MBTPS2 acts as a regulator of lipogenesis and cholesterol synthesis through SREBP signalling in prostate cancer.

BACKGROUND: Prostate cancer is the most common cancer in men in the developed world, with most deaths caused by advanced and metastatic disease which has no curative options. Here, we identified Mbtps2 alteration to be associated with metastatic disease in an unbiased in vivo screen and demonstrated its regulation of fatty acid and cholesterol metabolism. METHODS: The Sleeping Beauty transposon system was used to randomly alter gene expression in the PtenNull murine prostate. MBTPS2 was knocked down by siRNA in LNCaP, DU145 and PC3 cell lines, which were then phenotypically investigated. RNA-Seq was performed on LNCaP cells lacking MBTPS2, and pathways validated by qPCR. Cholesterol metabolism was investigated by Filipin III staining. RESULTS: Mbtps2 was identified in our transposon-mediated in vivo screen to be associated with metastatic prostate cancer. Silencing of MBTPS2 expression in LNCaP, DU145 and PC3 human prostate cancer cells reduced proliferation and colony forming growth in vitro. Knockdown of MBTPS2 expression in LNCaP cells impaired cholesterol synthesis and uptake along with reduced expression of key regulators of fatty acid synthesis, namely FASN and ACACA. CONCLUSION: MBTPS2 is implicated in progressive prostate cancer and may mechanistically involve its effects on fatty acid and cholesterol metabolism.

Phase II proof-of-concept study of atorvastatin in castration-resistant prostate cancer.

OBJECTIVES: To test for evidence of statin-mediated effects in patients with castration-resistant prostate cancer (CRPC) as post-diagnosis use of statins in patients with prostate cancer is associated with favourable survival outcome. PATIENTS AND METHODS: The SPECTRE trial was a 6-weeks-long proof-of-concept single-arm Phase II treatment trial, combining atorvastatin and androgen deprivation therapy in patients with CRPC (regardless of metastatic status), designed to test for evidence of statin-mediated effects in patients with CRPC. The primary study endpoint was the proportion of patients achieving a ≥50% drop from baseline in prostate-specific antigen (PSA) levels at any time over the 6-week period of atorvastatin medication (PSA response). Exploratory endpoints include PSA velocity and serum metabolites identified by mass spectrometry . RESULTS: At the scheduled interim analysis, one of 12 patients experienced a ≥50% drop in PSA levels (primary endpoint), with ≥2 patients satisfying the primary endpoint required for further recruitment. All 12 patients experienced substantial falls in serum cholesterol levels following statin treatment. While all patients had comparable pre-study PSA velocities, six of 12 patients showed decreased PSA velocities after statin treatment, suggestive of disease stabilization. Unbiased metabolomics analysis on serial weekly blood samples identified tryptophan to be the dominant metabolite associated with patient response to statin. CONCLUSIONS: Data from the SPECTRE study provide the first evidence of statin-mediated effects on CRPC and early sign of disease stabilization. Our data also highlight the possibility of altered tryptophan metabolism being associated with tumour response.

Sleeping Beauty screen reveals Pparg activation in metastatic prostate cancer.

Prostate cancer (CaP) is the most common adult male cancer in the developed world. The paucity of biomarkers to predict prostate tumor biology makes it important to identify key pathways that confer poor prognosis and guide potential targeted therapy. Using a murine forward mutagenesis screen in a Pten-null background, we identified peroxisome proliferator-activated receptor gamma (Pparg), encoding a ligand-activated transcription factor, as a promoter of metastatic CaP through activation of lipid signaling pathways, including up-regulation of lipid synthesis enzymes [fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), ATP citrate lyase (ACLY)]. Importantly, inhibition of PPARG suppressed tumor growth in vivo, with down-regulation of the lipid synthesis program. We show that elevated levels of PPARG strongly correlate with elevation of FASN in human CaP and that high levels of PPARG/FASN and PI3K/pAKT pathway activation confer a poor prognosis. These data suggest that CaP patients could be stratified in terms of PPARG/FASN and PTEN levels to identify patients with aggressive CaP who may respond favorably to PPARG/FASN inhibition.

Clustering of Toxoplasma gondii Infections Within Families of Congenitally Infected Infants.

BACKGROUND: Family clusters and epidemics of toxoplasmosis in North, Central, and South America led us to determine whether fathers of congenitally infected infants in the National Collaborative Chicago-Based Congenital Toxoplasmosis Study (NCCCTS) have a high incidence of Toxoplasma gondii infection. METHODS: We analyzed serum samples collected from NCCCTS families between 1981 and 2013. Paternal serum samples were tested for T. gondii antibodies with immunoglobulin (Ig) G dye test and IgM enzyme-linked immunosorbent assay. Additional testing of paternal serum samples was performed with differential-agglutination and IgG avidity tests when T. gondii IgG and IgM results were positive and serum samples were collected by the 1-year visit of the congenitally infected child. Prevalence of paternal seropositivity and incidence of recent infection were calculated. We analyzed whether certain demographics, maternal parasite serotype, risk factors, or maternal/infant clinical manifestations were associated with paternal T. gondii infection status. RESULTS: Serologic testing revealed a high prevalence (29 of 81; 36%) of T. gondii infection in fathers, relative to the average seropositivity rate of 9.8% for boys and men aged 12-49 years in the United States between 1994 and 2004 (P < .001). Moreover, there was a higher-than-expected incidence of recent infections among fathers with serum samples collected by the 1-year visit of their child (6 of 45; 13%; P < .001). No demographic patterns or clinical manifestations in mothers or infants were associated with paternal infections, except for sandbox exposure. CONCLUSIONS: The high prevalence of chronic and incidence of recent T. gondii infections in fathers of congenitally infected children indicates that T. gondii infections cluster within families in North America. When a recently infected person is identified, family clustering and community risk factors should be investigated for appropriate clinical management.

Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections.

Toxoplasma gondii persistently infects over two billion people worldwide. It can cause substantial morbidity and mortality. Existing treatments have associated toxicities and hypersensitivity and do not eliminate encysted bradyzoites that recrudesce. New, improved medicines are needed. Transductive peptides carry small molecule cargos across multiple membranes to enter intracellular tachyzoites and encysted bradyzoites. They also carry cargos into retina when applied topically to eyes, and cross blood brain barrier when administered intravenously. Phosphorodiamidate morpholino oligomers (PMO) inhibit gene expression in a sequence-specific manner. Herein, effect of transductive peptide conjugated PMO (PPMO) on tachyzoite protein expression and replication in vitro and in vivo was studied. Initially, sequence-specific PPMO successfully reduced transfected T. gondii's fluorescence and luminescence. PPMO directed against T. gondii's dihydrofolate reductase (DHFR), an enzyme necessary for folate synthesis, limited tachyzoite replication. Rescue with exogenous folate demonstrated DHFR PPMO's specificity. PPMO directed against enoyl-ACP reductase (ENR), an enzyme of type II fatty acid synthesis that is structurally distinct in T. gondii from ENR in mammalian cells was investigated. PPMO directed against plant-like Apetela 2 (AP2) domain transcription factor XI-3 (AP2XI-3), not present in human cells, was characterized. ENR and AP2XI-3 PPMO each restricted intracellular parasite replication validating these molecular targets in tachyzoites. DHFR-specific PPMO administered to infected mice diminished parasite burden. Thus, these antisense oligomers are a versatile approach to validate T. gondii molecular targets, reduce essential T. gondii proteins in vitro and in vivo, and have potential for development as curative medicines.

ALOX12 in human toxoplasmosis.

ALOX12 is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases. ALOX12 catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of ALOX12 are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined. Toxoplasma gondii is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as Toxo1 is critical for susceptibility or resistance to T. gondii infection in rats. An orthologous gene region with ALOX12 centromeric is also present in humans. Here we report that the human ALOX12 gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [P, <0.000309], rs312462 [P, <0.028499], rs6502998 [P, <0.029794], and rs434473 [P, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down ALOX12. In ALOX12 knockdown cells, ALOX12 RNA expression decreased and levels of the ALOX12 substrate, arachidonic acid, increased. ALOX12 knockdown attenuated the progression of T. gondii infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that ALOX12 influences host responses to T. gondii infection in human cells. ALOX12 has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role ALOX12 plays in T. gondii infection in humans.

Spiroindolone that inhibits PfATPase4 is a potent, cidal inhibitor of Toxoplasma gondii tachyzoites in vitro and in vivo.

Here, we show that spiroindolone, an effective treatment for plasmodia, is also active against Toxoplasma gondii tachyzoites. In vitro, spiroindolone NITD609 is cidal for tachyzoites (50% inhibitory concentration [IC50], 1µM) and not toxic to human cells at ≥10µM. Two daily oral doses of 100 mg/kg of body weight reduced the parasite burden in mice by 90% (P=0.002), measured 3 days after the last dose. This inhibition of T. gondii tachyzoites in vitro and in vivo indicates that spiroindolone is a promising lead candidate for further medicine development.

Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

Impaired innate immunity in mice deficient in interleukin-1 receptor-associated kinase 4 leads to defective type 1 T cell responses, B cell expansion, and enhanced susceptibility to infection with Toxoplasma gondii.

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a member of the IRAK family and has an important role in inducing the production of inflammatory mediators. This kinase is downstream of MyD88, an adaptor protein essential for Toll-like receptor (TLR) function. We investigated the role of this kinase in IRAK4-deficient mice orally infected with the cystogenic ME49 strain of Toxoplasma gondii. IRAK4(-/-) mice displayed higher morbidity, tissue parasitism, and accelerated mortality than the control mice. The lymphoid follicles and germinal centers from infected IRAK4(-/-) mice were significantly smaller. We consistently found that IRAK4(-/-) mice showed a defect in splenic B cell activation and expansion as well as diminished production of gamma interferon (IFN-γ) by T lymphocytes. The myeloid compartment was also affected. Both the frequency and ability of dendritic cells (DCs) and monocytes/macrophages to produce IL-12 were significantly decreased, and resistance to infection with Toxoplasma was rescued by treating IRAK4(-/-) mice with recombinant IL-12 (rIL-12). Additionally, we report the association of IRAK4 haplotype-tagging single nucleotide polymorphisms (tag-SNPs) with congenital toxoplasmosis in infected individuals (rs1461567 and rs4251513, P < 0.023 and P < 0.045, respectively). Thus, signaling via IRAK4 is essential for the activation of innate immune cells, development of parasite-specific acquired immunity, and host resistance to infection with T. gondii.

Prematurity and severity are associated with Toxoplasma gondii alleles (NCCCTS, 1981-2009).

BACKGROUND: Congenital toxoplasmosis is a severe, life-altering disease in the United States. A recently developed enzyme-linked immunosorbent assay (ELISA) distinguishes Toxoplasma gondii parasite types (II and not exclusively II [NE-II]) by detecting antibodies in human sera that recognize allelic peptide motifs of distinct parasite types. METHODS: ELISA determined parasite serotype for 193 congenitally infected infants and their mothers in the National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS), 1981-2009. Associations of parasite serotype with demographics, manifestations at birth, and effects of treatment were determined. RESULTS: Serotypes II and NE-II occurred in the United States with similar proportions during 3 decades. For persons diagnosed before or at birth and treated in infancy, and persons diagnosed after 1 year of age who missed treatment in infancy, proportions were similar (P = .91). NE-II serotype was more common in hot, humid regions (P = .02) but was also present in other regions. NE-II serotype was associated with rural residence (P < .01), lower socioeconomic status (P < .001), and Hispanic ethnicity (P < .001). Prematurity (P = .03) and severe disease at birth (P < .01) were associated with NE-II serotype. Treatment with lower and higher doses of pyrimethamine with sulfadizine improved outcomes relative to those outcomes of persons in the literature who did not receive such treatment. CONCLUSIONS: Type II and NE-II parasites cause congenital toxoplasmosis in North America. NE-II serotype was more prevalent in certain demographics and associated with prematurity and severe disease at birth. Both type II and NE-II infections improved with treatment. CLINICAL TRIALS REGISTRATION: NCT00004317.

Novel N-benzoyl-2-hydroxybenzamide disrupts unique parasite secretory pathway.

Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3ß, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.

P2X7 receptor-mediated killing of an intracellular parasite, Toxoplasma gondii, by human and murine macrophages.

The P2X7R is highly expressed on the macrophage cell surface, and activation of infected cells by extracellular ATP has been shown to kill intracellular bacteria and parasites. Furthermore, single nucleotide polymorphisms that decrease receptor function reduce the ability of human macrophages to kill Mycobacterium tuberculosis and are associated with extrapulmonary tuberculosis. In this study, we show that macrophages from people with the 1513C (rs3751143, NM_002562.4:c.1487A>C) loss-of-function P2X7R single nucleotide polymorphism are less effective in killing intracellular Toxoplasma gondii after exposure to ATP compared with macrophages from people with the 1513A wild-type allele. Supporting a P2X7R-specific effect on T. gondii, macrophages from P2X7R knockout mice (P2X7R-/-) are unable to kill T. gondii as effectively as macrophages from wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of NO production.

Cyclocreatine Suppresses Creatine Metabolism and Impairs Prostate Cancer Progression.

Prostate cancer is the second most common cause of cancer mortality in men worldwide. Applying a novel genetically engineered mouse model (GEMM) of aggressive prostate cancer driven by deficiency of the tumor suppressors PTEN and Sprouty2 (SPRY2), we identified enhanced creatine metabolism as a central component of progressive disease. Creatine treatment was associated with enhanced cellular basal respiration in vitro and increased tumor cell proliferation in vivo. Stable isotope tracing revealed that intracellular levels of creatine in prostate cancer cells are predominantly dictated by exogenous availability rather than by de novo synthesis from arginine. Genetic silencing of creatine transporter SLC6A8 depleted intracellular creatine levels and reduced the colony-forming capacity of human prostate cancer cells. Accordingly, in vitro treatment of prostate cancer cells with cyclocreatine, a creatine analog, dramatically reduced intracellular levels of creatine and its derivatives phosphocreatine and creatinine and suppressed proliferation. Supplementation with cyclocreatine impaired cancer progression in the PTEN- and SPRY2-deficient prostate cancer GEMMs and in a xenograft liver metastasis model. Collectively, these results identify a metabolic vulnerability in prostate cancer and demonstrate a rational therapeutic strategy to exploit this vulnerability to impede tumor progression. SIGNIFICANCE: Enhanced creatine uptake drives prostate cancer progression and confers a metabolic vulnerability to treatment with the creatine analog cyclocreatine.

THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer.

Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane-associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC.

NALP1 influences susceptibility to human congenital toxoplasmosis, proinflammatory cytokine response, and fate of Toxoplasma gondii-infected monocytic cells.

NALP1 is a member of the NOD-like receptor (NLR) family of proteins that form inflammasomes. Upon cellular infection or stress, inflammasomes are activated, triggering maturation of proinflammatory cytokines and downstream cellular signaling mediated through the MyD88 adaptor. Toxoplasma gondii is an obligate intracellular parasite that stimulates production of high levels of proinflammatory cytokines that are important in innate immunity. In this study, susceptibility alleles for human congenital toxoplasmosis were identified in the NALP1 gene. To investigate the role of the NALP1 inflammasome during infection with T. gondii, we genetically engineered a human monocytic cell line for NALP1 gene knockdown by RNA interference. NALP1 silencing attenuated progression of T. gondii infection, with accelerated host cell death and eventual cell disintegration. In line with this observation, upregulation of the proinflammatory cytokines interleukin-1ß (IL-1ß), IL-18, and IL-12 upon T. gondii infection was not observed in monocytic cells with NALP1 knockdown. These findings suggest that the NALP1 inflammasome is critical for mediating innate immune responses to T. gondii infection and pathogenesis. Although there have been recent advances in understanding the potent activity of inflammasomes in directing innate immune responses to disease, this is the first report, to our knowledge, on the crucial role of the NALP1 inflammasome in the pathogenesis of T. gondii infections in humans.

Unrecognized ingestion of Toxoplasma gondii oocysts leads to congenital toxoplasmosis and causes epidemics in North America.

BACKGROUND: Congenital toxoplasmosis presents as severe, life-altering disease in North America. If mothers of infants with congenital toxoplasmosis could be identified by risks, it would provide strong support for educating pregnant women about risks, to eliminate this disease. Conversely, if not all risks are identifiable, undetectable risks are suggested. A new test detecting antibodies to sporozoites demonstrated that oocysts were the predominant source of Toxoplasma gondii infection in 4 North American epidemics and in mothers of children in the National Collaborative Chicago-based Congenital Toxoplasmosis Study (NCCCTS). This novel test offered the opportunity to determine whether risk factors or demographic characteristics could identify mothers infected with oocysts. METHODS: Acutely infected mothers and their congenitally infected infants were evaluated, including in-person interviews concerning risks and evaluation of perinatal maternal serum samples. RESULTS: Fifty-nine (78%) of 76 mothers of congenitally infected infants in NCCCTS had primary infection with oocysts. Only 49% of these mothers identified significant risk factors for sporozoite acquisition. Socioeconomic status, hometown size, maternal clinical presentations, and ethnicity were not reliable predictors. CONCLUSIONS: Undetected contamination of food and water by oocysts frequently causes human infections in North America. Risks are often unrecognized by those infected. Demographic characteristics did not identify oocyst infections. Thus, although education programs describing hygienic measures may be beneficial, they will not suffice to prevent the suffering and economic consequences associated with congenital toxoplasmosis. Only a vaccine or implementation of systematic serologic testing of pregnant women and newborns, followed by treatment, will prevent most congenital toxoplasmosis in North America.

Rapid discovery of inhibitors of Toxoplasma gondii using hybrid structure-based computational approach.

Toxoplasma (T.) gondii, the causative agent of toxoplasmosis, is a ubiquitous opportunistic pathogen that infects individuals worldwide, and is a leading cause of severe congenital neurologic and ocular disease in humans. No vaccine to protect humans is available, and hypersensitivity and toxicity limit the use of the few available medicines. Therefore, safer and more effective medicines to treat toxoplasmosis are urgently needed. Using the Hybrid Structure Based (HSB) method, we have previously identified small molecule inhibitors of P. falciparum that seem to target a novel protein-protein interaction between the Myosin tail interacting protein and myosin light chain. This pathway has been hypothesized to be involved in invasion of host erythrocytes by the parasite and is broadly conserved among the apicomplexans. Guided by similar computational drug design approaches, we investigated this series of small molecules as potential inhibitors of T. gondii. Compound C3-21, identified as the most active inhibitor in this series, exhibited an IC(50) value ~500 nM against T. gondii. Among the 16 structural analogs of C3-21 tested thus far, nine additional compounds were identified with IC(50) values <10.0 µM. In vitro assays have revealed that C3-21 markedly limits intracellular growth of T. gondii tachyzoites, but has no effect on host cell human foreskin fibroblasts (HFF) at concentrations more than a log greater than the concentration that inhibits the parasites.

PPAR-gamma induced AKT3 expression increases levels of mitochondrial biogenesis driving prostate cancer.

Peroxisome Proliferator-Activated Receptor Gamma (PPARG) is one of the three members of the PPAR family of transcription factors. Besides its roles in adipocyte differentiation and lipid metabolism, we recently demonstrated an association between PPARG and metastasis in prostate cancer. In this study a functional effect of PPARG on AKT serine/threonine kinase 3 (AKT3), which ultimately results in a more aggressive disease phenotype was identified. AKT3 has previously been shown to regulate PPARG co-activator 1 alpha (PGC1α) localisation and function through its action on chromosome maintenance region 1 (CRM1). AKT3 promotes PGC1α localisation to the nucleus through its inhibitory effects on CRM1, a known nuclear export protein. Collectively our results demonstrate how PPARG over-expression drives an increase in AKT3 levels, which in turn has the downstream effect of increasing PGC1α localisation within the nucleus, driving mitochondrial biogenesis. Furthermore, this increase in mitochondrial mass provides higher energetic output in the form of elevated ATP levels which may fuel the progression of the tumour cell through epithelial to mesenchymal transition (EMT) and ultimately metastasis.

18F-Fluciclovine PET metabolic imaging reveals prostate cancer tumour heterogeneity associated with disease resistance to androgen deprivation therapy.

BACKGROUND: Prostate cancer is highly prevalent worldwide. Androgen deprivation therapy (ADT) remains the treatment of choice for incurable prostate cancer, but majority of patients develop disease recurrence following ADT. There is therefore an urgent need for early detection of treatment resistance. METHODS: Isogenic androgen-responsive (CWR22Res) and castration-resistant (22Rv1) human prostate cancer cells were implanted into the anterior lobes of the prostate in CD-1 Nu mice to generate prostate orthografts. Castrated mice bearing CWR22Res and 22Rv1 orthografts mimic clinical prostate cancer following acute and chronic ADT, respectively. 18F-Fluciclovine (1-amino-3-fluorocyclobutane-1-carboxylic acid) with a radiochemical purity of > 99% was produced on a FASTlab synthesiser. Ki67 staining in endpoint orthografts was studied. Western blot, quantitative RT-PCR and next-generation sequencing transcriptomic analyses were performed to assess the expression levels of amino acid transporters (including LAT1 and ASCT2, which have been implicated for Fluciclovine uptake). Longitudinal metabolic imaging with 18F-Fluciclovine-based positron emission tomography (PET) was performed to study tumour response following acute and chronic ADT. RESULTS: Both immunohistochemistry analysis of endpoint prostate tumours and longitudinal 18F-Fluciclovine imaging revealed tumour heterogeneity, particularly following ADT, with in vivo 18F-Fluciclovine uptake correlating to viable cancer cells in both androgen-proficient and castrated environment. Highlighting tumour subpopulation following ADT, both SUVpeak and coefficient of variation (CoV) values of 18F-Fluciclovine uptake are consistent with tumour heterogeneity revealed by immunohistochemistry. We studied the expression of amino acid transporters (AATs) for 18F-Fluciclovine, namely LAT1 (SLC7A5 and SLC3A2) and ASCT2 (SLC1A5). SLC7A5 and SLC3A2 were expressed at relatively high levels in 22Rv1 castration-resistant orthografts following chronic ADT (modelling clinical castration-resistant disease), while SLC1A5 was preferentially expression in CWR22Res tumours following acute ADT. Additional AATs such as SLC43A2 (LAT4) were shown to be upregulated following chronic ADT by transcriptomic analysis; their role in Fluciclovine uptake warrants investigation. CONCLUSION: We studied in vivo 18F-Fluciclovine uptake in human prostate cancer orthograft models following acute and chronic ADT. 18F-Fluciclovine uptakes highlight tumour heterogeneity that may explain castration resistance and can be exploited as a clinical biomarker.

Activation of ß-Catenin Cooperates with Loss of Pten to Drive AR-Independent Castration-Resistant Prostate Cancer.

Inhibition of the androgen receptor (AR) is the main strategy to treat advanced prostate cancers. AR-independent treatment-resistant prostate cancer is a major unresolved clinical problem. Patients with prostate cancer with alterations in canonical WNT pathway genes, which lead to ß-catenin activation, are refractory to AR-targeted therapies. Here, using clinically relevant murine prostate cancer models, we investigated the significance of ß-catenin activation in prostate cancer progression and treatment resistance. ß-Catenin activation, independent of the cell of origin, cooperated with Pten loss to drive AR-independent castration-resistant prostate cancer. Prostate tumors with ß-catenin activation relied on the noncanonical WNT ligand WNT5a for sustained growth. WNT5a repressed AR expression and maintained the expression of c-Myc, an oncogenic effector of ß-catenin activation, by mediating nuclear localization of NFκBp65 and ß-catenin. Overall, WNT/ß-catenin and AR signaling are reciprocally inhibited. Therefore, inhibiting WNT/ß-catenin signaling by limiting WNT secretion in concert with AR inhibition may be useful for treating prostate cancers with alterations in WNT pathway genes. SIGNIFICANCE: Targeting of both AR and WNT/ß-catenin signaling may be required to treat prostate cancers that exhibit alterations of the WNT pathway.