Kinesin Facilitates Phenotypic Targeting of Therapeutic Resistance in Advanced Prostate Cancer.

Understanding the mechanisms underlying resistance is critical to improving therapeutic outcomes in patients with metastatic castration-resistant prostate cancer (mCRPC). Previous work showed dynamic interconversions between epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) defines the phenotypic landscape of prostate tumors, as a potential driver of emergence of therapeutic resistance. In this study, we use in vitro and in vivo preclinical MDA PCa PDX models of resistant human prostate cancer to determine molecular mechanisms of cross-resistance between anti-androgen therapy and taxane chemotherapy, underlying the therapeutically resistant phenotype. Transcriptomic profiling revealed that resistant and sensitive prostate cancer C4-2B cells have a unique differential gene signature response to cabazitaxel. Gene pathway analysis showed that sensitive cells exhibit increase in DNA damage, while resistant cells express genes associated with protein regulation in response to cabazitaxel. These PDX specimens are from patients who have metastatic lethal CRPC, treated with androgen-deprivation therapy (ADT), antiandrogens and chemotherapy including 2nd line taxane chemotherapy, cabazitaxel. Immunohistochemistry revealed high expression of E-cadherin and low expression of vimentin resulting in re-differentiation toward an epithelial phenotype. Furthermore, the mitotic kinesin-related protein (HSET) involved in microtubule binding and the SLCO1B3 transporter (implicated in cabazitaxel intracellular transport), associated with resistance in these prostate tumors. Combinational targeting of kinesins (ispinesib) with cabazitaxel was more effective than single monotherapies in inducing cell death in resistant prostate tumors. Implications: Our findings are of translational significance in identifying kinesin as a novel target of cross-resistance, towards enhancing therapeutic vulnerability and improved clinical outcomes in patients with advanced prostate cancer.

Integrative Molecular Analyses of the MD Anderson Prostate Cancer Patient-derived Xenograft (MDA PCa PDX) Series.

PURPOSE: Develop and deploy a robust discovery platform that encompasses heterogeneity, clinical annotation, and molecular characterization and overcomes the limited availability of prostate cancer models. This initiative builds on the rich MD Anderson (MDA) prostate cancer (PCa) patient-derived xenograft (PDX) resource to complement existing publicly available databases by addressing gaps in clinically annotated models reflecting the heterogeneity of potentially lethal and lethal prostate cancer. EXPERIMENTAL DESIGN: We performed whole-genome, targeted, and RNA sequencing in representative samples of the same tumor from 44 PDXs derived from 38 patients linked to donor tumor metadata and corresponding organoids. The cohort includes models derived from different morphologic groups, disease states, and involved organ sites (including circulating tumor cells), as well as paired samples representing heterogeneity or stages before and after therapy. RESULTS: The cohort recapitulates clinically reported alterations in prostate cancer genes, providing a data resource for clinical and molecular interrogation of suitable experimental models. Paired samples displayed conserved molecular alteration profiles, suggesting the relevance of other regulatory mechanisms (e.g., epigenomic) influenced by the microenvironment and/or treatment. Transcriptomically, models were grouped on the basis of morphologic classification. DNA damage response-associated mechanisms emerged as differentially regulated between adenocarcinoma and neuroendocrine prostate cancer in a cross-interrogation of PDX/patient datasets. CONCLUSIONS: We addressed the gap in clinically relevant prostate cancer models through comprehensive molecular characterization of MDA PCa PDXs, providing a discovery platform that integrates with patient data and benchmarked to therapeutically relevant consensus clinical groupings. This unique resource supports robust hypothesis generation and testing from basic, translational, and clinical perspectives.

Conditional Deletion of ß-Catenin in the Mediobasal Hypothalamus Impairs Adaptive Energy Expenditure in Response to High-Fat Diet and Exacerbates Diet-Induced Obesity.

ß-Catenin is a bifunctional molecule that is an effector of the wingless-related integration site (Wnt) signaling to control gene expression and contributes to the regulation of cytoskeleton and neurotransmitter vesicle trafficking. In its former role, ß-catenin binds transcription factor 7-like 2 (TCF7L2), which shows strong genetic associations with the pathogenesis of obesity and type-2 diabetes. Here, we sought to determine whether ß-catenin plays a role in the neuroendocrine regulation of body weight and glucose homeostasis. Bilateral injections of adeno-associated virus type-2 (AAV2)-mCherry-Cre were placed into the arcuate nucleus of adult male and female ß-catenin flox mice, to specifically delete ß-catenin expression in the mediobasal hypothalamus (MBH-ß-cat KO). Metabolic parameters were then monitored under conditions of low-fat (LFD) and high-fat diet (HFD). On LFD, MBH-ß-cat KO mice showed minimal metabolic disturbances, but on HFD, despite having only a small difference in weekly caloric intake, the MBH-ß-cat KO mice were significantly heavier than the control mice in both sexes (p < 0.05). This deficit seemed to be due to a failure to show an adaptive increase in energy expenditure seen in controls, which served to offset the increased calories by HFD. Both male and female MBH-ß-cat KO mice were highly glucose intolerant when on HFD and displayed a significant reduction in both leptin and insulin sensitivity compared with controls. This study highlights a critical role for ß-catenin in the hypothalamic circuits regulating body weight and glucose homeostasis and reveals potential mechanisms by which genetic variation in this pathway could impact on development of metabolic disease.

Bone mimetic environments support engineering, propagation, and analysis of therapeutic response of patient-derived cells, ex vivo and in vivo.

Bone metastases are the most common milestone in the lethal progression of prostate cancer and prominent in a substantial portion of renal malignancies. Interactions between cancer and bone host cells have emerged as drivers of both disease progression and therapeutic resistance. To best understand these central host-epithelial cell interactions, biologically relevant preclinical models are required. To achieve this goal, we here established and characterized tissue-engineered bone mimetic environments (BME) capable of supporting the growth of patient-derived xenograft (PDX) cells, ex vivo and in vivo. The BME consisted of a polycaprolactone (PCL) scaffold colonized by human mesenchymal stem cells (hMSCs) differentiated into osteoblasts. PDX-derived cells were isolated from bone metastatic prostate or renal tumors, engineered to express GFP or luciferase and seeded onto the BMEs. BMEs supported the growth and therapy response of PDX-derived cells, ex vivo. Additionally, BMEs survived after in vivo implantation and further sustained the growth of PDX-derived cells, their serial transplant, and their application to study the response to treatment. Taken together, this demonstrates the utility of BMEs in combination with patient-derived cells, both ex vivo and in vivo. STATEMENT OF SIGNIFICANCE: Our tissue-engineered BME supported the growth of patient-derived cells and proved useful to monitor the therapy response, both ex vivo and in vivo. This approach has the potential to enable co-clinical strategies to monitor bone metastatic tumor progression and therapy response, including identification and prioritization of new targets for patient treatment.

Empowering Pacific Patients on the Weight Loss Surgery Pathway: A Co-designed Evaluation Study.

PURPOSE: Despite having the highest medical needs by population for weight loss treatment, Pacific patients in Aotearoa New Zealand face substantial levels of attrition in publicly funded weight loss surgery programs. In collaboration with the Auckland City Hospital bariatric surgery team, a Pacific-led preoperative weight loss surgery program was co-designed, delivered, and evaluated between 2020 and 2023. MATERIALS AND METHODS: This was a single-arm, prospective co-designed evaluation study that took place at Auckland City Hospital in Aotearoa New Zealand. Participants were Pacific patients (n = 14) referred to the weight loss surgery program. Survey and video diaries were analyzed to determine if the program had the potential to increase Pacific patient retention through the preoperative stage of weight loss surgery, increase surgery completion rates, and improve the quality of treatment experiences. RESULTS: Nine out of 14 participants attended all preoperative sessions. Six participants subsequently underwent weight loss surgery. Program components that had positive impacts on patient success and satisfaction were accessibility, information quality, having Pacific role models, cultural safety, and the group support system. The patients found the program to be culturally anchored and there was support for the implementation of the program going forward. CONCLUSION: This study demonstrated how a culturally anchored intervention can increase patient retention for those patients who may not respond to mainstream treatment. Adjusting existing preoperative weight loss surgery programs to integrate Pacific-led models of healthcare has the potential to increase Pacific patient resiliency to follow through with surgery.

Insights into the role of JAK2-I724T variant in myeloproliferative neoplasms from a unique cohort of New Zealand patients.

OBJECTIVES: This study aimed to compile bioinformatic and experimental information for JAK2 missense variants previously reported in myeloproliferative neoplasms (MPN) and determine if germline JAK2-I724T, recently found to be common in New Zealand Polynesians, associates with MPN. METHODS: For all JAK2 variants found in the literature, gnomAD_exome allele frequencies were extracted and REVEL scores were calculated using the dbNSFP database. We investigated the prevalence of JAK2-I724T in a cohort of 111 New Zealand MPN patients using a TaqMan assay, examined its allelic co-occurrence with JAK2-V617F using Oxford Nanopore sequencing, and modelled the impact of I724T on JAK2 using I-Mutant and ChimeraX software. RESULTS: Several non-V617F JAK2 variants previously reported in MPN had REVEL scores greater than 0.5, suggesting pathogenicity. JAK2-I724T (REVEL score 0.753) was more common in New Zealand Polynesian MPN patients (n = 2/27; 7.4%) than in other New Zealand patients (n = 0/84; 0%) but less common than expected for healthy Polynesians (n = 56/377; 14.9%). Patients carrying I724T (n = 2), one with polycythaemia vera and one with essential thrombocythaemia, had high-risk MPN. Both patients with JAK2-I724T were also positive for JAK2-V617F, found on the same allele as I724T, as well as separately. In silico modelling did not identify noticeable structural changes that would give JAK2-I724T a gain-of-function. CONCLUSION: Several non-canonical JAK2 variants with high REVEL scores have been reported in MPN, highlighting the need to further understand their relationship with disease. The JAK2-I724T variant does not drive MPN, but additional investigations are required to exclude any potential modulatory effect on the MPN phenotype.

Monitoring Glucocorticoid Receptor in Plasma-derived Extracellular Vesicles as a Marker of Resistance to Androgen Receptor Signaling Inhibition in Prostate Cancer.

Disease progression following androgen ablation was shown to be associated with upregulation of the glucocorticoid receptor (GR). Longitudinal monitoring of GR expression in circulating extracellular vesicles (EV) may reflect changes in the tumor cell and facilitates detection of acquired resistance. We utilized LNCaP, LREX cells and a patient-derived xenograft, MDA PDX 322-2-6a, for in vitro and in vivo experiments. Plasma-derived EVs were isolated from patients with localized high-risk prostate cancer undergoing androgen ablation. The mRNA levels of GR in EVs and their responsive genes were detected by transcriptome analysis, qRT-PCR and the protein levels by Western blot analysis. We detected changes in GR expression at mRNA and protein levels in EVs derived from LNCaP and LREX cells in in vitro studies. In in vivo experiments, LNCaP and the PDX MDA 322-2-6a-bearing mice were treated with enzalutamide. GR levels in plasma-derived EVs were increased only in those tumors that did not respond to enzalutamide. Treatment of mice bearing enzalutamide-resistant tumors with a GR inhibitor in combination with enzalutamide led to a transient pause in tumor growth in a subset of tumors and decreased GR levels intracellular and in plasma-derived EVs. In a subgroup of patients with high-risk localized prostate cancer treated with androgen signaling inhibition, GR was found upregulated in matching tissue and plasma EVs. These analyses showed that GR levels in plasma-derived EVs may be used for monitoring the transition of GR expression allowing for early detection of resistance to androgen ablation treatment. SIGNIFICANCE: Longitudinal monitoring of GR expression in plasma-derived EVs from patients with prostate cancer treated with androgen signaling inhibitors facilitates early detection of acquisition of resistance to androgen receptor signaling inhibition in individual patients.

ETV4 mediates dosage-dependent prostate tumor initiation and cooperates with p53 loss to generate prostate cancer.

The mechanisms underlying ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.

A role for ß-catenin in diet-induced skeletal muscle insulin resistance.

A central characteristic of insulin resistance is the impaired ability for insulin to stimulate glucose uptake into skeletal muscle. While insulin resistance can occur distal to the canonical insulin receptor-PI3k-Akt signaling pathway, the signaling intermediates involved in the dysfunction are yet to be fully elucidated. ß-catenin is an emerging distal regulator of skeletal muscle and adipocyte insulin-stimulated GLUT4 trafficking. Here, we investigate its role in skeletal muscle insulin resistance. Short-term (5-week) high-fat diet (HFD) decreased skeletal muscle ß-catenin protein expression 27% (p = 0.03), and perturbed insulin-stimulated ß-cateninS552 phosphorylation 21% (p = 0.009) without affecting insulin-stimulated Akt phosphorylation relative to chow-fed controls. Under chow conditions, mice with muscle-specific ß-catenin deletion had impaired insulin responsiveness, whereas under HFD, both mice exhibited similar levels of insulin resistance (interaction effect of genotype × diet p < 0.05). Treatment of L6-GLUT4-myc myocytes with palmitate lower ß-catenin protein expression by 75% (p = 0.02), and attenuated insulin-stimulated ß-catenin phosphorylationS552 and actin remodeling (interaction effect of insulin × palmitate p < 0.05). Finally, ß-cateninS552 phosphorylation was 45% lower in muscle biopsies from men with type 2 diabetes while total ß-catenin expression was unchanged. These findings suggest that ß-catenin dysfunction is associated with the development of insulin resistance.

A 3D Perfusable Platform for In Vitro Culture of Patient Derived Xenografts.

Many advanced cancer models, such as patient-derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor's heterogeneity and susceptibility to treatments, but face significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high-throughput screening (HTS). Complex multilineage co-cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein, the culture of multiple prostate cancer (PCa)-derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS-compatible multiwell microfluidic format, alongside bone marrow-derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal-based testing.

Response to BRAF-targeted Therapy Is Enhanced by Cotargeting VEGFRs or WNT/ß-Catenin Signaling in BRAF-mutant Colorectal Cancer Models.

The fact that 10% of colorectal cancer tumors harbor BRAF V600E mutations suggested targeting BRAF as a potential therapy. However, BRAF inhibitors have only limited single-agent efficacy in this context. The potential for combination therapy has been shown by the BEACON trial where targeting the EGF receptor with cetuximab greatly increased efficacy of BRAF inhibitors in BRAF-mutant colorectal cancer. Therefore, we explored whether efficacy of the mutant BRAF inhibitor vemurafenib could be enhanced by cotargeting of either oncogenic WNT/ß-catenin signaling or VEGFR signaling. We find the WNT/ß-catenin inhibitors pyrvinium, ICG-001 and PKF118-310 attenuate growth of colorectal cancer cell lines in vitro with BRAF-mutant lines being relatively more sensitive. Pyrvinium combined with vemurafenib additively or synergistically attenuated growth of colorectal cancer cell lines in vitro. The selective and potent VEGFR inhibitor axitinib was most effective against BRAF-mutant colorectal cancer cell lines in vitro, but the addition of vemurafenib did not significantly increase these effects. When tested in vivo in animal tumor models, both pyrvinium and axitinib were able to significantly increase the ability of vemurafenib to attenuate tumor growth in xenografts of BRAF-mutant colorectal cancer cells. The magnitude of these effects was comparable with that induced by a combination of vemurafenib and cetuximab. This was associated with additive effects on release from tumor cells and tumor microenvironment cell types of substances that would normally aid tumor progression. Taken together, these preclinical data indicate that the efficacy of BRAF inhibitor therapy in colorectal cancer could be increased by cotargeting either WNT/ß-catenin or VEGFRs with small-molecule inhibitors.

Genotype-to-Phenotype Associations in the Aggressive Variant Prostate Cancer Molecular Profile (AVPC-m) Components.

The aggressive variant prostate cancer molecular profile (AVPC-m), composed of combined defects in TP53, RB1 and PTEN, characterizes a subset of prostate cancers linked to androgen indifference and platinum sensitivity. To contribute to the optimization of the AVPC-m assessment for inclusion in prospective clinical trials, we investigated the status of the AVPC-m components in 28 patient tumor-derived xenografts (PDXs) developed at MDACC. We subjected single formalin-fixed, paraffin-embedded (FFPE) blocks from each PDX to immunohistochemistry (IHC), targeted next-generation genomic sequencing (NGS) and Clariom-S Affymetrix human microarray expression profiling. Standard validated IHC assays and a 10% labeling index cutoff resulted in high reproducibility across three separate laboratories and three independent readers for all tumor suppressors, as well as strong correlations with loss-of-function transcriptional scores (LOF-TS). Adding intensity assessment to labeling indices strengthened the association between IHC results and LOF-TS for TP53 and RB1, but not for PTEN. For TP53, genomic alterations determined by NGS had slightly higher agreement scores with LOF-TS than aberrant IHC, while for RB1 and PTEN, NGS and IHC determinations resulted in similar agreement scores with LOF-TS. Nonetheless, our results indicate that the AVPC-m components can be assessed reproducibly by IHC using various widely available standardized assays.

Bone Progenitors Pull the Strings on the Early Metabolic Rewiring Occurring in Prostate Cancer Cells.

Metastatic prostate cancer (PCa) cells soiling in the bone require a metabolic adaptation. Here, we identified the metabolic genes fueling the seeding of PCa in the bone niche. Using a transwell co-culture system of PCa (PC3) and bone progenitor cells (MC3T3 or Raw264.7), we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. In a Principal Component Analysis using transcriptomic data from human PCa samples (GSE74685), the altered metabolic genes found in vitro were able to stratify PCa patients in two defined groups: primary PCa and bone metastasis, confirmed by an unsupervised clustering analysis. Thus, the early transcriptional metabolic profile triggered in the in vitro model has a clinical correlate in human bone metastatic samples. Further, the expression levels of five metabolic genes (VDR, PPARA, SLC16A1, GPX1 and PAPSS2) were independent risk-predictors of death in the SU2C-PCF dataset and a risk score model built using this lipid-associated signature was able to discriminate a subgroup of bone metastatic PCa patients with a 23-fold higher risk of death. This signature was validated in a PDX pre-clinical model when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously, and appears to be under the regulatory control of the Protein Kinase A (PKA) signaling pathway. Secretome analyses of conditioned media showcased fibronectin and type-1 collagen as critical bone-secreted factors that could regulate tumoral PKA. Overall, we identified a novel lipid gene signature, driving PCa aggressive metastatic disease pointing to PKA as a potential hub to halt progression.

The minor allele of the CREBRF rs373863828 p.R457Q coding variant is associated with reduced levels of myostatin in males: Implications for body composition.

OBJECTIVE: The minor allele (A) of the rs373863828 variant (p.Arg457Gln) in CREBRF is restricted to indigenous peoples of the Pacific islands (including New Zealand Maori and peoples of Polynesia), with a frequency of up to 25% in these populations. This allele associates with a large increase in body mass index (BMI) but with significantly lower risk of type-2 diabetes (T2D). It remains unclear whether the increased BMI is driven by increased adiposity or by increased lean mass. METHODS: We undertook body composition analysis using DXA in 189 young men of Maori and Pacific descent living in Aotearoa New Zealand. Further investigation was carried out in two orthologous Arg458Gln knockin mouse models on FVB/NJ and C57BL/6j backgrounds. RESULTS: The rs373863828 A allele was associated with lower fat mass when adjusted for BMI (p < 0.05) and was associated with significantly lower circulating levels of the muscle inhibitory hormone myostatin (p < 0.05). Supporting the human data, significant reductions in adipose tissue mass were observed in the knockin mice. This was more significant in older mice in both backgrounds and appeared to be the result of reduced age-associated increases in fat mass. The older male knockin mice on C57BL/6j background also had increased grip strength (p < 0.01) and lower levels of myostatin (p < 0.05). CONCLUSION: Overall, these results prove that the rs373863828 A-allele is associated with a reduction of myostatin levels which likely contribute to an age-dependent lowering of fat mass, at least in males.

Fibroblast Growth Factor Receptor 1 Drives the Metastatic Progression of Prostate Cancer.

BACKGROUND: No curative therapy is currently available for metastatic prostate cancer (PCa). The diverse mechanisms of progression include fibroblast growth factor (FGF) axis activation. OBJECTIVE: To investigate the molecular and clinical implications of fibroblast growth factor receptor 1 (FGFR1) and its isoforms (α/ß) in the pathogenesis of PCa bone metastases. DESIGN, SETTING, AND PARTICIPANTS: In silico, in vitro, and in vivo preclinical approaches were used. RNA-sequencing and immunohistochemical (IHC) studies in human samples were conducted. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: In mice, bone metastases (chi-square/Fisher's test) and survival (Mantel-Cox) were assessed. In human samples, FGFR1 and ladinin 1 (LAD1) analysis associated with PCa progression were evaluated (IHC studies, Fisher's test). RESULTS AND LIMITATIONS: FGFR1 isoform expression varied among PCa subtypes. Intracardiac injection of mice with FGFR1-expressing PC3 cells reduced mouse survival (α, p < 0.0001; ß, p = 0.032) and increased the incidence of bone metastases (α, p < 0.0001; ß, p = 0.02). Accordingly, IHC studies of human castration-resistant PCa (CRPC) bone metastases revealed significant enrichment of FGFR1 expression compared with treatment-naïve, nonmetastatic primary tumors (p = 0.0007). Expression of anchoring filament protein LAD1 increased in FGFR1-expressing PC3 cells and was enriched in human CRPC bone metastases (p = 0.005). CONCLUSIONS: FGFR1 expression induces bone metastases experimentally and is significantly enriched in human CRPC bone metastases, supporting its prometastatic effect in PCa. LAD1 expression, found in the prometastatic PCa cells expressing FGFR1, was also enriched in CRPC bone metastases. Our studies support and provide a roadmap for the development of FGFR blockade for advanced PCa. PATIENT SUMMARY: We studied the role of fibroblast growth factor receptor 1 (FGFR1) in prostate cancer (PCa) progression. We found that PCa cells with high FGFR1 expression increase metastases and that FGFR1 expression is increased in human PCa bone metastases, and identified genes that could participate in the metastases induced by FGFR1. These studies will help pinpoint PCa patients who use fibroblast growth factor to progress and will benefit by the inhibition of this pathway.

Prostate cancer castrate resistant progression usage of non-canonical androgen receptor signaling and ketone body fuel.

Prostate cancer (PCa) that progresses after androgen deprivation therapy (ADT) remains incurable. The underlying mechanisms that account for the ultimate emergence of resistance to ADT, progressing to castrate-resistant prostate cancer (CRPC), include those that reactivate androgen receptor (AR), or those that are entirely independent or cooperate with androgen signaling to underlie PCa progression. The intricacy of metabolic pathways associated with PCa progression spurred us to develop a metabolism-centric analysis to assess the metabolic shift occurring in PCa that progresses with low AR expression. We used PCa patient-derived xenografts (PDXs) to assess the metabolic changes after castration of tumor-bearing mice and subsequently confirmed main findings in human donor tumor that progressed after ADT. We found that relapsed tumors had a significant increase in fatty acids and ketone body (KB) content compared with baseline. We confirmed that critical ketolytic enzymes (ACAT1, OXCT1, BDH1) were dysregulated after castrate-resistant progression. Further, these enzymes are increased in the human donor tissue after progressing to ADT. In an in silico approach, increased ACAT1, OXCT1, BDH1 expression was also observed for a subset of PCa patients that relapsed with low AR and ERG (ETS-related gene) expression. Further, expression of these factors was also associated with decreased time to biochemical relapse and decreased progression-free survival. Our studies reveal the key metabolites fueling castration resistant progression in the context of a partial or complete loss of AR dependence.

The CREBRF diabetes-protective rs373863828-A allele is associated with enhanced early insulin release in men of Maori and Pacific ancestry.

AIMS/HYPOTHESIS: The minor A allele of rs373863828 (CREBRF p.Arg457Gln) is associated with increased BMI, but reduced risk of type 2 and gestational diabetes in Polynesian (Pacific peoples and Aotearoa New Zealand Maori) populations. This study investigates the effect of the A allele on insulin release and sensitivity in overweight/obese men without diabetes. METHODS: A mixed meal tolerance test was completed by 172 men (56 with the A allele) of Maori or Pacific ancestry, and 44 (24 with the A allele) had a frequently sampled IVGTT and hyperinsulinaemic-euglycaemic clamp. Mixed linear models with covariates age, ancestry and BMI were used to analyse the association between the A allele of rs373863828 and markers of insulin release and blood glucose regulation. RESULTS: The A allele of rs373863828 is associated with a greater increase in plasma insulin 30 min following a meal challenge without affecting the elevation in plasma glucose or incretins glucagon-like polypeptide-1 or gastric inhibitory polypeptide. Consistent with this point, following an i.v. infusion of a glucose bolus, participants with an A allele had higher early (p < 0.05 at 2 and 4 min) plasma insulin and C-peptide concentrations for a similar elevation in blood glucose as those homozygous for the major (G) allele. Despite increased plasma insulin, rs373863828 genotype was not associated with a significant difference (p > 0.05) in insulin sensitivity index or glucose disposal during hyperinsulinaemic-euglycaemic clamp. CONCLUSIONS/INTERPRETATION: rs373863828-A allele associates with increased glucose-stimulated insulin release without affecting insulin sensitivity, suggesting that CREBRF p.Arg457Gln may increase insulin release to reduce the risk of type 2 diabetes.

Glucose regulates expression of pro-inflammatory genes, IL-1ß and IL-12, through a mechanism involving hexosamine biosynthesis pathway-dependent regulation of α-E catenin.

High glucose levels are associated with changes in macrophage polarisation and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose-induced increase in α-E catenin when hexosamine biosynthesis (HB) pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of HB pathway in this process. Then, we investigated the potential role of α-E catenin in glucose-induced macrophage polarisation. We find that the reduction in α-E catenin level using siRNA attenuates the glucose-induced changes of both IL-1ß and IL-12 mRNA levels under LPS-stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via HB pathway and also can modulate the glucose-induced gene expression of inflammatory markers such as IL-1ß and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.

Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK.

BACKGROUND: The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. METHODS: Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. RESULTS: We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. CONCLUSIONS: Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.

Efficacy of Providing the PI3K p110α Inhibitor BYL719 (Alpelisib) to Middle-Aged Mice in Their Diet.

BYL719 (alpelisib) is a small molecule inhibitor of PI3K p110α developed for cancer therapy. Targeted suppression of PI3K has led to lifespan extension in rodents and model organisms. If PI3K inhibitors are to be considered as an aging therapeutic, it is important to understand the potential consequences of long-term exposure, and the most practical way to achieve this is through diet administration. Here, we investigated the pharmacokinetics of BYL719 delivered in diet and the efficacy of BYL719 to suppress insulin signaling when administered in the diet of 8-month-old male and female mice. Compared to oral gavage, diet incorporation resulted in a lower peak plasma BYL719 (3.6 vs. 9.2 µM) concentration but similar half-life (~1.5 h). Consuming BYL719 resulted in decreased insulin signaling in liver and muscle within 72 h, and mice still showed impaired glucose tolerance and insulin sensitivity following 6 weeks of access to a diet containing 0.3 g/kg BYL719. However, consuming BYL719 did not affect food intake, body mass, muscle function (rotarod and hang time performance) or cognitive behaviors. This provides evidence that BYL719 has long-term efficacy without major toxicity or side effects, and suggests that administering BYL719 in diet is suitable for studying the effect of pharmacological suppression of PI3K p110α on aging and metabolic function.