Isomeric lipid signatures reveal compartmentalized fatty acid metabolism in cancer.

The cellular energy and biomass demands of cancer drive a complex dynamic between uptake of extracellular FAs and their de novo synthesis. Given that oxidation of de novo synthesized FAs for energy would result in net-energy loss, there is an implication that FAs from these two sources must have distinct metabolic fates; however, hitherto, all FAs have been considered part of a common pool. To probe potential metabolic partitioning of cellular FAs, cancer cells were supplemented with stable isotope-labeled FAs. Structural analysis of the resulting glycerophospholipids revealed that labeled FAs from uptake were largely incorporated to canonical (sn-) positions on the glycerol backbone. Surprisingly, labeled FA uptake also disrupted canonical isomer patterns of the unlabeled lipidome and induced repartitioning of n-3 and n-6 PUFAs into glycerophospholipid classes. These structural changes support the existence of differences in the metabolic fates of FAs derived from uptake or de novo sources and demonstrate unique signaling and remodeling behaviors usually hidden from conventional lipidomics.

Synthesis of a Unique Psammaplysin F Library and Functional Evaluation in Prostate Cancer Cells by Multiparametric Quantitative Single Cell Imaging.

The spirooxepinisoxazoline alkaloid psammaplysin F (1) was selected as a scaffold for the generation of a unique screening library for both drug discovery and chemical biology research. Large-scale extraction and isolation chemistry was performed on a marine sponge (Hyattella sp.) collected from the Great Barrier Reef in order to acquire >200 mg of the desired bromotyrosine-derived alkaloidal scaffold. Parallel solution-phase semisynthesis was employed to generate a series of psammaplysin-based urea (2-9) and amide analogues (10-11) in low to moderate yields. The chemical structures of all analogues were characterized using NMR and MS data. The absolute configuration of psammaplysin F and all semisynthetic analogues was determined as 6R, 7R by comparison of ECD data with literature values. All compounds (1-11) were evaluated for their effect on cell cycle distribution and changes to cancer metabolism in LNCaP prostate cancer cells using a multiparametric quantitative single-cell imaging approach. These investigations identified that in LNCaP cells psammaplysin F and some urea analogues caused loss of mitochondrial membrane potential, fragmentation of the mitochondrial tubular network, chromosome misalignment, and cell cycle arrest in mitosis.

Emergence of MicroRNAs as Key Players in Cancer Cell Metabolism.

BACKGROUND: Metabolic reprogramming is a hallmark of cancer. MicroRNAs (miRNAs) have been found to regulate cancer metabolism by regulating genes involved in metabolic pathways. Understanding this layer of complexity could lead to the development of novel therapeutic approaches. CONTENT: miRNAs are noncoding RNAs that have been implicated as master regulators of gene expression. Studies have revealed the role of miRNAs in the metabolic reprogramming of tumor cells, with several miRNAs both positively and negatively regulating multiple metabolic genes. The tricarboxylic acid (TCA) cycle, aerobic glycolysis, de novo fatty acid synthesis, and altered autophagy allow tumor cells to survive under adverse conditions. In addition, major signaling molecules, hypoxia-inducible factor, phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin/phosphatase and tensin homolog, and insulin signaling pathways facilitate metabolic adaptation in tumor cells and are all regulated by miRNAs. Accumulating evidence suggests that miRNA mimics or inhibitors could be used to modulate the activity of miRNAs that drive tumor progression via altering their metabolism. Currently, several clinical trials investigating the role of miRNA-based therapy for cancer have been launched that may lead to novel therapeutic interventions in the future. SUMMARY: In this review, we summarize cancer-related metabolic pathways, including glycolysis, TCA cycle, pentose phosphate pathway, fatty acid metabolism, amino acid metabolism, and other metabolism-related oncogenic signaling pathways, and their regulation by miRNAs that are known to lead to tumorigenesis. Further, we discuss the current state of miRNA therapeutics in the clinic and their future potential.

Provider Decisions and Patient Outcomes After Premature Metformin Discontinuation.

The purpose of this study was to evaluate the effects of alternative antihyperglycemic therapy after discontinuation of metformin due to documented declining renal function. This retrospective, single-site study evaluated patients who had metformin discontinued between 1 January 1999 and 30 September 2013. Medical records were evaluated for documented adverse events, subsequent glycemic control, and costs associated with the alternative therapy. Patients served as their own controls. A total of 179 patients met study entry criteria, and their peak A1C was significantly higher within the year after metformin discontinuation (P <0.001). After the provider added new medications to control patients' blood glucose, their A1C by the end of the first year after discontinuing metformin was similar to their A1C while taking metformin. Significant weight gain accompanied the use of the medications added to replace metformin, with an average increase of 3.81 kg (P <0.001). Additionally, after discontinuing metformin, more patients experienced hypoglycemia with the addition of other medications to control their blood glucose (P <0.001). As expected, the cost of therapy was significantly higher (P <0.0001) after metformin was discontinued because metformin was generically available, whereas the replacement medications frequently were not. Providers should consider the expanded recommendations for the use of metformin in patients with mild to moderate stable renal dysfunction to help such patients avoid weight gain, hypoglycemia, loss of blood glucose control, and increased costs.

Adverse effects of androgen-deprivation therapy in prostate cancer and their management.

OBJECTIVE: To provide an up-to-date summary of current literature on the management of adverse effects of androgen-deprivation therapy (ADT). PATIENTS AND METHODS: All relevant medical literature on men with prostate cancer treated with ADT from 2005 to 2014, and older relevant papers, were reviewed. Recent health advisory statements from the Australian government, societies and advocacy groups have been incorporated to the document. RESULTS: There are numerous adverse effects of ADT that require pro-active prevention and treatment. Ranging from cardiovascular disease, diabetes and osteoporosis, to depression, cognitive decline and sexual dysfunction, the range of adverse effects is wide. Baseline assessment, monitoring, prevention and consultation from a multidisciplinary team are important in minimising the harm from ADT. CONCLUSIONS: This review provides a series of practical recommendations to assist with managing the adverse effects of ADT.

Targeting valine catabolism to inhibit metabolic reprogramming in prostate cancer.

Metabolic reprogramming and energetic rewiring are hallmarks of cancer that fuel disease progression and facilitate therapy evasion. The remodelling of oxidative phosphorylation and enhanced lipogenesis have previously been characterised as key metabolic features of prostate cancer (PCa). Recently, succinate-dependent mitochondrial reprogramming was identified in high-grade prostate tumours, as well as upregulation of the enzymes associated with branched-chain amino acid (BCAA) catabolism. In this study, we hypothesised that the degradation of the BCAAs, particularly valine, may play a critical role in anapleurotic refuelling of the mitochondrial succinate pool, as well as the maintenance of intracellular lipid metabolism. Through the suppression of BCAA availability, we report significantly reduced lipid content, strongly indicating that BCAAs are important lipogenic fuels in PCa. This work also uncovered a novel compensatory mechanism, whereby fatty acid uptake is increased in response to extracellular valine deprivation. Inhibition of valine degradation via suppression of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) resulted in a selective reduction of malignant prostate cell proliferation, decreased intracellular succinate and impaired cellular respiration. In combination with a comprehensive multi-omic investigation that incorporates next-generation sequencing, metabolomics, and high-content quantitative single-cell imaging, our work highlights a novel therapeutic target for selective inhibition of metabolic reprogramming in PCa.

Epidermal growth factor potentiates EGFR(Y992/1173)-mediated therapeutic response of triple negative breast cancer cells to cold atmospheric plasma-activated medium.

Cold atmospheric plasma (CAP) holds promise as a cancer-specific treatment that selectively kills various types of malignant cells. We used CAP-activated media (PAM) to utilize a range of the generated short- and long-lived reactive species. Specific antibodies, small molecule inhibitors and CRISPR/Cas9 gene-editing approaches showed an essential role for receptor tyrosine kinases, especially epidermal growth factor (EGF) receptor, in mediating triple negative breast cancer (TNBC) cell responses to PAM. EGF also dramatically enhanced the sensitivity and specificity of PAM against TNBC cells. Site-specific phospho-EGFR analysis, signal transduction inhibitors and reconstitution of EGFR-depleted cells with EGFR-mutants confirmed the role of phospho-tyrosines 992/1173 and phospholipase C gamma signaling in up-regulating levels of reactive oxygen species above the apoptotic threshold. EGF-triggered EGFR activation enhanced the sensitivity and selectivity of PAM effects on TNBC cells. The proposed approach based on the synergy of CAP and EGFR-targeted therapy may provide new opportunities to improve the clinical management of TNBC.

Systematic review of antitumour efficacy and mechanism of metformin activity in prostate cancer models.

Metformin, the first line pharmacotherapy for type 2 diabetes has demonstrated favourable effects in prostate cancer (PCa) across a range of studies evaluating PCa patient outcomes amongst metformin users. However, a lack of rigorously conducted prospective studies has stalled clinical use in this setting. Despite multiple studies evaluating the mechanisms underpinning antitumour effects of metformin in PCa, to date, no reviews have compared these findings. This systematic review and meta-analysis consolidates the mechanisms accounting for the antitumour effect of metformin in PCa and evaluates the antitumour efficacy of metformin in preclinical PCa studies. Data were obtained through Medline and EMBASE, extracted by two independent assessors. Risk of bias was assessed using the TOXR tool. Meta-analysis compared in vivo reductions of PCa tumour volume with metformin. In total, 447 articles were identified with 80 duplicates, and 261 articles excluded based on eligibility criteria. The remaining 106 articles were assessed and 71 excluded, with 35 articles included for systematic review, and eight included for meta-analysis. The mechanisms of action of metformin regarding tumour growth, viability, migration, invasion, cell metabolism, and activation of signalling cascades are individually discussed. The mechanisms by which metformin inhibits PCa cell growth are multimodal. Metformin regulates expression of multiple proteins/genes to inhibit cellular proliferation, cell cycle progression, and cellular invasion and migration. Published in vivo studies also conclusively demonstrate that metformin inhibits PCa growth. This highlights the potential of metformin to be repurposed as an anticancer agent, warranting further investigation of metformin in the setting of PCa.

GelMA and Biomimetic Culture Allow the Engineering of Mineralized, Adipose, and Tumor Tissue Human Microenvironments for the Study of Advanced Prostate Cancer In Vitro and In Vivo.

Increasing evidence shows bone marrow (BM)-adipocytes as a potentially important contributor in prostate cancer (PCa) bone metastases. However, a lack of relevant models has prevented the full understanding of the effects of human BM-adipocytes in this microenvironment. It is hypothesized that the combination of tunable gelatin methacrylamide (GelMA)-based hydrogels with the biomimetic culture of human cells would offer a versatile 3D platform to engineer human bone tumor microenvironments containing BM-adipocytes. Human osteoprogenitors, adipocytes, and PCa cells are individually cultured in vitro in GelMA hydrogels, leading to mineralized, adipose, and PCa tumor 3D microtissues, respectively. Osteoblast mineralization and tumor spheroid formation are tailored by hydrogel stiffness with lower stiffnesses correlating with increased mineralization and tumor spheroid size. Upon coculture with tumor cells, BM-adipocytes undergo morphological changes and delipidation, suggesting reciprocal interactions between the cell types. When brought in vivo, the mineralized and adipose microtissues successfully form a humanized fatty bone microenvironment, presenting, for the first time, with human adipocytes. Using this model, an increase in tumor burden is observed when human adipocytes are present, suggesting that adipocytes support early bone tumor growth. The advanced platform presented here combines natural aspects of the microenvironment with tunable properties useful for bone tumor research.

Stochastic epithelial-mesenchymal transitions diversify non-cancerous lung cell behaviours.

Epithelial-mesenchymal plasticity (EMP) is a hallmark of cancer. By enabling cells to shift between different morphological and functional states, EMP promotes invasion, metastasis and therapy resistance. We report that near-diploid non-cancerous human epithelial lung cells spontaneously shift along the EMP spectrum without genetic changes. Strikingly, more than half of single cell-derived clones adopt a mesenchymal morphology. We independently characterise epithelial-like and mesenchymal-like clones. Mesenchymal clones lose epithelial markers, display larger cell aspect ratios and lower motility, with mostly unaltered proliferation rates. Stemness marker expression and metabolic rewiring diverge independently of phenotypes. In 3D culture, more epithelial clones become mesenchymal-like. Thus, non-cancerous epithelial cells may acquire cancer metastasis-associated features prior to genetic alterations and cancerous transformation.

TGFß and CIS Inhibition Overcomes NK-cell Suppression to Restore Antitumor Immunity.

Antibodies targeting "immune checkpoints" have revolutionized cancer therapy by reactivating tumor-resident cytotoxic lymphocytes, primarily CD8+ T cells. Interest in targeting analogous pathways in other cytotoxic lymphocytes is growing. Natural killer (NK) cells are key to cancer immunosurveillance by eradicating metastases and driving solid tumor inflammation. NK-cell antitumor function is dependent on the cytokine IL15. Ablation of the IL15 signaling inhibitor CIS (Cish) enhances NK-cell antitumor immunity by increasing NK-cell metabolism and persistence within the tumor microenvironment (TME). The TME has also been shown to impair NK-cell fitness via the production of immunosuppressive transforming growth factor ß (TGFß), a suppression which occurs even in the presence of high IL15 signaling. Here, we identified an unexpected interaction between CIS and the TGFß signaling pathway in NK cells. Independently, Cish- and Tgfbr2-deficient NK cells are both hyperresponsive to IL15 and hyporesponsive to TGFß, with dramatically enhanced antitumor immunity. Remarkably, when both these immunosuppressive genes are simultaneously deleted in NK cells, mice are largely resistant to tumor development, suggesting that combining suppression of these two pathways might represent a novel therapeutic strategy to enhance innate anticancer immunity.

Studying the Metabolism of Epithelial-Mesenchymal Plasticity Using the Seahorse XFe96 Extracellular Flux Analyzer.

The critical role of metabolism in facilitating cancer cell growth and survival has been demonstrated by a combination of methods including, but not limited to, genomic sequencing, transcriptomic and proteomic analyses, measurements of radio-labelled substrate flux and the high throughput measurement of oxidative metabolism in unlabelled live cells using the Seahorse Extracellular Flux (XF) technology. These studies have revealed that tumour cells exhibit a dynamic metabolic plasticity, using numerous pathways including both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to support cell proliferation, energy production and the synthesis of biomass. These advanced technologies have also demonstrated metabolic differences between cancer cell types, between molecular subtypes within cancers and between cell states. This has been exemplified by examining the transitions of cancer cells between epithelial and mesenchymal phenotypes, referred to as epithelial-mesenchymal plasticity (EMP). A growing number of studies are demonstrating significant metabolic alterations associated with these transitions, such as increased use of glycolysis by triple negative breast cancers (TNBC) or glutamine addiction in lung cancer. Models of EMP, including invasive cell lines and xenografts, isolated circulating tumour cells and metastatic tissue have been used to examine EMP metabolism. Understanding the metabolism supporting molecular and cellular plasticity and increased metastatic capacity may reveal metabolic vulnerabilities that can be therapeutically exploited. This chapter describes protocols for using the Seahorse Extracellular Flux Analyzer (XFe96), which simultaneously performs real-time monitoring of oxidative phosphorylation and glycolysis in living cells. As an example, we compare the metabolic profiles generated from two breast cancer sublines that reflect epithelial and mesenchymal phenotypes, respectively. We use this example to show how the methodology described can generate bioenergetic results that in turn can be correlated to EMP phenotypes. Normalisation of bioenergetic studies should be considered with respect to cell number, and to potential differences in mitochondrial mass, itself being an important bioenergetics endpoint.

Allele-Specific MicroRNA-Mediated Regulation of a Glycolysis Gatekeeper PDK1 in Cancer Metabolism.

BACKGROUND: Emerging evidence has revealed that genetic variations in microRNA (miRNA) binding sites called miRSNPs can alter miRNA binding in an allele-specific manner and impart prostate cancer (PCa) risk. Two miRSNPs, rs1530865 (G > C) and rs2357637 (C > A), in the 3' untranslated region of pyruvate dehydrogenase kinase 1 (PDK1) have been previously reported to be associated with PCa risk. However, these results have not been functionally validated. METHODS: In silico analysis was used to predict miRNA-PDK1 interactions and was tested using PDK1 knockdown, miRNA overexpression and reporter gene assay. RESULTS: PDK1 expression was found to be upregulated in PCa metastasis. Further, our results show that PDK1 suppression reduced the migration, invasion, and glycolysis of PCa cells. Computational predictions showed that miR-3916, miR-3125 and miR-3928 had a higher binding affinity for the C allele than the G allele for the rs1530865 miRSNP which was validated by reporter gene assays. Similarly, miR-2116 and miR-889 had a higher affinity for the A than C allele of the rs2357637 miRSNP. Overexpression of miR-3916 and miR-3125 decreased PDK1 protein levels in cells expressing the rs1530865 SNP C allele, and miR-2116 reduced in cells with the rs2357637 SNP A allele. CONCLUSIONS: The present study is the first to report the regulation of the PDK1 gene by miRNAs in an allele-dependent manner and highlights the role of PDK1 in metabolic adaption associated with PCa progression.

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.

Leptin antagonism inhibits prostate cancer xenograft growth and progression.

Hyperleptinaemia is a well-established therapeutic side effect of drugs inhibiting the androgen axis in prostate cancer (PCa), including main stay androgen deprivation therapy (ADT) and androgen targeted therapies (ATT). Given significant crossover between the adipokine hormone signalling of leptin and multiple cancer-promoting hallmark pathways, including growth, proliferation, migration, angiogenesis, metabolism and inflammation, targeting the leptin axis is therapeutically appealing, especially in advanced PCa where current therapies fail to be curative. In this study, we uncover leptin as a novel universal target in PCa and are the first to highlight increased intratumoural leptin and leptin receptor (LEPR) expression in PCa cells and patients' tumours exposed to androgen deprivation, as is observed in patients' tumours of metastatic and castrate resistant (CRPC) PCa. We also reveal the world-first preclinical evidence that demonstrates marked efficacy of targeted leptin-signalling blockade, using Allo-aca, a potent, specific, and safe LEPR peptide antagonist. Allo-aca-suppressed tumour growth and delayed progression to CRPC in mice bearing LNCaP xenografts, with reduced tumour vascularity and altered pathways of apoptosis, transcription/translation, and energetics in tumours determined as potential mechanisms underpinning anti-tumour efficacy. We highlight LEPR blockade in combination with androgen axis inhibition represents a promising new therapeutic strategy vital in advanced PCa treatment.

Revisiting Glycogen in Cancer: A Conspicuous and Targetable Enabler of Malignant Transformation.

Once thought to be exclusively a storage hub for glucose, glycogen is now known to be essential in a range of physiological processes and pathological conditions. Glycogen lies at the nexus of diverse processes that promote malignancy, including proliferation, migration, invasion, and chemoresistance of cancer cells. It is also implicated in processes associated with the tumor microenvironment such as immune cell effector function and crosstalk with cancer-associated fibroblasts to promote metastasis. The enzymes of glycogen metabolism are dysregulated in a wide variety of malignancies, including cancers of the kidney, ovary, lung, bladder, liver, blood, and breast. Understanding and targeting glycogen metabolism in cancer presents a promising but under-explored therapeutic avenue. In this review, we summarize the current literature on the role of glycogen in cancer progression and discuss its potential as a therapeutic target for cancer treatment.

Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.

Histone deacetylases (HDACs) drive innate immune cell-mediated inflammation. Here we identify class IIa HDACs as key molecular links between Toll-like receptor (TLR)-inducible aerobic glycolysis and macrophage inflammatory responses. A proteomic screen identified the glycolytic enzyme pyruvate kinase M isoform 2 (Pkm2) as a partner of proinflammatory Hdac7 in murine macrophages. Myeloid-specific Hdac7 overexpression in transgenic mice amplifies lipopolysaccharide (LPS)-inducible lactate and promotes a glycolysis-associated inflammatory signature. Conversely, pharmacological or genetic targeting of Hdac7 and other class IIa HDACs attenuates LPS-inducible glycolysis and accompanying inflammatory responses in macrophages. We show that an Hdac7-Pkm2 complex acts as an immunometabolism signaling hub, whereby Pkm2 deacetylation at lysine 433 licenses its proinflammatory functions. Disrupting this complex suppresses inflammatory responses in vitro and in vivo. Class IIa HDACs are thus pivotal intermediates connecting TLR-inducible glycolysis to inflammation via Pkm2.

Disruption of Glycogen Utilization Markedly Improves the Efficacy of Carboplatin against Preclinical Models of Clear Cell Ovarian Carcinoma.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses-of more than 10-fold lower than previously reported for other cancers-significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

Inhibition of inosine monophosphate dehydrogenase reduces adipogenesis and diet-induced obesity.

We previously described a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in lipid accumulation. Here we present data which demonstrate that IMPDH activity is required for differentiation of preadipocytes into mature, lipid-laden adipocytes and maintenance of adipose tissue mass. In 3T3-L1 preadipocytes inhibition of IMPDH with mycophenolic acid (MPA) reduced intracellular GTP levels by 60% (p<0.05) and blocked adipogenesis (p<0.05). Co-treatment with guanosine, a substrate in the salvage pathway of nucleotide biosynthesis, restored GTP levels and adipogenesis demonstrating the specificity of these effects. Treatment of diet-induced obese mice with mycophenolate mofetil (MMF), the prodrug of MPA, for 28 days did not affect food intake or lean body mass but reduced body fat content (by 36%, p=0.002) and adipocyte size (p=0.03) and number. These data suggest that inhibition of IMPDH may represent a novel strategy to reduce adipose tissue mass.

Vaginal Jade Eggs: Ancient Chinese Practice or Modern Marketing Myth?

OBJECTIVE: To identify evidence that vaginal jade eggs were recommended or used in sexual health practices or for pelvic muscle exercises in ancient Chinese culture. MATERIALS AND METHODS: A search of the online databases of 4 major Chinese art and archeology collections in the United States. RESULTS: More than 5000 jade objects were viewable in online databases. No vaginal jade eggs were identified. CONCLUSIONS: No evidence was found to support the claim that vaginal jade eggs were used for any indication in ancient Chinese culture.