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.
Synopsis
Resistance to androgen‐deprivation therapy (ADT) leads to the development of lethal castration‐resistant prostate cancer. This study highlights THEM6 as a clinically relevant protein that supports ADT‐induced rewiring of lipid metabolism and sustains UPR activation, thus promoting ADT resistance.
THEM6 is overexpressed in ADT‐resistant tumours and supports PCa tumour growth in androgen‐deprived conditions.
Loss of THEM6 significantly alters the cellular lipidome of CRPC cells, resulting in decreased levels of ether lipids.
THEM6 is an ER‐membrane protein that physically interacts with several components of the protein trafficking machinery.
Loss of THEM6 significantly affects ER function, reducing de novo sterol synthesis and impairing lipid‐mediated activation of the UPR, two processes that promote ADT resistance.
High THEM6 expression in tumour biopsies is associated with shortened patient survival and correlates with sustained UPR activation, thus highlighting the potential of THEM6 as a therapeutic target in CRPC.
Resistance to androgen‐deprivation therapy (ADT) leads to the development of lethal castration‐resistant prostate cancer. This study highlights THEM6 as a clinically relevant protein that supports ADT‐induced rewiring of lipid metabolism and sustains UPR activation, thus promoting ADT resistance.
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.
Toxoplasmosis causes significant morbidity and mortality, and yet available medicines are limited by toxicities and hypersensitivity. Because improved medicines are needed urgently, rational ...approaches were used to identify novel lead compounds effective against Toxoplasma gondii enoyl reductase (TgENR), a type II fatty acid synthase enzyme essential in parasites but not present in animals. Fifty-three compounds, including three classes that inhibit ENRs, were tested. Six compounds have antiparasite MIC90s ≤ 6 μM without toxicity to host cells, three compounds have IC90s < 45 nM against recombinant TgENR, and two protect mice. To further understand the mode of inhibition, the cocrystal structure of one of the most promising candidate compounds in complex with TgENR has been determined to 2.7 Å. The crystal structure reveals that the aliphatic side chain of compound 19 occupies, as predicted, space made available by replacement of a bulky hydrophobic residue in homologous bacterial ENRs by Ala in TgENR. This provides a paradigm, conceptual foundation, reagents, and lead compounds for future rational development and discovery of improved inhibitors of T. gondii.
Metastatic castration‐resistant prostate cancer (mCRPC) is a lethal form of treatment‐resistant prostate cancer and poses significant therapeutic challenges. Deregulated receptor tyrosine kinase ...(RTK) signalling mediated by loss of tumour suppressor Sprouty2 (SPRY2) is associated with treatment resistance. Using pre‐clinical human and murine mCRPC models, we show that SPRY2 deficiency leads to an androgen self‐sufficient form of CRPC. Mechanistically, HER2‐IL6 signalling axis enhances the expression of androgen biosynthetic enzyme HSD3B1 and increases SRB1‐mediated cholesterol uptake in SPRY2‐deficient tumours. Systemically, IL6 elevated the levels of circulating cholesterol by inducing host adipose lipolysis and hepatic cholesterol biosynthesis. SPRY2‐deficient CRPC is dependent on cholesterol bioavailability and SRB1‐mediated tumoral cholesterol uptake for androgen biosynthesis. Importantly, treatment with ITX5061, a clinically safe SRB1 antagonist, decreased treatment resistance. Our results indicate that cholesterol transport blockade may be effective against SPRY2‐deficient CRPC.
Synopsis
Tumour‐induced IL6 modulates host lipid metabolism to drive treatment‐resistant castration‐resistant prostate cancer (CRPC). Blocking tumoral cholesterol uptake using SRB1 antagonist or restoring systemic cholesterol homeostasis using statins decreases treatment resistance.
SPRY2 deficiency drives androgen self‐sufficient form CRPC though HER2‐mediated IL6.
IL6 mediates tumoural androgen synthesis by inducing HSD3B1 expression.
Systemic IL6 mediates host adipose lipolysis and stimulates hepatic cholesterol biosynthesis leading to elevated levels of circulating cholesterol.
SPRY2 loss‐induced IL6 mediates tumoural cholesterol uptake through the HDL receptor SRB1 to drive CRPC.
Using a clinically safe SRB1 antagonist (ITX5061) decreased treatment‐resistance.
Tumour‐induced IL6 modulates host lipid metabolism to drive treatment‐resistant castration‐resistant prostate cancer (CRPC). Blocking tumoral cholesterol uptake using SRB1 antagonist or restoring systemic cholesterol homeostasis using statins decreases treatment resistance.
Extracellular signal-regulated protein kinase 5 (ERK5) has been implicated during development and carcinogenesis. Nkx3.1-mediated Cre expression is a useful strategy to genetically manipulate the ...mouse prostate. While grossly normal at birth, we observed an unexpected phenotype of spinal protrusion in Nkx3.1:Cre;Erk5
(Erk5
) mice by ~6-8 weeks of age. X-ray, histological and micro CT (µCT) analyses showed that 100% of male and female Erk5
mice had a severely deformed curved thoracic spine, with an associated loss of trabecular bone volume. Although sex-specific differences were observed, histomorphometry measurements revealed that both bone resorption and bone formation parameters were increased in male Erk5
mice compared to wild type (WT) littermates. Osteopenia occurs where the rate of bone resorption exceeds that of bone formation, so we investigated the role of the osteoclast compartment. We found that treatment of RANKL-stimulated primary bone marrow-derived macrophage (BMDM) cultures with small molecule ERK5 pathway inhibitors increased osteoclast numbers. Furthermore, osteoclast numbers and expression of osteoclast marker genes were increased in parallel with reduced Erk5 expression in cultures generated from Erk5
mice compared to WT mice. Collectively, these results reveal a novel role for Erk5 during bone maturation and homeostasis in vivo.
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-3β, 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.
Background and Methodology: Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and ...cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3'(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3'-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested. Principal Findings and Conclusions: Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine. Significance: JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.
The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of ...peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.
The shikimate pathway is essential for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Seven enzymes of the shikimate pathway catalyze sequential conversion of ...erythrose 4-phosphate and phosphoenol pyruvate to chorismate. Chorismate is then used as a substrate for other pathways that culminate in production of folates, ubiquinone, napthoquinones, and the aromatic amino acids tryptophan, phenylalanine, and tyrosine. The shikimate pathway is absent from animals and present in the apicomplexan parasites Toxoplasma gondii, Plasmodium falciparum, and Cryptosporidium parvum. Inhibition of the pathway by glyphosate is effective in controlling growth of these parasites. These findings emphasize the potential benefits of developing additional effective inhibitors of the shikimate pathway. Such inhibitors may function as broad-spectrum antimicrobial agents that are effective against bacterial and fungal pathogens and apicomplexan parasites.
PDF
