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Background: We have recently demonstrated that human-CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5 days to 8.7 days in baboons. Intra-bone bone marrow transplantation (IBBMTx) in a pig-to-baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21 days from 1 to 4 days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx., Methods: Baboons received GalTKO-hCD47/hCD55Tg (n = 5) or -hCD55Tg (n = 1) or -hCD46/HLA-E Tg (n = 1) pig IBBMTx. Macrochimerism, anti-pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47- porcine lungs 1-3 months later., Results: All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30 days, and two maintained chimerism for >8 weeks. Notably, anti-pig antibody levels decreased over time and anti-pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14 days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1-4 days)., Conclusion: This is the first report demonstrating durable macrochimerism beyond 8 weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Despite recent progress in survival times of xenografts in non-human primates, there are no reports of survival beyond 5 days of histologically well-aerated porcine lung grafts in baboons. Here, we report our initial results of pig-to-baboon xeno-lung transplantation (XLTx)., Methods: Eleven baboons received genetically modified porcine left lungs from either GalT-KO alone (n = 3), GalT-KO/humanCD47(hCD47)/hCD55 (n = 3), GalT-KO/hD47/hCD46 (n = 4), or GalT-KO/hCD39/hCD46/hCD55/TBM/EPCR (n = 1) swine. The first 2 XLTx procedures were performed under a non-survival protocol that allowed a 72-hour follow-up of the recipients with general anesthesia, while the remaining 9 underwent a survival protocol with the intention of weaning from ventilation., Results: Lung graft survivals in the 2 non-survival animals were 48 and >72 hours, while survivals in the other 9 were 25 and 28 hours, at 5, 5, 6, 7, >7, 9, and 10 days. One baboon with graft survival >7 days, whose entire lung graft remained well aerated, was euthanized on POD 7 due to malfunction of femoral catheters. hCD47 expression of donor lungs was detected in both alveoli and vessels only in the 3 grafts surviving >7, 9, and 10 days. All other grafts lacked hCD47 expression in endothelial cells and were completely rejected with diffuse hemorrhagic changes and antibody/complement deposition detected in association with early graft loss., Conclusions: To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7 days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Recurrent chromosome breakpoints at 6q22.31, leading to truncation and potential loss-of-function of the NKAIN2 gene, in Chinese prostate cancer patients were previously identified. In this study we investigated genomic, methylation and expression changes of NKAIN2 in a large number of prostate cancer samples and determined its functional role in prostate cancer cells. Fluorescence in situ hybridization analysis confirmed that NKAIN2 truncation is specific to Chinese while deletion of the gene is frequent in both Chinese and UK prostate cancers. Significantly reduced expression of NKAIN2 was also detected at both RNA and protein levels. Somatic mutations of NKAIN2 in prostate cancer samples exist but at very low frequency, suggesting that it is a putative tumor suppressor gene (TSG) with haploid insufficiency. Our functional studies showed that overexpression of NKAIN2 in prostate cancer cells inhibits cellular growth by promoting cell apoptosis, and decreasing cell migration and invasion. Conversely, knockdown of NKAIN2 promotes prostate cancer cell growth by inhibiting cell apoptosis, and increasing cell migration and invasion. These data imply that NKAIN2 is a novel TSG whose activity is commonly reduced in prostate cancer. It may restrain the disease development and progression by inducing apoptosis and suppressing cancer cell growth, migration and invasion. This study provides new insights into prostate carcinogenesis and opportunities for development of novel therapies for prostate cancer.

While androgen and androgen receptor (AR) activity have been strongly implicated in prostate cancer development and therapy, the influence of the CAG repeat, which is found within the first exon of the AR gene, on prostate carcinogenesis is still unclear. We investigated the differences in the length of the CAG repeat between prostate cancer patients and controls in the Chinese population as well as between TMPRSS2:ERG fusion positive and negative samples. A general association between prostate cancer and either longer or shorter AR CAG repeat length was not observed in the Chinese population. However, our data suggest that certain CAG repeat lengths may increase or decrease prostate cancer risk. Shorter CAG repeat length was also not shown to be associated with a higher induction rate of TMPRSS2 and ERG proximity, an essential step for TMPRSS2:ERG fusion formation. However, samples with a CAG repeat of 17 were found more frequently in the TMPRSS2:ERG fusion positive than negative prostate cancer cases and mediated a higher rate of androgen-induced TMPRSS2 and ERG co-localisation than AR with longer (24) and shorter (15) CAG repeats. This suggests that 17 CAG repeats may be associated with TMPRSS2:ERG fusion positive prostate cancer, but may have a preventive role for prostate cancer in the Chinese population, which has a low TMPRSS2:ERG fusion frequency. This study suggests that different mechanisms for the association of CAG repeat length polymorphism and prostate cancer exist in different ethnic populations.

Although prostate cancer is the most common malignancy to affect men in the Western world, the molecular mechanisms underlying its development and progression remain poorly understood. Like all cancers, prostate cancer is a genetic disease that is characterized by multiple genomic alterations, including point mutations, microsatellite variations, and chromosomal alterations such as translocations, insertions, duplications, and deletions. In prostate cancer, but not other carcinomas, these chromosome alterations result in a high frequency of gene fusion events. The development and application of novel high-resolution technologies has significantly accelerated the detection of genomic alterations, revealing the complex nature and heterogeneity of the disease. The clinical heterogeneity of prostate cancer can be partly explained by this underlying genetic heterogeneity, which has been observed between patients from different geographical and ethnic populations, different individuals within these populations, different tumour foci within the same patient, and different cells within the same tumour focus. The highly heterogeneous nature of prostate cancer provides a real challenge for clinical disease management and a detailed understanding of the genetic alterations in all cells, including small subpopulations, would be highly advantageous.

Many human cancers present as multifocal lesions. Understanding the clonal origin of multifocal cancers is of both etiological and clinical importance. The molecular basis of multifocal prostate cancer has previously been explored using a limited number of isolated markers and, although independent origin is widely believed, the clonal origin of multifocal prostate cancer is still debatable. We attempted to address clonal origin using a genome-wide copy-number analysis of individual cancer and high-grade prostatic intraepithelial neoplasia (HGPIN) lesions. Using Affymetrix array 6.0 copy-number analysis, we compared the genomic changes detected in 48 individual cancer and HGPIN lesions, isolated from 18 clinically localized prostate cancer cases. Identical genomic copy-number changes, shared by all same-case cancer foci, were detected in all 13 informative cases displaying multiple tumor foci. In addition, individual HGPIN lesions in the two multifocal-HGPIN cases available shared identical genomic changes. Commonly known genomic alterations, including losses at 6q15, 8p21.3-8p21.2, 10q23.2-10q23.31, 16q22.3, 16q23.2-16q23.3 and 21q22.2-21q22.3 regions and gain of 8q24.3 were the most frequently detected changes in this study and each was detected in all same-case foci in at least one case. Microarray data were confirmed by fluorescence in situ hybridization in selected foci. Our high-resolution genome-wide copy-number data suggest that many multifocal cases derive from a single prostate cancer precursor clone and that this precursor may give rise to separate HGPIN foci and may further progress to multifocal invasive prostate cancer. These findings, which demonstrate the monoclonal origin of multifocal prostate cancer, should significantly enhance our understanding of prostate carcinogenesis., (Copyright © 2012 Wiley Periodicals, Inc.)

Prostate cancer, the most common male cancer in Western countries, is commonly detected with complex chromosomal rearrangements. Following the discovery of the recurrent TMPRSS2:ETS fusions in prostate cancer and EML4:ALK in non-small-cell lung cancer, it is now accepted that fusion genes not only are the hallmark of haematological malignancies and sarcomas, but also play an important role in epithelial cell carcinogenesis. However, previous studies aiming to identify fusion genes in prostate cancer were mainly focused on expression changes and fusion transcripts. To investigate the genes recurrently affected by the chromosome breakpoints in prostate cancer, we analysed Affymetrix array 6.0 and 500K SNP microarray data from 77 prostate cancer samples. While the two genes most frequently affected by genomic breakpoints were, as expected, ERG and TMPRSS2, surprisingly more known tumour suppressor genes (TSGs) than known oncogenes were identified at recurrent chromosome breakpoints. Certain well-characterised TSGs, including p53, PTEN, BRCA1 and BRCA2 are recurrently truncated as a result of chromosome rearrangements in prostate cancer. Interestingly, many of the genes residing at recurrent breakpoint sites have not yet been implicated in prostate carcinogenesis such as HOOK3, PPP2R2A and TCBA1. We have confirmed the generally reduced expression of selected genes in clinical samples using quantitative RT-PCR analysis. Subsequently, we further investigated the genes associated with the t(4:6) translocation in LNCaP cells and reveal the genomic fusion of SNX9 and putative TSG UNC5C, which led to the reduced expression of both genes. This study reveals another common mechanism that leads to the inactivation of TSGs in prostate cancer and the identification of multiple TSGs inactivated by chromosome rearrangements will lead to new direction of research for the molecular basis of prostate carcinogenesis.

Fusion genes play important roles in tumorigenesis. The identification of the high-frequency TMPRSS2 fusion with ERG and other ETS family genes in prostate cancer highlights the importance of fusion genes in solid tumor development and progression. However, the mechanisms leading to these fusions are unclear. We investigated whether androgen, through stimulating its receptor, could promote spatial genome reorganization and contribute to the generation of the TMPRSS2:ERG fusion. We show that treatment with androgen can induce the TMPRSS2:ERG fusion in both malignant and nonmalignant prostate epithelial cells. Although the fusion could be detected in malignant cells following 24-hour treatment, prolonged exposure to androgen was required to detect the fusion transcript in nonmalignant cells. We associated the fusion incidence with genetic factors, including androgen-induced gene proximity, androgen receptor exon1 CAG repeat length and expression of the PIWIL1 gene. This study demonstrates that fusions can be induced prior to malignant transformation and generation of the fusion is associated with both gene proximity and loss of the ability to prevent double-strand breaks.

The S. pombe Rad60 protein is required for the repair of DNA double strand breaks, recovery from replication arrest, and is essential for cell viability. It has two SUMO-like domains (SLDs) at its C-terminus, an SXS motif and three sequences that have been proposed to be SUMO-binding motifs (SBMs). SMB1 is located in the middle of the protein, SBM2 is in SLD1 and SBM3 is at the C-terminus of SLD2. We have probed the functions of the two SUMO-like domains, SLD1 and SLD2, and the putative SBMs. SLD1 is essential for viability, while SLD2 is not. rad60-SLD2Δ cells are sensitive to DNA damaging agents and hydroxyurea. Neither ubiquitin nor SUMO can replace SLD1 or SLD2. Cells in which either SBM1 or SBM2 has been mutated are viable and are wild type for response to MMS and HU. In contrast mutation of SBM3 results in significant sensitivity to MMS and HU. These results indicate that the lethality resulting from deletion of SLD1 is not due to loss of SBM2, but that mutation of SBM3 produces a more severe phenotype than does deletion of SLD2. Using chemical denaturation studies, FPLC and dynamic light scattering we show this is likely due to the destabilisation of SLD2. Thus we propose that the region corresponding to the putative SBM3 forms part of the hydrophobic core of SLD2 and is not a SUMO-interacting motif. Over-expression of Hus5, which is the SUMO conjugating enzyme and known to interact with Rad60, does not rescue rad60-SLD2Δ, implying that as well as having a role in the sumoylation process as previously described, Rad60 has a Hus5-independent function.

Prostate cancer is significantly more common in Western men than in Asian men, but the basis for this difference remains unknown. Because genomic studies of Asian prostate cancer are very limited, we used a genome-wide approach to reveal the genomic alterations in Chinese prostate cancers. We found a significant reduction in the frequency of certain somatic genomic changes that are commonly found in Western prostate cancers, including the 21q22.2-22.3 deletion, which involves the TMPRSS2:ERG fusion gene, and 10q deletion, which causes PTEN inactivation. Array results were confirmed by PCR-based molecular copy-number counting in selected samples. The different frequencies of these genomic changes were further evaluated by fluorescent in situ hybridization and immunohistochemistry analyses of tissue microarray samples. These alterations might be key genetic changes underlying the regional/ethnic difference in clinical incidence and might be induced by specific environmental and/or genetic risk factors that Western men are exposed to. Our findings suggest that tumors arise in Western and Chinese populations by alternative pathogenetic mechanisms., (Copyright 2010 AACR.)

Background: The development of cancer is accompanied by several genetic alterations. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation found in the human population. SNP arrays offer a high-resolution, high-throughput technology for genome-wide analysis, allowing the simultaneous detection of genotype and copy number changes. The power of SNP arrays as a research tool has accelerated our understanding of the genetic alterations in cancer, providing potential clinical applications., Objective: This manuscript reviews the use of SNPs in cancer research and discusses the potential clinical application of analysing SNPs for cancer predisposition analysis, diagnosis and prognosis. We also discuss potential future applications for the analysis of SNPs., Methods: In writing this review, we have reflected on our own extensive experience in the field of cancer genomics and have surveyed peer-reviewed articles focussing on the application of SNPs in cancer research. In addition, we have referred to product websites., Conclusion: Since its development, SNP array technology has been extensively applied in cancer research. Information generated from SNP array analysis has been providing valuable information. With the full understanding of the rich resources of SNPs and their effects on influencing cellular function, SNP arrays will revolutionise the clinical practice in cancer risk assessment, diagnosis and prognosis making the concept of personalised medicine a reality.

SUMOylation is a post-translational modification that affects a large number of proteins, many of which are nuclear. While the role of SUMOylation is beginning to be elucidated, it is clear that understanding the mechanisms that regulate the process is likely to be important. Control of the levels of SUMOylation is brought about through a balance of conjugating and deconjugating activities, i.e. of SUMO (small ubiquitin-related modifier) conjugators and ligases versus SUMO proteases. Although conjugation of SUMO to proteins can occur in the absence of a SUMO ligase, it is apparent that SUMO ligases facilitate the SUMOylation of specific subsets of proteins. Two SUMO ligases in Schizosaccharomyces pombe, Pli1 and Nse2, have been identified, both of which have roles in genome stability. We report here on a comparison between the properties of the two proteins and discuss potential roles for the proteins.