Introduction of Androgen Receptor Targeting shRNA Inhibits Tumor Growth in Patient-Derived Prostate Cancer Xenografts.

Patient-derived xenograft (PDX) models have been established as important preclinical cancer models, overcoming some of the limitations associated with the use of cancer cell lines. The utility of prostate cancer PDX models has been limited by an inability to genetically manipulate them in vivo and difficulties sustaining PDX-derived cancer cells in culture. Viable, short-term propagation of PDX models would allow in vitro transfection with traceable reporters or manipulation of gene expression relevant to different studies within the prostate cancer field. Here, we report an organoid culture system that supports the growth of prostate cancer PDX cells in vitro and permits genetic manipulation, substantially increasing the scope to use PDXs to study the pathobiology of prostate cancer and define potential therapeutic targets. We have established a short-term PDX-derived in vitro cell culture system which enables genetic manipulation of prostate cancer PDXs LuCaP35 and BM18. Genetically manipulated cells could be re-established as viable xenografts when re-implanted subcutaneously in immunocompromised mice and were able to be serially passaged. Tumor growth of the androgen-dependent LuCaP35 PDX was significantly inhibited following depletion of the androgen receptor (AR) in vivo. Taken together, this system provides a method to generate novel preclinical models to assess the impact of controlled genetic perturbations and allows for targeting specific genes of interest in the complex biological setting of solid tumors.

hSSB1 (NABP2/OBFC2B) modulates the DNA damage and androgen-induced transcriptional response in prostate cancer.

BACKGROUND: Activation and regulation of androgen receptor (AR) signaling and the DNA damage response impact the prostate cancer (PCa) treatment modalities of androgen deprivation therapy (ADT) and radiotherapy. Here, we have evaluated a role for human single-strand binding protein 1 (hSSB1/NABP2) in modulation of the cellular response to androgens and ionizing radiation (IR). hSSB1 has defined roles in transcription and maintenance of genome stability, yet little is known about this protein in PCa. METHODS: We correlated hSSB1 with measures of genomic instability across available PCa cases from The Cancer Genome Atlas (TCGA). Microarray and subsequent pathway and transcription factor enrichment analysis were performed on LNCaP and DU145 prostate cancer cells. RESULTS: Our data demonstrate that hSSB1 expression in PCa correlates with measures of genomic instability including multigene signatures and genomic scars that are reflective of defects in the repair of DNA double-strand breaks via homologous recombination. In response to IR-induced DNA damage, we demonstrate that hSSB1 regulates cellular pathways that control cell cycle progression and the associated checkpoints. In keeping with a role for hSSB1 in transcription, our analysis revealed that hSSB1 negatively modulates p53 and RNA polymerase II transcription in PCa. Of relevance to PCa pathology, our findings highlight a transcriptional role for hSSB1 in regulating the androgen response. We identified that AR function is predicted to be impacted by hSSB1 depletion, whereby this protein is required to modulate AR gene activity in PCa. CONCLUSIONS: Our findings point to a key role for hSSB1 in mediating the cellular response to androgen and DNA damage via modulation of transcription. Exploiting hSSB1 in PCa might yield benefits as a strategy to ensure a durable response to ADT and/or radiotherapy and improved patient outcomes.

Computationally efficient framework for diagnosing, understanding and predicting biphasic population growth.

Throughout the life sciences, biological populations undergo multiple phases of growth, often referred to as biphasic growth for the commonly encountered situation involving two phases. Biphasic population growth occurs over a massive range of spatial and temporal scales, ranging from microscopic growth of tumours over several days, to decades-long regrowth of corals in coral reefs that can extend for hundreds of kilometres. Different mathematical models and statistical methods are used to diagnose, understand and predict biphasic growth. Common approaches can lead to inaccurate predictions of future growth that may result in inappropriate management and intervention strategies being implemented. Here, we develop a very general computationally efficient framework, based on profile likelihood analysis, for diagnosing, understanding and predicting biphasic population growth. The two key components of the framework are as follows: (i) an efficient method to form approximate confidence intervals for the change point of the growth dynamics and model parameters and (ii) parameter-wise profile predictions that systematically reveal the influence of individual model parameters on predictions. To illustrate our framework we explore real-world case studies across the life sciences.

Modelling the tumor immune microenvironment for precision immunotherapy.

The complexity of the cellular and acellular players within the tumor microenvironment (TME) allows for significant variation in TME constitution and role in anticancer treatment response. Spatial alterations in populations of tumor cells and adjacent non-malignant cells, including endothelial cells, fibroblasts and tissue-infiltrating immune cells, often have a major role in determining disease progression and treatment response in cancer. Many current standard systemic antineoplastic treatments target the cancer cells and could be further refined to directly target commonly dysregulated cell populations of the TME. Recent developments in immuno-oncology and bioengineering have created an attractive potential to model these complexities at the level of the individual patient. These developments, along with the increasing momentum in precision medicine research and application, have catalysed exciting new discoveries in understanding drug-TME interactions, target identification, and improved efficacy of therapies. While rapid progress has been made, there are still many challenges to overcome in the development of accurate in vitro, in vivo and ex vivo models incorporating the cellular interactions that take place in the TME. In this review, we describe how advances in immuno-oncology and patient-derived models, such as patient-derived organoids and explant cultures, have enhanced the landscape of personalised immunotherapy prediction and treatment of solid organ malignancies. We describe and compare different immunological targets and perspectives on two-dimensional and three-dimensional modelling approaches that may be used to better rationalise immunotherapy use, ultimately providing a knowledge base for the integration of the autologous TME into these predictive models.

Chimeric Antigen Receptor T-Cell Therapy in Metastatic Castrate-Resistant Prostate Cancer.

Prostate cancer is the most commonly diagnosed solid-organ cancer amongst males worldwide. Metastatic castrate-resistant prostate cancer (mCRPC) is a rapidly fatal end-sequelae of prostate cancer. Therapeutic options for men with mCRPC are limited and are not curative in nature. The recent development of chimeric antigen receptor T-cell (CAR-T) therapy has revolutionised the treatment of treatment-resistant haematological malignancies, and several studies are underway investigating the utility of this technology in the treatment of solid tumours. In this review, we evaluate the current treatment options for men with mCRPC as well as the current landscape of preclinical and clinical trials of CAR-T cell therapy against prostate cancer. We also appraise the various prostate cancer-specific tumour-associated antigens that may be targeted by CAR-T cell technology. Finally, we examine the potential translational barriers of CAR-T cell therapy in solid tumours. Despite preclinical success, preliminary clinical trials in men with prostate cancer have had limited efficacy. Therefore, further clinically translatable preclinical models are required to enhance the understanding of the role of this investigational therapeutic in men with mCRPC. In the era of precision medicine, tailored immunotherapy administered to men in a tumour-agnostic approach provides hope to a group of men who otherwise have few treatment options available.

A chromosome-level genome of Antechinus flavipes provides a reference for an Australian marsupial genus with male death after mating.

The 15 species of small carnivorous marsupials that comprise the genus Antechinus exhibit semelparity, a rare life-history strategy in mammals where synchronized death occurs after one breeding season. Antechinus males, but not females, age rapidly (demonstrate organismal senescence) during the breeding season and show promise as new animal models of ageing. Some antechinus species are also threatened or endangered. Here, we report a chromosome-level genome of a male yellow-footed antechinus Antechinus flavipes. The genome assembly has a total length of 3.2 Gb with a contig N50 of 51.8 Mb and a scaffold N50 of 636.7 Mb. We anchored and oriented 99.7% of the assembly on seven pseudochromosomes and found that repetitive DNA sequences occupy 51.8% of the genome. Draft genome assemblies of three related species in the subfamily Phascogalinae, two additional antechinus species (Antechinus argentus and A. arktos) and the iteroparous sister species Murexia melanurus, were also generated. Preliminary demographic analysis supports the hypothesis that climate change during the Pleistocene isolated species in Phascogalinae and shaped their population size. A transcriptomic profile across the A. flavipes breeding season allowed us to identify genes associated with aspects of the male die-off. The chromosome-level A. flavipes genome provides a steppingstone to understanding an enigmatic life-history strategy and a resource to assist the conservation of antechinuses.

The long non-coding RNA GHSROS reprograms prostate cancer cell lines toward a more aggressive phenotype.

It is now appreciated that long non-coding RNAs (lncRNAs) are important players in orchestrating cancer progression. In this study we characterized GHSROS, a human lncRNA gene on the opposite DNA strand (antisense) to the ghrelin receptor gene, in prostate cancer. The lncRNA was upregulated by prostate tumors from different clinical datasets. Transcriptome data revealed that GHSROS alters the expression of cancer-associated genes. Functional analyses in vitro showed that GHSROS mediates tumor growth, migration and survival, and resistance to the cytotoxic drug docetaxel. Increased cellular proliferation of GHSROS-overexpressing PC3, DU145, and LNCaP prostate cancer cell lines in vitro was recapitulated in a subcutaneous xenograft model. Conversely, in vitro antisense oligonucleotide inhibition of the lncRNA reciprocally regulated cell growth and migration, and gene expression. Notably, GHSROS modulates the expression of PPP2R2C, the loss of which may drive androgen receptor pathway-independent prostate tumor progression in a subset of prostate cancers. Collectively, our findings suggest that GHSROS can reprogram prostate cancer cells toward a more aggressive phenotype and that this lncRNA may represent a potential therapeutic target.

Patient-Derived Explants as a Precision Medicine Patient-Proximal Testing Platform Informing Cancer Management.

Precision medicine approaches that inform clinical management of individuals with cancer are progressively advancing. Patient-derived explants (PDEs) provide a patient-proximal ex vivo platform that can be used to assess sensitivity to standard of care (SOC) therapies and novel agents. PDEs have several advantages as a patient-proximal model compared to current preclinical models, as they maintain the phenotype and microenvironment of the individual tumor. However, the longevity of PDEs is not compatible with the timeframe required to incorporate candidate therapeutic options identified by whole exome sequencing (WES) of the patient's tumor. This review investigates how PDE longevity varies across tumor streams and how this is influenced by tissue preparation. Improving longevity of PDEs will enable individualized therapeutics testing, and thus contribute to improving outcomes for people with cancer.

Culture of Bladder Cancer Organoids as Precision Medicine Tools.

Current in vitro therapeutic testing platforms lack relevance to tumor pathophysiology, typically employing cancer cell lines established as two-dimensional (2D) cultures on tissue culture plastic. There is a critical need for more representative models of tumor complexity that can accurately predict therapeutic response and sensitivity. The development of three-dimensional (3D) ex vivo culture of patient-derived organoids (PDOs), derived from fresh tumor tissues, aims to address these shortcomings. Organoid cultures can be used as tumor surrogates in parallel to routine clinical management to inform therapeutic decisions by identifying potential effective interventions and indicating therapies that may be futile. Here, this procedure aims to describe strategies and a detailed step-by-step protocol to establish bladder cancer PDOs from fresh, viable clinical tissue. Our well-established, optimized protocols are practical to set up 3D cultures for experiments using limited and diverse starting material directly from patients or patient-derived xenograft (PDX) tumor material. This procedure can also be employed by most laboratories equipped with standard tissue culture equipment. The organoids generated using this protocol can be used as ex vivo surrogates to understand both the molecular mechanisms underpinning urological cancer pathology and to evaluate treatments to inform clinical management.

The mitochondrial genome of the black-tailed dusky antechinus (Antechinus arktos).

In this study, we report the mitochondrial genome of the black-tailed antechinus (Antechinus arktos), a recently-discovered, endangered carnivorous marsupial inhabiting a caldera that straddles the border of Australia's mid-east coast. The circular A. arktos genome is 17,334 bp in length and has an AT content of 63.3%. Its gene content and arrangement are consistent with reported marsupial mitogenome assemblies.

The long non-coding RNA GHSROS facilitates breast cancer cell migration and orthotopic xenograft tumour growth.

Recent evidence suggests that numerous long non­coding RNAs (lncRNAs) are dysregulated in cancer, and have critical roles in tumour development and progression. The present study investigated the ghrelin receptor antisense lncRNA growth hormone secretagogue receptor opposite strand (GHSROS) in breast cancer. Reverse transcription­quantitative polymerase chain reaction revealed that GHSROS expression was significantly upregulated in breast tumour tissues compared with normal breast tissue. Induced overexpression of GHSROS in the MDA­MB­231 breast cancer cell line significantly increased cell migration in vitro, without affecting cell proliferation, a finding similar to our previous study on lung cancer cell lines. Microarray analysis revealed a significant repression of a small cluster of major histocompatibility class II genes and enrichment of immune response pathways; this phenomenon may allow tumour cells to better evade the immune system. Ectopic overexpression of GHSROS in the MDA­MB­231 cell line significantly increased orthotopic xenograft growth in mice, suggesting that in vitro culture does not fully capture the function of this lncRNA. This study demonstrated that GHSROS may serve a relevant role in breast cancer. Further studies are warranted to explore the function and therapeutic potential of this lncRNA in breast cancer progression.

The mitochondrial genome of the black-tailed dasyure (Murexia melanurus).

In this study, we report the mitochondrial genome of the black-tailed dasyure (Murexia melanurus) of New Guinea. The circular genome is 17,736 bp in length and has an AT content of 60.5%. Its gene content - 13 protein-coding genes (PCGs), 2 ribosomal (rRNA) genes, 21 transfer RNA (tRNA) genes, a tRNA pseudogene (tRNALys ), and a non-coding control region (CR) - and gene arrangement are consistent with previous marsupial mitogenome assemblies.

Limited short-term effects on human prostate cancer xenograft growth and epidermal growth factor receptor gene expression by the ghrelin receptor antagonist [D-Lys3]-GHRP-6.

PURPOSE: The ghrelin axis regulates many physiological functions (including appetite, metabolism, and energy balance) and plays a role in disease processes. As ghrelin stimulates prostate cancer proliferation, the ghrelin receptor antagonist [D-Lys3]-GHRP-6 is a potential treatment for castrate-resistant prostate cancer and for preventing the metabolic consequences of androgen-targeted therapies. We therefore explored the effect of [D-Lys3]-GHRP-6 on PC3 prostate cancer xenograft growth. METHODS: NOD/SCID mice with PC3 prostate cancer xenografts were administered 20 nmoles/mouse [D-Lys3]-GHRP-6 daily by intraperitoneal injection for 14 days and tumour volume and weight were measured. RNA sequencing of tumours was conducted to investigate expression changes following [D-Lys3]-GHRP-6 treatment. A second experiment, extending treatment time to 18 days and including a higher dose of [D-Lys3]-GHRP-6 (200 nmoles/mouse/day), was undertaken to ensure repeatability. RESULTS: We demonstrate here that daily intraperitoneal injection of 20 nmoles/mouse [D-Lys3]-GHRP-6 reduces PC3 prostate cancer xenograft tumour volume and weight in NOD/SCID mice at two weeks post treatment initiation. RNA-sequencing revealed reduced expression of epidermal growth factor receptor (EGFR) in these tumours. Further experiments demonstrated that the effects of [D-Lys3]-GHRP-6 are transitory and lost after 18 days of treatment. CONCLUSIONS: We show that [D-Lys3]-GHRP-6 has transitory effects on prostate xenograft tumours in mice, which rapidly develop an apparent resistance to the antagonist. Although further studies on [D-Lys3]-GHRP-6 are warranted, we suggest that daily treatment with the antagonist is not a suitable treatment for advanced prostate cancer.

No effect of unacylated ghrelin administration on subcutaneous PC3 xenograft growth or metabolic parameters in a Rag1-/- mouse model of metabolic dysfunction.

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1-/- mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 µg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1-/- mice on a high-fat diet, but did not significantly improve other metabolic parameters. We propose that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome.

Whole-Genome Sequence of the Metastatic PC3 and LNCaP Human Prostate Cancer Cell Lines.

The bone metastasis-derived PC3 and the lymph node metastasis-derived LNCaP prostate cancer cell lines are widely studied, having been described in thousands of publications over the last four decades. Here, we report short-read whole-genome sequencing (WGS) and de novo assembly of PC3 (ATCC CRL-1435) and LNCaP (clone FGC; ATCC CRL-1740) at ∼70 × coverage. A known homozygous mutation in TP53 and homozygous loss of PTEN were robustly identified in the PC3 cell line, whereas the LNCaP cell line exhibited a larger number of putative inactivating somatic point and indel mutations (and in particular a loss of stop codon events). This study also provides preliminary evidence that loss of one or both copies of the tumor suppressor Capicua (CIC) contributes to primary tumor relapse and metastatic progression, potentially offering a treatment target for castration-resistant prostate cancer (CRPC). Our work provides a resource for genetic, genomic, and biological studies employing two commonly-used prostate cancer cell lines.

Insights from engraftable immunodeficient mouse models of hyperinsulinaemia.

Hyperinsulinaemia, obesity and dyslipidaemia are independent and collective risk factors for many cancers. Here, the long-term effects of a 23% Western high-fat diet (HFD) in two immunodeficient mouse strains (NOD/SCID and Rag1 -/-) suitable for engraftment with human-derived tissue xenografts, and the effect of diet-induced hyperinsulinaemia on human prostate cancer cell line xenograft growth, were investigated. Rag1 -/-and NOD/SCID HFD-fed mice demonstrated diet-induced impairments in glucose tolerance at 16 and 23 weeks post weaning. Rag1 -/- mice developed significantly higher fasting insulin levels (2.16 ± 1.01 ng/ml, P = 0.01) and increased insulin resistance (6.70 ± 1.68 HOMA-IR, P = 0.01) compared to low-fat chow-fed mice (0.71 ± 0.12 ng/ml and 2.91 ± 0.42 HOMA-IR). This was not observed in the NOD/SCID strain. Hepatic steatosis was more extensive in Rag1 -/- HFD-fed mice compared to NOD/SCID mice. Intramyocellular lipid storage was increased in Rag1 -/- HFD-fed mice, but not in NOD/SCID mice. In Rag1 -/- HFD-fed mice, LNCaP xenograft tumours grew more rapidly compared to low-fat chow-fed mice. This is the first characterisation of the metabolic effects of long-term Western HFD in two mouse strains suitable for xenograft studies. We conclude that Rag1 -/- mice are an appropriate and novel xenograft model for studying the relationship between cancer and hyperinsulinaemia.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide.

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

Identification of a long non-coding RNA gene, growth hormone secretagogue receptor opposite strand, which stimulates cell migration in non-small cell lung cancer cell lines.

The molecular mechanisms involved in non­small cell lung cancer tumourigenesis are largely unknown; however, recent studies have suggested that long non-coding RNAs (lncRNAs) are likely to play a role. In this study, we used public databases to identify an mRNA-like, candidate long non-coding RNA, GHSROS (GHSR opposite strand), transcribed from the antisense strand of the ghrelin receptor gene, growth hormone secretagogue receptor (GHSR). Quantitative real-time RT-PCR revealed higher expression of GHSROS in lung cancer tissue compared to adjacent, non-tumour lung tissue. In common with many long non-coding RNAs, GHSROS is 5' capped and 3' polyadenylated (mRNA-like), lacks an extensive open reading frame and harbours a transposable element. Engineered overexpression of GHSROS stimulated cell migration in the A549 and NCI-H1299 non-small cell lung cancer cell lines, but suppressed cell migration in the Beas-2B normal lung-derived bronchoepithelial cell line. This suggests that GHSROS function may be dependent on the oncogenic context. The identification of GHSROS, which is expressed in lung cancer and stimulates cell migration in lung cancer cell lines, contributes to the growing number of non-coding RNAs that play a role in the regulation of tumourigenesis and metastatic cancer progression.

Laparoscopic nephrectomy for renovascular hypertension in a 6-month-old infant.

Laparoscopic procedures continue to gain popularity over traditional open operations for a variety of abdominal and retroperitoneal surgical procedures. With regard to urological surgery, the first laparoscopic nephrectomy was performed in an adult in 1991. In the following years, the feasibility of laparoscopic management of pediatric urological disorders was described, and in 1992 the first laparoscopic nephrectomy in an 8-month-old infant with a multicystic dysplastic kidney was reported. We report the feasibility of laparoscopic nephrectomy for the management of renovascular hypertension in a 6-month-old infant with a dysplastic left kidney.

Serum amylase and lipase alone are not cost-effective screening methods for pediatric pancreatic trauma.

BACKGROUND: Injury to the pancreas is rare in pediatric trauma. Identification of pancreatic injury relies on clinical, radiographic, and laboratory data. Serum screening for pancreatic injury frequently is used but has not proven to correlate well with pancreatic injury. This study investigated utility and cost effectiveness of serum assessment of amylase and lipase. METHODS: A retrospective study of 1,821 pediatric trauma patients over 64 months was conducted. A total of 293 (16%) of these patients suffered trauma to the torso 195 (11%) of whom had confirmed intraabdominal injury. Eight pancreatic injuries (4% of abdominal injuries) were identified; 5 underwent surgery for pancreatic ductal injury. One patient not operated on had a pseudocyst that required late drainage. RESULTS: Serum amylase or lipase levels (AMY/LIP) were measured in 507 (28%) patients. A total of 116 (23%) had elevated AMY/LIP levels. Six of 8 with proven pancreatic injury underwent AMY/LIP testing; 5 had elevated values. Forty-eight percent of patients with elevated AMY/LIP levels had no evidence of intraabdominal injury. Seventy-four of 116 (64%) with elevated AMY/LIP levels underwent abdominal and pelvic computed tomography (CT) scanning, yet 38 (51%) of these had completely normal scans. Many patients with elevated AMY/LIP levels (cost, $6 per test) underwent screening CT scans (cost, $592 per test) based on AMY/LIP alone. No patient with elevated AMY/LIP levels but without clinical suspicion was proven to have pancreatic injury. Cost data are presented. CONCLUSIONS: Serum amylase and lipase determinations may support clinical suspicion in the diagnosis of pediatric pancreatic trauma but are not reliable or cost effective as screening tools. Costs incurred from routine serum amylase and lipase or from imaging tests subsequent to elevated serum values may be significant and unjustified.