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N-acetyl-L-aspartyl-L-glutamate peptidase-like 2 (NAALADL2) is a member of the glutamate carboxypeptidase II family, best characterized by prostate-specific membrane antigen (PSMA/NAALAD1). Using immunohistochemistry (IHC), we have shown overexpression of NAALADL2 in colon and prostate tumours when compared with benign tissue. In prostate cancer, NAALADL2 expression was associated with stage and Grade, as well as circulating mRNA levels of the NAALADL2 gene. Overexpression of NAALADL2 was shown to predict poor survival following radical prostatectomy. In contrast to PSMA/NAALAD1, NAALADL2 was localized at the basal cell surface where it promotes adhesion to extracellular matrix proteins. Using stable knockdown and overexpression cell lines, we have demonstrated NAALADL2-dependent changes in cell migration, invasion and colony-forming potential. Expression arrays of the knockdown and overexpression cell lines have identified nine genes that co-expressed with NAALADL2, which included membrane proteins and genes known to be androgen regulated, including the prostate cancer biomarkers AGR2 and SPON2. Androgen regulation was confirmed in a number of these genes, although NAALADL2 itself was not found to be androgen regulated. NAALADL2 was also found to regulate levels of Ser133 phosphorylated C-AMP-binding protein (CREB), a master regulator of a number of cellular processes involved in cancer development and progression. In combination, these data suggest that changes in expression of NAALADL2 can impact upon a number of pro-oncogenic pathways and processes, making it a useful biomarker for both diagnosis and prognosis.

Androgen receptor (AR) signaling remains an important regulatory pathway in castrate-resistant prostate cancer, and its transcriptional downregulation could provide a new line of therapy. A number of small-molecule ligands have previously demonstrated the ability to stabilize G-quadruplex structures and affect gene transcription for those genes whose promoters contain a quadruplex-forming sequence. Herein, we report the probable formation of new G-quadruplex structure present in the AR promoter in a transcriptionally important location. NMR spectroscopy, circular dichroism, UV spectroscopy, and UV thermal melting experiments for this sequence are consistent with G-quadruplex formation. Fluorescence resonance energy transfer (FRET) melting studies have identified a novel compound, MM45, which appears to stabilize this G-quadruplex at submicromolar concentrations. The effects of MM45 have been investigated in prostate cancer cell lines where it has been shown to inhibit cell growth. A reporter assay intended to isolate the effect of MM45 on the G-quadruplex sequence showed dose-dependent transcriptional repression only when the AR promoter G-quadruplex sequence is present. Dose-dependent transcriptional repression of the AR by MM45 has been demonstrated at both a protein and mRNA level. This proof of concept study paves the route toward a potential alternative treatment pathway in castrate-resistant prostate cancer.

Background: Medical therapy for men with prostate cancer (PCa) is evolving, including recent evidence to support the use of chemotherapy for men with hormone sensitive disease. PI3K/AKT/mTOR pathway aberrations are common in patients with primary PCa and almost universal in metastatic tumours. The mTOR pathway acts as a central regulator of tumour cell metabolism, proliferation, cell cycle progression and immune regulation. The aim of this study was to investigate the effects of mTOR inhibitor AZD2014 on tumour growth and immune infiltration in a genetically engineered mouse model of PCa. Methods: PtenL/L;PB-Cre4 mice, which developed invasive hormone-sensitive prostate tumours between 10-14 months of age, were enrolled (n = 13-15 per group) and treated with AZD2014 (15mg/Kg) or vehicle orally for 14 doses (QD 5/7) with or without castration. Tumour volumes were assessed by ultrasound imaging. Tumour samples were collected within 4 hours post 14th dose for histological and molecular analysis. Results: AZD2014 was well tolerated, no overt toxicity was observed. Mean plasma concentrations of AZD2014 were 4.4±2.1μM at time of tumour sampling. AZD2014 alone or AZD2014+castration inhibited mTORC1 and mTORC2 activity as demonstrated by reduced p4EBP1(Thr37/46) 48%±27% (P Conclusion: These data confirm the anti cancer effect of AZD2014 in this PCa model. Furthermore, AZD2014+castration was more effective than either treatment as a single agent. The increased intra-tumoral T cell infiltration observed may have contributed to the anti-tumour effect. Further studies are ongoing to maximise this therapeutic effect and will inform current clinical studies in men with hormone sensitive, high risk prostate cancer. Citation Format: Chiranjeevi Sandi, Antonio Ramos-Montoya, Sergio L. Filisbino, Adina Hughes, Suzanne Mosely, Michelle Morrow, Robert W. Wilkinson, Sarah Jurmeister, Karan Wadhwa, Frances M. Richards, Duncan I. Jodrell, Sabina Cosulich, Barry R. Davies, Simon Pacey. The dual mTOR inhibitor, AZD2014, and castration increase intra-tumoral immune cell infiltration and anti-tumour activity in a genetically engineered mouse model of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 389.

Carbohydrate response element-binding protein (ChREBP) is a transcription factor that mediates glucose signaling in mammalian liver, leading to the expression of different glycolytic and lipogenic genes, such as pyruvate kinase (L-PK) and fatty acid synthase (FAS). The current model for ChREBP activation in response to sugar phosphates holds that glucose metabolization to xylulose 5-phosphate (X-5-P) triggers the activation of protein phosphatase 2A, which dephosphorylates ChREBP and leads to its nuclear translocation and activation. However, evidence indicates that glucose 6-phosphate (G-6-P) is the most likely signal metabolite for the glucose-induced transcription of these genes. The glucose derivative that is responsible for carbohydrate-dependent gene expression remains to be identified. The difficulties in measuring G-6-P and X-5-P concentrations simultaneously and in changing them independently have hindered such identification. To discriminate between these possibilities, we adapted a liquid chromatography mass spectrometry method to identify and quantify sugar phosphates in human hepatocarcinoma cells (Hep G2) and rat hepatocytes in response to different carbon sources and in the presence/absence of a glucose-6-phosphate dehydrogenase inhibitor. We also used this method to demonstrate that these cells could not metabolize 2-deoxyglucose beyond 2-deoxyglucose-6-phosphate. The simultaneous quantification of sugar phosphates and FAS and L-PK expression levels demonstrated that both X-5-P and G-6-P play a role in the modulation of gene expression. In conclusion, this report presents for the first time a single mechanism that incorporates the effects of X-5-P and G-6-P on the enhancement of the expression of carbohydrate-responsive genes. © 2012 the American Physiological Society., This study was supported by the projects SAF2009-12602 and SAF2011-25726 and by RD06/0020/0046 and RD06/0014/0025 from Red Temática de Investigación Cooperativa en Cáncer and Red de Centros FIS-RECAVA, respectively, from the Instituto de Salud Carlos III, both funded by the Ministerio de Ciencia e Innovación-Spanish government and European Regional Development Funds “Una manera de hacer Europa.” It has also received financial support from the European Union-funded project ETHERPATHS (FP7-KBBE-222639) (http://www.etherpaths.org/) and from the Agència de Gestió d'Ajuts Universitaris i de Recerca-Generalitat de Catalunya (2009SGR01308, 2006ITT-10007, and 2009CTP-00026).

Cyclin-dependent kinases CDK4 and CDK6 are essential for the control of the cell cycle through the G1 phase. Aberrant expression of CDK4 and CDK6 is a hall- mark of cancer, which would suggest that CDK4 and CDK6 are attractive targets for cancer therapy. Herein, we report that calcein AM is a potent specific inhibitor of CDK4 and CDK6 in HCT116 human colon adenocarcinoma cells, inhibiting retinoblastoma protein (pRb) phosphorylation and inducing cell cycle arrest in the G1 phase. The metabolic effects of calcein AM (the calcein acetoxymethyl-ester) on HCT116 cells were also evaluated and the flux between the oxidative and non-oxidative branches of the pentose phos-phate pathway was significantly altered. To elucidate whe-ther these metabolic changes were due to the inhibition of CDK4 and CDK6, we also characterized the metabolic profile of a CDK4, CDK6 and CDK2 triple knockout of mouse embryonic fibroblasts. The results show that the metabolic profile associated with the depletion of CDK4, CDK6 and CDK2 coincides with the metabolic changes induced by calcein AM on HCT116 cells, thus confirming that the inhibition of CDK4 and CDK6 disrupts the balance between the oxidative and non-oxidative branches of the pentose phosphate pathway. Taken together, these results indicate that low doses of calcein can halt cell division and kill tumor cells. Thus, selective inhibition of CDK4 and CDK6 may be of greater pharmacological interest, since inhibitors of these kinases affect both cell cycle progression and the robust metabolic profile of tumors.

Background: An estimated 220,800 cases and 27,540 deaths from prostate cancer (PCa) will occur in the USA during 2015. Altered PI3K/AKT/mTOR signalling contributes to prostate cancer progression and transition to androgen-independent disease, for example one study reported 42% of primary and 100% of metastatic PCa tumours exhibited mutations, altered expression or copy number variations within this pathway. First generation mTOR inhibitors (preferentially inhibit mTORC1), have had limited anti-cancer effect in patients with PCa, possibly due to negative feedback activation of the AKT pathway via mTORC2. The dual mTORC1/2 inhibitor, AZD2014, may overcome this liability. Using a genetically engineered PTEN conditional mouse model (Ptenloxp/loxp;PB-Cre4), we have investigated the effects of AZD2014. The studies complement a clinical trial (NCT02064608) of AZD2014, given to men before radical prostatectomy and are timed for when invasive prostate carcinomas develop in the model around 10-14 months prior to onset of resistance to castration through AKT pathway activation. AZD2014, 15mg/kg daily, oral (with or without castration) or vehicle were administered for 14 days. Results: AZD2014 was well tolerated with no overt toxicity observed. Pharmacokinetic (PK) analysis revealed mean concentrations of 4.4±2.1μM of AZD2014 in the plasma samples collected 4 hours after day 14 dose. AZD2014 alone or combined with castration inhibited mTORC1 and mTORC2 measured by reductions in p4EBP1(Thr37/46) by approximately 48%±27% (p Conclusions: Short term (14 days) treatment with AZD2014 with or without castration was associated with both pharmacodynamic and anti-tumour effects. The t-Cho/Cr ratio, previously reported as positively correlated with Gleason score in PCa patients, might be, in addition to our standard mTOR PD markers, utilised as a non-invasive biomarker of AZD2014 activity. The primary and phenotypic biomarker effects of monotherapy with AZD2014 in this relevant genetically engineered mouse model of prostate cancer will be compared with paired biopsies from the ongoing exploratory window study in the prostate cancer patients prior to prostatectomy, and may inform potential novel combination approaches that are translatable to the clinic. Citation Format: Chiranjeevi Sandi, Antonio Ramos-Montoya, Sergio L. Felisbino, Sarah Jurmeister, Basetti Madhu, Karan Wadhwa, John R. Griffiths, Frances M. Richards, Duncan I. Jodrell, David E. Neal, Sabina Cosulich, Barry Davies, Simon Pacey. Preclinical evaluation of dual mTOR inhibitor, AZD2014, in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A123.

The literature covering neurotensin (NT) and its signalling pathways, receptors, and biological profile is complicated by the fact that the discovery of three NT receptor subtypes has come to light only in recent years. Moreover, a lot of this literature explores NT in the context of the central nervous system and behavioral studies. However, there is now good evidence that the up-regulation of NT is intimately involved in cancer development and progression. This Review aims to summarize the isolation, cloning, localization, and binding properties of the accepted receptor subtypes (NTR1, NTR2, and NTR3) and the molecules known to bind at these receptors. The growing role these targets are playing in cancer research is also discussed. We hope this Review will provide a useful overview and a one-stop resource for new researchers engaged in this field at the chemistry−biology interface.

Tumour classification has traditionally focused on differentiation and cellular morphology, and latterly on the application of genomic approaches. By combining chromatin immunoprecipitation with expression array, it has been possible to identify direct gene targets for transcription factors for nuclear hormone receptors. At the same time, there have been great strides in deriving stem and progenitor cells from tissues. It is therefore timely to propose that pairing the isolation of these cell subpopulations from tissues and tumours with these genomics approaches will reveal conserved gene targets for transcription factors. By focusing on transcription factors (lineage-survival oncogenes) with roles in both organogenesis and tumourigenesis at multiple organ sites, we suggest that this comparative genomics approach will enable developmental biology to be used more fully in relation to understanding tumour progression and will reveal new cancer markers. We focus here on neurogenesis and neuroendocrine differentiation in tumours.

Metabolic control analysis predicts that effects on tumor growth are likely to be obtained with lower concentrations of drug, if an enzyme with a high control coefficient on tumor growth is being inhibited. Here we measure glucose-6-phosphate dehydrogenase (G6PDH) control coefficient on in vivo tumor growth using mice bearing Ehrlich ascites tumor cells. We used dehydroepiandrosterone-sulphate (DHEA-S), an uncompetitive inhibitor of this enzyme and the in situ cytochemical method to measure the enzyme activity changes that accompany changes on tumor cell growth. This method ensures that the enzyme activity determined is the one existing in the in situ conditions and enables computing a control coefficient in in situ conditions. From the data obtained on tumor cell number and the in situ enzyme activities in absence and presence of DHEA-S, a control coefficient of 0.41 for G6PDH on tumor cell growth was computed. This value is approximately the half of the transketolase control coefficient value of 0.9 previously reported. Moreover, the use of in situ methods to assess enzyme activities, applied for first time for the calculation of control coefficients in this study, opens new avenues to the use of uncompetitive inhibitors for the measurement of in situ control coefficients.

Background Castration-resistant prostate cancer is a heterogeneous conditon that is poorly characterised. Although the androgen receptor (AR) is of particular importance, other factors such as c-Myc and the E2F family also have a role in later stage disease. Hes6 is a transcription cofactor associated with stem-cell characteristics in neural tissue, but its role in cancer remains uncertain. We aimed to assess the role of Hes6 in castration-resistant prostate cancer and to identify its prognostic and therapeutic relavance. Methods We transduced an androgen-sensitive cell line (LNCaP) to constitutively overexpress Hes6 and luciferase to permit xenografting in NOD-SCID gamma mice and bioluminescent monitoring of tumour growth. We used microarray genomics and chromatin immunoprecipitation-sequencing to study the molecular environment underpinning the cellular effects of Hes6. We generated a 61-patient hormone relapsed prostate cancer tissue microarray and assessed 285 publicly available human prostate cancers in a meta-analysis to profile Hes6 and its associated regulome in aggressive human disease. Findings We found that Hes6 was upregulated in aggressive human prostate cancer with its expression controlled by c-Myc and AR. Hes6 drove castration-resistant tumour growth by enhancing AR transcriptional activity in the absence of ligand binding. The AR was preferentially directed to a regulatory network enriched for other transcription factors including E2F1. We found a physical interaction between E2F1 and both Hes6 and AR, with increased occupancy of AR at E2F1 target sites in the presence of Hes6. In the clinical setting, we uncovered a Hes6-associated signature that predicts poor outcome in prostate cancer on longitudinal follow-up of men after prostatectomy. We found, in our model of castration resistance, that this process can be pharmacologically targeted by inhibition of PLK1, part of the Hes6 and E2F1 regulome, with restoration of sensitivity to castration. Interpretation We have shown for the first time, to our knowledge, the crucial role of Hes6 in development of castration-resistant prostate cancer and identified its potential in patient-specific therapeutic strategies. Funding Cambridge Biomedical Research Centre, GlaxoSmithKline, Raymond and Beverley Sackler Studentship.

The AR signalling axis is central to Prostate Cancer (PrCa) biology, shown by its use in both screening (PSA) and treatment of the disease (androgen deprivation therapy). Identifying direct target genes of the AR will provide a better understanding the key signalling pathways controlled by the AR in PrCa and could lead to improved biomarkers and future therapeutic targets for the disease. To this end we have performed fine mapping of androgen responsive gene expression in prostate cancer cell lines using Illumina bead-arrays, coupled with AR chromatin-immunoprecipitation and Solexa sequencing technology (ChIP-seq). This approach generated over ten thousand candidate AR target genes. We have then undertaken large scale, cross-platform validation using BioTrove Realtime PCR panels which allow the measurement of >600 transcripts simultaneously. Using this combination of approaches we have identified and validated several hundred AR regulated genes. We found that metabolic enzymes and kinases were significantly enriched in the set of direct AR regulated genes. In nine independent clinical gene expression studies we found that the most consistently up-regulated AR target gene was a calcium regulated kinase. At the protein level this kinase showed increased expression in two independent patient cohorts. A small molecule inhibitor of this kinase reduced the growth of a panel of PrCa cell lines in vitro and tumour growth in xenograft models. Interestingly, we found that the levels of this AR regulated kinase were reduced in clinical PrCa following androgen deprivation therapy, but were raised in Castrate Resistant disease. Therefore, we have identified an AR regulate kinase which contributes to tumour growth and is a potential marker and therapeutic target in both hormone naïve and anti-androgen resistant disease. In summary, these data show that combining expression analysis, ChIP-seq and high through-put validation has identified a framework to understand the oncogenic functions of the AR in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1714.