elevated levels of E6AP in primary prostate cancer correlate with regional metastasis and demonstrated that E6AP promotes acquisition of mesenchymal features, migration potential, and ability for anchorage-independent growth. We identified the metastasis suppressor NDRG1 as a target of E6AP and showed it is key in E6AP induction of mesenchymal phenotype. We showed that treatment of prostate cancer cells with pharmacological agents upregulated NDRG1 expression suppressed E6AP-induced cell migration. We propose that the E6AP-NDRG1 axis is an attractive therapeutic target for the treatment of E6AP-driven metastatic prostate cancer.

Obesity Drives STAT-1-Dependent NASH and STAT-3-Dependent HCC.

Obesity is a major driver of cancer, especially hepatocellular carcinoma (HCC). The prevailing view is that non-alcoholic steatohepatitis (NASH) and fibrosis or cirrhosis are required for HCC in obesity. Here, we report that NASH and fibrosis and HCC in obesity can be dissociated. We show that the oxidative hepatic environment in obesity inactivates the STAT-1 and STAT-3 phosphatase T cell protein tyrosine phosphatase (TCPTP) and increases STAT-1 and STAT-3 signaling. TCPTP deletion in hepatocytes promoted T cell recruitment and ensuing NASH and fibrosis as well as HCC in obese C57BL/6 mice that normally do not develop NASH and fibrosis or HCC. Attenuating the enhanced STAT-1 signaling prevented T cell recruitment and NASH and fibrosis but did not prevent HCC. By contrast, correcting STAT-3 signaling prevented HCC without affecting NASH and fibrosis. TCPTP-deletion in hepatocytes also markedly accelerated HCC in mice treated with a chemical carcinogen that promotes HCC without NASH and fibrosis. Our studies reveal how obesity-associated hepatic oxidative stress can independently contribute to the pathogenesis of NASH, fibrosis, and HCC.

Biodosimetric transcriptional and proteomic changes are conserved in irradiated human tissue.

Transcriptional dosimetry is an emergent field of radiobiology aimed at developing robust methods for detecting and quantifying absorbed doses using radiation-induced fluctuations in gene expression. A combination of RNA sequencing, array-based and quantitative PCR transcriptomics in cellular, murine and various ex vivo human models has led to a comprehensive description of a fundamental set of genes with demonstrable dosimetric qualities. However, these are yet to be validated in human tissue due to the scarcity of in situ-irradiated source material. This represents a major hurdle to the continued development of transcriptional dosimetry. In this study, we present a novel evaluation of a previously reported set of dosimetric genes in human tissue exposed to a large therapeutic dose of radiation. To do this, we evaluated the quantitative changes of a set of dosimetric transcripts consisting of FDXR, BAX, BCL2, CDKN1A, DDB2, BBC3, GADD45A, GDF15, MDM2, SERPINE1, TNFRSF10B, PLK3, SESN2 and VWCE in guided pre- and post-radiation (2 weeks) prostate cancer biopsies from seven patients. We confirmed the prolonged dose-responsivity of most of these transcripts in in situ-irradiated tissue. BCL2, GDF15, and to some extent TNFRSF10B, were markedly unreliable single markers of radiation exposure. Nevertheless, as a full set, these genes reliably segregated non-irradiated and irradiated tissues and predicted radiation absorption on a patient-specific basis. We also confirmed changes in the translated protein product for a small subset of these dosimeters. This study provides the first confirmatory evidence of an existing dosimetric gene set in less-accessible tissues-ensuring peripheral responses reflect tissue-specific effects. Further work will be required to determine if these changes are conserved in different tissue types, post-radiation times and doses.

Exploring the oncoproteomic response of human prostate cancer to therapeutic radiation using data-independent acquisition (DIA) mass spectrometry.

INTRODUCTION: The development of radioresistance in prostate cancer (PCa) is an important clinical issue and is still largely uninformed by personalized molecular characteristics. The aim of this study was to establish a platform that describes the early oncoproteomic response of human prostate tissue to radiation therapy (RT) using a prospective human tissue cohort. METHODS: Fresh and fixed transperineal biopsies from eight men with clinically localized tumors were taken prior to and 14 days following a single fraction of high-dose-rate brachytherapy. Quantitative protein analysis was achieved using an optimized protein extraction pipeline and subsequent data-independent acquisition mass spectroscopy (DIA-MS). Ontology analyses were used to identify enriched functional pathways, with the candidates further interrogated in formalin-fixed paraffin-embedded tissue biopsies from five additional patients. RESULTS: We obtained a mean coverage of 5660 proteins from fresh tissue biopsies; with the principal post-radiation change observed being an increase in levels amongst a total of 49 proteins exhibiting abundance changes. Many of these changes in abundance varied between patients and, typically to prostate cancer tissue, exhibited a high level of heterogeneity. Ontological analysis revealed the enrichment of the protein activation cascades of three immunological pathways: humoral immune response, leukocyte mediated immunity and complement activation. These were predominantly associated with the extracellular space. We validated significant expression differences in between 20% and 61% of these candidates using the separate fixed-tissue cohort and established their feasibility as an experimental tissue resource by acquiring quantitative data for a mean of 5152 proteins per patient. DISCUSSION: In this prospective study, we have established a sensitive and reliable oncoproteomic pipeline for the analysis of both fresh and formalin-fixed human PCa tissue. We identified multiple pathways known to be radiation-responsive and have established a powerful database of candidates and pathways with no current association with RT. This information may be beneficial in the advancement of personalized therapies and potentially, predictive biomarkers.

Proteotranscriptomic Measurements of E6-Associated Protein (E6AP) Targets in DU145 Prostate Cancer Cells.

Prostate cancer is a common cause of cancer-related death in men. E6AP (E6-Associated Protein), an E3 ubiquitin ligase and a transcription cofactor, is elevated in a subset of prostate cancer patients. Genetic manipulations of E6AP in prostate cancer cells expose a role of E6AP in promoting growth and survival of prostate cancer cells in vitro and in vivo However, the effect of E6AP on prostate cancer cells is broad and it cannot be explained fully by previously identified tumor suppressor targets of E6AP, promyelocytic leukemia protein and p27. To explore additional players that are regulated downstream of E6AP, we combined a transcriptomic and proteomic approach. We identified and quantified 16,130 transcripts and 7,209 proteins in castration resistant prostate cancer cell line, DU145. A total of 2,763 transcripts and 308 proteins were significantly altered on knockdown of E6AP. Pathway analyses supported the known phenotypic effects of E6AP knockdown in prostate cancer cells and in parallel exposed novel potential links of E6AP with cancer metabolism, DNA damage repair and immune response. Changes in expression of the top candidates were confirmed using real-time polymerase chain reaction. Of these, clusterin, a stress-induced chaperone protein, commonly deregulated in prostate cancer, was pursued further. Knockdown of E6AP resulted in increased clusterin transcript and protein levels in vitro and in vivo Concomitant knockdown of E6AP and clusterin supported the contribution of clusterin to the phenotype induced by E6AP. Overall, results from this study provide insight into the potential biological pathways controlled by E6AP in prostate cancer cells and identifies clusterin as a novel target of E6AP.

E6AP promotes prostate cancer by reducing p27 expression.

Prostate cancer (PC) is the most common cancer in men. Elevated levels of E3 ligase, E6-Associated Protein (E6AP) were previously linked to PC, consistent with increased protein expression in a subset of PC patients. In cancers, irregular E3 ligase activity drives proteasomal degradation of tumor suppressor proteins. Accordingly, E3 ligase inhibitors define a rational therapy to restore tumor suppression. The relevant tumor suppressors targeted by E6AP in PC are yet to be fully identified. In this study we show that p27, a key cell cycle regulator, is a target of E6AP in PC. Down regulation of E6AP increases p27 expression and enhances its nuclear accumulation in PC. We demonstrate that E6AP regulates p27 expression by inhibiting its transcription in an E2F1-dependent manner. Concomitant knockdown of E6AP and p27 partially restores PC cell growth, supporting the contribution of p27 to the overall effect of E6AP on prostate tumorigenesis. Overall, we unravelled the E6AP-p27 axis as a new promoter of PC, exposing an attractive target for therapy through the restoration of tumor suppression.

Reduced abundance of the E3 ubiquitin ligase E6AP contributes to decreased expression of the INK4/ARF locus in non-small cell lung cancer.

The tumor suppressor p16INK4a, one protein encoded by the INK4/ARF locus, is frequently absent in multiple cancers, including non-small cell lung cancer (NSCLC). Whereas increased methylation of the encoding gene (CDKN2A) accounts for its loss in a third of patients, no molecular explanation exists for the remainder. We unraveled an alternative mechanism for the silencing of the INK4/ARF locus involving the E3 ubiquitin ligase and transcriptional cofactor E6AP (also known as UBE3A). We found that the expression of three tumor suppressor genes encoded in the INK4/ARF locus (p15INK4b, p16INK4a, and p19ARF) was decreased in E6AP-/- mouse embryo fibroblasts. E6AP induced the expression of the INK4/ARF locus at the transcriptional level by inhibiting CDC6 transcription, a gene encoding a key repressor of the locus. Luciferase assays revealed that E6AP inhibited CDC6 expression by reducing its E2F1-dependent transcription. Chromatin immunoprecipitation analysis indicated that E6AP reduced the amount of E2F1 at the CDC6 promoter. In a subset of NSCLC samples, an E6AP-low/CDC6-high/p16INK4a-low protein abundance profile correlated with low methylation of the gene encoding p16INK4a (CDKN2A) and poor patient prognosis. These findings define a previously unrecognized tumor-suppressive role for E6AP in NSCLC, reveal an alternative silencing mechanism of the INK4/ARF locus, and reveal E6AP as a potential prognostic marker in NSCLC.

Expression of growth differentiation factor 6 in the human developing fetal spine retreats from vertebral ossifying regions and is restricted to cartilaginous tissues.

During embryogenesis vertebral segmentation is initiated by sclerotomal cell migration and condensation around the notochord, forming anlagen of vertebral bodies and intervertebral discs. The factors that govern the segmentation are not clear. Previous research demonstrated that mutations in growth differentiation factor 6 resulted in congenital vertebral fusion, suggesting this factor plays a role in development of vertebral column. In this study, we detected expression and localization of growth differentiation factor 6 in human fetal spinal column, especially in the period of early ossification of vertebrae and the developing intervertebral discs. The extracellular matrix proteins were also examined. Results showed that high levels of growth differentiation factor 6 were expressed in the nucleus pulposus of intervertebral discs and the hypertrophic chondrocytes adjacent to the ossification centre in vertebral bodies, where strong expression of proteoglycan and collagens was also detected. As fetal age increased, the expression of growth differentiation factor 6 was decreased correspondingly with the progress of ossification in vertebral bodies and restricted to cartilaginous regions. This expression pattern and the genetic link to vertebral fusion suggest that growth differentiation factor 6 may play an important role in suppression of ossification to ensure proper vertebral segmentation during spinal development.

Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture.

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p<0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells.

Comprehensive tissue processing strategy for quantitative proteomics of formalin-fixed multiple sclerosis lesions.

Formalin-fixed (FF) autopsy tissue comprises the bulk of existing Multiple Sclerosis (MSc) pathology archives, providing a rich pool of material for biomarker discovery and disease characterization. Here, we present the development of a heat-induced extraction protocol for the proteomic analysis of FF brain tissue, its application to the study of lesion remyelination and its failure in MSc. A 4-round extraction strategy was optimized using FF tissue leading to a 35% increase in the number of proteins identified compared to a single extraction; and a 65% increase in proteins identified with ≥4 peptides. Histological staining of sections with oil red O and luxol fast blue-periodic acid Schiff, required to characterize MSc lesions was found to have minimal effect on LC-MS/MS. The application of the optimized protocol to chronic demyelinated and remyelinated FF MSc lesions and the adjacent periplaque white matter, isolated through laser guided manual dissection from 3 patients, identified 428 unique proteins (0.2% FDR) using LC-MS/MS. Comparison of the lesion types using iTRAQ and 2-D LC-MS/MS revealed 82 differentially expressed proteins. Protein quantitation by iTRAQ and spectral counting was well-correlated (r(s)= 0.7653; p < 10(-30)). The data generated from this work illustrates the scope of the methodology and provides insights into the pathogenesis of MSc and remyelination.